املقاومة املتعددة للمضادات احليوية من قبل البكترييا املعزولة من عينات من شواطئ غزة االستجمامية

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2 Islamic University - Gaza Deanship of Graduate Studies Faculty of Science Environmental Sciences Master Programme/Environmental Health اجلامعة اإلسالمية غزة شؤون البحث العلمي والدراسات العليا كلية العلوم برنامج ماجشتري العلوم البيئية الصحة البيئية Multiple Antimicrobial Resistances of Bacteria Isolated from Samples of Recreational Beach in Gaza Strip املقاومة املتعددة للمضادات احليوية من قبل البكترييا املعزولة من عينات من شواطئ غزة االستجمامية By Fatema Mohammed Elfarra Supervised by Prof. Dr. Abdelraouf A. Elmanama Prof. of Microbiology Dr. Kamal Elnabris Associate prof. of Marine Bio Environmental A Thesis Submitted in Partial Fulfillment of the Requirement for the Degree Master of Science in Environmental Sciences The Islamic University Gaza Palestine 215 AD AH

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4 بسم اهلل الرمحن الرحيم In the Name of Allah {ر ب أ و ز ع ن ي أ ن أ ش ك ر ن ع م ت ك ال ت ي أ ن ع م ت ع ل ي و ع ل ى و ال د ي و أ ن أ ع م ل ص ال ح ا ت ر ض اه و أ د خ ل ن ي ب ر ح م ت ك ف ي ع ب اد ك الص ال ح ين} اآلية: (النمل 91( O my Lord! So order me that I may be grateful for Thy favors which Thou have bestowed on me and on my parents, and that I may work the righteousness that will please Thee: and admit me, by Thy Grace, to the ranks of Thy Righteous Servants I

5 Dedication I dedicate this study to the soul of my beloved father, who always wished to see me standing here today, to the kindest heart ever, to my mother for her endless prayers for me to fulfill my ambition. With my respect and love Fatema El Farra II

6 Acknowledgment I fully praise Allah for all the blessings he grants us, I also deeply thank those ever encouraged me so finally I had my dream turned to be a bright fact. My special appreciation is for Prof. Dr. Abdelraouf Elmanama for his endless patience, guidance, support and valuable pieces of advice, which deeply enriched my research. I can never forget Dr. Kamal Elnabris rich suggestions that paved the way for my study to see the light. Deepest thanks for the staff of Environmental Science- post graduate program in the Environment and Earth Sciences Department at the Islamic University, for their academic and scientific supervision throughout my study. To all staff at the public health laboratory-ministry of health for all the help in doing the sample analysis and full access to their facilities that they provided me. To the Environmental and Rural research center- IUG for assisting me in sampling. This work would not have been possible without the extensive field Mr. Abdel Kareem Abu Hatab for helping me in sampling. Lastly, I offer my regards and respect to all those who ever supported me in any aspect during the completion of my project. III

7 Abstract The aim of the present study is a document the occurrence of pathogenic bacteria in seawater, beach sand and mussel samples collected from five locations along the coast of the Gaza strip and to determine the extent that these bacteria possess multi-drug resistance. Pathogens were isolated and identified by microbiological and biochemical methods and their susceptibility was determined against different antimicrobial agents by using the agar disc diffusion method and according to NCCLS (National Committee for Clinical and Laboratory Standard) guidelines. In most locations, cultivable fecal indicator bacteria such as fecal coliform (FC) and fecal streptococci (FS) were more abundant in the sand (an average of cfu/1 g) than in the sea water (579.5 cfu/1ml) and the number of FS in sand samples were higher than that of FC. Population density of both FC and FS was generally higher at Khan Yunis, South of Wadi Gaza outlet and North of Wadi Gaza outlet. Mussels were found to accumulate more FS than FC. The results also indicated that all sites comply with mandatory standards for water quality in the directive, and only the northern side of Wadi Gaza outlet do not achieve the more stringent guideline values for both FC and FS in water. Conventional biochemical tests identified 411 isolates belong to four bacterial groups of Enterobacteriaceae (42.4%), Enterococci (17.6%), Pseudomonas aeruginosa (23.7%) and Staphylococci (16.3%). Enterobacteriaceae, Enterococci, Pseudomonas aeruginosa and Staphylococci isolates were tested for their susceptibility to 1, 6, 8 and 1 antimicrobials respectively. The incidence of multiple resistances for the four bacterial groups from the different sample types (mussels, seawater and beach sand) and from the different sampling locations are presented. Amongst the Enterobacteriaceae isolates, 94.3% were resistant to at least one antimicrobial agent and 8.5% exhibited simultaneous resistance to more than one agent i.e., they are Multiple Antimicrobial Resistance (MAR). It also found that 1% of Enterococci isolates were resistant at least to one antimicrobial and 89.4% were MAR. For Pseudomonas aeruginosa isolates, 68.5% were resistant at least one agent and 43.8% were MAR. Staphylococci isolates showed that 98.4% were resistant to at least one agent and 88.6% were MAR. The greatest frequency of resistant in all Enterobacteriaceae isolates was found against Tetracycline (67.9%) and Amoxicillin-clavulanic (56%). For the Enterococci, the highest resistance was against Vancomycin (84.8%) and Ampicillin (8.3%). IV

8 Pseudomonas aeruginosa highest resistant was against Piperacillin (55.1%). The highest resistance of Staphylococci isolates was against Penicillin )91.8%(, Vancomycin )68.9%( and Oxacillin )62.3%(. All the calculated values of MAR index were higher than.2 demonstrating that the area of study is considered a high risk source of contamination environment where antimicrobial agents use are common. In conclusion, the results of the present study shows the widespread occurrence of antibiotic resistant bacteria in all types of samples tested and from all locations along the coast of the Gaza strip and their presence may become a potential human health hazard. Consequently efforts should be exerted to minimize the discharge of untreated or partially treated sewage from both domestic and hospitals sources which represent the prime source of antimicrobial agents in the marine environment in this area. Keywords: Antimicrobial resistance, multiple antimicrobial resistances, Enterobacteriaceae, Gaza beach, Enterococci, Staphylococci, Pseudomonas aeruginosa V

9 Abstract in Arabic language الملخص ر ذف ز ا ذساسخ إ ى ر ث ك ج د ا جىز ش ب ا شضخ ا مب خ ضبداد ا ىش ث خ ف ب ا جحش س بي ا شبطئ ػ بد ث ح ا جحش ا ز ر ج ؼ ب خ سخ الغ ػ ى ط ي سبح لطبع غضح رحذ ذ دسجخ ا زالن ز ا جىز ش ب مب خ ا زؼذدح ألد خ. ر ػضي ا جىز ش ب ا شضخ رؼش ف ب ثب طشق ا ىش ث ج خ ا ى بئ خ ا ح خ وز ه ر رحذ ذ مب ز ب ج ػخ خز فخ ا ضبداد ا ىش ث خ ثبسزخذا طش مخ شش األلشاص فمب ؼب ش ا ج خ ا ط خ م بسبد ا سش ش خ ا خجش خ.(NCCLS( ف ؼظ ا الغ وب ذ ثىزش ب ا م ا جشاص خ cfu( / 1 ج ( ب ف ب ا جحش cfu أوثش فشح ف ا ش بي )ث ز سظ (FS) ا ؼمذ بد ا جشاص خ (FC) /1 ) وب ػذد FSف ػ بد ا ش أػ ى.FC وب ذ وثبفخ وال FS FC أػ ى ف بطك خب س ج ة ادي غضح ش بي ادي غضح. ر ا ؼث س ػ ى أػذاد أوثش FS FC زشاو خ ف ث ح ا جحش. و ب أشبسد ا زبئج أ ج غ ا الغ رزفك غ ا ؼب ش اإل ضا خ ج دح ا ب أ ج غ ا الغ ب ػذا ا جب ت ا ش ب ادي غضح رحمك ا ؼب ش ا ز ج خ األوثش صشا خ ج دح ا ب ى.FS FC االخزجبساد ا ى بئ خ ا ح خ حذدد 411 ػض خ ر ز إ ى أسثغ ج ػبد وب ذ وب زب %42.4 ثىز ش خ ؼ خ %17.6 ى ساد ؼ خ %23.7 صائفخ ص جبس خ %16.3 ى ساد ػ م د خ. ر اخزجبس مب خ ا جىز ش ب ا ؼ خ ي 1 ضبداد ىش ث خ ا ى ساد ا ؼ خ ي 6 ضبداد ىش ث خ ا ضائفخ ا ض جبس خ ي 8 ضبداد ىش ث خ ا ى ساد ا ؼ م د خ ي 1 ضبداد ىش ث خ. أظ شد زبئج مب خ ا ضبداد ا ىش ث خ ا ؼض خ ا خ سخ الغ ا خز فخ أ 94.3 ا جىز ش ب ا ؼ خ مب خ احذ ضبداد ا ىش ثبد ػ ى األل أ 8.5 وب ذ مب خ الث أ أوثش ضبداد ا ىش ثبد. و ب جذ أ 1 ػضالد ا ى ساد ا ؼ خ وب ذ مب خ ضبد ىش ث احذ ػ ى األل 89.4 مب خ ألوثش ضبد ىش ث. وب ذ 68.5 ػضالد ا ضائفخ ا ض جبس خ مب خ ضبد ىش ث احذ ػ ى األل 43.8 مب خ ألوثش ضبد ىش ث. أظ شد ا ؼضالد أ %98.4 ا ى ساد ا ؼ م د خ وب ذ مب خ ضبد ىش ث احذ ػ ى األل 88.6 وب ذ راد مب خ زؼذدح. أظ شد ا جىز ش ب ا ؼ خ أػ ى مب خ ضذ ا ززشاس ى ) 67.9( أ وس س ا ىالف ال ه ) 56(. أظ شد ا ى ساد ا ؼ خ أػ ى مب خ ضذ فب ى س ) 84.8( األ ج س ) 8.3(. أظ شد ا ضائفخ ا ض جبس خ أػ ى مب خ ضذ ث ج شاس ) 55.1(. ث ب أظ شد ػضالد ا ى ساد ا ؼ م د خ أػ ى مب خ ضذ ا ج س ) 91.8( فب ى س ) 68.9( أ وسبس ) 62.3(. مذ وب ؤشش ا مب خ ا زؼذد ضبداد ا ىش ث خ )MARI( أػ ى.2 ف ج غ ا الغ ا ز ر اخزجبس ب ب ذي ػ ى أ ج غ ا الغ ا ذس سخ رؼزجش ثخ ثسجت االسزخذا ا شبئغ ضبداد ا جشاث. ف ا خزب رظ ش زبئج ز ا ذساسخ رظ ش ا زشبس اسغ جىز ش ب ا مب خ ضبداد ا ىش ث خ ف و أ اع ا ؼ بد ا ز ر اخزجبس ب و ا الغ ػ ى ط ي سبح لطبع غضح أ ج د ز ا جىز ش ب ا ى أ شى خطشا ػ ى صحخ اإل سب. ث بء ػ ى ر ه فئ جت ثزي ا ج د زم رصش ف ب ا صشف ا صح ا غ ش VI

10 ا ؼب جخ أ ا ؼب جخ جضئ ب ا صبدس ا ح خ ا سزشف بد ا ز ر ث ا صذس ا شئ س ضبداد ا ىش ث خ ف ا ج ئخ ا جحش خ. كلمات مفتاحية: مب خ ا ضبداد ا ىش ث خ ا مب خ ا زؼذدح ضبداد ا ىش ث خ ا جىز ش ب ا ؼ خ شبطئ غضح ا ى ساد ا ؼ خ ا ى ساد ا ؼ م د خ ا ضائفخ ا ض جبس خ. VII

11 Table of Contents Dedication... II Acknowledgment... III Abstract... IV Abstract in Arabic language... VI Table of Contents... VIII List of Tables... XI List of Figures... XII List of Abbreviations... XIII Chapter 1 Introduction Overview Research Problem Aim of the Study Significance of the Study... 7 Chapter 2 Literature Review General Description of Study Area Importance of Gaza Strip Beach Economic Importance Recreational Importance Biological Importance Sources of Pollutants Wastewater Treatment Plants Direct Sewage Discharges Solid Waste Wadi Gaza Agriculture Domestic Animals and Birds Bather sand Swimmers Bacteria from Stream Sediments Risks from Marine Pollution Microbiological Quality Indicators of Seawater Enterobacteriaceae Enterococci Staphylococcus aureus VIII

12 2.5.4 Pseudomonas aeruginosa Sea Quality Monitoring and Assessment of Gaza Beach Antimicrobial Overview of Antimicrobial Groups lactam antibiotics The fluoroquinolones Aminoglycosides The glycopeptides Mechanism of antibiotic action and resistance Mechanism of antibiotic Mechanism of antibiotic resistance Previous Studies Previous Study in Gaza Strip Chapter 3 Materials and Methods Sample Sites Sample Collection and Processing Preparation of Culture Media Seawater Beach Mussels Isolation of Bacteria Seawater Mussels Bacterial Identification Assessment of Beach Microbiological Quality Antimicrobial Susceptibility Testing Statistical analysis Chapter 4 Results Assessing Beach Microbiological Quality Antimicrobial Resistance The incidence of MAR in different sample types and sampling locations Enterobacteriaceae Generic Composition of Bacterial Isolates IX

13 Enterobacteriaceae Resistance Multiple Antimicrobials Resistance Index for Enterobacteriacae Enterococci Resistance of Isolated Enterococci Strains MARI for Enterococci Pseudomonas aeruginosa Resistance of Isolated Pseudomonas Strains Number of Antibiotic Resisted MAR Index for Pseudomonas Staphylococci Resistance of Isolated Staphylococci Strains Number of Antimicrobials Resisted Multiple Antimicrobials Resistance Index (MARI) for Staphylococci.. 81 Chapter 5 Discussion Beach Quality Assessment Antimicrobial Resistance Multiple Drug Resistance (MDR) Multiple Antimicrobial Resistances Index (MARI) Chapter 6 Conclusion and Recommendations Conclusions Recommendations... 1 References Annexes X

14 List of Tables Table 2.1 Summary of mechanisms of action of common antimicrobial agents 35 Table 3.1 Geographical and identification information of sampling sites 44 Table 3.2 Mandatory and guideline standards for fecal coliform and fecal streptococci set by European Union Bathing Directive 76/16/EE. 49 Table 3.3 Antimicrobials used in this study and their concentrations 5 Table 4.1 Table 4.2 Table 4.3 Table 4.4 Table 4.5 Table 4.6 Table 4.7 Table 4.8 Table 4.9 Table 4.1 Table 4.11 Table 4.12 Percentage of recorded observations that exceeded EU mandatory and guideline standards for fecal coliform (FC) and fecal streptococci (FS) in seawater samples collected from the studied locations. Incidence of multiple resistances for all bacteria isolated from mussels, seawater and sand beach. Incidence of multiple resistances for Bacteria isolated from five Locations Percentage of resistance of Enterobacteriaceae isolated from sand, seawater and mussel samples to different antimicrobial Percentage of resistance Enterobacteriaceae strains isolated from different locations at Gaza Strip shoreline to 1 antimicrobial (%). Resistance of Enterobacteriaceae according to source and sampling location along Gaza strip beach (Numbers in parentheses represent the numbers of isolate) Percentage of resistance of Enterococci isolate from sand, seawater and mussel samples to different Antimicrobial (%) Percentage of resistance of Enterococci strains isolated from different locations at Gaza strip shoreline to six antimicrobial (%) Percentage of resistance of P. aeruginosa isolate from sand, seawater and mussel samples to different Antimicrobial (%) Percentage of resistance of P. aeruginosa isolated from different locations at Gaza strip shoreline to six antimicrobial (%) Percentage of resistance of Staphylococci isolate from sand, seawater and mussel samples to different antimicrobials (%) Percentage of resistance of Staphylococci strains isolated from different locations at Gaza Strip shoreline to 1 antimicrobial (%) XI

15 List of Figures Figure 3.1: Geographic location of sampling sites Figure 4.1: Geometric means (cfu/1 ml seawater or 1g sand) of fecal coliform and fecal streptococci in the water and subsurface layer of wet sand at five locations Figure 4.2: Population density of fecal coliform and fecal streptococci in the water and sand at five locations Figure 4.3: The number of FC and FS in the mussels from locations 2 and Figure 4.4: A summary of bacteria belonging to Enterobacteriaceae family isolated from seawater sand and mussel samples and tested for antimicrobial resistance Figure 4.6: Multi-drug resistance among Enterobacteriaceae strains isolated from sand (n =81), seawaters (n = 66) and mussels (12) of Gaza strip shoreline Figure 4.7: Multiple Antimicrobial Resistance Index (MARI) for Enterobacteriaceae 67 Figure 4.8: Resistance isolation percentage of Enterococci strains isolated from mussel in and South of Wadi Gaza Outlet... 7 Figure 4.9: Multiple Antimicrobial resistance of Enterococci isolates from sand, mussels and seawater samples from the coast of the Gaza Strip Figure 4.1: Multiple Antimicrobial Resistance Index (MARI) for Enterococci Figure 4.11: Resistance isolation percentage of Pseudomonas aeruginosa isolated from mussel in and South of Wadi Gaza outlet Figure 4.12: Multiple Antimicrobial resistance of Pseudomonas aeruginosa isolated from sand, mussels and seawater samples from the coast of the Gaza Strip Figure 4.13: Multiple Antimicrobial Resistance Index for Pseudomonas aeruginosa. 76 Figure 4.14: Resistance isolation percentage of Staphylococci strains isolated from mussel in and South of Wadi Gaza outlet Figure 4.15: Multiple antimicrobial resistance of Staphylococcus aureus isolates from sand, mussels and seawater samples from the coast of the Gaza Strip... 8 Figure 4.16: Multiple Antimicrobial Resistance Index for Staphylococcus XII

16 List of Abbreviations APHA API ARB ARD ARG BHIB BOD BST BZU CDC CFU CFU CLSI CMWU DAHR DNA DO DOH EARSS EMA EPA EQA EU FAO FC FDA FIB FS GI GM GWWTP HHS HPA INH IUCN MARI MDR MEnA MLG MOA MOH MR-CoNS MRSA American Public Health Association Analytical Profile Index Antimicrobial Resistant Bacteria Acute Respiratory Disease Antibiotic Resistance Genes Brain-Heart Infusion Broth Biochemical Oxygen Demand Bacterial Source Tracking Birzeit University Palestine Centres for Disease Control and Prevention Colony Forming Units Cystic Fibrosis Clinical and Laboratory Standards Institute Coastal Municipalities Utility Dameer Association for Human Rights Deoxyribonucleic Acid Dissolved Oxygen Departments of Health European Antimicrobial Resistance Surveillance System European Medicines Agency Environmental Protection Agency Environment Quality Authority European Community Food and Agriculture Organization Fecal Coliform Food and Drug Administration Fecal Indicator Bacteria Fecal Streptococci Gastroenteritis Geometric Mean Gaza Wastewater Treatment Plant Health and Human Services Health protection agency Isonicotinic Acid Hydrazide International Union for Conservation of Nature Multiple Antibiotic Resistance Index Multiple Drug Resistant Ministry of Environmental affairs Ministry of Local Government Ministry of Agriculture Ministry of Health Methicillin Resistance Coagulase-Negative Staphylococci Methicillin Resistance Staphylococcus Aureus XIII

17 1 Chapter 1 Introduction 1.1 Overview The term antibiotics are most commonly used to refer to antimicrobial agents, produced by microorganisms, such as a fungi or bacteria and able at low concentrations to inhibit or kill other microorganisms. Since the first discovery of the antibiotic, penicillin in 1928 by Alexander Fleming, many antibiotic products have been developed mainly, for the purpose of treatment of infectious diseases in humans, animals, and plants. Currently, several antibiotic drugs are used worldwide, not only to combat diseases, but also to act as prophylactic agents to prevent diseases, to promote growth in agriculture and aquaculture, and to preserve food (Refsdal and Forsberg, 2). Antibiotics exhibit varying ranges of mechanisms to damage pathogens, for example, they may affect structures like the cell wall by inhibiting its synthesis or activating enzymes that destroy it, or increasing cell membrane permeability, interfering with proteins and nucleic acids synthesis or metabolism. The different antibiotics were designed to selectively target and inhibit the cells of invading organisms without harming the host cells (Kohanski et al., 21). In the past few decades however, certain human actions such as the intensive and often uncontrolled use of antibiotics in medicine, veterinary and agricultural practices have probably led to emergence and spread of antibiotic resistant bacteria. In this case, bacteria will be able to survive even after exposure to antibiotics to which it was originally sensitive. It has been estimated that more than one million metric tons of antibiotics has been released into the biosphere in the last 5 years (Linton, 1984). In fact, antibiotic resistance is not a new phenomenon. It has been known to be naturally present in the environment at earlier times, before the antibiotics were discovered, but apparently, the levels of resistance have increased during the past decades (Levy, 22). The emergence of resistance was described as extraordinarily complex processes, which require more understanding (Alanis, 25). Generally, bacteria may develop antibiotic 1

18 resistance by spontaneous chromosomal mutations in the bacterial genome or by acquiring resistance genes through horizontal gene transfer between different bacterial species through conjugation, transformation or transduction (Sengupta and Chattopadhyay, 212). The majority of resistance genes are carried by transmissible plasmids (R-factor or R-plasmid), transposons, and other elements capable of intergeneric and inter-specific mobility (Hirsch et al., 1999; HHS and CDC, 21). When bacteria become resistant to several kinds of antibiotics, they are termed as multiple antibiotics or multi drug resistant (MDR) bacteria. Such bacteria are commonly found among pathogenic bacteria and normal microbiota that inhabit our intestinal tract (Kümmerer, 29). This is the case for some strains of Staphylococcus aureus, Enterococcus faecalis, Neisseria gonorrhoeae, Haemophilus influenzae, Mycobacterium tuberculosis, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella dysenteria, Streptococcus pneumoniae Klebsiella pneumoniae, Clostridium difficile and Acinetobacter baumannii, all being important community or nosocomial pathogens. Acquisition of multi-drug resistance by pathogenic bacteria would essentially reduce the effectiveness of their treatment by commonly used antibiotics and increasing the risk of spreading resistance to other microorganisms. Unfortunately, in recent years, the situation of multi-drug resistance became the rule rather than the exception among resistant bacteria (Holmberg et al., 1984; Mudryk, 22; Davies and Davies, 21). The main mechanisms by which microorganisms exhibit resistance to antibiotics include; drug inactivation by hydrolysis or by modification, drug target site alteration, metabolic pathway alteration and finally prevention or reducing drug accumulation through efflux pump (Bockstael and Aerschot, 29). Antibiotics used in hospitals, domestically or in veterinary medicine will not remain in the human or animal body for long and will be finally excreted in some form in the urine or feces. Later, some of antibiotic resistant bacteria, residual antibiotics and antibiotic metabolites will eventually be introduced into various terrestrial and aquatic environments. Depending on the type of antibiotic, between 3% and 9% of an administered dose of most antibiotics given to humans and animals are excreted in the 2

19 urine or feces as the active substance and introduced to the sewage system, soil and water basins (Blyela et al., 24; Costanzo et al., 25). Studies have shown that the ocean is an important reservoir of antibiotic resistance bacteria and genes (Holmstrom et al., 23; Yang et al., 213; Agwu, 214). Sources of antibiotic resistant organisms and antibiotic residues in the marine environments include effluents from municipal wastewater treatment plants, industrial effluents (pharmaceutical industry), point and non-point source agricultural runoff, wild life and indigenous microorganisms. Aquaculture also represents a significant source of antibiotics contaminating marine environments, because large amounts antibiotics are administered via the feed to treat infections and improve aquaculture production in the high animal density cages (Lin et al., 24; Alpay-Karaoglu et al., 27). If suitable conditions are available, resistant bacteria could survive and multiply in the marine environment for relatively long periods while maintaining their resistance properties (Holmstrom et al., 23). They are also able to transfer their resistance genes to other bacterial species by horizontal gene transfer mechanisms, even after the death of the bacterial cells, thus increasing the percentages of antibiotic resistant bacteria in marine environments. Therefore, even if the specific antibiotic is no longer introduced into the environment, antibiotic-resistance genes will persist through the bacteria that have since replicated without continuous exposure. Sediments provide a particularly favorable environment for bacterial growth since they offer bacteria nutrients and protection from biotic and abiotic stress (OECD, 21). In contaminated marine systems, the high levels of antimicrobial-resistant bacteria (ARB) may result in dissemination of deleterious genes into the environment with potentially adverse effects on human and animal health as well as marine biodiversity. Their antimicrobial resistance genetic determinants can be passed to non-pathogenic native bacteria and/or to human, fish and animal pathogens (Kemper, 28; Zhang et al., 29). Furthermore, the persistence of antibiotic resistance genes (ARG) in non pathogenic marine microbes could provide a reservoir for further spreading of ARG in nature. Increasing antibiotic resistance in fish pathogens are the leading cause of seafood-borne illness and death in the United States. 3

20 testing methods currently uses microbiological quality and quantity as well as physico-chemical analyses of water as a mean to determine the health of aquatic systems, to spot polluted sites and also to estimate the extent of pollution. As they do not occur naturally in aquatic environments and are only found inhabiting the guts of warm-blooded animals, fecal coliform bacteria are usually used to assess the quality of aquatic environments. Other bacteria suggested as indicators of water quality include Pseudomonas aeruginosa, fecal streptococci, Enterococci, and staphylococci. Several studies have found correlations between pollution and the occurrence and distribution of ARB in the marine environments. While some of these studies were concerned with pathogenic bacteria in the marine environment, others were interested in free-living ARB. These studies suggested that antibiotic resistant bacteria could provide an important indicator of marine water quality (Oliveira and Pinhata, 28; Al-Bahry et al., 29; Oliveira et al., 21; Mudryk et al., 21) and as a risk assessment. Several calls have been made for antibiotic resistance to be considered when establishing bacteriological water quality criteria (Bell et al., 1983; El-Zanfaly, 1991). Recently, the concept of antibiotic resistance was used as a cost effective technique for tracking the source bacterial pollution in the environment i.e. Microbial Source Tracking. In the Gaza Strip, there is growing evidence that the misuse and overuse of antibiotics are the major contributors to the development and spread of ARB. In fact, no regulation exists regarding the use and dispense of antibiotics in the Gaza Strip. Although the problem of drug resistance is not new, the scale of the problem in the Gaza Strip however, has received little scientific attention, despite its importance. Previous studies of the antibiotic resistance problem have mainly focused on nosocomial infections caused by multi-drug resistant pathogens (Astal et al., 22; Astal, 25; Elmanama et al., 26b; Abu Elamreen et al., 27; Elmanama, 28; Al Jarousha, 28; Al Laham, 212; Elmanama and Abdelateef, 212). Historically, the beaches, located along the coast of the Gaza Strip has benefited local inhabitants by providing opportunities for water recreational activities such as swimming and diving, as well as non-water-contact activities, such as sunbathing, and 4

21 sand recreation, which is an especially attractive for children. The coast is also represent a vital economic resource for many people work in fields related to fishing, and boating as well as those who work in cafe shore restaurants that provide services for citizens during their visits to beach. However, because of rapid population growth, the rate of anthropogenic activities has increased, thus, deteriorating the quality of the very limited natural resources in the Gaza Strip including the marine environment. The most common potential sources of microbial pollution along the coast of the Gaza Strip include untreated or poorly/partially treated wastewater from various wastewater treatment plants, raw untreated sewage from Wadi Gaza and some sewage outlets from seaside residences, marine fish farms effluents and runoff from urban areas and agricultural lands. These are expected to be the main sources of spreading of antibiotics and ARB into the marine environment. The presence of pathogenic bacteria in marine environment, may pose a variety of health hazards such as gastrointestinal, respiratory, dermatologic, and ear, nose, and throat infections to people visiting beaches as a result of unintentional ingestion, inhalation and/or body contact. It may also leads to losses in economic activities that depend on coastal water quality, such as tourism and fishing due to closure of recreational beaches and fishing areas (NHMRC, 28; CDC, 211). In this context, monitoring the marine microbial pollution is a highly important task. Several studies reported that shellfish such as mussels may concentrate higher densities of pathogens in their bodies than the surrounding waters, and that bacteria may have higher survival rates inside the bivalves (De Donno et al., 28; Maalouf et al., 21; Martinez, 21). This may suggest that, these organisms might represent an interesting approach for evaluating the sanitary conditions of marine environment. Previous studies of the marine pollution in the Gaza Strip have primarily focused on the number and distribution of fecal coliforms in the seawater and the beach sand (Afifi, 1999; Afifi et al., 2; UNU/INWEH, 21; MEnA, 21; Elmanama et al., 25; Elmanama et al., 26a; El Jarousha, 26; Bahr, 27; EQA, 214a; EQA, 214b). 5

22 Although antimicrobial resistance against different kinds of antibiotics have been reported in different parts of the world, studies on the presence of ARB in marine environment of the Gaza Strip however, are absent. Therefore, the objective of the present study was to document and determine the occurrence and distribution of pathogenic antibiotic resistant bacteria in the seawaters and beach sands. Another objective of this study was to isolate, identify and enumerate pathogenic antibiotic resistant bacteria found in mussels collected from two selected stations. A comparison with surrounding water and beach sand in the same stations will be attempted. 1.2 Research Problem Gaza beach has socioeconomic significance and at the same time, it is the only recreational area in the Gaza strip and is subjected to pollution from different sources, mostly from untreated sewage. The extent of the problem is not fully known and is expected to vary with time. The impact on public health was not studied properly. International environmental organizations established standards and guidelines for seawater quality to minimize health risks associated with using such waters. Beach sand contains higher level of fecal contaminants and possibly pathogens but no country included standards for beach sand (Elmanama et al., 25). Hospital sewage contains large amount of various antimicrobials due to extensive use. In addition, it contains diverse groups of multiple drug resistant pathogens. This sewage is not treated in hospitals and instead it is discharged in a similar way to that of domestic sewage which eventually reach seawater. 1.3 Aim of the Study The primary aim of this study is the protection of public health, and is not to deter the use of recreational water environments but instead to ensure that they are operated as safely as possible in order that the largest possible population gets the maximum possible benefit. This will be done by investigating the antibiotic resistance pattern of pathogenic and indicator bacteria that will be isolated from the seawater, sand and Mussels samples in Gaza strip beach. 6

23 1.3.1 Objectives The following specific objectives were achieved: 1. Identify proper sampling locations for the study area (Gaza strip) along the beach. 2. Isolation and identification of bacterial species from seawater and sand samples. 3. Determination of antibiotic resistance pattern of bacteria that will be isolated from seawater and sand of Gaza beach. 4. Measuring the concentration of bacteria from mussels and to compare it with the concentration of bacteria in the surrounding seawater. 1.4 Significance of the Study This is the first study to generate data about the level and types of antimicrobial resistance of bacteria isolated from Gaza beach. The result of this investigative work will provide the local authorities with data that may prove useful in preparing plans to reduce the risk of acquiring MDR infection through direct or indirect contact with seawater. Since sewage from various sources is discharged to the sea, intestinal bacteria found in seawater may be considered representative model and therefore, concerned authorities may use the result of this study and other similar studies or monitoring programs to replace older models of monitoring of antimicrobial resistance, which depends on investigating larger geographical locations and larger number of samples. 7

24 2 Chapter 2 Literature Review 2.1 General Description of Study Area The Gaza strip is a narrow strip of land on the Mediterranean coast. It borders Occupied Palestine to the East and North and Egypt to the South. It is approximately 45 kilometers long, and 6-13 kilometers wide, with a total area of 378 square kilometers (UNDP, 29). Boundary near Beit Hanoun in the north, and Rafah on the Palestinian Egyptian border in the south, its width varies from 6 kilometer along the line trans versing through Deir El-Balah in the center, to 13 kilometer along the Egyptian boundary in the south. By the year 213, the number of the actually counted population in Gaza governorates is about 1.7 million distributed across five Governorates (PCBS, 213). Khan Yunis Governorate is located in the southern part of the Gaza Governorates. About 187 persons currently inhabit Khan Yunis City as the second largest city in Gaza strip. Total population of Khan Yunis Governorate is approximately 321 and the total population in Middle Zone is around 247 persons (PCBS, 213). Gaza strip consists of a coastal area and a strip of newly formed sandy beach. Have a semi-arid climate, dry in summer and moderate in winter. The average daily temperature range between 25 ºC in summer and 13 ºC in winter, the humidity percentage range between 65% daytime and 85% night time in summer and 6% daytime and 8% night time in winter (UNEP, 29). The prevailing winds in Gaza strip are northwesterly in the summer. The speed of these wind is variable reaching the speed of (3.9) m/s in the daytime and reduced to the half in the night time. The prevailing wind direction and speed change during the winter, as it turns to the southwesterly wind and speed increase up to 4.2 m/s with nonvolatile speed. Sometimes, it is noticed that there is blowing southwesterly winds increase up to 18 m/s. The mean annual rain in Gaza strip ranged between 2 to 45 mm (MLG, 24; PCBS, 213). 8

25 The seashore is the only recreational area in Gaza strip, usually is very crowded in summer season mostly with local inhabitants. The beaches of Gaza strip are potentially interesting for beach tourism, not only for local population but also for foreign tourists (Afifi et al., 2). A major problem, however, is the severe pollution of the seawater (Ajjour, 1997), and the beaches posing a major health risk for swimmers and marine life (Mukhallalati, 1997). The pollution is one of the problems being faced in the world all through 2 th century; the most important source of water pollution is the discharge of untreated wastewaters into the seawater (Carson et al., 21). Untreated or only partly treated wastewaters including industrial, agricultural and domestic wastes are frequently released into seawater, which affect water quality. Fecal indicator bacteria residing in the gastrointestinal tracts of humans and animals are commonly used to assess the microbiological safety of drinking and recreational waters. Although indicator bacteria do not necessarily cause illness, their presence indicates that the water has been contaminated by fecal material; implying the potential presence of pathogens (An et al., 22; Noble et al., 23). Fecal contamination of beaches can present significant public health risks, loss of recreational opportunities, and costly impacts for local economies (EC and EPA, 26). In Gaza strip, approximately 53.5% of the populations are connected with a collection sewage system (MOH, 21). The rest of the population relies on latrines connected to unsealed vaults. Gray water is disposed via narrow surface channels, the majority of which meets to form larger channels which in turn transport its content into the seawater. Vacuum tankers are used to remove solid septage from latrines and discharge it on soil surface, sometimes, near the sea, which may contribute to the increasingly polluted sea, especially during the rainfall (Afifi et al., 2). In Gaza strip, limited numbers of wastewater treatment facilities were established, most of which does not work properly or not at all. Recently, improved treated wastewater was produced by the SHIK EJLEEN wastewater treatment plant (known as Gaza Wastewater Treatment Plant or GWWTP). More than 5% of the raw sewage is discharged untreated into the sea, causing severe water pollution (Enshassi, 2). 9

26 The lack of sufficient wastewater treatment facilities makes wastewater that discharges into the sea the main source of pollution of the coastal zone of Gaza strip. There are more than 2 individual sewage drains, ending either on the beach or at a short distance away in the surf zone. Insufficient number of sewage treatment plants in operation, combined with poor operating conditions of available treatment plants, and the present disposal practices are likely to have an adverse effect on the quality of seawater (EQA and UNEP, 25). The population of Gaza strip continues to grow rapidly, which increase the amounts of poorly treated or untreated sewage being discharged into the coastal water. With the Palestinian population growth-rate of around 4.8% per annum, which would result in doubling of the population in 15 years, effective management and sustainable development of Gaza resources will be a huge challenge for the Palestinian Authority (UNEP, 23). Another major problem that increases the health risks from using seawater for recreation, is the limitation of treatment facilitates and method of effluent disposal. The sewage is either disposed near the seashore (sands) or few meters inside the seawater. This brings the bathers into a close contact with contaminated water, which is clearly a public health hazard (Elmanama et al., 25). The import restrictions have impeded the expansion and upgrading of Gaza s sewage infrastructure. Nearly 9 million litters of untreated or partially treated sewage are discharged into the sea every day. The contamination of seawater poses a serious health hazard (UN, 213). Due to strict travel limitation imposed by the Israelis on Palestinians and the limited recreational areas, a significant number of infrastructure installations are located along the beach in recent years to encourage local and foreign tourism. Unplanned infrastructural development is a part of the general pattern of growth of population growth and urban expansion. Human growth and mal-managed activity has almost been a major cause of pollution of the surrounding areas, which includes in this particular situation, the only recreational source in Gaza (Elmanama et al., 25). 1

27 2.2 Importance of Gaza Strip Beach Without seas and oceans, life on earth would not have been possible. Some people think that the importance of seas and oceans does not exceed being a source of sea food. Others think that it is a water body where ships travel from one place to another, or may be just a beach for swimmers. The importance and benefits of seas to humans can take many years to discuss. It is not surprising that the United Nations had named the year 1998, the year of seas and oceans. That year had witnessed various activities that focused on the importance and benefits of seas to humans and how we could preserve them as natural resource for many marine creatures (DAHR, 29). The beaches and shorelines are some of the more highly valued areas for recreation around the world, responsible, in many areas for significant tourism industry revenue (UNWTO, 21). The coastal zone is ecologically and economically important for the Palestinian Authority. The beach is the only accessible recreational area for the population in Gaza. At the same time, sand dunes in the coastal zone hold the best ground water resources in Gaza. Gaza coastal zone has multi functions and provides the area with different resources potentials which could be classified into economic social, and scientific potential (Elmanama et al., 25) Economic Importance A. Tourism Tourism in Gaza is expected to be a major source of future income or revenue. It depends on unspoiled areas and relatively cleans water. Its potential is considered medium, but limited by political constraints (Ajjour, 1997(. The beaches of Gaza strip are potentially interesting for beach tourism, not only for local population but also for foreign tourists (Afifi et al., 2). War and conflict are usually seen as major hindrances to travel and tourism (Isaaca, 21). B. Agriculture Agriculture is an essential component of the Palestinian national, cultural, economic and social fabric. In addition to its traditional significance for nations and states, agriculture is particularly important for Palestinians as it embodies their perseverance, 11

28 confrontation and adherence to their land under the threat of confiscation and settlement activities. It also provides a refuge and a source of income and food supplies at times of crises. In this context, a significant number of Palestinians have resorted to the agricultural activity (MOA, 213). The Gaza economy is largely dependent on agriculture; the coastal area in Gaza is a traditional zone for agricultural production for the large and rapidly growing population of Gaza. It is an important base for food supply and export. The zone has a certain potential for growth in the future (Ajjour, 1997; MOA, 213). C. Fishery The ability of people to gain a living from fishing activities has been severely undermined as a result of the gradual restrictions imposed by the Israeli authorities on the access of fishermen to sea areas along Gaza s coast (UN, 213). In the Gaza strip, there are approximately 25 fishermen. In addition, 12 Palestinians have jobs related to fishing, such as the manufacture and maintenance of boats and nets, the preparation ice blocks for fish storage and fish skinning and selling. Around 4-5 families, mostly women, from Al Shati refugee camp work in fish skinning for local traders or Israeli companies. They receive low wages for this job, despite long hours and health problems as a result of the work. They work in this profession in order to help improve the living conditions of their families. However, their work suffered a setback as the fish production in the Gaza strip sharply decreased, while living conditions of those families has rapidly deteriorated as their income from this work used to aid the easing the hard economic conditions under which they live (PCHR, 22). Fishermen in Gaza strip are working under undeveloped condition. In the future plan, fishery sector has a great value for food supply of the local people and for the exports to Israel or the West Bank. So it has to be developed in a proper way (special beaches areas, sheds / cottage, or daily transportation) to fulfill the national (Ajjour, 1997( Recreational Importance Residents of Gaza consider the surrounding marine environment as the only outlet available for recreation. The beaches of Gaza, during the summer time, are garnished with a large number of cafés that provide services for citizens during their leisure time. 12

29 Owners of horses, Camels and small boats take advantage of the season and do some business entertaining the crowds, especially children. Despite of this happy scene, there are other scenes that media and educated pens continuously describe; they keep on saying that the marine environment in the Gaza strip is polluted with wastewater, which makes a great part of the population abstain from spending their summer days on the beach. The current conditions of the marine environment represent a tough challenge for citizens and other concerned parties in the Gaza Strip (DAHR, 29). Gaza Strip beaches are the only major source of recreation for more than one and a half million inhabitants of Gaza's people. The area is in bad needs of public access, service, landscape improvement and new infrastructure. Its potential use connected with improvement of public transportation public service and safety of beach and water quality (Ajjour, 1997( Biological Importance Some areas in Palestine have special environmental significance: they are home to various important species of plants and animals not commonly found elsewhere and also have significant geophysical features. These areas should receive much care and protection. Moreover, the Palestinian population should have the chance to enjoy them (Zoi, 212). Gaza strip is considered rich in its marine biological resources including a high biodiversity of fisheries, plant and animal life like terrestrial. As a result, the coastal zone has been a very important asset for the attraction of national and international tourism and an important contributor to national economies. It also holds the last natural areas in Gaza, such as the wetlands or marshes the Wadi Gaza outlet, mobile sand dunes or sand cliffs (Ajjour, 1997). Wadi Gaza is one of the undeveloped natural areas in the Gaza Strip and serves as a natural habitat for migratory and endemic birds. Some bird species ranked as rare or endangered by international union for conservation of nature (IUCN) still find the valley an ideal place for feeding during migration. Risks to Wadi Gaza s environment stem mainly from wastewater and solid waste pollution, disposal of construction debris in the valley bed, encroaching urban development, and depletion of resources from overgrazing, tree cutting, hunting and the construction of a bridge (EQA, 29). The 13

30 white pelican passes over the region during its migration and is registered as a crossing bird. The common tern can be found in large numbers, reaching as many as 2 birds, some resident and others migratory. The great black-headed gull still lives on the shore (EQA, 26). Environmental situation of the Gaza coastal and marine environment is dire, regular national campaigns are needed to raise awareness among the public and policymakers as to the importance of biodiversity for the wellbeing of Palestinians. Daily practices among the bulk of the Palestinian population show a slowdown in care for biodiversity, principally as a result of the difficulties of daily survival, which make environmental issues secondary priorities (EQA, 26). 2.3 Sources of Pollutants Beaches worldwide provide recreational opportunities to hundreds of millions of people and serve as important components of coastal economies. Beach water is often monitored for microbiological quality to detect the presence of indicators of human sewage contamination so as to prevent public health outbreaks associated with water contact. However, growing evidence suggests that beach sand can harbor microbes harmful to human health, often in concentrations greater than the beach water (Sabino et al., 214). When recreational waters are contaminated with pathogenic microorganisms, it is important for public health officials to determine the source of the contamination to remedy the problem and prevent bather exposure to pathogenic microorganisms. Bacterial source tracking (BST) is often used to determine the source of fecal contamination in recreational waters (Garrido et al., 28). Diverse fecal contamination sources contribute to beach advisories, including point sources such as municipal wastewater effluents, and non-point sources such as agricultural run-off and wildlife droppings. It is important to identify the source of fecal contamination at beaches in order to better understand public health risks and correctly target fecal pollution prevention actions (Edge and Hill, 27). Seas receive great quantities of various contaminants that represent a threat to the marine life. The most outstanding source of marine pollution along the shore of Gaza 14

31 strip is discharging the raw wastewater and dumping solid waste and beach erosion. One of the main qualities of the marine environmental system is its ability to cleanse itself or self-disinfection ability. The marine environment is a hostile environment for bacteria. The seawater includes a number of microcosms; plants and animals especially the plankton plants that live on the water surface. All of the above creatures release active chemicals that provide seawater with important immunity against the germs resulted from dumping waste in the seawater (DAHR, 29). The seawater quality of Gaza strip has been highly polluted by sewage, sediments, nutrients pesticides, litter and marine debris, and toxic wastes during thirty years of Israeli occupation. This has left the coastal area in a bad and neglected state and consequently led to the deterioration of seawater quality. The beaches posed a major health risk for swimmers and marine life (MEnA, 21) Wastewater Treatment Plants A major problem, however, is the severe pollution of the seawater (Ajjour, 1997). Discharge of untreated wastewater along the shoreline is the main source of pollution in the coastal zone of Gaza. About 6% of wastewater that is generated in Gaza is currently discharged without treatment into the sea (5, cubic meters per day). The pollution presents major health risk for swimmers and marine life by affecting the coastal environment of complete marine food chain ranking (MEnA, 21; MOH, 21). The sewage is either disposed near the seashore (sand) or few contact with contaminated water, which is clearly a public health hazard (Elmanama et al., 25). Tourism development in the Gaza strip will largely depend on the extent to which the beaches and coastal cliffs will be cleared and stay clean from solid waste, and the extent to which the sewage effluent entering the surf zone of the coastal waters will be reduced (MEnA, 21). Adequate wastewater treatment prior to effluent discharge plays a critical role in minimizing public health risks. On-site wastewater disposal using septic tanks has also been an issue regarding pathogen entry into and transmission through water particularly groundwater (Fong et al., 27). 15

32 2.3.2 Direct Sewage Discharges Sewage may be treated, partially treated or untreated, and carry a variety of pathogenic microorganisms such as bacteria viruses and protozoan, which expose bathers to diseases when these pathogens reach beach waters (WHO, 1998), wastewater is the source of many human enteric pathogens (Nayak and Rose, 27) and often associated with swimming acquired illnesses in natural waters (Wade et al., 26). Higher percentage of the wastewater is generated in Gaza governorates are currently discharged without treatment into the sea from more than 2 individual sewage drains ending directly on either the beach or a short distance away in the surface zone without receiving any treatment (WHO, 23). Greatest volume of sewage discharged to the marine environment. Sewage includes pathogens, organic substances, heavy metals and trace elements. All of which, pose direct and indirect effects on ecosystems and organisms (Islam and Masaru 24). Direct discharge of crude, untreated sewage into recreational areas presents a serious risk to public health (WHO, 23) Solid Waste Dump sites for solid waste as well as littering contribute to the pollution of coast and seawater (MEnA, 2). The pollution caused by solid waste and construction rubble is one of the serious sources of endangering the environment; it damages the main environmental components. The marine environment in the Gaza strip was severely impacted by such pollution that has reached the beaches and the sand dunes along the coastal line (DAHR, 29). The variety of litter found in recreational water or washed up on the beach is considerable and includes, for example, discarded food, wrapping, bottles, cans, cigarette butts, dead fish, discarded sanitary towels, and syringes, needles and other medical wastes. Unlike most litter, medical waste and broken glass also represent hazards to health (MEnA, 2; WHO, 23). 16

33 2.3.4 Wadi Gaza Wadi Gaza has surface water all year- round, this may be due to the discharge of untreated water. Unfortunately, Wadi Gaza does not function as natural wadi anymore, it flooding in winter, caused by intense rainfall brings large amount of heavily polluted water into the marine environment, catchments area covers about 35 Km 2 of the Negev desert. Every day, raw sewage water is 1, cubic meters discharged from Gaza valley directly into the sea (BZU, 24; CMWU, 29) Agriculture Agricultural production makes a very important contribution to economy locally and nationally. Agriculture has major positive, with some negative, effects on the coastal environment. So, we need sustainable agricultural to limit the negative impacts of inland agriculture on coastal areas (FAO, 1998). Agricultural activities contribute about 5% of the total pollution source of surface water through the higher nutrient enrichment, mostly ammonium ion (NH 4 ) and NO 3 derived from agricultural inputs. Ammonia constitutes a major contributor to the acidification of the environment, especially in areas with considerable intensive livestock farming. Wastes, manures and sludge, through biological concentration processes, can supply soils with 1 times more hazardous products than do fertilizers for the equivalent plant nutrient content (Joly, 1993). Fertilizers are used extensively all over the world several decades in the past. Compost produced by cattle, pigs and other animals are used as organic fertilizer globally. Human excreta, especially in some Asian countries where animal and human excreta are traditionally used in fish culture as well as on soils, are added to this (Islam et al., 24). Fertilizers use in agriculture poses a warning to marine ecology, as they infect the marine environment through the run off. In addition, nitrate fertilizers of agriculture make up 7 % of the nitrate load in the Gaza groundwater resources (MEnA, 21). Pesticides usage has relation to pollution. Very small part of all applied pesticides becomes directly involved in pesticide systems, i.e., unless the compounds are quickly degradable, most of the pesticides find their way as remains in the environment 17

34 (Duursma and Marchand, 1974) and so, they are considered the most destructive agents for aquatic ecosystems and organisms affecting all stages of the food chain from the lowest up to the top level (Duursma and Marchand, 1974) Domestic Animals and Birds Fecal pollution of water resources enlarges as demographic densities increase. Fecal indicator bacteria are used to evaluate the microbial quality of water as they are not 1% disease finding but may be correlated with the company of many waterborne disease-causing organisms. An indicator of current fecal pollution recommended generally to be used for monitoring the microbiological quality of water is Escherichia coli, a thermo tolerant coliform found in the feces of warm-blooded animals (APHA, 1988). Animal feces are a major source of bacteria and so, the major microbial risk to human health came upon beaches and similar places that have contact with animal excreta, mainly from dogs (WHO, 23). Dynamic animal movements alongside the seashore were noticed; throughout the study period. This means that local people use the seashore as a road for those animals (Elmanama et al., 25). A mixture of bacteria is found in the digestive tracts and feces of wild and domestic animals and humans. Some of which, i.e. fecal coliforms, E. coli, and Enterococcus spp., are considered as pointers of fecal pollution in natural waters. Many urban lands use may implicate potential sources of fecal contamination in some cases (Young, 1999). Bird dung is a cause of FC to recreational waters, increasing the chance of disease spread (Elmanama et al., 25) Bather sand Swimmers The seasonal population grows in coastal regions during summer and so, the elevated organic loads are discharged into the water bodies, which are used as sewers (Oliveira, 28). Therefore, bathers can be a major source of pathogenic microorganisms (Calderon et al., 1991; Cheung et al., 1991). The danger of infection connected with swimming pools and other recreational water environments is related to fecal pollution of the water as bathers may release feces, especially afternoon if the toilet facilities are 18

35 not available, or the water source may be polluted, in case of outdoor pools, there may be direct animal contamination. If disinfection is not applied or is inadequate, the chance of infection will enlarge. The number of bathers affects that also (WHO, 21; WHO, 23). The high percentage of fecal indicators found in sand during summer indicates that there is a health risk to the bathers at these recreational areas (Sato et al., 25). As the number of tourists at beach resort areas increases the sewage discharge to the seawater too increases (Prüss et al., 1998) Bacteria from Stream Sediments Beach sands have been ignored from the public health point of view, despite the fact they are part of coastal environments with severe recreational use. But now, more care has been given to illustrate the micro biota in intertidal sediments and sands from beaches with recreational use (Elmanama et al., 26a; Oliveira et al., 27).Many studies have demonstrated that beach sands may stand as reservoirs and vectors for different of sicknesses, as sediments usually have higher concentrations of bacteria than the water column (Alm et al., 23; Whitman and Nevers, 23). Other studies have demonstrated extended persistence of culturable indicator bacteria in the sediments of environmental waters (Davies et al., 1995). Bacterial and viral pathogens discharged into water provisions are a danger to human and animal health, as the microbes may carry on for long periods and move long spaces. They adsorb to sand, clay and sediment particles that settle on the bottom resulting in the accumulation of microbes in river, lake and marine sediments (Greening, 24) 2.4 Risks from Marine Pollution Pollution from sewage livestock, urban runoff, and wildlife especially after rainfall and fecal transmitted pathogenic microorganisms affect fresh and marine water settings and so, recreational swimming can be a significant risk factor for many diseases including gastroenteritis (GI), acute respiratory disease (ARD), and eye, ear and skin infections (WHO, 23; Dwight et al., 24; Smith et al., 26). 19

36 Fecal indicator bacteria FIB pollution from human fecal material is usually considered to be a greater danger to human health as it is more likely to contain human enteric pathogens (Scott et al., 23). Fecal pollution at bathing beaches can be hazardous to humans as it may contain bacteria, viruses, and protozoa that can be ingested and result in intestinal disease. From1999 to 2, 59 disease outbreaks in the United States were referred to recreational water exposure, and 61% of these outbreaks were of gastroenteritis (Lee et al., 22). Bathers may catch illness from accidental swallowing or inhalation of water polluted with viral, bacterial, protozoan, or helminthes pathogens (Henrickson et al., 21). The rate of these diseases depends upon the level of pollution in the water, the nature and duration of exposure, and the immunological state of the bather (Oliveira, 28). Illnesses related to bathing contact in polluted recreational water are known as recreational water illness (RWI) (Saliba and Helmer, 199). In the case of diseases transmitted by the fecal oral route, this site is typically the alimentary canal. Other potential sites of infection contain the ears, eyes, nasal cavity and upper respiratory tract (WHO, 21). Spread of pathogens that can cause gastroenteritis is biologically plausible and is analogous to waterborne illness transmission in drinking-water, which is well documented. The association has been repeatedly mentioned in epidemiological studies (Kay et al., 1994; WHO, 21). Community gastroenteritis is a well-known disease in the developed world. It makes significant associated morbidity and economic costs through visits to medical practitioners and time taken off from work (Hellard et al., 23). Communicable disease observation predominantly spots the light on outbreaks of gastroenteritis (Victoria Department, 1998), a better understanding of the common sources of sporadic gastroenteritis is vital to ease targeting the interventions designed to decrease the occurrence of community gastroenteritis (Hellard et al., 23). Ocean swimmers exposed to dirty seawater are at high danger for a number of ailments compared to bathers who swim farther away from drains (Haile et al., 1999). Contact with polluted runoff has also been linked to RWI in surfers (Dwight et al., 24). Surfers may have higher exposure than swimmers because of the more frequent and 2

37 longer contact with fecal infected water (Schijven and de Roda Husman 26). In fact, standards for marine water contact in the United States and the United Kingdom are based upon risk to swimmers (Kay et al., 24). At the same time, surfers may include a disproportionately large division of marine bathers, mainly in some regions (CSDOC, 1996). The routes of exposure to waterborne pathogens are the same for surfers and swimmers (Dwight et al., 24). Beach sand is considered a site where sensitive individuals could contact pathogenic bacteria. Children usually play longer in recreational waters, and so they are more susceptible to infections than adults and are more expected to swallow recreational water. Quantitative microbial risk assessment (QMRA) models relating to the health effects and dose response curves developed for drinking water and food may be applied to the assessment of health risks from recreational waters (WHO, 21). 2.5 Microbiological Quality Indicators of Seawater Microbial disease is a public health concern; so a direct approach would be to monitor microbial pathogens in water. To do that, a large number of expensive and technically complicated assays would be required. While microarrays and other approaches to simultaneously assay numerous pathogens are under development (Maynard et al., 25; Hamelin et al., 26), there are troubles with sensitivity, specificity, and quantification, and techniques are not ready yet for common use (Field, 27) Enterobacteriaceae Enterobacteriaceae are facultative anaerobes or aerobes, ferment a wide variety of carbohydrates, possess a complex antigenic structure, and produce a variety of toxins and other virulence factors. The Enterobacteriaceae are a large, heterogeneous set of gram-negative rods. The family includes many genera (Escherichia, Shigella, Salmonella, Enterobacter, Klebsiella, Serratia, Proteus, and others) which have increased to be 44 genera and 176 named species. Some enteric organisms, e.g, Escherichia coli, are part of the normal flora and cause disease incidentally, while others, the Salmonella and Shigella, are regularly pathogenic for humans (Grimont and Grimont, 26; Nhung et al., 27). 21

38 Most species grow up well at 37 C, while others grow better at 25-3 C. They are facultative anaerobic, oxidase negative and catalase positive. They are spread all over the world and may be found in soil, water, plants and animals (HPA, 211). Morbidity and death in the developing world are mainly caused by diarrheal diseases and enteric infections. Epidemiology reports indicate that about 14 million people undergo from shigellosis with expected deaths of 6, per year all over the world. Nigeria and Bangladesh share children and young adults are at a higher risk (Wilson et al., 26). Enterobacteriaceae may account for 8% of clinically important isolates of Gramnegative bacilli and 5% of clinically significant bacteria in clinical microbiology. They account for approximately 5% of septicemia cases, over 7% of urinary tract infections UTI, a significant fraction of intestinal infections and some important pathogens like Klebsiella pneumoniae are included in hospital-acquired infections (Khan et al., 211). Enteric bacilli are frequently in charge for serious nosocomial infections such as sepsis and pneumonia in hospitalized and critical care patients (Deshpande et al., 26). Salmonella is one of the most important causes of human gastroenteritis globally. Consuming dirty pork and is one of the main causes for Salmonella infections (Guenther et al., 21). Enterobacteriaceae and Pseudomonadaceae are natural inhabitants of the guts of animals, humans, soil and water, on fruits, vegetables and cereals (Becker et al., 29), also from different clinical specimens, including blood, feces, sputum and wounds (Corti et al., 29). The wide division of these bacteria is economically and medically important for particular habitats e.g., weak and elderly persons, infants, immune compromised persons and those with chronically sickness, these bacteria represent a severe health risk (Zinkernagel, 21; Becker et al., 29). Some of the bacteria, i.e. fecal coliforms, E. coli (the predominant member of the fecal coliform group), and Enterococcus spp., are used as indicators of fecal contamination in natural waters. Relationships between indicator density and different urban land uses may implicate potential sources of fecal contamination (Whitlock et al., 22). Some of the symptoms of disease associated with fecal coliform pathogens are slight, such as upset stomach, diarrhea, ear infections, and rashes. Still, some pathogens, such as E. 22

39 coli hepatitis, and Salmonella, can have very harsh health impacts. Washington s water quality standard for fecal coliform bacteria is set to protect public health )WSDE, 25( Enterococci Enterococcus spp. is ubiquitous Gram-positive bacteria and is now the second cause of surgical and urinary tract infections and the third cause of bacteraemia. It can be found in soil, food, and water while making up a significant portion of the normal gut flora of humans and animals. It can be used as indicators of fecal contamination because they can withstand usual conditions of food matrix (ph, low/high temperature, and salinity). They pollute raw meats and heat-treated food materials (Ogier and Serror 28; Dogru et al., 21). The genus Enterococcus includes more than 17 species, but only a few species, including E. faecalis and E. faecium, cause the majority of clinical infections in humans (Devriese et al., 26), they account for more than 8% of clinical isolates (Low et al., 21), poultry have been associated with carriage of multi-resistant enterococci (Joseph et al., 21). Fecal indicators such as E. coli and enterococci are used as regulatory tools to monitor water with 24 h cultivation techniques for possible input of sewage or feces and presence of potential enteric pathogens yet their source (human or animal) cannot be determined with routine methods (Srinivasan, 211). Enterococci are recommended by the Environmental Protection Agency (EPA) for use in judging the health risk of recreational waters (EPA, 1986). These negative health effects are commonly associated with pathogens found in sewage and thus enterococci are used to protect human health in waters thought to be impacted by fecal pollution (Wade et al., 23). Lately, researchers have discovered that beach sediments can sustain populations of enterococci and are potential non-sewage sources of these indicator bacteria in recreational waters (Shibata et al., 24; Wright et al., 211). Results indicate a link between levels of enterococci in beach water and sands throughout South Florida s beaches and hint that the sands are one of the principal reservoirs of enterococci affecting beach water quality. Therefore, beaches with lower levels of enterococci in the 23

40 sand had fewer exceedances relation to beaches with higher levels of sand enterococci (Phillips et al., 211) High enterococci stages in beach water may signify an ongoing risk for bathers at these beaches (Fleisher et al., 21) and enterococci stages in sand have been shown to correlate with higher levels of human pathogens in beach sand (Shah et al., 211). And so, it is essential to judge the beach sediment s influence on the overall quality of the beach and associated recreational water. Departments of health (DOH) uses water quality measurements to evaluate the human health risk of recreational swimming at local beaches (Phillips et al., 211) Staphylococcus aureus Staphylococcus aureus is gram-positive cocci that can be found in the nose, throat, mouth, intestinal tract, on the skin of humans and warm-blooded animals. It is also isolated from a wide range of foodstuffs such as meat, cheese and milk, and from environmental sources such as soil, sand, air and water and it is an opportunistic pathogen (Prescott et al., 22; Chakraborty et al., 211). It causes many infections all over the world mainly in surgical wound infections. S. aureus causes superficial skin infections, endocarditis, sepsis and soft tissue, urinary tract, respiratory tract, intestinal tract skin abscesses, boils (furuncle), pustules and scalded skin syndrome and bloodstream infection. It has also been established that S. aureus can be transmitted among different species (Irlinger, 28; Polakowska, 212). The genus Staphylococci contains more than 3 species; only 18 of them are of potential hazard in food poisoning as they produce either coagulase, heat stable nuclease or enter toxins (Al-Tarazi, 29). S. aureus causes bacterial infections in patients on haemodialysis or continuous ambulatory peritoneal dialysis (Hetwaldt, 1998) Pseudomonas aeruginosa Pseudomonas aeruginosa is a Gram-negative, opportunistic, and pathogenic bacterium, which is present everywhere in the environment due to its ability to adapt to various adverse environmental conditions (Green et al., 1974). P. aeruginosa is an 24

41 environmental bacterium that has many lifestyles allowing it to live in water and sand (Bockelmann et al., 23). In fact, Pseudomonas is not dormant in the environment: it can replicate using a variety of food sources, either very simple molecules, such as acetate, or complex organic compounds as carbon sources (Clarke, 1982). Bacterial virulence is measure in its ability of bacterial strain to infect and inflict damage to a host. Historically, mice and rats have been used first to study Pseudomonas infections, but practical, financial and ethical considerations have led to the development of nonmammalian model hosts such as Drosophila flies, C. elegans nematodes, and more recently Dictyostelium amoebae (Lima et al., 211). The genus Pseudomonas is one of the most diverse and ubiquitous bacterial genera whose species have been isolated all over the world in all types of environments, present in sediments, clinical samples, and diseased animal specimens, water, soil, plant, deserts, etc (Peix et al., 29). P. aeruginosa microorganism rarely colonizes humans. However, the chance of colonization rises radically in hospitalized patients. Over 7% of Pseudomonas infections take place as nosocomial or healthcare-associated infections (Parkins et al., 21). In some hospitals, P. aeruginosa can be the first agent of infection, mainly in respiratory and urinary tract infections (Kerr and Snelling 29). The most serious infections caused by P. aeruginosa in humans range from acute infections like endophthalmitis, endocarditis, meningitis, and septicemia (Driscoll et al., 27) to chronic lung infections in cystic fibrosis patients (Gomez and Prince, 27). Many infections can be attributed to a common immune suppression such as in AIDS patients, burn victims, and neutropenic patients suffering chemotherapy (Steinstraesser et al., 24). A large death average happens in people with underlying illnesses like cystic fibrosis or cancer (Rowe et al., 25). Some risk aspects for Pseudomonas bacteremia have been illustrated as increased age, hemodialysis, solid organ transplant, neoplasms, heart disease, diabetes mellitus, and chronic obstructive airway disease. Nevertheless, these factors are permanent, and efforts should spotlight on suitable antibiotic therapy and prevention (Scheetz et al., 29). Most Cystic fibrosis (CF) patients suffer from chronic and, ultimately serious, pulmonary infections caused by 25

42 bacterial strains such as Staphylococcus aureus, Haemophilus influenzae, and P. aeruginosa (Stehling et al., 21). 2.6 Sea Quality Monitoring and Assessment of Gaza Beach Afifi ( ) carried out a nine-month monitoring program of 17 sampling sites down the Gaza coast. Outcomes showed that there is a climax counting of fecal streptococci as high as 233, per 1 ml for a discharge point north of Gaza city whereas the bathing water quality standards suggests less than 2 per 1 ml. The calculated Biological Oxygen Demand is high, up to 22 mg/l and the dissolved oxygen concentration was found 4.1 mg/l, at a water temperature of almost 3 degrees Celsius. Dissolved oxygen usually found to be around 5 mg/l for all measured locations. Orthophosphorus concentrations showed a maximum of.436 mg/l, which shows that a lot of nutrients enter into the sea, which cause eutrophication along Gaza coastal waters. The United Nation University/International Network on, Environment and Health (UNU/INWEH) (21) in teamwork with the Islamic University-Gaza and the Palestinian Higher Council for the Environment, developed a project for Gaza strip coastal and beach water quality. This project concluded that pollution of the seawater by untreated wastewater outflows causes widespread illness among users of popular coastal recreation beaches along the Gaza strip. The most polluted seawater was reported in middle of the Gaza strip, where sewage from Gaza city is discharged. Ministry of Environmental Affairs (MEnA) (21) stated that the Ministry of Health in Gaza strip has established a monitoring program for seawater quality in several localities alongside the beach of Gaza city. This program was performed to evaluate the microbiological pollution. The samples were collected from different selected locations along Gaza city beach from March to November during Results indicated significant microbiological pollution of the coastal waters that exceeded the global established standards especially in the sites located close to sewage outfalls. Data also indicated that the pollution caused by microbiological parameters decreased since This decrease was attributed to re-operation of Gaza wastewater treatment plant. 26

43 Elmanama et al., (25) conducted one year water quality monitoring of 5 locations along Gaza beach. Microbial presence was analysed for fecal coliform (FC) and fecal streptococci (FS) as well as Salmonella, Shigella and Vibrio. High levels of fecal indication were found in sand rather than in water approximately at all locations. The incidence of Salmonella and Vibrio separation found to be superior in sand than in water regardless of the details that just 1 grams of sand was analysed while one liter of water samples collected. Important and strong associations were found among fecal coliform and streptococci on one area of the shore as well as between Salmonella and Vibrio on the other area. The maximum concentration of microbiological parameters found to be in the vicinity that receiving urban runoff particularly during wet season. A study lasted for one year of 5 coastal water sites in the middle camps locality along Gaza shore was carried out during by Elmanama et al., (26a). samples were collected to evaluate the microbiological status and physiochemical parameters such as temperature, ph, electrical conductivity, dissolved oxygen, biological oxygen demand, total nitrogen, and ammonia. Outcomes showed seasonal and spatial differences in every measured parameter within the period of the study. The maximum level of contamination was noticed during winter, particularly after precipitation or after a release from Wadi Gaza. The associated sites with untreated wastewater discharges were having the maximum microbiological pollution levels. Nearly, all monitored values of ph were within the common level. DO concentrations varied from 3.9 to 8.2 mg/l and differed very much between different locations and within a certain location. The average values of BOD found to be 5 mg/l but at one location the average value was found to be 7.6 mg/l. Throughout the study, turbidity values varied at nearly all of the locations. El Jarousha (26) studied the incidence of few microbiological pointers in the seawater down Gaza coast throughout the period of October, 23 and September 24. The outcomes pointed out that the seawater along Gaza coast is polluted with respect to microbiological indicators. The total coliform mean values varied from 4,8 to 15, cfu/1ml, fecal coliform ranged from 2, to 12, cfu/1ml, and fecal streptococci ranged from 1,1 to 6,1 cfu/ml. The peak mean values of bacterial 27

44 indicators were obtained within winter season, while the lowest mean values were observed during summer season at all locations. The author concluded that the pollution of bathing water by fecal coliform was a serious public health alarm. Bahr (27) study focused on the impacts of the seawater pollution on total count, division and size of the species Ammonia beccarii. The aim of the research paper was to examine the seasonal distributions and sizes of Ammonia beccarii, in different selected stations from June 1997 to March The results of microbiological and chemical analysis showed that more than 9% of seawater samples in Gaza city and Northern area went over the suggested values for bathing water according to the WHO standards. The results also demonstrated that the usual size of the organism was 1.38 mm in the polluted site and.67 mm in non-polluted site. The study concluded that the seasonal variation had obvious effects on Total Count TC and size of Ammonia beccarii in the selected locations. (EQA, 214a) Environmental Quality Authority in collaboration with the Ministry of Health announced the results of monitoring water bathing beaches along the coast of the Sea of the Gaza strip in accordance to the Palestinian standards of the bathing water. Based on the samples obtained from the targeted areas, about 5% from the beaches are polluted and unsuitable for swimming. In another context the Environmental Quality Authority published in May 214 a map that shows locations where to swim and what to avoid under the title: Where to go this summer (See Annex I). (EQA, 214b) Environmental Authority of Gaza announced on 27 Jun 214 that the beaches of the besieged Gaza strip will be closed within days due to rising pollution levels prompted by tight Israeli restrictions. Pollution along Gaza's seashore has reached the highest levels in the last few years, according to a statement by Gaza's Environmental Quality Authority. The statement declared that with such high pollution levels, swimming poses health risks and the territory's fish wealth has come under threat. "Israel's ongoing siege of the Gaza Strip, which has led to the depletion of fuel reserves, has caused huge quantities of wastewater to spill directly into the sea," the authority said. The authority went on to call for swift action to prevent the wastewater spill from causing a potential health and environment crisis in the territory. 28

45 2.7 Antimicrobial Antimicrobial drugs have normally been derived into two groups; one includes the synthetic drugs, such as the sulfonamides and the quinolones, and the second, antibiotics, synthesized by microorganisms. Lately, growing numbers of semi-synthetic drugs have been developed which are chemical derivatives of antibiotics, thereby blurring between synthetic and natural antibiotics (Matthew and Levison, 24). Antibiotic resistance is an increasing trouble all over the world. Several studies were published on the mechanisms of bacterial resistance to antibiotic drugs. Similarly, several studies were published on the potential point source introduction of antibiotic resistant bacteria into the environment from agriculture, human waste, and the presence of already existing intrinsic resistance (Vanessa et al., 27) Overview of Antimicrobial Groups This is an overview of the main four antimicrobials groups - -lactam, fluoroquinolones, Aminoglycosides, glycopeptides- their mechanism of action, the way bacteria resist them and which kind of bacteria to treat lactam antibiotics -lactam antibiotics are relatively cheap drugs and are among the most commonly prescribed drugs in human and veterinary medicine. These antibiotics inhibit the activity of enzymes participating in the biosynthesis of the cell wall (Foster, 1983). The resistance of bacteria to -lactam antibiotics lies in their ability to synthesize three extracellular enzymes: -lactamase, acylase and penicillinase, which can limit the permeability of cytoplasmic membranes to those antibiotics or, by the hydrolysis, of the -lactam bond, transform them into antibiotically inactive penicilloic acid (Herwig et al., 1997). The number of bacterial strains producing an extended spectrum of - lactamases and other enzymes capable of hydrolyzing beta-lactam antibiotics is increasing both in United States and in Europe (Schwartz et al., 23). 29

46 The fluoroquinolones Fluoroquinolones are a family of synthetic, broad-spectrum antibacterial agents with bactericidal activity. The parent of the group is nalidixic acid, discovered in 1962 by Lescher and colleagues. The first fluoroquinolones were widely used because they were the only orally administered agents available for the treatment of serious infections caused by gram-negative organisms, including Pseudomonas species (Sharma et al., 29). The mechanism of action of quinolones is through the inhibition of bacterial gyrase, an enzyme involved in DNA replication, recombination and repair. By interfering with gyrase, quinolones arrest bacterial cell growth. The affinity of quinolones to metal ions seems to be an important prerequisite of their antibacterial activity: probably, quinolones bind to the DNA-gyrase-complex via magnesium ion (De Sarro et al., 21). Gram-positive and gram-negative bacteria have been reported to be resistant to quinolones. This resistance appears to be the result of one of three mechanisms: alterations in the quinolone enzymatic targets (DNA gyrase), decreased outer membrane permeability or the development of efflux mechanisms (Soni, 212) Aminoglycosides Aminoglycosides are used to treat infection caused by the aerobic, gram-negative and certain gram-positive organisms (Zhao et al., 25). The most commonly used aminoglycoside is Gentamicin. Generally, single daily dosing of aminoglycosides appeared to be safer, cost effective and efficacious. Prolonged use of the drugs will lead to side effects such as ototoxicity and nephrotoxicity. Resistance towards aminoglycoside is possible but it rarely happens (Abdul Rahim and Hau, 211). Aminoglycoside has been found 5 years ago and because of their excellent attainment, it was still used as a drug of choice to give various functions including rapid concentration-dependent bactericidal effect, clinical effectiveness, a low rate of true resistance and its low cost (Begg and Barclay, 1995). Aminoglycosides exert their antibacterial activities by two different mechanisms. The first is by inhibiting the translation of essential proteins for bacterial growth. The binding of aminoglycosides to bacterial 16S rrna, stops the translocation of the peptidyl-trna from site to the another site resulting in the misreading of the mrna. 3

47 Thereby, prevention of the production of the essential bacterial proteins leads to bacterial cell death (Magnet and Blanchard, 25). Secondly, aminoglycosides being highly positively charged, interact with negatively charged outer membranes of Gramnegative bacteria cells through electrostatic interaction and disrupt membrane integrity by displacing Mg 2+ and Ca 2+ bridges that connect neighboring lipopolysaccharides (Hancock, 1991). This creates temporary openings in the membrane and results in leaking of intracellular contents and increased antibiotic uptake through the membrane (Bryan, 1983). The resistance mechanisms can be located in mobile elements increasing the likelihood of spread of aminoglycoside resistance as well as co-resistance. Recently, a new type of mechanism, post-transcriptional methylation of the 16SrRNA, has been reported. This results in high-level resistance to aminoglycosides (EMA, 214) The glycopeptides They are narrow-spectrum agents that are active against gram-positive organisms, are bacteriostatic against staphylococci, streptococci, and Enterococci. Vancomycin was the first glycopeptide antibiotic to be discovered as early as 195 (Murray and Nannini, 21). The mechanism by which Vancomycin exerts its action is by preventing the synthesis of peptidoglycan precursors of the bacterial cell wall by blocking the transglycosylation step and subsequently affecting the transpeptidation step (Cetinkaya et al., 2; Courvalin, 25). Both the transglycosylation and transpeptidation steps are essential for bacterial cell wall cross-linking. Vancomycin resistance in enterococci was first reported by Uttley et al. in 1988 from Great Britain (Uttley et al., 1989). The basic mechanism of Vancomycin resistance in enterococci is the formation of peptidoglycan receptors with reduced glycopeptide affinity. This results in decreased binding of Vancomycin and decreased inhibition of cell wall synthesis. Peptidoglycan precursors with decreased binding to Vancomycin are responsible for this. Instead of the normally occurring peptidoglycan precursor D alanine-d alanine, precursors like D-ala-D-lactate or D-ala- D-serine are found on the cell wall of Vancomycin-resistant strains of Enterococci (Sujatha and Praharaj, 212). 31

48 2.7.2 Mechanism of antibiotic action and resistance Since the discovery of penicillin in 1929, other, more effective antimicrobials have been discovered and developed by elucidation of drug target interactions and by drug molecule modification. These efforts have greatly enhanced our clinical armamentarium. Antibiotic mediated cell death, however, is a complex process that begins with the physical interaction between a drug molecule and its specific target in bacteria, and involves alterations to the affected bacterium at the biochemical, molecular and ultra structural levels. The increasing prevalence of drug-resistant bacteria, as well as the increased means of gaining resistance, has made it crucial to better understand the multilayered mechanisms by which currently available antibiotics kill bacteria, as well as to explore and find alternative antibacterial therapies (Kohanski et al., 21) Mechanism of antibiotic In order to appreciate the mechanisms of resistance, it is important to understand how antimicrobial agents act. Antimicrobial agents act selectively on vital microbial functions with minimal effects or without affecting host functions. Different antimicrobial agents act differently. Antimicrobial agents may be described as either bacteriostatic or bactericidal. Bacteriostatic antimicrobial agents inhibit only the growth or multiplication of the bacteria giving the immune system of the host time to clear them from the system. Bactericidal agents kill the bacteria and therefore with or without a competent immune system of the host, the bacteria will be dead (Vranakis et al., 214). However, the mechanism of action of antimicrobial agents can be categorized further based on the structure of the bacteria or the function that is affected by the agents. These include generally the following: A. Inhibition of the cell wall synthesis The peptidoglycan layer is important for cell wall structural integrity, being the outermost and primary component of the wall. β- Lactam (beta-lactam) and glycopeptide antibiotics work by inhibiting or interfering with cell wall synthesis of the 32

49 target bacteria. β- Lactam antibiotics are a broad class of antibiotics that includes penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems. β-lactam antibiotics are bacteriocidal and act by inhibiting the synthesis of the peptidoglycan layer of bacterial cell walls. Glycopeptide antibiotics include vancomycin, teicoplanin, telavancin, bleomycin, ramoplanin, and decaplanin. Glycopeptide antibiotics inhibit the synthesis of cell walls in susceptible microbes by inhibiting peptidoglycan synthesis (Kohanski et al., 21). B. Inhibition of protein synthesis. A protein synthesis inhibitor is a substance that stops or slows the growth or proliferation of cells by disrupting the processes that lead directly to the generation of new proteins. It usually refers to substances, such as antimicrobial drugs, that act at the ribosome level. The substances take advantage of the major differences between prokaryotic and eukaryotic ribosome structures which differ in their size, sequence, structure, and the ratio of protein to RNA. The differences in structure allow some antibiotics to kill bacteria by inhibiting their ribosomes, while leaving human ribosomes unaffected. The following is a list of common antibacterial drugs and the stages which they target. Linezolid, Tetracyclines, Tigecycline, Aminoglycosides, Chloramphenicol, Macrolides, clindamycin, and Streptogramins (Kohanski et al., 21). C. Inhibition of nucleic acid synthesis Antimicrobial drugs can target nucleic acid (either RNA or DNA) synthesis. The antimicrobial actions of these agents are a result of differences in prokaryotic and eukaryotic enzymes involved in nucleic acid synthesis. Prokaryotic transcription is the process in which messenger RNA transcripts of genetic material are produced for later translation into proteins. The transcription process includes the following steps: initiation, elongation and termination. Antimicrobial drugs have been developed to target each of these steps. For example, the antimicrobial rifampin binds to DNA 33

50 dependent RNA polymerase, thereby inhibiting the initiation of RNA transcription (Kohanski et al., 21). D. Inhibition of Essential Metabolite Synthesis An antimetabolite is a chemical that inhibits the use of a metabolite, a chemical that is part of normal metabolism. Such substances are often similar in structure to the metabolite that they interfere with, such as antifolates that interfere with the use of folic acid. The presence of antimetabolites can have toxic effects on cells, such as halting cell growth or cell division. The three main types of antimetabolite antibiotics are antifolates, pyrimidine analogues and purine analogues (Kohanski et al., 21). E. Injuring the Plasma Membrane The plasma membrane or cell membrane is a biological membrane that separates the interior of all cells from the outside environment. Plasma membranes are involved in a variety of cellular processes such as cell adhesion, ion conductivity, and cell signaling. There are several types of antimicrobial drugs that function by disrupting or injuring the plasma membrane. One example is daptomycin, a lipopeptide which has a distinct mechanism of action, disrupting multiple aspects of bacterial cell membrane function. It appears to bind to the membrane causes rapid depolarization, resulting in a loss of membrane potential leading to inhibition of protein, DNA and RNA synthesis, which results in bacterial cell death. Another example is polymyxins antibiotics which have a general structure consisting of a cyclic peptide with a long hydrophobic tail. They disrupt the structure of the bacterial cell membrane by interacting with its phospholipids (Kohanski et al., 21). 34

51 Table 2.1: Summary of mechanisms of action of common antimicrobial agents Group of antimicrobial Effect on bacteria Mode of action in general Agents Penicillins Bactericidal Inhibition of cell wall synthesis Cephalosporins Bactericidal Inhibition of cell wall synthesis Carbanepems Bactericidal Inhibition of cell wall synthesis Polypeptide antibiotics Bactericidal Inhibition of cell wall synthesis Quinolones Bactericidal Inhibits DNA synthesis Metronidazole Bactericidal Inhibits DNA synthesis Rifamycins Bactericidal Inhibitions of RNA transcription Lincosamides Bactericidal Inhibition of protein synthesis Aminoglycosides Bactericidal Inhibition of protein synthesis Macrolides Bacteriostatic Inhibition of protein synthesis Tetracyclines Bacteriostatic Inhibition of protein synthesis Chloramphenicol Bacteriostatic Inhibition of protein synthesis Sulfonamides Bacteriostatic Competitive inhibition Mechanism of antibiotic resistance During the Second World War, penicillins have been viewed as miracle drugs. After the introduction of penicillin, isolation of new antibiotics proceeded rapidly and most of the major classes were isolated during the 194s to 196s (Walsh et al., 25). Recently, the miracle may be over as a result of increasing antibiotic resistance in bacteria, including multi resistant bacteria, which warns the earlier efficient treatment of bacterial infections. There are almost daily reports of bacteria developed resistance to familiar antibiotics. Besides, to all the different antibiotic classes existing, there exists at least one mechanism of resistance (Levy and Marshall, 24). The biggest dilemma is the appearance of multi-resistant bacteria, which make treatment especially complicated, costly, and in the end may be even impossible (Walsh and Amyes, 24). Even if antibiotic resistance appeared after short time of the development of antibiotics, the degree of the problem is presently increasing as both the formation and the transmission of antibiotic-resistant strains go up. Antibiotics act by restraining protein blending, cell wall building, or DNA replication (PIA, 1994). Bacterial resistance to antibiotics is a major concern in medical treatment. Bacteria have evolved several mechanisms to combat antibiotics (Wright, 21); these are often enzyme systems that are expressed 35

52 upon antibiotic challenge. A strategy for combating bacterial antibiotic resistance is to develop drugs that inhibit specifically these enzymes. This is not an easy matter as pointed out by Duclert et al., 29. A pathogen becomes resistant to an antibiotic used to treat it either through alteration or through the acquisition of a plasmid for resistance from another strain of bacteria (Neu, 1992). Resistant pathogens supply an antibiotic fruitless by either damaging or changing the antibiotic or preventing the antibiotic from recognizing or accessing its target. For example, microbes treated with the beta-lactam family of antibiotics (i.e., penicillins) have developed enzymes to destroy the betalactams, and as researchers change the organization of the beta-lactams a little, the bacterial enzyme alters to equal the altered antibiotic structure (Travis, 1994). Bacteria resist Antibiotics either genetically or biochemically; as shown below: A. Spontaneous Mutation and Gene Transfer Antibiotic tolerance in bacteria emerges as a result of error in DNA replication, a phenomenon known as spontaneous mutation having a frequency of one in 1 7 cells. During the epidemic of Shigella infections in Japan during 195s, it was noticed that bacteria could transfer copies of antibiotic resistance genes to susceptible bacteria thus making the latter antibiotic tolerant. The mechanism predominantly used for this purpose is conjugation1, a powerful gene-mobilizing mechanism (Sengupta and Chattopadhyay, 212). B. Drug Inactivation by Microbial Enzymes Among several mechanisms involved in the development of antibiotic resistance, drug modification plays a significant role in rendering many therapeutically useful drugs useless. For instance: lactamase, an enzyme elaborated by many Gram-positive and some Gram-negative bacteria, converts penicillin into penicilloic acid, which is therapeutically inactive (HHS and CDC, 21). C. Modification of Target Site The other mechanism of antibiotic-resistance e.g., modification of the target is best exemplified by streptomycin and erythromycin resistance of bacteria. Both of these 36

53 antibiotics act by ribosomal binding thereby inhibiting bacterial protein synthesis. Modification of the S12 protein of the 3S subunit of the ribosome makes the ribosome insensitive to streptomycin (HHS and CDC, 21). D. Reduction in Permeability to Antibiotics In some cases, emergence of mutants with reduced permeability of the cell membrane to antibiotics compared to that of the wild-type strain, causes tolerance to the antibiotic (Sengupta and Chattopadhyay, 212). E. Exclusion of Antibiotics from the Cell Among the mechanisms involved in the resistance of bacteria to Tetracycline, energy mediated efflux is a powerful strategy, which does not allow the drug to accumulate in sufficient concentration to exert its inhibitory effect (Sengupta and Chattopadhyay, 212). F. Overproduction of a Target Metabolite In some cases, the molecule, which is competitively antagonized by the antibiotic, is overproduced. For example, sulphonamides act by competitively inhibiting the enzyme dihydropteroate synthetase, which plays a crucial role in the synthesis of folate. Sulfonamides (or sulfa drugs) are structural analogs of p-aminobenzoic acid (PABA), the substrate of this enzyme (Sengupta and Chattopadhyay, 212). G. Gene Deletion Gene deletion is also known to contribute to the mechanism of antibiotic resistance. Deletion of the katg gene in some strains of Mycobaterium tuberculosis is associated with the tolerance of the organism to the anti-tubercular drug isonicotinic acid hydrazide (INH) (Sengupta and Chattopadhyay, 212) Previous Studies There has been a growing interest in the presence of many pharmaceutical substances, mostly antibiotics, in marine environment (Reinthaler et al., 23; Schwartz et al., 37

54 23). Antibiotics and their metabolites are discharged in different amounts into the aquatic environments as a result of indiscriminate use of those organic compounds in medical, veterinary, agriculture and aquaculture practices and animal husbandry (Lin et al., 24; Alpay-Karaoglu et al., 27). It had been established that antibiotics are generally, poorly riveted by the animal and human body, and therefore are excreted unchanged or transformed, by urine and feces mainly to soil and water (Gulkowska et al., 27). Though antibiotics are discharged into the water basins in very low concentrations these pharmaceuticals are of poor degradability and showed direct toxic effect to aquatic organisms, mainly bacteria (Batt et al., 26; Gulkowska et al., 27; Tamtam et al., 28). Antibiotic resistant bacteria in aquatic environments have increased dramatically as a result of the extensive use of these drugs by humans (Blyela et al., 24, Matyar et al., 27, Dang et al., 28). Morgan et al., (1976) isolated several gram-negative species that were resistant to combinations of ampicillin, chloramphenicol, kanamycin, sulfadiazine, streptomycin, and tetracycline. The Italian coastal waters yielded resistant Vibrio and Aeromonas spp., and resistance to ampicillin, erythromycin, and ciprofloxacin was detected in isolates of enterococci in samples collected from the coast of Greece (Arvanitidou et al., 21). In marine sediments from Norway, resistant Aeromonas spp., E. coli, and Vibrio spp. have been isolated (Anderson and aa, 1994). Samples collected from the coasts of Spain, Egypt, Puerto Rico, Maryland, North Carolina, and in the Gulf of Mexico, sites receiving runoff from sewage treatment plants, had a higher percentage of resistant organisms than sites with less human effect (Sabry et al., 1997). A study curried out in the waters of the Seine River (France). Two groups of Fecal indicator bacteria were discussed; E. coli and Enterococci. 42% of 214 E. coli were antibiotic resistance and 35% were multiple antimicrobial resistance. Enterococci were 83% antibiotic resistance and 49% were multiple antimicrobial resistance. E. coli and Enterococci were in point sources and non point sources. E. coli antimicrobial resistance, E. coli multiple antimicrobial resistance, Enterococci were all ranged as the 38

55 following: hospital, municipal, agricultural and forest wastewater in turn (Servais and Passerat, 29). In southern Baltic Sea shore, in Czolpino sand seashore, the bacterial demonstrated low degrees of antibiotics resistance.these microorganism were the most resistance to cefaclor and clindamycin and the most sensitive to clarithromycin, doxycycline, gentamycine, oxytetracycline. Most of the bacteria living in the sand of the studied beach were resistant to only one antibiotic out of the 18 tested ones in this study. The bacteria inhabiting the middle part of the beach and the dune were more antibiotic resistance than bacteria isolated from the seawater and shoreline-seawater contact zone. There was no clear or obvious difference in antibiotic resistance between bacteria isolated from the surface and subsurface sand layers (Mudryk et al., 21). Seawater and sand samples were gathered in southeastern Brazil, at Ilha Porchat and Gonzaguinha Beaches during summer (February 26). On Gonzaguinha, which is considered the most polluted beach, 72.3% of all isolated strains showed simple resistance, while 8.33 % had multiple resistances. The values found on Ilha Porchat beach, were 7.8% and 6.9% for simple and multiple resistances, respectively. On Guarau, the less polluted beach, only 35.3% of isolated strains had simple resistance while multiple resistances was not observed. The samples from Gonzaguinha and Ilha beach showed isolated strains resistant to seven and six different antimicrobial agents, respectively, samples from Gurarau beach were resistant to penicillin and erythromycin. The positive association obtained between the degree of seawater contamination and regularity and variability of bacterial resistance indicate that polluted marine recreational water and sands are sources of resistant bacteria contributing thus, to the dissemination of bacterial resistance (Oliveira and Pinhata, 28) Previous Study in Gaza Strip Astal et al., (22) attempted to characterize the microorganisms that cause urinary tract infections among adults and to investigate their resistance to fourteen selected antimicrobial agents. The conclusion showed resistant to amoxycillin (73.6%), doxycycline (68.6%) and trimethoprim-sulfamethoxazole (66.1%). The highest effective 39

56 drugs against all the isolates were ciprofloxacin (95.9%), amikacin (95.%) and ceftazidime (94.2%). A high rate of multiple-drug resistance was also clear for the majority of the isolates. Ciprofloxacin was weighed up along with other antibiotics against 48 clinical isolates found in urine samples collected from patients of Gaza Strip. Susceptibility tests were done by the Kirby Bauer method. The resistance rate to ciprofloxacin was 15.. Yet, high resistance to ciprofloxacin was detected among Acinetobacter haemolyticus (28.6%), Staphylococcus saprophyticus (25.%), Pseudomonas aeruginosa (2.%), Klebsiella pneumonia (17.6%) and Escherichia coli (12.%). This study points out upand-coming ciprofloxacin resistance through most UTI bacterial pathogens. Increasing resistance against ciprofloxacin demands coordinated monitoring of its activity and rational use of the antibiotics (Astal, 25). Elmanama et al., (26b) attempted to determine the most frequent bacterial causes of urinary tract infection in Gaza strip and to assess the sensitivity patterns to selected antimicrobials commonly used to heal such pathogens out of the 4778 processed urine samples were recorded as positive and only 1283 were tested for antimicrobial susceptibility. Escherichia coli were the most frequent uropathogen then Klebsiella spp. after that Pseudomonas spp., other Gram-negative bacilli. β-hemolytic streptococci, Enterococcus spp., other Staphylococcus spp. (including S. saprophyticus) Proteus S. aureus, in order. Resistance to antimicrobials was really dreadful. E. coli resistance to amoxicillin reached 97.9%, to piperacillin 78.3%, to doxycycline 9%, to sulfamethoxazole/trimethoprim 63.9% and to cefaclor 42.2%. Shigella and Salmonella-related diarrhea was studied within eight years among 357 children in Gaza, Palestine to find out the prevalence and the antimicrobial susceptibility of the isolates. The regularity of isolation showed 1.8% for Salmonella spp. and.8% for Shigella spp, with Shigella flexneri the most frequently isolated species. Most of the Shigella isolates were resistant to trimethoprim-sulfamethoxazole, ampicillin and chloramphenicol, in order. Most of the Salmonella isolates showed resistance to ampicillin, trimethoprim-sulfamethoxazole, chloramphenicol and cephalexin, in order (Abu Elamreen, 27). 4

57 In a study to investigate nosocomial pathogens in Gaza, Enterococci were prevalent in 1.9% of the entire nosocomial infected cases. The most effective antibiotics were Linzolid, Nitrofurantoin, Chloramphenicol and Ciprofloxacin, in order. While Amikacin, Gentamicin, Erythromycin and Lincomycin showed moderate activities, Methicillin, Ampicillin, Tetracycline, Aztreonam, Cefuroxime and Cefotaxim showed resistance. None of the available Cephalosporins, Monobactams, Aztreonam or semisynthetic Penicillins (Naficillin, Oxacillin) has any reaction against enterococci. The allocation of enterococci cases revealed highest rate in lower respiratory tract infection (4.5%), blood stream infection (3%), surgical wound infection (1.9%), and urinary tract infection (.8%) (Al Jarousha et al., 28). Al Laham (212) carried out a study to identify the popularity of bacterial infectivity, types, frequency and their antibiotic resistance pattern in operation theaters at major hospitals of Gaza strip. The most effective antibiotics were imipenem and tobramycin then amikacin, gentamicin, and ciprofloxacin. The highest resistance, though, was against ampicillin and amoxicillin followed by cefazolin. In contrast, Gram positive isolates were found to be highly resistant against penicillin and ampicillin. All isolates were totally sensitive with lack of resistant isolates against vancomycin. Low resistance pattern was revealed against rifampin, doxycycline and ciprofloxacin. Gram negative isolates contains extended spectrum beta-lactamase producers from Escherichia coli and Klebsiella spp. Methicillin resistance was detected in most of staphylococci isolates in methicillin resistance Staphylococcus aureus (MRSA) and methicillin resistance coagulase-negative staphylococci (MR-CoNS). Elmanama et al., (212) conducted a study to identify enteropathogenic bacteria, their antibiotic resistance and associated-risk factors in diarrheal patients in Gaza strip. Result showed the antimicrobial profile of all isolated enteropathogenic bacteria declared high resistance rates for the tested antimicrobials (E. coli, A. hydrophila, Y. enterocolitica, Shigella, and Salmonella spp. respectively. Moreover, the antimicrobial profile of the isolated enteropathogenic bacteria showed high resistance rate to rifampin, clindamycin, erythromycin, cephalexin and tetracycline, respectively. The isolated enteropathogenic bacteria declared high resistance rate to several antimicrobials. 41

58 In conclusion, a considerable number of studies on clinical isolates were performed and their results with regard to antimicrobial resistance are pessimistic. However, there are no studies or published materials regarding this issue in seawater. 42

59 3.1 Sample Sites 3 Chapter 3 Materials and Methods Seawater, sand and mussel samples were collected at five sites distributed along the shorelines of Khan Yunis and Middle governorates of the Gaza strip Figure (3.1). Sampling sites selection was based on a number of criteria including pre-existing studies and reports, site access and suitability for sampling and most importantly the different anthropogenic activities to which they are thought to be exposed. Figure 3.1: Geographic location of sampling sites. 43

60 While Khan Yunis sampling site receives partially treated sewage through the effluent of the newly established wastewater treatment plant, and Elzawaida sampling sites are open to the public for recreational activities, and believed to be least impacted sites by human activities in comparisons to other sites. The area of Wadi Gaza, especially, the northern side is directly impacted by continuous loads of untreated domestic sewage. All sampling sites were identified by geographic coordinates as determined by a hand-held Global Positioning System (Samsung Galaxy Note 1.1) (Table 3.1). A total of 138 samples including 58 seawater samples, 58 sand samples and 22 shellfish samples were collected for analysis. Samples were collected once a week, over a period of six month, from March to June and from October to November 213. All tests were carried out at the Public Health Laboratory for Food and -Ministry of health, Gaza Palestine. Table 3.1: Geographical and identification information of sampling sites Site Geographic Location Location Name Latitude Longitude Sample Type 1 Khan Yunis Besan Resort seawater, 2 Sonista Resort seawater, sand, mussel 3 Elzawaida Elzawayda Beach seawater, 4 El Nusairat Elwadi South seawater, sand, mussel 5 El Nusairat Elwadi North seawater, 3.2 Sample Collection and Processing Sampling was conducted according to the World Health Organization Manual for Recreational and Beach Quality Monitoring and Assessment WHO (1995). Seawater, beach sand and mussel samples were collected in sterile containers. All collected samples were kept immediately at 4 C in ice box packed with ice and transported to the laboratory for analysis within two hours of collection (WHO, 1995) Preparation of Culture Media In order to accomplish the objectives of the present study, several selective media were prepared. All media were obtained from HiMedia Ltd Co., India. M-FC Agar was used for the isolation of Fecal coliform and m-enterococcus agar was employed for the isolation of Enterococci. Pseudomonas aeruginosa, Staphylococcus aureus, and Vibrio 44

61 colonies were isolated on Citramide, Baird Parker, and Thiosulfate citrate Bile Salt (TCBS) Agar media respectively. Salmonella spp. and Shigella spp. were isolated on Xylose Lysine Desoxycholate (XLD) and Salmonella Shigella (SS) Agar media. All media were prepared a day before sampling and in accordance with the manufacturer s instruction depending on the volume needed. After preparation, media were allowed to cool, and then dispensed into Petri dishes Seawater Seawater samples were collected in 2L sterile bottles according world healt organisation while the sampler was standing in water at chest level (about 1.3 m). The bottles were rinsed 2-3 times with seawater from the same site before collection. The lid of the bottle was removed, turned upside down, gently lowered approximately 2-3 cm below the water surface in order to avoid the harmful effects of ultraviolet radiation and then turned so that mouth was pointing upward. When the bottle was approximately 2/3 filled, it was lifted above the surface and immediately the lid was placed back on the bottle (WHO, 1995) Beach Sterile, wide mouthed, 1 ml disposable plastic cups were used as a corer to collect sand samples from the swash zone. The lid of the cup was carefully removed, and then the cup was inverted and forced into the sand. In order to facilitate its removal from sand, large sterile spatula was used to remove the surrounding sand. The cup was then pulled together with the samples Mussels Mussels (Mytilus sp.) were sampled from the populations living on rocky outcroppings which found only at two sampling sites of and the southern side of Wadi Gaza. They were carefully detached by hand, collected in clean sterile cups and transported to the laboratory in a cooling box. 45

62 3.3 Isolation of Bacteria Seawater Under strict aseptic conditions, appropriate volumes of seawater samples were filtered through.45 µ membrane filters. The membrane filters were then placed on M-FC, m- Enterococci, Citramide and Baird-Parker media for growing of fecal coliform, Enterococci, Pseudomonas and Staphylococci respectively. The plates were incubated at C for h for fecal coliform, at 41±.5 C for 48 h for Enterococci, at 35 C for 24 h for Pseudomonas and at 35 ±.5 C for 48 ± 4 h for Staphylococcus. For the detection of Salmonella, Shigella and V. cholerae, which are usually present in very small numbers, specific volumes of seawater were first filtered through.45 µ membrane filters, then, filters were aseptically immersed in enrichment broth medium. While Selenite-F broth and Hajna Gram Negative (GN) broth were employed for the enrichment of Salmonella spp. and Shigella spp. respectively, Alkaline Peptone (APW) was used for Vibrio cholerae. Enriched cultures were incubated at C for 24 h and then streaked onto agar selective media. The APW was subcultured onto TCBS agar, whereas Selenite-F and Hajna (GN) broth cultures were subcultured onto XLD agar and SS agar. The plates were then incubated at C for up to 72 hours as many bacteria grow slowly. Discrete colonies with characteristic appearance were counted after 24 to 48h. The population density of the targeted bacteria, usually expressed as Colony Forming Unit per 1 ml (CFU/1 ml) in the original water sample, was calculated from the filtered volumes and dilutions used (WHO, 2) The first centimeter of the each sand sample was discarded, and 1 g from subsurface layer was suspended in 9 ml of.1% peptone water and mixed vigorously by vortex to detach the bacteria. Due to the expected high bacterial numbers, 3 serial tenfold dilutions (1-2, 1-3, and 1-4 ) of sand samples were prepared using sterile peptone water. Subsequently, 1 μl from each dilution was spread over the surface of agar media plates for fecal coliform, Enterococci, Staphylococci and Pseudomonas. For Salmonella, Shigella and Vibrio tests, 25g of sand were enriched as previously 46

63 described by Selenite-F, Hajna (GN) and alkaline peptone water broth media. Following 24±2 hours of incubation at 35 C, a loopful of each broth media was plated on their respective selective media. All plates were incubated as previously described and colonies were counted where possible and expressed as colony forming units per gram of sands (cfu g -1 ) (FDA, 21) Mussels The mussel samples were thoroughly washed using water and toothbrush to eliminate debris and fouling organisms from their surfaces. Bacteria in mussels were determined as described by the method No. 187 of the Nordic Committee of Food Analysis (NCFA 187, 27). They were aseptically opened using a flame-sterilized knife and the meat and intravalvular fluid were removed from the mussel and collected into sterile cups under aseptic conditions. Approximately 25 g of mussel tissues were homogenized with 225 ml (1:1) of.1 % neutralized bacteriological peptone water for 2 minutes by using a sterilized Stomacher homogenizer. The homogenate was further serially tenfold diluted with peptone water and 1 μl of all the samples (water and mussel homogenates) were plated on the surface of agar media plates for fecal coliform, Enterococci, NCFA (Nordic Committee on Food Analysis) 187, 27 and Pseudomonas and incubated in the same way as the water. On the other hand, Salmonella, Shigella and Vibrio were analyzed by using 25g of mussel homogenate and following the same enrichment processes previously described for sand samples. After the incubation period, the colonies grown were counted and expressed as Colony Forming Units per gram (CFU g -1 ) of mussels. 3.4 Bacterial Identification Presumptive identification of colonies for the purpose of enumeration was determined on the basis of morphological characteristics e.g., size, shape, color, consistency and pigment production on the selective media. Various shades of blue colonies on M-FC medium indicated fecal coliform, red colony with a metallic sheen on m-enterococci agar indicated Enterococci, yellow-green color on Citramide agar indicated Pseudomonas, smooth and entire colonies with grey to black coloration on Baird-Parker 47

64 agar indicated Staphylococcus. Pink colonies with or without black centers on xylose lysine desoxycholate (XLD) and black centered colonies with sometimes metallic sheen on Salmonella-Shigella agar (SSA) indicated Salmonella, red colonies on XLD and colorless colonies on SSA indicated Shigella, and finally, large yellow colonies on TCBS agar indicated Vibrio. On the other hand, for the purpose of antimicrobial susceptibility testing, singly growing, morphologically different colonies were picked up at random and purified by re-streaking on fresh media. Pure colonies were then subjected to biochemical tests such as Oxidase, Catalase and API-2E kit (BioMérieux- France) tests to confirm the identity of the isolated bacteria prior to subsequent antimicrobial susceptibility testing. Isolated bacteria may be either oxidase-positive or oxidase-negative for example; Enterococci, Staphylococcus, Enterobacteriaceae, Shigella and Salmonella are oxidase negative, but Pseudomonas and Vibrio are oxidase positive. Similarly, bacteria may be either catalase negative such as Enterococci or catalase positive such as Enterobacteriaceae, Staphylococcus, Pseudomonas, Salmonella, Shigella and Vibrio. The API 2E kit was used to complete the biochemical tests and for final identification of isolates. 3.5 Assessment of Beach Microbiological Quality The microbiological results of fecal indicator organisms (fecal coliform and fecal streptococci) in seawater samples collected at different sites were used to assess the seawater quality in the Gaza strip on the basis of compliance with microbiological standards set out by European Bathing s Directive (76/16/ EEC) (Table 3.2). The Directive sets two levels of compliance; "mandatory" which must be achieved, and "guideline" which should be aimed for. The guideline standards are twenty times more stringent than the mandatory. The mandatory standards are 2, cfu/1ml fecal coliform, 95% of samples should comply. Guideline standards are 1 cfu/1ml fecal coliform and fecal streptococci. Eighty percent of fecal coliform samples must comply and ninety percent of fecal streptococci samples should comply. The results were assessed against European standards to give an overall category of "guideline" pass, "mandatory" pass, or "fail" at the monitored sites. 48

65 Table 3.2 Mandatory and guideline standards for fecal coliform and fecal streptococci set by European Union Bathing Directive 76/16/EE. Organism Mandatory a, b Guideline a, b Fecal coliform 95% < 2 8% < 1 Fecal streptococci No level set 9% < 1 a Colony forming units (CFU) 1mL -1. b Minimum percentage of 2 samples required to meet standard in order to achieve compliance. 3.6 Antimicrobial Susceptibility Testing A total of 375 of identified bacterial isolate namely Enterobacteriaceae (159 isolates), Enterococci (66 isolates), Pseudomonas sp. (89 isolates) and Staphylococcus sp. (61 isolates) were tested for their susceptibility to antimicrobials using the disk diffusion method. Four hours culture of each isolate in Brain-Heart Infusion Broth (BHIB) was used to prepare a bacterial suspension which was adjusted to turbidity similar to that of.5 McFarland standards. A sterile cotton swab dipped into the culture suspension was used for inoculating the surface of solidified Mueller-Hinton agar (Oxoid, UK) plates. Then, antimicrobial discs, 6 disks per plate, were dispensed manually or with automatic dispenser. The plates were incubated at 37 C for 24h. The diameters of inhibition zones around the antimicrobial discs were measured to nearest mm and interpreted according to protocols standardized for the assay of antimicrobial compounds as guided by Clinical and Laboratory Standards Institute (CLSI). Based on inhibition zone, the isolates were categorized as resistant, intermediate or susceptible to the tested antimicrobials (CLSI, 211). 49

66 Staphylococcus spp. Pseudomonas sp. Enterococci Enterobacteriacae Disk potency Antimicrobial agent Table 3.3 Antimicrobials used in this study and their concentrations Amoxycillin/clavulinic acid (AMC, 3 µg) X Ampicillin (AMP, 1 µg) X Amikacin (AK, 3 µg) X Azithromycin (AZM, 15 µg) X Ciprofloxacin (CIP, 5 µg) X X X X Cefuroxime (CXM, 3 µg), X Ceftrazidime (CAZ, 3 µg) X X Ceftriaxone (CTR, 3 µg) X Doxycycline (DO, 3 µg) X Gentamicin (CN, 1 µg) X X X Meropenem (MER, 1 µg) X X Norfloxacin (NX, 1 µg) X X X Nitrofurantion (F, 3 µg) X Ofloxacine (OF, 5 µg) X Oxacillin (for methicillin) (OX, 1 µg) X Penicillin (P, 1 units) X Piperacillin (PRL, 1 µg) X X Trimethoprim (TM, 2.5 µg) X Tetracycline (TE, 3 µg) X X Trimethoprim/Sulfamethoxazole (SXT, 25 µg) X X Vancomycin (VA, 3 µg) X X Multiple Antimicrobial Resistance Index (MARI) The MAR index for each isolate was calculated by following the procedure described by Krumperman (1983) as: number of antimicrobials to which the isolate was resistant / Total number of antimicrobials against which isolate was tested. MAR indices for each sample site were calculated as the number of antimicrobials to which all isolates were resistant/ (number of antimicrobials tested number of isolates tested per site) (Kaspar, et al., 199). 5

67 3.7 Statistical analysis The geometric means of FC and FS densities in seawater, sand and mussel samples were determined and compared. Correlation analysis was used to explore relationships of fecal bacteria densities between seawater and sand at the same location. The resistance to antimicrobial agents was expressed as percentage. Results were used to calculate the Multiple Antibiotic Resistance index (MARI) for the different isolates and locations. Student t-tests was performed to test differences between Multiple Antibiotic Resistance index (MARI) values of mussel samples from the two locations and differences between MARI values of seawater and sand samples at the same location. One way analysis of variance (ANOVA) and Tukey post hoc test were performed to compare the MARI values between the five locations (regardless of sample type) and between seawater, sand and mussel samples at the same location. Probabilities <.5 were considered significant. Before analysis, all indices were arcsine transformed to improve normality. Statistical analyses in this study were carried out with SPSS (Statistic Package for the Social Science) software for windows (PASW Statistics 18). 51

68 4 Chapter 4 Results This chapter presents the main findings of the study, divided into two major sections. The first one is assessing beach microbiological quality while the second one is talking about Antimicrobial resistance. 4.1 Assessing Beach Microbiological Quality In this study, 116 samples (58 seawater and 58 beach-sand) were collected at 13 sampling occasions, from five stations, along the shoreline of Gaza strip, throughout the study period, which lasted March to June and from October to November 213. Mussel samples (N=22) were collected from only two locations at and south of Wadi Gaza outlet. Geometric means for FC and FS densities in seawater and sand samples collected from the five collections sites are presented in Figure (4.1). In most sites, cultivable fecal indicator bacteria were more abundant in the sand (ranging from 34 to 175 cfu/1g) than in the water (ranging from 14 to 2149 cfu/1ml), the average in sand was found cfu/1g, and in water was found cfu/1ml. Compared to water, FC counts in sand were 2.8, 4., 1.9, and 1.2 times higher at locations 1, 2, 3, and 4 respectively, while, at location 5 it was lower (.51). Similarly, FS counts in sand samples were 13.4, 3.3, 2.9, and 8.3 times greater than in the corresponding seawaters, considering the same order of locations as mentioned above with the exception of location five (.18). The numbers of fecal streptococci in sand samples were higher than that of FC at locations 1, 3 and 4, with geometric means of 3, 5 and 1518 cfu/1g for FS and 5, 182 and 495 cfu/1g respectively for FC. 52

69 Geometric mean (cfu/1 ml or 1g) sand water sand water sand water sand water sand water Fecal Coliform Fecal Streptococci Khan Yunis Elzawaida South of Wadi Gaza outlet North of Wadi Gaza outlet Sample location and type Figure 4.1: Geometric means (cfu/1 ml seawater or 1g sand) of fecal coliform and fecal streptococci in the water and subsurface layer of wet sand at five locations. Statistical analysis indicated a correlation between fecal bacteria densities in seawater and sand at location # 2 only with correlation coefficient values (r) of.65 and.81 for fecal coliform and fecal streptococci counts respectively. Population density of both fecal coliform and fecal streptococci was generally higher at locations 1, 4 and 5, suggesting that these locations are more polluted as compared to the other locations (Figure 4.2). The maximum populations of FC and FS in seawater samples were observed in the northern side of Wadi Gaza with geometric mean values of and cfu/1 ml respectively. 53

70 Geometric mean (cfu/g) Geometric mean (cfu/1 ml or 1g) 25 Average Khan Yunis Elzawaida South of Wadi Gaza outlet Locations North of Wadi Gaza outlet Figure 4.2: Population density of fecal coliform and fecal streptococci in the water and sand at five locations. The number of FC and FS in the mussel samples from locations 2 and 4 are presented in Figure 4.3. Mussels were found to accumulate more FS than FC. Compared to seawater from the same location, FS counts in mussels were and 17.6 times higher at Deir Al Balah and location to the south of Wadi Gaza outlet respectively. FC/FS ratio in mussel samples collected from locations 2 and 4 were as low as.19 and.2 respectively. Fecal Coliform Fecal Streptococci Sampling location South of Wadi Gaza outlet Figure 4.3: The number of FC and FS in the mussels from locations 2 and 4 54

71 Table 4.1 highlights the percentage of samples where fecal indicator organisms (fecal coliform and fecal streptococci) concentrations exceeded the EU mandatory and guideline standards for bathing water (Table 3.2). Proportions of samples that exceeded both mandatory and guideline standards were highly variable between the five locations. The percentage of samples that exceeded guideline standards at the five locations were ranged from 23.1 to 1% and from 46.2 to 1% for FC and FS respectively. On the other hand, the percentage of samples that exceeded mandatory standards for fecal coliform was ranged from. to 5%. A greater proportion (percentage) of samples from the northern side of Wadi Gaza outlet exceeded guideline standards for both FC and FS and mandatory standards for FC than other locations (Table 4.1). Table 4.1 Percentage of recorded observations that exceeded EU mandatory and guideline standards for fecal coliform (FC) and fecal streptococci (FS) in seawater samples collected from the studied locations. Locations % Exceeding guideline % Exceeding mandatory FC FS FC Khan Yunis Elzawaida South of Wadi Gaza outlet North of Wadi Gaza outlet Antimicrobial Resistance With the help of morphological as well as biochemical criteria a total of 375 of bacterial isolates from seawater, beach sand and mussel samples were identified and confirmed to at least the genus level and tested for their resistance to a selected list from 21 antimicrobial. These isolates were as follows; 159 (42.4%) Enterobacteriaceae, 66 (17.6%) Enterococci, 89 (23.7%) Pseudomonas and 61 (16.3%) Staphylococci. 55

72 4.2.1 The incidence of MAR in different sample types and sampling locations Summary statistics for the incidence of multiple resistance, that is, simultaneous resistance to more than one antimicrobial; in the different sample types and sampling locations are given in Table 4.2 and 4.3 respectively. The incidence of multiple resistance among the bacterial isolates from mussels, seawater and beach sand was 91.7%, 84.8% and 75.3% respectively for Enterobacteriaceae isolates, 75%, 91.1% and 92.3% for Enterococci isolates, 33.3%, 66.7% and 27.3% for P. aeruginosa isolates and 1%, 9.9% and 81.5% respectively for Staphylococcus isolates (Table 4.2). Table 4.2: Incidence of multiple resistances for all bacteria isolated from mussels, seawater and sand beach. Bacteria Mussels Enterobacteriaceae Enterococci P. aeruginosa Staphylococci Average More Than One % 75.3% 84.8% 91.7% Average More Than One % 92.3% 91.1% 75.% Average More Than One % 27.3% 66.7% 33.3% Average More Than One % 81.5% 9.9% 1.% Similarly, the incidence of multiple resistance for all isolates from the five sampling locations of Khan Yunis,, Elzawaida, South of Wadi Gaza Outlet and North of Wadi Gaza Outlet regardless of sample type (seawater, sand or mussels) were 82.8%, 8%, 6%, 88.4% and 81% respectively for Enterobacteriaceae, 1%, 76.9%, 9%, 88.2% and 94.1% for Enterococci isolates; 57.1%, 34.6%, 37.5%, 29.4% and 68.8% respectively for P. aeruginosa, isolates and 88.9%, 83.3%, 9.9%, 91.7% and 9.9% respectively for Staphylococcus isolates (Table 4.3). 56

73 Table 4.3: Incidence of multiple resistances for Bacteria isolated from five Locations Bacteria Enterobacteriace ae Enterococci P. aeruginosa Staphylococci Khan Deir al South of North of Elzawaida Yunis Balah Wadi Gaza Wadi Gaza Average > % Average > % Average > % Average > % Enterobacteriaceae In this study, 159 isolates of Enterococci were recovered from seawater, beach sand and mussel samples collected from Khan Yunis,, Elzawaida, South Wadi Gaza and North Wadi Gaza, and their resistance to ten different antimicrobial of AMC= Amoxicillin-clavulanic, PRL= Piperacillin, CN= Gentamicin, TE= Tetracycline, CTR= Ceftriaxone, CAZ= Ceftrazidime, CXM= Cefuroxime, MER= Meropenem, CIP= Ciprofloxacin, SXT= Trimethoprim/Sulfamethoxazole. The percentage of resistance isolates were 38.9%, whereas the percentages of intermediary sensitive and sensitive cases were and 45.5%, respectively Generic Composition of Bacterial Isolates Among the identified isolates, 81 isolates were from sand samples, 66 from seawater samples and 12 from mussel samples. These isolates were belonging to 1 different genera and 13 species. The predominant isolates in descending order of genus frequency were: E. coli (N = 75), Proteus spp. (N = 34) (22 of P. mirabilis, 7 of P. vulagaris and 5 of P. penneii), Klebsiella spp. (N = 25) (22 of K. pneumoniae and 3 of K. oxytoca), Citrobacter freundii (N = 1), 57

74 Salmonella typhi (N = 4), Serratia marcescens (N = 4), Morganella spp., (N = 3), Providencia stuartii (N = 2) and Pantoea spp. and Vibrio sp. (N = 1) (Figure 4.4). The predominant isolates from seawater samples in descending order according to their percentage (rate) of occurrence were: E. coli (5%), followed by P. mirabilis (13.6%), K. pneumonia (1.6%), P. vulagaris, C. freundii (6.1%), Morganella spp., P. stuartii (3%), and finally, P. penneii. K. oxytoca, Salomnella spp. and S. marcescens (1.5%). Similarly, enteric bacterial isolates recognized from beach sand samples revealed closely related arrangement to that of seawater, with E. coli, the most isolated species, constituting 45.7% of the total sand isolates. The second most common species was K. pneumonia (17.3%) followed by P. mirabilis (13.6%), P. penneii (4.9%), P. vulagaris, Salmonella spp., C. freundii (3.7%) K. oxytoca, S. marcescens (2.5%) and finally Morganella spp. (1.2%). The percentages of isolates from mussel samples were 41.7% E. coli, 25% C. freundii, 16.7% P. mirabilis and 1% for both K. pneumoniae and S. marcescens. Pantoea spp. and Vibrio cholerae were isolated only once from seawater and beach sand respectively. It is worth to mention that, E. coli was the most frequent species, constituting 5, 45.7 and 41.7% of the total isolates from seawater, sand and mussel samples respectively (Figure 4.4). Moreover, E. coli and K. pneumoniae were the only two species recovered from all sampling sites in the present study. 58

75 Percentage Seawater Mussels Microorganism Figure 4.4: A summary of bacteria belonging to Enterobacteriaceae family isolated from seawater sand and mussel samples and tested for antimicrobial resistance Enterobacteriaceae Resistance Isolates recovered from the five locations along the shoreline of the Gaza strip and confirmed as Enterobacteriaceae were tested for their susceptibilities to1 antimicrobial (Table 4.4). The total of resistance cases was 38.9%, whereas, the total of intermediary sensitive and sensitive cases were 15.5 and 45.5%, respectively. Bacterial isolates showed varying degrees of resistances to antimicrobial ranged from 15 to 68%. The highest percentage of resistance (> 5%) was observed for Tetracycline (67.9%), followed by Amoxicillin-clavulanic (56%) and Trimethoprim/Sulfamethoxazole (5.3%). In contrast, resistance to Piperacillin (49.1%), Ceftrazidime (39%), Cefuroxime (32.1%), Ceftriaxone (28.9%), Meropenem (27%) was classified as medium (25 5% resistance), whereas resistance to Gentamicin (23.9%) and Ciprofloxacin (15.1%) was low (<25% resistant) (Table 4.2). The results also showed that 68% of Enterobacteriaceae isolates were resistance to Tetracyclines, 5.3% to Trimethoprim/Sulfonamides (SXT), 41.6% to β-lactam (Amoxicillin-clavulanic, Piperacillin, Ceftriaxone, Ceftrazidime, Cefuroxime, and Meropenem), 24% to 59

76 aminoglycosides (Gentamicin) and 15% to fluoroquinolone (Ciprofloxacin) antimicrobial. Bacteria isolated from the beach sand, seawater and mussel samples showed differences in the level of resistance to studied antimicrobial (Table 4.4). The highest antimicrobial resistance was observed in bacteria isolated from the mussels (48.3%), followed by seawater (46.2%) and beach sand (31.6%). Bacteria isolated from the mussel samples were 83.3% resistance to Piperacillin, followed by Ceftrazidime, Trimethoprim/Sulfamethoxazole and Tetracycline (58.3), then by Amoxicillinclavulanic and Meropenem (5%), Ceftriaxone (41.7%), Gentamicin (33.3%), Ciprofloxacin and Cefuroxime (25%). On the other hand, bacteria isolated from the seawater were 71.2% resistance to Tetracycline, followed by Amoxicillin-clavulanic (62.1%), Trimethoprim/ Sulfamethoxazole (53%), Piperacillin (53%), Ceftrazidime (53%), Cefuroxime (4.9%), Ceftriaxone (36.4%), Meropenem (36.4%), Gentamicin (31.8%) and CIP (24.2%). Isolates from sand were 66.7% resistance to Tetracycline, 51.9, 46.9, 4.7, 25.9, 24.7, 21., 16., 16. and 6.2% resistant to Amoxicillin-clavulanic, Trimethoprim/ Sulfamethoxazole, Piperacillin, Cefuroxime, Ceftrazidime, Ceftriaxone, Meropenem, Gentamicin and Ciprofloxacin respectively. Table 4.4: Percentage of resistance of Enterobacteriaceae isolated from sand, seawater and mussel samples to different antimicrobial Sample Type Antimicrobials Seawater Mussels Average Amoxicillin-clavulanic Ceftrazidime Ciprofloxacin Cefuroxime Gentamicin Ceftriaxone Meropenem Piperacillin Trimethoprim/ Sulfamethoxazole Tetracycline Average

77 Comparison of isolates resistance at different locations showed little differences in the level of resistance to studied antimicrobial (Table 4.5). The highest resistant was observed for isolates from the southern side of Wadi Gaza (43.1%), followed by isolates from (39.2%), then, North Wadi Gaza (38.7%), Khan Yunis (36.4%) and finally Elzawaida (33.5%). Isolates from Khan Yunis, Elzawaida, South Wadi Gaza and North Wadi Gaza showed high resistant to Tetracycline and high susceptibility to Ciprofloxacin. Resistance against Amoxicillin-clavulanic and Trimethoprim/Sulfamethoxazole were more frequently observed in isolates from the Northern side of Wadi Gaza outlet (64 and 57% respectively). On the other hand, the highest resistance against Gentamicin, Ceftrazidime, Cefuroxime, and Ciprofloxacin were observed in isolates from South of Wadi Gaza, while resistance against Piperacillin and Meropenem were more frequently observed in isolates from and the highest resistance against Ceftriaxone was observed in isolates from Elzawaida. Table 4.5: Percentage of resistance Enterobacteriaceae strains isolated from different locations at Gaza Strip shoreline to 1 antimicrobial (%). Locations Khan Yunis Deir al Balah Elzawaida No of Isolation Antimicrobials % resistance AMC PRL CN TE CTR CAZ CXM MER CIP SXT Average South 43 Wadi Gaza North 42 Wadi Gaza Total Antimicrobial Abbreviations: AMC= Amoxicillin-clavulanic, PRL= Piperacillin, CN= Gentamicin, TE= Tetracycline, CTR= Ceftriaxone, CAZ= Ceftrazidime, CXM= Cefuroxime, MER= Meropenem, CIP= Ciprofloxacin, SXT= Trimethoprim/Sulfamethoxazole. 61

78 Resistant idolates (%) The antimicrobial resistance analysis performed for isolates from mussels showed that, isolates from were more frequently resistance than those isolates from the south of Wadi Gaza outlet (Figure 4.5). South Wadi Gaza CAZ CTR TE SXT PRL MER CIP CXM CN AMC Antimicrobials Figure 4.5: Resistance isolation percentage of Enterobacteriaceae strains isolated from mussel in and South of Wadi Gaza outlet. Antimicrobial Abbreviations: AMC= Amoxicillin-clavulanic, PRL= Piperacillin, CN= Gentamicin, TE= Tetracycline, CTR= Ceftriaxone, CAZ= Ceftrazidime, CXM= Cefuroxime, MER= Meropenem, CIP= Ciprofloxacin, SXT= Trimethoprim/Sulfamethoxazole Multiple Antibiotic Resistances The collection of bacterial isolates was analyzed for multiple antimicrobial resistances (Figure 4.6). Among the tested isolates, 13.8, 21.4, 12.6, 1.1, 8.8, 6.9, 7.5, 6.9 and 1.9% of the isolates showed resistance to one, two, three, four, five, six, seven, eight and nine antimicrobial respectively. It was only small fraction (4.4%) of the Enterobacteriaceae that appeared to be totally resistant to all ten antimicrobial tested. At the same time, only 5.7% demonstrated susceptibility to all antimicrobial; i.e., they did not have any acquired resistance to the antimicrobial tested. Strains isolated from seawater, sand beach and mussel samples were found to be respectively susceptible only to 3., 7.4 and 8.3% of all antimicrobial tested. 62

79 % of resistant strains The results also show that more than 94% of all isolates resistance to at least one antimicrobial. Moreover, 8.5, 59.1, 46.5, 36.5, 27.7, 2.8, 13.2, 6.4 and 4.4% of the isolates exhibited simultaneous resistance to more than one, two, three, four, five, six, seven, eight and nine antimicrobial respectively i.e., they are multi-drug or polydrug resistant. Mussels Average zero one two three four five six seven eight nine ten Number of antibiotics Figure 4.6: Multi-drug resistance among Enterobacteriaceae strains isolated from sand (n =81), seawaters (n = 66) and mussels (12) of Gaza strip shoreline Antibiotic Resistance Pattern According to Generic Composition of Bacterial Isolates The results of antimicrobial resistance analysis performed for each Enterobacteriaceae species are shown in Tables 4.6. Enteric bacterial species revealed variable resistance against the ten tested antimicrobials ranged from to 1% and the majority of them were multiple antimicrobial resistant (MAR). The highest antimicrobial resistance was attained by Pantoea spp. colonies (8%), followed by P. stuartii (6%), K. oxytoca (57%), P. vulgaris (53%), Salomnella sp. (4%), K. pneumonia (4%), E. coli (39%), P. mirabilis (36%), Morganella spp. (33%), S. marcescens (33%), C. freundii (3%), P. penneri (3%), and Vibrio cholerae (2%). E. coli, the predominant isolates recovered from sand, water and mussels were respectively 28.9, 49.7 and 4% resistant to the tested antimicrobial, and they were most 63

80 resistant against Tetracycline: 62.2% of the sand isolates, 72.7% of the seawater isolates and 8.% of the mussel isolates. The other antimicrobial revealed variable resistance and were 2.7%-43.2, and. 8% for sand, seawater and mussel isolates respectively. K. pneumonia isolates were most resistant against Amoxicillin-clavulanic; 57.1% of, 85.7% and 1% of the sand, seawater and mussel isolates respectively. P. mirabilis isolates from sands, seawaters and mussels were respectively %, -88.9% and -1% resistant against tested antimicrobial. The isolates of genus Proteus (n = 34) which include three species (P. mirabilis, P. penneri, P. vulgaris) from sand, seawater and mussel samples exhibited %,.-78.6% and -1% respectively resistant against antimicrobial. Isolates of C. freundii from sand, seawater and mussel were -66.7%, -5% and -66.7% respectively resistant to tested antimicrobial. 64

81 Table 4.6: Resistance of Enterobacteriaceae according to source and sampling location along Gaza strip beach (Numbers in parentheses represent the numbers of isolate) Bacterial isolates E. coli (75) Proteus spp. (34) Klebsiella spp. (25) C. freundii (1) Salmonella (4) sp. (4) S. marcescens (4) Morganella (3) spp. (3) (37) (33) Mussels (5) Total (75) (18) (14) Mussels (2) Total (34) (16) (8) Mussels (1) Total (25) (3) (4) Mussels (3) Total (1) (3) (1) Total (4) (2) (1) Mussels (1) Total (4) (1) (2) AMC CN CXM Antibiotics CIP MEM PRL SXT TE CTR CAZ Averag Total MARI e

82 Multiple Antimicrobials Resistance Index (MARI) for Enterobacteriacae Calculations of MAR index for each bacterial species revealed variation among the isolates (Table 4.6) with MARI values ranged from.2 to.8, in which Vibrio spp. show the lowest MARI value (.2) whereas Pantoea sp. showed the highest MARI value (.8). The MAR index values for the total isolates of other species were as follows;.3 for Citrobacter freundii,.33 for Serratia marcescens,.33 for Morganella morganii,.39 for Escherichia coli,.39 for Proteus spp.,.4 for Salmonella spp.,.42 for Klebsiella spp. and.6 for Providencia stuartii. For sand isolates, the highest MAR index value calculated was achieved by Salmonella (.5) followed by S. marcescens (.45), Klebsiella spp. (.43), Proteus spp. (.35), E. coli (.29), C. freundii (.23), M. morganii (.2) and finally Vibrio cholerae (.2). On the other hand, the most pronounced MAR index values of seawater isolates were recorded by Pantoea spp. (.8). This was followed by P. stuartii (.6), Klebsiella spp. (.55), E. coli (.5), Proteus spp. (.39), C. freundii (.3) and finally S. marcescens (.2). As regards mussel isolates, the highest MAR index value of 1. was calculated for Klebsiella spp., followed by.75 for Proteus spp., and then MARI decreased to.4,.37 and.2 for E. coli, C. freundii and S. marcescens respectively. The MAR index for sand, seawater and mussels isolates from the different sampling location was calculated separately (Figure 4.7). The overall MARI value for all isolates from all locations was found to be.39. The bacteria isolated from the Mussels demonstrated higher resistance to most of the tested antimicrobial (MARI =.48) than bacteria isolated from the seawater (MARI =.46) and sand beach (MARI=.32). The highest antimicrobial resistance was determined in the bacteria isolated from South of Wadi Gaza outlet (MARI.43), followed by North of Wadi Gaza outlet (MARI.39), (MARI.39) Khan Yunis (MARI.36), and finally Elzawaida (MARI.34). For seawater samples, MAR indices were.51,.49,.45,.42 and.41 for North of Wadi Gaza outlet, South of Wadi Gaza outlet,, Elzawaida and Khan Yunis. On the other hand, the MAR indices for sand samples were.36,.33,.33,.25 and.24 for South of Wadi Gaza outlet, North of Wadi Gaza outlet, Khan Yunis, Elzawaida and. 66

83 seawater sand seawater sand mussel seawater sand seawater sand mussel seawater sand MARI The MARI of mussel samples from (.52) was higher than that of South of Wadi Gaza Outlet (.46). Independent sample t-test however, could not reveal any significant differences between MARI of mussel samples from the two locations. Despite the observed differences among the MARI of isolates from different sample types (seawater, sand and mussels) at the four locations, it was only the northern side of Wadi Gaza outlet that showed significantly higher resistance in bacteria isolated from seawater samples (p <.5) compared to the sand samples from the same location. In addition, no significant differences were observed between the MARI from the five locations (ANOVA, p >.5). Among the 159 of Enterobacteriaceae isolates, 94 (59.1%) were found to have MAR values greater than Khan Yunis Elzawaida South of Wadi Gaza outlet Locations and sources North of Wadi Gaza outlet Figure 4.7: Multiple Antimicrobial Resistance Index (MARI) for Enterobacteriaceae 67

84 4.2.3 Enterococci In this study, sixty six isolates of Enterococci were recovered from seawater, beach sand and mussel samples collected from Khan Yunis,, Elzawaida, South Wadi Gaza and North Wadi Gaza, and their resistance to six different antimicrobial of Vancomycin (VA), Tetracycline (TE), Ciprofloxacin (CIP), Ampicillin (AMP), Norfloxacin (NX), and Nitrofurantion (F) were investigated. The total resistance was 36.6%, whereas the total of intermediary sensitive and sensitive cases was 11.4 and 52%, respectively Resistance of Isolated Enterococci Strains The resistance of the 66 Enterococci isolates against the six antimicrobials tested, from beach sand, seawater and mussel samples is shown in Table 4.7. Regardless of sample type (seawater, mussels or sand) Enterococci isolates showed high level of resistance (>5% resistant) to Vancomycin (84.8%), Ampicillin (8.3%) and Tetracycline (51.5%), and intermediate resistance (25 5% resistance) to Nitrofurantion (36.4%), Ciprofloxacin (33.3%) and Norfloxacin (25.8%). None of the enterococcal isolates was classified as low resistance (<25% resistant) to any of the tested clinical antibiotics. The results also showed that 84.8% of Enterococci isolates were resistance to glycopeptide (Vancomycin), 8.3% to β-lactam (Ampicillin), 51.5% to Tetracycline (Tetracycline) 36.4% to Nitrofurantion (Nitrofurantion), 33.3% to Fluoroquinolones (Ciprofloxacin), 25.8% to Norfloxacin (Norfloxacin). Data also showed differences in the level of resistance between beach sand, seawater and mussel samples to studied antibiotics (Table 4.7). The highest antimicrobial resistance was observed in bacteria isolated from the sand (59%), followed by mussels (54.2%) and water (49.6%). Except for Vancomycin, Enterococci isolates from sand displayed higher percentage of resistance against all antibiotics tested than isolates from seawater. Isolates from mussels on the other hand, displayed higher percentage of resistance against Tetracycline (62.5%), Ciprofloxacin (62.5%) and Norfloxacin (37.5%) than those from sand and seawater. 68

85 Table 4.7: Percentage of resistance of Enterococci isolate from sand, seawater and mussel samples to different Antimicrobial (%) Sample Type Antimicrobials Vancomycin Tetracycline Ciprofloxacin Ampicillin Norfloxacin Nitrofurantion Average Mussels Average Comparison of isolates resistance at different locations showed little differences in the level of resistance to studied antimicrobial (Table 4.8). The highest resistant was observed for isolates from the north side of Wadi Gaza (59%), followed by isolates from Khan Yunis (56%), then, south Wadi Gaza (52%), Elzawaida (47%) and finally (44 %). Table 4.8: Percentage of resistance of Enterococci strains isolated from different locations at Gaza strip shoreline to six antimicrobial (%) Antimicrobials % resistance Locations Khan Yunis Elzawaida South Wadi Gaza North Wadi Gaza Total No. of isolates VA TE CIP AMP NX F Average Antimicrobial abbreviations: VA= Vancomycin, TE= Tetracycline, CIP= Ciprofloxacin, AMP= Ampicillin, NX= Norfloxacin, F= Nitrofurantion. The antimicrobial resistance analysis performed for isolates from mussels showed that, isolates from were more frequently resistance than those isolates from the south of Wadi Gaza outlet (Figure 4.8). 69

86 Resistant idolates (%) Dier al Balah South Wadi Gaza VA TE CIP AMP NX F Antimicrobials Figure 4.8: Resistance isolation percentage of Enterococci strains isolated from mussel in and South of Wadi Gaza Outlet. Antimicrobial abbreviations: VA= Vancomycin, TE= Tetracycline, CIP= Ciprofloxacin, AMP= Ampicillin, NX= Norfloxacin, F= Nitrofurantion Number of Antibiotic Resisted Different antimicrobial resistance patterns were observed for isolates from beach sand, seawater and mussel samples. Generally, the data indicated that all Enterococci isolates were MAR (resistant to at least two antimicrobial). Nearly 11% of isolates were resistant to one antimicrobial, 3% were resistant to some level of two different antimicrobial and 25.8, 15.8 and 6.1% were resistant to three, four and five antimicrobial. Approximately, 12% of the isolates were resistant to all six antimicrobial used for testing (Figure 4.9). The results also show that approximately all isolates were resistance to at least one antimicrobial, whereas 89.4, 59.1, 33.3, 18.2 and 12.1% of the isolates exhibited simultaneous resistance to more than one, two, three, four and five antibiotics respectively. The highest bacterial resistant was found to two (35.6%) and three (31.1%) antimicrobial and was found among isolates from seawater. Bacteria resistant to one and four antimicrobial on the other hand, were more commonly found among isolates from mussels (25%) and sand (3.8%) respectively. While a lower percentage of isolates were found to be resistant to five and six antimicrobial, they were more commonly found among isolates from mussels (12.5%) and sand (15.4%), (Figure 4.9). 7

87 Percentage of strains Mussels Average one two three four five six Number of antibiotic Figure 4.9: Multiple Antimicrobial resistance of Enterococci isolates from sand, mussels and seawater samples from the coast of the Gaza Strip MARI for Enterococci For all sites, the average MAR index was.52 and ranged from.43 to.67. Enterococci isolates from the Northern side of Wadi Gaza outlet (MARI =.59) was higher than those from the other stations such as Khan Yunis (.56), South of Wadi Gaza Outlet (.52), Elzawaida (.47) and (.44). MAR indices calculated for Enterococci isolates from sand samples (.5-.67) were higher than seawater ( ) values (Figure 4.1). Moreover, MARI of mussel samples from South of Wadi Gaza Outlet had higher value of.57 than that of sampling stations of South of Deir al Balah (.5). Despite the observed variations, no significant differences in MARI values were detected among the six locations (ANOVA, p >.5). Also, no significant differences were observed in the MARI values among seawater, sand and mussel samples collected at the same location. Additionally, independent sample t-test could not reveal any significant differences between MARI of mussel samples from the two locations. Out of 66 Enterococci isolates, 59 (89.4%) were found to have MAR Indices more than.2 while only 1.6% (n = 7) were found to have MARI less than.2. 71

88 seawater sand seawater sand mussel seawater sand seawater sand mussel seawater sand MARI Khan Yunis Elzawaida South of Wadi Gaza outlet North of Wadi Gaza outlet Sample type and location Figure 4.1: Multiple Antimicrobial Resistance Index (MARI) for Enterococci Pseudomonas aeruginosa A total of 89 isolates from Pseudomonas aeruginosa were isolated to investigate bacterial resistance to 8 different antimicrobial; Amikacin (AK), Gentamicin, (CN), Ceftrazidime (CAZ), Piperacillin (PRL), Meropenem (MER), Ofloxacine (OF), Norfloxacin (NX) and Ciprofloxacin (CIP). The total of resistance cases were 23.7% whereas the total of intermediary sensitive and sensitive cases were 3.4% and 68.3%, respectively Resistance of Isolated Pseudomonas Strains Tables 4.9 and 4.1 show the percentage of resistance of P. aeruginosa isolates from mussels, seawater and sand samples to the eight antimicrobial used in the antimicrobial susceptibility testing. The data presented in Table 4.9 show that there were large differences between P. aeruginosa isolated from the different types of samples in their resistance to the antimicrobial used in this study. In general, isolates showed high resistance (>5% resistant) to Piperacillin (55.1%) only. In contrast, resistance to Ceftrazidime (28.1%), Norfloxacin (25.8%) and Ciprofloxacin (25.8%) was classified as medium (25 5%) resistance, whereas resistance to Ofloxacine (21.3), Gentamicin (18%), and Meropenem (15.7%) was low 72

89 (<25% resistant). On the other hand, P. aeruginosa isolates were found to be totally (1%) susceptible to Amikacin (AK). The results also showed that 32.9% of Pseudomonas aeruginosa isolates were resistance to β-lactam (Piperacillin, Ceftrazidime and Meropenem), 24.3% resistance to Fluoroquinolones (Norfloxacin, Ciprofloxacin, and Ofloxacine), 9% resistance to Aminoglycosides (Gentamicin, Amikacin). The percentage of antimicrobial resistant for isolates from seawater samples (39.6%) was higher than those isolated from mussels (19.4%) and sand (11.6%) samples. In seawater samples, a higher percentage of resistance was found to Piperacillin (72.2%), Ceftrazidime (55.6%) and Norfloxacin (5%). Other antibiotics (except Amikacin) exhibited medium resistance ranged from %. Isolates from sand samples on the other hand were all classified in the range of medium and low resistance only (4.5-41%). Isolates from mussel samples were found to be highly resistance to Piperacillin, 55.6% while the remaining was classified in the medium and low resistance ranges. Table 4.9: Percentage of resistance of P. aeruginosa isolate from sand, seawater and mussel samples to different Antimicrobial (%) Sample Type Antimicrobials Mussels Average Ceftrazidime Gentamicin Piperacillin Meropenem Ofloxacine Norfloxacin Ciprofloxacin Amikacin.... Average Percentage of resistance of P. aeruginosa isolated from the different locations at Gaza Strip shoreline to the six studied antimicrobial is shown in Table 4.1. The highest resistant was observed for isolates from the north side of Wadi Gaza (43.4%), followed by isolates from Khan Yunis (25%), then, Elzawaida (21.9%), South Wadi Gaza (2.6%) and finally (16.8%). 73

90 Resistant idolates (%) The results also showed that -18% of P. aeruginosa isolates were resistance to aminoglycosides (Amikacin and Gentamicin), 15.7% to β-lactams (Meropenem), % to Fluoroquinolones (Ofloxacine, Norfloxacin and Ciprofloxacin), 28% to Cephalosporins (Ceftrazidime) and 55% to Penicillins (PRL) antimicrobial. Table 4.1: Percentage of resistance of P. aeruginosa isolated from different locations at Gaza strip shoreline to six antimicrobial (%) Antimicrobials Locations No. of Isolates CAZ CN PRL MER OF NX CIP AK Average Khan Yunis Elzawaida South Wadi Gaza North Wadi Gaza Total Antimicrobial abbreviations: CAZ= Ceftrazidime, CN= Gentamicin, PRL= Piperacillin, MER= Meropenem, OF= Ofloxacine, NX= Norfloxacin, CIP= Ciprofloxacin, AK= Amikacin The antimicrobial resistance analysis performed for isolates from mussels showed that, isolates from were more frequently resistance than those isolates from the south of Wadi Gaza outlet (Figure 4.11) South Wadi Gaza CAZ CN PRL MER OF NX CIP AK Antimicrobials Figure 4.11: Resistance isolation percentage of Pseudomonas aeruginosa isolated from mussel in and South of Wadi Gaza outlet. Antimicrobial abbreviations: CAZ= Ceftrazidime, CN= Gentamicin, PRL= Piperacillin, MER= Meropenem, OF= Ofloxacine, NX= Norfloxacin, CIP= Ciprofloxacin, AK= Amikacin 74

91 Percentage of resistance Number of Antibiotic Resisted The collection of Pseudomonas aeruginosa isolates from the different types of samples and stations were analysed for multiple antimicrobial resistance (MAR) (Figure 4.12). The value of multiple antimicrobial resistances of the different types of samples was different. The data indicated that 68.5% of P. aeruginosa isolates were resistant to at least one antimicrobial at some level while the remaining isolates (n = 28, 31.5%) were found to be susceptible to all antimicrobial. Among the tested isolates, about 25 and 12% showed resistance for 1 and 2 antimicrobial respectively and only small percentages of studied isolates (2.2-9.%) showed resistance for 3 to 7 antibiotics of the 8 antimicrobial tested. The results also show that 31.5, 22, 5, 13.5, 7.9 and 2.2% of the isolates exhibited simultaneous resistance to more than two, three, four, five and six antimicrobial. None of the isolate appeared to be resistance against the all eight studied Antimicrobial collectively. The highest percentages of resistance of 22, 25 and 33% were for isolates from seawater, sand and mussel respectively were found to be resistant to a single antimicrobial. Mussels Average zero one two three four five six seven eight Antibiotics Figure 4.12: Multiple Antimicrobial resistance of Pseudomonas aeruginosa isolated from sand, mussels and seawater samples from the coast of the Gaza Strip. 75

92 sand water mussel sand water sand water mussel sand water sand water MARI MAR Index for Pseudomonas The MAR index of all isolates of P. aeruginosa was.24. Calculation of the MARI indicated considerable variation among the different types of samples from the different locations (Figure 4.13). The highest MAR index was determined in bacteria isolated from seawater ( ) compared to mussel (.8-.25) and sand (.5-.25) isolates. In addition, the level of resistance of P. aeruginosa isolated from the four stations, which represented different environmental conditions, was different. Analysis of MARI indicated that samples from North and Khan Yunis had higher MARI value of.39 and.25 respectively, followed by the sampling stations of Elzawaida (.22), South of Wadi Gaza outlet (.21) and finally (.17). Despite the observed variations, no significant differences in MARI values were detected among the six locations (ANOVA, p =.71). Significant differences however were observed between the MARI values of isolates from seawater, sand and mussel samples collected from the same locations of Khan Yunis,, North and South of Wadi Gaza outlet. On other hand no significant differences in antimicrobial resistance were seen among Elzawaida location (ANOVA, p =.145 >.5). Independent sample t-test couldn t reveal any significant differences between MARI of mussel samples from the, and South of Wadi Gaza outlet. Results obtained also indicated that only 4 (45%) out of the 89 P. aeruginosa isolates have MAR index greater than North of Wadi Gaza Outlets South of Wadi Gaza Outlets Elzawaida Khan Yunis Sample Type and Location Figure 4.13: Multiple Antimicrobial Resistance Index for Pseudomonas aeruginosa. 76

93 4.2.5 Staphylococci A total of 61 Staphylococcus isolates were tested for their susceptibility to ten antimicrobial agents, Penicillin (P), Azithromycin (AZM), Vancomycin (VA), Ciprofloxacin (CIP), Norfloxacin (NX), Doxycycline (DOX), Gentamicin (CN), Vancomycin (VA), Trimethoprim/Sulfamethoxazole (SXT), Oxacillin (OX) and Trimethoprim (TM). The total resistance cases were 36.6% whereas the totals of intermediary sensitive and sensitive cases were 9.3 and 54.1%, respectively Resistance of Isolated Staphylococci Strains Staphylococci isolates showed high level (>5%) of resistance to Penicillin (91.8%), Vancomycin (68.9%), and Oxacillin (62.3%). In contrast, resistance to Trimethoprim (37.7%), Azithromycin (29.5%), Trimethoprim/Sulfamethoxazole (26.2%) was classified as medium (25-5%), whereas resistance to Oxacilline (19.7%), Ciprofloxacin (16.4%), Norfloxacin (8.2%) and Gentamicin (4.9%) was low (< 25% resistant) (Table 4.11). The results also showed that 4.9% of Staphylococci isolates were resistance to Aminoglycosides (Gentamicin), 12.3% to Fluoroquinolones (Ciprofloxacin, Norfloxacin), 19.7% to Tetracyclines (Doxycycline), 29.5% to Macrolides (Azithromycin), 31.9% to Trimethoprim/Sulfonamides (SXT, Trimethoprim), to 68.9 to Glycopeptide (Vancomycin) and 77.1% to β-lactam (Penicillin, Oxacillin). Bacteria isolated from the beach sand, seawater and mussel samples were characterized by differences in the level of resistance to studied antimicrobial. The highest antimicrobial resistance was observed in bacteria isolated from the seawater (41.8%), followed by mussels (31.7%) and beach sand (29.9%). In seawater samples, the highest levels of resistance were seen in Penicillin (95.5%), Oxacillin (86.4%) and Vancomycin (77.3%). Trimethoprim, Doxycycline, Azithromycin and Trimethoprim/Sulfamethoxazole demonstrated medium levels of resistance ranged from 27.3 to 45.5%. Staphylococci isolates were least resistance to Ciprofloxacin (18.2%), Gentamicin (4.5%) and Norfloxacin (4.5%). 77

94 Similarly, isolates from sand samples were most resistance to Penicillin (85.2%) followed by Oxacillin (55.6%). Vancomycin and Trimethoprim/Sulfamethoxazole were moderately resistance against Staphylococci isolates. Trimethoprim, Azithromycin, Ciprofloxacin, Norfloxacin, Doxycycline and Gentamicin were found to be the most active (least resistance) antimicrobial against Staphylococci isolates. Staphylococci isolates from mussel samples on the other hand, demonstrated the highest levels of resistance to Vancomycin (1%) followed by Azithromycin and Trimethoprim (58.3%) while other antimicrobial demonstrated moderate to low activity against S. aureus. Isolates were found to be totally sensitive to Penicillin (% resistant). Table 4.11: Percentage of resistance of Staphylococci isolate from sand, seawater and mussel samples to different antimicrobials (%) % Resistance Isolate Antimicrobial Mussels Average Azithromycin Penicillin Vancomycin Ciprofloxacin Gentamicin Norfloxacin Trimethoprim Oxacillin Trimethoprim/Sulfamethoxazole Doxycycline Average Percentage of resistance of Staphylococcus aureus isolated from the different locations at Gaza Strip shoreline to the ten studied antimicrobial is shown in Table The highest percentage of resistant was observed for isolates from Elzawaida (4.9%) followed by isolates from the north side of Wadi Gaza (4%), (37.8%), Khan Yunis (56%) then, south Wadi Gaza (31.7%) and finally Isolates from Khan Yunis (25.6%). All locations showed higher resistant to Vancomycin, Penicillin, Oxacillin, Trimethoprim. 78

95 Resistant idolates (%) Table 4.12: Percentage of resistance of Staphylococci strains isolated from different locations at Gaza Strip shoreline to 1 antimicrobial (%) Antimicrobials Location No of Isolation AZM P VN CIP CN NX TM OX SXT DOX Average Khan Yunis Elzawaida South Wadi Gaza North Wadi Gaza Total Antimicrobial abbreviations: AZM= Azithromycin, P= Penicillin, VN= Vancomycin, CIP= Ciprofloxacin, CN= Gentamicin, NX= Norfloxacin, TM= Trimethoprim, OX= Oxacillin, SXT= Trimethoprim Sulfamethoxazole, DOX= Doxycycline. The antimicrobial resistance analysis performed for isolates from mussels showed that, isolates from were more frequently resistance to tested antimicrobial than those isolates from the south of Wadi Gaza outlet (Figure 4.14). Dier al Balah South Wadi Gaza AZM P VA CIP CN NX TM OX SXT DOX Antimicrobials Figure 4.14: Resistance isolation percentage of Staphylococci strains isolated from mussel in and South of Wadi Gaza outlet. Antimicrobial abbreviations: AZM= Azithromycin, P= Penicillin, VN= Vancomycin, CIP= Ciprofloxacin, CN= Gentamicin, NX= Norfloxacin, TM= Trimethoprim, OX= Oxacillin, SXT= Trimethoprim Sulfamethoxazole, DOX= Doxycycline. 79

96 Percent Number of Antimicrobials Resisted Multiple antimicrobial resistances of Staphylococcus aureus isolates from sand, mussels and seawater samples from the coast of the Gaza strip are shown in Figure Nearly 1% of S. aureus isolates were resistant to a single antimicrobial, 13% were resistant to two different antimicrobial and 28, 18 and 16.4% were resistant to three, four and five antimicrobial. Resistance to 6-9 antibiotics was ranged from 1.6-5%. The results also show that approximately 98.4% of isolates were resistance to at least one antibiotic, whereas 88.5, 75.4, 47.5, 29.5, 13.1, 8.2, 3.3 and 1.6 % of the isolates exhibited simultaneous resistance to more than one, two, three, four, five, six, seven and eight antibiotics respectively. None of the strain of staphylococci isolated in study was resistant to 1 antimicrobial. Different antimicrobial resistance patterns were observed for isolates from beach sand, seawater and mussel samples. The highest bacterial resistant was found to 2-6 antimicrobial with a range of %, % and % for sand, seawater and mussel samples respectively. In all samples, none of the isolates showed resistance to all (n = 1) of the antimicrobial used. Seawater Mussels Average zero one two three four five six seven eight nine ten Number of antibiotic resisted Figure 4.15: Multiple antimicrobial resistance of Staphylococcus aureus isolates from sand, mussels and seawater samples from the coast of the Gaza Strip. 8

97 sand water mussel sand water sand water mussel sand water sand water MARI Multiple Antimicrobials Resistance Index (MARI) for Staphylococci The overall MARI value for all Staphylococcus aureus isolates was.36. The MARI values for isolates from seawater (.41) were almost equal to that of mussels (.39) but higher than that of sand isolates (.3) (Figure 4.16). Except for isolates from Khan Yunis, (P =.33), no significant differences (Student t-test or ANOVA) were observed in the MAR index among the different types of samples (seawater, sand and mussel samples) at the same location. Although not statistically significant (ANOVA, p >.5), MARI values of isolates from the Northern side of Wadi Gaza outlet and Elzawaida was higher (.42) than those from the other locations. MARI values for the other locations were as follows; south of Wadi Gaza outlet.35,.33 and Khan Yunis,.28. Out of 61 Staphylococci isolates, 46 (75.4%) were found to have MAR Indices greater than.2, while those with MARI values less than.2 were 15 (24.6%). Analysis of MARI of mussel samples from had higher (.46) than the sampling stations of South of Wadi Gaza Outlet (.33). Independent sample t- test however, could not reveal any significant differences between MARI of mussel samples from North of Wadi Gaza outles South of Wadi Gaza outles Elzawaida Khan Yunis Sample type and location Figure 4.16: Multiple Antimicrobial Resistance Index for Staphylococcus. 81

98 5 Chapter 5 Discussion The aim of this chapter is to assess the microbiological beach quality in Khan Yunis,, Elzawaida, South of Wadi Gaza and North of Wadi Gaza. This chapter also measures the antimicrobial resistance. 5.1 Beach Quality Assessment Microbiological examination of marine samples for pathogenic organisms is of great importance in marine pollution studies, as it is a direct measurement of deleterious effect of microbiological marine pollution. Present investigation highlights the occurrence of fecal coliform and fecal streptococci bacteria in the collected water and sand samples. All locations exhibited variation in both sand and seawater content of fecal coliform (FC) and fecal streptococci (FS). In most of the tested sites, cultivable fecal indicator bacteria such as FC and FS were more abundant in the sand than in the water. The results of the present study were consistent with those reported by Elmanama et al., (25) who found more fecal indicator bacteria in the sand than in the in the overlying water. Several authors (Obiri and Jones, 2) have suggested that sediments act as a reservoir for fecal indicators. Many environmental variables may affect the abundance of fecal indicator in marine environment, but it is believed that sand may provide more protected environment for enteric bacteria than seawater. Deposition in beach sand for example would afford the microorganism s protection from exposure to sunlight (Alm et al., 23). Furthermore, the rich organic content and the retaining of substantial amounts of naturally occurring organic matter and nutrients favorable for microbial growth in sediments would promote the growth of such indicator bacteria in the sediments (Shah et al., 211). The relatively higher level of fecal bacteria in sand was also linked to longer survival of bacteria attached to the particulate matter in the sand (Sato et al., 25). From human health perspective, the high levels of fecal indicator bacteria, coliform and streptococci in sand may indicate a potential health risk associated with the exposure to the contaminated sand especially for children, who usually stay there for longer periods.present study, indicated higher densities of FS than that of FC in most locations 82

99 (e.g. 1, 3 and 4). Such variation in the different groups of microbial population may be attributed to the different rates of growth and survival of these pathogens in marine environment. The survival ability of FS for example was found to be higher at seawater environment compared with coliform group (Dufour, 1977). Hanes and Fragala, (1967) found that the survival of FS in marine water was 2.4 days while that of FC represented by E. coli was.8 days. Furthermore, it was suggested that FS populations in the sand are more resistant to the lytic effects of bacteriophages and to protozoan grazing than FC (Epstein and Shiaris, 1992). Also, E. coli degraded more rapidly with increased sunlight intensity than did Enterococci, a finding that was recently confirmed for bacterial samples from southern California (Noble et al., 21). Wastewater in Khan Yunis is collected in wastewater treatment plant, this water stays there for retention time from 1 days to 2 weeks. This lead to the fact that the ratio of FC/FS is less than 1. While on the other hand in north alwadi the ratio of FC/FS is more than 1 and this refers to the fact that wastewater of this area is pumped directly to the sea through pipes without being treated. This result ensures the previous mentioned information that FS long lives more than FC (Afifi, 215). FC/FS ratio was usually used to differentiate between the sources of fecal pollution whether it is human or animal. A ratio of 4 has been said to indicate a contamination of human origin whereas a ratio of <.7 is indicative of animal pollution (Geldreich and Kenner, 1969). The results of the present study couldn t reveal any general conclusion because of the variations in this ratio. Pourcher et al., (1991) indicated however, that this ratio does not hold true in many cases and is only valid for recent (24 hours) fecal contamination. Accordingly, some investigators have questioned its usefulness and the American Public Health Association (APHA) no longer recommends the use of the FC/FS ratio for the purpose of differentiating human from animal sources of pollution (APHA, 1998). The observed higher density of both fecal coliform and fecal streptococci at locations 1, 4 and 5, suggesting that these locations are more polluted as compared to the other locations, e.g., 3 and 4 (Fig. 4.2), Coliform are generally occurring in large number in coastal water, which is fecally contaminated. 83

100 In the Gaza strip, marine pollution is mainly attributed to the direct disposal of wastewater into seawater and beach sand without treatment or with partial treatment. Of all five locations, 4 and 5 were found to be the most polluted locations as indicated by the highest counts of FC and FS. This can be attributed to the continuous discharge of raw sewage from the Middle governorate and partly from the Gaza wastewater treatment Plant through Wadi Gaza into the seashore region of these locations. The least polluted locations were 1, 2, 3 and 4 because there are no direct sewage outlets of any kind in these locations except location 1. Other researchers conducted similar studies in the Gaza strip have also came to the same conclusion. Abudaya and Hararah, (213) found high levels of microbiological contamination (fecal coliform and fecal Enterococci) of seawater above internationally accepted limits at stations close to Wadi Gaza outlet station (known as station 4). Microbiological analysis of 94 seawater samples collected during summer and autumn seasons from different location sand over an area extended about 23 km along the Mediterranean Coast of the Gaza strip (from the Khan Yunis fishing port to Gaza fishing port), showed the presence of contamination (fecal coliform and fecal streptococci) in many of these samples and the pollution was concentrated in and surrounding the mouths of wastewater outfalls e.g. Wadi Gaza and El Shiekh Ejleen sewage outfall (Abualtayef et al., 214). On the other hand, Hilles et al., (214), studying parasitic contamination of shoreline seawaters in the Gaza strip detected large numbers of parasitic contamination in seawater at a zone termed as mouth of Wadi Gaza which correspond to locations 4 and 5 in our study. The great difference between the FC and FS populations in seawater of the northern side of Wadi Gaza and those of the southern side of the Wadi (Fig. 4.2) could be attributed to the natural coastal processes dominating the Palestinian coastline such as waves regimes and wave generated longshore current which are responsible for generating net longshore transport to the north, thus, carrying more pollutants toward the location at northern side of Wadi Gaza. By virtue of its feeding habits (i.e. filter feeders), the mussels tend to concentrate microorganisms present in the water. FS counts in mussels were found to be significantly higher (two sample t-test, p <.5) than that of the seawater in the two locations. On the other hand, no significant difference was found between the 84

101 concentrations of FC in mussels and water samples collected at the same locations. This also could be explained by the fact that FC is short lived in seawater environment. The mussel samples collected from location 4 showed the presence of fecal indicator bacteria in smaller numbers than location 2 in spite of the former coast being highly polluted than later one. These findings were similar to earlier study which found fewer numbers of enteric bacteria in mussels from highly polluted coast at Neendakara, India (Raveendran et al., 199). The high levels of fecal indicators in mussels from non contaminated area were attributed to their ability to concentrate bacteria from seawater even at considerable distance from the pollution source (Sasikumar and Krishnamoorthy, 21). The observed low FC/FS ratio in mussel samples collected from locations 2 and 4 were much lower than that used to interpret the origin of bacterial pollution (APHA, 1998). The suitability of all locations for bathing and other recreational activities were assessed using the European Community (EU) Bathing s Directive (76/16/ EEC) standards for fecal indicator organisms (fecal coliform and fecal streptococci) in waters. These standards were designed to protect the health of people who bathe in the waters. Compliance with the "mandatory" and "guideline" standards of fecal coliform at the five locations indicated that seawater quality at all sites has achieved the "mandatory" standards and four out of five locations have achieved the stricter "guideline" standard. It is only the northern side of Wadi Gaza outlet that failed to comply with the guideline standards for FC because all seawater samples (1%) from this location were found to exceed 1 fecal coliform/1 ml. Compliance with the "guideline" standards of fecal streptococci indicated similar results to "guideline" standards of fecal coliform, where only the northern side of Wadi Gaza outlet failed to meet the directive s guideline standards of fecal streptococci. The observed compliance failure of guideline standards of fecal coliform and fecal streptococciat location 5 is in accordance to the findings of Elmanama et al., (25), who found higher failure percentage at location 5 compared to other studied locations. Abudaya and Hararah, (213) also came to the same conclusion concerning stations lie at Wadi Gaza outlet. 85

102 5.2 Antimicrobial Resistance This study describes the resistance of four groups of pathogenic bacteria namely; Enterobacteriaceae, Enterococci, Staphylococcus aureus, and Pseudomonas aeruginosa, isolated from beach sand, seawater and mussel samples from the Mediterranean Sea environment of the Gaza strip to antimicrobial agents. The emergence of antimicrobial-resistant bacteria is a world-wide problem. Due to its global health importance, several monitoring programs for antimicrobial resistance were developed in the USA and Europe such as the National Antimicrobial Resistance Monitoring Systems (NARMS) (Fedorka et al., 22) in the USA and the European Antimicrobial Resistance Surveillance System (EARSS) (Fluit et al., 26) in Europe Determining microbial resistance from surrounding environment is of a major public health concern as drug resistant bacteria could be transferred to humans or to other organisms living in the same environment which then contributes to the spread and persistence of antimicrobial resistance bacteria in environment. According to Stewart and Koditschek, (198); Tendencia and Pena (21), the high levels of antibiotic resistance in pathogenic bacteria might result in dissemination of antibiotic resistance plasmids in the marine environment, thus, increasing the levels of resistance to antibiotics in marine bacteria. This information will be helpful for understanding the distribution of antimicrobial resistant pathogenic bacteria in coastal marine environment of the Gaza strip. Moreover, it may prove useful for developing new strategies to reduce their health risks in the future. Furthermore, the results of these studies will provide useful information for researchers working in marine pollution studies, as it is a direct measurement of deleterious effect of microbilogical coastal pollution on human health and to ensure the safety of seawater for recreational purposes. To date, the problem of resistance of pathogenic bacteria in the Gaza strip focused mainly on the prevalence of antimicrobial resistance bacteria from clinical specimens or in hospital environments (Astal, 25; Elmanama et al., 26 b ; Al Laham, 212; Elmanama and Abdelateef, 212). Only one study investigated the contribution of hospital effluents spreading of antibiotic resistance in the surrounding environment 86

103 (Elmanama et al., 26 b ). To our knowledge, this is the first study aimed to detect the presence of multiple antimicrobial resistant of pathogenic bacteria in seawater, sand and mussel samples collected from marine environment of the Gaza strip, although this problem is of a great significance in the public health risk. This study provides clear evidence of the wide presence and considerable spread of antimicrobial resistance among Enterobacteriaceae, Enterococci, Pseudomonas aeruginosa and Staphylococci isolates in the samples from mussels, seawater and sand beach from the different recreational beaches of Gaza strip. The presence of drug resistant bacteria in aquatic environment is a major public health concern as drug resistant bacteria could be transferred to humans, which then contributes to the spread and persistence of antimicrobial resistance bacteria in environment. In addition, the occurrence of pathogenic bacteria in aquatic environment with high resistance to antimicrobial may be an indication that the area is contaminated with antimicrobials which reflect human influence in the environment. The present study also indicated that marine organisms harbor antimicrobial resistance and therefore may serve as reservoirs for antimicrobial resistance genetic determinants. The overall pattern of resistance among Enterobacteriaceae for ten antimicrobial in descending order was found to be Tetracycline> Amoxicillin-clavulanic> Trimethoprim/Sulfonamides> Piperacillin> Ceftrazidime> Cefuroxime> Ceftriaxone> Meropenem> Gentamicin> Ciprofloxacin. Similarly, the trends of resistance for the tested antimicrobial among other bacterial groups, on the other hand were as follow: Vancomycin> Ampicillin> Tetracycline> Nitrofurantion> Ciprofloxacin> Norfloxacin for Enterococci, Piperacillin> Ceftrazidime> Ciprofloxacin> Norfloxacin> Ofloxacine> Gentamicin> Meropenem for Pseudomonas aeruginosa and Penicillin> Vancomycin> Oxacillin> Trimethoprim> Azithromycin> Trimethoprim/Sulfonamides> Doxycycline> Ciprofloxacin> Norfloxacin> Gentamicin for Staphylococci. These findings indicate a high level of misuse of these drugs. This high resistance rate was attributed to the widespread and prolonged use of these antibacterial drugs in the world including Gaza strip (Elmanama and Abdelateef, 212). 87

104 Enterobacteriaceae isolates showed a high resistance to Tetracycline (67.9%), followed by Amoxicillin-clavulanic (56%) and Trimethoprim/Sulfonamides (5.3%). The spread of antimicrobial resistance among Enterobacteriaceae in aquatic environment has been studied by various researchers from different types of aquatic environments, rivers (Tao et al., 21; Abo-State et al., 212), estuaries (Lourenc et al., 27), lakes (Pontes et al., 27) and coastal waters (Maloo et al., 214). The high value of resistance found among Enterobacteriaceae genera isolated from aquatic environment to Tetracycline was in general agreement with that reported by Abo-State et al., (212). Tetracycline class of antimicrobials agent is a broad-spectrum agent, widely used for the treatment of bacterial infections in human and veterinary medicine and as growth promoters in livestock industry (Speer et al., 1992). The widespread use of Tetracyclines can lead to the emergence of drug resistant bacteria and transfer of Tetracycline resistant genes between different species of bacteria. The usage of Tetracyclines was approximately 16,268 kg in the UK in 2 (Sarmah et al., 26), and account for 15.8% of 9.3 million kg of antibiotics used in animal feed in the United States, and nearly 56% of approximately 14,6 kg of antimicrobial used in animal food production in Kenya (Mitema et al., 21). In the Gaza Strip, may be Tetracyclines are also one of the most widely used antimicrobial. Enterococci are considered an important human pathogen due to their ability to acquire resistance genes and an important cause of nosocomial infections worldwide (Poeta et al., 27). Enterococci isolates showed high percentage of resistance to Vancomycin (84.8%), Ampicillin (8.3%) and Tetracycline (51.5%). Enterococci are known to be resistant to most antimicrobial used in clinical practice, yet, due to the fact that, it was frequently used worldwide for enterococcal infections in the past, an increased resistance to Vancomycin is observed today (Kimiran-Edrem et al., 27). Vancomycin-resistant Enterococci (VRE) were first reported in 1986, nearly 3 years after Vancomycin was clinically introduced. Olivera et al., (21) detected Vancomycin-resistant Enterococci in seawater and sand beach samples. Vancomycin resistant strains of Enterococci among non hospitalized individuals in Gaza strip were previously documented by Elmanama, 28 who found that 32.1% of the 84 obtained enterococcal isolates were resistant to this agent. 88

105 Enterococci susceptibility to Ampicillin is of great importance, since Ampicillin is the drug of choice in treatment of enterococcal infections (Barisic and Punda-Polić, 2). Increased Ampicillin resistance in Enterococci is attributable to either the production of beta-lactamase or alterations in the expression or structure of penicillin binding protein (Rice, 21).The spreading potential of resistance among Enterococci and from Enterococci to other species is an important issue since Enterococci are known to acquire antimicrobial resistance with relative ease and capable of spreading those resistance genes to other species (Klare et al., 23).Various rates of Ampicillin resistance in both clinical and environmental isolates of Enterococci isolates were previously reported in isolates from the Gaza strip (Al Jarousha, 28, Elmanama et al., 26 b ). It is well known that P. aeruginosa is ubiquitous in the environment (Ulah et al., 212), although most studies on antimicrobial resistance have been performed using strains of clinical origin (Vaz-Moreira et al., 212). The present study demonstrated high resistance rate (55.1%) of P. aeruginosa isolates from marine environment of Gaza strip towards Piperacillin. These findings were in agreement with that of Osundiya et al., (213), who detected high level of resistance to Piperacillin (44.1%). This high rate of resistance was attributed to ESBL (extended spectrum B-lactamase) and MBL (metallo B lactamase) encoding genes, together with the clonal spread of several specific clones (Picao et al., 29). P. aeruginosa exhibits remarkable ability to acquire resistance by mutation or acquisition of exogenous resistance determinants and can be mediated by several mechanisms (degrading enzymes, reduced permeability, active efflux and target modification (Lutz, 211). The results of the present study however were in contrast to the previously reported low resistance rates of P. aeruginosa from different aquatic environment to Piperacillin (Ulah et al., 212; Ezat et al., 214). The reason behind such difference may be due to the fact that Piperacillin is extensively used in Gaza for Pseudomonas treatment. The antimicrobial resistance patterns of Pseudomonas strains in a marine environment from Iskenderun Bay, Turkey, at northeast of Mediterranean Sea were found to be high to Nitrofurantion (86.2%), and Cefuroxime (71.7%) (Matyar et al., 21). 89

106 Staphylococcal isolates showed high level of resistance to Penicillin (91.8%), Vancomycin (68.9%), and Oxacillin (62.3%). S. aureus can be often carried asymptomatically as part of the normal flora on the human body, colonizing human nasal cavity (and on other mucous membranes) as well as on the skin. As an opportunistic pathogen however, the bacterium can cause infections that vary widely in their severity and in their susceptibility to antimicrobial treatment. Methicillin-resistant S. aureus (MRSA) strains have acquired a gene that makes them resistant to all beta lactam antimicrobial. The development of methicillin-resistant S. aureus is severely decreasing the usefulness of this antimicrobial to treat infections in humans. While this problem was initially confined to high risk populations e.g., intravenous drug users, people with chronic illnesses, over the last decade however, the frequency of community-acquired MRSA has become more common even in the general population with no health care exposure or known classical risk factors for MRSA infections. Several studies suggest that the oceans may be one pathway by which community acquired infections are transmitted. Charoenca and Fujioka (1995) found a correlation between seawater exposure and S. aureus infection rates. Contact with seawater has been associated with a four-fold increased risk of S. aureus skin infections in children (Roberts et al., 211) and high counts of S. aureus in recreational waters is often considered a risk factor for contracting many diseases that affect areas such as the skin, eyes, and ears (Gabutti et al., 2). These studies confirm the presence of pathogenic bacteria, including MRSA in recreational marine beaches that may act as potential reservoirs for transmission of these pathogenic strains to surrounding community and even to animals in marine environment (Faires et al., 29). There are several potential point and non-point sources possibly contributing to S. aureus and MRSA loads at marine environments. In addition to sewer overflows and urban runoff, previous studies documented that shedding of S. aureus from bathers into marine environment while swimming or engaging in other recreational activities are also contributing to S. aureus and MRSA loads at recreational beaches (Elmir et al.,27). In the present study, the presence of MRSA was screened by using Oxacillin disc of 1 µg concentration (Al Laham, 212). Results obtained from this study showed that 38 (62.3%) of the 61 Staphylococci isolated from marine recreational beaches were 9

107 Methicillin (Oxacillin) resistant which is extremely high. Previously, Abu Hujier and Sharif, (28) had reported MRSA prevalence of 22% from clinical isolates obtained from patients in Gaza strip. More recently however, Al Laham, (212) reported MRSA prevalence of 62.1% in operation theaters at main hospitals of Gaza strip. The observed higher rate of resistance among S. aureus isolates in this study may be due to the intergeneric transfer of resistance among different genera of Gram-positive cocci and between Bacillus species and Staphylococci and Streptococci (Schaberg and Zerros, 1986) which may increase in marine environment. Previous studies from marine environments from other localities around the world found much lower percentages than the current study (Yamahara et al., 212; Goodwin et al., 212). MRSA isolates often carry resistance genes that make them resistant to beta-lactam antibiotics. This may explain the high resistance of Staphylococci isolates to penicillins (91.8%) and suggesting the possibly that they are producers of penicillinase enzymes. High usage of Penicillin and other antimicrobials to treat staphylococcal infections in Gaza strip for many years may result in high rate of resistance among S. aureus isolates in this study. The results obtained in the present study revealed a high resistance to Vancomycin by S. aureus isolates (68.9%) In fact, from medicinal point of view, the glycopeptide agent, Vancomycin is the last resort and drug of choice to treat infections caused by MRSA isolates in the world (Rahimi et al., 213). Hence, the findings of the present study regarding the high resistance to Vancomycin could be an urgent warning for public health as well as for health authorities. However, these results were inconsistent with other studies in Gaza strip, which have reported very low prevalence (.66%) of Vancomycin resistant S. aureus isolates (Abu Hujier and Sharif, 28). This might be due to employment of improper diagnostic methods for Vancomycin resistance screening. As it has been recommended by CLSI, the standard methods for Vancomycin resistance screening in S. aureus are, Etest, broth dilution and agar dilution; therefore the results of disk diffusion test is not reliable (Rahimi et al., 213). Results presented in this study also showed that bacterial resistance to antimicrobial depends on their chemical structure. Enterobacteriaceae isolates were most resistant to 91

108 Tetracyclines, followed by Trimethoprim/Sulfonamides, β-lactam, aminoglycosides and finally fluoroquinolone antibiotics. Enterococci isolates were most resistance to glycopeptide, β-lactam, Tetracycline, Nitrofuran and Fluoroquinolones. Pseudomonas aeruginosa isolates were most resistance to β-lactam, Fluoroquinolones and Aminoglycosides. Staphylococci isolates were most resistance to Glycopeptide, β Lactam, Trimethoprim/Sulfonamides, Tetracyclines, fluoroquinolone and aminoglycosides. 5.3 Multiple Drug Resistance (MDR) Multi Drug Resistance (MDR) is a condition enabling disease causing microorganism (bacteria, virus, fungi or parasites) to resist distinct antimicrobial. In accordance with recent standard definitions (Magiorakos et al., 211), multi-drug resistance (MDR) was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories. It is a worldwide problem in clinical medicine affecting our global health system. The emergence and spread of MDR in the aquatic environment could be explained by the continual discharge of untreated domestic wastewater. Aquatic ecosystems which, received wastewater with pathogenic bacteria could serve as a reservoir of antimicrobial resistant genes. In this study the MDR character in Enterobacteriaceae, Enterococci, Staphylococcus aureus and Pseudomonas aeruginosa isolates were determined by subjecting the isolates to different antimicrobial agents. The study revealed that 8.5, 59.1, 46.5, 36.5, 27.7, 2.8, 13.2, 6.4 and 4.4% of Enterobacteriaceae isolates exhibited simultaneous resistance to more than one, two, three, four, five, six, seven, eight and nine antimicrobial agents respectively, whereas 89.4, 59.1, 33.3, 18.2 and 12.1% of the Enterococci isolates exhibited simultaneous resistance to more than one, two, three, four and five antimicrobial respectively. The results also showed that 31.5, 22, 5, 13.5, 7.9 and 2.2% of Pseudomonas aeruginosa isolates exhibited simultaneous resistance to more than two, three, four, five and six antimicrobial whereas 88.5, 75.4, 47.5, 29.5, 13.1, 8.2, 3.3 and 1.6 % of the Staphylococcus aureus isolates exhibited simultaneous resistance to more than one, two, three, four, five, six, seven and eight antimicrobial respectively. 92

109 This finding is generally consistent with those of other studies in which varying frequencies of antimicrobial resistance were detected among bacteria in natural environments including seawater and marine sediments (Neela et al., 27; Kimiran- Edrem et al., 27; Maloo et al., 214). Bacteria occurring in many water basins show multi-drug resistance, as has been reported by Qureshi and Qureshi (1992) and Mudryk (22). That means they are perfectly capable of neutralizing those antibacterial substances. The differences in the percentage of bacteria resistant to various antibiotics may reflect the history of antibiotic application and may serve as indicator for the high use of these antimicrobial (Hsu et al., 1992). The presence of multi-drug resistant strains is alarming because infection with such strains leads to a higher fatality rate than with antibiotic-sensitive strains (Holmberg et al., 1984). Presence of multiple drug resistance (MDR) in strains of Enterobacteriaceae, Enterococcus spp., Staphylococcus aureus and Pseudomonas aeruginosa isolated from different types of environments including marine environments has been reported previously. Maloo et al., (214) assessed the occurrence and distribution of multiple antimicrobial-resistant bacteria of Enterobacteriaceae family in surface and bottom waters along the Veraval coast, India and found that 97% of the isolates exhibited multi drug resistance character (identified as resistance to more than two antibiotics) to the antimicrobial tested. Kimiran-Edrem et al., (27) evaluated the resistance of a hundred Enterococci strains isolated from seawater samples collected from coastal areas of Istanbul to eleven antimicrobials. They reported that 1% of strains were resistant to all antimicrobials; mean while, 9, 28, 42, 14, 4 and 2% were resistant to one, two, three, four, five and six antimicrobial, respectively. Though they are well-known multi-drug resistance strains now a days, studies reporting the MDR condition in P. aeruginosa and S. aureus isolates from marine samples are limited and most of the available studies were concerned in the presence of these pathogens in marine environmental sources such as water, sediment and animals (Matyar et al., 21; Interaminense et al., 21; Unger et al., 214) 93

110 In Lebanon, 51% of the tested isolates of Staphylococcus aureus strains isolated from seawater, fresh water, sediments, and crab samples collected from representative communities along the coast of Lebanon have shown resistance to at least one of the five tested antimicrobials; with seawater isolates exhibiting the highest rates of antimicrobial resistance (Harakeh et al., 26). Isolates of various bacterial species from different types of samples (e.g. oviductal fluid during the egg-laying process in green turtles, the cloacal vent of the post egg laying turtles and sand samples near the turtle nests) from Ras Al-Hadd, Oman, showed that all the isolates, including P. aeruginosa and Staphylococcus spp. exhibited multiple resistance to more than one antibiotic. Among the isolates, the highest resistance to the fifteen antibiotics tested was P. aeruginosa, which showed resistance to twelve antibiotics (Al-Bahry et al., 211). Multi-drug resistance in Pseudomonas spp. was attributed to the outer membrane barrier (Nikaido, 1989) and to multi-drug efflux pumps which act as antimicrobial resistance mechanisms exploited by Pseudomonas spp. and affecting various antibiotic classes (Poole, 21). Recent studies showed that exposure to hypoxic conditions which spread dramatically over the last few decades induces the selective antibiotic resistance in P. aeruginosa (Schaible et al., 212), thus, increasing the threats to both marine organisms and humans incoastal marine environments. The accumulation of organic matter in deep waters after the occurrence of eutrophication in coastal marine systems and the consumption of dissolved oxygen due to respiration, combined with strong stratification and high residence time of bottom water are believed to be the reasons behind the formation of hypoxia or dead zones (Diaz and Rosenberg, 28). In the marine environment, Staphylococci (S. aureus and S. epidermidis) are broadly related to infections of wounds received in this environment (Isbister and Caldicott, 24). Contact with S. aureus contaminated seawater has been associated with a fourfold increase in the risk of S. aureus skin infections in children (Charoenca and Fujioka, 1995). The medical implications of staphylococci infections are serious because of the large number of strains recognized as being human pathogens (Stepanovic et al., 25). 94

111 5.4 Multiple Antimicrobial Resistances Index (MARI) The aim of this study was to establish the microbiological safety of marine coastal beaches of the Gaza strip and to provide data on resistance index, which may help in identifying the high risk contamination sites in this region. MAR index, is considered as a useful tool for risk assessment. A MAR index value >.2 is observed when isolates are exposed to high risk sources of human or animal contamination, where antimicrobial use is common; in contrast a MAR index value.2 observed when antimicrobials are seldom or never used (Krumperman, 1983; Matyar et al., 28). In this study the MAR indices of Enterobacteriaceae, Enterococcus spp., P.aerugenosa and Staphylococcus aureus isolates were determined. Isolates showed a variation in their MAR indices based on sample type and sampling location. Except in Enterococcus spp., higher percentages of multiple resistance and consequently higher MARI values for Enterobacteriaceae, Pseudomonas and Staphylococcus aureus isolates were observed in seawater than sand samples. These results were unexpected, since bottom sediments may act as long term reservoirs of drug resistant bacteria and drug residues (Thavasi et al., 27). Moreover, sediment is a favorable environment for microbes due to the prevailing stable environmental conditions and the accumulation of the nutrients (Thavasi et al., 27). This finding however, was in agreement with a pioneering study carried out by Sizemore and Colwell (1977) who found that sediment samples from the Atlantic Ocean off the southeastern coast of the United States contained smaller populations of resistant strains as compared with the seawater samples examined. The increase in the percentage of antibiotic-resistant bacteria in seawater samples as compared with the sediment samples however, may be attributed to plasmid exchange which may occurs more frequently in more disturbed or less stable seawater than sediments. The results of the present study also could not reveal clear difference in the frequency of antimicrobial resistance and MARI in Enterobacteriaceae, Enterococcus spp., Pseudomonas aerugenosa and Staphylococcus aureus isolated from the mussel samples from the two locations and between mussel and seawater samples from the same locations. This result was in harmony with early observations made by Cooke, (1976), 95

112 who found a very similar incidence of antibiotic resistance among the coliform and fecal coliform isolates from freshwater mussels (Hydridellamenziesii) samples from Lake Rotoiti which is subject to more pollution and that of the less polluted Lake Rotoroa, South Island, New Zealand. Calculation of MARI values for each bacterial group of Enterobacteriaceae, Enterococcus spp., Pseudomonas aerugenosa and Staphylococcus aureus from north of Wadi Gaza, south of Wadi Gaza, KhanYunis, Elzawaida and revealed variable patterns of antimicrobial resistance among the different sampling locations. The MARI values for the different bacterial groups from the five locations can be ranked as follows: South of Wadi Gaza > North of Wadi Gaza > Khan Yunis> Deir al Balah> Elzawaida for Enterobacteriaceae, North of Wadi Gaza > South of Wadi Gaza> Elzawaida> > Khan Yunis for Enterococcus spp., North of Wadi Gaza> Khan Yunis> Southof Wadi Gaza> Elzawaida> for Pseudomonas aerugenosa and North of Wadi Gaza> Elzawaida> Southof Wadi Gaza> > Khan Yunis for Staphylococcus aureus. In all cases, the Northern side of Wadi Gaza Outlets which is thought to be the most polluted site, occupied the first (in case of Enterococcus spp., Pseudomonas aerugenosa and Staphylococcus aureus) or the second rank (in case of Enterobacteriaceae) after the Southern side of Wadi Gaza Outlets (less polluted), which in turn occupied the first, second, and third rank in case of Enterobacteriaceae, Enterococcus spp., Pseudomonas aerugenosa and Staphylococcus aureus isolates respectively. Other locations occupied second, third, fourth and fifth rank. The differences in antimicrobial resistance and MAR indices among bacterial isolates from contaminated and non-contaminated sites in aquatic environments have been previously documented, but led to contradictory conclusions. Some investigators correlated the increased number of antimicrobial resistance bacteria in a given environment with the pollution status in that environment (Chaturvedi et al., 28; Chitanand et al., 21). Kimiran-Erdem et al., (27) found such direct correlation between pollution and bacterial resistance to antibiotics in Marmara Sea and Black Sea waters. In another study of antimicrobial resistance of heterotrophic marine bacteria 96

113 isolated from seawater and sands of recreational beaches with different organic pollution levels in south eastern Brazil, the multiple antimicrobial resistance of heterotrophic marine bacteria was found to be highest at most polluted beach of Gonza guinha and not observed at all in less polluted beach of Guaraú, (Oliveira et al., 21). On the other hand, calculation of MARI values for all isolates from north of Wadi Gaza, south of Wadi Gaza, Khan Yunis, Elzawaida and was found to be.43,.39,.35,.34 and.33 respectively, which is more clearly correlated to the pollution status of the different locations. MARI value of isolates from the Northern side of Wadi Gaza, which represents the most polluted location, was found to be significantly higher (ANOVA, p <.5) than that of and Elzawaida (least polluted sites). No significant difference however were detected among the isolates from the Northern side of Wadi Gaza and those of the Southern side and Khan Yunis regarding their MARI values (p >.6), as these sites were documented as polluted locations. The high incidence of resistance among bacteria isolated from sites seemingly exposed to less anthropogenic impact has been also described by number of earlier studies. Multivariate analyses of the resistance profiles of native bacteria isolated from seven sites along the Yarra River in south-eastern Australia, that spanned a range of land-use practices, as well as from two reservoirs and from the effluent of three sewage treatment facilities couldn t reveal consistent spatial pattern in the incidence of resistance (Boon and Cattanach, 1999). The incidence of antibiotic resistance among bacteria isolated from the water of Windermere (English Lake District) was higher than those isolated from sewage effluent entered the lake and lower than in those isolated from two remote upland tarns (Jones et al., 1986). A high incidence of antibiotic resistance in an environment virtually free from anthropogenic influence is also observed (Magee and Quinn1991). In north eastern Victoria, bacteria isolated from rivers were more resistant to antibiotics than those isolated from billabongs (branch of a river forming a backwater or stagnant pool) (Boon, 1992). It is possible that the source of such multiple antimicrobial resistant bacteria could be from sources other than the direct discharges from wastewater treatment facilities. The excretion of wild and domestic animals and the presence of litter on the beaches can also be considered as important factors contributing to the emergence of bacterial 97

114 resistance (EPA, 213). Recreational activities themselves which, are intensified during holiday season and the consequent increase in produced litter may also positively affect the proportion of resistant bacteria. There was also a suggestion that resistance to antibiotics may be affected by climatic conditions (Silva et al., 26). Boon and Cattanach, (1999) attributed the occurrence of higher incidence of antibiotic resistance among aquatic bacteria isolated from less contaminated sites to the production of antibiotics by the native bacteria themselves. Thus, different parameters should be taken into consideration to examine this suggestion. The result of the present study may indicate some variation between the counts of fecal indicators and the percentage of multiple antibiotic resistant isolates. It is worth to mention that there is no correlation between the number of such indicators and antibiotic resistance test. Some studies have found that wastewater treatment could reduce the total number of enteric bacteria in sewage, but may increase the proportion of antibiotic resistant coliform in effluent water (Silva et al., 26). The obtained results on antimicrobial resistance confirmed the presence of the multidrug resistance i.e. the majority of strains were resistant to most antimicrobials tested. The prime source of antimicrobials in this area may be the hospitals; aquaculture and agriculture were antimicrobial are widely used as therapy for bacterial infections in humans and in animals as growth promoters as well as for treatment. The coastal waters of Gaza strip receive drug residues as well as microbial pathogens from multiple routes such as wastewater and aquaculture effluents, which may contain drug residues in substantial amounts. In addition, the effluents of Wadi Gaza which mainly consist untreated sewage are discharged directly into the shore. 98

115 6 Chapter 6 Conclusion and Recommendations In all the tested locations, bacteria strain showed high percentage of resistance to different antimicrobials.in addition, most of pathogenic bacteria isolates were multi antimicrobial resistant. Going through these findings, many conclusions and reccomendations were drawn as following: 6.1 Conclusions This study underscores the widespread occurrence of pathogenic and antibiotic resistant microorganisms in the Gaza beach environment. The anthropogenic impact in this region is very high, and the majority of pathogenic isolates are resistant to at least one antibiotic. Moreover, the continued introduction of resistant organisms to the aquatic environment from human sources magnifies the reservoirs of resistant strains over time. The following conclusions were drawn from the results obtained during this study: 1. North of Al Wadi, South of Al Wadi and Khan Yunis were the most contaminated locations in the order listed. 2. According to EU guideline, North of Al Wadi showed (1%) failure, South of Al Wadi showed (66.5%) failure, Khan Yunis which showed a failure of (77.8%). 3. Geometric mean of fecal coliform showed higher rates than fecal streptococci in sand while it was the opposite in water. 4. Fecal streptococci and fecal coliform were found to be higher in sand and mussel than water. 5. High resistance rate was found among potentially pathogenic isolated from the five tested locations along Gaza strip beach. 6. MAR was found in 8% of Enterobacteriaceae and resistance against Tetracycline was (67.9%) and (56%) against amoxicillin-clavulanic acid. 7. MAR of Enterococci isolates was 89%. The greatest resistant against Vancomycin (84.8%) followed by Ampicillin (8.3%). 99

116 8. Pseudomonas aerugenosa isolates showed 46% MAR. The highest resistant was against Piperacillin (55.1%) while all isolates were sensitive to Amikacin. 9. Staphylococci isolates showed 88% MAR. The highest resistance among Staphylococci isolates was found to be against Penicillin (91.8%) followed by Vancomycin (68.9%) and Oxacillin (62.3%). 1. Multi Antimicrobial Resistance Index (MARI) was higher than (.2) in all tested locations, that's mean all locations was contaminated. 6.2 Recommendations The problem of antibiotic resistance will not be solved with the creation of many more, or stronger, bactericidal antimicrobials. Past history is anyhow a good interpreter of the future coming history, and so, microorganisms will consistently continue to adapt to their environment by developing resistance to newer antibiotics and serious infections caused by these bacteria will continue to create a major challenge to the practicing clinician and to patients as well as to the general public health. In light of the above listed conclusions and the results of this study, the following recommendations are suggested: 1. Seawater sampling program should include antimicrobial resistance profile of the four studies pathogens ( Enterobacteriaceae, Enterococci, Staphylococci, and Pseudomonas aerugenosa. 2. The results of antimicrobial resistance profiles of bacterial pathogens should be used as a general measure of the resistance pattern in Gaza strip. 3. Establishing a National Antimicrobial Resistance Monitoring System (NARMS) to monitor the susceptibility of most pathogenic bacteria to antimicrobial agents of medical importance in order to help assess the impact of antimicrobial use on human health. 4. Enhancement of the surveillance system by adapting a computerized laboratory databases and electronic medical records data, and develop a web-based program that facilitates data collection from public health sites which will facilitate surveillance at the healthcare level and so increase surveillance capacity. 1

117 5. Establishing wastewater treatment plants and improves the existing ones to ensure proper wastewater treatment before discharging to Gaza beach. 6. Wastewater generated from health care institution should be treated to levels that ensure the absence of such pathogens before it is discharged to the public sewage system. 7. Healthcare institutions need robust infection control programs and antibiotic stewardship programs to prevent transmission of resistant bacteria and to decrease the selective pressure for resistance. 8. Support of new and effective vaccines to prevent infections caused by some of the most serious infections. 9. Further studies about the concentration of antimicrobial agents and patterns of bacterial resistance in marine habitats are of crucial importance, as well as those about the transfer of bacterial resistance in these ecosystems. 1. Educational campaigns for prudent antibiotics use should be launched for the public. 11. Foster international research collaborations, special regional studies, and national pilot projects to characterize unique and common elements in the epidemiology of antimicrobial-resistant in different countries. 12. Issue and enforce laws to regulate the use of antimicrobials in order to prevent misuse, abuse and over use of these vital drugs. 11

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142 ANNEX ANNEX I 126

143 This map provides bathers of the polluted areas and clean ones (EQA, 214) 127

144 ANNEX II The Main Result of Microbiological Quality Beach 128

145 1. Result of Microbiological Quality Beach Assessment # Location T. Colifo F.Colif F.Strepto Salmonela Shigilla Psedom Staphylo Vibrio

146 2. Food microbiology reports 13

147 131

148 132

149 3. microbiology reports. 133

150 134

151 135

152 ANNEX III The Main Result of Antimicrobial Resistance 136

153 A. Bacteria Resistance 1. Enterobacteriaceae Resistance (1 sensetive, 2 intermediat, 3 rseietance) # AMC CN CXM CIP MER PRL SXT TE CTR CAZ

154 2. Enterococcus Resistance (1 sensetive, 2 intermediat, 3 rseietance) # VA TE CIP AMP NX F

155 3. Pseudomonas aeruginosa Resistance (1 sensetive, 2 intermediat, 3 rseietance) # CAZ CN PRL MER OF NX CIP AK

156 4. Staphylococcus aureus Resistance # AZM P VN CIP CN NX TM OX SXT DOX

157 B. Multible Antimicrobial Resistance Index 1. Multible Antimicrobial Resistance Index of Enterobacteriaceae Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Mussels Mussels Mussels Mussels Mussels Mussels Mussels Resistance MARI Average SD SE Average SD SE Average SD SE Average SD SE Average SD SE 141

158 2. Multible Antimicrobial Resistance Index of Enterococcus Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Resistance MARI MARI average SD SQRT SE average SD SQRT SE average SD SQRT average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE 142

159 3. Multible Antimicrobial Resistance Index of Pseudomonas aeruginosa Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Resistance MARI MARI average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE 143

160 4. Multible Antimicrobial Resistance Index of Staphylococcus aureus Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Khan Yunis Elzawaida Elzawaida Elzawaida Elzawaida Elzawaida Elzawaida Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi South Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Elwadi North Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Mussels Resistance MARI MARI average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE average SD SQRT SE 144

161 ANNEX IV Photo 145

162 Sampling Sampling 146

163 Mussel Sampling Subculture 147

164 Mussels After Washing Opining the Mussels 148

165 Mussels After Opening Colonies Counting 149

166 Identification of Bacteria by API 2 Susceptibility Testing 15

167 Results of Susceptibility Fishers on the Beach 151