High Antibiotic Resistance Pattern Observed in Bacterial Isolates from a Tertiary Hospital in South East Nigeria

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1 International Journal of Research in Pharmacy and Biosciences Volume 3, Issue 1, February 2016, PP 1-6 ISSN (Print) & ISSN (Online) High Antibiotic Resistance Pattern Observed in Bacterial Isolates from a Tertiary Hospital in South East Nigeria Chijioke A. Nsofor 1, Anyanwu N.C 2, Ogbulie T.E 1 1 Department of Biotechnology, Federal University of Technology, Owerri, Nigeria 2 Department of Microbiology, Imo Sate University Owerri Nigeria ABSTRACT Antibiotics resistance in bacteria has continued to pose threat in both human and animal medicine. This study, investigated the distribution and antibiotic susceptibility pattern of bacterial isolates from a tertiary hospital in Owerri, southeast Nigeria. The bacterial isolates were identified by the conventional microbiological techniques and antibiotic susceptibility determined by modified Kirby-Bauer method. In all 506 bacterial isolates were identified, including 325 Gram-negative bacterial strains (64.2%) and 175 Gram-positive bacterial strains (35.8%). Staphylococcus aureus were Methicillin-resistant and 52.9% of coagulase-negative staphylococci were Methicillin-resistant, but susceptible to vancomycin. About three isolates of Enterococcus faecium and 3 Enterococcus faecalis strains were resistant to vancomycin. Also, 62.1% of Escherichia coli and 56.23% Klebsiella pneumoniae were extended spectrum β-lactamases (ESBLs) positive, and carbapenem showed high activity against both bacteria (resistant rates < 10%). This study has shown that there is high rate of antibiotic resistance among bacterial isolates from this hospital. Thus, there is need for urgent and necessary measures to promote systematic, continuous and high-quality bacterial-resistance surveillance in the region. Keywords: Antibiotic resistance Pattern; Bacterial profile; Pathogen INTRODUCTION Data on the overall patterns of antibiotic use in hospitals have frequently appeared in the literature in the last several decades. The general thrust of such data indicates that from 25% to 40% of hospitalized patients receive systemic antibiotics at any given time (Bolaji et al, 2011). Furthermore, a sequentially obtained data suggests that there is a trend toward increasing, rather than decreasing, antibiotic use in hospitals. Continuous and sharp increases in the use of extended- spectrum cephalosporin, vancomycin, metronidazolee, and amphotericin B have been observed (Akortha and Ibadin 2008). Such massive use of antibacterial drugs in hospitals, whether appropriate or inappropriate, has profound effects on both the hosts who receive these drugs and the bacteria exposed to them. The global antibiotic market was valued at over 20billion dollars in 1994 (Kahlmeter, 2003), and this could well double in the years ahead. There is enormous pressure on the part of the pharmaceutical industry to increase antibiotic use, both to recoup the cost of new drug development, and to accomplish the very legitimate goal of making profit, and these have encouraged irrational use of antibiotics which in turn favor the development of resistance. Every major class of bacterial pathogens has so far demonstrated an ability to develop resistance to one or more commonly used antimicrobial agents (Nwadioha et al., 2010). In the mid 1940s, shortly after the introduction of Penicillin G, it was recognized that certain strains of Staphylococci elaborated a potent β-lactamase; an enzymatic inactivator of penicillin, and that Penicillin G had no therapeutic activity in patients with infections caused by such staphylococci. This recognition came as a major disappointment but not a total surprise to the scientific and medical community. Since that time, it has become abundantly clear that the major nosocomial pathogens are either naturally resistant to clinically useful antimicrobial drugs or possess the ability to acquire resistance. The best-known examples are the staphylococci and aerobic gram-negative bacilli, which together regularly account for most nosocomial infections (Nsofor et al., 2013). *Address for correspondence International Journal of Research in Pharmacy and Biosciences V3 I2 February

2 Origin of antibiotics resistance has been grouped into genetic and no genetics sources. Genetic sources involve; chromosomal mutation which gives rise to change in structural receptor in the pathogen thereby making it difficult for the drug to attach, and possession of extra chromosomal elements called plasmids some of which carries resistance genes (Jawetx et al., 2004). Some of these plasmids mediate for production of ß- lactamases, which are responsible for resistance in most of ß- lactam antibiotics as well as cross-resistance in other classes of antibiotics. Indiscriminate use of antibiotics for medical purposes has taken the brunt of the blame, namely, use by those physicians who prescribe antibiotics for viral infections to make their patients feel comfortable when antibiotics are known to be useless against viruses. In fact, all antibiotic use, whether medical, agricultural, and necessary or not, leads to increased resistance. Studies on antibiotic residues in hospitals and in other environmental niches have been conducted mostly in high-income countries, while studies in low- and middle-income settings are few and sparsely distributed. It is against this background that this study set out to identify the bacteria pathogens and their antibiotic resistant pattern among out-patients and hospitalized patients attending Federal Medical Centre Owerri, a tertiary health facility in South-Eastern Nigeria. It is our belief that the result of this study will show the current trend of antibiotic susceptibility pattern of the pathogens in our setting, a necessary step towards defining locally effective empirical treatment of infectious diseases. METHODS Sample Collection, Cultivation and Identification of Bacterial Strains Clinical specimens were collected from out-patients and in-patients with bacterial infections who were given a bacteriological examination. Pathogenic bacteria were cultured and isolated with appropriate media and growth environment. Isolates were identified by conventional microbiological techniques and biochemical tests. Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC and Staphylococcus aureus ATCC were included as quality control strains. Antibiotic Susceptibility Test Antibiotic susceptibility of the bacteria isolates was assayed according to the Kirby Bauer disc diffusion method (Bauer et al., 1996). All the plates were incubated for 20 minutes before inoculation and placement of antibiotic disc to allow excess moisture to dry. After the drying, a single loop of each isolate was inoculated into sterile normal saline and compare with 0.5 McFarland standard, the suspension was aseptically swabbed on the surface of Mueller Hinton plates and antibiotic sensitivity disc that contains the appropriate antibiotic was aseptically laid on the surface of plates. The plates were incubated at 35 0 C for 24 hours. After the incubation, zone of growth of inhibition around each disc was measured and used to classify the organisms as sensitive or resistant to an antibiotic according to the interpretive standard of the Clinical and Laboratory Standards Institute (CLSI, 2006). Data analysis The results of the antimicrobial susceptibility tests were interpreted according to CLSI. Data were analyzed using analysis of variance ANOVA. The same strain from the same type of specimens from one patient was counted once toward the non-repetitive strain counting. RESULTS AND DISCUSSION Generally, a total of 506 single strains were identified, including 325 Gram-negative bacterial strains (64.2%), and 175 Gram-positive bacterial strains (35.8%). The Gram-positive strains mostly consist of Staphylococcus aureus, coagulase negative staphylococci, Enterococcus faecium and Enterococcus faecalis. The main Gram-negative bacterial strains ware Acinetobacter baumannii, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae (Table 1). The strains were isolated from respiratory tract specimens (sputum, throat.) (51.24%), genitourinary tract 2 International Journal of Research in Pharmacy and Biosciences V3 I2 February 2016

3 specimens (urine, vaginal secretions) (13.60%), wound secretions (13.07%), pus (9.16%), blood (7.65%) and others (5.28%). Table1. Percentages of Bacteria Isolated from the Patients attending the Hospital Bacterial pathogens Percentage Acinetobacter baumannii 08.28% Pseudomonas aeruginosa 15.23% Escherichia coli 10.26% Klebsiella pneumoniae 10.00% Staphylococcus aureus 11.12% Coagulase negative staphylococci 5.35% Enterococcus faecium 2.49% Enterococcus faecalis 4.21% Haemophilus influenzae 0.87% Proteus mirabilis 0.82% Streptococcus pneumoniae 0.65% Klebsiella oxytoca 0.60% Streptococcus Feacalis 0.41% Streptococcus agalactiae 0.39% The result of this study indicated that strains of bacteria isolated in this study were resistant to most of the tested antibiotics, and this showed that they have become multi-resistant to these therapeutic agents, thus rendering these drugs ineffective as treatments of choice for infections caused by these pathogens. This is obvious from the results of percentage of the isolates that are resistant to all the tested antibiotics. For Staphylococcus aureus or coagulase negative staphylococci, Methicillinresistant Staphylococcus aureus (MRSA) and Methicillin-resistant coagulase negative staphylococci (MRCNS) were 64.63% and 73.39% but all strains were fully susceptible to vancomycin, (Table 2). Staphylococcus aureus was susceptible to doxycycline (76%) and chloramphenicol (83.62%), but highly resistant (resistant rate over 60%) to penicillin, ceftazidime, cefazolin, erythromycin and azithromycin (Table 2). Table2. Antibiotic Susceptibility Pattern of Staphylococci Isolates Antibacterial Staphylococcus aureus Coagulase negative staphylococci agents Penicillin 2.14% 97.86% 12.29% 87.71% Oxacillin 41.37% 58.63% 30.61% 69.39% Cefazolin 23.33% 76.67% 35.03% 64.97% Ceftazidime 36.67% 63.33% 45.01% 54.99% Vancomycin % 0.00% % 0.00% Erythromycin 31.25% 68.75% 22.83% 77.17% Tetracycline 33.97% 76.57% 47.31% 52.69% Doxycycline 82.89% 11.47% 77.93% 32.07% Ciprofloxacin 39.61% 60.39% 34.85% 65.15% Levofloxacin 40.39% 59.61% 42.10% 57.90% Chloramphenicol 83.62% 20.39% 77.38% 22.62% Rifampicin 48.14% 51.86% 69.29% 30.71% Linezolid % 0.00% % 0.00% Key: S, susceptibility; R, resistance. Enterococcus faecium and Enterococcus faecalis were fully susceptible to teicoplanin and linezolid, but four Enterococcus faecium and three Enterococcus faecalis strains were resistant to vancomycin (Table 3). Enterococcus faecium were more resistant to almost all tested agents than Enterococcus faecalis, except tetracycline (Table 3). Enterococcus faecalis were highly susceptible to ampicillin (77.21%) and penicillin (74.92%) (Table 3). Table3. Antibiotic Susceptibility Pattern of Enterococcus spp Antibacterial Enterococcus faecium Enterococcus faecalis agents Penicillin 10.29% 89.71% 77.92% 15.08% Ampicillin 9.86% 91.14% 74.21% 7.79% International Journal of Research in Pharmacy and Biosciences V3 I2 February

4 Vancomycin 90.00% 09.01% 86.98% 13.02% Erythromycin 08.33% 91.67% 20.80% 99.2o% Tetracycline 11.67% 68.33% 11.11% 88.89% Ciprofloxacin 24.05% 75.95% 77.59% 22.41% Levofloxacin 27.14% 72.86% 86.13% 13.87% Linezolid % 0.00% % 0.00% Key: S, susceptibility; R, resistance. The majority of Escherichia coli strains (72.82%) and Klebsiella pneumoniae strains (66.24%) were ESBLs positive, but carbapenem antibiotics are very effective, with resistant rates below 10% (Table 4). Escherichia coli and Klebsiella pneumoniae were susceptible to the β-lactamase inhibitor compounds cefoperazone/sulbactam, but highly resistant to ampicillin (> 90%), and also resistant to cefazolin, cefuroxime, cefotaxime and ceftriaxone (Table 4). Acinetobacter baumannii were resistant to ceftriaxone (91.73%), trimethoprim/sulfamethoxazole (81.39%), cefotaxime (78.92%) and ciprofloxacin (76.10%), but susceptible to amikacin (84.20%), (Table5). Pseudomonas aeruginosa were susceptible (> 50%) to all tested antibacterial agents, amikacin and ciprofloxacin were the top 2 potential agents (Table 5). Table4. Antibiotic Susceptibility Pattern of Escherichia coli and Klebsiella pneumoniae Isolates Antibacterial Escherichia coli Klebsiella pneumoniae Agents Ampicillin 7.16% 92.63% 0.00% % Ampicillin/Sulbactam 24.27% 50.82% 35.86% 50.00% Cefazolin 20.54% 79.46% 26.25% 61.95% Cefepime 51.18% 42.45% 50.63% 26.64% Cefotaxime 30.99% 62.06% 27.50% 59.38% Ceftriaxone 25.34% 63.42% 46.88% 52.73% Cefotetan 93.99% 4.29% 91.72% 6.17% Ceftazidime 56.48% 27.70% 47.03% 33.67% Cefuroxime 17.32% 74.66% 25.55% 67.42% Aztreonam 48.46% 51.25% 41.88% 51.64% Imipenem 98.07% 1.72% 95.63% 2.19% Meropenem 92.63% 2.36% 92.50% 6.02% Gentamicin 42.95% 56.41% 54.77% 44.22% Tobramycin 45.88% 23.05% 57.27% 23.44% Amikacin 89.69% 4.80% 86.64% 12.58% Levofloxacin 37.87% 57.98% 74.14% 18.20% Ciprofloxacin 35.86% 62.49% 67.50% 26.88% Note: S, susceptibility; R, resistance. Table5. Antibiotic Resistance Patterns of Acinetobacter baumannii and Pseudomonas aeruginosa Antibacterial Acinetobacter baumannii Pseudomonas aeruginosa Agents Ampicillin/Sulbactam 41.37% 58.63% - - Piperacillin/Tazobactam 19.11% 80.89% 62.69% 37.31% Ceftazidime 21.60% 78.04% 67.17% 32.83% Cefepime 23.24% 76.76% 67.63% 32.37% Aztreonam % 45.88% Cefotaxime 21.08% 78.92% - - Ceftriaxone 8.27% 91.73% - - Imipenem 29.81% 70.19% 65.46% 34.55% Gentamicin 18.58% 81.42% 56.69% 39.75% Tobramycin 31.58% 68.42% 67.11% 26.76% Amikacin 84.20% 25.80% 75.81% 20.44% Ciprofloxacin 23.90% 76.10% 68.16% 26.70% Levofloxacin 38.61% 61.39% 62.69% 24.13% Ofloxacin % 43.77% Trimethoprim/Sulfameth oxazole 18.45% 81.55% - - Note: S, susceptibility; I, insensitivity; R, resistance; -, no break point in CLSI. 4 International Journal of Research in Pharmacy and Biosciences V3 I2 February 2016

5 The use of antibiotics in medicine and veterinary practice has aroused some concern about the incidence and spread of antibiotic resistance among bacteria populations. As a result of the usage of antibiotics in medical or veterinary practice, selected for resistant bacteria, these bacteria have inevitably entered the naturally environment. This is particularly true when transfer occurs in environments such as hospitals where the human population is at risk (Farajnia et al., 2009). The use, misuse and under-use of antibiotics are responsible for resistance development to bacterial antimicrobials worldwide. Lateef (2004), reported that in developing countries, drugs are available to the public and thus people may practice self administration of antibiotics and further increase the prevalence of drug resistant bacteria. There have been many surveys of the occurrence of antibiotic resistant E. coli in animals (Nsofor and Iroegbu 2012), (Matyar et al., 2004). Chong et al., (1990) found that 204 of 400 fecal samples from human sources containing E. coli were resistant to one or more antibiotics at a rate of 83%. The relatively high level of resistance to antimicrobial agents recorded in this study is a reflection of misuse or abuse of these agents in hospitals. Multiple drug resistance is an extremely serious public health problem and it has been found associated with the outbreak of major epidemic throughout the world. Thus, the multiple drug resistance shown by these pathogens are worrisome and of public health concern Encountering multiple antibiotic resistant bacteria in this study is therefore not a surprise but worrisome. Therefore, the occurrence of multiple antibiotic resistant pathogenic bacteria encountered in this study represents a well-known phenomenon that carries a negative impact for public health, an observation that it is in consonance with the reports of Jombo et al., (2011). In this study, the percentage of antibiotic resistant bacteria recorded for all the tested antibiotic is dreadful because most of the isolates were resistant to at least one or more of the antibiotics that are commonly use in medicine and agriculture for prevention and treatment of diseases. These bacteria, like Pseudomonas aeruginosa, are common environmental organisms, which act as opportunistic pathogens in clinical cases where the defense system of the patient is compromised (Ohi and Luther, 2011). Because of the prevalent of multiple antibiotic resistant bacteria, search for new antibiotics that will be effective against multi-drug resistant pathogenic bacteria is presently an important area of antibiotic research. CONCLUSION This study reinforces the need for continuous local surveillance of bacterial antimicrobial resistance. There is also a need for a comprehensive Antibiotics Surveillance Programs in our hospital setting while the public should be educated on the consequences of indiscriminate use of antibiotics. Finally, government at all tiers should endeavor to sponsor researches on development of new antibiotics that could be relevant in the treatment of severe infections caused by antibiotic resistant bacteria. REFERENCES [1] Akortha EE, Ibadin OK. (2008). Incidence and antibiotic susceptibility pattern of Staphylococcus aureus amongst patients with urinary tract infection (UTI) in UBTH Benin City, Nigeria. Afr J Biotechnol; 7: [2] Bolaji A.S, Akande I.O, Iromini F.A, Adewoye S.O and Opasola O.A (2011) Antibiotic resistance pattern of bacteria spp isolated from hospital wastewater in Ede South Western, Nigeria. European Journal of Experimental Biology 1 (4):66-71 [3] Bauer A.W, Kirby WMM, Sherris JC, and Turc M (1996). Antibiotics testing for bacterial pathogens J. Clinic Pathol.45, [4] Chong Y., Lee K and Kwoh., (1993), Antibiotics susceptibility pattern of bacterial isolated in a hospital environment. International Journal of Antimicrobial Agent, 3; [5] Clinical and Laboratory Standards Institute Performance standards for antimicrobial disk susceptibility tests; approved standard 9 th edition: M2-A9.Clinical and Laboratory Standards Institute, Wayne, PA. [6] Farajnia S, Alikhani MY, Ghotaslou R, Naghili B, Nakhlband A. Causative agents and antimicrobial susceptibilities of urinary tract infections in the northwest of Iran. Int J Infect Dis 2009; 13:140-4 International Journal of Research in Pharmacy and Biosciences V3 I2 February

6 [7] Jombo GT, Emanghe UE, Amefule EN, Damen JG. (2011). Urinary tract infections at a Nigerian university hospital: Causes, patterns and antimicrobial susceptibility profile. J Microbiol Antimicrob 3: [8] Kahlmeter G, ECO.SENS. (2003). An international survey of the antimicrobial susceptibility of pathogens from uncomplicated urinary tract infections: The ECO.SENS Project. J Antimicrob Chemother; 51: [9] Lateef A., (2004). Antibiotics use and misuse in developing countries. World Journal of Microbiology. 20, [10] Nsofor C. A., and Iroegbu C.U., (2012). Antibiotic resistance profile of Escherichia coli isolated from apparently healthy domestic livestock in South East Nigeria. Journal of Cell and Animal Biology 6(6), [11] Nsofor C.A, Iroegbu C.U., Eze I.O and Nwabia B.C (2013). Nasal Carriage of Multidrug- Resistant Staphylococcus aureus among School Children in Elele Nigeria. International Journal of Microbiology and Immunology Research 1(2), [12] Nwadioha SI, Nwokedi EO, Ikeh I, Egesie J, Kashibu E. (2010). Antibiotic susceptibility pattern of uropathogenic bacterial isolates from AIDS patients in a Nigerian tertiary hospital. J Med Med Sci 1: [13] Ohi CA, and Luther VP. (2011). Antimicrobial stewardship for inpatient facilities. J Hosp Med; 6 Suppl 1:S International Journal of Research in Pharmacy and Biosciences V3 I2 February 2016

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