ABSTRACT. * 1 Wemambu I.I, 1 Ajose D.J., 2 Akomolafe E.U INTRODUCTION

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1 Bells University Journal of Applied Sciences and Environment (BUJASE) Vol. 1 No 2, December 2018, Pp Copyright College of Natural and Applied Sciences, Bells University of Technology, Ota ISSN: (Print); (Online) The prevalence and antibiotic susceptibility pattern of methicillin-resistant Staphylococcus aureus (MRSA) amongst apparently healthy students in a private University in Ota, Ogun State, Nigeria * 1 Wemambu I.I, 1 Ajose D.J., 2 Akomolafe E.U 1 Department of Biological Sciences, College of Natural and Applied Sciences, Bells University of Technology, Ota, Ogun State, Nigeria. 2 Dept. of Biological Sciences, Oduduwa University, Ipetumodu, Ile Ife, Osun State, Nigeria *Corresponding author: wems5@yahoo.com; +234(0) Received: 31 December 2018 Accepted: 31 January 2019 Published online: ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections that are becoming increasingly difficult to combat because of emerging resistance to all current antibiotic classes and its prevalence in the community is on the increase. This study was designed to determine the prevalence of MRSA amongst students of a private university. Sixty samples were collected from the nose, skin and ear of some apparently healthy students selected at random and screened for the presence of Staphylococcus aureus that are methicillin resistant using standard microbiological procedures. Two hundred isolates were obtained of which 52% were coagulase positive Staphylococcus aureus; a majority of 34% was isolated from the male students while the remaining 18% were from female students. Of all the isolates obtained only 2% were found to be methicilin resistant. 46% of the isolates were other species of Staphylococcus; 15% of which were from the male and the remaining 31% were from the female students. The MRSA strains (2%) were all obtained from the samples collected from male students. It was observed that 40% of the isolates were from the nasal region, 38% from the skin and the remaining 22% from the ear. The emergence of MRSA amongst private University students is a danger signal to the spread of the bacterium among student population which could eventually lead to an outbreak of an epidemic and even nosocomial infection in the school hospital if an effective measure to combat the organism is not put in place. Keywords: Methicillin-Resistant Staphylococcus aureus (MRSA); Antibiotic susceptibility INTRODUCTION Staphylococcus aureus is the most important pathogenic member of the genus Staphylococcus. It has shown resistance to most of the antibiotics that have been introduced against it in the recent years (Rajaduraipandi et al., 2006). The commercial introduction of β-lactamase resistant semisynthetic penicillin in the 1960s contributed momentary alleviation which ended just after methicillin became commercially accessible for therapeutic utilization (Kaleem et al., 2010). Staphylococcus aureus has been a prominent cause of nosocomial infection for many years. Its role diminished markedly in the 1960s when the drug methicillin and similar drugs were introduced, as most strains of Staphylococcus at that time were susceptible. Methicillin was the first antibiotic in this class to be used (it was introduced in 1959), but only two years later, the first case of Methicillin-resistant S. aureus (MRSA) was reported in England (Jevons, 1961). Despite this, MRSA generally remained an uncommon finding even in hospital settings until the 1990s when there was an explosion in MRSA prevalence in hospitals where it is now endemic. Williams (1963) described the first carriage of Staphylococcus aureus by healthy persons and cases of Methicilin-resistant Staphylococcus aureus infection presenting from the community were first described in the early 1980s. MRSA is a bacterium responsible for difficult-totreat infections in humans. It may also be referred to as multiple-resistant Staphylococcus aureus or oxacillin-resistant Staphylococcus aureus (ORSA) (Hena and Sudha, 2011). It is a strain of S. aureus that is distinguished from other S. aureus strains 74

2 by its resistance to the special Beta-lactam drugs (called the methicillin-like drugs) that are usually used to treat these organisms (Palavecino, 2007). MRSA can affect people in different ways. People can carry the organism in the nose, ear or on the skin without showing any symptoms of illness (CFSPH, 2016). Carrier types vary and they include persistent, occasional, and transient carriers. The persistent carriers are those who keep a particular organism for a long time and persistent carriage is more common in children than in adults. Occasional carriers sporadically harbour the organism intermittently, while the transient carriers are those which harbour a particular strain for a certain period and then change to another. Some factors have been thought to be involved in the carrier state and this involved the immune system of the host. People with an active immune system harbour the organism more readily and are likely to be persistent carriers (Mackie and McCartney, 1985). The carriage of Staphylococcus aureus by healthy persons was first described by Williams (1963). In Nigeria, the prevalence of MRSA has also been reported (Taiwo et al., 2004; Onanuga et al., 2005). This study was therefore carried out to determine the prevalence, and incidence of MRSA isolates in the nose, skin and ear of students of a private university in Ota, Ogun State, South West Nigeria. MATERIALS AND METHODS Samples were collected using a sterile swab stick, from the ear, nose and skin of randomly selected students of a private University in Ota, Ogun State. Students who had previously taken antibiotics in less than three months and those with any form of known infection of any of the body parts of choice were excluded from this research. This research was carried out between the months of January - May Sample collection and culture Samples were collected by wiping the skin with the sterile swab stick, cleaning the ear with the swab stick and bending their head back in other to collect the specimen deep down the nasal passage using the swab stick. Demographic factors were recorded and indicated on the sample sticks. The samples collected were cultured on to Mannitol salt agar (Oxoid, CM0085) and incubated at 37 C for hours, for the isolation of Staphylococcus aureus (Tuba et al., 2017). Golden to dark yellow colonies observed on the mannitol salt agar after incubation was suspected to be Staphylococcus aureus and they were sub-cultured onto nutrient agar (Oxoid, CM003). The plates were incubated at 37 C for 24 hours and were sub-cultured onto nutrient agar slant and incubated for 24 hours at 37 C. The slants were stored in the refrigerator at about 2-8 C for further investigations. Screening and identification of isolates This was done following the method of Tuba et al. (2017). Macroscopy Colonies showing golden-yellow colour with average zone diameter of about 2-3 mm on mannitol salt agar were considered presumptive for Staphylococcus aureus. The yellowish colouration observed is an indication of mannitol fermentation. Gram staining was performed on the presumptive isolates and only positive samples with typical Staphylococcal appearance were further characterised. Catalase test The isolates that appeared Gram-positive and cocci after examination under the microscope were tested for the enzyme catalase. A drop of hydrogen peroxide was placed on a clean grease free slide and a colony was aseptically picked with the aid of a wireloop and placed in the solution on the slide. The production of gas bubbles indicated positive reaction (Cruickshank et al., 1980). This test is used to differentiate those bacteria that produce the enzyme catalase, such as Staphylococci from noncatalase producing bacteria such as Streptococci. Catalase acts as a catalyst in the breakdown of hydrogen peroxide to oxygen and water (Cheesebrough, 2000). Coagulase test All Gram positive cocci colonies that were positive to the catalase test were tested for the presence of the enzyme coagulase. Coagulase is an enzyme that causes the plasma to clot by converting fibrinogen to fibrin. EDTA pooled anticoagulated human plasma was diluted 1/10 with normal saline. To 0.5 ml volume, 5 drops of overnight broth culture of the test organism were aseptically added in a test tube and incubated at 37 C in a water bath for 4 hours. A positive result was indicated by the formation of a clot. However, tubes that showed negative results within this duration were further incubated overnight to enable the growth of some Staphylococcus aureus strains that produce coagulase slowly (Cruickshank et al., 1980, 75

3 Cheesebrough, 2000). All organisms that were negative for coagulase after overnight incubation were regarded as coagulase negative. Antibiogram sensitivity testing of the test isolates The antimicrobial susceptibility profile of the isolates was determined using the agar disc diffusion technique in accordance with the Clinical and Laboratory Standard Institute (CLSI) recommendations (CLSI, 2007). With a sterile wire loop, few colonies of each of the isolates were separately but aseptically emulsified in 5 ml of sterile peptone water to a turbidity corresponding to 0.5 McFarland standards (corresponding to approximately 10 8 cfu/ml). Then 0.5 ml of each inoculum was dispensed unto the surface of dried Mueller Hinton agar (MHA) (Oxoid, CM0337) plate using sterile Pasteur pipette. These were spread evenly on the agar surface with sterile swab sticks. The excess inocula were drained off. The inoculated plates were kept on the bench for 3 minutes to dry. The antibiotics multidisc (Oxoid, CM0337) (ceftazidine (30 µg), cefuroxim (30 µg), gentamicin (10 µg), lincomycin (2 µg), methicillin (5 µg), cloxacillin (5 µg), ofloxacin (5 µg), and augmentine (30 µg)) was then aseptically placed centrally on the inoculated plates using a sterile forcep. The preparations were incubated aerobically for 24 hours at 37 o C. The diameter of the zone of inhibition produced by each of the discs was measured, recorded and the isolates were classified as resistant ( 10 mm) or sensitive ( 13 mm) based on the standard interpretative chart as recommended by the Clinical and Laboratory Standard Institute (CLSI, 2007). This was done in triplicate and mean average readings recorded. RESULTS Sample statistics are represented in Table 1. Two hundred isolates were obtained; about fifty -four percent (54%) of the isolates were Staphylococcus aureus; 36% from male and 18% from female. The remaining forty-six per cent (46%); 15% from male and 31% from female samples were other species of Staphylococcus (Figure 1). It was also observed that 40% of the isolates were from the nasal region, 22% from the ear and the remaining 38% from the skin (Figure 2). The frequency of methicillin-resistant Staphylococcus aureus (MRSA) strains amongst the Staphylococcus aureus isolates was about 2% in relation to non-methicilin resistant Staphylococcus aureus which was about 52%. This is represented in Figure 3 while Figure 4 shows the antibiotic susceptibility pattern of all isolates. Antibiotic sensitivity test on the Staphylococcus aureus species revealed that 2% of the strains were resistant to methicilin, ofloxacin and augmentin while others were sensitive. This indicates that methicilin, ofloxacin and augmentin were the most effective antibiotic tested against the isolated strains of Staphylococcus aureus. Over 70% of the Staphylococcus aureus strains resisted ceftazidine and more than 60% of the Staphylococcus aureus population resisted cloxacilin, making these two antibiotics the most resisted. The pattern of antibiotic sensitivity is shown is Figure 4. Table 1: Demographic Study Pattern SEX Number of S. aureus (SA) No (%) Other Staph. (OS) MRSA (%) samples (%) MALE FEMALE TOTAL Key: SA - Staphylococcus aureus OS - Other Staphylococcus MRSA - Methicilin-resistant Staphylococcus aureus Fig. 1: Percentage frequency of occurrence of Staphylococcus aureus and the Methicilin resistant strains with respect to the source 76

4 Fig. 2: Percentage frequency of occurrence of Staphylococcus aureus from the different body parts Fig. 3: Percentage distribution of all isolates Key: SA Staphylococcus aureus OS Other Staphylococcus MRSA Methicilin Resistant Staphylococcus aureus Fig. 4: Antibiotic sensitivity distribution pattern Key: CAZ Ceftazidine CRX Cefuroxine GEN Gentamicin LIN Lincomycin MET Methicilin CXC Cloxacilin OFL Ofloxacin AUG Augmentin 77

5 DISCUSSION Methicillin-resistant S. aureus is rampant in our society today and as such caused a major threat to the health care system (Akhter et al., 2009). In this study, the carrier rate of Staphylococcus aureus is in agreement with some of the earlier authors who estimated between 40 to70% carrier rates of pathogenic Staphylococci particularly among hospital personnel (Liu et al., 2015). In this present study, 54% of the study population harboured Staphylococcus aureus in their nostrils, ear and on their skin. The reported carrier rates of the pathogenic coagulase positive organisms in healthy individuals vary from one community to the other (CFSPH, 2016) and the differences within carrier rate of the community are dependent upon the hygiene and sanitation of the community as well as the knowledge of the epidemiology of infection of organisms involved. Globally, it has been reported that there is an upsurge in infections caused by Community-associated Methicilinresistant Staphylococcus aureus (CA-MRSA) (Pickett, 2009). It was observed that the carrier rate was higher in the male than in the female which could probably result from the hygienic level of the gender involved. The males are more involved in activities which bring them together and thus facilitate spread either by direct or indirect contact with each other and their active engagement in sporting activities cannot be overlooked as this leads to the production of sweat on the skin thus the propagation of S. aureus because it has an affinity for sodium chloride and metabolizes speedily in the presence of sodium chloride (Cheesebrough, 2000). The isolation of Staphylococcus aureus from the skin of some individuals could be because S. aureus is a normal flora of the skin and could also be due to indirect and direct contact with carriers. Staphylococcus species are among the hardest of all the non- spore-forming bacteria to combat. They remain alive for months and can be cultured from samples of dried pus; some strains are heat resistant, being capable of withstanding a temperature of 60 C for one hour (Maria et al., 2017). The occurrence of Staphylococcus aureus in the study community was higher in the nasal cavity of carriers which may be directly related to the contamination of the environment (Liu et al., 2015). Liu et al. (2015) observed that Staphylococcus aureus in the air and on objects and surface areas increased when a carrier spoke, sneezed or coughed. The occurrence of S. aureus may also increase through inhalation and contact of contaminated air by individuals, thus leading to an increase in the carrier rate. This is responsible for the contamination of food and formites which can serve as vehicles for transmission. Omoregbe and Igbinovia (1992) suggested that the high carrier rate of Staphylococcus species particularly among hospital staff is due to the predominance of Methicilin-resistant Staphylococcus aureus and this may not only serve as a source of further infections within the hospitals but can constitute an obvious risk to the community. From this study only 2% of the two hundred isolates obtained were MRSA and these strains were observed only amongst the male students, this is in contrast to earlier studies in Nigerian women that recorded 71.2% (Onanuga et al., 2005). In a multicenter study conducted by Hafiz et al. (2002), a low prevalence rate of MRSA (2%) was reported in a city in Pakistan. Though it has been reported that MRSA prevalence is ever increasing (Voss, 2006), the lower prevalence observed from this study may be attributed to the fact that antibiotics are not sold over the counter within the study site thus curbing the inappropriate use of antibiotics and as well checking the spread of antibiotic resistance. Students who are ill are compelled to report at the clinic for appropriate medical attention. Again, the low prevalence observed in this study in comparison to the report of other researchers (Shafiq et al., 2011, Tuba et al., 2017) may be subjected to differences in geographical distribution. However, the methicilin-resistant Staphylococcus aureus isolated from the students of study site is of serious therapeutic implication as these resistant strains gradually replace other strains which cannot synthesize beta lactamase - the enzyme responsible for methicilin resistance. If an infected individual/ carrier happens to be admitted to a hospital, there is a possibility of infecting other patients who then can serve as vehicles of transmission to the larger community. Besides this, students who are carriers can even spread these resistant strains among other students who are non - carriers by day to day direct and indirect contact and the spread can be accelerated by overcrowding and the abuse of methicillin drugs. CONCLUSION The major problem threatening the public is the prevalence and high rate of occurrence of methicilin-resistant Staphylococcus aureus in hospitals and the community at large. In view of this, 78

6 regular prophylactic use of antibiotics should be stopped to avoid the elimination of non-resistant strains and recolonization by the resistant strains, routine laboratory examination of students in the university clinic should be carried out prior to commencement of treatment or admission to ensure that the student is not a carrier and if a carrier patient, should be treated immediately to avoid further spread; the maintenance of a clean, healthy and less crowded environment should be encouraged in the school and amongst students to reduce the rate of occurrence and the maintenance of a sterile environment and work with a nose and mouth mask (if the patient is coughing) is suggested in other to control the spread of this pathogen. REFERENCES Akhter, R., Khan, K. M. A. and Hasan, F. (2009). Isolation and antimicrobial susceptibility pattern of methicillin resistant and methicillin sensitive Staphylococcus aureus. J. Surg. Pak., 14(4): Centre for Food Security and Public Health. Methicillin Resistant Staphylococcus aureus. Cheesbrough, M. (2000). Biochemical Tests to identify bacteria. In: District Laboratory Practice in Tropical Countries, Part 2: low price edn. Cambridge University Press, United Kingdom. Pp CLSI, (2007). Performance Standards for Antimicrobial Susceptibility Testing, Fifteenth Information Supplement, CLSI document M100-S16. vol. 26-3; M7-A7, Vol. 26-2; M2-A9, Vol Wayne, PA USA. Cruikshank, R., Duguid, J. P., Marmion, B. P. and Swain, R. H. A. (1980). Medical Microbiology, 12 th ed. Churchill Livingstone Pub. Co. Ltd. Edinburgh, London, pp Hafiz, S., Hafiz, A. N., Ali, L., Chughtai, A. S., Memon, B., Ahmed, A. (2002). Methicillin resistant Staphylococcus aureus: a multicentre study. JPMA, 52: Hena, J. V. and Sudha, S. S. (2011). RFLP Analysis of Clinical MRSA Isolates. International Journal of Pharmacy and Biological Sciences, 2(1), Jevons, M. P. (1961). Celbenin resistant Satphylococci. BMJ 1: Kaleem, F., Usman, J., Hassan, A., Omair, M., Khalid, A. and Uddin, R. (2010). Sensitivity pattern of methicillin resistant Staphylococcus aureus isolated from patients admitted in a tertiary care hospital of Pakistan. Iranian Journal of Microbiology, 2(3): 143. Liu, C. M., Lance, B. P., Bruce, A. H., Alison, G. A., Lisbeth, A. L., Kaare, C., Marc, S., Robert, S. and Paal, S. A. (2015). Staphylococcus aureus and the ecology of the nasal microbiome. Sci. Adv. 1 (5), e DOI: /sciadv Mackie and McCartney. (1985). Microbial infections. Medical Microbiology 13 th ed. Longman Group Limited, London pp236 Maria, N. Y., Charis, A. and Tri, Y. B. (2017). The effect of temperature and Pasteurization time on Staphylococcus aureus isolates from dairy products. American Institute of Physics (AIP) Conference Proceedings 1908, ; doi: / Omoregbe, R. E. and Igbinovia, O. (1992). Prevalence of Staphylococcus and Streptococcus species among food handlers in Edo State University, Ekpoma, Nigeria. J. App. Biol. 4: Onanuga, A., Oyi, A.R. and Olayinka, B.U. (2005). Prevalence of community associated multiresistant Staphylococcus aureus among healthy women in Abuja, Nigeria. Afr. J. Biotech. 4: Palavecino, E. (2007), Clinical, epidemiological, and laboratory aspects of methicillinresistant Staphylococcus aureus (MRSA) infections, Methods in Molecular Biology, Pickett A., Wilkinson M., Menoch M., Snell J., Yniguez R. and Bulloch B. (2009). Changing incidence of methicillin-resistant Staphylococcus aureus skin abscesses in a pediatric emergency department. Pediatr Emerg Care. 25(12) : Rajaduraipandi, K., Mani, K., Panneerselvam, K., Mani, M., Bhaskar, M. and Manikandan, P. (2006). Prevalence and antimicrobial susceptibility pattern of methicillin resistant Staphylococcus aureus: A multicentre study. Indian J. Med. Microbiol., 24(1):

7 Shafiq, R., Shahina, M., Ahmad, J., Syed, H. and Malala, R. (2011). Incidence of Methicillin resistant Staphylococcus in Peshawar. J. Ayub. Med. College Abbotabad, 23(1): Taiwo, S. S., Onile, B. A. and Akanbi, I. (2004). Methicillin-resistant Staphylococcus aureus (MRSA) isolates in Ilorin. Afr J Clin Exp Microbiol. 5(2): Tuba, S., Iyad, N. M., Muhammad, N. K., Shazia, N., Lubna, B., Nosheen, S., Rida, M., Aatka, A., Sakina, F. and Talat, N. (2017). MRSA: Prevalence and susceptibility pattern in health care setups of Karachi. Pak. J. Pharm. Sci., 30(6), pp Voss, A. and Doebbeling, R. (2006). The world wide prevalence of methicillin resistant Staphylococcus aureus. Int J Antimicrob. Agents 5: Willaims, R. E. O. (1963). Healthy carriage of Staphylococcus aureus: its prevalence and importance. Bacteriol. Rev. 27: Submit your next manuscript to Bells University Journal of Applied Sciences and Environment (BUJASE), and we will help you through every stage. We accept manuscripts at any time. Our customer support is available round the clock. We assure you of thorough and timely peer review. The Editorial Board Members are national and international experts. Being open access journal (print and online), you are sure to get maximum visibility for your research. Submit your manuscript to editor.bujase@gmail.com and editor.bujase@bellsuniversity.edu.ng 80