13J Contemp Med Sci | Vol. 1, No. 1, Winter 2015:13–16 Research Objective Carriage of Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA) is a problem within healthcare organizations and in the community. The aim of the study was to screen S. aureus carriage and their susceptibility to cefoxitin and oxacillin among medical students. Methods A total of 100 nasal swabs were collected. Isolation and identification of the isolate as S. aureus was done using Gram stain, coagulase test and catalase test. S. aureus isolates were confirmed as MRSA using cefoxitin (30 μg) disc and oxacillin (30 μg) disc by Kirby- Bauer disc diffusion method on Mueller-Hinton agar. From 100 nasal swabs, 76 were coagulase negative Staphylococci and 20 were coagulase positive Staphylococci. Results From 100 nasal swabs, 76 were coagulase negative Staphylococci and 20 were coagulase positive Staphylococci. From these, 60% and 40% were oxacillin- and cefoxitin-resistant isolates, respectively. The data obtained from this study revealed that there were carriers of MRSA among the medical students. Keywords MRSA, coagulase, oxacillin, cefoxitin Screening of nasal carriage for Staphylococcus aureus and their resistance to oxacillin and cefoxitin among medical students in Karbala University Suhad Hadi Mohammed, Mohammed Neama Hmood, Areej Abbas Abd, Seham Aaid Obaid, Baraa Ahmed Fahad & Fatemah Hadi Kadhem Introduction Staphylococcus aureus has long been recognized as an important human pathogen.1 The anterior nares represent the primary ecological reservoir of S. aureus in humans, and nasal carriage is a major risk factor for a variety of infections.2 Three patterns of nasal carriages are known (persistent carrier, intermittent, and non-carrier). Approximately 20% of the individuals almost always carry one type of strain and they are called persistent carriers. A large proportion of the population (60%) harbours S. aureus intermittently, and the strains change with varying frequencies. Such persons are called intermittent carriers. Finally, a minority of the people (20%) almost never carry S. aureus and they are called non- carriers. A persistent carriage is more common in children than in adults, and many people change their pattern of carriage between the age of 10 and 20 years. The reasons for these differences in the colonization patterns are unknown.3 Healthcare workers (HCWs) constitute an important reservoir of S. aureus. Several studies have reported that the rate of the nasal carriage of S. aureus among the HCWs ranges from 16.8% to 56.1%.4–7 Treatment of infection caused by S. aureus has become more problematic since the development of antimicrobial- resistant S. aureus (MRSA). As the MRSA strains are resistant to all β-lactam antibiotics, the treatment options are limited significantly. The incidence of nosocomial infection caused by MRSA continues to increase worldwide. Infections caused by MRSA strains are associated with longer hospital stay, prolonged antibiotic administration and higher costs than infections caused by methicillin-susceptible S. aureus (MSSA) strains.8 By definition, MRSA strains harbours the mecA gene, which encodes the low-affinity penicillin-binding protein (PBP) designated as PBP2a.9,10 The Clinical and Laboratory Standards Institute (CLSI) states that the oxacillin and cefoxitin disk tests are equivalent in sensitivity and specificity for the detection of mecA- mediated resistance in S. aureus.11 The screening of the nasal carriage in HCWs is an important component in the control of MRSA in any healthcare facility. The identification of the colonized staff members allows an appropriate management of these persons to prevent the spread of organism within hospitals or in communities. Because medical students belong to the HCW in future, the aim of our study is to screen S. aureus nasal carriage of these individuals, and to identify the prevalence of oxacillin and cefoxitin resistance among the isolated S. aureus. Materials and Methods Specimen Collections Anterior nasal swabs were taken from 100 healthy students from third and fourth stage whom they had internship programme in hospitals during summer from October 2014 to March 2015. Sterile swab was moistened with sterile normal saline and was rotated at least 5 times in both nares, then placed in the transport media, using standard methods.12–14 Specimens Processing All specimens were directly inoculated from transport media into plates of Mannitol salt agar (MSA) and blood agar and incubated at 37°C for 24 h (Fig. 1). All colonies from primary cultures were subcultured onto MSA and incubated at 37°C for 24 h.15 S. aureus were identified depending on the morphological features on culture media (beta-hemolytic on blood agar and mannitol fermentation on MSA) and biochemical tests (catalase positive test and coagulase positive Department of Clinical Laboratories, College of Applied Medical Sciences, Karbala University, Karbala, Iraq. Correspondence to Suhad Hadi Mohammed (email: shm.med.school@gmail.com). (Submitted: 03 January 2015 – Revised version received: 26 February 2015 – Accepted: 01 March 2015 – Published online: Winter 2015) ISSN 2413-0516 14 J Contemp Med Sci | Vol. 1, No. 1, Winter 2015:13–16 Detection of oxacillin and cefoxitin resistance in Staphylococcus aureus Research Suhad Hadi Mohammed et al. were resistant to cefoxitin and 12 (60%) were resistant to oxacillin. There is positive significant correlation between the two drugs (Table 2). Distribution of the Resistant Strains Among Sex and Age The prevalence of S. aureus nasal car- riage was higher among the older age group individuals (70%) than the younger age group (30%). Concerning the resistance profile, there were 62.5% (5/8) of cefoxitin-resistant isolates, and 66.6% (8/12) of oxacillin-resistant iso- lates found among the older than the younger volunteers (Table 3). The cur- rent study revealed that 62.5% (5/ 8) of cefoxitin-resistant isolates and 50% (6/12) of oxacillin-resistant isolates were found in females. Discussion The primary reservoir of S. aureus in humans is the anterior nares. Nasal carriage is recognized as a major risk factor for the development of both community-acquired and nosocomial infections.17,18 This appears to play a key role in the epidemiology and pathogenesis of infection.17,18 The factors that distinguish between a carrier and a non-carrier are still unknown. Enhanced adhesion of S. aureus, to cell associated and cell-free secretions, along with the induction of reduced mucociliary activity, could well explain the nasal colonization by S. aureus. It is imperative that nasal carriage due to S. aureus strains should be prevented in order to stem the rate of infection, and in preventing the transmission of resistant strains of the organism.8 Although nasal carriage of S. aureus is harmless in healthy individuals, they can become carriers who could pose the risk of spreading infections to the community at large, and since the section of individuals under this study were medical students, their interaction and exposure to hospital environment could cause major risks in transmitting to patients Fig. 1 A: Blood agar plate; B: beta hemolytic isolates; C: Mannitol salt agar; D: Mannitol salt agar plate with S. aureus isolates; E: catalase test; F: coagulase test. A D B E C F Fig. 2 A: Mueller-Hinton agar without antibiotic disc with bacterial colonies (control); B: Mueller-Hinton agar with antibiotic disc. A B Table 1. Culture results of nasal swabs Culture results Isolates N (%) Culture positive S. aureus 20 (%) Coagulase negative Staphylococci 76 (%) Culture positive Micrococcus 3 Other bacteria Bacillus 1 Table 2. Screening for MRSA Antibiotic Resistant N (%) Sensitive N (%) Total N (%) Oxacillin 12 (60) 8 (40) 20 (100) Cefoxitin 8 (40) 12 (60) 20 (100) Correlation r = 0.667 P value = 0.001* MRSA: methicillin-resistant S. aureus. *Correlation significant at 0.01 level. test) also gram staining showed gram- positive grape-like clusters (Fig. 1). Antibiotic Susceptibility Test All S. aureus isolates were tested for cefoxitin and oxacillin susceptibility by Kirby-Bauer method on Mueller-Hinton agar (MHA) (Hi-media). Plates were incubated at 37°C for 24 h. Following the incubation, the inhibition zone diameter was measured. Identification of MRSAs were done by following CLSI.16 Isolates were considered suscep- tible to oxacillin and cefoxitin if the zone of inhibition around the disks was ≥22 mm, and resistant if the zone was ≤21 mm (Fig. 2). Statistical Analysis IBS, SPSS version 20 was used in the analysis of the present data. Results A total of 100 nasal swab samples were collected and screened during this study. From these, a total of 20 (20%) were identified as coagulase positive S. aureus isolates and 80 (80%) were identified as coagulase negative Staphylococci isolates (Table 1). The coagulase positive isolates were then tested to demonstrate their resistances to methicillin by using oxacillin and cefoxitin discs, 8 (40%) 15J Contemp Med Sci | Vol. 1, No. 1, Winter 2015:13–16 Research Detection of oxacillin and cefoxitin resistance in Staphylococcus aureusSuhad Hadi Mohammed et al. and spreading nosocomial infections. Therefore, it is necessary to detect S. aureus carriage in medical students. The current study revealed that out of the 100 samples collected from medical students, 96 were identified as Staphylococcus while 4 specimens showed no growth of Staphylococcal colonies, instead they showed growth of Micrococcus sp. and Bacillus. Twenty samples out of 96 (20.8%) were coagulase positive S. aureus isolates and 76 (79.1%) were coagulase negative Staphylococci. The prevalence of S. aureus carriage has been reported in healthy populations in several countries; 43.2% of S. aureus in nasal cavity of adults in Iraq, 17.3% in nasal cavity of Turkish children, 36% in nares of Japanese adults and 32.4% in nasal cavity of adults in the United States.19–22 Pant and Rai’s (2007)23 findings revealed higher S. aureus nasal colonization rate (43.8%) in staffs of teaching hospital in Nepal. Also, in Abia state of Nigeria, Chigbu and Ezeronye (2003)24 reported 50% nasal colonization in both hospital and nonhospital subjects. Chatterjee et al. (2009)25 showed that the overall prevalence of S. aureus nasal colonization was 52.3%. Whereas Onanuga and Temedie (2011)26 showed that 33.3% S. aureus isolates were obtained from 120 nares specimens screened. Whilst, Adesida et al. (2007)27 reported a much lower (14.0%) nasal colonization in medical students in Lagos, Nigeria. These variations may be attributed to the characteristics of the population under study. A population that is on antibiotics at the time of sampling may yield a much lower prevalence of S. aureus while a population from hospital settings may yield a much higher prevalence because of the high prevalence of infectious patients in that environment. Other factors that can cause variations may be sampling and culture techniques. Table 3. Distribution of S. aureus isolates among age group S. aureus N (%) Oxacillin sensitivity Cefoxitin sensitivity Sensitive Resistance Sensitive Resistance Age groups Younger 6 (30) 2 4 3 3 Older 14 (70) 6 8 9 5 Sex Male 10 (50) 4 6 7 3 Female 10 (50) 4 6 5 5 Total 20 (100) 8 12 12 8 Concerning the detection of MRSA, all the 20 S. aureus isolates were tested for cefoxitin and oxacillin resistance using a disk diffusion method and 60% and 40% were found to be susceptible to oxacillin and cefoxitin, respectively. Higher incidence of MRSA was found in another study in Iraq/Baghdad among HCWs and patients in hospital.28 In another study, resistant percentage was found to be of 90.9%.29 Fey et al. (2003)30 stated that the resistance to methicillin was 81%, while Jain et al. (2008)31 observed about 75.26% of isolates were methicillin-resistant. These observed differences may due to the variation in the geographic area, sources of clinical specimens, genetic background and the collection site of the isolates.31 Conclusion The data obtained from this study revealed that there were reservoirs or carriers of MRSA in medical students. Screening for resistant strains of Staphylococci in medical students should be adopted as a protocol in medical colleges, in order to curb the spread of drug-resistant Staphylococci from the hospital to the community. This will also help in monitoring the HCWs population who might pose a risk to patients and hospital personnel and the community, at large.  References 1. Rongpharpi SR, Hazarika NK, Kalita H. The prevalence of nasal carriage of Staphylococcus aureus among healthcare workers at a tertiary care hospital in Assam with special reference to MRSA. J Clin Diagn Res. 2013 Feb;7(2):257–60. doi: http://dx.doi.org/10.7860/JCDR/2013/4320.2741 PMID: 23543837 2. van den Akker EL, Nouwen JL, Melles DC, van Rossum EF, Koper JW, Uitterlinden AG, et al. Staphylococcus aureus nasal carriage is associated with glucocorticoid receptor gene polymorphisms. J Infect Dis. 2006 Sept 15; 194(6):814–8. doi: http://dx.doi.org/10.1086/506367 PMID: 16941349 3. Kluytmans J, van Belkum A, Verbrugh H. Nasal carriage of Staphylococcus aureus: epidemiology, underlying mechanisms and associated risks. Clin Microbiol Rev. 1997 July;10(3):505–20. PMID: 9227864 4. Dan M, Moses Y, Poch F, Asherov J, Gutman R. Carriage of methicillin- resistant Staphylococcus aureus by non-hospitalized subjects in Israel. Infection. 1992 Nov;20(6):332–35. doi: http://dx.doi.org/10.1007/ bf01710678 PMID: 1293052 5. Duncan IB, Collins AM, Neelin EM, Roy TE. Nasal carriage of Staphylococcus pyogenes by student nurses. Can Med Assoc J. 1957 Dec 1;77(11):1001–9. PMID: 13489588 6. McAnally TP, Lewis MR, Brown DR. Effect of rifampin and bacitracin on nasal carriers of Staphylococcus aureus. Antimicrob Agents Chemother. 1984 April; 25(4):22–6. PMID: 6732212 7. Paul MO, Lamikanra A, Aderibigbe DA. Nasal carriers of coagulase-positive staphylococci in a Nigerian hospital community. Trans R Soc Trop Med Hyg. 1982 Jan;76(3):319–23. doi: http://dx.doi.org/10.1016/0035-9203(82)90180- 8 PMID: 7112654 8. Kumar P, Shukla I, Varshney S. Nasal screening of healthcare workers for nasal carriage of coagulase positive MRSA and prevalence of nasal colonization with Staphylococcus aureus. Biology and Medicine. 2011;3(2) Special Issue: 182–6. 9. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. Eighteenth informational supplement. CLSI document M100-S22. Wayne, PA: Clinical and Laboratory Standards Institute; 2012. 10. Chambers HF, Sachdeva M. Binding of beta-lactam antibiotics to penicillin- binding proteins in methicillin-resistant Staphylococcus aureus. J Infect Dis. 1990 Jun;161(6):1170–6. 11. Clinical and Laboratory Standards Institute: Performance Standards for Antimicrobial Susceptibility Testing. Fifteenth Informational Supplement January 2005. Approved Standard, M100-S15, Vol. 25, No. 1. Wayne, PA. 12. Kloos WE, Bannerman TL. Staphylococcus and Micrococcus. In: Murry PR, Baron EJ, Pfaller M, Tenover AFC, Yolken RK, editors. Manual of clinical microbiology, 7th ed. Washington, DC: American Society for Microbiology; 1999. pp. 264–82. 13. Monsen T, Rönnmark M, Olofsson C, Wiström, J. An inexpensive and reliable method for routine identification of staphylococcal species. Eur J Clin Microbiol Infect Dis. 1998 May;17(5):327–35. doi: http://dx.doi.org/10.1007/bf01709455 PMID: 9721961 14. Jain A, Agarwal A, Verma RK. Cefoxitin disc diffusion test for detection of meticillin-resistant staphylococci. J Med Microbiol. 2008 Aug;57 (Pt 8):957–61. doi: http://dx.doi.org/10.1099/jmm.0.47152-0 PMID: 18628495 15. Talan DA, Staatz D, Staatz A, Overturf GD. Frequency of Staphylococcus intermedius as human nasopharyngeal flora. J Clin Microbiol. 1989 Oct; 27(10):2393 PMID: 2584388 16 J Contemp Med Sci | Vol. 1, No. 1, Winter 2015:13–16 Detection of oxacillin and cefoxitin resistance in Staphylococcus aureus Research Suhad Hadi Mohammed et al. 16. Clinical Laboratory Standards Institute. Performance standard for antimicrobial susceptibility testing: seventeenth informational supplement M100-S17. Clinical Laboratory Standards Institute, Wayne, PA, USA; 2007. 17. Boelaert JR, Van Landuyt HW, De Baere YA, Deruyter MM, Daneels RF, Schurgers ML, et al. Staphylococcus aureus infections in hemodialysis patients: pathophysiology and use of nasal mupirocin for prevention. J Chemother. 1995 Jul;7 Suppl 3:49–53. PMID: 8609538 18. Koziol-Montewka M, Chudnicka A, Ksiazek A, Majdan M. Rate of Staphylococcus aureus nasal carriage in immunocompromised patients receiving hemodialysis treatment. Int J Antimicrob Agents. 2001 Aug;18(2):193–6. doi: http://dx.doi. org/10.1016/s0924-8579(01)00350-8 PMID: 11516945 19. Moellering RC Jr. Problems with antimicrobial resistance in gram-positive cocci. Clin Infect Dis. 1998 May;26(5):1177–8. PMID: 9597248 20. Uemura E, Kakinohana S, Higa N, Toma C, Nakasone N. Comparative characterization of Staphylococcus aureus isolates from throats and nose of healthy volunteers. Jpn J Infect Dis. 2004 Feb;57(1):21–4. PMID: 14985632 21. Soysal A, Sahin H, Yagci A, Barlan I, Bakir M. The low rate of methicillin- resistant Staphylococcus aureus in Turkish children. Jpn J Infect Dis. 2006 Jun;59(3):195–6. PMID: 16785704 22. Onanuga A, Onaolapo J.A. Antimicrobial susceptibility of community associated Staphylococcus aureus isolates from healthy women in Zaria, Nigeria. Trop J Pharmaceutical Res. 2008 Apr 3;7(1):929–39. doi: http:// dx.doi.org/10.4314/tjpr.v7i1.14679 23. Pant J, Rai SK. Occurrence of Staphyloccous aureus in hospital environment and staffs in teaching hospital in Katmandu, Nepal. J Nepal Assoc. Medi Lab Sci. 2007;8:72–3. 24. Chigbu CO, Ezeronye OU. Antibiotic resistant Staphylococcus aureus in Abia state of Nigeria. Afr J Biotech. 2003 Oct 31;2(10):374–8. doi: http://dx.doi. org/10.5897/ajb2003.000-1077 25. Chatterjee SS, Ray P, Aggarwal A, Das A, Sharma M. A community-based study on nasal carriage of Staphylococcus aureus. Indian J Med Res. 2009 Dec;130(6):742–8. PMID: 20090137 26. Onanuga A, Temedie TC. Nasal carriage of multi-drug resistant Staphylococcus aureus in healthy inhabitants of Amassoma in Niger delta region of Nigeria. Afr Health Sci. 2011 Jun;11(2):176–81. PMID: 21857847 27. Adesida SA, Abioye OA, Bamiro BS, Brai BIC, Smith SI, Amisu KO, et al. Associated risk factors and pulsed field gel electrophoresis of nasal isolates of Staphylococcus aureus from medical students in a tertiary hospital in Lagos, Nigeria. Braz J Infect Dis. 2007 Feb;11(1):63–9. doi: http://dx.doi. org/10.1590/s1413-86702007000100016 PMID: 17625730 28. Ali M. Al-Dahbi, Harith J. Al-Mathkhury. Distribution of methicillin resistant Staphylococcus aureus in Iraqi patients and healthcare workers. Iraqi J Sci. 2013;54(2):293–300. 29. Al-Geobory HA. Comparative study between methicillin resistant Staphylococcus aureus (MRSA) and methicillin sensitive Staphylococcus aureus (MSSA), and detect the antimicrobial effects of some plant extracts on them [MSc thesis]. Baghdad, Iraq: College of Science, Baghdad University; 2011. 30. Fey PD, Saïd-Salim B, Rupp ME, Hinrichs SH, Boxrud DJ, Davis CC, et al. Comparative molecular analysis of community or hospital-acquired methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother. 2003 Jan;47(1):196–203. 31. Jain A, Agarwal A, Verma RK. Cefoxitin disc diffusion test for detection of meticillin-resistant staphylococci. J Med Microbiol. 2008 Aug;57(pt 8): 957–61. doi: http://dx.doi.org/10.1099/jmm.0.47152-0 PMID: 18628495