Iraqi J Pharm Sci, Vol.23(1) 2014 Nasal carriage of MRSA 83 Determine Nasal Carriage of Methicillin Resistant Staphylococcus aureus MRSA in Young Adult College Student Ebtihal N. Saeed *,1 ,Hanan I.Omer Al-Deen * , Maysoon A. Merdaw * * Department of Clinical Laboratory Sciences , College of Pharmacy , University of Baghdad , Baghdad, Iraq Abstract Present study was carried out to find prevalence of MRSA in healthy individual of second stage students, college of pharmacy/Baghdad University. A total of 74 student selected between age 18-23 years old were included in this study, nasal swabs collected and subjected to many diagnostic standard bacteriological identification methods. Culture, colonial morphology, Gram stain, mannitol fermentation, coagulase ,gelatinasetest, DNAase, MR/VP and antimicrobial susceptibility test was performed on tryptic soy agar by modified Kirby-Bauer muller hinton disc diffusion method and the result show that out of 74 nasal swabs,67(90.5%) were MRSA positive isolates, 21(31.4%) of them were mannitol ferment and 46(68.6%) non mannitol fermenter, among these isolates 33(44.6%)male and 41(55.4%) female, there was no significant sex difference in the prevalence of Staph. aureus, while show decrease in prevalence with age group,(54%),(27%),(9.5%) alternatively, MRSA positive isolates indicated relatively high rate of resistance to antibiotics, so to vancomycin 3(4.5%),ciprofloxacin 4(6%) , tetracyclin 9(13.5%) ,gentamycin 6(9%), erythromycin 15(22.5%) and keflex 20(30%). This study show a high prevalence of MRSA carriage in young adult college student (healthy people) that indicating the spread of MRSA in the community which consider high risk of spreading infections also we isolate non mannitol fermenter (MRSA) Staph. aureus that need further moleculer analysis to prove it. Keywords: Staphylococcus aureus , MRSA, Modified Kirby-Baur methods. ليه في سيت المقاومت للمضاد الحيىي مثفتحديد الحامليه لبكتريا المكىراث العىقىديت االو البالغيه االصحاء مه طالب الجامعت وىري سعيد ابتهال ،*1 عمر الديه ،حىان ابراهيم * مرداو ، ميسىن عبد الزهرة * * العلْم الوختبشٗة الغشٗشٗة ، كل٘ة الص٘ذلة ، جبهعة بغذاد . فشع الخالصة (MRSA)اجشٗت ُزٍ الذساعة لوعشفة هعذل اًتشابس بتتشٗاب الوتاْسال العٌيْدٗاة الويبّهاة للولابد الل٘إْ الو غال٘ي ّ الوغاوب 47فٖ الوجتوع ب٘ي الٌبط االصلبء ّ رلك ببخذ عٌ٘بل عشْائ٘ة لطالة الوشحلة الذساع٘ة ال بًَ٘ فاٖ كل٘اة الصا٘ذلَ / جبهعاة بغاذاد بعاذد ( ّ قااذ اان عااضل البتتشٗااب ّ شخ٘صااِب باابلطشا البتت٘شٗااة الو بل٘ااة هااي دساعااة الوااضسّع البتت٘ااشٕ ّ اات 32-81هغاالة باا٘ي االعواابس %( لْٕ علٔ بتتشٗاب الوتاْسال 9..5 74عضلة , 47التشام ّ الفعبل٘بل الببْٗك٘و٘بّٗة , ّقذ ّ جذ اًَ هي ب٘ي الوغتعوشال ّ صبغة %( ُاٖ ي٘اش هخوااش 71.7 77هٌِاب ُاٖ هخواش لغاتش الوابًتْل بٌ٘واب %(28.7 38( ّببًاَ (MRSAالعٌيْدٗاة الويبّهاة للو غال٘ي ( عاٌة لإْ اعلأ ًغابة هاي 85-81يبسًة بوجتوعبل اخشٓ , كوب ّجذ ببى االعوبس ب٘ي لغتش الوبًتْل ّ عتبش ُزٍ الٌغبة عبل٘ة ببلو %( هواب 5.9عاٌة بٌغابة 38%( ّهي ثن االعوبس التٖ ُٖ ضوي الفئة العوشٗة االكبش هاي 34( عٌة 38-.3%( ّ ب٘ي 97العضالل جٌظ ال ٗؤثش فاٖ الٌغابة ح٘اي الْٗجاذ فاشا با٘ي الازكْس ّ االًاب الاى ٗذل علٔ ٌبقص هعذل التْاجذ للبتتشٗب هع التيذم ببلعوش كوب ب٘ي %( , 7.9كوااااب ا ِااااشل الذساعااااة اى البتتشٗااااب الوعضّلااااة اِااااش بعاااار الويبّهااااة لولاااابدال ح٘ب ٘ااااة اخااااشٓ ه اااا الفبًتْهبٗغاااا٘ي %( . .2التفلتااظ %( ّ اخ٘ااشا 33.9%( ّاالسثشّهبٗغاا٘ي 82.9%( , التتشاعاابٗتل٘ي 5%( الجٌتبهبٗغاا٘ي 7البغبشّفلْكغبعاا٘ي باااا٘ي الذساعااااة خطااااْس اصدٗاااابد ًغاااابة اًتشاااابس البتتشٗااااب فااااٖ الوجتوااااع ّ التااااٖ بااااذّسُب ضٗااااذ هااااي ًغاااابة االصاااابببل الوتتغاااابة فااااٖ .ّ كوااب باا٘ي الذساعااة اًتشاابس ًااْع هااي ُاازٍ البتتشٗااب ي٘ااش الوخوااش لغااتش الواابًتْل Hospital acquired infectionالوغتشااف٘بل .Molecular analysisالتٖ عتبش عتش جذٗذ ّ لتبج بذّسُب الٔ دساعة جضٗئ٘ة لت ب٘تِب ّالويبّهة للو غل٘ي ّ . عدلتي بىر المب، طريقت كير المقاومت للمضاذ الحيىي مثسليه المكىراث العىقىديتالكلماث المفتاحيت: Introduction Staphylococcus aureus is a major pathogen responsible for nosocomial and community acquired infections (1, 2) . Methicillin resistant Staph. aureus (MRSA) has emerged as a nosocomial pathogen of a major worldwide importance and is an increasingly frequent cause significant morbidity and mortality (3) , colonized person can serve as a reservoir for the nosocomial spread of (MRSA) . Active surveillance and timely identification of (MRSA) colonization of patients is an important infection control activity that help to prevent nosocomial spread and is cost effective (4,5) . Acquired infection with Staph. aureus have until very recently Corresponding author e. mail: ibtihalnoori@gmail.com Received:6/10/2013 Accepted:4/5/2014 Iraqi J Pharm Sci, Vol.23(1) 2014 Nasal carriage of MRSA 84 been reliably treated with B-Lactam antibiotics, B-lactam antibiotics normally bind to penicillin binding proteins (PBPS) in the cell wall, resulting in the disruption of synthesis of the peptidoglycan layer and death of bacterium. Since a lactam cannot bind to low affinity PBPS; synthesis of the peptidoglycan layer and cell wall synthesis are able to continue cited by (2) MRSA infection often require systematic antibiotic therapy and are an important health care burden since they increase treatment costs and patient morbidity ,MRSA carriage in many communities is largely un- known and it varies in different geographical regions ,so to control the spread of disease continuing study is needed to asses geographical distribution and epidemiology of infection and develop strategies to that ( 2 ,6) . The spread of MRSA can also be potentially minimized by prevention of risk factors such as previous antibiotic use, day care attendance , contact with a health care workers or nursing home resident, residence in a long - term care facility, diabetes mellitus ,hospitalization, admission to intensive care unit, intravenous drug use, invasive indwelling devices, hemodialysis or peritoneal dialysis, naso gastric tube, gastrostomy ,and external feeding ,mechanical ventilation, endotracheal tube, tracheostomy tube ,surgical procedures ,immune suppression ,chronic illness (2,7) . The anterior nares are the primary reservoir of Staph. aureus in both adult and children (8) Nasal carriage of Staph . aureus is important because most of Staph .aureus infection occur in person who are colonized with this organisms, this Staph .aureus carriage has been assumed to be one of the risk factors for subsequent infection (8) ,there for recognition of persons colonized orinfected with Staph .aureus is recommended for preventing the spread of the organisms within hospitalsor communities (4,8) . Staphylococcus aureus —methicillin resistant mannitol fermenter negative strain were first isolated and reported as subtype of "antario epidemic" strain (MRSA-1) known In Canada lab.2003 by using un- enriched media such as Brain Heart Infusion (BHI) and incubate for 24-48 hours then sub cultured on blood agar, improve isolation and detection of these un usual strain which first reported by antario researchers need further molecular analysis (9, 10) . So the aim of this study is to estimate the frequency of MRSA Staph .aureus in community and control the spread of disease which need continuing study to asses geographical distribution and epidemiology of infection and develop strategies to that (3) . Materials and Methods Sample collection: nasal swabs of 74 student within age limit 21-23 years were collected for purpose of the study during a period from (October--Feb.2010), the specimens were collected with sterile cotton swabs available commercially The swabs was introduced 2-3 centimeter in the nasal cavity and rotated 4-5 times both clock wise and anti- clock wise before with draw. Each sample was labeled with code number and various other information including age, sex, location, etc. were recorded. The sample was transported to laboratory of microbiology in sidethe college of pharmacy and immediately inoculate on the special media (BHI, MSA, Blood Agar Media) for diagnosis (2, 11, 12). Culture and Sensitivity To be sure that none of the Staph. aureus were lost, Nasal swabs were inoculated into brain heart infusion broth, non selective media containing 0.5% salt,75 mg/Loztreonam, 5mg/L ceftizoxime (9,10) overnight incubation for 24hrs,37C°, then subcultured on blood agar and mannitol salt agar to indicate the type of hemolysin and fermentation of mannitol. Both hemolytic mannitol fermenter yellowish colonies and non hemolytic, non mannitol fermentor pink colonies, are subsequently identified by the gram stain, catalase test, slide and tube coagulase test, gelatinase test, MR/VP test (1,2,12,13) . Also, the colonies subcultured on tryptic soy agar (TSA) media for further examination. Antibiotic Susceptibility test: All the identified Staph. aureus isolates from nasal swabs subjected to in vitro susceptibility test Modified Kirby-Bauer disc diffusion methods (2,14) antibiotics used in the study were Methicillin (10mcg), Tetracycline (30 mcg), Ciprofloxacin (30mcg), Erythromycin (15mcg), Vancomycin (30mcg), Gentamycin (10mcg) and Keflex (30 mcg), all the disc were obtained from (oxoid) . Quality control for the test: in this study the accuracy of the overall testingprocedure was monitored by using Stap. aureus ATCC 25923 as reference strain. Statistical analysis The computer programmer SSPS (Statistical Package for Social Sciences) version 17.0 was employed to manipulate the statistical analysis of present study results. The results were presented as percentage frequencies. Significant differences were assessed by Student T-Test in which P<0.05 was considered significant. Result Table 1 and 2 show that among 74 student under study, 33(44.5%) male and 41 (55.5%) Iraqi J Pharm Sci, Vol.23(1) 2014 Nasal carriage of MRSA 85 female, Staph. aureus could be isolated from 67 (90.5%) nasal swabs sample, among these isolates 29(43.3%) from male, while 38 (56.7%)from female. There was no significant sex difference in colonization of Staph .aureus between male and female, while the study showed that highest colonization of Staph .aureus in age group 18-19 years old (54%) follow by 20-21, age group (27%) and over 21 years old (9%), and only 7 which equalto (9.5%) show negative result means no Staph. aureus isolates, 3 (4.1%) 0f them male and 4 (5.4%) female . Table( 1) Number of Nasal swabs in correlation with age group and sex Age group yrs. Number of swabs No % Male No % Female No % 18-19 43(58) 22(29.7) 21(28.3) 20-21 24(32) 6(8.1) 18(24.3) 22> 7(10) 5(6.8) 2 (2.7) Total No. 74(100) 33(44.7) 41(55.3) Table (2) Frequency of Staphylococcus aureus(MRSA) isolates , Nasal carriage by age and sex group Age group yrs. Number of MRSAisolated No % Male No % Female No % 18-19 40(54.0) 20(27.0) 20(27.0) 20-21 20(27.0) 5(6.75) 15(20.2) 22> 7(9.5) 5(6.75) 2(2.70) Total No. 67(90.5) 30(40.5) 37(50.0) Total no. of sample 74 , no. of negative isolates 7(9.54) , male 3(4.1%) and female 4(5.4%) Table 3-show the identification methods for Staph. aureus, and we can see all the mannitol fermenter Staph. aureus isolates show strong slide and tube coagulase test DNAase test, gelatinase test, MR/VP test as mention in reference lab. while mannitol fermenter negative 46 (68.6%) give weakly positive tube coagulase test andmostly negative for other test, which means that n.m.f. Staph aureus are different in some properties (new strain reported in 2003) (10) . Table 4- show the sensitivity pattern according to number of Staph aureus isolates, 67 (90.5%)were methicillin resistant MRSA which divided in to21(31%) mannitol fermenter and 46 (68.6%) non mannitol fermenter, indicated relatively some resistant to other antibiotics such as for Keflex (kf) 30%, Erylhromycin (E) 22.5%,Tetracyclin (Te) 13.5%,Vancomycine (V) 4.5% and for Ciprofloxacin (Cp) 6%. Table (3) Identification methods used to diagnose Staphylococcus aureus Test used Type and no. result Tube coagulase 21 (+) 46(w+) * Slide coagulase 67 (+) - Mannitolferment 21 (+) 46(-) Gelatinase 21 (+) 46(-) DNA ase 21 (+) 46(-) Catalase 67 (+) - MR/VP 67 +/- *W=weakly positive non mannitol ferment 46 Table( 4 ) Sensitivity pattern according to no. of isolated Staphylococcus aureus (MRSA) Antibiotics used Mannitolfermention total No. 21(31.4%) Non Mannitolfermention 46(68.6%) Sensitivity No % Resistance No. % Sensitivity No % Resistance No. % Vancomycin 19 28.4 2 2.9 45 67.2 1 1.49 Tetracyclin 19 28.4 2 2.9 39 58.2 7 10.4 Erythromycin 16 23.9 5 7.5 36 53.7 10 14.9 Keflex 14 20.9 7 10.4 33 49.2 13 19.4 Gentanycin 20 29.9 1 1.5 41 61.2 5 7.5 Cifrofloxacin 20 29.9 1 1.5 43 64.2 3 4.4 Iraqi J Pharm Sci, Vol.23(1) 2014 Nasal carriage of MRSA 86 Discussion Study on Staphylococcus aureus nasal carriage rates in various populations have been investigated in the developed countries with temperate climate (10) , but nosuch study among healthy population had been reported, in India so far researchers reported that nasal carriage of Staph. aureus varied in different communities, (10) manyinvestigators have reported an increasing incidence of CA-MRSA infections in community settings (8,15) such increase without the usual risk factors related to MRSA infection or colonization have given the surveillance for Staph. aureus greater significance. So due to association between the carriages of Staph.aureus and subsequent infection (8) evolution of colonization prevalence may be useful for the estimation of potential Staph.aureus causing disease (8) . The result of present study showed that nasal carriage of Staphylococci was as high as (90.5%) among 74 nasal swabs which are under study table (1, 2), this result is slightly higher than what reported in previous studies on college student in Brazil (40.8%) (l6) ,and in more recent studies in India reported that the rate of approximately (88.2 %)found to be positive Staphylococcus and (52.2%) of them MRSA were isolated (15) , other found different ratio such as in two studies with pre-clinical medical student showed that (35. 2%) and (29%) were Staph. aureus nasal carriers (11,12) . The prevalence of MRSA in the apparently healthy community of Eask Sikkim was estimated to be (11.1%)a total of 129 (46.1%) among 280 healthy individual screened were nasal carriers of Staph. aureus ,similar findings were reported by Anwar et al, cited by Majuandar D et al (23) . In their study in Lahore, Pakistan who screened 1024 and 636 apparently healthy persons from urban and rural area respectively for nasal carriage of Staph. aureus and MRSA and reported thaturban area prevalence of nasal carriers of Staph. aureus was estimated to be(16.99%) but rural areas was (11.32%) and the prevalence of MRSA in urban areas was found to be(22.98%) against ( 11.11%) in rural areas (8) . In a study by Lamikanra and colleagues (15) it was observed that (56.4%) of healthy Nigerian students were nasal carriers of Staph. aureus, Tanakaetal. While studying Staph. aureus in healthy individuals in Japan reported (24.3%) of them to be nasal carriers (11) , another study conducted at university of Taxas, reported that 99 (58%) of 170 isolates are Staphylococcus aureus (22) . Also we found that there was no statistically significant difference in the prevalence of Staph. aureus between male and female subjected in the present study, this finding was contrary to that observed in the study done in Nigreian population where females harbored Staph. aureus more often than males (15) , but most studies agree with our result that no significant sex differences (15, 19, 20, 21) and all of them agree with that increasing of colonization on healthy population in older adult between 18-40 years old which affected by many factors some of them depend on sampling, quality of culture media, using enriched media, population under study, geographical area,civilization of the country little knowledge about factors that make one person to be chronic carriers or transient carriers (2) , so it need further investigation and epidemiological studies including genotyping to understand the dynamics of spread of MRSA in the community in more details. Table (3) shows that from the 67 (90.5%) MRSA isolates 21, (31.4%) were mannitol fermenter and 45 (68.6%) were non mannitol fermenter Staph. aureus, and this strain was first reported as subtype of epidemic strain C MRSA-1 in Canada research lab. (10) , this strain of n.m.f. Staph. aureus which is community strain found to be different in there biochemical activity from m.f. MRSA Staph. aureus such as weakly positive tube coagulase test and gelatinase negativeand MR/VP variable this strain need further molecular analysis because it show high prevalence in community so further study is need. In table (4) the sensitivity pattern of MRSA isolates 67 (90.5%) to antibiotics show rate of resistance toward some antibiotics, Keflex(30%), Erythromycin(22.5%), Tetracycline (13.5%), Gentamycin (9%), Ciprofloxacin (6%) and Vancomycin (4.5%). PantandRai (17) reported rate of resistant for Staph. aureus, some antibiotics, Ampicillin (38.1), Erythromycin (33.3%1, Cloxacilin ( 14.3%), Gentamycin (9.5%)and Methicillin (9.5%) respectively. In comparison to these result we can see that our value nearly equal to some values, while other study like in Nepal medical study show high rate of resistant for MRSA isolates It show resistance toward cloxacilin (68.8%) , follow by ofloxacin (40.7%), Tetracycline (15.6%), Erythromycin (9.4%),ciprofloxacin (6.3%) , and Vancomycin (3.1%). The different in antibiotic resistant pattern may be due to different in '' community strain, personal factors such as antibiotic therapy. Iraqi J Pharm Sci, Vol.23(1) 2014 Nasal carriage of MRSA 87 References 1. Skov R, Smyth R, Larsen AR, Bolmostrom A, Karlsson A. Phenotypic detection of methicillin resistance in Staphylococcus aureus by disk diffusion testing and E test on Muller-Hinton agar. J. Clin. Microbiol. 2006; 44: 4395- 9 . 2. Rijal KR, Phari N, Shrestha BK, Nepal AK, Paudel B, and Skalko N, prevalence of methicillin resistant Staphylococcus aureus in school children of Pokara-Nepal. Med.coll. J. 2003; 10(3): 192-195. 3. Rubin RJ, Harrington CA, Poon A, Dietrich k, Green JA, and Moiduddin A. (1999).The economic impact of Staphylococcus aureus infection in New York City hospital . Emerg. lnfect. Dis. 5, 9-17. 4. Muto CA, Jernigan JA, Ostrowsky BE, Richet HM, Jarvis WR, Boyce JM, and Farr BM. (2003). SHEA Guideline for preventing nosocomial transmission. Epidemiol. 24, 362-386 .(Cross Ref.) . 5. Wernitz MH, Keck S, Swidsinki S, Schulz S, &Vei SK. (2005). Cost analysis of hospital wide selective screening program for methicillin-resistant Staphylococcus aureus (MRSA) carriers in the context of diagnosis related (DRG) payment Clin. Microbiol.infect.11,466-471 .(Cross Ref.) (Medline). 6. SuSuan V , Leonie C , Auton Y petal . carriage of methicillin resistant Staphylococcus aureusin a Queens land indigenous community Med. 1 Agust. 2006; 184:556-9 . 7. Cohen PR. Community -aquired Methicillin-Resistant Staphylococcus aureus skin infections; areview of epidemiology, clinical features management, and prevention. lntel.J.Dermatol.2007 ;46:1-11 . 8. Sooko K, Lee JY, Baek JY, Peck KR, Rhee jy, Kwon KT, Heo ST, Ahn KM and Song JH. Characterization of Staphylococcus aureus.Nasal carriage from children attending an outpatient clinic .in Seoul,Korea.Microbial . Drug .resistance volume14, Number 1,2008. 9. Martel c, Ramotark, Ouellet C, etal.2001 Mannitol-negative methicillin-resistant Staphylococcus aureus(mn MRSA):Are you missing them abstracts J3.69th.Annual Meeting of the Canadian Association of Clinical Microbiology and lnfectious Diseases. Victoria ,B . C. 10. CMPT M032-3 Nasal swab: MRSA positive-Un graded Educational Challenge (mannitol-negative strain of MRSA,subtype of ( MRSA-1) .Clinical Microbiology Proficiency Testing,August 2003. 11. Brown DFJ, Hawkey PM, Edwards Dl etal, Guidelines for the laboratory diagnosis and susceptibility testing of Methicillin Resistant Staphylococcus aureus(MRSA).J. Antimicrobial Chemotherapy 2005;56:1000-18. 12. Collee JG, Marmion BP, FraserAGetal. Mackie and McCartney practical Medical Microbiology, Ed.l4th.Churchill Livingstone, United Kigdom,1996 . 13. Forbes BA, Sahm DF ,Weissfeld AS. Bailey &Scotts Diagnostic Microbiology ,10th edition ,Mos by lnc.USA 1998. 14. World Health Organization Guidelines For Antimicrobial Susceptibility testing WHO Coilaborating Center For Surveillance of Antimicrobial Resistance.Egypt,1996. 15. LamiKanar A, Paul BD , Akinwole OB , Paul Mo . Nassal carriage of Staphylococcus aureus in a population of healthy Nigerian students .j. Med. Microbial .1995 19 ,211-216 . 16. Kaplan Sl, HultenKG ,Gonzalez BE etal. Three years surveillance of community- aquired Staphylococcus aureus infection in children. clin.lnfect.Dis. 2005;40:1785-91. 17. PanT j ,RaiSK Occurrence of Staphylococcus aureus in Hospital Environment and Staffs in Teaching Hospital in Katmandu, NepalAssos. Medi. Lab .Sci2007 ;8:7 2_7 3 . 18. Soysal A, Sahim ,Yagci A, Barlant I,Bakir M. the low rate of methicillin – resistant staphylococcus aureus in Turkish chidren .Japanse J.infect Dis.2006;59:195 – 6 . 19. 1r a 20-Uemura E, Kakinohana S, Higa N, Toma C, Nakason N, Comparative characterization of Staphylococcus aureus isolates from throat and nose of healthy volunteers. Japanse J .lnfect. Dis.2004;57:2t-24 . 20. Lo. WT. linWJ , Tseng MH etal . Nasal carriage of singl clone of community acquired methicillin- – resistant Staphylococcus aureus among kindergarten attendees northern Taiwan ,Bimed central Infect . Dis 2007 , 15-4 . Iraqi J Pharm Sci, Vol.23(1) 2014 Nasal carriage of MRSA 88 21. Al faro C ,Mascher- Denen M, Fergie J . etal prevalence of methicillin- resistant Staphylococcus aureus Nasal carriage in patients admitted to driscoll children’s hospital , pediatr infect . Dis. J.2006;25:459-61 . 22. Partes KA , Tories AM ; Gracia LB . etal Nasal carriage of methicillin- resistant Staphylococcus aureus in university student . Braz J. infect. Dis. Vol. 14 no. 3 Salvador may / June 2010. 23. Majuandar D. BARUA A, Paul B. Prevalence of nasal carriage of methicillin- resistant Staphylococcus aureus in healthy population of GanGtokEast SIKKIM JIMAS Octobers – December 2008.Vol.21,No.4 .