|Year : 2014 | Volume
| Issue : 1 | Page : 39-43
Nasal carriage of Methicillin-resistant Staphylococcus aureus among healthy population of Kashmir, India
BA Fomda1, MA Thokar1, A Khan1, JA Bhat1, D Zahoor1, G Bashir1, A Majid2, P Ray3
1 Department of Microbiology, Sher i Kashmir Institute of Medical Sciences, Soura, Srinagar, Kashmir, India
2 Department of Community Medicine, Sher i Kashmir Institute of Medical Sciences, Soura, Srinagar, Kashmir, India
3 Department of Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, Punjab, India
|Date of Submission||08-Oct-2013|
|Date of Acceptance||04-Sep-2013|
|Date of Web Publication||4-Jan-2014|
B A Fomda
Department of Microbiology, Sher i Kashmir Institute of Medical Sciences, Soura, Srinagar, Kashmir
Source of Support: Indian Council of Medical Research,, Conflict of Interest: None
Background: Nasal colonisation with community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) is being increasingly reported, especially in places where people are in close contact and where hygiene is compromised. The aim of this study was to find out prevalence of methicillin resistant S.aureus (MRSA) colonising anterior nares of healthy subjects. Materials and Methods: Nasal swabs of healthy subjects were collected aseptically and cultured using standard microbiological protocols. Antibiotic susceptibility was done by Kirby-Bauer disc diffusion method according to CLSI guidelines. Methicillin resistance was detected by cefoxitin disc diffusion method and confirmed by minimum inhibitory concentration (MIC) and amplification of mecA gene by PCR. Strain typing of MRSA strains was done by PFGE. Results: Out of 820 samples, S.aureus was isolated from 229 (27.92%) subjects. Of the 229 isolates, 15 were methicillin resistant. All S. aureus isolates were susceptible to vancomycin. Nasal carriage of MRSA was found to be 1.83% among healthy population. The isolates were found to be polyclonal by PFGE analysis. Conclusion: High prevalence of MRSA is a cause of concern and strategies to interrupt transmission should be implemented.
Keywords: Community, methicillin-resistant S. aureus, minimum inhibitory concentration, nasal carriage, pulsed field gel electrophoresis typing
|How to cite this article:|
Fomda B A, Thokar M A, Khan A, Bhat J A, Zahoor D, Bashir G, Majid A, Ray P. Nasal carriage of Methicillin-resistant Staphylococcus aureus among healthy population of Kashmir, India. Indian J Med Microbiol 2014;32:39-43
|How to cite this URL:|
Fomda B A, Thokar M A, Khan A, Bhat J A, Zahoor D, Bashir G, Majid A, Ray P. Nasal carriage of Methicillin-resistant Staphylococcus aureus among healthy population of Kashmir, India. Indian J Med Microbiol [serial online] 2014 [cited 2020 Jan 26];32:39-43. Available from: http://www.ijmm.org/text.asp?2014/32/1/39/124296
| ~ Introduction|| |
Staphylococcus aureus is one of the most prevalent and clinically significant pathogen, causing wide variety of infections ranging from mild skin and soft-tissue infections to serious life-threatening infections.  Multi-drug resistant strains of S. aureus have been reported with increasing frequency worldwide. Methicillin-resistant S. aureus (MRSA) infections account for 40-60% of all nosocomial S. aureus infections in many centres across the world. 
MRSA was an important cause of healthcare-associated infections since 1970s.  In 1990s, explosion in the number of MRSA infections were reported for populations without prior healthcare contact. This increase has been associated with the recognition of new MRSA strains, often called community- associated MRSA (CA-MRSA) strains. 
S. aureus is a member of commensal microflora and readily colonises the anterior nares. Nasal carriage of S. aureus acts as an endogenous reservoir for clinical infections in the colonised individuals or as a source of cross-colonisation for community spread.  The present study was aimed at assessing the prevalence and antimicrobial resistance profile of S. aureus, particularly MRSA, from anterior nares of healthy inhabitants of Kashmir Valley.
| ~ Materials and Methods|| |
Multistage sampling procedures were used for sample collection. Two districts were randomly selected and list of blocks from these two districts were framed out of which two blocks were randomly selected. Samples were collected from two villages of each block. From each village, 10% population was sampled; thus a total of 820 specimens were collected from healthy subjects in a defined area. Ethical clearance was obtained from institute's ethical clearance committee. Detailed history like age, sex, location, risk factors like antibiotic intake, previous hospitalisation was recorded on pretested proforma. Subjects having history of antibiotic intake and previous hospitalisation were excluded from the study. Sterile cotton tipped swab was moistened in a culture tube containing 2 ml of 0.1% buffered Tween-80. Swabs were wrung out within the tube, swirled inside the anterior-nares of subjects five times while applying an even pressure and rotating the swab without interruption, reintroduced into the culture tube and wrung out. The specimens were transported to the laboratory without delay for further processing. These specimens were inoculated on blood agar and mannitol salt agar and incubated overnight at 37°C. After the organism was identified as gram-positive cocci in bunches by gram staining, various biochemical tests such as catalase, coagulase (slide and tube), phosphatase and DNase tests were performed. 
Antibiotic susceptibility testing
The antibiotic susceptibility testing was performed by using the Kirby-Bauer disc diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines.  The following antibiotic discs cefoxitin-30 μg, oxacillin-1μg, erythromycin-15μg, tetracycline-30μg, gentamicin-10μg, vancomycin-30μg, linezolid-30μg, penicillin-30μg, cotrimoxazole (1.25/23.75 μg), ciprofloxacin-5μg and clindamycin-2μg procured from Hi-media Mumbai India were used.
Minimum inhibitory concentration determination
Minimum inhibitory concentration (MIC) of various antibiotics like tetracycline, vancomycin, gentamicin, clindamycin, erythromycin, ciprofloxacin, oxacillin and cefoxitin (Sigma St. Louis, Mo, USA) against S. aureus was determined by using micro broth dilution method according to CLSI guidelines.  The concentration range of antibiotics was as follows: Oxacillin 0.12-256 μg/ml, tetracycline 0.06-128 μg/ml, vancomycin 0.06-32 μg/ml, gentamicin 0.06-128 μg/ml, clindamycin 0.015-8 μg/ml, erythromycin 0.06-128 μg/ml, co-trimoxazole 8-128 μg/ml, ciprofloxacin 0.06-128 and cefoxitin 0.06-32 μg/ml.
Polymerase chain reaction methodology
Amplification of targeted genes was carried out by a polymerase chain reaction (PCR)-assay using template deoxyribonucleic acid (DNA).  Bacterial DNA was extracted by using 10 mg/ml lysostaphin. PCR was carried out for mecA (for detection of methicillin resistance) and nucA (for detection of S. aureus) by using following primers mec-A1 (5'- AAA ATC GAT GGT AAA GGT TGC C-3'), mec-A2 (5'- AGT TCT GCA GTA CCG GAT TTG C- 3'), nuc-A1 (5'- GCG ATT GAT GGT GAT ACG GTT-3'), nuc-A2 (5'- AGC CAA GCC TTG ACG AAC TAA AGC- 3'. Multiplex PCR was performed for, mecA and nuc A gene in a 25 μl reaction volume (200 μlPCR vial) with 1XPCR buffer containing 10 mM Tris-HCL pH-8.3, 50 mM KCL, 1.5 mM MgCl 2 , 200 μM concentration of each deoxynucleoside-triphosphate (dNTPs), 2.5U of taq polymerase, 0.2 μM concentration of each primer and 2.5 μl template DNA. Thermo cycling was carried out in a (Bio-metra) thermo cycler and the conditions were as follows: Denaturation at 94°C for 10 min, followed by 30 cycles of 94°C for 30 seconds, annealing at 59°C for 30 seconds and extension at 72°C for 30 seconds with a final extension of 10 min at 72°C. Electrophoresis at 100 Vfor 40 min was performed to separate the products on 1.5% 1XTBE (8.9M boric acid) and [0.2 M Ethylenediaminetetraacetic acid (EDTA)] agarose gel. Gel was stained with 5 μg/ml ethidium bromide and photograph was taken under gel documentation system (Alpha Innotech Corporation U.S.A).
Pulsed-field gel electrophoresis typing
Pulsed field gel electrophoresis (PFGE) typing of SmaI (Fermentas, UK)-digested DNA was carried out by a modification of a protocol described previously by Bannerman et al.  Electrophoresis was performed in 0.5 X TBE buffer by a contour-clamped homogeneous electric field method using a CHEF MAPPER system (Bio-Rad) as described previously. The gels were stained with ethidium bromide for 1 h, visualised under ultraviolet (UV) light and photographed using a SynGeneBioimaging System. The banding patterns were interpreted visually and the relatedness of the strains was determined according to the recommendation of Tenover et al.  Strains showing the same PFGE pattern were grouped as a pulsotype and assigned alphabetically (A, B, C etc).
Frequencies were obtained and percentages were calculated for study variables. Demographic characteristics were compared with the use of Chi square and Fisher's exact test (two-tailed) with the Statistical Package for Social Sciences (SPSS) statistical program (version 15). All reported P values are two-sided and a P value of less than or equal to 0.05 was considered to be statistically significant.
| ~ Results|| |
Nasal colonisation with S. aureus
A total of 229 S. aureus isolates were isolated from nares of 820 subjects in the study. The nasal carriage rate of S.aureus was 27.92%. The nasal carriage of S. aureus was 107 (46.72%) in males and 122 (53.27%) in females. The difference between nasal carriage in male and female was not statistically significant (P > 0.05). Highest nasal carriage, 45 (17.85%) of S. aureus was recorded in the age group of 30-39 yrs followed by 34 (15.17%), 32 (14.28%), 31 (13.8%), 30 (13.39%), 24 (10.7%), 21 (9.37%) and 12 (5.35%) in the age groups of 20-29 yrs, 40-49 yrs, 50-59 yrs, 10-19 yrs, 60-69 yrs, 70-79 yrs, 1-9 yrs, respectively. There was statistical significant difference (P < 0.05) between ages 30-39 yrs, 60-69 yrs, 70-79 yrs and 1-9 yrs.
Susceptibility pattern of S. aureus to various antibiotics is shown in [Table 1]. All S. aureus isolates were susceptible to vancomycin.
Isolation of methicillin-resistant S. aureus
Out of 229 S. aureus isolates, 15 (6.55%) were MRSA by cefoxitin disc diffusion method and further confirmed by MIC for cefoxitin. The prevalence of nasal carriage was 1.83% (15/820). Out of fifteen MRSA isolates, 8 (53%) were detected in males and 7 (46%) in females. Among the 15 isolates, resistance to erythromycin, clindamycin, gentamicin, tetracycline, ciprofloxacin and cotrimoxazole was found in 4 (26.7%), 5 (33.3%), 14 (93.3%), 2 (13.3%), 3 (20%) and 9 (60%) isolates respectively. Resistance to erythromycin, cotrimoxazole, gentamicin and penicillin was greater for MRSA when compared to MSSA and the difference was statistical significant [Table 1].
Polymerase chain reaction assay
Multiplex PCR was done for amplification of mecA and nucA genes in all the isolates. Both genes were amplified in MRSA isolates. However, nuc gene was amplified in all S. aureus isolates [Figure 1].
|Figure 1: Agarose gel electrophoresis of PCR amplifi ed products with mec-A and nuc-A specifi c primers. Lane M: 100 bp ladder and Lane 1-5: Showing mec-A gene (533 bp) and nuc-A gene (275 bp)|
Click here to view
Pulsed-field gel electrophoresis typing
The banding patterns of MRSA isolates on PFGE were different from one another suggesting that the CA-MRSA isolates were polyclonal.
| ~ Discussion|| |
S. aureus is an important pathogen associated with nosocomial and community-acquired infection. Nasal colonisation by S. aureus has been considered an important risk factor for infections that could threaten a carrier's life. 
Our study shows an overall prevalence of 27.92% of S. aureus in the nostrils of the healthy subjects. The prevalence of S. aureus nasal carriage among healthy adults ranges from approximately 20% to 30%, with higher prevalence in overcrowded population. ,, These variations in carriage rates may be attributed to the characteristics of the population under study. The variation in reported rates results, at least partly, from differences in study populations, and sampling and culture techniques. The isolation of S. aureus with a single sample may further result in under estimating the number of intermittent carriers. In order to improve the Isolation of S. aureus, multiple nasal swabs should be taken from both anterior nares.
The prevalence of MRSA nasal colonisation was 1.83% in present study. A high rate of S. aureus nasal colonisation in the 5-15 yr age group and an alarming rate (3.89%) of community-acquired methicillin-resistant S. aureus nasal colonisation was reported by Chatterji et al., in an Indian community setting of rural, urban and semi-urban slums.  Studies in the developed world suggest that factors associated with CA-MRSA carriage include prior antibiotic usage, contact with health care facility, poor socio-economic conditions and overcrowding. ,
The overall susceptibility test result showed co-trimoxazole to be least effective drug against S. aureus with a resistant rate of 32.75%, but the resistance to all the other antibiotics was low. Resistance rate for penicillin was also low (21.39%). This finding is in agreement with another study conducted by Chatterjee et al. This is of clinical significance as infections caused by such strains in the community may also be susceptible to multiple antimicrobial agents including penicillin.  However, our data contradicts several studies reporting low rates of resistance to trimethoprim-sulphamethoxazole for S. aureus isolates recovered from the nares, , but is in agreement with another study by Jimei Du et al.  Review of antimicrobial susceptibility patterns also demonstrated that both clindamycin (33.3%), co-trimoxazole (60%) and erythromycin (26.7%) resistance is high among our CA-MRSA isolates, which is in contrast with previous studies of CA-MRSA isolates from hospitalised children or adults, and colonised children. , The moderately high resistance observed with ciprofloxacin, erythromycin and clindamycin may also be as a result of their uncontrolled usage in the community which favours the increasing number of resistant strains due to selection pressure.
PCR-based methods have previously been evaluated for rapid diagnosis of MRSA colonisation directly from nasal swabs.  We used multiplex PCR for detection of MRSA and methicillin sensitive S. aureus (MSSA) isolates from nasal samples of healthy subjects, which has proved more sensitive, accurate, time saving and reliable method. Cefoxitin disk diffusion method also gave equivalent results to the multiplex PCR, thus can be used as an alternative to PCR for the detection of MRSA in those labs were PCR is not available.
PFGE is considered the gold standard method for epidemiological typing of a variety of bacterial species including S. aureus since it is a highly discriminatory, stable and reproducible method.  PFGE was performed to predict strain types and to discern the most closely related clonal type among MRSA strains. Our study showed tremendously high level of genetic diversity among CA-MRSA strains. This is in agreement with other studies. ,,
Our study reports a relatively high prevalence of MRSA isolates from anterior nares of healthy individuals. This finding is of great concern as carriage of S. aureus in the nose appears to play a key role in the epidemiology and pathogenesis of infection. Strategies to interrupt transmission of S. aureus by elimination of nasal carriage and, thereby, preventing subsequent infection should be implemented. Continuing surveillance is needed to more accurately assess the prevalence, geographic distribution and epidemiology of community acquired infections. The results emphasise the need to improve personal hygiene and discourage antibiotics abuse so as to prevent the return of the consequences of pre-antibiotic era.
| ~ Acknowledgment|| |
We are highly thankful to Indian Council of Medical Research (ICMR) for financial assistance in this study.
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