Journal of Hospital Infection (2009) 73, 253e263

Available online at www.sciencedirect.com

www.elsevierhealth.com/journals/jhin

Clinical and molecular characteristics ofnosocomial meticillin-resistant Staphylococcusaureus skin and soft tissue isolates from threeIndian hospitals

R. Gadepalli a, B. Dhawan a,*, A. Kapil a, V. Sreenivas b, M. Jais c,R. Gaind d, R. Chaudhry a, J.C. Samantaray a, E.E. Udo e

a Department of Microbiology, All India Institute of Medical Sciences, New Delhi, Indiab Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, Indiac Department of Microbiology, Lady Hardinge Medical College and associated Hospitals, New Delhi, Indiad Department of Microbiology, Vardhaman Mahavir Medical College and Safdarjang Hospital,New Delhi, Indiae Department of Microbiology, Faculty of Medicine, Kuwait University, Safait, Kuwait

Received 19 December 2008; accepted 5 July 2009Available online 25 September 2009

KEYWORDSMeticillin-resistantStaphylococcusaureus;Multilocus sequencetyping;Nosocomial infection;Pulsed-field gelelectrophoresis;Skin and soft tissueinfection;Staphylococcalcassette chromosome

* Corresponding author. Address: DTel.: þ91 11 26588500x6444; fax: þ

E-mail address: b_neha2002@yaho

0195-6701/$ - see front matter ª 200doi:10.1016/j.jhin.2009.07.021

Summary We analysed risk factors for nosocomial meticillin-resistantStaphylococcus aureus (MRSA) skin and soft tissue infections (SSTIs) inthree Indian hospitals. We also determined antimicrobial resistancepatterns and genotypic characteristics of MRSA isolates using pulsed-fieldgel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphy-lococcal cassette chromosome (SCCmec) typing. Medical records of 709patients admitted to three tertiary hospitals with nosocomial S. aureusSSTIs were clinically evaluated. Antimicrobial susceptibility testing of pa-tient isolates was performed in accordance with Clinical and LaboratoryStandards Institute guidelines, with meticillin and mupirocin resistanceconfirmed by multiplex polymerase chain reaction. PFGE analysis of220 MRSA isolates was performed, followed by MLST and SCCmec typingof a selected number of isolates. MRSA was associated with 41%, 31% and7.5% of infections at the three hospitals, respectively. Multiple logistic re-gression analysis identified longer duration of hospitalisation [odds ratio(OR): 1.78; OR: 2.83 for �20 days], intra-hospital transfer (OR: 1.91),

epartment of Microbiology, All India Institute of Medical Sciences, New Delhi-29, India.91 11 26588641/26588663.o.co.in

9 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

254 R. Gadepalli et al.

non-infectious skin conditions (3.64), osteomyelitis (2.9), neurologicaldisorders (2.22), aminoglycoside therapy (1.74) and clindamycin therapy(4.73) as independent predictors for MRSA SSTIs. MRSA isolates from allthree hospitals were multidrug resistant, with fifteen clones (IeXV) recog-nised. A majority of the strains possessed type III cassette. The commonsequence type (ST) 239 was considered the signature MLST sequencefor PFGE clone III. This major MRSA clone III was closely related to theUK EMRSA-1 and was significantly more resistant to antibiotics. Dissemina-tion of multidrug-resistant MRSA clones warrants continuous tracking ofresistant genotypes in the Indian subcontinent.ª 2009 The Hospital Infection Society. Published by Elsevier Ltd. All rightsreserved.

Introduction

Skin and soft tissue infections (SSTIs) are commonnosocomial infections, forming a major cause ofmorbidity among hospitalised patients. These in-fections are caused by a wide variety of bacteria.Staphylococcus aureus is the main aetiologicalagent and the most frequently isolated micro-organism in various SSTIs reported from differentparts of the world including India.1e4

SSTIs are almost always treated with antimicro-bial agents. However, hospital-acquired S. aureusstrains have become resistant to multiple anti-microbial agents.5 The widespread antibiotic resis-tance in bacteria found in SSTIs complicatestreatment management, resulting in prolongedhospital stay, increased trauma care and treat-ment costs.6

Effective prevention and control measures forreducing the hospital reservoir of MRSA dependinitially on knowing the risk factors of the exposedpopulation for MRSA acquisition. Prevention andcontrol of MRSA infection also includes analysis ofisolates by discriminatory bacterial DNA typing.Understanding the epidemiology and clonality ofMRSA infections has important implications forfuture efforts to control the emergence of multi-drug-resistant strains and the spread of pathogenicMRSA clones.

To gain an insight into the epidemiology andclonality of MRSA in Indian hospitals, we analysedhospitalised patients with nosocomial S. aureusSSTIs. The present communication describes theclonal diversity of MRSA isolates as determinedby antimicrobial resistance patterns, PFGE,MLST and SCCmec typing from three tertiarycare hospitals in the city of New Delhi. In addi-tion, we attempted to assess the risk factor pro-file of MRSA SSTIs in hospitalised patients from allhospitals.

Methods

Setting

This study was conducted at (i) the All IndiaInstitute of Medical Sciences (AIIMS), (ii) LadyHardinge Medical College (LHMC) and (iii) Safdar-jang Hospital (SFJH), which are all teaching hos-pitals providing tertiary care, located in the city ofNew Delhi, India. AIIMS is a National Institute withan average admission of 130 000 patients perannum. It receives patients from the Northernregion of India. SFJH and LHMC cater for the localpopulation of Delhi with annual admissions of110 000 and 30 000 respectively.

The study was approved by each hospital’sethics committee.

Patients

It is routine practice in all three study hospitals tosubmit soft tissue specimens for culture from allcases of SSTIs. During the period July 2005 to June2007, a total of 781 patients had cultures positivefor S. aureus. Only the first isolate from eachpatient representative of the different clinicalunits was analysed. Patients were enrolled intothe study if they fulfilled the following inclusioncriteria: presented with clinical signs and symp-toms of SSTIs; had cultures positive for S. aureus>48 h after hospital admission; isolates wereneither from samples marked ‘nasal’, ‘axilla’ or‘perineal swabs’ nor from polymicrobial infec-tions. No recognised MRSA outbreak occurred inthe hospitals during the study period.

A total of 781 patients were eligible for studyenrolment, but 48 were unavailable to studypersonnel because of discharge before contactingthem. Of the 733 patients who completed the

Nosocomial MRSA in Indian hospitals 255

informed consent process, 709 patients (96.7%)consented and 24 (3.3%) refused.

Bacterial strains

Of the 709 S. aureus isolates, AIIMS contributed409, LHMC 200 and SFJH 100. All staphylococciwere identified by standard biochemical testsand confirmed as S. aureus by detection of thefemB gene using multiplex polymerase chainreaction (PCR).7

Clinical data collection

The medical records of inpatients with clinical andmicrobiological evidence of nosocomial S. aureusSSTIs were reviewed for patient demographicsand clinical information.

Definitions

S. aureus isolates were classified as those causinginfection according to established diagnostic cri-teria for nosocomial infections.8 S. aureus SSTIwas defined as a case associated with isolation ofpure growth of S. aureus from a soft tissue speci-men. A nosocomial case was defined as a patientwithout any evidence of infection on admissionand who was culture positive >48 h afteradmission.9

Antimicrobial susceptibility testing

Susceptibility to oxacillin was determined using thecefoxitin (30 mg) disc diffusion method and themecA gene multiplex PCR assay was used for confir-mation.7,10 Disc diffusion method was used to testthe susceptibility to amikacin (30 mg), amoxicillin/clavulanic acid (20/10 mg), chloramphenicol(30 mg), ciprofloxacin (5 mg), clindamycin (2 mg),co-trimoxazole (25 mg), erythromycin (15 mg), fu-sidic acid (10 mg), imipenem (10 mg), linezolid(30 mg), mupirocin (5 and 200 mg), netilmicin(30 mg), penicillin G (10 IU), piperacillin/tazobac-tam (100/10 mg), quinupristin/dalfopristin (15 mg),rifampicin (5 mg), teicoplanin (30 mg), tetracycline(30 mg), and vancomycin (30 mg) (Hi Media Mumbai,India), and inducible resistance to clindamycin inmacrolide-resistant strains of S. aureus was de-tected by a standardised disc approximationtest.10,11 A vancomycin (6 mg/mL) agar screen wasalso used to detect intermediate resistance to van-comycin.10 The minimum inhibitory concentrations(MICs) of teicoplanin and mupirocin for teicoplanin-and mupirocin-resistant isolates were determined

by the E-test (AB Biodisk, Solna, Sweden). Highlevel mupirocin resistance was further confirmedby the presence of the ileS-2 gene by multiplexPCR.7

Multidrug resistance was defined as resistanceto three or more non-b-lactam antibiotic classes.

MLST

MLST was performed with selected isolates asspecified by Enright et al.12 The sequencesobtained were compared with the sequences atthe MLST website (http://www.mlst.net) toassign a sequence type (ST).

SCCmec typing

SCCmec was typed by PCRs that determined theclass of mec complex with primers describedpreviously.13,14

Pulsed-field gel electrophoresis (PFGE)

Molecular typing of MRSA isolates was performedby PFGE using SmaI (Sigma Chemical Co., St Louis,MO, USA)-digested DNA as described previously byUdo et al.14

Data analysis

PFGE patterns were analysed by the use ofBionumerics software version 4.5 (Applied-Maths,Sint-Martens-Latem, Belgium). Isolates wereconsidered to be genetically related if their mac-rorestriction DNA patterns differed by seven bandsor fewer and the Dice coefficient of correlationwas �75%.15,16

Statistical analysis

All statistical tests were two-tailed, with P� 0.05considered as significant. Analysis was imple-mented on Stata 9.1 (Stata Corp., College Station,TX, USA).

Results

Patient demographics

S. aureus isolates were cultured from 467 men(65.9%) and 242 women (34.1%). The mean age ofAIIMS patients (34.2� 19.6) was more than thatin the other two participating hospitals. The inten-sive care units at AIIMS had a significantly higher

Table I Association of study characteristics of patients with MSSA and MRSA skin and soft tissue infections(N¼ 709)

Characteristic MSSA(N¼ 488)

MRSA(N¼ 221)

OR (95% CI) P

Age (years) 28.4� 19.9 32.3� 19.4 1.01 (1.00e1.01) 0.01<15 29.6 20.6 1.0015e44 44.1 50.0 1.62 (1.08e2.44) 0.02�45 26.3 30.0 1.62 (1.03e2.54) 0.03

SexMale 63.0 72.6 1.00Female 37.1 27.4 0.64 (0.45e0.90) 0.01

Duration of hospital stay (days)before culture

14.2� 0.33 17.9� 7.31

�10 34.4 16.0 1.0010e19 42.2 44.7 2.27 (1.47e3.52) <0.001�20 23.4 39.3 3.61 (2.28e5.72) <0.001

Patient locationMedical 61.2 52.5 1Surgical 24.5 17.8 0.84 (0.55e1.29) 0.44Intensive care units 14.3 29.7 2.42 (1.62e3.61) < 0.001

Risk factors associatedEndotracheal intubations

No 87.6 80.4 1.00Yes 12.4 19.6 1.71 (1.12e2.63) 0.01

Intra-hospital transferNo 89.2 79.0 1.00Yes 10.8 21.0 2.19 (1.42e3.38) <0.001

Previous hospitalisation (<6 months)No 64.5 55.2 1.00Yes 35.5 44.8 1.47 0.02

Intravenous cathetersNo 91.0 88.6 1.00Yes 9.0 11.4 0.67 (0.18e2.45) 0.54

Urinary cathetersNo 92.4 89.5 1.00Yes 7.6 10.5 1.44 (0.83e2.48) 0.19

DialysisNo 96.5 94.1 1.00Yes 3.5 5.9 1.76 (0.84e3.68) 0.14

Organ graftsNo 98.0 95.4 1.00Yes 2.0 4.6 2.30 (0.94e5.60) 0.07

Surgical treatmentNo 69.8 60.7 1.00Yes 30.2 39.3 1.49 (1.07e2.08) 0.02

Underlying conditions/diseases (%)Diabetes mellitus 12.4 14.6 1.20 (0.76e1.91) 0.43Multi-organ failure 5.5 8.7 1.62 (0.88e2.99) 0.11Bone fractures 16.1 8.7 1.46 (0.80e2.65) 0.22Osteomyelitis 6.1 11.4 1.98 (1.13e3.45) 0.02Non-infectious dermatosisa 28.4 39.3 1.63 (1.17e2.28) <0.01Sepsis 4.5 5.0 1.12 (0.54e2.36) 0.76HIV infection 2.0 2.7 1.35 (0.49e3.77) 0.56Myeloma/leukaemia 1.8 0.9 0.49 (0.11e2.30) 0.37Renal and urinary tract diseases 2.4 5.0 2.11 (0.91e4.85) 0.08Gastrointestinal diseases 14.9 3.2 0.64 (0.27e1.51) 0.31Neurological disorders 6.9 12.8 1.97 (1.16e3.33) 0.01

256 R. Gadepalli et al.

Table I (continued )

Characteristic MSSA(N¼ 488)

MRSA(N¼ 221)

OR (95% CI) P

Recent antimicrobial therapyb (%)b-Lactams 36.3 33.8 0.89 (0.64e1.25) 0.52Aminoglycosides 24.9 33.8 1.54 (1.09e2.18) 0.02Metronidazole 16.8 12.8 0.74 (0.47e1.18) 0.20Quinolones 9.4 15.1 1.71 (1.06e2.76) 0.02Glycopeptides 6.1 10.5 1.80 (1.02e3.18) 0.04Clindamycin 3.3 13.2 4.52 (2.40e8.52) <0.001Mupirocin 3.7 1.4 0.36 (0.11e1.25) 0.11

No. of classes of antibiotics (%)Monotherapy 19.2 18.7 1.00Dual combination therapy 40.4 46.1 1.31 (0.85e2.04) 0.22Triple or multiple combination therapy 21.4 20.1 1.11 (0.67e1.83) 0.69No information available 19.0 15.1 1.17 (0.67e2.05) 0.57

Mortality 2.5 4.0 1.12 (0.45e2.82) 0.81

MRSA, meticillin-resistant Staphylococcus aureus; MSSA, meticillin-susceptible Staphylococcus aureus; OR, odds ratio; CI, confi-dence interval; HIV, human immunodeficiency virus.The percentages are not mutually exclusive, as one patient may have more than one condition.

a For example, psoriasis, eczema, etc.b Preceding antibiotics used for the condition for which the patient was hospitalised.

Nosocomial MRSA in Indian hospitals 257

number of patients (32.3%) infected with S. aureuswhereas isolates of S. aureus were most frequentlyisolated from the internal medicine unit at SFJHand the cardiothoracic surgery unit at LHMC. Thesedifferences were statistically significant.

Prevalence of MRSA

Prevalence of MRSA was 31.2% (95% confidenceinterval: 27.5e34.3). There was considerable var-iation in the prevalence of MRSA-SSTIs betweenthe three study hospitals (AIIMS: 41.3%; LHMC:7.5%; SFJH: 35.0%; P< 0.001).

Table II Multiple logistic regression analysis offactors associated with MRSA infection

Characteristic OR 95% CI P

Duration of hospital stay (days) before culture10e19 1.78 1.09e2.90 0.02�20 2.83 1.67e4.78 <0.001

Intra-hospital transfer 1.91 1.17e3.12 0.01Non-infectious dermatosis 3.64 2.40e5.52 <0.001Osteomyelitis 2.90 1.55e5.44 0.001Neurological disorders 2.22 1.24e3.97 <0.01Antimicrobial therapy

with aminoglycosides 1.74 1.18e2.56 <0.01with clindamycin 4.73 2.38e9.40 <0.001

MRSA, meticillin-resistant Staphylococcus aureus; OR, oddsratio; CI, confidence interval.Multiple logistic regression adjusting for source hospital.

Risk factors for nosocomial MRSA-SSTIs

The associations of MRSA and MSSA with variouscharacteristics are presented in Table I. Stepwisemultiple logistic regression analysis adjusting forthe differences among the source hospitals identi-fied six independent factors to be significantlyassociated with MRSA-SSTIs, namely: longer dura-tion of hospital stay, intra-hospital transfer, non-infectious dermatosis, osteomyelitis, neurologicaldisorders, recent use of aminoglycosides and clin-damycin (Table II).

Antimicrobial susceptibility

All the isolates were susceptible to vancomycin andlinezolid (Table III). However, two strains from AIIMS(both MRSA isolates) showed reduced susceptibilityto teicoplanin (MICs: 16 and 32 mg/mL). MRSAisolates from all three hospitals were multidrugresistant. Low rates of chloramphenicol and rifam-picin resistance were found among MRSA in all threehospitals. High level mupirocin resistance (MuH) inS. aureus isolates was identified in all three hospi-tals, with rates ranging from 3.7% at AIIMS to 2% atSFJH. The range of mupirocin MICs was8e�1024 mg/mL. MuH was more prevalent amongmeticillin-resistant compared with meticillin-susceptible isolates of S. aureus (6.4% vs 1.8%).There were no discrepancies observed betweenthe disc diffusion and E-test MIC values and ileS-2PCR.

Table III Antimicrobial resistance patterns (%) of Staphylococcus aureus strains from three Indian hospitals (N¼ 709)

MSSA MRSA Total

AIIMS(N¼ 238)

LHMC(N¼ 185)

SFJH(N¼ 65)

P AIIMS(N¼ 171)

LHMC(N¼ 15)

SFJH(N¼ 35)

P MSSA(N¼ 488)

MRSA(N¼ 221)

Amikacin 12.5 2.2 3.1 <0.001 47.3 20.0 62.9 0.02 37.3 47.9Amoxicillin/clavulanic acid 12.5 13.5 6.1 0.29 NA NA NA e 12.0 NAChloramphenicol 4.2 1.6 7.7 0.07 8.9 0.0 2.9 0.34 3.7 7.3Ciprofloxacin 26.2 22.2 12.3 0.05 64.5 73.3 68.6 0.79 22.7 65.7Co-trimoxazole 33.3 28.1 12.3 <0.01 72.8 73.3 82.6 0.50 28.6 74.4Clindamycin 14.6 4.3 21.5 <0.001 52.1 60.0 91.4 <0.001 11.6 59.0Erythromycin 28.3 45.9 15.4 <0.001 84.0 86.7 91.4 0.57 33.3 85.4Fusidic acid 6.7 2.2 1.5 0.05 16.6 0.0 8.6 0.14 4.3 14.2Imipenem 7.5 1.1 6.1 <0.01 NA NA NA e 4.9 NAMupirocin (5 mg) 2.5 5.4 1.5 0.24 11.2 6.7 2.9 0.34 3.5 9.6Mupirocin (200 mg) 0.83 3.2 1.5 0.12 7.7 0.0 2.9 0.51 1.8 6.4Netilmicin 5.4 1.1 3.1 0.04 26.6 13.3 20.0 0.44 3.5 24.7Piperacillin/tazobactam 8.3 1.6 7.7 <0.01 NA NA NA e 5.7 NAQuinupristin/dalfopristin 11.2 15.7 16.9 0.27 41.4 26.6 26.7 <0.001 13.7 47.5Rifampicin 9.6 1.1 6.1 <0.001 27.8 6.7 607 0.01 5.9 23.3Tetracycline 14.6 7.6 9.2 0.07 65.1 65.1 60.0 0.01 11.2 61.6Teicoplanin 0.0 0.0 0.0 e 1.2 0.0 0.0 e 0.0 0.9

MSSA, meticillin-susceptible Staphylococcus aureus; MRSA, meticillin-resistant Staphylococcus aureus; AIIMS, All India Institute of Medical Sciences; LHMC, Lady Hardinge MedicalCollege; SFJH, Safdarjang Hospital; NA, not applicable.All S. aureus resistant to oxacillin have been considered resistant to amoxillin/clavulanic acid, imipenem and piperacillin/tazobactam as per Clinical and Laboratory Standards Instituteinterpretive criteria.10

All strains were uniformly susceptible to vancomycin (30 mg) and linezolid (30 mg).

258R.

Gadepalli

et

al.

Nosocomial MRSA in Indian hospitals 259

MRSA isolates from AIIMS tended to be more re-sistant to rifampicin (P¼ 0.01). However, resistanceto amikacin (P¼ 0.02), clindamycin (P< 0.001) andquinupristin/dalfopristin (P< 0.001) was higher in iso-lates from SFJH. MSSA isolates from AIIMS also tendedto be more resistant to most antimicrobial agentstested.

Clonal diversity among isolates of MRSAbetween hospitals

The SmaI macrorestriction fragment profiles of 220MRSA isolates were determined by PFGE. Fifteenclones (IeXV) were identified. Each clone hadtwo to five subtypes except clone XI, which hadno subtype. Thirteen clones (IeX, XIIeXIV) wereidentified from AIIMS, eight from SFJH (I, IIIeVI,XIIIeXV) and four clones (I, III, IV and XV) fromLHMC strains. Clones I, III and IV were observedin all three hospitals. Clone III was the most fre-quent (40%), followed by clone I (20.5%). Clone IIIappeared to be widespread in all three hospitals(38.8% in AIIMS, 40% in SFJH and 53.3% in LHMCisolates).

MLST and SCCmec types

Selected strains from the major PFGE clones IeIVwere processed for MLST and SCCmec typing, andthe data are presented in Table IV. Four isolateseach from clones III, II and I belonged to sequencetype (ST) 239. Two isolates from clone IV and oneisolate from clone III were ST 241, which has yqiLallele 30 and is a single-locus variant of yqiL 3(MLST database). SCCmec typing was done forthe above isolates; 13 isolates carried the type IIIcassette, three isolates carried the type II cas-sette. Four isolates of PFGE clone III and three iso-lates of PFGE clone I belonged to the ST 239 typeIII, whereas one isolate of PFGE clone III was ST241 type III. The common sequence type 239 was

Table IV SCCmec and MLST typing for selected strains of

No. of strains SCCmectypes

MLSTa (no. of isolates)

12 III 2e3e1e1e4e4e3 (9)3 II 2e3e1e1e4e4e30(3)

2e3e1e1e4e4e31 III 2e3e1e1e4e4e115UK EMRSA-1b NT 2e3e1e1e4e4e3

MLST, multilocus sequence typing; PFGE, pulsed-field gel electropa Allelic profile based on the DNA sequences of seven housekeepb UK EMRSA-1 (NCTC11939) Brazilian/Hungarian clone.

considered the signature MLST sequence for PFGEclone III.

PFGE types compared with internationalclones

PFGE was performed with epidemic MRSA strains(EMRSA)-1, -15 and -16 from the UK and Mu-50(vancomycin-intermediate S. aureus) along withstrains of clones IeV (Figure 1). The predominantclone III showed close similarity (similarity coeffi-cient: 74%) in DNA pattern to that of the epidemicUK EMRSA-1, the member of a lineage that includesthe Brazilian clone.

Antimicrobial resistance patterns amongdifferent clones

The profile of antibiotic resistance of MRSA isolatesalong with their clonality is shown in Table V. Theresistance to various antimicrobials varied fromclone to clone. Clone III showed more resistancecompared with other clones, with w80% or moreresistance to four of the antimicrobial agentstested, namely, erythromycin, ciprofloxacin, co-trimoxazole and tetracycline. The two teicoplanin-resistant isolates belonged to clone III, and thechloramphenicol-resistant isolates belonged toclones I and III.

Discussion

This is the first prospective multicentre study fromIndian hospitals aimed at defining the magnitude,antimicrobial susceptibility patterns and genotypiccomposition of nosocomial MRSA strains amongSSTIs.

The prevalence of MRSA-SSTIs varied betweenstudy hospitals. This is consistent with the earlierreports that MRSA prevalence varies not only from

the major PFGE clones

ST PFGE clones (no. of isolates)

239 III(4), I(3), II(2)241 IV(2),III(1)239 II(2), I(1)

1294 I(1)239 e

horesis; ST, sequence type; NT, non-typeable.ing genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL).

50

PFGE PFGE

60 70 80 90 100

Clone I

NCTC 8325

UK EMRSA-1

655U

Clone III

UK EMRSA-16Clone VClone IXMu-50 (VISA)

UK EMRSA-15

Clone IIClone IV

Figure 1 Dendrogram based on the similarities derived from the unweighted pair group method with arithmeticmean (UPGMA) and Dice coefficients, using Gel ComparII (4.5 version) software. The scale at the top represents sim-ilarity. Pulsed-field gel electrophoretic patterns of SmaI macrorestriction fragments of EMRSA-1, -15, -16, 655U, Mu-50along with representative strains of the MRSA clones IeV and IX.

260 R. Gadepalli et al.

region to region, but also from institution toinstitution within a region.5

Our study demonstrates the risk factors fornosocomial MRSA-SSTIs in hospitalised patients.Longer length of stay before infection is a well-known risk factor for antimicrobial resistance. Thisfactor may represent chronic illness, exposure toantibiotics as well as opportunities for resistantorganisms to colonise.17 Intra-hospital transfer re-sults in cross-infection and our observation of sig-nificantly higher risk of MRSA with intra-hospitaltransfer may be a reflection of this.18 Underlyingconditions such as non-infectious dermatosis maypresent opportunities for the patient to be colon-ised with MRSA, leading to subsequent infection.Osteomyelitis is associated with chronic morbidityand a refractory response to prolonged antimicro-bial therapy, predisposing S. aureus to developmeticillin resistance. Perhaps that is the reasonfor MRSA being the most frequently isolated organ-ism in osteomyelitis.19 Patients with underlying

neurological disorders tend to have surgical inter-ventions performed, which has been previouslyidentified as a risk factor for MRSA.20

Antimicrobial use and a number of differentclasses of antibiotics have been associated withMRSA.21,22 In the present study, use of aminoglyco-sides and clindamycin were independently associ-ated with MRSA. Our observation of associationbetween aminoglycosides is in agreement withearlier reports.9,22 The relationships between anti-microbial use and MRSA are complex. Nonetheless,most studies reiterate the fact that, in addition togood infection control practice, the prudent use ofantimicrobial agents is one of the major steps toreduce the growing problem of MRSA.

All our isolates were susceptible to vancomycin,thereby suggesting that vancomycin resistance hasyet to be widely disseminated in hospitals of ourregion. The most active non-glycopeptide anti-microbial agents against MRSA were chloramphen-icol and rifampicin. This could be due to the

Table V Antimicrobial resistance pattern (%) of different antimicrobial agents according to pulsed-field gelelectrophoretic clones

Antimicrobial agents Major clones of MRSAa P

I(N¼ 45)

II(N¼ 23)

III(N¼ 88)

IV(N¼ 20)

V(N¼ 12)

Othersb

(N¼ 32)

Amikacin 60.0 73.9 51.1 30.0 16.7 25.8 <0.01Chloramphenicol 26.7 0.0 4.5 0.0 0.0 0.0 <0.01Ciprofloxacin 48.9 60.9 93.2 45.0 16.7 48.4 <0.01Co-trimoxazole 84.4 91.3 89.8 45.0 16.7 45.2 <0.01Clindamycin 53.3 43.9 45.4 40.0 33.3 25.8 0.26Erythromycin 95.6 86.9 95.4 100 25.0 54.8 <0.001Fusidic acid 15.6 8.7 21.6 0.0 0.0 9.7 0.07Mupirocin (200 mg) 8.9 0.0 4.5 10.0 25.0 3.1 0.08Netilmicin 37.8 26.1 29.5 5.0 8.3 9.7 <0.01Quinupristin/dalfopristin

62.2 39.1 46.6 60.0 41.7 29.0 0.07

Rifampicin 33.3 4.3 25.0 22.0 8.3 25.8 0.09Tetracycline 64.4 95.6 79.5 15.0 16.7 29.0 <0.01Teicoplanin 0.0 0.0 2.2 0.0 0.0 0.0 e

MRSA, meticillin-resistant Staphylococcus aureus.a Roman numbers indicate the major clonal types.b These include the clonal types from VI to XV.

Nosocomial MRSA in Indian hospitals 261

absence of selective pressure since chloramphen-icol and rifampicin are used sparingly in ourhospitals. The newer anti-Gram-positive anti-biotics, namely, linezolid and quinupristin-dalfo-pristin, represent alternative treatment optionsfor serious MRSA infections.23 All our isolates weresusceptible to linezolid; however, only 52.5% ofMRSA strains were susceptible to quinupristin-dalfopristin. This is in contrast to reports fromEuropean countries where MRSA strains resistantto quinupristin-dalfopristin have only been sporad-ically observed.24

The prevalence (6.9%) of mupirocin resistanceamong S. aureus in this study is quite similar whencompared to the overall resistance rates reportedin the literature.25e27 Resistance to mupirocincompounds the issue of antibiotic resistance inS. aureus and makes infection control all themore difficult.

Genotyping of Indian S. aureus strains has previ-ously been reported.28,29 Arakere et al. have iden-tified a novel type III staphylococcal cassettechromosome mec (SCCmec) among MRSA isolatesfrom South India.30 The strength of the present in-vestigation is the number of strains genotyped. Incontrast to other studies which have shown thata small number of clones of MRSA are responsiblefor epidemic spread, in our study population 15different clones were responsible for all cases ofSSTIs.31,32 However, despite this diversity amongthe isolates of MRSA, PFGE types III and I were

responsible for 60.5% of all cases of nosocomialMRSA-SSTIs, indicating their widespread existence.The predominant clone III showed genetic related-ness with epidemic international MRSA lineages.

The sequence types reported in this study, STs239 and 241, have also been detected in China andTaiwan, as well as in India.29,32 The MLST, ST andSCCmec typing results for the selected strains ofmajor PFGE clones are also in concordance withsimilar patterns for the Hungarian and the Brazil-ian clones, where ST 239 and SCCmec type III pre-dominate.29 Both MLST and SCCmec typingsuggested a very limited divergence between theisolates included in this study, but PFGE generateda more diverse pattern.

From the magnitude of the frequency of cloneIII in all three hospitals together with its propensityfor resistance to different antimicrobials, it ap-pears that clone III is gaining footage in the NorthIndian hospitals studied. Apparently, we are facedwith highly multidrug-resistant MRSA strains andstrategies to prevent further spread are urgentlyneeded.

In a hospital setting, knowledge of the riskfactors for MRSA SSTIs, clonal spread and resis-tance patterns are particularly useful in patientmanagement and maintenance of infection pre-vention measures. Because this study was limitedto three hospitals in a specific geographic locationand to MRSA isolates from SSTIs, the problem ofmultidrug-resistant MRSA identified might only

262 R. Gadepalli et al.

represent the proverbial tip of an iceberg. Furtherstudies on these aspects from different regions ofthe country from time to time will help to monitorclonal dynamics of MRSA in Indian hospitals.

Acknowledgements

We thank Dr M. Mulvey and his team (NationalMicrobiology Laboratory, Winnipeg, Canada) forgeneration of dendrograms.

Conflict of interest statementNone declared.

Funding sourceFinancial support was received from Depart-ment of Biotechnology, Government of India,New Delhi.

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