Carrier (38) and disease (30) isolates were collected from rural, urban out patient and urban in patient environments and analysis is presented in Table 1.

¹New spa types reported to the data base; ² 1 isolate is agr negative.

Twenty six percent of carrier isolates and sixty percent of disease isolates were MRSA. All MRSA carried SCCmec type IV or V. Total of 15 STs were present among all the 68 isolates characterized. All but one sequence type were present in carrier isolates. ST 22, 772, 30, 121, 1208, 199, 672, and 45 were present among disease isolates. ST 5, 6, 7, 39, 72, and 291were present only among carriers. Antibiotic sensitivity to five antibiotics -oxacillin, cefoxitin, erythromycin, gentamicin, and tetracycline were tested on all the strains (data not presented). Isolates belonging exclusively to carrier STs were sensitive to all the antibiotics tested. Predominant methicillin resistant STs were 22 (68%) and 772 (69%) along with small percentage of isolates belonging to ST30, 672 and 1208 carrying 1.5, 3.0 and 4.4 percent of isolates respectively as MRSA. Carrier MRSA isolates were limited to ST22, 772, 30 and 1208 while disease MRSA isolates in addition included ST672. All carrier and disease isolates of ST22 and 772 lineage were PVL and egc positive.

Twelve S. aureus CC (15 STs) were identified with three of the clones detected in more than 10% of the isolates (ST22, ST772 and ST121) (Table 1). New or recently emerging clones were also detected (ST1208 and ST672). Figure 1 shows the eBURST analysis and lineages of all sequence types. Details of all the STs follow as given below. CC and STs of MSSA were much more diverse than those of MRSA (12 for MSSA, 5 for MRSA). Isolates belonged to all the 4 agr types. New spa types were detected among MRSA and MSSA isolates of lineages ST672, 772, 45, 121 and 6. PVL genes were detected in 69% of the isolates and egc in 84%. Microarray analysis was performed for representative carrier and disease isolates from each sequence type to determine the virulent factors and toxins.

Factors which were common to all isolates when analyzing the microarray results, were as follows: virulence factor genes- α, γ, δ haemolysins, staphylococcal complement inhibitor (scn), aureolysin, sspA, sspB and sspP; MSCRAMMS genes- fnbA, fib, ebpS, vwb, sdrC; Clumping factors A and B; bbp (bone sialo-protein binding protein); map (major histocompatibility complex class II analog protein) and immune-evasion genes- isaB, isdA, imrP, mprF, hysA1, hysA2, set 6, ssl9 were present in all except in one isolate of ST199 and one isolate of ST22, ssl7 absent only in one isolate of ST121. The patterns of presence and absence of virulence and immune evasion factors strictly followed the sequence type. Carrier and disease isolates belonging to a particular ST type had the same patterns. Raw microarray data of 33 isolates is provided as an Additional file 1. In a few cases where results were ambiguous, results have been confirmed with PCRs.

Figure 2A represents PFGE patterns of one representative isolate from each ST and 2B the dendrogram of PFGE depicting the relatedness of patterns based on the similarities derived from the UPGMA and dice coefficients using the Quantity one software. All profiles were different from each other and were distinct patterns characteristic of the ST.

ST22 is the major clone detected in 28% of the isolates present in both carrier and disease isolates. Methicillin resistance was detected in 68% in both groups, and the MRSA isolates had a SCCmec IV element. PFGE patterns of all ST22 isolates resembled classical EMRSA-15 patterns with 3–4 band differences and were related variants 10. Spa types from MSSA isolates differed from those of MRSA. ST22 is the clone most resistant to antibiotics with resistance to gentamicin and erythromycin, in MRSA as well as MSSA, both in carriers and infected patients. This clone was agr type I, capsular type 5, PVL and egc positive.

This is the second major clone present in our collection detected in 19% of the isolates both in carrier and disease isolates. Methicillin resistance was detected in 69% in both groups and the isolates had a SCCmec V element. Isolates with resistance to gentamicin and erythromycin were found in MRSA only, but both in carriers and infected patients. Spa types from MSSA isolates differed from MRSA. This clone was agr type II, capsular type 5, PVL and egc positive.

The ST120/121 clones were detected in 10% of the isolates both in carriers and patients. Methicillin resistance as well as resistance to other antibiotics was not detected in any of the isolates. This clone was agr type IV, capsular type 8, PVL and egc positive.

We are reporting a new sequence type from India, which appears to have the potential to be a founder clone. This clone was detected in 6% of the isolates in both carrier and disease isolates. Methicillin and gentamicin resistance was detected in 2 disease isolates with a SCCmec V element. Spa types from MSSA isolates differed from those of MRSA. This clone was agr type I, capsular type 8, PVL negative and egc and seb positive.

ST1208 is a new single locus variant (SLV) of ST8 and ST72 is a double locus variant (DLV). One ST1208 isolate was PVL positive. All three ST1208 MRSA isolates and one ST72 MSSA isolate were resistant to gentamicin and erythromycin. These clones were agr type I, and capsular polysaccharide type 5.

CC30 was represented by 4 isolates from the community and the hospitals belonging to ST30 and one ST39 carrier isolate (SLV of ST30). Methicillin and erythromycin resistance was detected in one ST30 carrier isolate with SCCmec type IVc. All isolates were agr type III. This is the only SCCmec type IVc isolate belonging to agr type III in our collection with a distinct PFGE pattern different from EMRSA-15. Except for one carrier ST39 MSSA isolate, all isolates were PVL and egc positive and belonged to capsular polysaccharide type 8.

This is the first report of two carrier MSSA isolates which are related to S. aureus from bovine origin. ST291 is a DLV of ST398 and spa types t937 and t3096 differed by one repeat unit. No antibiotic resistance was detected. PFGE patterns of these two isolates were very closely related with one band difference. These two isolates contained exotoxin D (etD) and edinB (epidermal cell differentiation inhibitor B) unlike other isolates and were negative for PVL and tst and contained capsular polysaccharide type 5.

These five other STs included 14 isolates with various characteristics. Methicillin resistant isolates were not detected among these STs, as well as other antibiotic resistance determinants. The PVL genes were detected in two isolates. While ST6, 7, 45, and 199 had capsular polysaccharide type 8, CC5 contained type 5.

Table 2 represents the PCR and microarray data for all MRSA (A) and representative carrier and disease isolates belonging to SCCmec type IV and V (B and C) respectively. After determination of mecA gene in all 68 samples, multiplex PCRs were performed for determination of the mec and ccr complexes using primers for amplification of ΔmecR1, IS1272, dcs, ccrA2B2, ccrC, mec C2 complex, subtypes of SCCmec type IV from IVa to IVd and IVh only for MRSA isolates. Various regions of SCCmec type V element from known sequences were also amplified by PCR to further identify SCCmec type V isolates.

* Second ccr C present in SCCmecZH47.

Isolates carrying SCCmec type IV cassettes did not amplify primers specific for IVa, IVb, IVc, IVd and IVh. Previous work from our laboratory has shown several variants of classical EMRSA-15 in PFGE patterns, and the J regions could be different from the known ST22, EMRSA-15 isolates 10. One ST30 carrier isolate carrying SCCmec type IV has a different PFGE pattern from that of ST22 (Figure 2) and amplified primers specific for SCCmec type IVc.

SCCmec type V elements were present in three different classes of STs-772, 672 and 1208. PCRs to identify different regions of type V elements (using strain WIS (WBG8318), Genbank accession no. AB121219) and microarray of selected isolates pointed to two different variants of type V element as shown in Table 2 (B and C). CcrC, mecA and ugpQ (Glycerophosphoryl-diester-Phosphodiesterase next to mecA) were present in all type V isolates while only isolates belonging to ST772 and ST672 carried a second ccrC region in the SCCmecZH47 in the microarray from the mosaic cassette ZH47 reported by Heuser et al 15. This region was positive by PCR using primers specific for the second ccrC in the SCCmecZH47 region with a size of 435 bp and is identical in sequence to isolates containing composite cassettes of SCCmec type V (5&5 C2). Type V isolates belonging to CC8 did not carry the second ccrC region. SCCmecZH47 also contain ccrA2ccrB2 and a very small truncated mecR region which did not amplify in our ST772 and ST672 isolates by PCR and microarray. Apart from amplifying the mecC2 complex upstream of mecA, none of the primers designed for several different regions of SCCmec type V based on sequences from WIS strain, amplified DNA from our type V isolates indicating that the J regions could be different. All isolates belonging to ST672 and 772 amplified primers for both hsdR and hsdM regions while ST1208 isolates did not amplify the hsdR region indicating there could be changes in this region as well (Table 2A). No DNA fragments targeting hsdS, which determine the specificity of restriction modification system, were amplified with DNAs of all isolates. The other genes indicated in Table 2C are selected from the microarray data to examine the differences among isolates belonging to different STs.

Sixty eight S. aureus isolates were included in this study, 38 from healthy nasal carriers and 30 from infection sites. Isolates collected from nasal carriers from rural community and urban population between 2006 and 2008 were cultured. Carriers had no identified risk factors for MRSA acquisition which included prior hospitalization, use of antibiotics, and surgeries in the past year. Nasal swabs were collected after explaining the prepared questionnaire and with consent of the subjects. Isolates recovered from infected sites were from wounds, pleural fluid and blood cultures collected in patients from hospitals in Bengaluru, Mumbai, Delhi, and Hyderabad. Data on community origin of these isolates is limited to a few as the isolates were sent to us from physicians from different hospitals. Ethical clearances and written consents for publication were obtained from the respective hospitals.

S. aureus isolates were selected after growth on chromogenic agar medium (chromAgar, bioMérieux, Marcy-L’Etoile, France) and identified after characterization by Gram staining, detection of catalase, coagulase and DNAse as described elsewhere 26.

Susceptibility testing was performed by Kirby-Bauer disc diffusion according to the guidelines recommended by the CLSI (formerly NCCLS) on Mueller-Hinton agar plates at 37°C using antibiotic discs. Minimum Inhibitory Concentration (MIC) for oxacillin and cefoxitin was determined by the broth dilution method in Mueller-Hinton Broth after 24 hrs of incubation at 37°C in micro titer plates 27.

Chromosomal DNA was extracted according to previously published procedures using lysostaphin 7.

SCCmec typing by determination of mec and ccr complexes for types IV and V SCCmec elements was carried out by multiplex PCR 282930. Subtyping of type IV SCCmec was performed according to the procedure of Zhang et al and Milherico et al 3132.

The four agr alleles were determined by a multiplex PCR as described in Gilot et al 33.

The presence of PVL genes was detected by PCR using the published primers and procedure 34. Presence of staphylococcal entero-toxins A, B, C, D and E, exfoliating toxins A and B and toxic shock syndrome toxin tst (TSST-1) and enterotoxin gene cluster (egc) cluster were detected by several multiplex PCRs using published procedures 3536.

MLST and spa typing were done as described earlier 3738.

PFGE was performed as described before 7.

Clonal relationship of the isolates was determined by using eBURST v3 program with the entire MLST database.

Microarray was performed for selected isolates from each of the clonal complexes. Diagnostic DNA microarray based on the Array/Tube platform (CLONDIAG, Jena, Germany) were utilized as described by Monecke et al 14. The micro-array covers 185 distinct genes and about 300 alleles there of, including species- specific controls, agr alleles, genes including virulence factors, and microbial surface components recognizing adhesive matrix molecules (MSCRAMMS), capsule- type specific genes, as well as resistance determinants and immune evasion factors.