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 ~  Abstract
 ~ Introduction
 ~ Methods
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
 ~  Article Figures
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 3  |  Page : 326-336
 

Clonal clusters and virulence factors of methicillin-resistant Staphylococcus Aureus: Evidence for community-acquired methicillin-resistant Staphylococcus Aureus infiltration into hospital settings in Chennai, South India


1 Department of Microbiology, University of Madras, Chennai; Department of Microbiology, Food Analysis Laboratory, Tamil Nadu Food Safety and Drugs Administration, Madurai, Tamil Nadu, India
2 Institute of Microbiology, Rajiv Gandhi Government General Hospital, Chennai, Tamil Nadu, India
3 Department of Thoracic Medicine, Government Hospital of Thoracic Medicine, Chennai, Tamil Nadu, India
4 Institute for Hygiene and Medical Microbiology, University of Wuerzburg, Wuerzburg, Germany; Department of Medical Microbiology, University Medical Center, Groningen, Netherlands
5 Department of Microbiology, University of Madras, Chennai, Tamil Nadu, India

Date of Submission06-Nov-2018
Date of Decision21-Jan-2019
Date of Acceptance18-Nov-2019
Date of Web Publication29-Jan-2020

Correspondence Address:
Dr. Padma Krishnan
Department of Microbiology, Dr ALM PGIBMS, University of Madras, Taramani, Chennai - 600 113, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_18_271

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 ~ Abstract 


Background and Objective: Staphylococcus aureus is one of the major pathogens of nosocomial infections as wells as community-acquired (CA) infections worldwide. So far, large-scale comprehensive molecular and epidemiological characterisation of S. aureus from very diverse settings has not been carried out in India. The objective of this study is to evaluate the molecular, epidemiological and virulence characteristics of S. aureus in both community and hospital settings in Chennai, southern India. Methods: S. aureus isolates were obtained from four different groups (a) healthy individuals from closed community settings, (b) inpatients from hospitals, (c) outpatients from hospitals, representing isolates of hospital–community interface and (d) HIV-infected patients to define isolates associated with the immunocompromised. Antibiotic susceptibility testing, multiplex polymerase chain reactions for detection of virulence and resistance determinants, molecular typing including Staphylococcal cassette chromosome mec (SCCmec) and agr typing, were carried out. Sequencing-based typing was done using spa and multilocus sequence typing (MLST) methods. Clonal complexes (CC) of hospital and CA methicillin-resistant S. aureus (MRSA) were identified and compared for virulence and resistance. Results and Conclusion: A total of 769 isolates of S. aureus isolates were studied. The prevalence of MRSA was found to be 7.17%, 81.67%, 58.33% and 22.85% for groups a, b, c and d, respectively. Of the four SCCmec types (I, III, IV and V) detected, SCCmec V was found to be predominant. Panton-Valentine leucocidin toxin genes were detected among MRSA isolates harbouring SCCmec IV and V. A total of 78 spa types were detected, t657 being the most prevalent. 13 MLST types belonging to 9 CC were detected. CC1 (ST-772, ST-1) and CC8 (ST238, ST368 and ST1208) were found to be predominant among MRSA. CA-MRSA isolates with SCCmec IV and V were isolated from all study groups including hospitalised patients and were found to be similar by molecular tools. This shows that CA MRSA has probably infiltrated into the hospital settings.


Keywords: Community-acquired methicillin-resistant Staphylococcus aureus, HIV, hospital-acquired methicillin-resistant Staphylococcus aureus, innate immune evasions, MLST, microbial surface component recognising adhesive matrix molecules, spa typing, ST 772


How to cite this article:
Abimannan N, Sumathi G, Krishnarajasekhar O R, Sinha B, Krishnan P. Clonal clusters and virulence factors of methicillin-resistant Staphylococcus Aureus: Evidence for community-acquired methicillin-resistant Staphylococcus Aureus infiltration into hospital settings in Chennai, South India. Indian J Med Microbiol 2019;37:326-36

How to cite this URL:
Abimannan N, Sumathi G, Krishnarajasekhar O R, Sinha B, Krishnan P. Clonal clusters and virulence factors of methicillin-resistant Staphylococcus Aureus: Evidence for community-acquired methicillin-resistant Staphylococcus Aureus infiltration into hospital settings in Chennai, South India. Indian J Med Microbiol [serial online] 2019 [cited 2020 Dec 3];37:326-36. Available from: https://www.ijmm.org/text.asp?2019/37/3/326/274091





 ~ Introduction Top


The number of serious infections with Staphylococcus aureus is increasing worldwide; this is true for both community-acquired (CA) and hospital-acquired (HA) methicillin-resistant S. aureus (MRSA) infections. The first case of nosocomial MRSA infection in India was reported in 1988.[1] Subsequently, the Indian hospitals were identified to be endemic for MRSA with the attributed nosocomial infections ranging from 45% to 70%. Only 15 years later, in 2003, CA-MRSA was first reported from India.[2] Thereafter, only a few case reports and hospital-based studies have documented CA-MRSA in India with recent reports demonstrating CA-MRSA infections from hospitalised patients with limited diversity in India.[3],[4]

Molecular characterisation and epidemiological typing of MRSA involving a large and diverse population has not been done in India. Such studies are urgently required to not only understand the current molecular epidemiology of the S. aureus in the country, but also to understand the impact of virulence and resistance determinants in hospital and community settings and in treatment outcomes. Hence, this study was aimed at performing a comprehensive phenotypic and molecular characterization of S. aureus obtained from various settings including community and hospital settings, their interface and from immunocompromised hosts in Chennai, South India. The results of this study showed unexpectedly high levels of CA-MRSA infiltration onto the hospitals, throwing light on the changing demographics of the S. aureus in India and the need for revision of screening and treatment strategies.


 ~ Methods Top


Study population

This is a cross-sectional study conducted between January 2010 and January 2014. S. aureus isolates from four different groups were included in this study: (a) healthy individuals from closed community settings to represent CA-MRSA (CA), (b) inpatients from hospitals that constitute HA-MRSA (HA), (c) outpatients from hospitals that represent CA–HA interface (CHI) and (d) HIV infected patients to represent isolates from immunocompromised individuals (HIV). Inpatient isolates were obtained from patients admitted in post-operative wards of hospitals for wound infection. Outpatient group represents patients visiting these tertiary hospitals for skin and soft tissue infections. S. aureus isolates were obtained from HIV patients visiting Government hospital for Thoracic Medicine for treatment of HIV and secondary infections. S. aureus isolates from individuals with prior treatment history for MRSA infections or history of long stay in hospital or association with hospital by any means (employed, patient attendee) were excluded from the study Groups a and c. The study was approved by Institutional ETHICAL committees of Dr ALM PG Institute of Basic Medical Sciences, Madras Medical College and Government Hospital for Thoracic Medicine, Tambaram. Informed consent was obtained from all the study participants.

Sample collection

Nasal swabs were obtained from individuals of various community settings including old age homes, orphanages and sports teams and were included in this group. Pus and pus swabs from various pyogenic infections were collected by standard procedures. The patient details including name, age, sex, date and hours of hospital admission, underlying clinical condition, previous medical history and antibiotic treatment if any were noted using a sample request form cum questionnaire.

Microbiological methods

All reference strains of S. aureus were obtained from Institute of Hygiene and Microbiology, University of Wuerzburg. The cultures were maintained as glycerol stocks at −80°C. Specimens were processed using standard microbiological methods.[5]

Culture and biochemical tests

The clinical specimens were processed for bacteria culturing in blood agar followed by Gram's staining, catalase, slide and tube coagulase test and growth on mannitol salt agar.

Antibiotic susceptibility testing

Antibiotic susceptibility testing was carried out by Kirby Bauer disc diffusion method and interpreted using CLSI guidelines. Inducible clindamycin resistance was detected by disc approximation test.[6]S. aureus ATCC 43300 (MRSA) and S. aureus ATCC 25923 methicillin sensitive S. aureus (MSSA) were employed as positive control and negative control, respectively.

Molecular methods

S. aureus cultures were grown overnight in 45 mL of LB broth at 37°C in shaker incubator. The cells were pelleted by centrifugation at 8,000 rpm for 15 min. The cells were lysed using lysostaphin (15 mg/mL) (Sigma) by incubation at 37°C for 15 min. DNA was extracted from lysed cells using the QiagenDNAeasy Extraction kit following manufacturer's protocol and was stored at 4°C until use.

Staphylococcal cassette chromosome mec typing and detection of virulence factors

Multiplex polymerase chain reactions (PCRs) were used for the specific detection of MRSA, panton valentine leucocidin (pvl) genes[7] and Staphylococcal cassette chromosome mec (SCCmec) typing[8],[9] and virulence genes such as enterotoxins, exfoliative toxins, hemolysins and leucocidins.[10],[11],[12],[13]S. aureus COL, S. aureus BK2464, S. aureus ANS 46, S. aureus MW2 and S. aureus WIS were used as reference strains for SCCmec typing, ccr typing and mec gene complex detection. S. aureus MW2, S. aureus N315, S. aureus COL, S. aureus RN 6607, S. aureus ATCC 27664 and S. aureus USA300 FPR3757 were used as reference strains for the detection of virulence factors. All the PCR methods were carried out using Eppendorf Mastercycler PCR and Taq DNA polymerase (NEB).

Genotyping

Genotyping of S. aureus was done by agr typing, spa typing and MLST using standard protocols.[14],[15],[16]

Statistical methods

The data were stored in MS excel and analysed using Minitab software v-15. ANOVA and Mann–Whitney tests were done for comparison of various groups included in the study.


 ~ Results Top


Staphylococcus aureus in study groups

A total of 769 non-duplicate isolates of S. aureus were collected from four different study populations: Hospital-associated S. aureus isolates (HA; n = 251) included isolates from inpatients of septic wards (n = 99), dermatology wards (n = 80), orthopaedic wards (n = 37) and ENT wards (n = 35). Community-associated clinical isolates (CA) of S. aureus were from outpatients attending the dermatology (n = 156), general surgery (n = 23) and ENT (n = 46) departments. S. aureus isolates (HIV) from HIV-infected patients were collected from the inpatient and outpatient settings. Carrier isolates of S. aureus included isolates from healthy individuals (CHI) and were obtained from three different closed communities at risk namely orphanages128/356 (35.95%), sportspersons 67/225 (29.77%) and old age homes 28/251 (11.15%). Of 852 healthy individuals from various communities, 223 (26.17%) were found to be carriers of S. aureus. The demographic characteristics of the groups are presented in [Table 1].
Table 1: Demographic Characteristics of study groups

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Prevalence of methicillin resistant Staphylococcus aureus and staphylococcal cassette chromosome mec types among various groups

The overall prevalence of MRSA as identified by PCR among the S. aureus isolates included in this study was found to be 48% (368/769). The prevalence of MRSA was the highest among HA infections (82%; 205/251) followed by CA infections (58%; 131/225). Among HIV patients, 23% (16/70) were MRSA, which was at least three times higher than what is observed in the community (CA-7%; 16/223).

Presence of SCCmec is the defining characteristic of MRSA, which encodes resistance for β-lactam antibiotics. Multiplex PCR of study MRSA isolates identified four types of SCCmec types: SCCmec type I, III, IV and V. Other SCCmec types such as SCCmec II, VI, VII–XIII were not detected in any of the MRSA isolates included in the study. SCCmec types V dominated in all the settings with 51%, 72%, 63% and 81% distribution among HA, CHI, HIV and CA-MRSA isolates. Next to SCCmec types V, SCCmec type IV was present in all study settings (41% – HA, 28% – CHI, 31% – HIV and 13% – CA). Four isolates from HA-MRSA and one isolate from CA-MRSA harboured SCCmec type I. Only one isolate among all the study isolates, present in HIV-MRSA group carried SCCmec type III, making it a rare type to be found among South Indian MRSA isolates. Comparison of study groups showed that there was a significant difference in the prevalence of SCCmec type V and SCCmec type IV between groups (f = 5.55, P = 0.001 and f = 12.59, P = 0.000).

Antibiotic resistance among Staphylococcus aureus isolates from various groups

All the tested S. aureus isolates from the four different settings were found to be susceptible to linezolid and vancomycin. MRSA isolates showed higher resistance compared to MSSA irrespective of the settings [Table 2].
Table 2: Antibiotic resistance among MRSA and MSSA

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A total of 70 (19.23%) isolates including 51 from HA, 14 from CHI, 2 from group HIV and 3 from CA-MRSA showed inducible clindamycin resistance (iMLSB). Comparison of inducible clindamycin resistance among MRSA isolates from different group by ANOVA showed that there was a significant difference between inpatient and outpatient isolates (f = 5.16, P = 0.002). Both constitutive and inducible clindamycin-resistant isolates were found to harbour the ermA gene by PCR. All MRSA isolates included in this study were susceptible to fusidic acid. Four MSSA isolates showed resistance to fusidic acid and were found to be positive for fusC gene by PCR. Forty-three (23.71%) of 205 MRSA isolates from hospital setting showed high-level mupirocin resistance by disc diffusion method (mupirocin 200 μg). All the isolates which showed mupirocin resistance were found to be positive for the mupA gene by PCR.

Virulence determinant genes

A total of40 virulence determinant genes including 15 enterotoxins, 3 exfoliative toxins, 5 hemolysins, 3 leucocidins, tsst-1, pvl, 3innate immune evasions and 9 microbial surface component recognising adhesive matrix molecules (MSCRAMMs) were detected. All toxin genes except leukocidin M (lukM) and exfoliative toxinD (etd) were present [Table 3]. No single virulence factor was found to be exclusive for any group of isolates included in this study. However, varying prevalence was detected for some of the virulence genes tested (pvl, sea, sec and sel). Toxigenic virulence factors such as pvl, sea and sec were highest for HA infections and MSCRAMMS such as clf, fib and fnbA were slightly high for CA infections. S. aureus isolates from asymptomatic carriers of community settings and infected HIV patients showed lowest prevalence of toxigenic virulence factors.
Table 3: Virulence determinants among various groups

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Genotyping

All four types of accessory gene regulator subtypes were detected among the S. aureus isolates included in the study. The predominant agr type was agr II (n = 427), followed by agr I (n = 208), agr III (n = 105) and ag rIV (n = 16). 13 isolates were found to be negative for the agr. Agr types I and III were predominant among MSSA isolates, while agr type II was predominant among MRSA isolates. The major agr subtype in this study was found to be agr II. Agr typing showed good discriminatory power in relation to the virulence determinants.

A total of 78 different spa types were obtained. The major spa types (n ≥ 10) obtained in this study were typed for sequence type using MLST. The BURP analysis of obtained spa sequence types (ST) i shown in [Figure 1].
Figure 1: Snapshot of BURP showing the spa clusters obtained in this study; Blue dot – represents the founders of the clonal complex; black dot – members of the clonal complexes; Size of the cluster corresponds to the number of isolates in the cluster. The major clonal complexes in this study were spaCC359, spaCC005 and spaCC037

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The representative ST and their clonal complexes (CC) were ST1; 1-1-1-1-1-1-1 (CC1), ST772 (CC1), ST5 (CC5), ST6 (CC6), ST239 (CC8), ST 368 (CC8), ST1208 (CC8), ST20 (CC20), ST22 (CC22), ST30 (CC30), ST45 (CC45), ST109, ST672 and ST120 (CC121). The characteristics of major CC of S. aureus identified in this study were compared [Table 3].


 ~ Discussion Top


In recent years, the prevalence of MRSA in Indian hospitals is continuously increasing.[17] The current study observed that the prevalence of MRSA among hospitalised patients which hits a new high to 82% and is so far the highest compared to all reports of HA-MRSA in India and other parts of the world including developed countries.[17],[18],[19] One would expect a concomitant increase in CA-MRSA as, 1990 onwards, increasing CA-MRSA infections have been reported across the world. In this study, the prevalence of MRSA among community-associated skin and soft tissue infections was found to be 58%, which is lower compared to previous and recent reports from South India studying superficial and deep-seated infections[18],[20] and is significantly lower compared to HA-MRSA. This observed prevalence of CA-MRSA is lower than the current prevalence in the USA and other developed countries.[21] Recently, it has been reported that HIV-infected patients have increased rates of S. aureus colonisation and skin and soft tissue infections.[22] Interestingly, the prevalence of MRSA infections among HIV-infected patients included in this study was detected as 22.85%, which is again, very low when compared to reports from developed countries.[23],[24] The prevalence of MRSA among HIV-infected patients was found to be significantly lower compared to both hospital- and community associated infections (P < 0.005).

Endogenous infections are common in healthcare settings and similar circumstances may occur in communities like sports, orphanages, day care centres and prisoners, where there is crowding of people. Recent studies report nasal carriage of MRSA among healthy individuals from various communities at risk indicating it as a source of CA-MRSA infections. The overall carriage rate of S. aureus among healthy individuals included in this study was found to be 26.17%, which is in the reported range of 20%–30% by different studies.[25] The prevalence of MRSA among carrier isolates of S. aureus in the current study was found to be 7.17%, which is comparatively lower than the recent reports from India.[20] Overall, our study shows that while HA-MRSA is increasing at an alarming rate in Chennai, the CA-MRSA and MRSA infections observed in immunosuppressed individuals are much lower than expected/reported levels, indicating a need for public health efforts to focus on hygiene practices in hospitals.

The prevalence of SCCmec types among MRSA from both hospital and community settings were reported to vary with geographical location. In this study, 4 different SCCmec types, namely SCCmec type I, type III, type IV and type V were detected. SCCmec type V was found to be the predominant SCCmec type irrespective of the source of the MRSA. Among MRSA isolates from hospitalised patients, SCCmec type I and III were exclusively associated, whereas only SCCmec t ype IV and V were present in CA-MRSA.

The study found that only 41% of HA-MRSA isolates harboured SCCmec type III, the predominant SCCmec type among HA-MRSA isolates endemic in hospitals of South Asian, European and American countries.[17] The presence of SCCmec V and SCCmec IV among HA-MRSA isolates indicates that the CA-MRSA strains are gradually infiltrating into the Indian hospitals. The results of this study were in agreement with the results from recent reports of MRSA among hospitalised patients.[4],[23]

An interesting finding of this study was that among CA-MRSA, SCCmec type V was predominant unlike reports from developed countries such as USA where SCCmec type IV was reported as the predominant type among CA-MRSA infections.[10],[24] Our results show that the SCCmec type V is predominant in CA-MRSA indicating changing virulence patterns.[3],[20]

In the present study, results of SCCmec typing of MRSA isolates from HIV-infected patients showed that SCCmec type V (81.25%) to be the predominant type followed by SCCmec type IV (12.5%). MRSA from healthy individuals from various communities at risk, showed three SCCmec types (I, IV and V). SCCmec type V was predominant and was detected in 62.5% of MRSA, followed by SCCmec type IV (31.25%). The above findings were in agreement with a recent study from India,[20] reporting 52.63% of CA-MRSA with SCCmec V. Interestingly, for the first time in India, we identified SCCmec type I from healthy individuals of community settings. Insights into all study groups showed that SCCmec type V was significantly higher among MRSA isolates from carriers, non-hospitalised patients and HIV-infected patients when compared to MRSA from hospitalised patients (P = 0.001), revealing that in this geographical location, SCCmec V type is predominant.

MRSA gained much importance mainly because of its potential multi-drug resistance to antibiotics. Recent reports show that the CA-MRSA strains entered into hospitals and were found to be multi-drug resistant. Among tested antibiotics, MRSA isolates showed highest resistance to erythromycin (100%), followed by ciprofloxacin (72.55%), gentamicin (67.93%), ofloxacin (64.94%), TMP-SMX (60.86%), amikacin (50.5%), tetracycline (47.82%) and netilmicin (39.13%). Comparatively, low level resistance was observed for rifampicin (17.39%) and mupirocin (15.21%). Resistance to aminoglycosides, fluoroquinolones, tetracycline and erythromycin were found to be high in MRSA isolates, when compared to previous reports. A community-based study[25] on the nasal carriage of S. aureus from North India has reported about 13% of MSSA to be erythromycin resistant, which is in concordance with the erythromycin resistance of carrier isolates of MSSA from healthy community in our study. Furthermore, there was a significant difference in the prevalence of erythromycin resistance of MSSA between the carrier isolates and the clinical isolates from hospitalised and community patients (P < 0.005).

Our study reports 3% of MRSA causing clinical infections to be positive for constitutive clindamycin resistance. Deotale et al.[26] from North India has shown 7.3% clinical isolates of MRSA to be clindamycin resistant, which is higher than our study. Studies from developed countries report a high percentage of HA-MRSA to be constitutively resistant to clindamycin and also a significant difference in constitutive clindamycin resistance between HA-MRSA and CA-MRSA. Our study reports 19% of MRSA and 12.21% of MSSA isolates to show inducible clindamycin resistance, which is comparable with the 2011 study[27] from south India, which reports 14.67% and 4.92% of MRSA and MSSA, respectively. There was a significant difference in iMLSB phenotype between MRSA isolates from hospitalised patients (24.67%) and community patients (10.68%) (P < 0.005). In this study, second only to erythromycin, highest resistance was observed for ciprofloxacin among MRSA isolates causing infections of hospitalised patients (93.17%), HIV-infected patients (75%) and among MRSA isolates from healthy carriers (93.75%). In a study from Mumbai,[3] about 98% HA-MRSA and 87% CA-MRSA isolates causing infections were found to be ciprofloxacin resistant which is higher than the present study. The prevalence of rifampicin resistance is significantly higher among MRSA from HIV-infected patients compared to the MRSA from community patients and hospitalised patients (P < 0.005). The highest rifampicin resistance among MRSA from HIV-infected patients may be due to the extensive use of rifampicin in HIV/TB co-infected patients to treat the tuberculosis infection. No significant difference on rifampicin resistance was observed between HA-MRSA and CA-MRSA isolates.

CA-MRSA isolates containing pvl genes have been epidemiologically linked to recurrent and often severe skin and soft tissue infections. The prevalence of pvl-positive CA-MRSA varies considerably from one continent to another. European countries have the least prevalence of approximately 1%–3%, the USA has up to 50% of clinical isolates causing community associated skin and soft tissue infections. 100% pvl prevalence among CA-MRSA has been reported from developed countries including France, Switzerland, USA, Oceania, South West pacific and Australia. We observed pvl gene among MRSA and MSSA isolates from all the four study groups. Our study reports 57% of MRSA from hospital-associated infections, 58% of MRSA from community-associated infections, 94% of MRSA from HIV-infected patients and 75% of MRSA from anterior nares of healthy individuals to be positive for pvl genes. A hospital based study from Mumbai[3] reports 56.7% of MRSA to be pvl positive which is in concordance with the prevalence of pvl among MRSA from hospital inpatients of our study. A community based study from several cities in India,[20] reports about 72% of clinical isolates of MRSA from community to be positive for pvl, which is comparatively higher than our study (58%). The same study also reports 50% of carrier isolates of MRSA to be positive for pvl gene, which is comparatively lower than our study. No significant difference on prevalence of pvl was observed between MRSA from community and hospital-associated infections. However, significant difference in the prevalence of pvl was seen between carrier isolates of MRSA and MRSA from HIV, community and hospital-associated infections.

Of the other virulence factors studied, among clinical isolates of S. aureus, lukD was always present with lukE. 62% of clinical isolates from hospitalised patients, 57% of isolates from community patients and 32.8% of isolates from HIV-infected patients were found to be positive for lukD-E genes. The prevalence of lukD-E was low, when compared to studies from USA, Japan and European countries. Among carrier isolates, the presence of lukD and lukE varied and was found to be 26% and 28% respectively. This was in agreement with the previous reports, which also have shown varying prevalence rates of lukD and lukE.[18]

Of the enterotoxins studied, sea gene for staphylococcal enterotoxin A was frequently detected in 64% from hospital-associated infections, 39% from community-associated infections, 40% from infections of HIV-infected patients and 8% from healthy carriers. The prevalence of sea (63.6%) among S. aureus from hospitalised patients of our study is high, when compared to a nationwide hospital-based study from Japan, which reports 9.6% of MRSA to be sea positive. The prevalence of sea among carrier isolates of S. aureus in this study was found to be lower when compared to studies from developed countries.[18] In a worldwide study of CA-MRSA, Vandenesch et al.[28] reported about 79% of CA-MRSA isolates from USA to be positive for sea gene. No significant difference on prevalence of sea was observed between S. aureus from community and HIV-associated infections; while, the prevalence of sea was significantly high among clinical isolates of S. aureus from hospitalised patients than the clinical isolates from community and HIV-infected patients (P < 0.005).

The type of agr polymorphism has been reported to be associated with the clinical significance, specific virulence factors (pathogenicity) and resistance. Our study reports agr I and agr II in 74% of the isolates, which shows their high prevalence in this geographical location. In this study, 31% of MRSA from hospital associated infections, 12% of MRSA from community-associated infections and 6% isolates from HIV-infected patients belonged to the agr-I and 67% of MRSA causing hospital-associated infections, 87% of isolates causing community associated infections, 94% of isolates from HIV-infected patients and 62% of isolates from healthy carriers belonged to the agr-II subtype. This shows that in this geographical location, the antibiotic resistance is more common among the agr I and II. All the isolates with agr-IV harboured the exfoliative toxins ETA and/ETB, which is in agreement with the previous reports.[12] 90% of the isolates belonging to agr-III were found to be MSSA.

Genotyping

Spa typing

A total of 78 spa types were detected in this study. Among S. aureus isolates from hospitalised patients, 26 different spa types were found, which constituted 33% of the total spa types detected. Majority of the S. aureus isolates from hospital-associated infections, belonged to spa types t657, t037, t1223 and t852 and were also found to be MRSA. S. aureus causing community-associated infections belonged to 23 different spa types constituting 29% of total spa types. The majority of isolates from community-associated infections belonged to spa types t657, t064, t1154 and t852 which were also found to be MRSA. The major spa types of MSSA isolates from community-associated infections were found to be t3841 and 3204. The results were comparable with the previous report from India.[20]

S. aureus isolates causing infections among HIV-infected patients belonged to 19 different spa types, which constituted 24% of the total spa types obtained in this study.

Carrier isolates of S. aureus included in this study showed high diversity belonging to 58 different spa types, which constituted 75% of total spa types obtained in this study. Spa types t4615, t272, t159, t021, t005, t015, t3841, t701, t937 and t9037 were found to be common among carrier isolates of S. aureus in this study.

MLST types and clonal complexes

When coupled with SCCmec typing, MLST is used to discriminate between different clones of MRSA. In this study, a total of 15 ST were detected among major spa types (n > 10) subjected to MLST types. The CC and STs included CC1 - (ST1, ST772), CC5 - (ST5), CC6 - (ST6), CC8 - (ST239, ST1208, ST368), CC20 - (ST20), CC22 - (ST22), CC30 - (ST30), CC45 - (ST45), CC121 - (ST120) and CC9 - (ST109). This indicates a high diversity of MRSA and MSSA clone in community as well as in the hospital settings. No clonal complex was found for ST672. This is the first Indian study to compare the ST of S. aureus from hospital-associated infections, community associated infection, infections of HIV-infected patients and healthy carriers. The representative CC in this study was compared for their spa types, resistance, SCCmec types, agr types, virulence factors and infections [Table 4].
Table 4: Characteristics of the MLST . Clonal complexes (CC) obtained in this study

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Clonal complex CC1

ST772 - also called as subcontinent clone or Bengal Bay Clone was the predominant CA - MRSAclone, which is single locus variant of ST-1. MRSA ST-772 clone was found to be highly pathogenic associated with abscess infections and acquired resistance determinants and belonged to single spa type 657. Other members were found to be t347 (pvl MSSA); t1931 (MSSA).

Clonal complex CC8

ST239 was the predominant multidrug-resistant HA–MRSA clone endemic in hospitals almost worldwide. It is the second major MRSA clone among HA infections, detected in this study. It was found to be mupirocin and inducible clindamycin resistant strain.

ST368– another MDR HA–MRSA clone of the study belonging to this clonal complex with spa type t425/agr-I with a prevalence of 6% of MRSA causing HA infections mostly among diabetic patients. It has been previously reported from Sri Lanka. Other members of this clonal complex include ST1208 MRSA-III and ST1208-MRSA-V with spa types – t1223 and t064, respectively. Clones with t064 were found to be typical CA-MRSA and were causing middle ear, skin and soft tissue infections. The clones with spa type t1223 were found to be HA-MRSA and were associated with post-operative wound infections and were recently reported from India.

Clonal complex CC-22

ST22-MRSA-IV was the second major CA-MRSA in this study constituting 9% of total MRSA isolates with spa types – t005/t852 with PVL. ST22is an international clone of CA-MRSA, is found circulating in most parts of the world. In this study, these CA-MRSA isolates were found to be causing skin and soft tissue infections. It is highly prevalent in Europe and India. We find gentamicin resistance among these MRSA clones.

Clonal complex CC-5

ST5 is highly virulent with super-antigen enterotoxins, leucocidins and is resistant to multiple antibiotics. It is a pvl positive MSSA and was found to cause community associated ear, skin and soft tissue infections, belonged to spa type t448 and agr type III. Along with the egc gene cluster, all the 10 isolates of t448 carried the plasmid pIB485 with enterotoxins sed and sej. MRSA isolates of this clone were reported from Australia, Ireland and Germany.

Single pvl-positive CA-MRSA (CC30; ST30-MRSA-IV) was isolated from breast abscess. It has been previously reported from developed countries.

Clonal complexes of methicillin sensitive Staphylococcus aureus

CC9; ST109 clone-A total of 13 methicillin sensitive S. aureus isolates were obtained from healthy carriers and were positive for exfoliative toxin B. It has been previously reported predominantly among veterinary isolates.

Clones with methicillin resistant Staphylococcus aureus and methicillin sensitive Staphylococcus aureus

ST-672-carried both MRSA and MSSA isolates with spa type t3841. Five of these isolates were found to be MRSA, which carried SCCmec I and were found to be causing HA infections, while one MSSA isolate was from healthy carrier. ST 672 was first reported from South India in 2010.[20]


 ~ Conclusion Top


This study comparing molecular characteristics of resistance and virulence determinants of MRSA from four different populations has shown that the MRSA isolates causing hospital-associated infections were significantly different in their genotypes from MRSA causing community-associated infections, HIV-associated infections and carrier isolates of high-risk community. Virulence factors were significantly high among community and hospital-associated S. aureus compared to clinical isolates from HIV-infected patients. Antibiotic resistance was significantly high among HA-MRSA compared to MRSA from community-associated infections and HIV-infected patients. Antibiotic resistance was significantly lower among carrier isolates compared to clinical isolates, but carrier isolates also showed resistance to antibiotics especially to erythromycin, tetracycline and cefoxitin. Genotyping by spa typing showed that the carrier S. aureus isolates from community were highly diverse compared to clinical isolates. Prevalence of MRSA (2%) among healthy individuals from various high risk communities indicates that CA-MRSA isolates are circulating in the Indian community settings asymptomatically. The emergence of MDR-CA-MRSA among hospitalised patients shows that CA-MRSA has acquired additional drug resistance determinant on entering the hospital settings. This study has shown that in the Indian scenario, CA-MRSA isolates have infiltrated into the hospital settings, acquired multiple drug resistance determinants and have become endemic in hospitals.

Acknowledgement

We do not have any conflicts of interest in the subject area of the research discussed. We thank all the study participants. This work was supported by an Indian Council of Medical Research (ICMR–BMBF Indo-German Collaborative project) grant provided by the Government of India (grant no. INDO/FRC/610/09-IHD). We thank Dr. C. Prabha for her support in drafting the manuscript. We thank Dr. G. Sivakumar, Department of General Surgery, MMC, and GH, Chennai, for his support in collecting the clinical samples and Dr. J. Suriakumar, GHTM, Tambaram for his support in collecting isolates. We also thank SivanandaGurukulam, Chennai, AnbuIllam, Chennai, AnandamIllam, Chennai and Alpha arts and Science College for consenting to collect the nasal swabs.

Financial support and sponsorship

ICMR - BMBF.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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2004 - Indian Journal of Medical Microbiology
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