The Prevalence and the Risk Factors Which are Associated with Staphylococcus aureus and Methicillin-Resistant S. aureus Which Harboured the Panton-Valentine- Leukocidin Gene in Sikkim

1. Lecturer, Department of Microbiology, Sikkim Manipal Institute of Medical Sciences. 2. Professor & HOD, Department of Microbiology, Sikkim Manipal Institute of Medical Sciences.

Abstract

Background: The clinical sequelae of PVL-positive S. aureus infection tend to be more severe than those of the PVL-negative ones. The strong association of the PVL toxin in the MRSA isolates suggests the methcillin-resistance which contributes to the success of these PVL-positive isolates.
Aims: To estimate the burden and the risk factors which were associated with S. aureus and MRSA which harboured the pvl gene in Sikkim.
Settings and Design: A point prevalence study was conducted during a period of one year from August 2010 to 2011 in the teaching hospitals.
Materials and Methods: A total of 119 clinical strains of S. aureus were subjected to monoplex (fem-A) and multiplex PCR (mec-A and pvl) respectively.
Statistical Analysis: The data were analyzed by using Pearson’s Chi-square test. The p-value of <0.05 was taken as statistically significant.
Results: Out of 119 isolates of S. aureus, 117 (98.31%), 49 (41.17%) and 54 (45.37%) isolates were positive for the fem-A (internal control), mec-A and the pvl genes respectively. Out of the 117 fem-A positive isolates, 47 (40.17%) and 52 (44.44%) were found to be mec-A (MRSA) and pvl positive. Among the 54 pvl positive isolates, 47 (87.03%), 5 (9.25%) and 2 (3.7%) were reported from the MRSA (fem-A and mec-A positive), MSSA (fem-A positive and mec-A negative), and the MRCoNS (fem-A negative and mec-A positive) isolates respectively. The PVL toxin was significantly higher in the MRSA (87.03%), as compared to the MSSA (9.25%). Among the risk factors which were studied, patients with a previous history of antibiotic intake and hospitalization and who attended the surgery and the burn/ICU units were more positive for the MRSA and the PVLS. aureus infections as compared to the negative patients.
Conclusion: The high prevalence of the PVL toxin among the S. aureus population, which was mainly reported from the MRSA isolates, irrespective of their types i.e. CA or HA-MRSA, revealed that PVL was not a reliable marker for the CA-MRSA strains. Rather, it may be hypothesized that the MRSA strains may be important reservoirs of the PVL toxin and that it may be slowly acquired by the MSSA strains.

MRSA, PVL, Risk-factor, S.aureus, Sikkim

Introduction
Staphylococcus aureus expresses a variety of virulence factors, including Panton-Valentine-leukocidin (PVL). It is a cytotoxin that causes leucocyte destruction and tissue necrosis, which is produced by fewer than 5% of the S. aureus strains (1). PVL positive S. aureus causes diverse clinical syndromes, ranging from superficial skin and soft tissue infections to various deep-seated infections such as pneumonia, infective endocarditis, osteomyelitis, and enterocolitis (1). Reports have suggested the strong association of the PVL toxin in the community-associated (CA) MSSA, and the CA-MRSA infections (2). However, in recent years, the strains of MRSA which carried the pvl gene are increasingly being reported in hospitalassociated (HA) infections (3), and to a lesser extent in CA- infections (4), thus revealing that the pvl gene is no longer a reliable marker for the CA-MRSA infections. The established risk factors for the MRSA infection include recent hospitalization or surgery, residence in a long-term-care facility, dialysis, and indwelling percutaneous Original Article Metabolic Section medical devices and catheters. Recently, cases of MRSA have been documented in healthy, community-dwelling persons who did not have the established risk factors for the MRSA acquisition. Because they are apparently acquired in the community, they are often referred to as community-acquired MRSA (5). The present study was conducted to determine the burden of the PVL toxin in the general S. aureus population and in the MRSA strains in Sikkim, and to determine the risk-factors which are associated with it. Secondly, it was done to investigate whether the pvl gene carriage in CA-MRSA still hold true for its differentiation from the HA-MRSA isolates.

Material and Methods

Design and Settings of Study
Point prevalence study which was conducted on 119 S. aureus strains which were isolated from various clinical specimens during a period of one year from August 2009 to 2010 in teaching hospitals.
Case Definition and Source of the Data
Hospital-associated S. aureus isolates, as ones which were cultured from clinical specimens, were obtained at > 72 hours after the hospital admission of the patients or from patients with a history of hospitalization within six months of S. aureus isolation date. Community-associated S. aureus isolates which were cultured during the first 72 hours of a patient’s hospital admission, from outpatients or from patients with no h/o hospitalization within six months of the S. aureus isolation date were used. The data of the patients were obtained from the laboratory investigation registers and from the medical record files. The data of the study subjects included the basic demographic profiles, the date of hospital admission and discharge, clinical findings, culture site, status of the patients (IP/OPD), clinical history, and the department which was visited.
Identification of the S. aureus Isolates
All the isolates were identified as S. aureus by using conventional techniques, including the slide and the tube coagulase test, the DNase test (Hi-Media), the Phosphatase test (Hi-Media) and the Modified OF test (Hugh-Leifson test). The S. aureus strains were inoculated into semi-solid nutrient agar in screw capped vials and they were stored at -20ºC for further molecular analysis.
DNA Isolation
The test inoculum was prepared by inoculating two to three isolated colonies of S. aureus into 3 to 4 ml of BHI broth (Hi- Media), and it was incubated overnight at an ambient temperature of 35-37 ºC. Then, DNA was extracted by using the HiPurA TM Bacterial and Yeast Genomic DNA Miniprep Purification Spin kit (Hi-Media).The DNA concentration was determined as micrograms per milliliter according to the A260 values by using a Nanodrop ND-1000 Spectrophotometer.
Monoplex PCR for the Detection of the fem-A gene
To validate, all the isolates which were tested was S. aureus. The primers, GFEMAR-1(5’-AAA AAAGCACATAACAAGCG-3’) and GFEMAR-2 (5’-GATAAAGAAGAAACCAGCAG-3’),with a 132 bp amplicon size for the amplification of the fem-A gene (internal control) were selected from a published sequence of Mehrotra et al (6), and they were checked for their specificity against available S. aureus genomes by using the BLAST utility which was available through the National Center for Biotechnology Information website (www.ncbi.nim.nih.gov) under GenBank Accession no (X17688.1). They were commercially obtained from Sigma-Aldrich, Bangalore. PCR was performed by using the Taq PCR Master Mix Kit (Qiagen). A 25-μl final reaction volume mixture was prepared, which consisted of 12.5 µl of the master mix, 1µl of each of the forward and reverse primers (0.4µM), 7.5 µl of RNase free water and 3µl of the DNA template. PCR was performed by using a thermocycler (Biometra) with the thermocycling conditions of initial denaturation (95ºC,5 min), followed by 35 cycles of the three step cycling conditions of denaturation ( 94ºC, 30 sec), annealing (57ºC, 1 min) and extension (72ºC, 30 sec), followed by the final extension (72ºC,7 min ) and soaking at 4ºC. Then, 5µl of the amplified products were mixed with 2µl of ethidium bromide (Fermentas) and loaded on a 2% agarose gel (Amresco) along with GeneRuler TM 100 bp Plus DNA Ladder, (Fermentas), followed by electrophoresis at 100 volts for 50-60 min. The gel was visualized under a UV transilluminator (Bio- Doc analyzer, Biometra).
Multiplex PCR for the Detection of the mec-A and the pvl Genes
The primers for the amplification of the mec-A and the pvl genes were MECAP4 (5’-TCCAGATTA CAACTTCACCAGG-3’), and MECAP7(5’-CCACTTCATATCTTGTAACG-3’) under (GenBank Accession No-Y00688) with a 162 bp amplicon size for the mec-A gene as was described by Oliveria et al (7) and luk-PV-1 (5’-ATCATTAGGTAAAATGTC TGGACATGATCCA-3’) and luk- PV-2 (5’-GCATCAAGTGTATTGGATAGCAAAAGC-3’) under (GenBank accession no-X72700) with a 433 bp amplicon size as was described by McClure et al (8) respectively. The primers were blasted and commercially obtained (Sigma-Aldrich, Bangalore). PCR was performed by using a Qiagen Multiplex PCR kit with slight modifications. A 25-μl final reaction volume mixture was prepared, which consisted of 12.5 µl of the mastermix, 2.5 µl of the primer mix (0.2µM of each primer), 3µl of DNA template and 7µl of RNase free water. The DNA samples were subjected to thermocycling conditions, with the initial inactivation step (95oC,15min), the three step cycling condition of denaturation (94oC, 30 sec), annealing (60oC, 90 sec) and extension (72oC, 90sec) for 35 cycles, with the final extension (72oC,10 min) and soaking at 4oC. Then, 5µl of the amplified products were mixed with 2µl of ethidium bromide (Fermentas) and loaded on a 2% agarose gel (Amresco) along with GeneRulerTM 100bp Plus DNA Ladder (Fermentas), followed by electrophoresis at 100 volts for 50-60min. The gel was visualized under a UV transilluminator (Bio-Doc analyzer, Biometra).
Statistical Analysis
The categorical variables were analyzed by using Pearson’s Chisquare test. A p-value of <0.05 was taken as statistically significant.

Results

A total of 119 conventionally identified isolates of S. aureus, 117 (98.31%), 49 (41.17%) and 54 (45.37%) were positive for the fem-A (internal control), mec-A and the pvl genes, as was detected by monoplex [Table/Fig 1] and multiplex PCR [Table/Fig 2] respectively. Out of the 117 fem-A positive S. aureus isolates, 47 (40.17%) and 52 (44.44 %) were found to be mec-A (MRSA) and pvl positive. Among the 54 pvl- S. aureus isolates, 47 (87.03%), 5 (9.25%) and 2 (3.7%) were reported to be mec-A positive (MRSA), mec-A negative (MSSA) and fem-A negative, mec-A positive (MRCoNS) S. aureus isolates respectively. Out of the 117 S. aureus isolates respectively, 34 (29.05%) and 13 (11.11%) were categorized as CA- and HA-MRSA, and 50 (42.73%) and 20 (17.09%) were categorized as CA- and HAMSSA respectively. All the CA- and HA-MRSA isolates were positive for the pvl gene, whereas in comparison, the pvl gene were detected in only four (8%) and one (5%) isolates among the CA- and the HA-MSSA isolates [Table/Fig 3].
Comparison of the Patients’ Characteristics with the MRSA and the MSSA Infections
No significant difference was seen in the MRSA patients’ demographic (gender, age-group and ethnicity) and clinical (status of the patients, settings for acquiring infections and clinical characteristics) profiles in comparison to those of the MSSA patients. The mean age ± SD of the patients with the MRSA and the MSSA infections were found as 25.23 ±16.86 and 23.59 ± 17 respectively. However, a significant number of patients with the MRSA infections had a history of antibiotic intake (36.17%) and hospitalization (34.04%) against 7 (10%) and 5(7.14%) in the MSSA patients. A majority of the infections were reported from the ICU and the burn units. In comparison, the MSSA infections were reportedly higher in the patients who were attending the orthopaedic and other units of the hospitals, whereas the incidence of both the MRSA and the MSSA infections were found to be highest in patients who attended the surgery department. A significant difference was also seen in the distribution of the PVL toxin in the MRSA (100%) and the MSSA (7.14%) isolates [Table/Fig 4].
Comparison of the Patients’ Characteristics with the PVL-MRSA and the PVL-MSSA Infections
Both the PVL positive MRSA and MSSA isolates were found to be higher in males (63.82% and 80%) as against the females (36.17% and 20%).The mean ages in both the groups were also found to lie within the similar range, i.e. 25- 30 years. (25.23±16.86 and 30± 17.39). However, the PVL-MRSA were found to be higher in the hospitalized patients (57.44%), and the PVL-MSSA were more in the out-patients (80%). The PVL-MRSA was found to be prevalent in different ethnicities and age groups, as against the PVL-MSSA [Table/Fig 5]. The clinical profiles of the PVL-MRSA patients were varied in comparison to those of PVL-MSSA, which was reported from patients with abscess (60%) and bone and joint infections (40%) only. Both the PVL-MRSA and the MSSA isolates were found to be higher in community-acquired infections (72.34% and 80%) in comparison to their incidence in hospital-acquired infections (27.65% and 20%). The highest number of PVLMRSA were isolated from the patients who attended the surgery department (51.06%), followed by the burn /ICU units (25.53%), the orthopaedic unit (12.76%), and other departments (10.63%). The PVL-MSSA isolates were present only in the patients who visited the surgery (60%) and the orthopaedic (40%) departments [Table/Fig 5].
Comparison of the Patients’ Characteristics with the PVL Positive and Negative S. aureus Infections
The prevalence of PVL positive and negative S. aureus isolates, in terms of gender, ethnicity, mean age ± SD, settings of the infections acquired, status of patients (OP/IP) and age-group wise was found to be comparable. [Table/Fig 6]. However, the PVL positive S. aureus was significantly higher in patients with a previous h/o antibiotic intake (32.69%) and hospitalization (30.76%) and in patients who were admitted in the burn/ICU units in comparison to the PVL negative group [Table/Fig 6].

Discussion

The data on the prevalence of MRSA among the Indian isolates of S. aureus is extensive in the literature [9,10]. However, the screening for the PVL toxin among them was initiated recently (11). Due to the increased infiltration of the CA-MRSA strains into the hospital setting in the recent years, and their association, mainly with the skin and soft-tissue infections, has gained importance in the detection of the PVL toxin in the MRSA population (12). In this study, the prevalence of the PVL toxin among the general S. aureus population was found to be 44.44% (52/117), which was very high as compared to that which was reported by McClure et al (8) in <5%of S. aureus population. But it was comparable to the rate (37.20%) that was reported by Lina et al (64 out of 172) (1). The three major genotypic markers that distinguish the CA from the HA-MRSA isolates: their genetic lineage (ST), the architecture of their mobile genetic element (SCCmec type) and th presence of the PVL toxin (13). Epidemiologic and clinical data have also suggested that the high virulence potential of CA-MRSA is associated with the bearing of the PVL toxin (2). Therefore, the PVL locus represents a stable genetic marker of the CA-MRSA strains (13). This study has evaluated the presence of the PVL toxin as a marker to validate the genotypic definition of the CA-MRSA isolates. The PVL toxin was detected in all the MRSA isolates, irrespective of their types. On the contrary, several studies have reported the presence of the PVL toxin in all the CA-MRSA isolates, whereas its presence was not reported from HA-MRSA , thus concluding that the PVL toxin was a reliable marker of the CA-MRSA infections (2),(11),(13). However, the findings of this study were in agreement with few reports which said that the presence of the PVL toxin could not be used as a sole marker for CA-MRSA (3),(4). Our findings suggested that though the prevalence of the PVL toxin varied in different geographical regions, in our region, the MRSA strains may be important reservoirs of the PVL toxin, which was now being slowly acquired by the MSSA strains. Among the risk factors which were studied, a previous history of hospitalization and antibiotic intake were found to be significantly associated with the MRSA infections. Similarly, many studies reported prior antibiotic use (9),(10),(14), a prior h/o of hospitalization (10),(14) as an important risk factors for the acquisition of the MRSA infections. In our study, the MRSA infections were also significantly higher in the patients who were admitted to the surgery and the burn/ICU units. Similarly, Narezkina et al found that MRSA were significantly higher in the surgery and the burn/ ICU units (15). A recent study from New Delhi too reported that MRSA accounted for ≥95% of all the S. aureus infections in burn patients (16). On the contrary, a study from Assam (17), reported that the rate of the MRSA infections was highest among the patients in the orthopaedic unit (34%), followed by the surgery department (18%). We also noticed a high difference in the distribution of the PVL toxin in the MRSA (100%) and the MSSA (7.14%) populations, whereas Campbell and colleagues (18), in 2008, reported that the presence of the PVL toxin was very high in both the groups, the MRSA (89%) and the MSSA (75%) isolates respectively. However, this distribution was found to be statistically insignificant. An increase in the incidence of the MRSA infection among the patients in the surgery and the ICU/burn units, may be due to the high association of the MRSA strains in the skin and soft tissue infections. The presence of the PVL toxin may serve as an added virulence factor, complicating the MRSA infections in patients who were admitted to these critical units (ICUs/burn) of the hospitals. In this study, we too evaluated the risk factors which were associated with the PVL-MRSA and the MSSA infections. We found that PVL-MRSA was higher in males than in females ( 63.82% and 36.17%) and mainly in younger adults (25.23±16.86 years). Similarly, some studies have reported a higher incidence of PVLMRSA in male patients than in females (4),(19). It was more prevalent in young adults, with the mean or median age ranging from 20-25 years (4),(19),(20). On the contrary, Denis et al (20), in 2005, reported a higher prevalence of PVL-MRSA in females than in males (56% and 44%). Our findings suggested that young male adults were more vulnerable to the PVL-MRSA infections, than older people and children. As in our study, Roosney et al (4), in 2007, found that a majority of the PVL-MRSA were associated with skin and soft-tissue infections, particularly from abscesses (52%). However, they reported a slightly higher incidence in Blood stream infections (16% vs. 10.63%) and a lesser incidence in pneumonia (4% vs 14.89%) than in our study. Boakes et al, in (19), found that very less PVL-MRSA was associated with bacteraemia (1.75%) and pneumonia (2.63%), as compared to that in our study. Similarly, Denis et al (20), in 2005, in Belgium, found that a majority of the PVL-MRSA were present in subcutaneous abscesses (50%), than in cellulitis (6.25%),and bacteraemia (6.25%). In agreement to this, a recent study by Muttaiyah et al (21), in 2010, too mentioned that a majority of the PVL-positive MSSA infections were reported from SSTI (48%), and bone and joint diseases (31%) and that only a very less percentage of catheter-related infections (1%), primary bacteraemia and endocarditis (0%) were reported. This data indicated that both the PVL-MRSA and MSSA were mainly associated with skin and softtissue infections, mainly abscesses. However, severe infections like BSI and pneumonia are more likely to be associated with PVLMRSA than PVL-MSSA, suggesting that the methicillin resistance in the PVL-S. aureus had contributed to the success of these strains in being associated with life-threatening infections (BSI and pneumonia) as compared to the relatively mild skin and soft-tissue infections. Based on the settings of acquiring infections, we found that a majority of the PVL-MRSA infections were community-acquired (72.34%) than hospital acquired infections (27.65%). This was comparable to a finding from the study of Roosney et al (2007) in which the community acquired infections were 76% and the hospital acquired infections were 24% (4). On the contrary, Denis et al, in 2005, in Belgium, found that a very high percentage of PVL-MRSA were community-acquired (97.56%) and that merely 2.44% were hospital-acquired (20). The changing epidemiology of the PVL-MRSA strains over the years, had begun to be reported from the HA-infections as well, may be due to their preexisting colonization with the PVL positive CA-MRSA isolates that had found a portal of entry during the invasive procedures which were performed in the hospitals. The majority of the PVLMRSA patients had a h/o of trauma and accident, hospitalization, antibiotic intake and self-reported diabetic mellitus. However, none of these risk factors were reported from the compared group. A study reported that 31.25% patients with PVL-MRSA had a h/o previous antibiotic therapy, which was comparable to our finding (34.04%), whereas 12.5% had a h/o of hospitalization, which is comparatively less as compared to our findings (36.17%) (20). These were the established risk factors for the acquisition of the MRSA infections were and found to be associated with the PVLMRSA infections as well. These showed no significant alterations in the established risk factors, even with the acquisition of the PVL toxin in these groups (MRSA and MSSA), but the presence of the PVL toxin may serve as an added virulence factor, complicating the MRSA infections. However, the incomparable numbers of PVL-MRSA and MSSA may limit these findings, which need to be further validated. In the present study, the prevalence of PVL-S. aureus was found to be 44.44%. In comparison, a higher prevalence of over 90%. was reported by Campbell et al (18) and a lower prevalence of < 5% was reported by McClure et al (8). No significance difference was seen in the patient’s demographic and clinical profiles, except that as compared to the above groups, the patients with a previous h/o antibiotic intake, hospitalization and those who were admitted to the surgical and burn/ICU units were more prone to the PVL-S. aureus infections. The mean age of the patients was comparable in both the groups, mainly those of younger adults. On the contrary, in a recent study by Muttaiyah et al (2010), ethnicity, younger age, and community-onset infections, were all found to be independently associated with the presence of the PVL-positive MSSA infections (21). In conclusion, the high prevalence of the PVL toxin among the S. aureus population and a higher prevalence which was reported from the MRSA isolates irrespective of their types ie. communityassociated (CA) or hospital-associated(HA) MRSA, suggested that the presence of the PVL toxin was no longer a reliable marker for the CA-MRSA infections. Rather, it may be hypothesized that the MRSA strains may be the important reservoirs of the PVL toxin which was slowly being acquired by the MSSA strains. This study also indicated that PVL appeared to be a possible virulence factor that caused the success of these MRSA strains in being associated with infections which ranged from mild skin and softtissue infections to life-threatening infections like BSI and burn infections. Therefore, specific therapeutic approaches which could target the PVL toxin may be helpful in treating the severe PVLMRSA infections in these vulnerable groups.

Acknowledgement

We would like to thank the Dean, Sikkim Manipal Institute of Medical Sciences. We would also like to acknowledge Mr. Arum Prakash (CEO), Dr. Samar Husain Naqvi and Ms Shilpie Biswas of Genetix-Biotech, New Delhi.

DOI and Others

DOI: JCDR/2012/3981:1986

Financial OR OTHER COMPETING INTERESTS:
None.


Date of Submission: Sep 24, 2011
Date of Peer Review: Dec 31, 2011
Date of Acceptance: Jan 17, 2012
Date of Publishing: May 01, 2012