Distribution of virulence genes and SCCmec types among methicillin-resistant Staphylococcus aureus of clinical and environmental origin: a study from community of Assam, India

This study was designed to discover the dissemination of virulence genes in Methicillin-resistant Staphylococcus aureus from clinical, community and environmental settings. This study includes 1165 isolates collected from hospital, community and environmental settings. Among them sixty three were confirmed as MRSA with varied SCCmec types viz; type I, type II, type III, type IV, type V, type VI, type VII, type VIII and type XII. The virulence gene such as sea (n = 54), seb (n = 21), eta (n = 27), etb (n = 2), cna (n = 24), ica (n = 2) and tst (n = 30) was also revealed from this study. The study underscores coexistence of resistance cassette and virulence genes among clinical and environment isolates which is first of its kind from this part of the world.


Introduction
Incidence of community associated as well as hospital acquired Staphylococcus aureus has spread its infections over last few decades. S. aureus has the ability to cause a wide variety of infections ranging from septicaemia to toxic shock syndrome [1]. In 2011, a study from a high altitude area of Northeast India showed 18.4% MRSA among clinical specimen and carrier screening samples [2]. Another study from Northeast India found 47% of MRSA, of which 63.8% were hospital acquired and 36.2% were community acquired [3]. A previous study from our research laboratory showed 21.8% occurrence rate of MRSA from Hospital settings [4]. Rich diversification in mobile genetic element Staphylococcus cassette chromosome mec (SCCmec) leads to the expansion of antibiotic resistance determinants as well as virulence factors, which is naturally designed for the stable maintenance of the core genome environment. These virulence determinants are the genes that facilitate the successful colonisation and endurance of the organism or interrupt with the defence system of the host. [5] The dissemination of SCCmec types and along with virulence determinants in Methicillin Resistant S.aureus isolates among the hospital as well as environmental settings constitute a vast reservoir for potential spread of infection. It is important to study isolates from hospital, community and environment as this would give an insight of potential transmission route from hospital-community-environment and vice-versa. In India, SCCmec III, IV and V types were reported in 2010 from Mumbai within hospital and community acquired MRSA strains (ST239, ST22 and ST772). SCCmec types IV & V were related to community associated MRSA [6]. In a recent study from Chennai, SCCmec types I, III, IV, V were detected of which SCCmec type V was predominant [7]. The study showed 7.17% prevalence rate of MRSA in community settings whereas 81.67% from hospital environment [7]. Accumulation of toxins, virulence factors, surface proteins and enzymes have became an attributing factor of S. aureus along with the rapid development of multidrug resistance. Most of the S.aureus' pathogenicity depends on the surface proteins which help in attachment and colonisation, while the cellular protein, proteases and toxins helps in inhibiting phagocytosis thus leaving immune response ineffective [1]. The enterotoxins in S.aureus are most commonly found toxin associated with staphylococcal food poisoning which is regulated by two enterotoxin genes, sea and seb [8]. A serine protease enzyme such as exfoliative toxin harbouring gene eta and etb which is associated with the loss of keratinocytes, cell-cell adhesion, blister formation etc. [9]. Collagen binding adhesion (CNA) which is a virulence factor belongs to the microbial surface component recognizing adhesive matrix molecule (MSCRAMM) adhesions family, which functions in adhesion to the molecules regulated by cna gene [10]. Biofilm is generally associated with antimicrobial resistance. The development of biofilm is synthesised by Intercellular adhesion cluster harbouring ica gene [11]. Virulence factor, toxic shock syndrome toxin (TSST) is an exoprotein that belongs to pyrogenic toxin super antigen family (PTAgs) which is encoded by tst gene. The presence of which affects the cells' immune system which eventually leads to death [12]. Lastly, the virulence factor fibronectin protein encoded by two genes fnbA and fnbB plays a very important role in adhesion to cells as well as also favours internalization by cells which leads to intercellular persistence and chronic staphylococcal infections [13]. So far no study had been carried out to explore the detail in involvement of virulence gene in S.aureus in this study area. Therefore, a comparative analysis on prevalence of virulence genes in the Methicillin Resistant S. aureus isolates among hospital, community as well as environmental setting is undertaken in the present study.

Study location
Isolates were collected from hospital, community and environment from three districts of Southern Assam, India. A total of 1165 isolates were collected (550 isolates from hospital and 329 isolates from community settings and 286 isolates from the environmental setting) for the study. Clinical isolates were collected from the patient with wound infections, UTI, Post surgical infections from various wards and those visited the clinic of Silchar Medical College and Hospital, Assam, India while the environmental samples were collected from the soil (90 samples from 30 different sites), water (50 samples from 10 different sites) and sewage water (146 samples from 40 different sites) of the study area for a period of one year from June 2017 to May 2018.

Identification and characterization of Isolates
Isolates were cultured on Mannitol Salt broth for specifying the growth of Staphylococcal isolates and incubated for 24 h for observing the visible growth of the organism. Organisms are then spread onto MRSA Chrom agar, Baird Parker agar (Himedia) and the plates were incubated overnight. The DNA of the isolates was extracted by using the phenol chloroform method [14]. The presumptive S. aureus isolates were confirmed through 16 s rDNA sequence analysis. Additionally phenotypic characterization was performed through gram staining, rapid coagulase (Rapid Hiaureus Coagulase confirmation kit, Himedia) and latex agglutination testing (HiStaph ™ Latex Test Kit, Himedia). Screening of the methicillin resistance was performed by cefoxitin agar disk diffusion and further genotypically confirmed by coa, nuc, femB, mecA and mecC PCR [15][16][17][18][19]. The reaction conditions of the following genes are described in Table 1.

Determination of virulence factor by multiplex PCR assay
Genes encoding virulence factors such as sea (entero-toxinA), seb (enterotoxinB), eta (exfoliative toxin A), etb (exfoliative toxin B), cna (collagen binding adhesion), fnb (fibronectin binding protein), ica (intercellular adhesion protein), tst (toxic shock syndrome) were studied by multiplex PCR amplification for all the MRSA isolates. Virulence specific primers were used for the study (Table 1). Three sets of multiplex PCR were employed for the study which was initiated with the 25 μl of the reaction mixture which includes (12.5 μl of the Go green Taq mixture (Promega, India), 10 pmol of each primer, nuclease free water and 50 pmol of the DNA product) with varied reaction conditions, the details of which is given in Table 1.

Distribution of toxin encoded virulence genes among SCCmec types:
SCCmec typing was done for the isolates by a set of three multiplex PCR assays. The details of the assays performed are as described in the previous study [21].

Multilocus sequence typing of SCCmec types carrying Virulence genes:
To establish the relatedness of SCCmec types with the virulence factors, amplification of MLST gene was performed according to Enright et al. 2000 for all the MRSA isolates carrying SCCmec types in combination with virulence gene. Seven housekeeping genes were targetted for the MLST study and the primers were used for amplification by targeting the genes viz; arcC, aroE, glp, gmk, pta, tpi and yqiL. The sequence were obtained and analysed in Centre for Genomic Epidemiology MLST 2.0 website (https ://cge.dtu.dk/servi ces/MLST-2.0) [22].

Result
Among 1165 samples collected from different location, 330 staphylococcal isolates were isolated, of which 123 isolates were confirmed as S. aureus. Further, presence of femB and mecA genes was also observed and sixty three isolates exhibited methicillin resistance. Antibiotic susceptibility testing showed linezolid (66.6%) was the  1%). It was also observed that, environmental sources acted as reservoir as majority of the sea gene (47.6%) was prevailing, followed by tst (28.57%) and cna (26.98%) ( Table 2). The MRSA isolates having combination of the virulence genes were also focused in this study and it was found that twenty nine different combination of virulence  genes coexisted. Moreover it was also observed from the current study that three isolates were having a combination of six virulence genes, seven isolates with a combination of five virulence genes, and thirteen isolates showed different combinations with four virulence genes whereas combination three virulence genes were observed in 19 isolates. (Additional file 1: Table S1).

Discussion
Virulence factors play a key step for pathogenic invasion leading to staphylococcal infection in the hospital, community and in environmental settings. Rich diversity of virulence associated genes within S. aureus is reported worldwide [23,24]. It was observed in the present study that the virulence determinants such as sea, tst and eta gene were found to be more predominant in the study isolates.  [26,27]. Studies from different countries revealed that sea is the most common enterotoxin recovered followed by seb and sed along with different Staphylococcal enterotoxins (SE's) associated with outbreaks [28]. Studies from Korea by Lim et al. 2010, stated that Staphylococcal enterotoxin (seg, sei, sec) genes and toxic shock syndrome toxin (tst) genes were most common in clinical MRSA isolates [29].  [33,34]. Current study observed sixty three MRSA isolates of which 85.71% of the isolates were found to harbour enterotoxin A (sea) gene. This rate is found to be higher than the previous studies [35,36] [6]. Also in a recent study from Chennai, reported the presence of SCCmec types I, III, IV & V of which SCCmec type V was predominant [7]. This study also showed high prevalence rate of MRSA (81.67%) in the hospital environment as compared to community (7.17%). While our study from Northeastern part of India detected the presence of SCCmec types I, II, III, IVa, V, VI, VII, VIII & XII from hospital, community and environmental settings. Another study by Carvalho in 2019, from Brazil has detected SCCmec types I, IVa, & V and virulence genes from Intensive Care Unit (ICU), equipment surfaces and healthy children [37]. Mobile genetic element (SCCmec) carries both resistance as well as virulence genes. It has been found from the study of Lim et al. 2010 that 97.6% of SCCmec type II carried sec, seg, sei and tst gene; 73% of SCCmec type III strains carried sea gene and 89.7% of SCCmec type IV strain carried sec, seg, sei genes [29]. While our study reveals that 28.2% of SCCmec type II isolates carried sea, seb, cna, tst and eta genes, 10% of SCCmec type III carried sea, seb, cna and tst gene, 34.1% of SCCmec type V was found to carry sea, seb, ica, cna, tst eta and etb genes and 20% of SCCmec type VII carried sea, seb, cna, tst, eta and etb genes. However the rest isolates with SCCmec types I, IV, VI, VIII and XII showed less prevalence rate as compared to the above. The study conducted by Liang et al. 2016 showed that ST239-SCCmecAIII-t37 clone was more prevalent one as reported from china whereas our current study showed that ST2884-SCCmec type V was more predominant than the other sequence type [33].
This study involved a comparative analysis of virulence genes found to be prevailing in hospital, community as well as in environmental settings. It was recorded that majority of the isolates containing virulence gene were found in the environmental sources which is in contrast to the other studies where virulence genes were observed in clinical settings [23,34] and underscores the risk of acting environmental sources as reservoir.

Limitations
This study warrants a proteomic approach to analyse the mode of transfer of virulence gene which may be from environment setting to the hospital and community origin and vice versa.