Microflora of the fish in retail outlets are a reflection of general contamination in the aquatic environment as well as post-harvest handling contamination.¹ Harvesting, storage, distribution, processing, and preservation of seafood are the major handling practice responsible for the transmission of microbes from fish to human and vice versa.^2-6 The possibility of transmission of MRSA between humans and food has been described.^7,8 Similarly, augmented reports have established MRSA in various food-producing industries. Since 1961, MRSA has been a huge problem in the health care settings owing to it gaining resistance to the most potent antibiotics.⁹ In seafood, MRSA enters by post-harvest contamination and flourishes as a persistent pathogen. However, genetic level comparison of the MRSA isolates revealed that uniqueness existed between the hospital, community, livestock, and environmental sectors.^6,10-12 Hence, specific strains of MRSA have become persistent in a particular population or environment.⁵ In contrast to the S. aureus which is already an established bacterial population in the environment, MRSA is recently evolving as a persistent population in the food sector. Hence, there is a possibility of using this microbial population as a virtual networking tool, and thereby link the MRSA transmission in the seafood supply chain as well as assigning geographical identity. Even though bacterial source tracking studies are implemented for various indicator bacteria and gram-negative bacteria, benefit of using MRSA as source tracking tool was not performed.¹³

Based on MRSA strain, its origin and dissemination can be investigated through local epidemiological approach. Generally, micro-variation analyses are being used for local epidemiological studies in a particular environment/population and in outbreak investigations.¹⁴ In the case of MRSA, genetic typing tools available for local and global epidemiology are staphylococcal protein A (spa) typing, clumping factor B (Clfb) typing, capsular protein (CP) typing. accessory gene regulator (agr) typing pulsed-field gel electrophoresis (PFGE) and Multi-locus sequence typing (MLST).^5,15-18 However, the tools become more popular because of their user friendliness and economic viability.¹⁹ The applicability of these tools for microbial source tracking of contamination was proven in hospital sector and in seafood.^5,20,21

Hence, in the present investigation, spa typing, virulence factor typing (clfb + CP) and agr typing were used as characterization tools as well as for tracking the contamination. The key idea behind this study is to validate the tools for source tracking the MRSAs in the seafood with earlier documented different sets of landing centre and retail markets samples⁵ which can be used as the virtual networking tool for a better predicting the origin of the food from geographically distinct locations based on the strain identification.

The sampling plan consisted of two traditional fish landing centres and three retail fish markets which include unorganized roadside as well as organized markets of Kottayam District, Kerala, India,⁵ not covered in the earlier study (Figure 1). The distances between fish landing centre and retail fish markets ranged from 4.5 km to 29 km. Sampling was carried out early in the morning at fish landing centre followed by fish markets at random. The study was carried out in four batches with an average of 7 samples of water and fishes from fish landing centre and 7 samples from the retail market. The sampling strategy, based on the availability, envisages to include one finfish, shellfish, mollusc, water and ice used at every landing centre and retail fish market. Totally, 72 samples were collected during the entire study, which includes finfish, crustaceans, molluscs, water and ice (Table 1). Samples were collected in sterile bags (labelled landing centre and fish market wise) and brought to the laboratory in the insulated icebox and processed within four hours of sample collection.

Table (1):
Details of samples collected in Batch I to IV between fish Landing centres and retail markets of Kottayam District, Kerala

MRSA was isolated and identified from the seafood samples as per the Bacteriological Analytical Manual with modifications.^3,22 Initially, all the isolates were confirmed conventionally by Oxacillin agar dilution method²³ then molecularly by multiplex PCR (mPCR) targeted genes specific for S. aureus (nuc), MRSA (mec) and 16S rDNA for Staphylococci.²⁴ Further, the molecular tools viz. spa typing, virulence Factor typing (clfb + CP) and agr typing were used to characterize the strains of MRSA isolated. In all the PCR assays, S. aureus ATCC 43300 was used as MRSA positive control, S. aureus ATCC 29213 was used as negative control for methicillin resistance, and no template control was used for reaction negative control.

Using spa-1113f and spa-1514r primers, the variable region within the conserved region of the spa gene was amplified for detecting the presence of spa gene of variable sizes.²⁵ The amplified region was eluted and outsourced for sanger sequencing (Genespec Pvt. Ltd., India) and the spa types were deduced from ridom server as well as Fortinbras software available in the public domain and same was used to characterize the isolates from all the sectors of the source without any modification.⁴

A virulence factor typing was carried out with the combination of clfb and cap to characterize the MRSA. The pathogenesis is significantly influenced by capsular polysaccharide (CP), which is produced by S. aureus. More than 80% of clinical isolates produce either CP5 or CP8 capsular polysaccharides.^26,27 DNA templates (3 µl) were added to 22 µl of PCR mix containing 1x conc. of Master Mix (Thermo Scientific) and 0.4 µM of each primer. PCR conditions included an initial denaturation stage at 94 °C for 5 minutes, 35 cycles of 94 °C for 30 seconds, {49.0 °C for 30 seconds (cap5 gene)/46.0 for 30 seconds (cap8 gene)}, and 72 °C for 1 minute, and a final extension step at 72 °C for 7 minutes comprised the amplification cycle.

PCR was performed for agr typing for all isolates using primers agr I, agr II, agr III, and agr IV in a 25 µl reaction volume containing 0.4 mM dNTP’s, 0.6 µM concentration of each primer (agr pan F, agr pan I, agr pan II, agr pan III and agr pan IV, respectively), 2.5 µl of 10X buffer, 3 mM MgCl₂, 1 U/µl of EX Taq DNA polymerase (Takara) and template DNA of 3 µl. PCR conditions included an initial denaturation step at 94 °C for 5 minutes, 35 cycles of 94 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 1 minute, and a final extension step at 72 °C for 10 minutes.^28,29

The discriminatory power of each method in delineating seafood environment MRSA were calculated using the method described.³⁰ Briefly calculating how many types of MRSA can be categorized from each typing method with the total number of strains used for characterization was calculated based on the formula described below,

D = 1 – 1/(N(N-1) ∑^s_j=1 x_j (x_j -1)

Where D = Index of Discriminatory Power, N = Number of unrelated strains tested, S = Number of different types and x_j = Number of strains belonging to the j^th type”.

To check for the existence of MRSA isolates, 74 samples from different fish landing centres and retail fish marketplaces were used in this investigation. The multiplex PCR were used for the molecular confirmation of MRSA by targeting 16S rRNA of 756 bp (Genus specific for Staphylococcus), nuc of 279 bp (Species specific for S. aureus), and mecA of 310 bp (a gene for methicillin resistance).²⁴ The isolates which showed all three genes were confirmed as MRSA.

A total of 12.16% of samples showed positive result for MRSA. This included 6.75% from the fish landing centres (LC) and 5.40% from the retail fish markets (RM). Among that 1.35% of samples were isolated from LC2, 1.35% from RM1, 5.40% from LC1 and 4.05% from RM3. LC2 and RM1 showed the lowest number of MRSA positive samples. The study indicates that MRSA is becoming persistent in the seafood niche and understanding its diversity on a larger scale might be potentially useful for tracking studies (Table 2). Before the strains of MRSA get established as a persistent population in the seafood sector, the strain assignment would provide more scope for the networking of food transmission.

The presence of clfB and cap5 or cap8 indicates the potential colonization of these MRSA’s in the vestibulum nasi of human beings, adhesion to squamous epithelium, adhesion to a or b chains of human fibrinogen and also aids the organism to survive from the uptake and killing by host neutrophils.^31,32 Based on the presence of virulence factors (cap and clfb), the MRSA were categorized to cap5 & clfb and cap8 & clfb (Table 2). Accessory gene regulatory (agr) genes are the important regulator for controlling the expression of virulence factors in S. aureus and based on the sequence variations in the genes, agr are divided into agrC (autoinducing peptide) and agrD (cyclic AIP), and further categorized into four groups (agr I, agr II, agr III, and agr IV). Based on the agr typing, the MRSAs isolated in this study belonged to only agrI (Table 2).

Table (2):
Spa Types identified in fish landing centre and retail markets

The samples of fish/clam or ice detected with MRSA and their spa types; Combination virulence and agr profiles identified in two landing centres and two retail markets

Sanger sequence analysis showed 5 different spa types among the 36 isolates of MRSA viz., spa types t3841, t334, t127, t304 and t311. According to the spa typing results, t3841, t334 and t304 were the predominant spa types. The strains t3841 and t304 were found in both LC1 and LC2 and in RM1 and RM3 samples. Additionally, LC1 harboured t334 and t311 spa types and RM3 harboured t127 and t334 spa types. Pearl spot from LC1, Indian prawns from LC2 and False Trevally from RM3 carried 2 types of MRSA clones in a single seafood sample (Figure 2). This shows the diversity of the pathogenic organism in a single sample itself and t304 was present in both the landing centres and retail markets. This implicates the dissemination of microorganisms from the landing centre to the adjoining retail markets. t311 isolated from pearl spot collected from LC1 showed no evidence of dissemination; similarly, t127 isolated from False Trevally also showed no evidence of the origin of the source.

Clfb typing along with spa typing have the capability of discriminating isolates within clonal groups. ^{13, 33} The most predominant capsular polysaccharide serotypes are CP5 and CP8 ³⁴ and in the study both of these types were identified as CP5 (t334) at 25% and CP8 (t304, t3841, t304 and t127) at 75% incidence.

Among MRSA isolates, spa typing had the highest discriminating power (D = 0.758), followed by agr typing (D = 0.4) and cap typing (D = 0.3555). A D value of 0.0 in clfb typing implies that each and every member of a strain population belonged to the same type (Table 3). Poor hygiene and inadequate sanitization during the processing leads to MRSA contamination.³⁵ in ready to eat, ready to cook and fresh seafood.^3-6,36,37 MRSA is evolving in the seafood environment and it is becoming a persistent population.

Table (3):
Discriminatory Index of Each Typing Methods

One of the best methods for controlling and preventing the spread is molecular typing. The ideal molecular typing test for epidemiological research should be simple to use, affordable, highly reproducible, quick, and simple to interpret.³⁸ The present study represents the simultaneous tool comparison for molecular source tracking of foodborne antibiotic-resistant pathogen (MRSA) between landing centres and retail markets, and this provides an insight into the diversity of genotypes of MRSA. The various MRSA typing tools such as PFGE, MLST, SCCmec typing are expensive, time-consuming and may be difficult to interpret.²⁵ But the spa typing of MRSA allows speed, costs low and provides comparability of data which are the essential factors for the source tracking of MRSA in seafood chain. clfB, CP (5&8) and agr typing methods could explain the clinical characteristics of the MRSA isolates.³⁹ The combined results of spa typing and clfB typing are highly stable and has high discriminatory power comparatively to PFGE and whole-genome microarray analysis.¹⁴ The CP typing plays a major role in the immunotherapy against pathogen and the prevalence of capsular types 5 and 8 varied between the isolates.⁴⁰ There is an obvious correlation between capsular type and genotype, according to the findings of the CP and spa typing tests. Some researchers have found that the expression of the agr gene is differentially regulated in various S. aureus groups and is involved in the regulation of the production of bacterial virulence factors. Agr I type isolates make up 77.77% of all isolates in this investigation. The key idea behind this study is to assess the seafood contamination by setting MRSA as candidate organism and spa typing as a molecular marker to assess the diversity and also transmission between the landing centre and its adjoining retail markets. Once the diversity of all the fish landing centres and retail fish markets are established for MRSA, it can be virtually networked for understanding the movement of the food between geographical locations using MRSA and spa typing tool. Our previous study was carried out as source tracking the contamination of MRSA between landing centre and retail market of Kottayam and Ernakulam districts with the help of MLST and spa typing tool, however in the present study only Kottayam district landing centres and retail markets were studied with spa typing and other PCR based virulence markers tool.⁴

The bacterial persistence was initially observed in Staphylococci with penicillin exposure in early 1942 and the surviving bacterium became the persistent population over the years.⁴¹ So, the persistent population can rapidly adapt to the antibiotic stress by triggering gene responses linked with various stressors.⁴² The increased use of antibiotics eventually leads to the sudden spread of MRSA and its possible transmission is greatly dependent on its persistence in the environment.⁴³ The rapid increase of MRSA clones in the aquatic environment could be an indication that MRSA clones are getting established as persistent and therefore can be used as markers. Only a comprehensive investigation to identify the source and dissemination pattern of contamination could assure seafood safety.

In this persistent MRSA, diversity is documented completely between various landing centres and retails markets in the country. In this virtual networking process, the genetic identity of this persistent and evolving MRSA needs to be documented in over 1500 fish landing centres across India along with their adjoining retail fish markets so that it will provide valuable information. This study also compares the tools to study the molecular epidemiology of MRSA isolates that can be used as a molecular source tracking indicator. This study indicates 12.16% of samples contain MRSA isolates and can be used as a benchmark for future studies. In the extended period of time, the same pathogen can be used as an indicator pathogen for molecular source tracking. Individual molecular markers could not provide 100% source identification due to a lack of sensitivity and specificity.⁴⁴ Although MRSA does not constitute the normal flora of fresh seafood, it could become a part of persistent populations.⁴⁵ By considering the current population of MRSA as a molecular marker, the emergence and transformation of MRSA as a persistent population can be identified. The use of spa typing alone could provide evidence of the prevalence, diversity, and dissemination of MRSA clones between the landing centres and retail markets. Very few studies have focused on MRSA in seafood and many of them concentrated only on the prevalence and diversity of MRSA.^46,47 Other studies mainly explain the antibiotic resistance, epidemiological types and virulence profile of MRSA isolated from marine fish.⁴⁸ Source tracking study explicitly used on animal samples for phylogenetic tracking of LA-MRSA.⁴⁹ Hence, in this study, we have used this organism as an indicator organism and we compared molecular tools for characterizing them to strain type, and also tried to document these types of MRSA available in the sector. This molecular tool not only documents diversity but also confirms the movement of the seafood from one landing centre to retail markets. This would make this study unique and relevant from various existing studies. This is the first study to conceptualize the food transmission with the bacterial source tracking tool being MRSA and its molecular typing tools.

The study was conducted for 74 samples representing two fish landing centres and three retail fish markets, in order to better understand the local epidemiological picture. However, understanding the broader epidemiology, i.e. between the districts and between the states, there shall be a network project encompassing wider geographical regions covering all the major fish landing centres and retail fish markets representing the major points of seafood harvest and distribution. Covering wider geographical region with representative landing centres and retail markets will validate this tool’s suitability in the virtual networking of seafood transmission. Also, the study may be explored for the use of several phenotypic fingerprinting tools like antimicrobial resistance to supplement the source tracking protocol.