Molecular characterization and antimicrobial resistance of Enterococcus faecalis isolated from seafood samples

Abstract Background Enterococcus faecalis is considered an opportunistic foodborne pathogen. The present study aimed to assess the prevalence, antimicrobial resistance, virulence characters, and molecular typing of E. faecalis strains isolated from seafood samples. Methods Two hundred and seventy‐six seafood samples were collected. E. faecalis was isolated from samples using bacterial culture. Furthermore, the disk diffusion assessed their antimicrobial resistance. Also, the distribution of virulence factors was determined using polymerase chain reaction (PCR) assay. Random amplified polymorphic DNA (RAPD) method was used for their molecular typing. Results Fifty‐six of 276 (20.2%) seafood samples were contaminated with E. faecalis. Fish harboured the highest contamination rate (30.0%). Isolates harboured the highest resistance rate towards oxacillin (100%), tetracycline (100%), erythromycin (100%), cefoxitin (89.2%), cefazolin (87.5%), trimethoprim‐sulfamethoxazole (85.7%), rifampin (69.6%), clindamycin (69.6%), and gentamicin (64.2%) antimicrobials. Efa (100%), ebpA (89.2%), ebpB (58.9%), ebpC (53.5%), and esp (51.7%) were the most commonly detected virulence factors among E. faecalis isolates. RAPD–PCR analysis showed 11 different molecular clusters considering the closeness of more than 80%. Conclusion Seafood samples were considered reservoirs of virulence and resistant E. faecalis strains. Different molecular clusters of isolates may reflect their diverse sources of contamination.

E. faecalis strains cause serious infections, including gastrointestinal and urinary tract infections, meningitis, bacteraemia, and periodontitis (Abat et al., 2016;Ma et al., 2021;Prajsnar et al., 2013;Said et al., 2021). The severity and pathogenicity of diseases caused by these bacteria are higher in the presence of well-defined virulence factors and toxins (Goh et al., 2017;Wu et al., 2020). Clinical investigations showed that enterococcal surface protein (esp), Fsr regulator locus responsible for bacterial quorum sensing (encoded by fsrA, fsrB, and fsrC genes), structural pilin genes (ebpA, ebpB, and ebpC), cytolysin (cylL and cylS), and endocarditis-specific antigen (efa) are the most important virulence factors of the bacterium responsible for adherence, colonization, evasion, enzymes extracellular production, biofilm development and pathogenicity, and severity of subsequent infections (Goh et al., 2017;Bin-Asif & Abid Ali, 2019). Destructiveness variables of Enterococcus spp. may contribute to competition with other microbes, colonization of the have, resistance against defence instruments of the have, and generation of obsessive changes specifically through the generation of poisons or by implication through acceptance of aggravation (Kayaoglu & Ørstavik, 2004). Infections caused by E. faecalis are mainly hard to treat by common antimicrobials (Shiadeh et al., 2019). Surveys showed the high resistance rate of E. faecalis clinical strains towards commonly used antimicrobials, particularly penicillins, tetracyclines, aminoglycosides, phenicols, cephalosporins, penicillins, and macrolides (Ahmed & Baptiste, 2018). Therefore, the assessment of antimicrobial resistance of E. faecalis strains can directly introduce the most suitable antimicrobial agents for further therapeutic options (Johnston & Jaykus, 2004;Perera et al., 2020).
According to the high pathogenicity of E. faecalis as an opportunist foodborne pathogen and the absence of epidemiological surveys in this field, the present research was performed to evaluate the prevalence, antimicrobial resistance, virulence factors distribution, and molecular typing of E. faecalis bacteria isolated from seafood samples.
A programmable DNA thermo-cycler (Eppendorf Mastercycler 5330; Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany) was used in all PCR reactions. Ten microliters of PCR product were exposed to elec-

Molecular typing
Random amplified polymorphic DNA (RAPD)-PCR analysis was done using the primer M13(5′-GAGGGTGGCGGTTCT-3′) as described previously (Versalovic & Lupski, 2002). Grouping of the RAPD-PCR patterns was performed using the UPGMA cluster analysis. The strains grouping coefficients of similarity of 80% for RAPD typing were applied.

Statistical analysis
Data obtained in this survey were analyzed using the SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). For this purpose, data were analyzed by χ 2 test and Fisher's exact two-tailed tests, and significant relationships and differences between data were determined. p-Value < 0.05 was considered as a statistically significant level (Dehkordi et al., 2011b;Dehkordi, Parsaei, et al., 2012;Dehkordi, Haghighi Borujeni, et al., 2013;Dehkordi, Khamesipour, et al., 2014;Dehkordi, Tirgir, et al., 2017).   shown, all isolates had resistance to at least two different antibiotic agents. Findings showed that 30.35% of E. faecalis had resistance against more than six antibiotic agents.   Figure 3 shows the RAPD-PCR-based typing of E.

RAPD-PCR typing
faecalis isolates. In the analysis of 18 isolates with RAPD marker, the isolates were placed in 11 profiles considering the closeness of more than 80%, among which isolates 3, 12-18 were placed in a separate profile. Profile A with five isolates 2, 5-8 is considered as the dominant clone. Isolates 5 and 6 in this category have 100% affinity.

DISCUSSION
The present study was performed to assess the prevalence, antimi-  Most isolates in this study were resistant to common antimicrobial agents used in Iran, particularly oxacillin, tetracycline, erythromycin, cefoxitin, cefazolin, trimethoprim-sulfamethoxazole, rifampin, clindamycin, and gentamicin. Irregular and unauthorized prescription of antimicrobial agents is the probable reason for the high resistance rate.
As some isolates harbored a high resistance towards human-based antimicrobial agents (those are basically used to treat human infectious diseases), it can be indirectly concluded that these isolates originated from infected staffs of seafood sales and processing centres and tetracycline (50%-70%) antimicrobials. Badul et al. (2021) Han et al. (2011) and Barbosa et al. (2014). Jahan and Holley (2014) reported the high distribution of esp, efa, gelE, ace, and agg virulence determinants in E.
faecalis recovered from meat products. Most isolates in this research harbored esp and efa virulence factors. Both factors are responsible for bacterial persistence and colonization in host cells. Esp gene was also detected in some isolates. This gene is responsible for the pathogenicity island, biofilm formation, and dynamics of antibiotic release (Leavis et al., 2004). Esp gene also had some effects in the occurrence of ampicillin, ciprofloxacin, and imipenem resistance (Billström et al., 2008) and vancomycin resistance (Ochoa et al., 2013) in Enterococcus strains.
The role of fsr complexes in biofilm formation was also reported previously (Cybulska et al., 2020). In an Indian survey (Biswas et al., 2019), isolates of the present survey. The applied RAPD-PCR method was also reported to be sensitive and practical for the molecular typing of clinical isolates of E. faecalis (Banerjee, 2013;Emaneini et al., 2016).

CONCLUSION
E. faecalis strains were detected in fish, shrimp, and lobster samples collected from the Persian Gulf. Isolates harbored both antibiotic resistance and virulence markers, which may show their high pathogenicity. In addition, isolates were classified into 11 different genetic clus-