Biofilm-associated virulence in Enterococcus isolates from poultry: Comparative gene distribution and statistical gene-phenotype associations

Introduction

Enterococci are Gram-positive, facultatively anaerobic microorganisms that commonly inhabit the intestinal tract of poultry and other animals. During poultry slaughter and processing, contamination of meat products may occur, frequently facilitated by exposure to intestinal microflora. Owing to their ability to survive adverse environmental conditions, such as extreme temperatures, high salinity, and desiccation, these organisms are capable of thriving during processing, storage, and even under conditions of inadequate sanitation. Their biofilm-forming capability provides additional survival advantages, particularly on foodcontact surfaces such as stainless steel and plastic used in processing lines. These biofilms can protect bacterial cells against commonly applied sanitizers and stressors, facilitating persistence and potential cross-contamination (Chavez, 2007; Son et al., 2024) [2,17] . Several virulence genes have been implicated in the pathogenicity and survival of Enterococcus within food systems. Among these gelE, encoding gelatinase, has been widely reported; asa1 contributes to aggregation during colonization; esp encodes enterococcal surface protein, which enhances adherence and biofilm development; while cylA is associated with cytolysin production, which may support colonization under stress. However, the expression and regulatory impact of these genes on biofilm development remain poorly understood. Previous studies often described descriptive associations between virulence markers and biofilm phenotype but lacked statistical support, reducing biological inference reliability. Therefore, this study aimed to investigate not only the presence of virulence-associated genes but also their correlation with biofilm formation through statistical assessment.

Materials and Methods

Sampling

A total of 150 poultry meat samples were collected aseptically from retail outlets over six months. Samples were transported under refrigeration conditions and processed within four hours to minimize contamination bias.

Isolation and Biochemical Identification

Isolation of enterococci was performed using selective media including Slanetz-Bartley agar and Citrate Azide Tween Carbonate agar. Colonies exhibiting characteristic morphology were subjected to biochemical tests such as PYR and catalase reaction for further confirmation.

Molecular Confirmation and PCR Analysis

Genomic DNA from Enterococcus isolates was extracted using the snap-chill method. PCR amplification of the tuf gene (genus-level) followed published protocols by Dutka-Malen et al. (1995) [4] . Primer sequences and specifications are listed in Table 1. PCR reactions (25 µl) contained 12.5 µl 2X master mix, 1 µl of each primer (10 pmol), 5 µl DNA template, and nucleasefree water. Cycling conditions for tuf included: 94 °C for 5 min; 30 cycles of 94 °C for 30 s, 50 °C for 90 s, 72 °C for 60 s; final extension at 72 °C for 10 min. PCR products were resolved on 2% agarose gels and visualized using a gel documentation system. (1999) [7]

Optimization of PCR for Virulence Associated Genes of Enterococcus species isolates

The isolates of Enterococcus spp. were screened for the detection of major virulence-associated genes by targeting asa1, gelE, cylA, esp and hyl genes (Table 2). The DNA from isolates was extracted by boiling method. A loopful of pure culture was suspended in 100 μl of DNAse and RNAse free Milli-Q water in a sterilized eppendorf tube. The suspension was vortexed and then heated at 95˚C for 10 min in the thermal cycler. This was then centrifuged at 10000 rpm for 6 min in order to settle down the cell debris. The upper aqueous phase was transferred to another eppendorf tube and this was used as a DNA template for PCR. The reaction mixture was prepared in 200 μl PCR tubes which contained 12.5μl PCR master mix (2X), 1 μl each of forward and reverse primer (10pmol), 5.5 μl nuclease free water and 5 μl templates. The details of thermal profiling of PCR are mentioned in Table 3described by Pangtey (2017) [12] with slightly modifications. The final amplified product was analysed by agarose gel electrophoresis on 2% agarose gel and visualized under gel documentation system.

Biofilm Detection

Biofilm formation was assessed using the Congo Red Agar method. Colonies were visually examined and categorized as strong biofilm producers if black and dry in appearance, moderate producers when exhibiting slightly black mucoid colonies, and weak or non-producers upon presentation of red or smooth colony morphology.

Statistical Analysis

Statistical analysis was performed using Chi-square test, and Fisher's exact test when cell frequencies were fewer than five, to determine whether virulence gene presence significantly influenced biofilm formation. A threshold of p<0.05 was used to measure statistical significance.

Results

Prevalence of Enterococcus

Using cultural methods for primary isolation, the overall prevalence of Enterococcus spp. in poultry meat samples was found to be 56% (84/150) (Fig. 1 and2). Comparable findings were reported by Banik et al. (2018) [1] , who documented a prevalence of 62.5%. In contrast, a considerably higher prevalence of 91.60% was observed by Naas et al. (2021) [10] , likely attributable to inadequate hygienic practices during meat processing (Laban et al., 2021) [8] . Conversely, a lower prevalence of 39.21% (40/102) reported by Talebi et al. (2015) [18] may reflect the implementation of improved hygienic standards during slaughter and processing. Of the 84 culturally confirmed Enterococcus isolates, 80 (95.23%) were further verified as Enterococcus spp. by PCR (Fig. 3). The prevalence observed in the present study is comparable to the findings of Manson et al. (2019) [9] , who reported a 98.23% detection rate of Enterococcus spp. in chicken meat. In contrast, moderate prevalence levels such as 49% have been reported by Rozanska et al. (2015) [13] , while Onaran et al. (2019) [14] recorded a lower prevalence of 30%, likely attributable to improved hygienic practices during meat processing.

Virulence Gene Distribution

Among the confirmed isolates, detection of the gelE gene was highest (71/80; 88.75%), followed by asa1 34/80 (40.47%), esp12/80 (15%), and cylA 9/80 (11.25%), while the hyl gene was not detected in any isolate.

Biofilm Formation

Biofilm testing revealed that out of 80 isolates, 72 (90%) demonstrated biofilm formation, with a majority exhibiting strong production. Correlation between virulence gene prevalence and biofilm phenotype was analyzed through contingency analysis.

Results showed that gelE, asa1, and esp, despite their presence among biofilm-positive isolates, did not exhibit statistically significant correlation, while cylA was significantly associated with biofilm development (p = 0.042). The contingency values used to calculate these results were presented in Table 4. The actual statistical metrics, including Chi-square and Fisher's exact test values, were reported in table 5, [17] , who reported similarly high biofilm prevalence in poultry-derived isolates. However, the absence of significant association between gelE and biofilm contrasts with Yang et al. (2023) [20] , suggesting geographic, strain-level, or method-related discrepancies. It is possible that gene expression and regulatory controls differ between isolates, and PCR-based detection does not capture transcription or activity level, limiting interpretation. These findings hold important implications for food processing safety, as biofilm-forming enterococci may withstand cleaning procedures and consequently contribute to cross-contamination. Given that poultry meat remains one of the most widely consumed protein sources, the persistence of biofilm-producing strains represents a potentially underestimated risk. The reliance on qualitative biofilm detection methods such as CRA also presents limitations, and future research should incorporate quantitative assessment techniques such as micro-titer plate assays coupled with absorbance analysis.

Conclusion

This study demonstrated that enterococci isolated from poultry meat predominantly possess virulence traits and exhibit strong biofilm-forming ability; however, only the presence of the cylA gene was statistically associated with biofilm formation. The high prevalence of gelE and asa1 did not translate into significant biofilm predictive reliability. These findings highlight the need for more advanced molecular diagnostics and surface decontamination strategies in poultry processing units. Quantitative biofilm evaluation and genome-level analysis are recommended for future studies to improve mechanistic understanding and accurately characterize risk potential.

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