Assessment of Biofilm Formation and Shiga-like Toxin Genes in ESBL-producing E. coli Isolates Recovered from Retail Salad Vegetables in Mirzapur District, Uttar Pradesh, India

The present study assessed the occurrence and biofilm forming potential of ESBL-producing Escherichia coli isolates recovered from raw salad vegetables collected from 32 retail vegetable shops of Mirzapur district, Uttar Pradesh, India (n=224).

Standard bacteriological culture methods using cefotaxime-supplemented EMB agar were used for initial isolation of cefotaximeresistant E. coli which were subjected to phenotypic detection of ESBL production by Kirby-Bauer disc diffusion test. Subsequently, all the phenotypically positive ESBL-producing E. coli isolates were subjected to determine their biofilm-producing ability by Congo red agar (CRA) assay and microtiter plate assay (MPA) followed by genotypic confirmation of biofilm production by PCR assay targeted at fim-H, Sfa and papC genes. Additionally, the isolates were genotypically explored for a potential presence of Shiga-like toxin genes (stx1 and stx2).

Altogether, 276 cefotaxime-resistant E. coli isolates were recovered, of which, 99.27% (274/276) were observed to be phenotypically positive for ESBL production. The brown, dry and rough (BDAR) and red, dry and rough (RDAR) colony morphotypes were observed by 70.80% (194/274) and 29.19% (80/274) of the isolates, respectively by CRA method revealing their potential to form biofilms. Biofilm formation was evident in 94.16% (258/274) of the isolates by MPA method, of which 79.45% (205/258), 17.05% (44/258) and 3.48% (9/258) were considered as weak, moderate and strong biofilm producers, respectively. The fimH gene was found to be the predominant genetic determinant of biofilm formation which was detected in 77% (211/274) of the tested isolates followed by Sfa gene (11.31%) and papC gene (4.01%). A significant difference (p<0.05) was evident between CRA and MPA methods as well as phenotypic and genotypic methods for detection of biofilm formation among the recovered isolates. In addition, significant differences (p<0.05) were also observed upon vegetable-wise comparison of ESBL-producing E. coli isolates and the presence of biofilm producing genes. The stx1 and stx2 genes were found to be present in one isolate recovered from a carrot sample and two isolates from coriander samples, respectively. The findings of this study highlight the widespread occurrence of ESBL-producing E. coli from raw salad vegetables with significant biofilmforming potential posing a public health risk. The detection of Shiga-like toxin genes in some isolates further underscores the need for stringent surveillance and improved hygiene practices to mitigate contamination risk in fresh produce.