Aim: Enterotoxigenic Escherichia coli (ETEC) is one of the most widely recognized diarrhoeal pathogens in developing countries. The advancement of ETEC vaccine development depends on the antigenic determinants of the ETEC isolates from a particular geographical region. So, the aim here was to comprehend the distribution of virulence determinants of the clinical ETEC strains of this region. Additionally, an attempt was made to find any correlation with the antimicrobial response pattern. Methods and results: Multiplex PCR was employed to identify virulence determinants followed by confirmatory singleplex PCR. For observation of antibiotic response, the Kirby-Bauer method was used. Out of 379 strains, 46% of strains harboured both the enterotoxins ST and LT, whereas 15% were LT only. Among the major colonization factors (CFs), CS6 (41%) was the most prevalent followed by CFA/I (35%) and CFA/ III was the lowest (3%). Among the minor CFs, CS21 (25%) was most prevalent, while CS15 showed the lowest (3%) presence. Among the non-classical virulence factors, EatA (69%) was predominant. ETEC strains harbouring CS6 showed resistance towards the commonly used drug Ciprofloxacin (70%). Conclusion: CS6 and elt+est toxin genes co-occurred covering 51% of the isolates. CS21 was found in most strains with est genes (43%). EatA was found to occur frequently when ST was present alone or with LT. CS6-harbouring strains showed an independent correlation to antimicrobial resistance. Significance and Impact of the Study: This study would aid in identifying the commonly circulating ETEC isolates of Kolkata, India, and their prevalent virulence determinants. Knowledge of antibiotic resistance patterns would also help in the appropriate use of antibiotics. Furthermore, the study would aid in identifying the multivalent antigens suitable for region-specific ETEC vaccines with maximum coverage.

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Enterotoxigenic Escherichia coli (ETEC) is a major pathogen of acute watery diarrhoea. The pathogenicity of ETEC is linked to adherence to the small intestine by colonization factors (CFs) and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). CS6 is one of the most common CFs in our region and worldwide. Iron availability functions as an environmental cue for enteropathogenic bacteria, signalling arrival within the human host. Therefore, iron could modify the expression of CS6 in the intestine. The objective of this study was to determine the effect of iron availability on CS6 expression in ETEC. This would help in understanding the importance of iron during ETEC pathogenesis. ETEC strain harbouring CS6 was cultured under increasing concentrations of iron salt to assess the effect on CS6 RNA expression by quantitative RT-PCR, protein expression by ELISA, promoter activity by β-galactosidase activity, and epithelial adhesion on HT-29 cells. RNA expression of CS6 was maximum in presence of 0.2 mM iron (II) salt. The expression increased by 50-fold, which also reduced under iron-chelation conditions and an increased iron concentration of 0.4 mM or more. The surface expression of CS6 also increased by 60-fold in presence of 0.2 mM iron. The upregulation of CS6 promoter activity by 25-fold under this experimental condition was in accordance with the induction of CS6 RNA and protein. This increased CS6 expression was independent of ETEC strains. Bacterial adhesion to HT-29 epithelial cells was also enhanced by five-fold in the presence of 0.2 mM iron salt. These findings suggest that CS6 expression is dependent on iron concentration. However, with further increases in iron concentration beyond 0.2 mM CS6 expression is decreased, suggesting that there might be a strong regulatory mechanism for CS6 expression under different iron concentrations.