Association of Putative Virulence Genes with HEp-2 Cell Adherence and Biofilm Production in Enteroaggregative Strains Isolated from Acute Diarrheal and Healthy Children, India.

Enteroaggregative  (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhea in developed and industrialized countries in children. EAEC colonizes the human intestine and this ability to form colonies and biofilm is an important step in pathogenesis. Here, we investigated the relationship between known or putative 22 EAEC virulence genes and biofilm formation in isolates derived from acute diarrhea and healthy children and their aggregative adherence (AA) pattern with Hep-2 cell lines. A total of 138 EAEC isolates were recovered from 1210 stool samples from children (age < 10 years) suffering from acute diarrhea and 33 EAEC strains isolated from 550 healthy children (control group) of different Anganwadi centers in Chandigarh region were included. Polymerase chain reaction using the primer pair pCVD432 identified  isolates as EAEC. A total of 22 virulence-related genes have been identified using M-PCR chain reactions. The crystal violet method was used for the quantitative biofilm assay. Aggregative adherence assay was also studied using HEp-2 cell lines. Of 138 EAEC isolates from the acute diarrheal group, 121 (87.6%) EAEC isolates produced biofilm. In our findings, typical EAEC (62%) isolates were strong biofilm producers (37.5%) in the diarrheal group. Among adhesive variants, 4A (39.6%) and A (21.6%) were the most common and were statistically significant ( = 0.01 and  = 0.03 respectively). We reported that R gene along with the typical AA pattern was present in 71.4% of the EAEC strains in the diarrheal group, whereas it was present in 44% of the control group. Other R non-dependent genes like ORF3 and A may also lead to biofilm formation. In conclusion, there is significant heterogeneity in putative virulence genes of EAEC isolates from children and biofilm formation is associated with the combination of many genes.