1. Molecular detection of Diarrheagenic E. coli pathotypes: A total of 334 bacterial isolates were recovered from the faecal samples of pediatric diarrheal patients. The genomic and plasmid DNA isolated from 334 diarrheagenic E. coli isolates were subjected to the PCR based virulent marker detection assays. Among the isolates, 31.44% (105 of 334) isolates carried pathogenic virulent genes and 68.56% (229 of 334) isolates were non-pathogenic which does not carry the selected virulent genes confirmed by multiplex PCR using gene-specific primers (Table-1). Pathogenic isolates were further classified into two types based on the virulence and cytological effects on cell monolayers such as non-toxigenic adherent pathotypes (20.05%) which include EAEC, EPEC, EIEC, and DAEC and adherent toxigenic pathotypes (11.37%) which included the strains of STEC, ETEC, and EHEC.
In this study, we have used four different types of adherent pathogenic isolates which were classified based on the virulent genes and adherence pattern on the cell monolayer. The first type isolates are EPEC 14.97% (50 of 334) which have both eaeA and bfpA genes containing EAF plasmid; this EPEC has been further classified into two subtypes such as typical EPEC (tEPEC) 8.68% (29 of 334), which carried both eaeA and bfpA genes and atypical EPEC (aEPEC) 6.28%, (21 of 334) which carried the eaeA gene alone. The Second type was EIEC 2.40% (8 of 334) which carried virulent gene ial which was a part of the plasmid of invasion (pInv). The third and fourth types were DAEC 0.90% (3 of 334) and EAEC 1.80% (6 of 334) which carried the virulent genes daaE for diffuse adherence and pCVD432 occurred in DAEC and EAEC respectively (Fig. 1A).
2. Screening of pathological interaction between diarrheagenic E. coli pathotypes and cell monolayers: In cell monolayer adhesion assay, all the adherent pathogenic isolates showed strong adhesion against HeLa and CHO-k1 cell lines. The pattern of isolates adherence to monolayers of human cancer (HeLa) and non- cancerous (CHO-k1) cells were classified into four types such as localized (LA), localized-like (LAL), diffuse (DA), or aggregative (AA) patterns (Table-2) (Fig-1A). The AA pattern was observed from 8.96% (6 of 67) of EAEC isolates on HeLa and CHO-k1 monolayer and 1.49% (1 of 67) DAEC on HeLa monolayer only. The LAL pattern was observed from both atypical and typical EPEC. The atypical EPEC on HeLa monolayer was 32.8% (22 of 67 isolates) and the CHO-k1 monolayer was 31.3% (21 of 67 isolates). The typical EPEC on HeLa monolayer was (4.47%, 3 of 67 isolates) and the CHO-k1 monolayer was 1.49%, (1 of 67 isolates). Similarly, the LA pattern was also observed from both atypical, typical EPEC, and EIEC. The atypical EPEC observed at 10.4% (7 of 67 isolates), typical EPEC was detected at 26.8% (18of67isolates), and EIEC (4.47%, 3 of 67 isolates) on HeLa monolayer. Atypical EPEC at 11.9% (8 of 67 isolates), typical EPEC at 29.8% (20 of 67 isolates), and EIEC (1.49%, 1 of 67 isolates) on the CHO-k1 monolayer were detected with LA pattern. The DA pattern was observed from 2.98% (2of67) of DAEC isolates, 7.46% (5of67) of EIEC isolates on HeLa monolayer and 4.47% (3 of 67) of DAEC isolates, 10.4% (7 of 67) EIEC isolates were observed on CHO monolayer (Fig. 1B).
3. Serogrouping of the pathogenic E. coli isolates based on ‘O’ antigen: The diarrheagenic E. coli of this study mostly belongs to the following serotypes including O22, O141, O124, O128, O86, O5, O96, and UT. The O antigen serotypes of all the strains included in this study with their virulence profile, origin, antibiotic resistance profile are shown in table- 2.
4. Enumeration of Bacterial adhesion: The percentage of an adherent pattern of different pathotypes varies from cancerous (HeLa) and non-cancerous (CHO-k1) cell monolayers in in-vitro adhesion assay. The bundle forming pilus containing typical EPEC isolates showed stronger adhesion (Adh++) in both HeLa and CHO-k1 cell lines but the adherent percentage was high in cancerous HeLa cells (60%) when compared with normal CHO-K1 cells (20%). Similarly, the pCVD432 gene carried EAEC isolates showed an adhesive adhesion (Adh+) in both HeLa and CHO-k1 cell lines but the adherent percentage was high in cancerous HeLa cells (20%) when compared with normal CHO-K1 cells (10%). DAEC and EIEC isolates were showed non-adhesion (Adh-) on HeLa monolayer and CHO-k1 monolayers (Fig. 3) (Table-3).
5. Effect of Minimum inhibitory concentration of antibiotics against bacterial adhesion: Adhesion inhibition of different pathotypes was observed in two different methods of inhibition in-vitro that by cell monolayers and Luria Bertani broth dilution assay. In this study, we used a different concentration of antibiotics, buttermilk, and skimmed milk powder. The adhesion inhibition of these antibiotics, buttermilk, and skimmed milk was dose-dependent in in-vitro cell monolayer adhesion assay and Luria Bertani broth dilution assay but the MIC of antibiotics, buttermilk, and skimmed milk was varied depending on cell types and adhesion pathotypes. The adhesion inhibition (MIC) of Buttermilk in HeLa cell monolayer containing EACE, EPEC, DAEC, and EIEC was 69.5, 73.7, 87.0 and 83.7 µg/ml, respectively and CHO-k1 cell monolayer containing EACE, EPEC, DAEC, and EIEC was 69.5, 73.7, 87.0 and 83.7 µg/ml, respectively. Similarly, the adhesion inhibition (MIC) of cephalexin in HeLa cell monolayer containing EACE, EPEC, DAEC, and EIEC was 67.7, 67.5, 69.2, and 68.9 µg/ml, respectively, and CHO-k1 cell monolayer containing EACE, EPEC, DAEC, and EIEC were 75.3, 72.5, 79.9 and 84.8 µg/ml, respectively. In Luria Bertani broth dilution assay, MIC of buttermilk on EACE, EPEC, DAEC, and EIEC was 4, 8, 8, and 4 µg/ml respectively which is very low in comparison with the results got from the cell lines. Similarly, MIC of cephalexin on EACE, EPEC, DAEC, and EIEC was 0.75, 8, 8, and 8 µg/ml respectively and MIC of human skimmed milk on EACE, EPEC, DAEC, and EIEC was 0.5, 16, 12, and 24 µg/ml respectively.
In adhesion inhibition (MIC) of human skimmed milk on HeLa cell monolayer containing EACE, EPEC, DAEC, and EIEC was 50.2, 53.4, 72.7, and 59.1 µg/ml, respectively and on CHO-k1 cell monolayer containing EACE, EPEC, DAEC and EIEC were 190.9, 259.7, 130.3 and 83.8 µg/ml respectively. As a whole, the result revealed that the minimal inhibitory concentration required by all the components was greatly lower in broth medium as compared to cell lines especially against the normal cell lines (CHO-k1 monolayers).
Further, this study revealed that human skimmed milk has a lethal effect on adhesion inhibition of EPEC pathotypes in both CHO-k1 and HeLa cell lines. Followed by cephalexin and buttermilk showed adhesion inhibition with higher concentrations in both cell lines. Further, this study revealed that the immune/chemical components of human skimmed milk have the maximum potential to inhibit EAEC pathotypes with 50.25 µg/ml followed by cephalexin (67.7µg/ml) and buttermilk (69.6µg/ml) in both cell lines with minor concentration differences.