Exploring the use of Congo red agar in improving the serotyping of non-serotypeable Shigella with In-silico evidence.

OBJECTIVES: To assess if congo red could make non-serotypeable Shigella strains serotypeable and to employ molecular docking to determine the basis of the same phenomenon. METHODS: We used 42 bacterial strains of non-serotypeable Shigella collected from 2012 to 2019 for this study. Each bacterial strain was freshly inoculated onto congo red agar and incubated at 37° C for 18-24 h. Bacterial colonies obtained were re-subjected to biochemical tests followed by serotyping and serogrouping. In-silico studies to investigate the interaction between MxiC protein of T3SS and O-antigen LPS with congo red were performed. RESULTS: Of the total 42 non-serotypeable Shigella studied, (26/42)62% were capable of being serotyped following the use of congo red agar, 65% were Shigella flexneri, 19% were Shigella dysenteriae, while 2 strains (7%) each of Shigella boydii and Shigella sonnei were detected. We observed no change in their biochemical properties. The in-silico molecular docking studies revealed high binding affinity between congo red and the B-Chain of Mxi C. Out of the 5 chains of the O-Antigen, congo red showed robust binding with the B-chain with the involvement of a cluster of hydrophobic interactions between them. This may have a crucial role in the conversion of non-serotypeable strains to serotypeable strains on exposure to congo red as observed in our study. CONCLUSION: Congo red agar as a medium converts a sizeable percentage of non-serotypeable Shigella strains to serotypeable Shigella strains.

CRISPR-cas heterogeneity and plasmid incompatibility types in relation to virulence determinants of Shigella.

Introduction. Virulence factors (VFs) are the most potent weapon in the molecular armoury of Shigella. In bacteria, the mobile genetic elements (MGEs) are contributors to the evolution of different types of clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-cas) variants and plasmid incompatibility types. The present study explored the virulence potential of Shigella in relation to the CRISPR-cas pattern and incompatibility types among the isolates.Hypothesis/Gap Statement. The profile of the CRISPR-cas systems among clinical isolates of Shigella in India has not been reported earlier. Limited knowledge is available on the pattern of plasmid incompatibility groups among clinical isolates Shigella. The bias is always towards studying the genetic elements associated with AMR, but the present study highlights CRISPR-cas and incompatibility types among Shigella in association with virulence.Aim. We aimed to investigate the distribution of virulence factors, CRISPR-cas pattern followed by plasmid incompatibility types among Shigella isolates.Methodology. Between 2012-2017, a total of 187 isolates of Shigella were included in the study. The virulence genes' distribution was carried out. CRISPR-cas profiling followed by analysis of the repeats and spacers was carried out. PCR-based replicon typing was used to determine the incompatibility types. The interplay was statistically determined using STATA.Results. The distribution of virulence genes showed varied pattern with ipaH present in all the isolates followed by ompA (93.6 %), virF (66.8 %), ial and sen (60.4 %), set1A (39.6 %) and set1B (39 %). CRISPR 1, CRISPR 3 and Cas6-Cas5 region were dominantly conserved. Twenty-two types of spacers were identified. The CRISPR3 repeat appeared to have a highly conserved sequence. CRISPR2 being the least common CRISPR type showed a strong association with an array of virulence genes (ial-set1A-set1B-virF) while CRISPR1 being the most dominant showed the least association with virulence genes (sen-virF). The dominant plasmids were found to be belonging to the inc FII group. The incompatibility groups FII, IncIγ, U, FIIS, FIIK, K, A/C, I1alpha was found to be associated with a greater number of virulence genes.Conclusion. The isolates showed increasing diversity in their gene content that contributes to increasing heterogeneity among the isolates, which is a known virulence strategy among pathogens.