Differential response of PLE-positive and PLE-negative Vibrio cholerae O1 to CRISPR/cas-positive and CRISPR/cas-negative ICP1 bacteriophages in Bangladesh

The interaction between Vibrio cholerae O1 and bacteriophage ICP1 is critical for cholera epidemiology. V. cholerae O1 uses Phage Inducible Chromosomal Island Like Element (PLE) to defend against the ICP1 infection, while ICP1 bypasses the PLE-mediated defense with its CRISPR/cas system. In this study we isolated and characterized 16 ICP1 strains from cholera patient stool samples ( n = 8) and surface water ( n = 8) from 2 regions of Bangladesh. Additionally, ICP2 ( n = 6) and ICP3 ( n = 4) were recovered from the same samples. To determine PLE prevalence, we selected 92 V. cholerae O1 isolates from Dhaka ( n = 52) and Mathbaria ( n = 40) between 2012 and 2018. Thirty-eight V. cholerae O1 strains (PLE+ n = 28, PLE− n = 10) were tested for phage resistance via plaque assays against our characterized ICP1. All ICP1 isolates from Dhaka harbored CRISPR/cas, whereas only 50% of the ICP1 strains from Mathbaria carried CRISPR/cas. ICP2 and ICP3 were recovered predominantly from environmental sources, with ICP3 found exclusively in the environment. Overall, 80% ( n = 74) of V. cholerae O1 isolates carried PLE1, indicating widespread PLE acquisition. Two recent isolates lacked the Int4 site but contained PLE1, suggesting alternative chromosomal integration warranting further genomic surveillance. Plaque assays demonstrated differential susceptibility of PLE-positive and PLE-negative isolates to ICP1 infection. These findings highlight ongoing co-evolution between V. cholerae O1 and ICP1, and provide insights into cholera surveillance and outbreak mitigation in endemic regions.