Targeting antibiotic resistance genes in Vibrio cholerae using a phage-encoded CRISPR-Cas system to improve efficacy of phage prophylaxis

PROJECT SUMMARY The incessant rise of multidrug resistance in bacterial pathogens has created a dire situation that necessitates development of new modalities for preventing and treating infectious diseases. Phage prophylaxis and phage therapy represent one such approach, since phages are not affected by antibiotic resistance phenotypes. The causative agent of cholera, Vibrio cholerae, has become extensively drug resistant (XDR) in just the past decade due to indiscriminate and widespread antibiotic use in the community in low- and middle-income countries. We recently reported a phage product, comprised of virulent phages ICP1, ICP2 and ICP3, that effectively prevents cholera in animal models, which could be used to reduce infection rates those at-risk. However, that work used an antibiotic-sensitive, pre-1980 strain of V. cholerae. Here, we seek to test two hypotheses that, if substantiated, could dramatically improve phage prophylaxis for cholera and would lend itself to improving phage products for other diseases caused by multidrug-resistant bacteria. The first hypothesis is that incorporating CRISPR spacers into ICP1, which specifically target antibiotic resistance genes in XDR V. cholerae, can improve the ability of ICP1 to kill these strains both in vitro and in animal models, thereby better protecting the animals from infection. The second hypothesis is that by targeting antibiotic resistance genes for cleavage by CRISPR-Cas, we can dramatically reduce their frequencies of horizontal gene transfer.