Combating chlorine-resistant marine Klebsiella pneumoniae biofilms with chlorine-tolerant bacteriophages

Biofilm formation presents a significant challenge in health care, food industries, water distribution systems, etc. In addition to their inherent resistance to various stresses and biocides, emerging resistance against widely used biocides like chlorine is a growing concern. The strong link between chlorine resistance and the development of antibiotic resistance among microbes further exacerbates this issue. Therefore, it is highly desirable to devise a method to mitigate the problems associated with biofilms formed by Chlorine Resistant Bacteria (CRB). In this study, a highly chlorine resistant, biofilm-forming Klebsiella pneumoniae was isolated from the cooling water system of a nuclear power plant employing continuous chlorination for biofilm control. Interestingly, K. pneumoniae was found to enhance biofilm formation under the influence of increasing concentrations of chlorine, highlighting the limitations of chlorination-based biofilm control measures. As a remedial measure, chlorine resistant bacteriophages specific to K. pneumoniae were successfully isolated from the same water sample. These bacteriophages effectively inhibited planktonic growth biofilm formation and removed preformed biofilms. Whole-genome sequencing of two of the promising bacteriophages confirmed their identity as novel bacteriophages specific to K. pneumoniae. The absence of any antibiotic-resistant gene, virulent factor(s), or gene associated with the lysogenic life cycle further supports their suitability for environmental applications. This study provides valuable insights into the prevalence of chlorine resistant, pathogenic bacteria in cooling water distribution systems. It also highlights the promising application of bacteriophages to mitigate chlorine resistant bacteria and their biofilms.