Phage Infection Kinetics Outpace Chlorine Inactivation to Enable Control of Biocide-Resistant <i>Klebsiella quasipneumoniae</i> Biofilms in Cooling Water Systems

Industrial cooling water systems that rely on continuous chlorination increasingly select for biofilm-forming bacteria with elevated tolerance to oxidizing biocides, enabling biofilms to persist despite sustained disinfectant exposure. To address this, we evaluated whether bacteriophages isolated from a chlorinated seawater system can be deployed against the model organism, biocide-resistant Klebsiella quasipneumoniae . Three phages were isolated that effectively suppressed planktonic growth and biofilm formation; however, phage treatment alone rapidly selected resistant mutants. Drawing on the rationale of phage-antibiotic synergy, we assessed phage-biocide combinations to minimize resistance emergence. Although all phages exhibited measurable sensitivity to chlorine in host-free assays, time-kill analysis revealed that phage replication occurs on a comparable or shorter time scale than chlorine-mediated inactivation in the presence of the host. Short latent periods (30–45 min) and high burst sizes enabled a fraction of the phage population to complete productive infection cycles before substantial loss of infectivity, sustaining phage activity under chlorinated conditions. This interaction translated into improved control outcomes: combined phage-chlorine treatment achieved >90% biofilm reduction and considerably reduced the emergence of phage-resistant mutants compared with phage monotherapy. These findings identify infection-inactivation kinetics as a rational criterion for designing compatible phage-biocide combinations for biofilm control in chlorinated industrial water systems.