Bacteriophage Treatment before Chemical Disinfection Can Enhance Removal of Plastic-Surface-Associated Pseudomonas aeruginosa

Abstract

Opportunistic pathogens can linger on surfaces in hospital and building plumbing environments, leading to infections in at-risk populations. Furthermore, biofilm-associated bacteria are protected from removal and inactivation protocols such as disinfection. Bacteriophages show promise as tools to treat antibiotic-resistant infections. As such, phages may also be useful in environmental applications to prevent newly acquired infections. In the current study, the potential of synergies between bacteriophage and chemical disinfection against the opportunistic pathogen Pseudomonas aeruginosa was assessed under various conditions. Specifically, surface-associated P. aeruginosa was treated with various concentrations of phages (P1 or JG004), chemical disinfectants (sodium hypochlorite or benzalkonium chloride), or combined sequential treatments under three distinct attachment models (spot inoculations, dry biofilms, and wet biofilms). Phages were very effective at removing bacteria in spot inoculations (>3.2 log₁₀ removal) and wet biofilms (up to 2.6 log₁₀ removal), while phages prevented the regrowth of dry biofilms in the application time. In addition, phage treatment followed by chemical disinfection inactivated P. aeruginosa cells under wet biofilm conditions better than either treatment alone. This effect was hindered when chemical disinfection was applied first, followed by phage treatment, suggesting that the additive benefits of combination treatments are lost when phage is applied last. Furthermore, we confirm previous evidence of greater phage tolerance to benzalkonium chloride than to sodium hypochlorite, informing choices for combination phage-disinfectant approaches. Overall, this paper further supports the potential of using combination phage and chemical disinfectant treatments to improve the inactivation of surface-associated P. aeruginosa. IMPORTANCE Phages are already utilized in the health care industry to treat antibiotic-resistant infections, such as those on implant-associated biofilms and in compassionate-care cases. Phage treatment could also be a promising new tool to control pathogens in the built environment, preventing infections from occurring. This study shows that phages can be combined effectively with chemical disinfectants to improve the removal of wet biofilms and bacteria spotted onto surfaces while preventing regrowth in dry biofilms. This has the potential to improve pathogen containment within the built environment and drinking water infrastructure to prevent infections by opportunistic pathogens.

Keywords: biofilm; opportunistic pathogens; phage therapy.

FIG 1

Log reduction of P. aeruginosa in spot tests. The data represent the averages and standard deviations from four replicates of the log₁₀ reduction of the intact-cell counts of P. aeruginosa treated with sodium hypochlorite (a), benzalkonium chloride (b), phage JG004 (c), or phage P1 (d). The dotted lines at the bottom of panels c and d represent the lower limit of quantification (LLOQ) of the FCM measurements. The applied phage concentrations correspond to multiplicities of infection (MOIs) of approximately 2 × 10⁻⁵ to 20.

FIG 2

Log reduction of P. aeruginosa in dry biofilms after rehydration. The data represent the averages and standard deviations from four replicates of the log₁₀ reduction of the intact-cell counts of P. aeruginosa treated with 10⁶ PFU/ml of phage P1 for various treatment times compared with no-treatment control biofilms.

FIG 3

Log reduction of P. aeruginosa in wet biofilms by phage and chlorine. The data represent the averages and standard deviations from five replicates of the log₁₀ reduction of the intact-cell counts of P. aeruginosa in wet biofilms due to various sequential treatments with phages and sodium hypochlorite compared with phage-only and chemical-only treatment control biofilms: phage JG004 followed by chlorine (a), chlorine followed by phage JG004 (b), phage P1 followed by chlorine (c), and chlorine followed by phage P1 (d). Phages were used at concentrations of 10¹ to 10⁹ PFU/ml (corresponding to multiplicities of infection of 10⁻⁶ to 10), while sodium hypochlorite was used at 200 ppm.