Bacteriophage treatment of carbapenemase-producing <em>Klebsiella pneumoniae</em> in a multispecies biofilm: a potential biocontrol strategy for healthcare facilities
Ariel J. Santiago, Maria Burgos-Garay, Leila Kartforosh, Mustafa Mazher, Rodney M. Donlan
Abstract
The p-traps of hospital handwashing sinks represent a potential reservoir for antimicrobial-resistant organisms of major public health concern, such as carbapenemase-producing KPC+ <em>Klebsiella pneumoniae</em> (CPKP). Bacteriophages have reemerged as potential biocontrol agents, particularly against biofilm-associated, drug-resistant microorganisms. The primary objective of our study was to formulate a phage cocktail capable of targeting a CPKP strain (CAV1016) at different stages of colonization within polymicrobial drinking water biofilms using a CDC biofilm reactor (CBR) p-trap model. A cocktail of four CAV1016 phages, all exhibiting depolymerase activity, were isolated from untreated wastewater using standard methods. Biofilms containing <em>Pseudomonas aeruginosa</em>, <em>Micrococcus luteus</em>, <em>Stenotrophomonas maltophilia</em>, <em>Elizabethkingia anophelis</em>, <em>Cupriavidus metallidurans</em>, and <em>Methylobacterium fujisawaense</em> were established in the CBR p-trap model for a period of 28 d. Subsequently, CAV1016 was inoculated into the p-trap model and monitored over a period of 21 d. Biofilms were treated for 2 h at either 25 °C or 37 °C with the phage cocktail (10<sup>9</sup> PFU/ml) at 7, 14, and 21 d post-inoculation. The effect of phage treatment on the viability of biofilm-associated CAV1016 was determined by plate count on m-Endo LES agar. Biofilm heterotrophic plate counts (HPC) were determined using R2A agar. Phage titers were determined by plaque assay. Phage treatment reduced biofilm-associated CAV1016 viability by 1 log<sub>10</sub> CFU/cm<sup>2</sup> (p < 0.05) at 7 and 14 d (37 °C) and 1.4 log<sub>10</sub> and 1.6 log<sub>10</sub> CFU/cm<sup>2</sup> (p < 0.05) at 7 and 14 d, respectively (25 °C). No significant reduction was observed at 21 d post-inoculation. Phage treatment had no significant effect on the biofilm HPCs (p > 0.05) at any time point or temperature. Supplementation with a non-ionic surfactant appears to enhance phage association within biofilms. The results of this study suggest the potential of phages to control CPKP and other carbapenemase-producing organisms associated with microbial biofilms in the healthcare environment.