Bacteriophage treatment of carbapenemase-producing Klebsiella pneumoniae in a multispecies biofilm: a potential biocontrol strategy for healthcare facilities.

Ariel J Santiago,Rodney M Donlan,Leila Kartforosh,Maria L Burgos-Garay,Mustafa Mazher

doi:10.3934/microbiol.2020003

Ariel J Santiago, Rodney M Donlan + Show 3 more

Open Access

https://doi.org/10.3934/microbiol.2020003

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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+ Klebsiella pneumoniae (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 Pseudomonas aeruginosa, Micrococcus luteus, Stenotrophomonas maltophilia, Elizabethkingia anophelis, Cupriavidus metallidurans, and Methylobacterium fujisawaense 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 (109 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 log10 CFU/cm2 (p < 0.05) at 7 and 14 d (37 °C) and 1.4 log10 and 1.6 log10 CFU/cm2 (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.

Highlights

Handwashing sinks and associated premise wastewater plumbing in healthcare facilities have been identified as a source of transmission for antibiotic-resistant (AR) organisms from the environment to the patient [1]
Both growth and treatment media were supplemented with liquid handsoap primarily to simulate conditions that would be encountered in the p-trap environment and secondly, because preliminary experiments indicated that phage-mediated disruption of the biofilm biomass was not impacted in the presence of handsoap
An average biofilm biomass reduction of approximately 35% was observed across the range of multiplicities of infection (MOI) tested (Figure 1)

Summary

Introduction

Handwashing sinks and associated premise wastewater plumbing in healthcare facilities have been identified as a source of transmission for antibiotic-resistant (AR) organisms from the environment to the patient [1]. Typically associated with a surface, encased in a self-produced, extracellular polymeric substance matrix (EPS) that provides structural support and defense against environmental challenges [3] These sink and p-trap biofilms can become colonized by AR organisms [4,5] providing them some level of protection against disinfection [6,7] and increasing the likelihood that transmission to the patient may occur, leading to complications commonly referred to as healthcare-associated infections (HAIs). Carbapenem-resistant Enterobacteriaceae (CRE) are considered a serious public health threat due to decades of poor antibiotic stewardship, increasing trans-global dissemination, and a dwindling pipeline of available drugs to keep up with evolving molecular mechanisms of resistance [10,11,12,13] These organisms have been associated with colonization of the healthcare sink environment and their transmission thereafter [14,15]. These characteristics highlight the potential that phages have as biofilm control agents

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