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

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.

Census of the viral metagenome within an activated sludge microbial assemblage.

The viral metagenome within an activated sludge microbial assemblage was sampled using culture-dependent and culture-independent methods and compared to the diversity of activated sludge bacterial taxa. A total of 70 unique cultured bacterial isolates, 24 cultured bacteriophages, 829 bacterial metagenomic clones of 16S rRNA genes, and 1,161 viral metagenomic clones were subjected to a phylogenetic analysis.

The effect of hydroxylamine on the activity and aggregate structure of autotrophic nitrifying bioreactor cultures.

Addition of hydroxylamine (NH2OH) to autotrophic biomass in nitrifying bioreactors affected the activity, physical structure, and microbial ecology of nitrifying aggregates. When NH2OH is added to nitrifying cultures in 6-h batch experiments, the initial NH3-N uptake rates were physiologically accelerated by a factor of 1.4-13. NH2OH addition caused a 20-40% decrease in the median aggregate size, broadened the shape of the aggregate size distribution by up to 230%, and caused some of the microcolonies to appear slightly more dispersed. Longer term NH2OH addition in fed batch bioreactors decreased the median aggregate size, broadened the aggregate size distribution, and decreased NH3-N removal from >90% to values ranging between 75% and 17%. This altered performance is explained by quantitative fluorescence in situ hybridization (FISH) results that show inhibition of nitrifying populations, and by qPCR results showing that the copy numbers of amoA and nxrA genes gradually decreased by up to an order-of-magnitude. Longer term NH2OH addition damaged the active biomass. This research clarifies the effect of NH2OH on nitrification and demonstrates the need to incorporate NH2OH-related dynamics of the nitrifying biomass into mathematical models, accounting for both ecophysiological and structural responses.