Supplemental nutrients stimulate the amplification of carbapenemase-producing Klebsiella pneumoniae (CPKP) in a sink drain in vitro biofilm reactor model.

Liquid wastes (LW) disposed in hospital handwashing sinks may affect colonization of sink P-traps by carbapenemase-producing Klebsiella pneumoniae (CPKP), causing CPKP dispersal into the patient care environment. This study aimed to determine the effect of LW on biofilm formation and CPKP colonization in a P-Trap model (PTM). PTMs containing polymicrobial biofilms grown in autoclaved municipal tap water (ATW) supplemented with 5% dextrose in water (D5W), nutritional shake (Shake), sugar-based soft drink (Soda), or ATW were inoculated with K. pneumoniae ST258 KPC+ (ST258) or K. pneumoniae CAV1016 (CAV1016) and sampled after 7, 14, and 21 d. Biofilm bio-volume, mean thickness, and heterotrophic plate counts were significantly reduced and roughness coefficient significantly increased by Soda compared with D5W, Shake, or ATW. CPKP were significantly reduced by Soda but significantly amplified by D5W (ST258; CAV1016, 7 d) and Shake (ST258) suggesting that reducing LW disposal in sinks may reduce CPKP dispersal into patient care environments.

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.

Pythium and Phytopythium Species in Two Pennsylvania Greenhouse Irrigation Water Tanks.

Two commercial greenhouses producing potted plants in Pennsylvania using recycled irrigation water in an ebb-and-flood system have incurred significant crop losses due to Pythium aphanidermatum. In cooperation with the greenhouses, one or more of their water tanks was monitored continuously (128 tank samplings) for Pythium spp. by baiting. Nine species of Pythium and three species of Phytopythium were recovered, representing clades A, B, E, and K, but none was P. aphanidermatum. The recovered Pythium spp. were (i) P. rostratifingens, (ii) isolates identical to Pythium sp. nov. OOMYA1702-08 (clade B2), (iii) P. coloratum, (iv) P. middletonii, (v) and (vi) two new species in clade E2, (vii) a new species in clade B2, (viii) isolates very similar to Pythium sp. nov. OOMYA1646-08 (clade E2), and (ix) a new species in clade A. The Phytopythium spp. recovered were (i) Phytopythium litorale, (ii) P. helicoides, and (iii) P. chamaehyphon. This article illustrates the different communities of Pythium and Phytopythium spp. found in each greenhouse over 10 months. Some of the baited species display resistance to the oomycete fungicide active ingredient, mefenoxam. P. helicoides and the new species in clade B2 were pathogenic on seedlings in potting mix with fertilizer added.