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Sink drains in healthcare facilities may provide an environment for antimicrobial-resistant microorganisms, including carbapenemase-producing Klebsiella pneumoniae (CPKP).
We investigated the colonization of a biofilm consortia by CPKP in a model system simulating a sink-drain P-trap. Centers for Disease Control (CDC) biofilm reactors (CBRs) were inoculated with microbial consortia originally recovered from 2 P-traps collected from separate patient rooms (designated rooms A and B) in a hospital. Biofilms were grown on stainless steel (SS) or polyvinyl chloride (PVC) coupons in autoclaved municipal drinking water (ATW) for 7 or 28 days.
Microbial communities in model systems (designated CBR-A or CBR-B) were less diverse than communities in respective P-traps A and B, and they were primarily composed of β and γ Proteobacteria, as determined using 16S rRNA community analysis. Following biofilm development CBRs were inoculated with either K. pneumoniae ST45 (ie, strain CAV1016) or K. pneumoniae ST258 KPC+ (ie, strain 258), and samples were collected over 21 days. Under most conditions tested (CBR-A: SS, 7-day biofilm; CBR-A: PVC, 28-day biofilm; CBR-B: SS, 7-day and 28-day biofilm; CBR-B: PVC, 28-day biofilm) significantly higher numbers of CAV1016 were observed compared to 258. CAV1016 showed no significant difference in quantity or persistence based on biofilm age (7 days vs 28 days) or substratum type (SS vs PVC). However, counts of 258 were significantly higher on 28-day biofilms and on SS.
These results suggest that CPKP persistence in P-trap biofilms may be strain specific or may be related to the type of P-trap material or age of the biofilm.
Background: Hand-hygiene sink drains in healthcare facilities may provide an environment for the survival and dissemination of various multidrug-resistant organisms (MDROs), including carbapenemase-producing Klebsiella pneumoniae (CPKP). We developed a sink model system to establish and test native drinking water biofilms containing CPKP in the p-traps of hand-hygiene sink drains. Methods: A handwashing sink gallery was designed to consist of 6-wall mounted stainless-steel sink basins connected to the same municipal water line. Each sink’s plumbing included a chrome-plated brass p-trap. Healthcare facility conditions were simulated to include handwashing events with the addition of hand-soap and municipal water 4 per day, and nutritional shake (simulating liquid waste) 1 per day. Resultant biofilms in the p-traps of each sink were harvested after 28 days for community analysis. Microbial community analyses were performed on selected biofilm samples using 16S rRNA sequencing of the V4 hypervariable region of genomic DNA. Another experiment evaluated 28-day p-trap biofilm inoculated with CPKP CAV1016 (10 mL 7.010E 7 CFU/mL) and was assessed over 14 days. Heterotrophic plate counts (HPCs) were determined on R2A medium (7 days of incubation at 25C). CPKP was quantified on mEndo selective medium (48 hours of incubation at 36C). Results: Biofilms developed in all p-traps, but biofilm HPC (5.78 mean log CFU/cm2, range 4.35–7.16) and community diversity (15–20 genera per p-trap) varied with sink position. Community analysis showed similarities in bacterial community composition and diversity between sinks 1 and 2, and between sinks 3, 5 and 6, but with differences between the 2 groups. The most abundant family in sinks 3, 5, and 6 was Erythrobacteriaceae (76%, 78%, and 55% of the total reads, respectively), whereas sinks 1 and 2 were dominated by Sphingomonadaceae (63% and 36%) and Methylobacteriaceae (19% and 55%). Also, 16S sequencing revealed the presence of potential opportunistic pathogens in the biofilms, including reads attributed to Pseudomonas and Acinetobacter. CPKP CAV1016 inoculated into 28-day p-trap biofilms colonized and persisted in all 6 sinks for 12 days after inoculation. Conclusions: Despite all 6 sinks sharing an incoming water line, soap, and carbon and energy source, there was a significant variation in the bacterial community composition observed between the sinks. CPKP can colonize and persist in the p-trap biofilms; however, additional work is needed to achieve a reproducible model system. Once this is achieved, the sink gallery will be used to investigate interventions to mitigate colonization or persistence of CPKP in p-trap biofilms.
Background: Carbapenem-resistant Enterobacteriaceae (CRE) are multidrug-resistant bacteria that persist in healthcare environments, particularly in wastewater reservoirs where they can pose risks for patients. Healthcare-associated outbreaks of carbapenemase-producing (CP) CRE can be propagated via a single bacterial strain and/or mobile genetic element (MGEs) harboring a carbapenemase gene. Unlike chromosomally encoded carbapenemases, CP-MGEs can rapidly facilitate the spread of these carbapenemase genes across bacterial strains. From July 2017 to December 2018, the Florida Department of Health in Orange County investigated an outbreak of patients colonized with various bacterial genera of CP-CRE carrying the Klebsiella pneumoniae carbapenemase gene (blaKPC), indicating a potential MGE reservoir. WGS was performed to identify transmission pathways and linked cases, beyond what traditional testing provides. Methods: We selected a subset of blaKPC-harboring isolates for WGS on short- and long-read platforms (MiSeq, PacBio, MinION) to achieve high quality, complete genome and plasmid assemblies. Laboratory, clinical, and epidemiological data were combined to identify possible transmission events, common sources, and common MGEs. Results: Eleven clinical isolates from 5 genera (Citrobacter, Enterobacter, Klebsiella, Morganella, Providencia, and Serratia), and 10 environmental isolates collected from the pharmacy and medication room, ICU, and patient rooms and comprising 4 genera (Citrobacter, Enterobacter, Klebsiella, and Serratia) underwent WGS. Although short-read WGS elucidated additional subsets of closely related strains, high genomic diversity was also observed within some species: Citrobacter freundii: 13,483 single-nucleotide variants (SNVs), 67% core genome; Enterobacter spp: 3–18,563 SNVs; 34%; and K. pneumoniae: 8–18,460 SNVs, 80%. Further analysis using long-read hybrid assemblies revealed 2 unique blaKPC-harboring plasmids. The first plasmid, pDHQP20145-KPC3 (50 kb), contained the blaKPC-3 gene and was detected in both patient and environmental isolates across 3 of the 5 sequenced genera. The second plasmid, pDHQP201745-KPC2 (180 kb), contained the blaKPC-2 gene, and was found across 2 CP-CRE genera isolated from both patients and the environment, including isolates from the medication room sink drain and a patient who received compounded oral medications. Conclusion: WGS identified 2 blaKPC-harboring plasmids, including pDHQP20145-KPC3, which was found across 3 genera of CP-CRE isolated from patients and the environment, supporting prolonged transmission of KPC-producing CRE in this facility, and a CP-MGE driving transmission. The rapid spread of emerging, potentially mobile, antimicrobial resistance has increased our need to further explore the genomic environment of promiscuous MGEs. WGS can contribute to infection control beyond traditional subtyping methods, such as pulsed-field gel electrophoresis (PFGE), as MGEs increasingly represent an important driver of transmission.
Background: Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a frequent cause of healthcare-associated infections (HAIs). The CDC Emerging Infections Program (EIP) conducted population and laboratory-based surveillance of CRPA in selected areas in 8 states from August 1, 2016, through July 31, 2018. We aimed to describe the molecular epidemiology and mechanisms of resistance of CRPA isolates collected through this surveillance. Methods: We defined a case as the first isolate of P. aeruginosa resistant to imipenem, meropenem, or doripenem from the lower respiratory tract, urine, wounds, or normally sterile sites identified from a resident of the EIP catchment area in a 30-day period; EIP sites submitted a systematic random sample of isolates to CDC for further characterization. Of 1,021 CRPA clinical isolates submitted, 707 have been sequenced to date using an Illumina MiSeq. Sequenced genomes were classified using the 7-gene multilocus sequence typing (MLST) scheme, and a core genome MLST (cgMLST) scheme was used to determine phylogeny. Antimicrobial resistance genes were identified using publicly available databases, and chromosomal mechanisms of carbapenem resistance were determined using previously validated genetic markers. Results: There were 189 sequence types (STs) among the 707 sequenced genomes (Fig. 1). The most frequently occurring were high-risk clones ST235 (8.5%) and ST298 (4.7%), which were found across all EIP sites. Carbapenemase genes were identified in 5 (<1%) isolates. Overall, 95.6% of the isolates had chromosomal mutations associated with carbapenem resistance: 93.2% had porinD-associated mutations that decrease membrane permeability to the drugs; 24.8% had mutations associated with overexpression of the multidrug efflux pump MexAB-OprM; and 22.9% had mutations associated with overexpression of the endogenous β-lactamase ampC. More than 1 such chromosomal resistance mutation type was present in 37.8% of the isolates. Conclusions: The diversity of the sequence types demonstrates that HAIs caused by CRPA can arise from a variety of strains and that high-risk clones are broadly disseminated across the EIP sites but are a minority of CRPA strains overall. Carbapenem resistance in P. aeruginosa was predominantly driven by chromosomal mutations rather than acquired mechanisms (ie, carbapenemases). The diversity of the CRPA isolates and the lack of carbapenemase genes suggest that this ubiquitous pathogen can readily evolve chromosomal resistance mechanisms, but unlike carbapenemases, these cannot be easily spread through horizontal transfer.
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