Genomic Analysis of Hospital Plumbing Reveals Diverse Reservoir of Bacterial Plasmids Conferring Carbapenem Resistance

Abstract

The hospital environment is a potential reservoir of bacteria with plasmids conferring carbapenem resistance. Our Hospital Epidemiology Service routinely performs extensive sampling of high-touch surfaces, sinks, and other locations in the hospital. Over a 2-year period, additional sampling was conducted at a broader range of locations, including housekeeping closets, wastewater from hospital internal pipes, and external manholes. We compared these data with previously collected information from 5 years of patient clinical and surveillance isolates. Whole-genome sequencing and analysis of 108 isolates provided comprehensive characterization of bla_KPC/bla_NDM-positive isolates, enabling an in-depth genetic comparison. Strikingly, despite a very low prevalence of patient infections with bla_KPC-positive organisms, all samples from the intensive care unit pipe wastewater and external manholes contained carbapenemase-producing organisms (CPOs), suggesting a vast, resilient reservoir. We observed a diverse set of species and plasmids, and we noted species and susceptibility profile differences between environmental and patient populations of CPOs. However, there were plasmid backbones common to both populations, highlighting a potential environmental reservoir of mobile elements that may contribute to the spread of resistance genes. Clear associations between patient and environmental isolates were uncommon based on sequence analysis and epidemiology, suggesting reasonable infection control compliance at our institution. Nonetheless, a probable nosocomial transmission of Leclercia sp. from the housekeeping environment to a patient was detected by this extensive surveillance. These data and analyses further our understanding of CPOs in the hospital environment and are broadly relevant to the design of infection control strategies in many infrastructure settings.IMPORTANCE Carbapenemase-producing organisms (CPOs) are a global concern because of the morbidity and mortality associated with these resistant Gram-negative bacteria. Horizontal plasmid transfer spreads the resistance mechanism to new bacteria, and understanding the plasmid ecology of the hospital environment can assist in the design of control strategies to prevent nosocomial infections. A 5-year genomic and epidemiological survey was undertaken to study the CPOs in the patient-accessible environment, as well as in the plumbing system removed from the patient. This comprehensive survey revealed a vast, unappreciated reservoir of CPOs in wastewater, which was in contrast to the low positivity rate in both the patient population and the patient-accessible environment. While there were few patient-environmental isolate associations, there were plasmid backbones common to both populations. These results are relevant to all hospitals for which CPO colonization may not yet be defined through extensive surveillance.

Keywords: antimicrobial resistance; carbapenem resistant; carbapenemase-producing organisms; environment; infection control; plasmid-mediated resistance; plumbing; wastewater.

FIG 1

Connections identified on the basis of the genome sequence similarity of CPOs isolated from the environment and patients. Each rectangle in the outer ring represents a sequenced bla_KPC-positive isolate and is color coded on the basis of the source. Black, patient; red, wastewater manhole; light gray, housekeeping equipment; green, housekeeping closet drain; purple, high-touch surface; dark blue, sink; yellow, wastewater pipe. Four patients have multiple isolates, and these are clustered and denoted by additional black bars. Orange arcs indicate >99.90% ANI between patient and environmental isolates. Blue arcs indicate >99.90% ANI between environmental isolates. Arc color saturation does not have meaning and is used solely to aid the visual distinction of links. Redundant arcs between environmental isolates are excluded for simplicity. The isolate name is provided only if an environmental connection was found. Icon credits: Alonzo Design, Kathy Konkle, cihanterlan, pialhovik, Panpty, istrejman/Getty Images under license.

FIG 2

Antimicrobial susceptibility data on bla_KPC/bla_NDM PCR-positive Enterobacteriaceae from patients and the environment between January 2012 and December 2016. Enterobacteriaceae isolates were interpreted as intermediate or resistant to the antimicrobial agents on the basis of CLSI M100 guidelines (62). Black bars are isolates from the environment, and gray bars are isolates from patients. The values below the bars are the total numbers of isolates tested. Pip/Tazo, piperacillin-tazobactam; Trimeth/Sulfa, trimethoprim-sulfamethoxazole. (*, two-tailed P < 0.05; **, P < 0.001; Fisher’s exact test.)

FIG 3

CPOs are able to persist and recombine in sink drains. (A) In February 2011, E. cloacae carrying two bla_KPC⁺ plasmids was cultured from patient Y. This organism may have also colonized a sink in the patient’s room. (B) Eleven months later (January 2012), an isolate from patient A, who was colonized with bla_KPC⁺ K. pneumoniae upon admission, was likely introduced into the same sink (KPNIH27). (C) KPNIH27’s pKPC-39c plasmid in a sink drain isolate is hypothesized to have horizontally transferred to the sink drain isolate from patient Y, generating strain ECNIH2. Plasmids carrying the bla_KPC gene are colored, and bla_KPC genes are marked by blue (KPC-2) or pink (KPC-3) circles. An insertion in the pKPC-39c plasmid is black. Non-KPC plasmids are gray. Selected plasmids are labeled with their size in kilobases. E. cloacae and K. pneumoniae isolates are orange and blue, respectively.

FIG 4

Venn diagram of bla_KPC-containing plasmids detected in the environment and patients. The presence of plasmids in isolates from patients and/or the environment was based on a k-mer inclusion approach for which a plasmid was considered present within a genome assembly if ≥95% of the plasmid k-mers were contained within the MiSeq genome assembly k-mers. Each value is the number of isolates detected in this study that carry the specified bla_KPC-containing plasmid. In the overlapping region, the ratio indicates the number of environmental isolates/number of patient isolates. Plasmids are drawn to scale. The inner circle represents the bla_KPC allele, and the outer circle represents the transposon. The bla_KPC alleles are blue for bla_KPC2 and pink for bla_KPC3. Color was used to differentiate the flanking sequence as follows: orange, Tn4401a; purple, Tn4401b; red, Tn4401b-IS630; gray, Tn4401d; green, IS26-TnpA; light blue, Ahyd_WCHAH01; black, not determined.

FIG 5

Genetic diversity of the pENT-e56-family of plasmids. Alignment of genetically similar bla_KPC-negative (top three) and bla_KPC-positive (bottom five) plasmids. Gray ribbons indicate regions of homology (>99.90% sequence similarity). Antimicrobial resistance genes are yellow, tra genes are dark green, transposase/resolvase genes are brown, and hypothetical genes are gray. Differences between the annotations of aligned regions are largely due to changes in the annotation pipeline and databases; genomes were annotated at the time of sequencing by using PGAP (versions 2.1 to 4.2).

FIG 6

Phylogenetic tree of Leclercia spp. based on core genome alignment (ParSNP v 1.2). The tree includes eight genomes from this study (LSNIH1 to -8), two publicly available environmental isolates (NCBI accession no. JWJV01 and MUFS01), and two L. adecarboxylata reference strains (ATCC 23216, USDA-ARS-USMARC-60222). Isolation sources are indicated in parentheses. Core genome alignments supporting this tree covered 36% of the reference sequence.