Molecular dissection of an outbreak of carbapenem-resistant enterobacteriaceae reveals Intergenus KPC carbapenemase transmission through a promiscuous plasmid

Abstract

Carbapenem-resistant Enterobacteriaceae (CRE) have emerged as major causes of health care-associated infections worldwide. This diverse collection of organisms with various resistance mechanisms is associated with increased lengths of hospitalization, costs of care, morbidity, and mortality. The global spread of CRE has largely been attributed to dissemination of a dominant strain of Klebsiella pneumoniae producing a serine β-lactamase, termed K. pneumoniae carbapenemase (KPC). Here we report an outbreak of KPC-producing CRE infections in which the degree of horizontal transmission between strains and species of a promiscuous plasmid is unprecedented. Sixteen isolates, comprising 11 unique strains, 6 species, and 4 genera of bacteria, were obtained from 14 patients over the first 8 months of the outbreak. Of the 11 unique strains, 9 harbored the same highly promiscuous plasmid carrying the KPC gene bla(KPC). The remaining strains harbored distinct bla(KPC) plasmids, one of which was carried in a strain of Klebsiella oxytoca coisolated from the index patient and the other generated from transposition of the bla(KPC) element Tn4401. All isolates could be genetically traced to the index patient. Molecular epidemiological investigation of the outbreak was aided by the adaptation of nested arbitrary PCR (ARB-PCR) for rapid plasmid identification. This detailed molecular genetic analysis, combined with traditional epidemiological investigation, provides insights into the highly fluid dynamics of drug resistance transmission during the outbreak. IMPORTANCE The ease of horizontal transmission of carbapenemase resistance plasmids across strains, species, and genera of bacteria observed in this study has several important public health and epidemiological implications. First, it has the potential to promote dissemination of carbapenem resistance to new populations of Enterobacteriaceae, including organisms of low virulence, leading to the establishment of reservoirs of carbapenem resistance genes in patients and/or the environment and of high virulence, raising the specter of untreatable community-associated infections. Second, recognition of plasmid-mediated outbreaks, such as those described here, is problematic because analysis of resistance plasmids from clinical isolates is laborious and technically challenging. Adaptation of nested arbitrary PCR (ARB-PCR) to investigate the plasmid outbreak facilitated our investigation, and the method may be broadly applicable to other outbreaks due to other conserved mobile genetic elements. Whether infection control measures that focus on preventing transmission of drug-resistant clones are effective in controlling dissemination of these elements is unknown.

FIG 1

Pulsed-field gel electrophoresis of outbreak isolates of the same species. (A) All three K. oxytoca isolates are a single strain. (B) Four K. pneumoniae isolates are three distinct strains. (C) Six E. cloacae isolates are four distinct strains.

FIG 2

Plasmid electrophoresis and Southern blot analysis of all unique outbreak clones. (A) Plasmid DNA electrophoresis resolved on a 0.6% agarose gel stained with ethidium bromide. Southern blot of gel A with a bla_KPC-specific probe (B), sg1-specific probe (C), or sg2-specific probe (D). Lanes: 1, Kpn1014 (negative control); 2 to 4, K. pneumoniae; 5 to 7, E. cloacae; 8, E. asburiae; 9, C. freundii; 10, K. oxytoca; 11, E. coli. Supercoiled and relaxed bla_KPC-hybridizing plasmid DNA was obtained from E. coli strain Eco1036.

FIG 3

Southern blot analysis of KpnI-digested plasmid DNA of representative outbreak clones and resistance plasmid transformants. (A) Restriction digests of plasmid DNA electrophoresis resolved on a 0.6% agarose gel stained with ethidium bromide. (B to D) Southern blot of gel A with a bla_KPC-specific probe (B), sg1-specific probe (C), or sg2-specific probe (D). Lanes: L, ladder; 1, Kpn1014 (negative control); 2, Kpn1016; 3, Ecl1027; 4, Cfr1047; 5, Kox1028; 6, Eco1036; 7, blank; 8, AMGH01-T; 9, AMGH07-T.

FIG 4

Hypothesized possible route(s) of spread of bla_KPC between patients based on plasmid profiles and epidemiological risk for CRE acquisition. Each number denotes a patient. Shape and color denote the plasmid type: blue circle, pUVA01; red triangle, pUVA02; black diamond, pUVA03. Solid arrows denote a possible route of spread based on shared plasmids and epidemiological risk factors. Dashed arrows denote a possible route of spread without a shared plasmid type. Note that arrows do not necessarily signify direct patient-to-patient transmission; the spread of CRE may have occurred to a source that shared the same epidemiological space as the infected patient.