Nested Russian Doll-Like Genetic Mobility Drives Rapid Dissemination of the Carbapenem Resistance Gene blaKPC

Abstract

The recent widespread emergence of carbapenem resistance in Enterobacteriaceae is a major public health concern, as carbapenems are a therapy of last resort against this family of common bacterial pathogens. Resistance genes can mobilize via various mechanisms, including conjugation and transposition; however, the importance of this mobility in short-term evolution, such as within nosocomial outbreaks, is unknown. Using a combination of short- and long-read whole-genome sequencing of 281 blaKPC-positive Enterobacteriaceae isolates from a single hospital over 5 years, we demonstrate rapid dissemination of this carbapenem resistance gene to multiple species, strains, and plasmids. Mobility of blaKPC occurs at multiple nested genetic levels, with transmission of blaKPC strains between individuals, frequent transfer of blaKPC plasmids between strains/species, and frequent transposition of blaKPC transposon Tn4401 between plasmids. We also identify a common insertion site for Tn4401 within various Tn2-like elements, suggesting that homologous recombination between Tn2-like elements has enhanced the spread of Tn4401 between different plasmid vectors. Furthermore, while short-read sequencing has known limitations for plasmid assembly, various studies have attempted to overcome this by the use of reference-based methods. We also demonstrate that, as a consequence of the genetic mobility observed in this study, plasmid structures can be extremely dynamic, and therefore these reference-based methods, as well as traditional partial typing methods, can produce very misleading conclusions. Overall, our findings demonstrate that nonclonal resistance gene dissemination can be extremely rapid, presenting significant challenges for public health surveillance and achieving effective control of antibiotic resistance.

FIG 1

Diversity of bacterial species, strains, plasmids, and Tn4401 variants. For each species, a phylogeny was generated from mapping to a species-specific chromosomal reference, after the deduplication of closely related isolates from the same patient (see Materials and Methods). Distinct strains are defined by a cutoff of ∼500 SNVs (see Materials and Methods); strains found in more than one patient are shaded gray. Circles show plasmid “presence” as determined from Illumina data, with the fill color indicating uncertainty about whether the plasmid contains bla_KPC. Boxes show plasmid structures determined from long-read PacBio sequencing of 17 randomly chosen isolates, as well as the previously sequenced isolates from index patient B (37). Where the PacBio-sequenced isolate was excluded from the phylogeny as a patient duplicate, the plasmid structure of the corresponding closely related isolate from the same patient is shown. Tn4401 and bla_KPC variants (Table 2) are indicated by large and small squares, respectively. The likely sources of bla_KPC acquisition, as determined from epidemiological data, are indicated by text color.

FIG 2

pKPC_UVA01-like plasmids identified through long-read PacBio sequencing. The reference pKPC_UVA01 sequence is shown together with all 11 pKPC_UVA01-like plasmids identified through long-read PacBio sequencing, including the 6 that do not contain bla_KPC. Arrows indicate predicted open reading frames; Tn4401 is purple. Pink shading indicates regions of identity between adjacent sequences, and SNVs are indicated by red lines.

FIG 3

Tn4401 is commonly integrated into a Tn2-like element. Tn4401 and the surrounding region (i.e., partial plasmid sequence, except for pKPC_CAV1320) are shown for each distinct bla_KPC plasmid. Variants of the same plasmid backbone (Table 1) are not shown. Arrows indicate predicted open reading frames; Tn4401 is purple. Pink shading indicates regions of identity between adjacent sequences, SNVs are indicated by red lines, and short indels (1 or 2 bp) are indicated by blue lines. At the very top is the Tn2* reference sequence from AY123253 (49).

FIG 4

Ward contacts between patients with genetically related isolates. Each horizontal line represents a different strain (top) or Tn4401 variant (bottom). Filled circles indicate patients who had previous ward contact with another patient on the same horizontal line (i.e., possible patient-to-patient transmission). As Tn4401b-1 is present in two-thirds of the patients, many coincidental ward contacts may be expected to occur, resulting in a substantial overestimate of the number of transmission events. Therefore, the total number of Tn4401 acquisitions explainable by direct ward contact is indicated, as well as that with Tn4401b-1-carrying patients excluded. The vertical line indicates the onset of routine patient screening.

FIG 5

bla_KPC spreads at multiple genetic levels, resulting in a high level of diversity in bla_KPC-positive Enterobacteriaceae isolates.