Covert dissemination of carbapenemase-producing Klebsiella pneumoniae (KPC) in a successfully controlled outbreak: long- and short-read whole-genome sequencing demonstrate multiple genetic modes of transmission

Abstract

BackgroundCarbapenemase-producing Enterobacteriaceae (CPE), including KPC-producing Klebsiella pneumoniae (KPC-Kpn), are an increasing threat to patient safety.ObjectivesTo use WGS to investigate the extent and complexity of carbapenemase gene dissemination in a controlled KPC outbreak.Materials and methodsEnterobacteriaceae with reduced ertapenem susceptibility recovered from rectal screening swabs/clinical samples, during a 3 month KPC outbreak (2013–14), were investigated for carbapenemase production, antimicrobial susceptibility, variable-number-tandem-repeat profile and WGS [short-read (Illumina), long-read (MinION)]. Short-read sequences were used for MLST and plasmid/Tn4401 fingerprinting, and long-read sequence assemblies for plasmid identification. Phylogenetic analysis used IQTree followed by ClonalFrameML, and outbreak transmission dynamics were inferred using SCOTTI.ResultsTwenty patients harboured KPC-positive isolates (6 infected, 14 colonized), and 23 distinct KPC-producing Enterobacteriaceae were identified. Four distinct KPC plasmids were characterized but of 20 KPC-Kpn (from six STs), 17 isolates shared a single pKpQIL-D2 KPC plasmid. All isolates had an identical transposon (Tn4401a), except one KPC-Kpn (ST661) with a single nucleotide variant. A sporadic case of KPC-Kpn (ST491) with Tn4401a-carrying pKpQIL-D2 plasmid was identified 10 months before the outbreak. This plasmid was later seen in two other species and other KPC-Kpn (ST14,ST661) including clonal spread of KPC-Kpn (ST661) from a symptomatic case to nine ward contacts.ConclusionsWGS of outbreak KPC isolates demonstrated blaKPC dissemination via horizontal transposition (Tn4401a), plasmid spread (pKpQIL-D2) and clonal spread (K. pneumoniae ST661). Despite rapid outbreak control, considerable dissemination of blaKPC still occurred among K. pneumoniae and other Enterobacteriaceae, emphasizing its high transmission potential and the need for enhanced control efforts.

Distinct evolutionary dynamics of horizontal gene transfer in drug resistant and virulent clones ofKlebsiella pneumoniae

“…While these data are subject to the biases of the underlying databases within which clinically relevant (MDR and virulence) plasmids are overrepresented, they are also consistent with the findings above regarding overall pan--genome diversity. These data imply that MDR clones frequently acquire and lose plasmids, consistent with the high plasmid diversity reported previously for ST258 52,53 and several others 7 , and with data from recent investigations of Kp circulating in hospitals which showed that individual plasmids transferred frequently between clones 54,55 . In contrast, the hypervirulent clones were associated with comparatively low plasmid diversity, mirrored by generally narrow plasmid load distributions.…”

“…This is particularly worrying from a hospital infection control perspective since many of the MDR clones investigated here appear well adapted to transmission and colonisation in the human population, and are frequent causes of hospital outbreaks 7,8 . Given the mounting evidence that MDR clones can carry multiple plasmids at limited fitness cost [64][65][66] and frequently exchange plasmids with other bacteria 54,55 , it seems these MDR clones may also be the perfect hosts for consolidation and onwards dissemination of MDR and virulence determinants. The greatest concern is that these determinants will be consolidated onto a single mobile genetic element; indeed mosaic Kp plasmids carrying AMR genes plus iuc and rmpA2 have already been reported in an MDR Kp clone 30 , and Escherichia coli plasmids bearing iuc, rmpA and AMR genes have been detected in Kp 27 .…”

Klebsiella pneumoniae as a key trafficker of drug resistance genes from environmental to clinically important bacteria

“…This suggests K. pneumoniae receives DNA from a wider diversity of HGT partners; indeed lowest common ancestor analysis of K. pneumoniae accessory genes has implicated >20 distinct genera as DNA donors, including numerous other members of the Enterobacteriaceae but also diverse groups such as Acinetobacter, Burkholderia, Streptomyces, Vibrio, Xanthomonas and Xyella [9]. More direct evidence for K. pneumoniae engaging in inter--species HGT can be found in recent genomic comparisons of carbapenem--resistant Enterobacteriaciae in hospitals, which captured identical or highly similar carbapenemase encoding plasmids and/or transposons from K. pneumoniae and other species originating from the same ward and/or patient (E. coli, E. cloacae, Enterobacter asburiae, Citrobacter freundii, Klebsiella oxytoca, Raoultella ornothinolytica) [73][74][75].…”

Antimicrobial resistant Klebsiella pneumoniae carriage and infection in specialized geriatric care wards linked to acquisition in the referring hospital

“…SNVs with which to identify lineages and AMR genes, bolstered by long-read sequencing to resolve plasmids and AMR gene context and to maximize resolution for detecting transmission. While this strategy has been used in other studies [30][31][32][33] , ours is the first to adopt multiplex nanopore sequencing and hybrid assembly to rapidly and cost-efficiently complete genome sequences of interest in a high-throughput manner [11,12] (Tables S3, S4).…”