Klebsiella quasipneumonaiae provides a window into carbapenemase gene transfer, plasmid rearrangements and nosocomial acquisition from the hospital environment
1Division of Infectious Disease and International Health. Department of Medicine, University of Virginia Health System. Charlottesville, Virginia, USA.
2Clinical Microbiology Laboratory. Department of Pathology, University of Virginia Health System, Charlottesville, Virginia, USA.
3Modernizing Medical Microbiology Consortium, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK.
4NIHR Health Protection Research Unit in Healthcare Associated Infection and Antimicrobial Resistance at University of Oxford in partnership with Public Health England, Oxford, UK.
5Health Information & Technology, University of Virginia Health System, Charlottesville, Virginia, USA.
6Department of Information Technology Research, School of Medicine, University of Virginia, Charlottesville, USA
7Icahn Institute and Department of Genetics and Genomic Sciences, Icahn School of Medicine, Mount Sinai, New York, New York, USA
#Corresponding Author: Amy Mathers, M.D. Associate Professor of Medicine, Division of Infectious Diseases & International Health, P.O. Box 800255, Charlottesville, VA, USA 22908-1361, Phone: (434) 982-4814, Fax: (434)-924-0075, Email : ajm5b@virginia.eduAbstract
Several emerging pathogens have arisen as a result of selection pressures exerted by modern healthcare. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intra-species transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, blaKPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10 and five-year periods respectively. There were 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from eight patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subspecies quasipneumoniae (n=23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then probable transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique blaKPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases.
Introduction
In the last 50 years transformations in healthcare have created new niches for microorganisms such as Acinetobacter baumannii complex and Candida auris to arise from obscurity and emerge as important pathogens. Similarly, we have seen an increasing number of highly resistant Klebsiella pneumoniae strains which have been successfully transmitted worldwide(1). Klebsiella pneumoniae has proven to be an important contributor to the modern antibiotic resistance epidemic with its ability to acquire and carry antimicrobial resistance plasmids, as well as being successful a human pathogen. More recently, whole-genome sequencing has revealed that many isolates classified as K. pneumoniae actually encompass three related but distinct species – K. pneumoniae, K. variicola and K. quasipneumoniae(1, 2). K. quasipneumoniae was originally thought to be largely confined to agriculture and the environment, however it appears that it may also be prominent in human disease(3), and several recent reports have demonstrated that it harbors virulence factors and acquires clinically relevant genes of antimicrobial resistance(4, 5). Although there have been relatively few reports of K. quasipneumoniae to date, the true prevalence of this organism is likely underestimated as it is not generally distinguished from K. pneumoniae in routine testing of clinical laboratories(2).
Bacterial evolution via horizontal gene transfer is central to the ongoing crisis of antimicrobial resistance among clinically relevant bacteria. Hospital wastewater is being increasingly recognized as an ideal reservoir for resistance gene exchange and amplification, with ongoing antimicrobial selection pressure exerted through antimicrobials excreted in patient waste(6). Premise plumbing can be seeded by antimicrobial resistance genes in diverse bacterial strains and species, and represents a difficult-to-treat reservoir for ongoing gene exchange, creating successful drug-resistant bacteria that can thrive in both the environmental and human niches(7).
Whole-genome sequencing studies have demonstrated that our understanding of the interplay between antimicrobial resistance plasmids and their host strains/species is limited(8). The host range of a plasmid is critical for acquisition and persistence in specific species, but it appears that some bacterial strains are better equipped than others to prevent acquisition of or destroy foreign plasmid DNA(9). The durability of plasmid acquisition events and the creation of new highly resistant strains reflects complex dynamics which depend on the characteristics of the plasmid in question as well as host strain tolerance(10, 11). Seldom do we have the opportunity to witness strains acquiring plasmids in vivo or in the environment and inferences about genetic re-arrangements are often highly speculative. However, understanding the mechanisms and frequency of resistance gene transfer events occurring in real world contexts can provide important insights into the wider evolutionary landscape creating modern multidrug resistant bacteria which cannot be effectively modeled in lab experiments(12).
Within our institution we have seen ongoing transmission of diverse carbapenemase-producing organisms for the last decade, driven by genetic exchange of the Klebsiella pneumoniae carbapenemase (KPC) gene (blaKPC) in patients and the environment(13, 14). This has enabled us to understand specific pathways of genetic mobility involving numerous different mobile genetic elements and host bacterial species(13, 15). Herein we examine blaKPC acquisition and associated genetic rearrangements within K. quasipneumoniae as a real-life representation of an emerging pathogen associated with the hospital wastewater environment.
Results
From our collection of blaKPC-positive isolates from patients (2007-2017) and the hospital environment (2013-2017), there were a total of 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory (Table 1). Twenty-three of these were K. quasipneumoniae subspecies quasipneumoniae (KpIIA) (ten patient isolates from four patients and 13 environmental isolates from seven rooms) and nine were K. quasipneumoniae subspecies similipneumoniae (KpIIB) (five patient isolates from four patients and four environmental isolates from four rooms). The KpIIA and KpIIB isolates were separated by >100,000 single nucleotide variants (SNVs). We identified a single strain of KpIIA and four strains of KpIIB differing from each other by >20,000 SNVs (Fig. 1).
Within the KpIIA strain, there were two subclades separated by ~150 SNVs (Fig. 1a). The first subclade contained two isolates separated by 10 SNVs (Fig. 1a). CAV1360 was from patient 1 in November 2009 and CAV2279 was identified in early 2014 (shortly after environmental sampling began) from room B that patient 1 had occupied in May 2009.
The second subclade of KpIIA contained isolates from three patients (patients 2-4) and six rooms (rooms A, C-G). The earliest of these was from patient 2 in November 2013. Patient 2 was in the hospital with a prolonged stay in the Surgical Trauma and Burn Intensive Care Unit (STBICU) following complications of a liver transplant (Figure 2). Patient 2 was noted to be first colonized with blaKPC-positive KpIIA in November 2013. KpIIA was not found in the STBICU environment prior to closure for remediation of KPC-contamination of the drains in December 2013. Following drain exchange and unit re-opening patient 2 was immediately moved back into the STBICU and subsequently occupied rooms C, D, E and G in the STBICU, suggesting that the KpIIA isolates in these rooms originated from patient 2 (Figure 2). Patient 3 was admitted to the STBICU at the same time as patient 2 and thus could have acquired KPC-KpIIA directly from patient 2 without environmental transmission. Patient 4 was later admitted to STBICU room E for 28 days and discharged before he was found to have KpIIA. He was never on a ward at the same time as any other patients known to carry KpIIA, suggesting acquisition from the hospital environment.
There were four patients (patients 5-8) carrying four distinct strains of blaKPC-KpIIB seen over a five year period (Fig. 1b, Table 1). For patient 7, the same KpIIB strain (~80 SNV differences) was also seen in sinks from two rooms in the Medical Intensive Care Unit (MICU) (rooms H-I) and two rooms in the STBICU (rooms J-K) in December 2013 when environmental sampling first began; this preceded detection in the patient in February 2014. Patient 7 was admitted to the MICU, but did not stay in rooms H or I. The other three patients with KpIIB each had a unique blaKPC strain, none of which were identified in another patient or the environment. Patient 6 had a prolonged hospital stay and was also colonized/infected with another blaKPC-positive species (K. pneumoniae).
Three patients developed infections with KPC-KpIIA (Table 1). Patient 1 died of ventilator-associated pneumonia with KPC-KpIIA following a complicated heart transplant. Patient 2 had both ventilator-acquired pneumonia, which was successfully treated, and a subsequent untreatable intraabdominal infection with KPC-KpIIA bacteremia, which contributed to the patient’s death after a long hospital stay with a complicated liver transplant. Patient 4 had a successfully treated complicated KPC-KpIIA urinary tract infection. Patient 3 did not develop an infection with KpIIA. None of the patients with KpIIB developed K. quasipneumoniae infections, however two of the patients did develop infections with other species carrying blaKPC (K. pneumoniae for patient 6 and Serratia marcescens for patient 8) (Table 2).
Genetic variation and rearrangements within KpIIA
All KpIIA isolates were closely related at the core chromosome level, with a maximum divergence of <180 SNVs. If blaKPC were acquired only once in this lineage then any sequence variation within the 10 kb blaKPC transposon Tn4401 would be the result of mutational change, which is expected to be rare. Surprisingly, the Illumina sequence data revealed a great deal of sequence variation within Tn4401 (Fig. 1a). Two sites (positions 8015 and 9663 in the Tn4401b reference) showed variation at the single-nucleotide level, and one isolate had a deletion at positions 7075-7153. Interestingly, several isolates showed mixtures at one or both of the variable sites, indicating two or more different versions of Tn4401 in the same isolate. This included mixtures at position 8015, which is located within the blaKPC gene and differentiates blaKPC-2 and blaKPC-3, indicating that there were isolates with both blaKPC alleles.
Similarly, if a single blaKPC plasmid were acquired and stably maintained within KpIIA, then we would expect to see a single flanking sequence context for Tn4401. On the contrary, there was significant diversity in Tn4401 flanking regions, with eight and seven different 5 bp sequences on the left and right sides of Tn4401 respectively, suggesting active transposition of Tn4401 within KpIIA and/or multiple plasmid acquisitions.
To better understand the origin of the genetic diversity within and surrounding Tn4401, we performed long-read PacBio sequencing on three of the KpIIA isolates (CAV2013 from patient 2, CAV1947 from room C and CAV2018 from room C), as well as a S. marcescens isolate from patient 2 (CAV1761). The room C isolates were chosen because this room only became positive after admission of patient 2 following sink trap exchange in the STBICU, hence they are expected to be descended from the patient 2 KpIIA.
Both patient 2 isolates had a single blaKPC plasmid each (Figure 3a-b). The KpIIA isolate had a 47,095 bp “RepA” blaKPC-3 plasmid, and the S. marcescens isolate had a 69,158 bp IncU/IncX5 blaKPC-2 plasmid (16). Both plasmids contained Tn4401b, however there were two SNV differences within the Tn4401b sequence, one at position 8015 (differentiating blaKPC-2 and blaKPC-3) and one at position 9663.
The KpIIA isolates from room C (CAV1947 and CAV2018) had three and two blaKPC plasmids respectively (Fig. 3c-d). Both isolates harbored the IncU/IncX5 blaKPC plasmid from the patient 2 S. marcescens isolate, indicating likely blaKPC plasmid transfer from S. marcescens to K. quasipneumoniae (Fig. 3e). In CAV1947, the plasmid sequence was identical to the patient isolate, CAV1761, with the exception of two large indels (Fig. 4a). One of these was a 16,315 bp deletion immediately adjacent to Tn4401, presumably as a result of intramolecular transposition, that converted the left flanking sequence from TTTTT to ACAAT and removed the IncU replicon sequence (Fig. 3g). In CAV2018, the plasmid sequence was identical to CAV1761, except for a single 5,923 bp deletion that truncated part of the Tn4401 sequence (Fig. 3h, 4a).
Both isolates also harboured the ancestral RepA blaKPC plasmid from the patient 2 KpIIA isolate, with several SNVs and large indels (Fig. 4b). Interestingly, in CAV2018, one of the SNVs was located within Tn4401, such that the CAV2018 RepA plasmid contained blaKPC-2 rather than blaKPC-3. Given that there was plasmid transfer of the IncU/IncX5 blaKPC-2 plasmid from S. marcescens, we infer that the blaKPC-2-containing RepA plasmid most likely arose as a result of homologous recombination between these two different plasmids flanking the blaKPC region (Fig. 3f, k). The Illumina data also revealed similar patterns of homologous recombination in other isolates (notably CAV2983, CAV2984, CAV3444, CAVp64 and CAVp275, which all have the TTTTT IncU/IncX5 plasmid flanking sequences, but with the C8015T blaKPC-3 mutation and without the T9663C mutation), suggesting frequent exchange of Tn4401 variants between different blaKPC plasmids within the same host bacterium (Fig. 1, 3k).
CAV1947 also harboured a third blaKPC plasmid, representing transposition of Tn4401 into a 4,095 bp non-typeable plasmid that was present in the CAV2013 ancestor from patient 2 (Fig. 3i, 4c).
K. quasipneumoniae has acquired blaKPC on multiple occasions
Within KpIIB, there were four divergent strains separated by >20,000 SNVs, suggesting four separate acquisitions of blaKPC in this subspecies. Within KpIIA, there were two subclades separated by ~180 SNVs. Given that Tn4401 variation and flanking sequences were different between the two subclades (apart from the GTTCT flanking sequence which is known to be present in many different blaKPC plasmids)(13); and that there was no epidemiological overlap, it is most likely that the subclades acquired blaKPC independently. Additionally, as described above, the second subclade likely acquired blaKPC on two occasions, with the second acquisition originating from S. marcescens. Therefore, overall there were likely seven acquisitions of blaKPC by K. quasipneumoniae, three in KpIIA and four in KpIIB.
Interestingly, there was evidence that one of the acquisitions in KpIIB also originated from S. marcescens, indicating the compatibility of these two species in exchanging plasmids. This was in the patient 8 KpIIB lineage. Patient 8 was first colonised with blaKPC-S. marcescens carrying Tn4401b with a T9663C mutation and TTTTT/TTTTT flanking sequences. Four months later, blaKPC-KpIIB was identified with the same Tn4401 mutation and flanking sequences, suggesting plasmid transfer from S. marcescens to K. quasipneumoniae within this patient.
Discussion
We describe the behaviour of nosocomial blaKPC-positive K. quasipneumoniae strains within a single-hospital setting, observing their propensity to uptake multiple carbapenemase plasmids from other species, and disseminate between patients and sink drains. Our study also suggests that rapid genetic rearrangement occurs in the mobile genetic elements carrying blaKPC in KpIIA.
There is increasing recognition that the hospital environment is an important potential reservoir in the transmission of carbapenemase-producing Enterobacteriaceae (CPE), but delineating transmission chains is often challenging(17, 18). Through our K. quasipneumoniae example we provide compelling evidence for patient-to-drain and drain-to-patient transmission, as has been observed in other studies(7). We also provide evidence supporting the ability of K. quasipneumoniae to be maintained in the environment for a long period of time, with the first subclade of KpIIA detected in the environment on initial sampling, even though it had not been seen in a patient nor had that patient been in the room for over three years. The costly closure of the STBICU and exchange of all the sink drain plumbing pipes had a limited effect on environmental contamination with CPE; instead it appears to have provided an environment for immediate new seeding and establishment of previously unobserved carbapenem-resistant strains. Understanding the dynamics and natural history of colonization of premise plumbing with CPE will be important in designing effective interventions to limit transmission(19).
Although there have only been a handful of reports of K. quasipneumoniae since its definition as a species in 2014, it does appear that this organism is widespread(2, 5, 20, 21). As seen here, it is not readily distinguished from K. pneumoniae with current clinical microbiology techniques and thus the true prevalence is unknown(2, 22). On the evolutionary time scale, modern medicine has provided a novel ecology with immunocompromised patients, widespread antimicrobial use, newly circulating antimicrobial resistance genes and the design of the modern hospital providing new microbiologic niche for organisms to emerge(7, 23). As seen here we provide evidence for K. quasipneumoniae to be sustained in both a human host and the environment encountering several different species which may be relatively new in the evolutionary tree of Klebsiella sp. As a consequence of these encounters transfer of mobile DNA occurs via traceable carbapenemase plasmids. We found evidence for seven independent acquisitions of blaKPC by K. quasipneumoniae, suggesting that this species is amenable to plasmid uptake from other species of Enterobacteriaceae. Given the difficulties in accurately identifying K. quasipneumoniae, this species may therefore be more significant in the context of blaKPC dissemination than has previously been recognised.
Within K. quasipneumoniae, there was surprising variability in mobile elements carrying blaKPC, which was the result of several different processes observed amongst a limited number of highly related isolates(n=23). Specifically, there were multiple independent blaKPC plasmid acquisitions, homologous recombination between different blaKPC plasmids, transposition of Tn4401 into new plasmids, intramolecular transposition of Tn4401, a deletion within Tn4401 and a deletion truncating Tn4401. This high degree of genetic mobility has been similarly observed in other small studies(25, 26), and highlights the difficulty in developing an accurate understanding of the transmission epidemiology of important drug resistance genes which can be rapidly mobilized by multiple independent genetic modalities.
Within KpIIA, there were multiple acquisitions of blaKPC within the same lineage, such that a blaKPC-positive KpIIA strain acquired a second, unrelated blaKPC plasmid from S. marcescens. Consequently, there were then two different blaKPC plasmids, with different Tn4401 sequences and different blaKPC alleles, within the same host bacterium. This situation facilitated multiple rearrangements via homologous recombination between the different plasmids, resulting in the generation of new combinations of Tn4401 SNVs and host plasmids. Multiple acquisition of resistance plasmids followed by rearrangements between those plasmids is likely to be important in the generation of adaptive allelic combinations which contribute to the amplification of cross-class antimicrobial resistance within strains. High-risk clones with a propensity for uptake of antimicrobial resistance plasmids may represent important targets for intervention(24).
This study has several limitations. Most notably, it is a small retrospective series, preventing a full understanding of the role of the environment. Also, the order of genetic rearrangements is also not completely known given the limited number of long-read sequenced isolates and inability to capture all isolates from the environment over time. We offer however, that this is higher resolution than seen in many studies, and the analysis does contribute to the greater understanding of rapid rearrangement and mechanisms at play around mobility of genetic elements harbouring genes of antibiotic resistance in Enterobacteriaceae.
In summary, we demonstrate the relevance of K. quasipneumoniae as a species fit for nosocomial transmission in the modern era that is capable of acquiring and maintaining relevant resistance elements.
Methods
Setting
Isolates were collected at the University of Virginia, a 619-bed tertiary care hospital, from August 2007-May 2017. A robust K. pneumoniae carbapenemase-producing organism (KPCO) prevention program existed throughout the study period as previously described(27), and included perirectal screening beginning in April 2009 in the medical intensive care unit (MICU) and surgical intensive care unit (STBICU), and weekly screening of all patients in the MICU and STBICU as well as units where any known KPCO-colonized patient was present(28). Screening was performed as previously described(28). Clinical Enterobacteriales and Aeromonadaceae isolates, as identified by MALDI-TOF or VITEK2 (Biomerieux, Durham, NC), with an elevated ertapenem or meropenem minimum inhibitory concentration (MIC) by VITEK2 (Biomerieux, Durham, NC) immediately underwent CarbaR (Cepheid Sunnyvale, CA) carbapenemase PCR testing. All species identification was performed using a combination of VITEK2, VITEK-MS (Biomerieux, Durham, NC). Clinical data was gathered by retrospective electronic medical record review under University of Virginia Health Sciences IRB#13558 with waiver of consent.
In September 2013 sink trap sampling for KPCO began using previously described techniques(14) with a swab for drain collection and p-trap water. Following identification of KPCO in the hospital environment, the STBICU was closed to patient care in December 2013. Over the following 9 weeks all sink drain pipes were removed and replaced with sink traps that eliminated overflows on the sink bowl. Patients were readmitted to the surgical intensive care unit in February 2014. Bleach, hydrogen peroxide and ozone impregnated water (2ppm) were applied weekly from February-May 2014 in the STBICU (following drain exchange and sink bowl overflow closure and removal) and from March-May 2014 in the MICU (without drain exchange or sink bowl overflow removal).
Whole-genome sequencing
Illumina sequencing was performed as described previously(29). PacBio long-read sequencing and assembly were performed as previously described(13).
Broad level species classification was performed using Kraken(30). To identify K. quasipneumoniae isolates, we queried all isolates initially classified as K. pneumoniae against reference sequences representing each of the four clades in Holt et al(1). We arbitrarily selected a single reference sequence for each clade; these were: ERR025521 (KpI), ERR025986 (KpIIA), ERR025528 (KpIIB) and ERR025573 (KpIII). We used mash v1.1.1(31) with parameters “-r -m 5” to compare Illumina data for each of our isolates to these reference sequences. Each isolate was then assigned to one of the four Kp clades according to the reference with the lowest distance value. All isolates assigned to KpIIA or KpIIB were included in the analysis. In addition, we also included any other KPCO isolates from patients carrying K. quasipneumoniae.
To identify chromosomal single-nucleotide variants (SNVs), Illumina reads for each K. quasipneumoniae isolate were mapped to the CAV2013 chromosome sequence (derived from long-read sequencing), with mapping and variant calling performed as described previously (32). A phylogeny was generated using IQ-TREE v1.3.13 (33) from an alignment of variable sites where at least 70% of samples had a high-quality reference/variant call (i.e. we excluded sites where >30% of samples had an “N” call). This was run with parameters “-blmin 0.000000001 -nt 4 -m GTR”, with -fconst used to specify the number of invariant sites.
To identify Tn4401 variation and flanking sequences from Illumina data, we used TETyper with published parameters(34).
Plasmid Inc typing was performed using the February 2018 version of the PlasmidFinder Enterobacteriaceae database(16), with an identity threshold of 95% and minimum length 60%.
Funding
This work was funded in part by a contract from the Centres for Disease Control and Prevention (CDC) Broad Agency Announcement BAA 200-2017-96194. ASW, DWC, ASW are affiliated to the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Healthcare Associated Infections and Antimicrobial Resistance at University of Oxford in partnership with Public Health England (PHE) [grant HPRU-2012-10041] and are supported by the Oxford NIHR Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health or Public Health England.