Clinical and Molecular Description of a High-Copy IncQ1 KPC-2 Plasmid Harbored by the International ST15 Klebsiella pneumoniae Clone

In many parts of the world, carbapenem resistance is a serious public health concern. In Brazil, carbapenem resistance in Enterobacterales is mostly driven by the dissemination of KPC-2-producing K. pneumoniae clones. Despite being endemic in this country, only a few reports providing both clinical and genomic data are available in Brazil, which limit the understanding of the real clinical impact caused by the dissemination of different clones carrying blaKPC-2 in Brazilian hospitals. Although several of these KPC-2-producer K. pneumoniae isolates belong to the clonal complex 258 and carry Tn4401 transposons located on large plasmids, a concomitant emergence and silent dissemination of small high-copy-number blaKPC-2 plasmids are of importance, as described in this study. Our data identify a small high-copy-number IncQ1 KPC plasmid, its clinical relevance, and the potential for conjugative transfer into several K. pneumoniae isolates, belonging to different international lineages, such as ST258, ST101, and ST15.

clonal complex 258 and carry Tn4401 transposons located on large plasmids, a concomitant emergence and silent dissemination of small high-copy-number bla  plasmids are of importance, as described in this study. Our data identify a small high-copy-number IncQ1 KPC plasmid, its clinical relevance, and the potential for conjugative transfer into several K. pneumoniae isolates, belonging to different international lineages, such as ST258, ST101, and ST15. KEYWORDS Gram-negative bacteria, IncQ1, KPC-2, Klebsiella pneumoniae, ST15, bloodstream infections, carbapenemase, plasmid-mediated resistance C arbapenem resistance in Enterobacterales represents a serious threat to modern medicine and the global health system, as stressed by international agencies (1). KPC-producing Klebsiella pneumoniae infections are responsible for a severe burden in health care systems, particularly in North America, Latin America, Southern and Eastern Europe, Israel, and China (2). K. pneumoniae sepsis rates have been rising in recent years; according to PHE (Public Health England, including Wales and Northern Ireland), the rate of Klebsiella species bacteremia increased from 12 cases in 2009 to 17 cases in 2018 per 100,000 population (3).
The Brazilian Health Surveillance Agency (ANVISA) ranked K. pneumoniae as the most frequent pathogen (19.0%) causing central catheter-related bloodstream infections (CR-BSI) among adult intensive care unit (ICU) patients in 2017, with an increasing carbapenem resistance rate of 44.1% (4). This high rate is mostly due to the dissemination in Brazilian hospitals of various KPC-2-producing K. pneumoniae clones, belonging to the clonal complex (CC) 258, such as ST437 (a tonB31 single-allele variant of ST258), ST11, and ST340. Recently, the international KPC clone ST258 (clade 2, KL107, a hybrid clone resulting from genomic recombination events between ST11 and ST442) has been identified as a main driver of KPC-2 dissemination (5)(6)(7)(8). Other lineages include non-CC258 KPC-producing clones such as ST101, ST307, and ST16 (8). KPC-3producing clones have been reported in Latin America, mainly in Colombia, but are not disseminated in Brazil (9).
In this study, we report the clinical and molecular characterization of a K. pneumoniae ST15 clone, associated with high mortality rates in a Brazilian hospital, including its bla KPC-2 -bearing IncQ1 plasmid.
(This study was presented in part at the European Congress of Clinical Microbiology and Infectious Diseases, Amsterdam, The Netherlands, 13 to 16 April 2019, abstract O0917 [25].)

RESULTS
Clinical description. Within a retrospective cohort of 165 KPC-2-producing K. pneumoniae BSI cases in a tertiary Brazilian hospital during the 2014 to 2016 period, six cases were due to isolates displaying a clonal pulsotype (data not shown) and were assigned to ST15 group by in silico multilocus sequence typing (MLST). The clinical description of these six cases is provided in Table 1. The patients were hospitalized in diverse wards throughout the hospital, and five out of six were admitted initially at the Emergency Department ICU. The overall 3-day and 30-day crude mortality was 20% (2/6 patients) and 85% (5/6 patients), respectively. Half of these patients presented with septic shock. There was one primary catheter-related BSI, and in the remaining cases the BSI were secondary to ventilator-acquired pneumonia (n ϭ 2) or abdominal (n ϭ 2) or urinary (n ϭ 1) infections. Four out of six patients were treated with a triple antibiotic combination irrespective of in vitro susceptibility. In all six cases, the combination included polymyxin B, but the median number of in vitro active antimicrobials given to these patients was 1 (interquartile range [IQR], 1;2). The only surviving patient (case three), who had been admitted at the hospital with a urinary sepsis complicating an indwelling urethral catheter, was initially empirically treated with meropenem and ertapenem (dual carbapenem therapy) in association with polymyxin B. Antimicrobial susceptibility testing. Antimicrobial susceptibility results revealed that all six KPC-2-producing ST15 isolates were highly resistant to meropenem (MICs, 32 to 128 mg/liter) but remained 100% susceptible to amikacin (MICs, 2 to 4 mg/liter) and ceftazidime-avibactam (MICs at 0.5 mg/liter). All isolates had tigecycline MICs of 1 mg/ liter, while three isolates showed resistance to polymyxin B (MICs, 0.125 to 64 mg/liter; 50% susceptible).
bla KPC-2 mobilization. To test the mobilization of bla KPC-2 -IncQ1 plasmids, we performed mating-out assays first into Escherichia coli J53 and then into various K. pneumoniae recipients, belonging to high-risk clones. It showed bla KPC-2 -IncQ1 conjugation at high frequency (5 ϫ 10 Ϫ4 ) into J53. Both IncQ1 and IncL/M plasmids were transferred, as verified by PCR of 10 independent transconjugants, suggesting comobilization of the IncQ plasmid. Indeed, pP35-IncL/M (and the 100% similar pP16-IncL/M) contains a complete repertoire of genes belonging to the type IV secretion system (T4SS), with both Dtr and Mpf genes (pP16-IncL/M accession number CP053039), suggesting that this 53-kb plasmid provides the Mpf machinery (T4SS) allowing comobilization of the IncQ plasmid. Subsequently, we assessed the transmissibility of the bla KPC-2 -IncQ1 plasmid (using P16-KPC-TC as donor) into clinical isolates belonging to ST258, ST101, and ST15 (Table 2). Interestingly, the higher conjugation frequency (10 Ϫ6 ) was observed in ST258 and ST101, in accordance with the predominant role of these clones in the global acquisition and dissemination of KPC. The expected increase in the meropenem MICs ranged from 3 to 9 log 2 dilutions dependent upon the recipient isolate (Table 2). Altogether, these data confirm the potential for comobilization of this IncQ1 plasmid into E. coli and into several epidemiologically important K. pneumoniae clones.

DISCUSSION
To date, few ST15 isolates carrying the bla KPC-2 gene have been reported (21,22). This study reinforces our knowledge of K. pneumoniae ST15 as a multidrug-resistant clone facilitating the spread of carbapenemase genes worldwide. The clinical characteristics of the KPC-K. pneumoniae ST15-infected patients were similar to those encountered for other KPC-K. pneumoniae infections: mainly severely ill patients (high Charlson score) predominantly from ICUs. Though most isolates retained susceptibility to at least one antimicrobial prescribed for Gram-negative BSI treatment, a fatal outcome was observed in 85% of cases. The analysis of virulence factors identified the accessory iron uptake system kfuABC, a known invasiveness determinant generally found in ST15 lineage. Currently, there is little information available on the role of the KL112 capsule in virulence.
These ST15 isolates harbored bla KPC-2 on a small IncQ1 mobilizable high-copynumber plasmid. Interestingly bla KPC-2 -bearing IncQ1 plasmids have been described only on rare occasions (8,(26)(27)(28). We show here that this plasmid carries bla KPC-2 embedded within an NTE KPC element of class Ic that has successfully established itself within K. pneumoniae ST15 and spread silently in tertiary Brazilian hospitals.
Over the last 5-year period, IncQ1 plasmids carrying bla KPC-2 have been reported in several different pathogens in Brazil including Klebsiella quasipneumoniae (1 isolate, BSI), K. pneumoniae ST340 (CC258) (1 isolate, no clinical data), and Pseudomonas aeruginosa ST2584 (1 isolate, BSI), as shown in Fig. 3 (29-31). This current outbreak added a further six additional cases and suggests that IncQ1 plasmids can act as efficient bla KPC-2 carriers. The comparison of the genetic organization of IncQ1 plasmids found in geographically and temporally unrelated isolates (Fig. 2C) suggests indepen- dent parallel events rather than clonal horizontal dissemination of a unique cloneplasmid pair. These small IncQ1 plasmids (5.1 to 14.0 kb) have been shown to have the broadest host range of any known plasmids in both Gram-negative and Gram-positive bacteria; they typically replicate independently of the host chromosome and have high copy number (32)(33)(34). This combination of high copy number, broad host range, and common comobilization means that IncQ1 plasmids are typically highly promiscuous (35). Recently, IncQ1 plasmids were reported to be involved in the tet(X4)-mediated tigecycline resistance dissemination in farm animals in China (36), as well as in the spread of bla CMY-4 , bla GES-1 , bla IMP-27 , strA-strB, and sul2 gene clusters (37)(38)(39)(40). At the same tertiary hospital, an IncQ1 plasmid was previously described carrying the carbapenemase bla BKC-1 in K. pneumoniae isolates belonging to ST11 and ST442 (2010 to 2012) (unpublished data). We also identified a common IncL/M coresident helper plasmid that was responsible for the mobilization of these IncQ plasmids (34). Besides IncL/M plasmids, IncP, IncF, IncI, IncX, IncN, and IncW plasmids have also been described aiding IncQ1 mobilization (35).
In conclusion, we have presented here a cryptic outbreak of a K. pneumoniae ST15 clone that was carbapenem resistant due to an IncQ1 plasmid-carried bla KPC-2 gene. The outbreak resulted in several fatalities and highlights the importance of IncQ1 plasmids in the spread of the KPC carbapenemase gene. The ubiquitous presence of IncQ plasmids among both enteric and nonfermentative Gram-negative bacteria together with acquisition of KPC-2 suggests this combination of carbapenemase gene and promiscuous plasmid deserves particular attention and should be closely monitored.

MATERIALS AND METHODS
Study population. The present study involves a 3-year (2014 to 2016) retrospective cohort of KPC-producing K. pneumoniae bloodstream infections (BSI), from a Brazilian public teaching hospital located in the city of São Paulo, published by our collaborative group (8). This cohort included the microbiological and genetic characterization of unique KPC-K. pneumoniae BSI adult cases. The study was approved by the Hospital São Paulo/Federal University of São Paulo (UNIFESP) Ethics Committee for Clinical Research (protocol number 1.814.158). Epidemiological and clinical data were extracted from the medical records in a standardized case form, as previously described (8).
Isolates selection and microbiological analysis. Six clonally related isolates, belonging to ST15, were selected for the detailed analysis presented here. In addition, two carbapenem-susceptible K. pneumoniae ST15 isolates from the same hospital collection, bla KPC negative (HSP32 and P21), were selected for comparative genomic analysis. Isolate identification was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using a Microflex LT mass spectrometer and Biotyper 3.3 software (Bruker Daltonics) according to the manufacturer's recommendations. MICs of meropenem, amikacin, gentamicin, tigecycline, and ceftazidime-avibactam were determined by agar dilution, while the broth microdilution technique was used to determine the polymyxin B MICs. Susceptibility testing results were performed and interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations (41).
WGS and bioinformatics analysis. The isolates were sequenced using the Illumina MiSeq platform (Illumina Inc.). DNA libraries were prepared for paired-end sequencing (2 ϫ 300 cycles) using Nextera XT (Illumina Inc.). Quality control of raw sequence reads included FastQC (0.11.2), and adaptor trimming was performed using Trim Galore (0.4.3). K. pneumoniae genome assembly was performed using Spades (version 3.8.0), with the k-mer length increased to 127 (42). Multilocus sequence type (MLST), antimicrobial resistance (AMR) determinants, and plasmid replicons were identified using the MLST 2.0, ResFinder 3.1, and PlasmidFinder online tools (Center for Genomic Epidemiology) setting cutoff values of 90% identity and 80% minimum coverage (10 September 2018 database) (43). Virulence genes were analyzed with Geneious 10.6.1 using an in-house data set (80% minimal coverage, 75% identity) (8). Assembled genomes were submitted to the Kaptive platform, and capsular loci (KL) were determined using Klebsiella K locus primary as a reference (44). In addition, two isolates (P35 and P16) were selected for complete assembly (chromosome and plasmids). For these, total genomic DNA was extracted and sequenced using long-read (MinION; Oxford Nanopore Technologies), in combination with MiSeq Illumina raw short-read, hybrid de novo assembly using Unicycler (v0.4.0). This strategy enabled the generation of complete circularized sequences of both chromosomes and plasmids (45). Plasmid copy number was obtained based on the ratio of long reads containing bla KPC-2 divided by the mean of chromosomal single-copy tonB-and gapA-containing reads.
Mating-out (conjugation) experiments. To evaluate and compare the transferabilities of plasmidborne bla KPC-2 , conjugation assays were carried out with an ST15 donor isolate into the E. coli J53 azide-resistant strain. Subsequently, a sequence-verified J53-derived transconjugant, named P16-KPC-TC, was used as donor for a secondary conjugation set into selected K. pneumoniae isolates. Briefly, mid-log cultures of donor and recipient strains were mixed in LB broth. The mating culture was then incubated overnight at 37°C, appropriately diluted in physiological saline, and plated onto UTI agar (16636 HiCrome UTI agar; Sigma-Aldrich) containing 0.5 mg/liter meropenem for assessing the colony count. After incubation, for each conjugation, at least 5 (when available) putative transconjugant colonies were tested by restreaking onto meropenem 0.5-mg/liter UTI agar plates and the putative transconjugants were further tested by PCR for bla KPC-2 . Conjugative frequency was calculated as the ratio of transconjugant CFU per donor. Isolates were considered unable to transfer bla KPC-2 into the recipient species if the transfer frequency was 10 Ϫ9 or lower (46)(47)(48).
Data availability. Whole-genome sequences of the studied K. pneumoniae ST15 isolates have been deposited in the NCBI database under nucleotide accession numbers CP053035 to CP053041 and JABEPV000000000, JABEPW000000000, JABEPX000000000, JABEPY000000000, JABEPZ000000000, and JABENA000000000).
Julius Thorn Trust Foundation (Switzerland). Sequencing data were supported by Cardiff University.