Genomic and clinical characterisation of multidrug-resistant carbapenemase-producing ST231 and ST16 Klebsiella pneumoniae isolates colonising patients at Siriraj hospital, Bangkok, Thailand from 2015 to 2017

Eligible patients included all hospitalised patients aged ≥18 years who had CPE recovered from clinical specimens submitted to the microbiology laboratory. The participants were classified as CPE-colonised patients or CPE-infected patients at enrolment. CPE-colonised patients were defined according to whether or not CPE carriage was found, but its presence was not associated with any symptoms or clinical disease. CPE-infected patients were defined according to whether or not CPE isolates were found at sites of infection diagnosed as CPE infections by clinicians. Rectal swabs were collected from the patients within 48 h of enrolment and then once a week until the specimens were CPE-negative for 3 consecutive weeks (at the time of study there was no routine CPE screening procedure). One hundred and nineteen patients met the eligibility criteria, including 69 CPE-colonised patients and 50 CPE-infected patients at enrolment. Forty-five randomly selected and deidentified rectal swab samples from 33 patients, who were admitted for treatment between December 2015 and April 2017, were used for bacterial isolation and subsequent whole genome sequencing. Clinical information from each patient was collected, including information on patient demographics, clinical diagnosis, as well as their treatment during hospitalisation and outcome. The clinical outcomes of the CPE-infected patients at the end of treatment were classified as either ‘favourable response’ (absence or improvement of all clinical signs and symptoms of CPE infection) or ‘unfavourable response’ (worsening or persistence of clinical signs and/or symptoms of CPE infection, superinfection, or death).

Rectal swabs were inoculated onto MacConkey agar (BD, USA) supplemented with ceftriaxone (4 mg/L) and the plates incubated at 37 °C for 18 h. Bacterial identification was performed using Biotyper MALDI-TOF MS (Bruker Daltonics, Germany) according to the manufacturer’s protocol. Colonies identified as Enterobacteriaceae were tested for antimicrobial susceptibility using standard methods and following the guidelines for the disk-diffusion method [15]. Confirmation of suspected carbapenemase production in Enterobacteriaceae-positive specimens was performed using a modified carbapenem inactivation method [16]. Phenotypic screening for the presence of carbapenemases was performed using a double-disc synergy approach with phenylboronic acid or ethylenediaminetetraacetic acid with meropenem as previously described [17]. Colistin resistance was tested using the broth microdilution method with cation-adjusted Mueller–Hinton II broth [16]. Susceptibility to tigecycline was not tested at Siriraj Hospital during the study period.

DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, USA) and the multiplexed Illumina NexteraXT-generated libraries prepared from it were sequenced on the NextSeq® 500 platform (Illumina, USA) using a 2 × 150-bp paired-end kit with an average target coverage of 100-fold. Each read set was de novo assembled using SPAdes [18] and annotated with NCBI’s prokaryotic genome annotation pipeline (PGAP) [19] as described previously [20, 21]. For phylogenetic analysis, reads were mapped to the K. pneumoniae QS17–0029 (GCA_003073235.1) reference genome, which was previously identified in Thailand and known to carry mcr-1 [22], using Snippy v3.0 (https://​github.​com/​tseemann/​snippy). FastTree v2.1 [23] was used to generate an approximate maximum-likelihood phylogenetic tree. Metadata and phylogenetic trees were visualised using iTOL v4 [24]. To identify the bacterial sequence types, AMR genes, virulence loci and plasmid replicon types, the assembled contigs were analysed by multilocus sequence typing (MLST)1.8 [25], ResFinder3.1.0 [26], the Comprehensive Antibiotic Research Database [27], Kleborate3.0 (https://​github.​com/​katholt/​Kleborate), and PlasmidFinder2.0 [28], respectively. Virulence loci (yersiniabactin, ybt; colibactin, clb; salmochelin, iro; aerobactin, iuc; hypermucoidy, rmpA1, rmpA2; Klebsiella capsule K-locus, KL; and ICEKp-associated virulence loci in CPKP) were identified using Kleborate v3.0 (https://​github.​com/​katholt/​Kleborate). The virulence scores, which ranged from 0 to 5, were calculated as follows: 0 = no virulence loci; 1 = yersiniabactin only; 2 = yersiniabactin and colibactin, or colibactin only; 3 = aerobactin and/or salmochelin only (without yersiniabactin or colibactin); 4 = aerobactin and/or salmochelin with yersiniabactin (without colibactin); and 5 = yersiniabactin, colibactin and aerobactin and/or salmochelin.

Thirty-three patients had their CPE isolates whole genome sequenced for this study. Of these, 15 patients were found to be colonised with CPE, while the others had a confirmed CPE infection at the time of enrolment. Nineteen of the 33 patients (57.6%) were female. The mean age of all the patients was 62.8 years (IQR, interquartile range: 47–81 years) (Table 1). Most CPE-colonised patients (n = 13/15) had experienced prolonged hospitalisation before CPE was detected in their rectal swabs (median stay: 21 days, IQR 0–34 days), and 39% of them were admitted to the intensive care unit. Thirty-two patients had underlying conditions such as diabetes mellitus or chronic kidney disease and had received antibiotic treatment within the month prior to enrolment, of which two thirds (n = 23) received carbapenems (Supplementary Table 1). Among the 15 CPE-colonised patients, three (16.7%) had CPE colonisation detected prior to developing CPE infection during their hospital stay (median 16 days, IQR; 3–31.75 days) (Supplementary Table 1). We still included these three patients in the CPE-colonised group. Among the CPE-infected patients, ventilator-associated pneumonia (VAP) was the most common consequence of CPE infection, followed by urinary tract infection and primary bacteraemia (Supplementary Table 1). Only one CPE-infected patient received colistin monotherapy; all 17 of the other patients received colistin-based combination therapy, with the median duration of antibiotic treatment lasting 11 days (IQR: 7–14 days) (Supplementary Table 1). Colistin–fosfomycin was the most common antimicrobial combination regimen (45%, n = 9/20) followed by colistin–piperacillin/tazobactam (15%, n = 3/20) (Table 1). Colistin–fosfomycin was the first treatment option for patients with carbapenem-resistant infections because this combination has been shown to afford higher microbiological eradication rates than colistin monotherapy in Siriraj Hospital [29]. Regarding the local antibiogram, because CPE was more susceptible to piperacillin–tazobactam than to imipenem and meropenem, the second most common combination regime was colistin–piperacillin/tazobactam. Unfavourable clinical outcomes were observed in 52.4% of all the CPE-infected patients (n = 21), including three who were initially colonised with CPE but later developed CPE infections, and seven of these patients experienced superinfections with different bacterial species at the end of their antibiotic regimes (Table 1). There was no statistically significant mortality observed between the CPE-infected patients (47.6%, n = 10/21) vs. the CPE colonised ones (33.3%, n = 4/12; chi-square test; p = 0.43).

We isolated 39 K. pneumoniae, four Escherichia coli, and one isolate each of Enterobacter hormaechei subsp. steigerwaltii and K. quasipneumoniae subsp. similipneumoniae from the 33 patients in our study (Table 1). All 45 isolates displayed meropenem resistance, only 20% of them (n = 9) were susceptible to amikacin, and 17.8% (n = 8) were susceptible to fosfomycin (Table 1). All of the CPE isolates were resistant to ciprofloxacin, cefoxitin, ceftriaxone, ceftazidime, piperacillin–tazobactam, ertapenem and imipenem (Table 1). Only five of the isolates, all K. pneumoniae, showed resistance to colistin with MIC values ranging between 32 and 64 mg/L (Table 1). No significant differences between the antimicrobial susceptibility patterns of isolates from CPE-colonised patients and those from CPE-infected patients were observed, indicating that colonising and disease-causing strains show very similar AMR profiles, although this finding may also be attributed to the relatively small sample size available.

Our genomic analysis showed that bla_OXA-232 was the most dominant carbapenemase gene family and was found in 34 of 39 K. pneumoniae and two of the four E. coli isolates we sequenced (Supplementary Table 2). The two most common sequence types (STs) identified in K. pneumoniae were ST16 (n = 15) and ST231 (n = 14), from which 12 ST16 isolates carried bla_OXA-232 and bla_NDM-1, whereas almost all of the ST231 (n = 13) isolates carried only bla_OXA-232 (Fig. 1, Supplementary Table 2). In addition, all of the CPE isolates carrying β-lactamase genes also carried genes encoding other AMR genes, including aminoglycosides (aac(6)-I, aph [3]), fluoroquinolones (qnrB, qnrS), and fosfomycins (fosA6, UhpT) (Supplementary Table 2). None of the five colistin-resistant isolates harboured mcr-genes, although they were highly resistant to colistin (Table 1), We found a pmrB (D150Y) mutation in KPCTRPRTH02 and KPCTRPRTH04, mgrB disruptions were observed in KPCTRPRTH03 (W20*) and KPCTRPRTH01 (Q30*). In isolate KPCTRPRTH05, a S60L mutation in YcaR was also identified (Brinkac et al., manuscript in preparation).

We identified the following range of incompatibility (Inc) plasmid groups in the CPE isolates: FIA, FIB (pQil), FII, HI2B, N2 and R (Fig. 1). We were particularly interested in the presence of IncFIB and the small-sized Col plasmid group in our CPE dataset because these two plasmid groups are reported to be most commonly found in clinical samples and are associated with the spread of AMR genes [30]. Interestingly, in our dataset, all cases where ST231-CPKP was present (n = 14) and nearly half of those with ST16-CPKP (n = 7) contained an IncFIB (pQil)-like plasmid (Fig. 1). Additionally, genomic analysis indicated that all bla_OXA-232-containing CPE isolates were predicted to contain ColKp3 plasmid replicons (Fig. 1).

We searched the K. pneumoniae genomes for the virulence genes previously found in hvKP strains, including those encoding siderophores for the biosynthesis and uptake of iron (ybt, iuc and iro) and genes for the regulator of mucoid phenotype (rmpA1/rmpA2) [13]. The ybt locus, encoding the siderophore yersiniabactin, was present in 38/39 of the CPKP genomes. The most common allele, ybt14 (located on ICEKp5), was identified in 19 isolates, while the second most common allele, ybt9 (located on ICEKp3), was identified in 17 isolates, and the rest two isolates had ybt8 (located on ICEKp9) and ybt10 (located on ICEKp4), respectively (Fig. 2). Notably, iuc5, encoding the siderophore aerobactin, was only detected in ST231 (n = 14). We detected six distinct K locus (KL) types among 39 CPKP isolates, the most frequent ones being KL51 (n = 28), KL2 (n = 5), and KL17 (n = 3) (Fig. 2). Virulence plasmid-associated loci such as iro, encoding the siderophore salmochelin, colibactin and rmpA1 and rmpA2 were not present in the investigated CPKP genomes. We also found that wzi50 was more common in the ST16 isolates, whereas wzi104 was only found in the ST231 isolates, and capsular antigen KL51 was found in ST16 and ST231 isolates alike.

We identified nine patients who had more than one CPE isolate isolated throughout their hospital stay (Table 1). Six of them, despite receiving appropriate treatment, had > 2 follow-up isolates that were the same bacterial species with the same sequence type and similar antibiogram pattern (Table 1). Genomic analysis also confirmed that the bacterial isolates from the same patient were identical with only 0–1 single-nucleotide polymorphism (SNP) difference.

We noted that the bla_OXA-232 carbapenemase-encoding ColKP3 plasmid was present in different strains of K. pneumoniae as well as in E. coli (Fig. 1), indicating the possibility of horizontal interspecies spread of this plasmid and possibly resulting in a polyclonal outbreak within our hospital. Although ST231 and ST16 were the two main clones associated with invasive disease and poor outcomes in our study, we did not identify any particular STs that were found only in CPE-colonised patients or only in CPE-infected patients.