Emergence of an XDR and carbapenemase-producing hypervirulent Klebsiella pneumoniae strain in Taiwan

Emergence of an XDR and carbapenemase-producing hypervirulent Klebsiella pneumoniae strain in Taiwan Abstract Background Carbapenemase-producing Klebsiella pneumoniae causes high mortality owing to the limited therapeutic options available. Here, we investigated an emergent carbapenem-resistant K. pneumoniae strain with hypervirulence found among KPC-2-producing strains in Taiwan. Methods KPC-producing K. pneumoniae strains were collected consecutively from clinical specimens at the Taipei Veterans General Hospital between January 2012 and December 2014. Capsular types and the presence of rmpA/rmpA2 were analysed, and PFGE and MLST performed using these strains. The strain positive for rmpA/rmpA2 was tested in an in vivo mouse lethality study to verify its virulence and subjected to WGS to delineate its genomic features. Results A total of 62 KPC-2-producing K. pneumoniae strains were identified; all of these belonged to ST11 and capsular genotype K47. One strain isolated from a fatal case with intra-abdominal abscess (TVGHCRE225) harboured rmpA and rmpA2 genes. This strain was resistant to tigecycline and colistin, in addition to carbapenems, and did not belong to the major cluster in PFGE. TVGHCRE225 exhibited high in vivo virulence in the mouse lethality experiment. WGS showed that TVGHCRE225 acquired a novel hybrid virulence plasmid harbouring a set of virulence genes (iroBCDN, iucABCD, rmpA and rmpA2, and iutA) compared with the classic ST11 KPC-2-producing strain. Conclusions We identified an XDR ST11 KPC-2-producing K. pneumoniae strain carrying a hybrid virulent plasmid in Taiwan. Active surveillance focusing on carbapenem-resistant hypervirulent K. pneumoniae strains is necessary, as the threat to human health is imminent. Introduction The emergence of carbapenem-resistant Klebsiella pneumoniae is a serious threat to public health worldwide.1 KPC, a class A β-lactamase, is able to hydrolyse penicillins, cephalosporins and carbapenems. KPC-producing K. pneumoniae, initially isolated in 1996 in the USA, has spread worldwide2 with a mortality rate of up to 40%.3 The carbapenemase-producing K. pneumoniae strain ST258 was found to be nearly avirulent experimentally,4 and the high mortality was mainly associated with the immunocompromised status of the patients and the lack of therapeutic options available for the MDR organism.1 Hypervirulent K. pneumoniae is known to cause life-threatening and community-acquired pyogenic infections in immunocompetent hosts and has the capacity to metastatically spread.5 Capsular type K1 and K2 strains are well-known hypervirulent strains, owing to their strong anti-phagocytic ability and serum resistance.6 The hypermucoviscosity phenotype is considered to be strongly associated with the virulence of K. pneumoniae strains and is regulated by the plasmid-borne regulator of the mucoid phenotype gene (rmpA)—a determinant controlling the expression of capsular polysaccharide synthesis (cps) genes and capsule production.7 There is no precise definition of hypervirulent K. pneumoniae, but strains with virulent capsular types and/or carriage of rmpA/rmpA2 genes are considered hypervirulent. These hypervirulent strains, especially those with K1 or K2 types, are usually susceptible to commonly used antibiotics and rarely resistant to antibiotics, aside from their intrinsic resistance to ampicillin.5 The emergence of carbapenem-resistant K. pneumoniae strains with increased virulence makes the distinction between carbapenem-resistant K. pneumoniae and hypervirulent populations difficult,8 and the convergence of carbapenem resistance and hypervirulence in K. pneumoniae is a worrisome threat.9 ST11 carbapenem-resistant hypervirulent K. pneumoniae strains that were highly transmissible, causing a fatal outbreak in China, were recently reported to pose a substantial threat to public health.10 It is suggested that other countries with a high prevalence of K. pneumoniae should be vigilant for the emergence of carbapenem-resistant hypervirulent strains.9 However, no survey of carbapenem-resistant hypervirulent K. pneumoniae strains had been undertaken in Taiwan. In this study, we aimed to determine the capsular types and carriage of rmpA/rmpA2 genes among KPC-2-producing K. pneumoniae strains. The genomic features of a carbapenem-resistant hypervirulent K. pneumoniae strain were investigated. Materials and methods KPC-producing K. pneumoniae isolates and data collection KPC-producing K. pneumoniae isolates were collected consecutively from clinical specimens in the microbiological laboratories at the Taipei Veterans General Hospital between January 2012 and December 2014. Only the first culture was included for patients with two or more cultures that tested positive for KPC-producing strains. Ethics The study protocol was approved by the Institutional Review Board at the Taipei Veterans General Hospital (2016-05-009CC). Informed consent was waived. Bacterial identification and antimicrobial susceptibility testing The identification of K. pneumoniae was performed using the Vitek 2 System (bioMérieux) or by MALDI-TOF MS (bioMérieux). MICs were determined using the Vitek 2 System except for those of trimethoprim/sulfamethoxazole, tigecycline and colistin. The MICs of tigecycline and trimethoprim/sulfamethoxazole were determined using the Etest method (bioMérieux), as previously described,11,12 and the MIC of colistin was determined using the recommended standard broth microdilution method. MICs, except those of tigecycline and colistin, were interpreted according to CLSI breakpoints.13 EUCAST breakpoints were used for tigecycline and colistin susceptibility (http://www.eucast.org/clinical_breakpoints). Microbiological characteristics and in vivo virulence study Capsular genotyping, detection of rmpA/rmpA2 genes and colony mucoviscosity testing were performed as previously described.14 Genes encoding for carbapenemase were determined by PCR.15 The resistance mechanism for tigecycline, fluoroquinolone and colistin was tested as previously described.11,12,16,17 Carbapenem-resistant hypervirulent K. pneumoniae and other control strains were subjected to a mouse lethality study to determine the 50% lethal dose (LD50). Female C57BL/6 mice (6–8 weeks old) were administered an intraperitoneal injection of K. pneumoniae at various concentrations of inoculum, as previously described.11,18 All animal care procedures and protocols were approved by the institutional animal care and use committee of National Yang-Ming University. PFGE and MLST We performed PFGE for all KPC-producing strains as per a previous report.19 The Dice coefficient (1% tolerance and 0.5% optimization) was used to calculate the similarities and the unweighted pair-group method with the arithmetic mean was used for the cluster analysis using GelCompar II software version 6.5 (Applied Maths, St-Martens-Latem, Belgium). Isolates with >80% similarity were considered similar. S1-PFGE was performed as previously described.20 All KPC-producing strains were subjected to MLST according to the protocol described on the K. pneumoniae MLST website (http://bigsdb.pasteur.fr/klebsiella/primers_used.html) and the results analysed using the international K. pneumoniae MLST database created in 2005 at the Pasteur Institute (Paris, France).21 Bacterial conjugation A plasmid conjugation assay was performed using K. pneumoniae TVGHCRE225 as the donor strain (imipenem resistant) and Escherichia coli J53 (sodium azide resistant) as the recipient strain. The experiment was modified as described in a previous study.22 About 1 × 108 cfu of both donor strain and recipient strain were mixed and dotted on sterilized filter paper, which was then incubated on an LB agar plate for 18 h at 37°C. Transconjugants were selected by LB agar plates supplemented with 0.125 mg/L meropenem and 100 mg/L sodium azide. PCR for blaKPC-2 and rmpA/rmpA2 was performed on the transconjugants and the carbapenem MICs were determined. DNA extraction, sequencing and data analysis The DNA of the carbapenem-resistant hypervirulent K. pneumoniae strain was extracted using a Gentra Puregene Yeast/Bact kit (Qiagen) and subjected to PacBio SMRT sequencing. The data analysis, nucleotide sequence accession numbers and phylogenetic analysis of the K. pneumoniae ST11 strains based on SNPs in the core genome are shown in the Supplementary Methods (available as Supplementary data at JAC Online). Results Clinical characteristics of a case infected with an XDR and carbapenemase-producing hypervirulent K. pneumoniae During the study period, a total of 63 KPC-producing K. pneumoniae strains were identified in our hospital. Most of these carried blaKPC-2 (n = 62) and only one strain exhibited blaKPC-3. All strains with KPC-2 belonged to ST11 and capsular genotype K47. We found only one strain (TVGHCRE225) harbouring rmpA and rmpA2 genes. This strain also displayed the hypermucoviscous phenotype, as evaluated by the string test. This strain was isolated from an intra-abdominal abscess of an 83-year-old woman in August 2014. This patient had Parkinson’s disease and had not travelled outside Taiwan within the 3 months prior to this admission. The intra-abdominal abscess was caused by a fistula located between the splenic flexure of the colon and spleen. The patient underwent percutaneous ultrasound-guided catheter drainage for the abscess. The initial pus culture showed ESBL-phenotype K. pneumoniae, E. coli and Enterococcus faecium. The patient received ceftazidime and tigecycline but the disease progressed. The second pus culture from the drainage tube obtained 10 days later showed the growth of a carbapenem-resistant K. pneumoniae strain (TVGHCRE225) with imipenem MIC ≥16 mg/L, as evaluated using the Vitek 2 System. This strain also showed resistance to cephalosporins and fluoroquinolones and was susceptible to amikacin (MIC ≤2 mg/L). Amikacin was administered but septic shock ensued and the patient died 6 days after this strain had been obtained. The MICs of tigecycline and colistin for this strain were 8 and 4 mg/L, respectively, indicating TVGHCRE225 to be an XDR strain. Microbiological characteristics and in vivo virulence of TVGHCRE225 The detailed MICs for TVGHCRE225 are shown in Table 1. Further experimental work to evaluate the resistance mechanism revealed overexpression of the efflux pump gene acrB (8.8 ± 1.5-fold) and its regulatory gene ramA (21.6 ± 5.9-fold) relative to control strain KP 478 (expression = 1; tigecycline MIC, 0.25 mg/L) by quantitative RT-PCR, which was associated with tigecycline resistance. An insertion sequence ISKpn26 in acrR (negative regulatory genes for acrB) and a missense mutation (G104C and T580A) in ramR (negative regulatory genes for ramA) were also identified. Elevated expression of the pmrHFIJKLM operon increased modification of lipopolysaccharide and usually contributed to colistin resistance.17 This strain displayed a significantly higher expression level of pmrH mRNA (9.3 ± 7.4-fold) as compared with the colistin-susceptible strain NTUHK2044 (expression = 1; colistin MIC, 1 mg/L); an insertion sequence element, ISKpn26, in mgrB was responsible for colistin resistance. We also investigated the resistance mechanism of fluoroquinolones, and mutations in the quinolone resistance-determining regions of gyrA and parC were identified. Amino acid substitutions were observed in two codons of GyrA: 83 (Ser→Ile) and 87 (Asp→Gly). In the ParC subunit, substitutions were also identified in one codon: 80 (Ser→Ile). We also found qnrA1, one of the plasmid-mediated quinolone resistance determinants in this strain. Table 1. MICs for K. pneumoniae TVGHCRE225 Antibiotic MIC (mg/L) Cefazolin ≥64 Cefuroxime ≥64 Cefmetazole ≥64 Cefoxitin ≥64 Flomoxef ≥64 Ceftriaxone ≥64 Cefepime ≥64 Piperacillin/tazobactam ≥128 Gentamicin ≥16 Amikacin ≤2 Ciprofloxacin ≥4 Levofloxacin ≥8 Ertapenem ≥8 Imipenem ≥16 Trimethoprim/sulfamethoxazole ≥32 Colistin 4 Tigecycline 8 Antibiotic MIC (mg/L) Cefazolin ≥64 Cefuroxime ≥64 Cefmetazole ≥64 Cefoxitin ≥64 Flomoxef ≥64 Ceftriaxone ≥64 Cefepime ≥64 Piperacillin/tazobactam ≥128 Gentamicin ≥16 Amikacin ≤2 Ciprofloxacin ≥4 Levofloxacin ≥8 Ertapenem ≥8 Imipenem ≥16 Trimethoprim/sulfamethoxazole ≥32 Colistin 4 Tigecycline 8 MICs were determined using the Vitek 2 System, except those of trimethoprim/sulfamethoxazole and tigecycline, which were determined using the Etest method, and that of colistin, which was determined using broth microdilution. Table 1. MICs for K. pneumoniae TVGHCRE225 Antibiotic MIC (mg/L) Cefazolin ≥64 Cefuroxime ≥64 Cefmetazole ≥64 Cefoxitin ≥64 Flomoxef ≥64 Ceftriaxone ≥64 Cefepime ≥64 Piperacillin/tazobactam ≥128 Gentamicin ≥16 Amikacin ≤2 Ciprofloxacin ≥4 Levofloxacin ≥8 Ertapenem ≥8 Imipenem ≥16 Trimethoprim/sulfamethoxazole ≥32 Colistin 4 Tigecycline 8 Antibiotic MIC (mg/L) Cefazolin ≥64 Cefuroxime ≥64 Cefmetazole ≥64 Cefoxitin ≥64 Flomoxef ≥64 Ceftriaxone ≥64 Cefepime ≥64 Piperacillin/tazobactam ≥128 Gentamicin ≥16 Amikacin ≤2 Ciprofloxacin ≥4 Levofloxacin ≥8 Ertapenem ≥8 Imipenem ≥16 Trimethoprim/sulfamethoxazole ≥32 Colistin 4 Tigecycline 8 MICs were determined using the Vitek 2 System, except those of trimethoprim/sulfamethoxazole and tigecycline, which were determined using the Etest method, and that of colistin, which was determined using broth microdilution. We subjected these 63 KPC-producing strains to PFGE and identified a major cluster (>80% similarity) (Figure S1). The carbapenem-resistant hypervirulent strain (TVGHCRE225) did not belong to this major cluster. The in vivo virulence of TVGHCRE225 was compared with that of other strains using a murine model of septicaemia generated by intraperitoneal injection. Upon intraperitoneal infection in mice, this hypervirulent XDR strain showed high virulence, with an LD50 value of 1.1 × 105 cfu. The LD50 of the other carbapenem-resistant ST11 KPC-2-producing strain (TVGHCRE161) belonging to the major cluster in our hospital was 5 × 107 cfu. Two hypervirulent strains with capsular type K1 and K2, rmpA/A2 gene and WT resistance showed an LD50 value of 100 cfu. Genomic comparative analysis The XDR hypervirulent K. pneumoniae strain (TVGHCRE225) was sequenced using the PacBio RS II platform (Pacific Biosciences, USA), generating a library containing 79 892 single reads with an average length of 13 246 bp and a 128-fold average coverage. Reads were assembled using HGAP version 3.0, which returned four contigs. Comparative analysis of this strain with one recently identified carbapenem-resistant hypervirulent ST11 strain (hvkp-5)10 and two classic ST11 strains (HS11268 and JM45) showed highly similar inter-strain genome content, with only a few regions unidentified in the latter three strains (Figure 1). Figure 1. View largeDownload slide Comparative chromosome maps of four ST11 carbapenem-resistant K. pneumoniae strains. Comparative chromosome maps of the hypervirulent carbapenem-resistant K. pneumoniae strain (TVGHCRE225), one recently reported hypervirulent carbapenem-resistant K. pneumoniae strain (hvkp-5) and two publicly available classic ST11 strains (HS11286 and JM45) using TVGHCRE225 as the reference. The circular map was generated using the BLAST Ring Image Generator. Sequence comparison revealed that the four ST11 strains were highly similar; JM45 and HS11286 showed 97% coverage and 99% identity with TVGHCRE225. The layout of the circular diagram implies that TVGHCRE225 may provide a single contig representing the most completely assembled chromosomal sequence among ST11 strains. For instance, the region around the 1000th kbp on the TVGHCRE225 chromosome showed obvious higher GC bias and GC skewness as compared with other regions, and other genome assemblies revealed a pattern of very fragmented alignments. The hvkp-5 chromosome assembly was consistent with that of TVGHCRE225 in other regions that genome assemblies of JM45 and HS11286 may miss, such as the 2750th, 3050th, 4250th and 4700th kbp regions. However, the genome assembly of hvkp-5 was very fragmented, as it was assembled from Illumina short-read sequencing results; furthermore, there were as many as 249 contigs in the NCBI GenBank record. Figure 1. View largeDownload slide Comparative chromosome maps of four ST11 carbapenem-resistant K. pneumoniae strains. Comparative chromosome maps of the hypervirulent carbapenem-resistant K. pneumoniae strain (TVGHCRE225), one recently reported hypervirulent carbapenem-resistant K. pneumoniae strain (hvkp-5) and two publicly available classic ST11 strains (HS11286 and JM45) using TVGHCRE225 as the reference. The circular map was generated using the BLAST Ring Image Generator. Sequence comparison revealed that the four ST11 strains were highly similar; JM45 and HS11286 showed 97% coverage and 99% identity with TVGHCRE225. The layout of the circular diagram implies that TVGHCRE225 may provide a single contig representing the most completely assembled chromosomal sequence among ST11 strains. For instance, the region around the 1000th kbp on the TVGHCRE225 chromosome showed obvious higher GC bias and GC skewness as compared with other regions, and other genome assemblies revealed a pattern of very fragmented alignments. The hvkp-5 chromosome assembly was consistent with that of TVGHCRE225 in other regions that genome assemblies of JM45 and HS11286 may miss, such as the 2750th, 3050th, 4250th and 4700th kbp regions. However, the genome assembly of hvkp-5 was very fragmented, as it was assembled from Illumina short-read sequencing results; furthermore, there were as many as 249 contigs in the NCBI GenBank record. The result of the virulence gene analysis is shown in Table 2. We found that all KP strains (NTUHK2044, CG43, HS11286, JM45, TVGHCRE225 and hvkp-5) harboured several conserved virulence genes, namely entB, fimABCDEFGHIK, iroE, iutA, kpn, mrkABCDFHIJ and ycfM. TVGHCRE225 carried iroBCDN, iucABCD, rmpA and rmpA2, and iutA, whereas hvkp-5 displayed iucABCD, rmpA2 and iutA. This set of virulence genes, including iroBCDN, iucABCD, rmpA, rmpA2 and iutA, was also located in the virulence plasmids pLVPK and pK2044 but not in the other two classic ST11 strains. These observations were consistent with the results of the pan-genome analysis, wherein the virulence plasmid-related genes were unique to ST11 carbapenem-resistant hypervirulent K. pneumoniae but not classic ST11 carbapenem-resistant K. pneumoniae strains. Table 2. Virulence gene analysis of K. pneumoniae strains Virulence gene K. pneumoniae strain NTUHK2044 (K1, ST23) CG43 (K2, ST86) TVGHCRE225 (K47, ST11) hvkp-5 (K47, ST11) HS11286 (K47, ST11) JM45 (undefined K type, ST11) clbB 0 0 0 0 0 0 clbJ 0 0 0 0 0 0 clbK 0 0 0 0 0 0 entB 98.94b,c 98.59b 100 100 100c 100 fimA 100c 100 100 100 100c 100 fimB 97.52b,c 100b 100 100 100c 100 fimC 100c 100 100 100 100c 100 fimD 98.51b,c 99.2b 99.89b 99.89b 100c 100 fimE 97.52b,c 99.01b 100 100 100c 100 fimF 98.86b,c 100b 100 100 100c 100 fimG 100c 100 100 100 100c 100 fimH 98.01b,c 98.01b 100 100 100c 100 fimI 100c 100 100 100 100c 100 fimK 100c 100 100 100 100c 100 iroBa 100 100b 100 0 0 0 iroCa 100 100b 99.92b 0 0 0 iroDa 100 100b 100 0 0 0 iroE 99.36b,c 99.68b 99.68b 99.68b 99.44b,c 99.68b iroNa 100 100b 100 0 0 0 irp1 100 0 100 99.97b 100 0 irp2 100 0 100 100 100 0 iucAa 100 100b 100 100 0 0 iucBa 100 100b 100 100 0 0 iucCa 100 100b 99.83b 100 0 0 iucDa 100 100 100 100 0 0 iutAa 100 100b 100 100 98.5b,c 98.5b,c kpn 89.62b,c 99.45b 100 100 100c 100 mrkA 100c 100 100 100 100c 100 mrkB 100c 100 100 100 100c 100 mrkC 100c 100 100 100 100c 100 mrkD 100c 100 100 100 100c 100 mrkF 100c 100 100 100 100c 100 mrkH 100c 100 100 100 100c 100 mrkI 100c 100 100 100 100c 100 mrkJ 100c 100 100 100 100c 100 rmpA2a 100 100b 99.17b 100b 0 0 rmpAa 100 100b 99.53b 0 0 0 traTa 0 0 86.01 0 98.35 98.35 ycfM 99.53b,c 100 100 100 100b,c 100 Virulence gene K. pneumoniae strain NTUHK2044 (K1, ST23) CG43 (K2, ST86) TVGHCRE225 (K47, ST11) hvkp-5 (K47, ST11) HS11286 (K47, ST11) JM45 (undefined K type, ST11) clbB 0 0 0 0 0 0 clbJ 0 0 0 0 0 0 clbK 0 0 0 0 0 0 entB 98.94b,c 98.59b 100 100 100c 100 fimA 100c 100 100 100 100c 100 fimB 97.52b,c 100b 100 100 100c 100 fimC 100c 100 100 100 100c 100 fimD 98.51b,c 99.2b 99.89b 99.89b 100c 100 fimE 97.52b,c 99.01b 100 100 100c 100 fimF 98.86b,c 100b 100 100 100c 100 fimG 100c 100 100 100 100c 100 fimH 98.01b,c 98.01b 100 100 100c 100 fimI 100c 100 100 100 100c 100 fimK 100c 100 100 100 100c 100 iroBa 100 100b 100 0 0 0 iroCa 100 100b 99.92b 0 0 0 iroDa 100 100b 100 0 0 0 iroE 99.36b,c 99.68b 99.68b 99.68b 99.44b,c 99.68b iroNa 100 100b 100 0 0 0 irp1 100 0 100 99.97b 100 0 irp2 100 0 100 100 100 0 iucAa 100 100b 100 100 0 0 iucBa 100 100b 100 100 0 0 iucCa 100 100b 99.83b 100 0 0 iucDa 100 100 100 100 0 0 iutAa 100 100b 100 100 98.5b,c 98.5b,c kpn 89.62b,c 99.45b 100 100 100c 100 mrkA 100c 100 100 100 100c 100 mrkB 100c 100 100 100 100c 100 mrkC 100c 100 100 100 100c 100 mrkD 100c 100 100 100 100c 100 mrkF 100c 100 100 100 100c 100 mrkH 100c 100 100 100 100c 100 mrkI 100c 100 100 100 100c 100 mrkJ 100c 100 100 100 100c 100 rmpA2a 100 100b 99.17b 100b 0 0 rmpAa 100 100b 99.53b 0 0 0 traTa 0 0 86.01 0 98.35 98.35 ycfM 99.53b,c 100 100 100 100b,c 100 Each value in the table represents the percentage of homology of one virulence gene in the K. pneumoniae strains when compared with the reference alleles in BIGSdb. ‘0’ means the absence of this gene in the specific strain analysed and ‘100’ means the presence of an identical gene in the specific strain. The results may not be completely consistent either with those generated by the BIGSdb online tool or with those revealed in previous literature. This might be partly because we ran NCBI BLASTP in a repeat masking disabled mode, and thus the additional repeat-containing virulence factors could be identified. For example, the full-length rmpA sequence of TVGHCRE225 could be completely aligned to a repeat-containing allele retrieved from BIGSdb. a This gene is located in the plasmid. All of the genes, except for traT located in the KPC-2-carrying plasmid, are located in the pVirCRE225, pLVPK, pK2044 or pLVPK-like plasmid. b Results differ from those generated by performing BLAST on the BIGSdb web site. c Results differ from the literature. Table 2. Virulence gene analysis of K. pneumoniae strains Virulence gene K. pneumoniae strain NTUHK2044 (K1, ST23) CG43 (K2, ST86) TVGHCRE225 (K47, ST11) hvkp-5 (K47, ST11) HS11286 (K47, ST11) JM45 (undefined K type, ST11) clbB 0 0 0 0 0 0 clbJ 0 0 0 0 0 0 clbK 0 0 0 0 0 0 entB 98.94b,c 98.59b 100 100 100c 100 fimA 100c 100 100 100 100c 100 fimB 97.52b,c 100b 100 100 100c 100 fimC 100c 100 100 100 100c 100 fimD 98.51b,c 99.2b 99.89b 99.89b 100c 100 fimE 97.52b,c 99.01b 100 100 100c 100 fimF 98.86b,c 100b 100 100 100c 100 fimG 100c 100 100 100 100c 100 fimH 98.01b,c 98.01b 100 100 100c 100 fimI 100c 100 100 100 100c 100 fimK 100c 100 100 100 100c 100 iroBa 100 100b 100 0 0 0 iroCa 100 100b 99.92b 0 0 0 iroDa 100 100b 100 0 0 0 iroE 99.36b,c 99.68b 99.68b 99.68b 99.44b,c 99.68b iroNa 100 100b 100 0 0 0 irp1 100 0 100 99.97b 100 0 irp2 100 0 100 100 100 0 iucAa 100 100b 100 100 0 0 iucBa 100 100b 100 100 0 0 iucCa 100 100b 99.83b 100 0 0 iucDa 100 100 100 100 0 0 iutAa 100 100b 100 100 98.5b,c 98.5b,c kpn 89.62b,c 99.45b 100 100 100c 100 mrkA 100c 100 100 100 100c 100 mrkB 100c 100 100 100 100c 100 mrkC 100c 100 100 100 100c 100 mrkD 100c 100 100 100 100c 100 mrkF 100c 100 100 100 100c 100 mrkH 100c 100 100 100 100c 100 mrkI 100c 100 100 100 100c 100 mrkJ 100c 100 100 100 100c 100 rmpA2a 100 100b 99.17b 100b 0 0 rmpAa 100 100b 99.53b 0 0 0 traTa 0 0 86.01 0 98.35 98.35 ycfM 99.53b,c 100 100 100 100b,c 100 Virulence gene K. pneumoniae strain NTUHK2044 (K1, ST23) CG43 (K2, ST86) TVGHCRE225 (K47, ST11) hvkp-5 (K47, ST11) HS11286 (K47, ST11) JM45 (undefined K type, ST11) clbB 0 0 0 0 0 0 clbJ 0 0 0 0 0 0 clbK 0 0 0 0 0 0 entB 98.94b,c 98.59b 100 100 100c 100 fimA 100c 100 100 100 100c 100 fimB 97.52b,c 100b 100 100 100c 100 fimC 100c 100 100 100 100c 100 fimD 98.51b,c 99.2b 99.89b 99.89b 100c 100 fimE 97.52b,c 99.01b 100 100 100c 100 fimF 98.86b,c 100b 100 100 100c 100 fimG 100c 100 100 100 100c 100 fimH 98.01b,c 98.01b 100 100 100c 100 fimI 100c 100 100 100 100c 100 fimK 100c 100 100 100 100c 100 iroBa 100 100b 100 0 0 0 iroCa 100 100b 99.92b 0 0 0 iroDa 100 100b 100 0 0 0 iroE 99.36b,c 99.68b 99.68b 99.68b 99.44b,c 99.68b iroNa 100 100b 100 0 0 0 irp1 100 0 100 99.97b 100 0 irp2 100 0 100 100 100 0 iucAa 100 100b 100 100 0 0 iucBa 100 100b 100 100 0 0 iucCa 100 100b 99.83b 100 0 0 iucDa 100 100 100 100 0 0 iutAa 100 100b 100 100 98.5b,c 98.5b,c kpn 89.62b,c 99.45b 100 100 100c 100 mrkA 100c 100 100 100 100c 100 mrkB 100c 100 100 100 100c 100 mrkC 100c 100 100 100 100c 100 mrkD 100c 100 100 100 100c 100 mrkF 100c 100 100 100 100c 100 mrkH 100c 100 100 100 100c 100 mrkI 100c 100 100 100 100c 100 mrkJ 100c 100 100 100 100c 100 rmpA2a 100 100b 99.17b 100b 0 0 rmpAa 100 100b 99.53b 0 0 0 traTa 0 0 86.01 0 98.35 98.35 ycfM 99.53b,c 100 100 100 100b,c 100 Each value in the table represents the percentage of homology of one virulence gene in the K. pneumoniae strains when compared with the reference alleles in BIGSdb. ‘0’ means the absence of this gene in the specific strain analysed and ‘100’ means the presence of an identical gene in the specific strain. The results may not be completely consistent either with those generated by the BIGSdb online tool or with those revealed in previous literature. This might be partly because we ran NCBI BLASTP in a repeat masking disabled mode, and thus the additional repeat-containing virulence factors could be identified. For example, the full-length rmpA sequence of TVGHCRE225 could be completely aligned to a repeat-containing allele retrieved from BIGSdb. a This gene is located in the plasmid. All of the genes, except for traT located in the KPC-2-carrying plasmid, are located in the pVirCRE225, pLVPK, pK2044 or pLVPK-like plasmid. b Results differ from those generated by performing BLAST on the BIGSdb web site. c Results differ from the literature. The strain TVGHCRE225 harboured three plasmids. S1-PFGE confirmed the size of the three plasmids analysed by WGS (Figure S2). The IncR/IncFII/IncN plasmid (103 454 bp) carrying the antimicrobial resistance gene blaKPC-2 conferring resistance to carbapenems shared 99% identity and 72% query coverage with plasmid pKPC-LK30 from a KPC-2-producing K. pneumoniae strain in Taiwan (Figure S3). The other IncA/C2-type plasmid (159 072 bp) had antimicrobial resistance genes aadA2, aac(3)-IId, strA, strB, sul1, sul2, qnrA1 and blaTEM-1B, which conferred resistance to aminoglycosides, sulphonamide, fluoroquinolones and β-lactams. The IncHI1B/IncFIB plasmid (297 984 bp) encoded 306 predicted ORFs. The set of virulence genes, including iroBCDN, iucABCD, rmpA and rmpA2, and iutA, were located in this plasmid, whereas no antimicrobial resistance gene was identified. Comparison of this virulent plasmid, designated as pVir, and other plasmids with the use of the Basic Local Alignment Search Tool (BLAST) revealed its identity with pK2044 (49% coverage, 99% identity), pLVPK (47% coverage, 99% identity) and pPMK-NDM (61% coverage, 99% identity). TVGHCRE225 pVir was larger in size than the other two virulent plasmids (pK2044: 224 152 bp; pLVPK: 219 385 bp) in K. pneumoniae. pPMK-NDM is an IncHI2-type resistance plasmid harboured by the NDM-producing K. pneumoniae strain that caused an outbreak in a Nepali neonatal unit. Sequence analysis of TVGHCRE225 pVir suggests that this is a novel hybrid plasmid comprising a part of the virulence plasmid pLVPK and pPMK-NDM. The first region was highly similar to the CG43 (pLVPK), hvkp-5 and NTUH-K2044 plasmids, while the second region was highly similar to the PMK1 (pPMK1-NDM) plasmid (Figure 2). Figure 2. View largeDownload slide Gene map of four virulence plasmids and pPMK1-NDM harboured by K. pneumoniae strains. Alignment of the four virulent plasmids recovered from our strain TVGHCRE225, ST11 carbapenem-resistant hypervirulent K. pneumoniae strain hvkp-5, capsular K1 hypervirulent K. pneumoniae NTUH-K2044 and capsular K2 hypervirulent K. pneumoniae CG43, and a plasmid harboured by strain PMK1. The circular genome comparison diagram was generated with the BLAST Ring Image Generator. TVGHCRE225 pVir could be roughly divided into two distinct regions based on its similarity to different plasmids. The first region, 1st to ∼130th kbp, was highly similar to the CG43 (pLVPK), hvkp-5 and NTUH-K2044 plasmids, while the second region, 130th to ∼300th kbp, was highly similar to the PMK1 (pPMK1-NDM) plasmid. Virulence factors such as iroBCDN, iucABCD, rmpA and rmpA2, and iutA are all located in the first region corresponding to the pLVPK-like plasmid region. Figure 2. View largeDownload slide Gene map of four virulence plasmids and pPMK1-NDM harboured by K. pneumoniae strains. Alignment of the four virulent plasmids recovered from our strain TVGHCRE225, ST11 carbapenem-resistant hypervirulent K. pneumoniae strain hvkp-5, capsular K1 hypervirulent K. pneumoniae NTUH-K2044 and capsular K2 hypervirulent K. pneumoniae CG43, and a plasmid harboured by strain PMK1. The circular genome comparison diagram was generated with the BLAST Ring Image Generator. TVGHCRE225 pVir could be roughly divided into two distinct regions based on its similarity to different plasmids. The first region, 1st to ∼130th kbp, was highly similar to the CG43 (pLVPK), hvkp-5 and NTUH-K2044 plasmids, while the second region, 130th to ∼300th kbp, was highly similar to the PMK1 (pPMK1-NDM) plasmid. Virulence factors such as iroBCDN, iucABCD, rmpA and rmpA2, and iutA are all located in the first region corresponding to the pLVPK-like plasmid region. Phylogenetic analysis of the K. pneumoniae ST11 strains based on SNPs in the core genome To estimate the genetic relationships among two hypervirulent carbapenem-resistant strains (TVGHCRE225 and hvKP-5) and other classic ST11 strains (JM45, HS11286, FJ8 and FJ10), we performed phylogenetic analysis based on core genome SNPs. This revealed that four ST11 strains, including TVGHCRE225, FJ10 (from Hong Kong), FJ8 (from Hong Kong) and hvkp-5 (from Hangzhou, China), formed a clade that is distinct from the other two ST11 strains, HS11286 (from Shanghai, China) and JM45 (from Hangzhou, China). In this clade, FJ10, FJ8 and hvkp-5 were more closely related, whereas TVGHCRE225 alone forms another branch (Figure S4). The branch length of the TVGHCRE225 node is longer than that of the other three ST11 strains, suggesting that TVGHCRE225 is genetically more divergent in this clade (Figure S4). This result suggested that TVGHCRE225 was evolutionarily closer to the hvkp-5 than to the other two ST11 strains (HS11286 and JM45). Conjugation experiment A conjugation assay was performed to examine whether the plasmid carrying KPC-2 could be transferred. The conjugation frequency was determined to be 2.65 × 10−7. The imipenem MIC was 0.125 mg/L for E. coli J53 and 6 mg/L for the transconjugant. The carbapenem resistance phenotype of TVGHCRE225 could be transferred to E. coli strain J53. The transconjugant carried the blaKPC-2 gene, which was located on the IncR/IncFII/IncN plasmid, but the virulent determinant rmpA/rmpA2 located on pVir could not be detected in these transconjugants. Discussion We identified one XDR hypervirulent K. pneumoniae strain, TVGHCRE225, and WGS showed that this strain acquired a virulence plasmid harbouring rmpA/rmpA2 as compared with the classic ST11 KPC-2-producing K. pneumoniae strain. This XDR hypervirulent K. pneumoniae strain was isolated in Taiwan in 2014, earlier than that isolated in China recently reported by Gu et al.10 The distribution of the carbapenem-resistant hypervirulent K. pneumoniae strain is broader than expected, suggesting the emergence of a strain with high virulence and antimicrobial resistance. Capsular polysaccharide is an important virulence factor in K. pneumoniae. K1/K2/K5/K20/K54/K57 types were considered virulent and responsible for a variety of community-onset pyogenic infections.14,23 In the present study, all KPC-2-producing strains belong to capsular type K47. K47 is the most common capsular type among KPC-producing K. pneumoniae isolates in China.24 Carbapenem-resistant hypervirulent K. pneumoniae strains have been mostly reported in China,8 but were also recently identified in the USA.25 Most strains from China belong to K1 or K2 capsular type and carried rmpA/rmpA2 genes,26–29 and it has been speculated that the horizontal transfer of resistance plasmids contributes to carbapenem resistance.9,In vitro experiments demonstrated that KPC-carrying plasmids were successfully conjugated and retained by a virulent K2 K. pneumoniae recipient isolate and that transconjugants are able to maintain high serum resistance and murine lethality.30 These findings provided the rationale that hypervirulent strains evolved to become carbapenem resistant. Gu et al.10 recently reported the emergence of ST11 carbapenem-resistant hypervirulent K. pneumoniae strains in China, different from K1 or K2 capsular types, owing to the acquisition of a pLVPK-like virulence plasmid. In our study, genomic analyses showed that the XDR hypervirulent strain TVGHCRE225 acquired a novel hybrid virulence plasmid containing a set of virulence genes on pLVPK and pK2044. Moreover, TVGHCRE225 was resistant to tigecycline and colistin, which has never been reported previously among carbapenem-resistant hypervirulent strains. The patient received tigecycline but not colistin before the isolation of TVGHCRE225. The evolution to a hypervirulent strain even occurred with an XDR background. Our results correspond to the report by Gu et al. and provide evidence that virulence plasmids are not restricted only to hypervirulent K. pneumoniae backgrounds, suggesting a change in the host–plasmid relationship in the ST11 KPC-2-producing K. pneumoniae background.9 Thus, the possibility of transfer of plasmid-encoded virulence genes into carbapenem-resistant isolates represents a different mechanism for the interplay between virulence and antimicrobial resistance. Only two studies have detailed the genomic analyses of carbapenem-resistant hypervirulent K. pneumoniae strains10,25 using Illumina sequencing platforms. The short-read assembly is unlikely to resolve abundant regions containing repetitive elements, which may be found in most genomes. In addition, the GC content bias featured in regions with horizontal gene transfers may be associated with low sequencing coverage or be completely lost during sequencing with the Illumina short-read technology. In this study, we used a third-generation sequencing platform, PacBio, to build a complete sequence map for TVGHCRE225. With a fully assembled genome, it is straightforward to delineate the structural features of the chromosome and three plasmids and determine the locations of virulence factors and drug resistance genes. It is notable that the virulence plasmid, TVGHCRE225 pVir, was clearly identified to comprise two evolutionarily distinct regions likely derived from different plasmids. In addition, the phylogenetic tree showed that TVGHCRE225 was evolutionarily close to the hvkp-5 isolated in China recently. However, our TVGHCRE225 was found in 2014, which was earlier than hvkp-5 (isolated in 2016), and, therefore, TVGHCRE225 might have the ability to evolve independently. More ST11 genomes to elucidate the evolution of the carbapenem-resistant hypervirulent K. pneumoniae strains are necessary. Zhu et al.31 recently reported the low in vivo virulence of seven carbapenem-resistant hypermucoviscous K. pneumoniae isolates in a mouse lethality assay in a teaching hospital in China between January 2015 and December 2016. Only two of the seven strains carried the rmpA gene. Zhu et al.’s results indicated that defining hypervirulent K. pneumoniae only by hypermucoviscosity phenotype was far from sufficient. The absence of specific genomic markers to define hypervirulent K. pneumoniae strains has been criticized previously.32 A recent review article proposed further testing of virulence behaviour among the carbapenem-resistant strains with hypermucoviscosity in a suitable animal model.8 In this study, we detailed the genetic content of virulence determinants and demonstrated the virulence of TVGHCRE225 using a mouse lethality assay by comparing it with the WT capsular K1/K2 and classic ST11 strains. The acquisition of the virulence plasmid in this XDR strain resulted in higher virulence compared with the classic ST11 strain; however, this strain was not as virulent as capsular K1/K2 strains in the mouse lethality assay. The virulent plasmids hvkp-5, pLVPK-like, pLVPK and pK2044 were highly similar, but the virulence plasmid in TVGHCRE225 was a novel hybrid plasmid containing a set of virulence genes. The plasmid spread between strains contributed to the confluence of hypervirulence and carbapenem resistance. Therefore, it is necessary to understand the molecular mechanisms underlying the plasmid–host expansion and adaptation with respect to antimicrobial resistance and virulence.9 The major limitation of this study is the limited number of KPC-producing strains investigated. A large-scale screening of the capsular types and for the presence of rmpA among all carbapenem-resistant K. pneumoniae strains carrying other resistance mechanisms is ongoing to elucidate the virulence characteristics and strain background of these newly emerged strains. In conclusion, we identified an XDR ST11 KPC-2-producing K. pneumoniae strain carrying a virulent plasmid for the first time in a country of the Asia Pacific rim. This strain was different from the major cluster of KPC-2-producing strains in our hospital. Active surveillance focusing on both the antimicrobial resistance and virulence characteristics of K. pneumoniae strains is necessary, as the threat to human health is imminent. Further studies regarding carbapenem-resistant hypervirulent K. pneumoniae worldwide to explore the evolutionary relationship between virulence and resistance in K. pneumoniae are encouraged. Acknowledgements Partial results of this manuscript were presented as a paper poster (P0919) at the Twenty-eighth European Congress of Clinical Microbiology and Infectious Diseases, Madrid, Spain, 2018.  We acknowledge the Medical Science and Technology Building of the Taipei Veterans General Hospital for providing experimental space and facilities. We thank Professor Po-Liang Lu for PFGE assistance. We thank the GenoInfo Core Facility (C1) funded by the NCFPB of the Ministry of Science and Technology in Taiwan (MOST106-2319-B-010–001) for technical support regarding genome annotation. We thank National Core Facility for Biopharmaceuticals (NCFB, MOST 106-2319-B-492-002) and the National Center for High-performance Computing (NCHC) of National Applied Research Laboratories (NARLabs) of Taiwan for providing computational resources and storage resources. Funding This work was supported by grants from the Ministry of Science and Technology in Taiwan (MOST103-2314-B-075-078-MY2, MOST105-2628-B-010-015-MY3, MOST105-2320-B-010-006 and MOST106-2320-B-010-019) and the Taipei Veterans General Hospital (V104B-001, V105B-001, V106B-001 and V107C-081). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Transparency declarations None to declare. Supplementary data Supplementary Methods and Figures S1–S4 are available as Supplementary data at JAC Online. References 1 Lee CR , Lee JH , Park KS et al. Global dissemination of carbapenemase-producing Klebsiella pneumoniae: epidemiology, genetic context, treatment options, and detection methods . Front Microbiol 2016 ; 7 : 895 . Google Scholar PubMed 2 Doi Y , Paterson DL. Carbapenemase-producing Enterobacteriaceae . Semin Respir Crit Care Med 2015 ; 36 : 74 – 84 . Google Scholar CrossRef Search ADS PubMed 3 Gomez-Simmonds A , Nelson B , Eiras DP et al. Combination regimens for treatment of carbapenem-resistant Klebsiella pneumoniae bloodstream infections . Antimicrob Agents Chemother 2016 ; 60 : 3601 – 7 . Google Scholar CrossRef Search ADS PubMed 4 Tzouvelekis LS , Miriagou V , Kotsakis SD et al. KPC-producing, multidrug-resistant Klebsiella pneumoniae sequence type 258 as a typical opportunistic pathogen . Antimicrob Agents Chemother 2013 ; 57 : 5144 – 6 . Google Scholar CrossRef Search ADS PubMed 5 Shon AS , Bajwa RP , Russo TA. Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: a new and dangerous breed . Virulence 2013 ; 4 : 107 – 18 . Google Scholar CrossRef Search ADS PubMed 6 Siu LK , Yeh KM , Lin JC et al. Klebsiella pneumoniae liver abscess: a new invasive syndrome . Lancet Infect Dis 2012 ; 12 : 881 – 7 . Google Scholar CrossRef Search ADS PubMed 7 Hsu CR , Lin TL , Chen YC et al. The role of Klebsiella pneumoniae rmpA in capsular polysaccharide synthesis and virulence revisited . Microbiology 2011 ; 157 : 3446 – 57 . Google Scholar CrossRef Search ADS PubMed 8 Arena F , Henrici De Angelis L , D'Andrea MM et al. Infections caused by carbapenem-resistant Klebsiella pneumoniae with hypermucoviscous phenotype: a case report and literature review . Virulence 2017 ; 8 : 1900 – 8 . Google Scholar CrossRef Search ADS PubMed 9 Chen L , Kreiswirth BN. Convergence of carbapenem-resistance and hypervirulence in Klebsiella pneumoniae . Lancet Infect Dis 2018 ; 18 : 2 – 3 . Google Scholar CrossRef Search ADS PubMed 10 Gu D , Dong N , Zheng Z et al. A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study . Lancet Infect Dis 2018 ; 18 : 37 – 46 . Google Scholar CrossRef Search ADS PubMed 11 Lin YT , Huang YW , Huang HH et al. In vivo evolution of tigecycline-non-susceptible Klebsiella pneumoniae strains in patients: relationship between virulence and resistance . Int J Antimicrob Agents 2016 ; 48 : 485 – 91 . Google Scholar CrossRef Search ADS PubMed 12 Juan CH , Huang YW , Lin YT et al. Risk factors, outcomes, and mechanisms of tigecycline-nonsusceptible Klebsiella pneumoniae bacteremia . Antimicrob Agents Chemother 2016 ; 60 : 7357 – 63 . Google Scholar PubMed 13 Clinical and Laboratory Standards Institute . Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Fourth Informational Supplement M100-S24 . CLSI , Wayne, PA, USA , 2014 . 14 Lin YT , Wang YP , Wang FD et al. Community-onset Klebsiella pneumoniae pneumonia in Taiwan: clinical features of the disease and associated microbiological characteristics of isolates from pneumonia and nasopharynx . Front Microbiol 2015 ; 9 : 122 . Google Scholar CrossRef Search ADS PubMed 15 Wu PF , Chuang C , Su CF et al. High minimum inhibitory concentration of imipenem as a predictor of fatal outcome in patients with carbapenem non-susceptible Klebsiella pneumoniae . Sci Rep 2016 ; 6 : 32665 . Google Scholar CrossRef Search ADS PubMed 16 Cheng YH , Lin TL , Lin YT et al. Amino acid substitutions of CrrB responsible for resistance to colistin through CrrC in Klebsiella pneumoniae . Antimicrob Agents Chemother 2016 ; 60 : 3709 – 16 . Google Scholar CrossRef Search ADS PubMed 17 Cheng YH , Lin TL , Pan YJ et al. Colistin resistance mechanisms in Klebsiella pneumoniae strains from Taiwan . Antimicrob Agents Chemother 2015 ; 59 : 2909 – 13 . Google Scholar CrossRef Search ADS PubMed 18 Lin YT , Pan YJ , Lin TL et al. Transfer of CMY-2 cephalosporinase from Escherichia coli to virulent Klebsiella pneumoniae causing a recurrent liver abscess . Antimicrob Agents Chemother 2015 ; 59 : 5000 – 2 . Google Scholar CrossRef Search ADS PubMed 19 Lin YT , Wang FD , Chan YJ et al. Clinical and microbiological characteristics of tigecycline non-susceptible Klebsiella pneumoniae bacteremia in Taiwan . BMC Infect Dis 2014 ; 14 : 1 . Google Scholar CrossRef Search ADS PubMed 20 Xie L , Dou Y , Zhou K et al. Coexistence of blaOXA-48 and truncated blaNDM-1 on different plasmids in a Klebsiella pneumoniae isolate in China . Front Microbiol 2017 ; 8 : 133 . Google Scholar PubMed 21 Diancourt L , Passet V , Verhoef J et al. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates . J Clin Microbiol 2005 ; 43 : 4178 – 82 . Google Scholar CrossRef Search ADS PubMed 22 Borgia S , Lastovetska O , Richardson D et al. Outbreak of carbapenem-resistant Enterobacteriaceae containing blaNDM-1, Ontario, Canada . Clin Infect Dis 2012 ; 55 : e109 – 17 . Google Scholar CrossRef Search ADS PubMed 23 Chuang C , Fan WC , Lin YT et al. The emergence of Klebsiella pneumoniae liver abscess in non-diabetic patients and the distribution of capsular types . Gut Pathog 2016 ; 8 : 46 . Google Scholar CrossRef Search ADS PubMed 24 Liu Y , Liu PP , Wang LH et al. Capsular polysaccharide types and virulence-related traits of epidemic KPC-producing Klebsiella pneumoniae isolates in a Chinese university hospital . Microb Drug Resist 2017 ; 23 : 901 – 7 . Google Scholar CrossRef Search ADS PubMed 25 Krapp F , Morris AR , Ozer EA et al. Virulence characteristics of carbapenem-resistant Klebsiella pneumoniae strains from patients with necrotizing skin and soft tissue infections . Sci Rep 2017 ; 7 : 13533 . Google Scholar CrossRef Search ADS PubMed 26 Zhang Y , Zeng J , Liu W et al. Emergence of a hypervirulent carbapenem-resistant Klebsiella pneumoniae isolate from clinical infections in China . J Infect 2015 ; 71 : 553 – 60 . Google Scholar CrossRef Search ADS PubMed 27 Zhang R , Lin D , Chan EW et al. Emergence of carbapenem-resistant serotype K1 hypervirulent Klebsiella pneumoniae strains in China . Antimicrob Agents Chemother 2015 ; 60 : 709 – 11 . Google Scholar CrossRef Search ADS PubMed 28 Yao B , Xiao X , Wang F et al. Clinical and molecular characteristics of multi-clone carbapenem-resistant hypervirulent (hypermucoviscous) Klebsiella pneumoniae isolates in a tertiary hospital in Beijing, China . Int J Infect Dis 2015 ; 37 : 107 – 12 . Google Scholar CrossRef Search ADS PubMed 29 Wei DD , Wan LG , Deng Q et al. Emergence of KPC-producing Klebsiella pneumoniae hypervirulent clone of capsular serotype K1 that belongs to sequence type 11 in Mainland China . Diagn Microbiol Infect Dis 2016 ; 85 : 192 – 4 . Google Scholar CrossRef Search ADS PubMed 30 Siu LK , Huang DB , Chiang T. Plasmid transferability of KPC into a virulent K2 serotype Klebsiella pneumoniae . BMC Infect Dis 2014 ; 14 : 176. Google Scholar CrossRef Search ADS PubMed 31 Zhu J , Yin X , Xi W et al. Emergence of low virulent carbapenem-resistant hypermucoviscous Klebsiella pneumoniae in China . J Infect 2017 ; 75 : 469 – 72 . Google Scholar CrossRef Search ADS PubMed 32 Zhang Y , Ma Y , Ye L et al. Prevalence and antimicrobial susceptibility of hypervirulent Klebsiella pneumoniae isolates in China . Clin Infect Dis 2014 ; 58 : 1493 – 4 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Emergence of an XDR and carbapenemase-producing hypervirulent Klebsiella pneumoniae strain in Taiwan

Journal of Antimicrobial Chemotherapy , Volume Advance Article (8) – May 24, 2018

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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10.1093/jac/dky164
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Abstract

Abstract Background Carbapenemase-producing Klebsiella pneumoniae causes high mortality owing to the limited therapeutic options available. Here, we investigated an emergent carbapenem-resistant K. pneumoniae strain with hypervirulence found among KPC-2-producing strains in Taiwan. Methods KPC-producing K. pneumoniae strains were collected consecutively from clinical specimens at the Taipei Veterans General Hospital between January 2012 and December 2014. Capsular types and the presence of rmpA/rmpA2 were analysed, and PFGE and MLST performed using these strains. The strain positive for rmpA/rmpA2 was tested in an in vivo mouse lethality study to verify its virulence and subjected to WGS to delineate its genomic features. Results A total of 62 KPC-2-producing K. pneumoniae strains were identified; all of these belonged to ST11 and capsular genotype K47. One strain isolated from a fatal case with intra-abdominal abscess (TVGHCRE225) harboured rmpA and rmpA2 genes. This strain was resistant to tigecycline and colistin, in addition to carbapenems, and did not belong to the major cluster in PFGE. TVGHCRE225 exhibited high in vivo virulence in the mouse lethality experiment. WGS showed that TVGHCRE225 acquired a novel hybrid virulence plasmid harbouring a set of virulence genes (iroBCDN, iucABCD, rmpA and rmpA2, and iutA) compared with the classic ST11 KPC-2-producing strain. Conclusions We identified an XDR ST11 KPC-2-producing K. pneumoniae strain carrying a hybrid virulent plasmid in Taiwan. Active surveillance focusing on carbapenem-resistant hypervirulent K. pneumoniae strains is necessary, as the threat to human health is imminent. Introduction The emergence of carbapenem-resistant Klebsiella pneumoniae is a serious threat to public health worldwide.1 KPC, a class A β-lactamase, is able to hydrolyse penicillins, cephalosporins and carbapenems. KPC-producing K. pneumoniae, initially isolated in 1996 in the USA, has spread worldwide2 with a mortality rate of up to 40%.3 The carbapenemase-producing K. pneumoniae strain ST258 was found to be nearly avirulent experimentally,4 and the high mortality was mainly associated with the immunocompromised status of the patients and the lack of therapeutic options available for the MDR organism.1 Hypervirulent K. pneumoniae is known to cause life-threatening and community-acquired pyogenic infections in immunocompetent hosts and has the capacity to metastatically spread.5 Capsular type K1 and K2 strains are well-known hypervirulent strains, owing to their strong anti-phagocytic ability and serum resistance.6 The hypermucoviscosity phenotype is considered to be strongly associated with the virulence of K. pneumoniae strains and is regulated by the plasmid-borne regulator of the mucoid phenotype gene (rmpA)—a determinant controlling the expression of capsular polysaccharide synthesis (cps) genes and capsule production.7 There is no precise definition of hypervirulent K. pneumoniae, but strains with virulent capsular types and/or carriage of rmpA/rmpA2 genes are considered hypervirulent. These hypervirulent strains, especially those with K1 or K2 types, are usually susceptible to commonly used antibiotics and rarely resistant to antibiotics, aside from their intrinsic resistance to ampicillin.5 The emergence of carbapenem-resistant K. pneumoniae strains with increased virulence makes the distinction between carbapenem-resistant K. pneumoniae and hypervirulent populations difficult,8 and the convergence of carbapenem resistance and hypervirulence in K. pneumoniae is a worrisome threat.9 ST11 carbapenem-resistant hypervirulent K. pneumoniae strains that were highly transmissible, causing a fatal outbreak in China, were recently reported to pose a substantial threat to public health.10 It is suggested that other countries with a high prevalence of K. pneumoniae should be vigilant for the emergence of carbapenem-resistant hypervirulent strains.9 However, no survey of carbapenem-resistant hypervirulent K. pneumoniae strains had been undertaken in Taiwan. In this study, we aimed to determine the capsular types and carriage of rmpA/rmpA2 genes among KPC-2-producing K. pneumoniae strains. The genomic features of a carbapenem-resistant hypervirulent K. pneumoniae strain were investigated. Materials and methods KPC-producing K. pneumoniae isolates and data collection KPC-producing K. pneumoniae isolates were collected consecutively from clinical specimens in the microbiological laboratories at the Taipei Veterans General Hospital between January 2012 and December 2014. Only the first culture was included for patients with two or more cultures that tested positive for KPC-producing strains. Ethics The study protocol was approved by the Institutional Review Board at the Taipei Veterans General Hospital (2016-05-009CC). Informed consent was waived. Bacterial identification and antimicrobial susceptibility testing The identification of K. pneumoniae was performed using the Vitek 2 System (bioMérieux) or by MALDI-TOF MS (bioMérieux). MICs were determined using the Vitek 2 System except for those of trimethoprim/sulfamethoxazole, tigecycline and colistin. The MICs of tigecycline and trimethoprim/sulfamethoxazole were determined using the Etest method (bioMérieux), as previously described,11,12 and the MIC of colistin was determined using the recommended standard broth microdilution method. MICs, except those of tigecycline and colistin, were interpreted according to CLSI breakpoints.13 EUCAST breakpoints were used for tigecycline and colistin susceptibility (http://www.eucast.org/clinical_breakpoints). Microbiological characteristics and in vivo virulence study Capsular genotyping, detection of rmpA/rmpA2 genes and colony mucoviscosity testing were performed as previously described.14 Genes encoding for carbapenemase were determined by PCR.15 The resistance mechanism for tigecycline, fluoroquinolone and colistin was tested as previously described.11,12,16,17 Carbapenem-resistant hypervirulent K. pneumoniae and other control strains were subjected to a mouse lethality study to determine the 50% lethal dose (LD50). Female C57BL/6 mice (6–8 weeks old) were administered an intraperitoneal injection of K. pneumoniae at various concentrations of inoculum, as previously described.11,18 All animal care procedures and protocols were approved by the institutional animal care and use committee of National Yang-Ming University. PFGE and MLST We performed PFGE for all KPC-producing strains as per a previous report.19 The Dice coefficient (1% tolerance and 0.5% optimization) was used to calculate the similarities and the unweighted pair-group method with the arithmetic mean was used for the cluster analysis using GelCompar II software version 6.5 (Applied Maths, St-Martens-Latem, Belgium). Isolates with >80% similarity were considered similar. S1-PFGE was performed as previously described.20 All KPC-producing strains were subjected to MLST according to the protocol described on the K. pneumoniae MLST website (http://bigsdb.pasteur.fr/klebsiella/primers_used.html) and the results analysed using the international K. pneumoniae MLST database created in 2005 at the Pasteur Institute (Paris, France).21 Bacterial conjugation A plasmid conjugation assay was performed using K. pneumoniae TVGHCRE225 as the donor strain (imipenem resistant) and Escherichia coli J53 (sodium azide resistant) as the recipient strain. The experiment was modified as described in a previous study.22 About 1 × 108 cfu of both donor strain and recipient strain were mixed and dotted on sterilized filter paper, which was then incubated on an LB agar plate for 18 h at 37°C. Transconjugants were selected by LB agar plates supplemented with 0.125 mg/L meropenem and 100 mg/L sodium azide. PCR for blaKPC-2 and rmpA/rmpA2 was performed on the transconjugants and the carbapenem MICs were determined. DNA extraction, sequencing and data analysis The DNA of the carbapenem-resistant hypervirulent K. pneumoniae strain was extracted using a Gentra Puregene Yeast/Bact kit (Qiagen) and subjected to PacBio SMRT sequencing. The data analysis, nucleotide sequence accession numbers and phylogenetic analysis of the K. pneumoniae ST11 strains based on SNPs in the core genome are shown in the Supplementary Methods (available as Supplementary data at JAC Online). Results Clinical characteristics of a case infected with an XDR and carbapenemase-producing hypervirulent K. pneumoniae During the study period, a total of 63 KPC-producing K. pneumoniae strains were identified in our hospital. Most of these carried blaKPC-2 (n = 62) and only one strain exhibited blaKPC-3. All strains with KPC-2 belonged to ST11 and capsular genotype K47. We found only one strain (TVGHCRE225) harbouring rmpA and rmpA2 genes. This strain also displayed the hypermucoviscous phenotype, as evaluated by the string test. This strain was isolated from an intra-abdominal abscess of an 83-year-old woman in August 2014. This patient had Parkinson’s disease and had not travelled outside Taiwan within the 3 months prior to this admission. The intra-abdominal abscess was caused by a fistula located between the splenic flexure of the colon and spleen. The patient underwent percutaneous ultrasound-guided catheter drainage for the abscess. The initial pus culture showed ESBL-phenotype K. pneumoniae, E. coli and Enterococcus faecium. The patient received ceftazidime and tigecycline but the disease progressed. The second pus culture from the drainage tube obtained 10 days later showed the growth of a carbapenem-resistant K. pneumoniae strain (TVGHCRE225) with imipenem MIC ≥16 mg/L, as evaluated using the Vitek 2 System. This strain also showed resistance to cephalosporins and fluoroquinolones and was susceptible to amikacin (MIC ≤2 mg/L). Amikacin was administered but septic shock ensued and the patient died 6 days after this strain had been obtained. The MICs of tigecycline and colistin for this strain were 8 and 4 mg/L, respectively, indicating TVGHCRE225 to be an XDR strain. Microbiological characteristics and in vivo virulence of TVGHCRE225 The detailed MICs for TVGHCRE225 are shown in Table 1. Further experimental work to evaluate the resistance mechanism revealed overexpression of the efflux pump gene acrB (8.8 ± 1.5-fold) and its regulatory gene ramA (21.6 ± 5.9-fold) relative to control strain KP 478 (expression = 1; tigecycline MIC, 0.25 mg/L) by quantitative RT-PCR, which was associated with tigecycline resistance. An insertion sequence ISKpn26 in acrR (negative regulatory genes for acrB) and a missense mutation (G104C and T580A) in ramR (negative regulatory genes for ramA) were also identified. Elevated expression of the pmrHFIJKLM operon increased modification of lipopolysaccharide and usually contributed to colistin resistance.17 This strain displayed a significantly higher expression level of pmrH mRNA (9.3 ± 7.4-fold) as compared with the colistin-susceptible strain NTUHK2044 (expression = 1; colistin MIC, 1 mg/L); an insertion sequence element, ISKpn26, in mgrB was responsible for colistin resistance. We also investigated the resistance mechanism of fluoroquinolones, and mutations in the quinolone resistance-determining regions of gyrA and parC were identified. Amino acid substitutions were observed in two codons of GyrA: 83 (Ser→Ile) and 87 (Asp→Gly). In the ParC subunit, substitutions were also identified in one codon: 80 (Ser→Ile). We also found qnrA1, one of the plasmid-mediated quinolone resistance determinants in this strain. Table 1. MICs for K. pneumoniae TVGHCRE225 Antibiotic MIC (mg/L) Cefazolin ≥64 Cefuroxime ≥64 Cefmetazole ≥64 Cefoxitin ≥64 Flomoxef ≥64 Ceftriaxone ≥64 Cefepime ≥64 Piperacillin/tazobactam ≥128 Gentamicin ≥16 Amikacin ≤2 Ciprofloxacin ≥4 Levofloxacin ≥8 Ertapenem ≥8 Imipenem ≥16 Trimethoprim/sulfamethoxazole ≥32 Colistin 4 Tigecycline 8 Antibiotic MIC (mg/L) Cefazolin ≥64 Cefuroxime ≥64 Cefmetazole ≥64 Cefoxitin ≥64 Flomoxef ≥64 Ceftriaxone ≥64 Cefepime ≥64 Piperacillin/tazobactam ≥128 Gentamicin ≥16 Amikacin ≤2 Ciprofloxacin ≥4 Levofloxacin ≥8 Ertapenem ≥8 Imipenem ≥16 Trimethoprim/sulfamethoxazole ≥32 Colistin 4 Tigecycline 8 MICs were determined using the Vitek 2 System, except those of trimethoprim/sulfamethoxazole and tigecycline, which were determined using the Etest method, and that of colistin, which was determined using broth microdilution. Table 1. MICs for K. pneumoniae TVGHCRE225 Antibiotic MIC (mg/L) Cefazolin ≥64 Cefuroxime ≥64 Cefmetazole ≥64 Cefoxitin ≥64 Flomoxef ≥64 Ceftriaxone ≥64 Cefepime ≥64 Piperacillin/tazobactam ≥128 Gentamicin ≥16 Amikacin ≤2 Ciprofloxacin ≥4 Levofloxacin ≥8 Ertapenem ≥8 Imipenem ≥16 Trimethoprim/sulfamethoxazole ≥32 Colistin 4 Tigecycline 8 Antibiotic MIC (mg/L) Cefazolin ≥64 Cefuroxime ≥64 Cefmetazole ≥64 Cefoxitin ≥64 Flomoxef ≥64 Ceftriaxone ≥64 Cefepime ≥64 Piperacillin/tazobactam ≥128 Gentamicin ≥16 Amikacin ≤2 Ciprofloxacin ≥4 Levofloxacin ≥8 Ertapenem ≥8 Imipenem ≥16 Trimethoprim/sulfamethoxazole ≥32 Colistin 4 Tigecycline 8 MICs were determined using the Vitek 2 System, except those of trimethoprim/sulfamethoxazole and tigecycline, which were determined using the Etest method, and that of colistin, which was determined using broth microdilution. We subjected these 63 KPC-producing strains to PFGE and identified a major cluster (>80% similarity) (Figure S1). The carbapenem-resistant hypervirulent strain (TVGHCRE225) did not belong to this major cluster. The in vivo virulence of TVGHCRE225 was compared with that of other strains using a murine model of septicaemia generated by intraperitoneal injection. Upon intraperitoneal infection in mice, this hypervirulent XDR strain showed high virulence, with an LD50 value of 1.1 × 105 cfu. The LD50 of the other carbapenem-resistant ST11 KPC-2-producing strain (TVGHCRE161) belonging to the major cluster in our hospital was 5 × 107 cfu. Two hypervirulent strains with capsular type K1 and K2, rmpA/A2 gene and WT resistance showed an LD50 value of 100 cfu. Genomic comparative analysis The XDR hypervirulent K. pneumoniae strain (TVGHCRE225) was sequenced using the PacBio RS II platform (Pacific Biosciences, USA), generating a library containing 79 892 single reads with an average length of 13 246 bp and a 128-fold average coverage. Reads were assembled using HGAP version 3.0, which returned four contigs. Comparative analysis of this strain with one recently identified carbapenem-resistant hypervirulent ST11 strain (hvkp-5)10 and two classic ST11 strains (HS11268 and JM45) showed highly similar inter-strain genome content, with only a few regions unidentified in the latter three strains (Figure 1). Figure 1. View largeDownload slide Comparative chromosome maps of four ST11 carbapenem-resistant K. pneumoniae strains. Comparative chromosome maps of the hypervirulent carbapenem-resistant K. pneumoniae strain (TVGHCRE225), one recently reported hypervirulent carbapenem-resistant K. pneumoniae strain (hvkp-5) and two publicly available classic ST11 strains (HS11286 and JM45) using TVGHCRE225 as the reference. The circular map was generated using the BLAST Ring Image Generator. Sequence comparison revealed that the four ST11 strains were highly similar; JM45 and HS11286 showed 97% coverage and 99% identity with TVGHCRE225. The layout of the circular diagram implies that TVGHCRE225 may provide a single contig representing the most completely assembled chromosomal sequence among ST11 strains. For instance, the region around the 1000th kbp on the TVGHCRE225 chromosome showed obvious higher GC bias and GC skewness as compared with other regions, and other genome assemblies revealed a pattern of very fragmented alignments. The hvkp-5 chromosome assembly was consistent with that of TVGHCRE225 in other regions that genome assemblies of JM45 and HS11286 may miss, such as the 2750th, 3050th, 4250th and 4700th kbp regions. However, the genome assembly of hvkp-5 was very fragmented, as it was assembled from Illumina short-read sequencing results; furthermore, there were as many as 249 contigs in the NCBI GenBank record. Figure 1. View largeDownload slide Comparative chromosome maps of four ST11 carbapenem-resistant K. pneumoniae strains. Comparative chromosome maps of the hypervirulent carbapenem-resistant K. pneumoniae strain (TVGHCRE225), one recently reported hypervirulent carbapenem-resistant K. pneumoniae strain (hvkp-5) and two publicly available classic ST11 strains (HS11286 and JM45) using TVGHCRE225 as the reference. The circular map was generated using the BLAST Ring Image Generator. Sequence comparison revealed that the four ST11 strains were highly similar; JM45 and HS11286 showed 97% coverage and 99% identity with TVGHCRE225. The layout of the circular diagram implies that TVGHCRE225 may provide a single contig representing the most completely assembled chromosomal sequence among ST11 strains. For instance, the region around the 1000th kbp on the TVGHCRE225 chromosome showed obvious higher GC bias and GC skewness as compared with other regions, and other genome assemblies revealed a pattern of very fragmented alignments. The hvkp-5 chromosome assembly was consistent with that of TVGHCRE225 in other regions that genome assemblies of JM45 and HS11286 may miss, such as the 2750th, 3050th, 4250th and 4700th kbp regions. However, the genome assembly of hvkp-5 was very fragmented, as it was assembled from Illumina short-read sequencing results; furthermore, there were as many as 249 contigs in the NCBI GenBank record. The result of the virulence gene analysis is shown in Table 2. We found that all KP strains (NTUHK2044, CG43, HS11286, JM45, TVGHCRE225 and hvkp-5) harboured several conserved virulence genes, namely entB, fimABCDEFGHIK, iroE, iutA, kpn, mrkABCDFHIJ and ycfM. TVGHCRE225 carried iroBCDN, iucABCD, rmpA and rmpA2, and iutA, whereas hvkp-5 displayed iucABCD, rmpA2 and iutA. This set of virulence genes, including iroBCDN, iucABCD, rmpA, rmpA2 and iutA, was also located in the virulence plasmids pLVPK and pK2044 but not in the other two classic ST11 strains. These observations were consistent with the results of the pan-genome analysis, wherein the virulence plasmid-related genes were unique to ST11 carbapenem-resistant hypervirulent K. pneumoniae but not classic ST11 carbapenem-resistant K. pneumoniae strains. Table 2. Virulence gene analysis of K. pneumoniae strains Virulence gene K. pneumoniae strain NTUHK2044 (K1, ST23) CG43 (K2, ST86) TVGHCRE225 (K47, ST11) hvkp-5 (K47, ST11) HS11286 (K47, ST11) JM45 (undefined K type, ST11) clbB 0 0 0 0 0 0 clbJ 0 0 0 0 0 0 clbK 0 0 0 0 0 0 entB 98.94b,c 98.59b 100 100 100c 100 fimA 100c 100 100 100 100c 100 fimB 97.52b,c 100b 100 100 100c 100 fimC 100c 100 100 100 100c 100 fimD 98.51b,c 99.2b 99.89b 99.89b 100c 100 fimE 97.52b,c 99.01b 100 100 100c 100 fimF 98.86b,c 100b 100 100 100c 100 fimG 100c 100 100 100 100c 100 fimH 98.01b,c 98.01b 100 100 100c 100 fimI 100c 100 100 100 100c 100 fimK 100c 100 100 100 100c 100 iroBa 100 100b 100 0 0 0 iroCa 100 100b 99.92b 0 0 0 iroDa 100 100b 100 0 0 0 iroE 99.36b,c 99.68b 99.68b 99.68b 99.44b,c 99.68b iroNa 100 100b 100 0 0 0 irp1 100 0 100 99.97b 100 0 irp2 100 0 100 100 100 0 iucAa 100 100b 100 100 0 0 iucBa 100 100b 100 100 0 0 iucCa 100 100b 99.83b 100 0 0 iucDa 100 100 100 100 0 0 iutAa 100 100b 100 100 98.5b,c 98.5b,c kpn 89.62b,c 99.45b 100 100 100c 100 mrkA 100c 100 100 100 100c 100 mrkB 100c 100 100 100 100c 100 mrkC 100c 100 100 100 100c 100 mrkD 100c 100 100 100 100c 100 mrkF 100c 100 100 100 100c 100 mrkH 100c 100 100 100 100c 100 mrkI 100c 100 100 100 100c 100 mrkJ 100c 100 100 100 100c 100 rmpA2a 100 100b 99.17b 100b 0 0 rmpAa 100 100b 99.53b 0 0 0 traTa 0 0 86.01 0 98.35 98.35 ycfM 99.53b,c 100 100 100 100b,c 100 Virulence gene K. pneumoniae strain NTUHK2044 (K1, ST23) CG43 (K2, ST86) TVGHCRE225 (K47, ST11) hvkp-5 (K47, ST11) HS11286 (K47, ST11) JM45 (undefined K type, ST11) clbB 0 0 0 0 0 0 clbJ 0 0 0 0 0 0 clbK 0 0 0 0 0 0 entB 98.94b,c 98.59b 100 100 100c 100 fimA 100c 100 100 100 100c 100 fimB 97.52b,c 100b 100 100 100c 100 fimC 100c 100 100 100 100c 100 fimD 98.51b,c 99.2b 99.89b 99.89b 100c 100 fimE 97.52b,c 99.01b 100 100 100c 100 fimF 98.86b,c 100b 100 100 100c 100 fimG 100c 100 100 100 100c 100 fimH 98.01b,c 98.01b 100 100 100c 100 fimI 100c 100 100 100 100c 100 fimK 100c 100 100 100 100c 100 iroBa 100 100b 100 0 0 0 iroCa 100 100b 99.92b 0 0 0 iroDa 100 100b 100 0 0 0 iroE 99.36b,c 99.68b 99.68b 99.68b 99.44b,c 99.68b iroNa 100 100b 100 0 0 0 irp1 100 0 100 99.97b 100 0 irp2 100 0 100 100 100 0 iucAa 100 100b 100 100 0 0 iucBa 100 100b 100 100 0 0 iucCa 100 100b 99.83b 100 0 0 iucDa 100 100 100 100 0 0 iutAa 100 100b 100 100 98.5b,c 98.5b,c kpn 89.62b,c 99.45b 100 100 100c 100 mrkA 100c 100 100 100 100c 100 mrkB 100c 100 100 100 100c 100 mrkC 100c 100 100 100 100c 100 mrkD 100c 100 100 100 100c 100 mrkF 100c 100 100 100 100c 100 mrkH 100c 100 100 100 100c 100 mrkI 100c 100 100 100 100c 100 mrkJ 100c 100 100 100 100c 100 rmpA2a 100 100b 99.17b 100b 0 0 rmpAa 100 100b 99.53b 0 0 0 traTa 0 0 86.01 0 98.35 98.35 ycfM 99.53b,c 100 100 100 100b,c 100 Each value in the table represents the percentage of homology of one virulence gene in the K. pneumoniae strains when compared with the reference alleles in BIGSdb. ‘0’ means the absence of this gene in the specific strain analysed and ‘100’ means the presence of an identical gene in the specific strain. The results may not be completely consistent either with those generated by the BIGSdb online tool or with those revealed in previous literature. This might be partly because we ran NCBI BLASTP in a repeat masking disabled mode, and thus the additional repeat-containing virulence factors could be identified. For example, the full-length rmpA sequence of TVGHCRE225 could be completely aligned to a repeat-containing allele retrieved from BIGSdb. a This gene is located in the plasmid. All of the genes, except for traT located in the KPC-2-carrying plasmid, are located in the pVirCRE225, pLVPK, pK2044 or pLVPK-like plasmid. b Results differ from those generated by performing BLAST on the BIGSdb web site. c Results differ from the literature. Table 2. Virulence gene analysis of K. pneumoniae strains Virulence gene K. pneumoniae strain NTUHK2044 (K1, ST23) CG43 (K2, ST86) TVGHCRE225 (K47, ST11) hvkp-5 (K47, ST11) HS11286 (K47, ST11) JM45 (undefined K type, ST11) clbB 0 0 0 0 0 0 clbJ 0 0 0 0 0 0 clbK 0 0 0 0 0 0 entB 98.94b,c 98.59b 100 100 100c 100 fimA 100c 100 100 100 100c 100 fimB 97.52b,c 100b 100 100 100c 100 fimC 100c 100 100 100 100c 100 fimD 98.51b,c 99.2b 99.89b 99.89b 100c 100 fimE 97.52b,c 99.01b 100 100 100c 100 fimF 98.86b,c 100b 100 100 100c 100 fimG 100c 100 100 100 100c 100 fimH 98.01b,c 98.01b 100 100 100c 100 fimI 100c 100 100 100 100c 100 fimK 100c 100 100 100 100c 100 iroBa 100 100b 100 0 0 0 iroCa 100 100b 99.92b 0 0 0 iroDa 100 100b 100 0 0 0 iroE 99.36b,c 99.68b 99.68b 99.68b 99.44b,c 99.68b iroNa 100 100b 100 0 0 0 irp1 100 0 100 99.97b 100 0 irp2 100 0 100 100 100 0 iucAa 100 100b 100 100 0 0 iucBa 100 100b 100 100 0 0 iucCa 100 100b 99.83b 100 0 0 iucDa 100 100 100 100 0 0 iutAa 100 100b 100 100 98.5b,c 98.5b,c kpn 89.62b,c 99.45b 100 100 100c 100 mrkA 100c 100 100 100 100c 100 mrkB 100c 100 100 100 100c 100 mrkC 100c 100 100 100 100c 100 mrkD 100c 100 100 100 100c 100 mrkF 100c 100 100 100 100c 100 mrkH 100c 100 100 100 100c 100 mrkI 100c 100 100 100 100c 100 mrkJ 100c 100 100 100 100c 100 rmpA2a 100 100b 99.17b 100b 0 0 rmpAa 100 100b 99.53b 0 0 0 traTa 0 0 86.01 0 98.35 98.35 ycfM 99.53b,c 100 100 100 100b,c 100 Virulence gene K. pneumoniae strain NTUHK2044 (K1, ST23) CG43 (K2, ST86) TVGHCRE225 (K47, ST11) hvkp-5 (K47, ST11) HS11286 (K47, ST11) JM45 (undefined K type, ST11) clbB 0 0 0 0 0 0 clbJ 0 0 0 0 0 0 clbK 0 0 0 0 0 0 entB 98.94b,c 98.59b 100 100 100c 100 fimA 100c 100 100 100 100c 100 fimB 97.52b,c 100b 100 100 100c 100 fimC 100c 100 100 100 100c 100 fimD 98.51b,c 99.2b 99.89b 99.89b 100c 100 fimE 97.52b,c 99.01b 100 100 100c 100 fimF 98.86b,c 100b 100 100 100c 100 fimG 100c 100 100 100 100c 100 fimH 98.01b,c 98.01b 100 100 100c 100 fimI 100c 100 100 100 100c 100 fimK 100c 100 100 100 100c 100 iroBa 100 100b 100 0 0 0 iroCa 100 100b 99.92b 0 0 0 iroDa 100 100b 100 0 0 0 iroE 99.36b,c 99.68b 99.68b 99.68b 99.44b,c 99.68b iroNa 100 100b 100 0 0 0 irp1 100 0 100 99.97b 100 0 irp2 100 0 100 100 100 0 iucAa 100 100b 100 100 0 0 iucBa 100 100b 100 100 0 0 iucCa 100 100b 99.83b 100 0 0 iucDa 100 100 100 100 0 0 iutAa 100 100b 100 100 98.5b,c 98.5b,c kpn 89.62b,c 99.45b 100 100 100c 100 mrkA 100c 100 100 100 100c 100 mrkB 100c 100 100 100 100c 100 mrkC 100c 100 100 100 100c 100 mrkD 100c 100 100 100 100c 100 mrkF 100c 100 100 100 100c 100 mrkH 100c 100 100 100 100c 100 mrkI 100c 100 100 100 100c 100 mrkJ 100c 100 100 100 100c 100 rmpA2a 100 100b 99.17b 100b 0 0 rmpAa 100 100b 99.53b 0 0 0 traTa 0 0 86.01 0 98.35 98.35 ycfM 99.53b,c 100 100 100 100b,c 100 Each value in the table represents the percentage of homology of one virulence gene in the K. pneumoniae strains when compared with the reference alleles in BIGSdb. ‘0’ means the absence of this gene in the specific strain analysed and ‘100’ means the presence of an identical gene in the specific strain. The results may not be completely consistent either with those generated by the BIGSdb online tool or with those revealed in previous literature. This might be partly because we ran NCBI BLASTP in a repeat masking disabled mode, and thus the additional repeat-containing virulence factors could be identified. For example, the full-length rmpA sequence of TVGHCRE225 could be completely aligned to a repeat-containing allele retrieved from BIGSdb. a This gene is located in the plasmid. All of the genes, except for traT located in the KPC-2-carrying plasmid, are located in the pVirCRE225, pLVPK, pK2044 or pLVPK-like plasmid. b Results differ from those generated by performing BLAST on the BIGSdb web site. c Results differ from the literature. The strain TVGHCRE225 harboured three plasmids. S1-PFGE confirmed the size of the three plasmids analysed by WGS (Figure S2). The IncR/IncFII/IncN plasmid (103 454 bp) carrying the antimicrobial resistance gene blaKPC-2 conferring resistance to carbapenems shared 99% identity and 72% query coverage with plasmid pKPC-LK30 from a KPC-2-producing K. pneumoniae strain in Taiwan (Figure S3). The other IncA/C2-type plasmid (159 072 bp) had antimicrobial resistance genes aadA2, aac(3)-IId, strA, strB, sul1, sul2, qnrA1 and blaTEM-1B, which conferred resistance to aminoglycosides, sulphonamide, fluoroquinolones and β-lactams. The IncHI1B/IncFIB plasmid (297 984 bp) encoded 306 predicted ORFs. The set of virulence genes, including iroBCDN, iucABCD, rmpA and rmpA2, and iutA, were located in this plasmid, whereas no antimicrobial resistance gene was identified. Comparison of this virulent plasmid, designated as pVir, and other plasmids with the use of the Basic Local Alignment Search Tool (BLAST) revealed its identity with pK2044 (49% coverage, 99% identity), pLVPK (47% coverage, 99% identity) and pPMK-NDM (61% coverage, 99% identity). TVGHCRE225 pVir was larger in size than the other two virulent plasmids (pK2044: 224 152 bp; pLVPK: 219 385 bp) in K. pneumoniae. pPMK-NDM is an IncHI2-type resistance plasmid harboured by the NDM-producing K. pneumoniae strain that caused an outbreak in a Nepali neonatal unit. Sequence analysis of TVGHCRE225 pVir suggests that this is a novel hybrid plasmid comprising a part of the virulence plasmid pLVPK and pPMK-NDM. The first region was highly similar to the CG43 (pLVPK), hvkp-5 and NTUH-K2044 plasmids, while the second region was highly similar to the PMK1 (pPMK1-NDM) plasmid (Figure 2). Figure 2. View largeDownload slide Gene map of four virulence plasmids and pPMK1-NDM harboured by K. pneumoniae strains. Alignment of the four virulent plasmids recovered from our strain TVGHCRE225, ST11 carbapenem-resistant hypervirulent K. pneumoniae strain hvkp-5, capsular K1 hypervirulent K. pneumoniae NTUH-K2044 and capsular K2 hypervirulent K. pneumoniae CG43, and a plasmid harboured by strain PMK1. The circular genome comparison diagram was generated with the BLAST Ring Image Generator. TVGHCRE225 pVir could be roughly divided into two distinct regions based on its similarity to different plasmids. The first region, 1st to ∼130th kbp, was highly similar to the CG43 (pLVPK), hvkp-5 and NTUH-K2044 plasmids, while the second region, 130th to ∼300th kbp, was highly similar to the PMK1 (pPMK1-NDM) plasmid. Virulence factors such as iroBCDN, iucABCD, rmpA and rmpA2, and iutA are all located in the first region corresponding to the pLVPK-like plasmid region. Figure 2. View largeDownload slide Gene map of four virulence plasmids and pPMK1-NDM harboured by K. pneumoniae strains. Alignment of the four virulent plasmids recovered from our strain TVGHCRE225, ST11 carbapenem-resistant hypervirulent K. pneumoniae strain hvkp-5, capsular K1 hypervirulent K. pneumoniae NTUH-K2044 and capsular K2 hypervirulent K. pneumoniae CG43, and a plasmid harboured by strain PMK1. The circular genome comparison diagram was generated with the BLAST Ring Image Generator. TVGHCRE225 pVir could be roughly divided into two distinct regions based on its similarity to different plasmids. The first region, 1st to ∼130th kbp, was highly similar to the CG43 (pLVPK), hvkp-5 and NTUH-K2044 plasmids, while the second region, 130th to ∼300th kbp, was highly similar to the PMK1 (pPMK1-NDM) plasmid. Virulence factors such as iroBCDN, iucABCD, rmpA and rmpA2, and iutA are all located in the first region corresponding to the pLVPK-like plasmid region. Phylogenetic analysis of the K. pneumoniae ST11 strains based on SNPs in the core genome To estimate the genetic relationships among two hypervirulent carbapenem-resistant strains (TVGHCRE225 and hvKP-5) and other classic ST11 strains (JM45, HS11286, FJ8 and FJ10), we performed phylogenetic analysis based on core genome SNPs. This revealed that four ST11 strains, including TVGHCRE225, FJ10 (from Hong Kong), FJ8 (from Hong Kong) and hvkp-5 (from Hangzhou, China), formed a clade that is distinct from the other two ST11 strains, HS11286 (from Shanghai, China) and JM45 (from Hangzhou, China). In this clade, FJ10, FJ8 and hvkp-5 were more closely related, whereas TVGHCRE225 alone forms another branch (Figure S4). The branch length of the TVGHCRE225 node is longer than that of the other three ST11 strains, suggesting that TVGHCRE225 is genetically more divergent in this clade (Figure S4). This result suggested that TVGHCRE225 was evolutionarily closer to the hvkp-5 than to the other two ST11 strains (HS11286 and JM45). Conjugation experiment A conjugation assay was performed to examine whether the plasmid carrying KPC-2 could be transferred. The conjugation frequency was determined to be 2.65 × 10−7. The imipenem MIC was 0.125 mg/L for E. coli J53 and 6 mg/L for the transconjugant. The carbapenem resistance phenotype of TVGHCRE225 could be transferred to E. coli strain J53. The transconjugant carried the blaKPC-2 gene, which was located on the IncR/IncFII/IncN plasmid, but the virulent determinant rmpA/rmpA2 located on pVir could not be detected in these transconjugants. Discussion We identified one XDR hypervirulent K. pneumoniae strain, TVGHCRE225, and WGS showed that this strain acquired a virulence plasmid harbouring rmpA/rmpA2 as compared with the classic ST11 KPC-2-producing K. pneumoniae strain. This XDR hypervirulent K. pneumoniae strain was isolated in Taiwan in 2014, earlier than that isolated in China recently reported by Gu et al.10 The distribution of the carbapenem-resistant hypervirulent K. pneumoniae strain is broader than expected, suggesting the emergence of a strain with high virulence and antimicrobial resistance. Capsular polysaccharide is an important virulence factor in K. pneumoniae. K1/K2/K5/K20/K54/K57 types were considered virulent and responsible for a variety of community-onset pyogenic infections.14,23 In the present study, all KPC-2-producing strains belong to capsular type K47. K47 is the most common capsular type among KPC-producing K. pneumoniae isolates in China.24 Carbapenem-resistant hypervirulent K. pneumoniae strains have been mostly reported in China,8 but were also recently identified in the USA.25 Most strains from China belong to K1 or K2 capsular type and carried rmpA/rmpA2 genes,26–29 and it has been speculated that the horizontal transfer of resistance plasmids contributes to carbapenem resistance.9,In vitro experiments demonstrated that KPC-carrying plasmids were successfully conjugated and retained by a virulent K2 K. pneumoniae recipient isolate and that transconjugants are able to maintain high serum resistance and murine lethality.30 These findings provided the rationale that hypervirulent strains evolved to become carbapenem resistant. Gu et al.10 recently reported the emergence of ST11 carbapenem-resistant hypervirulent K. pneumoniae strains in China, different from K1 or K2 capsular types, owing to the acquisition of a pLVPK-like virulence plasmid. In our study, genomic analyses showed that the XDR hypervirulent strain TVGHCRE225 acquired a novel hybrid virulence plasmid containing a set of virulence genes on pLVPK and pK2044. Moreover, TVGHCRE225 was resistant to tigecycline and colistin, which has never been reported previously among carbapenem-resistant hypervirulent strains. The patient received tigecycline but not colistin before the isolation of TVGHCRE225. The evolution to a hypervirulent strain even occurred with an XDR background. Our results correspond to the report by Gu et al. and provide evidence that virulence plasmids are not restricted only to hypervirulent K. pneumoniae backgrounds, suggesting a change in the host–plasmid relationship in the ST11 KPC-2-producing K. pneumoniae background.9 Thus, the possibility of transfer of plasmid-encoded virulence genes into carbapenem-resistant isolates represents a different mechanism for the interplay between virulence and antimicrobial resistance. Only two studies have detailed the genomic analyses of carbapenem-resistant hypervirulent K. pneumoniae strains10,25 using Illumina sequencing platforms. The short-read assembly is unlikely to resolve abundant regions containing repetitive elements, which may be found in most genomes. In addition, the GC content bias featured in regions with horizontal gene transfers may be associated with low sequencing coverage or be completely lost during sequencing with the Illumina short-read technology. In this study, we used a third-generation sequencing platform, PacBio, to build a complete sequence map for TVGHCRE225. With a fully assembled genome, it is straightforward to delineate the structural features of the chromosome and three plasmids and determine the locations of virulence factors and drug resistance genes. It is notable that the virulence plasmid, TVGHCRE225 pVir, was clearly identified to comprise two evolutionarily distinct regions likely derived from different plasmids. In addition, the phylogenetic tree showed that TVGHCRE225 was evolutionarily close to the hvkp-5 isolated in China recently. However, our TVGHCRE225 was found in 2014, which was earlier than hvkp-5 (isolated in 2016), and, therefore, TVGHCRE225 might have the ability to evolve independently. More ST11 genomes to elucidate the evolution of the carbapenem-resistant hypervirulent K. pneumoniae strains are necessary. Zhu et al.31 recently reported the low in vivo virulence of seven carbapenem-resistant hypermucoviscous K. pneumoniae isolates in a mouse lethality assay in a teaching hospital in China between January 2015 and December 2016. Only two of the seven strains carried the rmpA gene. Zhu et al.’s results indicated that defining hypervirulent K. pneumoniae only by hypermucoviscosity phenotype was far from sufficient. The absence of specific genomic markers to define hypervirulent K. pneumoniae strains has been criticized previously.32 A recent review article proposed further testing of virulence behaviour among the carbapenem-resistant strains with hypermucoviscosity in a suitable animal model.8 In this study, we detailed the genetic content of virulence determinants and demonstrated the virulence of TVGHCRE225 using a mouse lethality assay by comparing it with the WT capsular K1/K2 and classic ST11 strains. The acquisition of the virulence plasmid in this XDR strain resulted in higher virulence compared with the classic ST11 strain; however, this strain was not as virulent as capsular K1/K2 strains in the mouse lethality assay. The virulent plasmids hvkp-5, pLVPK-like, pLVPK and pK2044 were highly similar, but the virulence plasmid in TVGHCRE225 was a novel hybrid plasmid containing a set of virulence genes. The plasmid spread between strains contributed to the confluence of hypervirulence and carbapenem resistance. Therefore, it is necessary to understand the molecular mechanisms underlying the plasmid–host expansion and adaptation with respect to antimicrobial resistance and virulence.9 The major limitation of this study is the limited number of KPC-producing strains investigated. A large-scale screening of the capsular types and for the presence of rmpA among all carbapenem-resistant K. pneumoniae strains carrying other resistance mechanisms is ongoing to elucidate the virulence characteristics and strain background of these newly emerged strains. In conclusion, we identified an XDR ST11 KPC-2-producing K. pneumoniae strain carrying a virulent plasmid for the first time in a country of the Asia Pacific rim. This strain was different from the major cluster of KPC-2-producing strains in our hospital. Active surveillance focusing on both the antimicrobial resistance and virulence characteristics of K. pneumoniae strains is necessary, as the threat to human health is imminent. Further studies regarding carbapenem-resistant hypervirulent K. pneumoniae worldwide to explore the evolutionary relationship between virulence and resistance in K. pneumoniae are encouraged. Acknowledgements Partial results of this manuscript were presented as a paper poster (P0919) at the Twenty-eighth European Congress of Clinical Microbiology and Infectious Diseases, Madrid, Spain, 2018.  We acknowledge the Medical Science and Technology Building of the Taipei Veterans General Hospital for providing experimental space and facilities. We thank Professor Po-Liang Lu for PFGE assistance. We thank the GenoInfo Core Facility (C1) funded by the NCFPB of the Ministry of Science and Technology in Taiwan (MOST106-2319-B-010–001) for technical support regarding genome annotation. We thank National Core Facility for Biopharmaceuticals (NCFB, MOST 106-2319-B-492-002) and the National Center for High-performance Computing (NCHC) of National Applied Research Laboratories (NARLabs) of Taiwan for providing computational resources and storage resources. Funding This work was supported by grants from the Ministry of Science and Technology in Taiwan (MOST103-2314-B-075-078-MY2, MOST105-2628-B-010-015-MY3, MOST105-2320-B-010-006 and MOST106-2320-B-010-019) and the Taipei Veterans General Hospital (V104B-001, V105B-001, V106B-001 and V107C-081). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Transparency declarations None to declare. Supplementary data Supplementary Methods and Figures S1–S4 are available as Supplementary data at JAC Online. References 1 Lee CR , Lee JH , Park KS et al. Global dissemination of carbapenemase-producing Klebsiella pneumoniae: epidemiology, genetic context, treatment options, and detection methods . Front Microbiol 2016 ; 7 : 895 . Google Scholar PubMed 2 Doi Y , Paterson DL. Carbapenemase-producing Enterobacteriaceae . Semin Respir Crit Care Med 2015 ; 36 : 74 – 84 . 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: May 24, 2018

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