IMP-38-Producing High-Risk Sequence Type 307 Klebsiella pneumoniae Strains from a Neonatal Unit in China

We described the genome and resistome characterization of a carbapenem-resistant Klebsiella pneumoniae ST307 strain carrying blaIMP-38 in China. This report highlights that the high-risk ST307 clone continues to acquire different antimicrobial resistance genes, posing significant challenges to clinical practice, and should be closely monitored.

(ST11) and ST258 and closely related STs (5). Meanwhile, other high-risk clones contributing to the global spread of CRKP have also been frequently reported. One example is K. pneumoniae ST307, which is an emerging global antimicrobial drugresistant clone (6)(7)(8). In certain regions, ST307 even showed a tendency to replace CG258. A U.S. study sequenced 1,777 extended-spectrum-beta-lactamase (ESBL)producing K. pneumoniae strains collected over 4 years and found that the CG307 strains caused more infections (36.2%, 644/1,777) than the widely studied CG258 epidemic strains (26.7%, 474/1,777) (9). In addition, a recent study reported a regional spread of OXA-181-producing ST307 CRKP in South Africa (8) that was associated with a promiscuous IncX3 plasmid harboring bla  . Moreover, NDM-producing ST307 CRKP strains have also been reported to cause hospital outbreaks elsewhere (10). Those studies indicated that the high-risk ST307 strains, harboring various carbapenemase (bla KPC , bla NDM , bla  , and bla OXA-181 ) and/or ESBL (e.g., bla CTX-M-15 ) genes, have spread widely in different regions in the world. However, IMP-producing or VIMproducing ST307 strains remain infrequently reported.
IMP carbapenemase, a member of the metallo-␤-lactamases capable of hydrolyzing almost all ␤-lactams, was first described in Japan in the 1990s (11). In 2011, our group first identified a novel IMP-38 variant in the neonatology department in our hospital (12). In this study, we examined the prevalence of bla IMP-38 -harboirng K. pneumoniae isolates in our hospital and determined their genetic backgrounds. The genomic characteristics of a representative IMP-38-procuding ST307 CRKP clinical strain are further described.
Notably, 13 of the 14 IMP-38-positive strains were isolated from patients with neonatal sepsis in the neonatal ward, while the remaining strain was from a patient with persistent pneumonia in the pediatric neurology ward, which is adjacent to the neonatal ward. Specific clinical characteristics are described in Table 1. All 14 patients were preterm infants and had undergone endotracheal intubation, which appeared to be one of major risk factors associated with neonatal sepsis among our patients. We suspected that the infections by IMP-38-producing CRKP might have been associated with endotracheal intubation and might have been caused either by contaminated equipment or by staff contact transmission. Previously published studies also suggested that endotracheal intubation was an independent risk factor for infections caused by multidrug-resistant K. pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa (13)(14)(15). However, since these clinical isolates were retrospectively collected, we were unable to recover CRKP from endotracheal tube cultures and therefore cannot confirm whether the presence of IMP-38-producing CRKP was due to endotracheal tube contamination. Antibiotic susceptibilities and molecular detection. All 14 isolates exhibited susceptibility to amikacin, levofloxacin, ciprofloxacin, and trimethoprim-sulfamethoxazole but showed resistance to cefepime, ceftriaxone, ceftazidime, aztreonam, ertapenem, and meropenem, while 13 isolates were susceptible to imipenem (MIC Յ 1 g/ ml). Ten isolates were resistant to piperacillin-tazobatam (Table 1).
PCR detection of carbapenemase and ESBL genes revealed that all 14 strains carried bla IMP-38 , bla CTX-M-3 , bla SHV-2a , bla SHV-28 , and bla TEM-1 . Pulsed-field gel electrophoresis (PFGE) analysis showed that these isolates shared highly similar profiles (see Fig. S1 in the supplemental material), suggesting a clonal spread of the same bla IMP-38 -harboring strains.
Multilocus sequence type (MLST) analysis showed that all 14 bla IMP-38 -harboring K. pneumoniae strains belonged to ST307. As mentioned above, ST307 strains have been associated with different carbapenemase genes (bla KPC , bla NDM , bla OXA-48 , and bla OXA-181) and ESBL genes (e.g., bla CTX-M-15 ); however, bla IMP has not been reported in ST307 strains. To the best of our knowledge, this is the first report of the presence of IMP metallo-␤-lactamase in this high-risk clone. To reveal the genomic characteristics of IMP-38-produicng K. pneumoniae ST307 strains, we selected one strain, WCGKP294, for whole-genome sequencing using the combination of PacBio and Illumina HiSeq sequencing.
Genomic characterization of K. pneumoniae strain WCGKP294. Strain WCGKP294 was isolated from a sputum sample from a preterm infant in 2016. The combination of PacBio SMRT and Illumina HiSeq sequencing generated the complete closure of the WCGKP294 genome. WCGKP294 harbors a 5,540,574-bp circular chromosome and two plasmids, pWCGKP294-1 and pWCGKP294-2. The WCGKP294 chromosome harbors the oqxA and oqxB genes, which have been linked to quinolone resistance; the bla SHV-28 gene, conferring cephalosporin resistance; and the fosA6 gene, which has been linked to fosfomycin resistance. Strain WCGKP294 carried the same K and O loci (KL102, associated with wzi allele 173, and O2v2) as other global ST307 strains, as well as the second putative capsule synthesis locus (Cp2) and a -fimbria-encoding gene cluster (16). However, inspection of the chromosomal quinolone resistance-determining regions (QRDR) failed to identify amino acid substitutions at GyrA codon 83 or 87 and at ParC codon 80 or 84, which appears to be one of the major genetic differences from other global ST307 genomes (17). Previous study of 95 global ST307 genomes showed that the ParC 80I and GyrA 83I mutations were 100% conserved among all the genomes, with a small cluster of strains carrying an additional GyrA 87N mutation (17). The QRDR mining results in WCGKP294 were in agreement with our susceptibility testing results indicating that all our strains were susceptible to ciprofloxacin and levofloxacin (Table 1).
In order to describe the relationship between IMP-38-producing strain WCGKP294 and other global ST307 strains obtained from different countries and sources, a hierarchical Bayesian clustering analysis of ST307 genomes was performed (Fig. 1). Similarly to the results of a previous study (8), the analysis divided ST307 into 6 distinct clades: clades I to VI. Strain WCGKP294 was found to belong to clade V, which is distantly related to clades I to IV (the U.S. Texas clades) and clade VI (the South African OXA-181 clade). Clades I to IV and clade VI represent strains that have been locally isolated over a long period in the United States and South Africa, while the clade V is more like a global lineage and includes isolates from Australia, Brazil, Cambodia, Cameroon, China, Colombia, France, Guinea, Iran, Italy, Nepal, Netherlands, Nigeria, Norway, Pakistan, Thailand, the United Kingdom, and the United States (8).
Characteristics of bla IMP-38 -harboring plasmid pWCGKP294-2. WCGKP294 was found to contain two plasmids, pWCGKP294-1 and pWCGKP294-2. Plasmid pWCGKP294-1 (GenBank accession number CP046613) was found to be 237,090 bp in length and to belong to the IncFIB incompatibility group, carrying three copies of tandemly repeated IS26-bla SHV-2A -deoR-ygbJ-ygbK-fucA-IS26 composite transposon elements (see Text S1 in the supplemental material). Plasmid pWCGKP294-2 (GenBank accession number CP046614) was found to be 230,910 bp in length with GC content of 47%. Sequence analysis indicated that plasmid pWCGKP294-2 belonged to the IncHI5 incompatibility group (Fig. 2). BLAST analysis showed that pWCGKP294-2 had high similarity to bla IMP-38 -harboring pA324-IMP (MF344566), recovered from a K. pneumoniae strain in Beijing, with 97% query coverage and Ͼ99.9% identity, and to bla IMP-4 -harboring pIMP-LL34 (CP025964), isolated from a K. pneumoniae in Chengdu, with 91% query coverage and Ͼ99.9% identity (Fig. 2). In China, the main IMP type of carbapenemase was found to be IMP-4 (18,19), which differs from IMP-38 with an amino acid substitution at Ambler position 262 (S262G, serine to glycine) (12). It is likely that IMP-38 evolved from IMP-4 from amino acid substitution. The S262G substitution was previously identified as important for substrate specificity according to studies of other IMP variants, especially IMP-6, which differs from IMP-1 by the same S262G substitution (20)(21)(22). The S262G substitution led to a reduction in the imipenem MIC but to higher levels of meropenem and doripenem resistance in IMP-6. We therefore suspected that the imipenem-susceptible but meropenem-resistant profiles found in our IMP-38-producing ST307 CRKP strains may have been due to the S262G substitution (Table 1). Further enzyme kinetic analysis is ongoing to confirm the hypothesis.
Similarly to pA324-IMP, plasmid pWCGKP294-2 had two transposons, designated Tn6381 and Tn6382. The Tn6381 transposon was located in an ARI-B island, identified as a Tn6535 derivative, while the Tn6382 transposon was within an ARI-A island, identified as a Tn1696 derivative (23). The bla IMP-38 gene was carried by a class I integron within Tn6382 with the gene cassette of bla IMP-38 -qacG2-aacA4-Δcat3 on pWCGKP294-2. In addition to bla IMP-38 , several antibiotic resistance genes have also been identified on pWCGKP294-2, including ␤-lactamase genes bla CTX-M-3 and bla TEM-1B , aminoglycoside resistance gene aacA4, and chloramphenicol resistance gene catB3. In comparison to pIMP4_LL34 and pA324-IMP, pWCGKP294-2 contained an approximately 44-kb deletion that extended from the umuD gene to a hypothetical protein gene located upstream of hns. This deleted region included the conjugation-encoding cpl, rlx, and tivF genes, which is consistent with the inability of conjugation of pWCGKP294-2 seen during the conjugation experiment (data not shown).
In conclusion, we have reported here the identification of IMP-38-producing highrisk ST307 K. pneumoniae strains in a neonatal unit in China. The bla IMP-38 gene is harbored by a Tn6382 transposon on an IncHI5 plasmid. Unlike the other global ST307 strains, our IMP-38-producing ST307 strain does not harbor QRDR fluoroquinolone resistance-associated mutations and was not associated with the ESBL bla CTX-M-15 gene on the IncFIIK/IncFIBK plasmid, suggesting that IMP-38-producing ST307 strains may have a different evolutionary path and are under different antibiotic selection pressures. Nevertheless, our study showed that this high-risk clone continues to acquire various antimicrobial resistance genes, posing significant challenges to clinical practice, and should be closely monitored.

MATERIALS AND METHODS
Bacterial isolates and antimicrobial susceptibility testing. Clinical isolates were collected from January 2013 to November 2016 in a tertiary care hospital in Changsha located in the middle of China. A total 515 unique CRKP isolates (one isolate per patient) were recovered from clinical specimens. MICs of 12 antimicrobial agents were determined for all 515 clinical isolates using the microdilution method, according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (24). The study was approved by the institutional review board (IRB) at Xiangya Hospital of Central South University.
Detection of bla IMP-38 gene. All CRKP isolates were screened for the presence of the bla IMP gene using a previously described PCR protocol (25)(26)(27). PCR amplifications were carried out on an ABI 2720 thermal cycler (Applied Biosystems, USA). The amplicons from IMP-positive strains were then sequenced to determine bla IMP-38 variants.
MLST. MLST was performed on clinical bla IMP-38 -harboring isolates using a method described previously by Diancourt et al. (27). The ST was determined using the database maintained by the MLST Web server (https://pubmlst.org/bigsdb?dbϭpubmlst_mlst_seqdef).
Whole-genome sequencing and bioinformatics analysis. The genomic DNA was extracted from IMP-38-producing ST307 strain WCGKP294 using the SDS method (28). The DNA was subsequently sequenced using single-molecule sequencing (PacBio RS) and an Illumina HiSeq system. The sequencing reads were assembled using Unicycler v0.4.8 (29). The acquired antimicrobial resistance genes were identified using ResFinder 3.0 (30), and the plasmid replicons in the sequenced isolates were identified using PlasmidFinder 2 (31). A core single nucleotide polymorphism (SNP) phylogenetic tree of WCGKP294 and of 708 global ST307 isolates (from 19 countries) was constructed using a method described previously (8). The phylogenetic tree was annotated in iTOL (32). A total of 21 IncHI5 plasmids were downloaded from GenBank, followed by alignment using Mauve 2.4.0 (33). Conserved regions were extracted from Mauve alignment, and a maximum likelihood (ML) phylogenetic tree was generated using FastTree 2.1 (34).
Data availability. The complete genome sequences of WCGKP294 were submitted to GenBank under accession numbers CP046612 to CP046614.