KPC-2-Producing Carbapenem-Resistant Klebsiella pneumoniae of the Uncommon ST29 Type Carrying OXA-926, a Novel Narrow-Spectrum OXA β-Lactamase

We isolated and characterized a carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical strain from blood carrying a novel blaOXA gene, blaOXA–926, and belonging to ST29, an uncommon CRKP type. The strain, 130002, was genome sequenced using both short- and long-read sequencing and has a 94.9-kb self-transmissible IncFII plasmid carrying blaKPC–2. K. pneumoniae genomes of the ST29 complex (ST29 and its single-allele variants) were retrieved and were subjected to single nucleotide polymorphism-based phylogenomic analysis. A total of 157 genomes of the ST29 complex were identified. This complex is commonly associated with extended-spectrum β-lactamase-encoding genes, in particular, blaCTX–M–15 but rarely has carbapenemase genes. The novel plasmid-encoded β-lactamase-encoding gene blaOXA–926 was identified on a 117.8-kb IncFIA-IncFII plasmid, which was transferrable in the presence of the blaKPC–2-carrying plasmid. blaOXA–926 was cloned and MICs of β-lactams in the transformants were determined using microdilution. OXA-926 has a narrow spectrum conferring reduced susceptibility only to piperacillin, piperacillin-tazobactam, and cephalothin. Avibactam cannot fully inhibit OXA-926. blaOXA–926 and its variants have been seen in Klebsiella strains in Asia and Brazil. OXA-926 is the closest in sequence identity (89.9%) to a chromosome-encoding OXA-type enzyme of Variovorax guangxiensis. In conclusion, OXA-926 is novel plasmid-borne narrow-spectrum β-lactamase that cannot be fully inhibited by avibactam. It is likely that blaOXA–926 originates from a species closely related to V. guangxiensis and was introduced into Klebsiella > 10 years ago.


INTRODUCTION
Resistance to β-lactam agents such as penicillins, cephalosporins, and carbapenems in the Enterobacteriaceae, one of the most common human pathogens, is mainly due to the production of hydrolyzing enzymes called β-lactamases. β-Lactamases can be divided into classes A, B, C, and D based on amino acid homology (Hall and Barlow, 2005). Class A, C, and D enzymes are also termed serine β-lactamases as they possess a serine at the active site, while class B enzymes require a metal ion for activity and are therefore called metallo-β-lactamases. OXA (oxacillinase) is a large group of class D β-lactamases with a remarkably varied spectrum against β-lactam agents from narrow-spectrum (hydrolyzing penicillins only, e.g., OXA-1) to extended-spectrum (with ability to hydrolyze 3rd generation cephalosporins, e.g., OXA-11) and carbapenemases (e.g., OXA-23 and OXA-48) (Evans and Amyes, 2014). A number of bacterial species, e.g., Acinetobacter baumannii and Pseudomonas aeruginosa contain intrinsic OXA-encoding genes bla OXA in their chromosomes, while many bla OXA genes are carried by plasmids (Evans and Amyes, 2014). In this study, we found a bla OXA gene encoding a novel OXA enzyme in a carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical strain and determined its active spectrum. We also found that this strain belongs to ST29, an uncommon CRKP type. CRKP has emerged worldwide as a significant human health challenge (World Health Organization, 2017). The global dissemination of CRKP is mainly due to certain high-risk clones, in particular, ST 258 (Adler et al., 2014) and ST11 in China (Qi et al., 2011), but new CRKP lineages are continuously emerging. We, therefore, analyzed all available genomes of ST29 and found that this ST is commonly associated with the carriage of extended-spectrum β-lactamase-encoding genes rather than carbapenemases genes.

MATERIALS AND METHODS
The Study, the Strain, and Susceptibility Testing Strain 130002 was recovered from the blood of an ICU patient in 2020 at West China Hospital as part of routine care. MICs of aztreonam, ceftazidime, ceftazidime-avibactam, cefepime, ertapenem, imipenem, meropenem, piperacillin-tazobactam, and The allele differences are highlighted.
colistin were determined using the broth microdilution method of the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2020). This study has been approved by the Ethical Committee of West China Hospital without the requirement of an informed consent due to the fact that no patient information is needed.

Short-and Long-Read Whole Genome Sequencing and Analysis
Strain 130002 was subjected to whole genome sequencing using both a HiSeq X10 sequencer (Illumina; San Diego, CA, United States; 200×) and a MinION Sequencer (Nanopore; Oxford, United Kingdom). Genomic DNA was prepared using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Both short (Illumina) and long (Nanopore) reads were utilized to generate a de novo hybrid assembly using Unicycler (Wick et al., 2017) and Pilon (Walker et al., 2014). Sequence type (ST) was determined by querying the multilocus sequence typing database 1 , while capsule (KL) typing was performed using Kleborate (Wyres et al., 2016). Antimicrobial resistance genes were identified from the genome sequences using the ABRicate program 2 to query the ResFinder database 3 . Replicon sequence types of IncF plasmids were determined using the pMLST 4 . Plasmid comparison was performed using BRIG (Alikhan et al., 2011) in the default settings. Insertion sequences were identified using ISFinder 5 .

Protein Analysis
The secondary structure of OXA-926 β-lactamase was predicted using the neural network based web service JPred4 (Drozdetskiy et al., 2015) with the default settings. The origin of OXA-926 was investigated using BlastP 6 .

Phylogenomic Analysis of the ST29 Complex
All complete and draft genomes of K. pneumoniae belonging to the ST29 complex including ST29 and its single-allele variants, i.e., ST193, 465, 711, 723, 985, 1161, and 1271 (the allele profile of these STs is shown in Table 1) were retrieved from GenBank including the SRA database (accessed during May 2020). The genome sequences were mapped against the complete genome of 130002 for single nucleotide polymorphisms (SNP) calling using Snippy v4.6.0 7 with the default settings. Gubbins v2.4.1 (Croucher et al., 2015) was used for recombination filtering prior to the phylogenomic reconstruction using RAxML v8.2.12 (Stamatakis, 2014) under the GTRGAMMA model and a 1,000-bootstrap test. Trees were annotated and viewed in iTOL v5.7 (Letunic and Bork, 2019) and FigTree v1.4.4 8 . As there are up to 1,048 SNPs between the genomes of ST29 and its single-allele variants (see below), suggesting a significant phylogenetic divergence, we, therefore, did not include the double-allele variants of ST29 for analysis. Strain 130002 was resistant to aztreonam, ceftazidime, cefepime, ertapenem, imipenem, meropenem, and piperacillin-tazobactam but was susceptible to ceftazidime-avibactam and was intermediate to colistin (Table 2). The complete genome sequence of strain 130002 was obtained by de novo hybrid assembly of both short (Illumina) and long (Nanopore) reads and had a 5.3-Mb circular chromosome with three plasmids (  Frontiers in Microbiology | www.frontiersin.org genomes were identified and strains of the ST29 complex have been identified in all continents but Antarctica (Supplementary Datasheet 1). Most strains (57.3%, 90/157) of the ST29 complex carried genes encoding extended-spectrum β-lactamases (ESBL), in particular, bla CTX−M−15 (n = 69), while only four strains had carbapenemase genes (bla KPC−2 , bla KPC−3 , or bla IMP−19 ; Supplementary Datasheet 1). Strains of the ST29 complex were assigned to 13 capsular types (KL2,10,19,24,28,30,31,33,54,62,63,107,and 113; Figure 1), while 130002 is the only strain of KL62. There was a maximum of 1,048 SNPs between strains of the ST29 complex (Supplementary Datasheet 2), suggesting that the complex is diverse in clonal background. Strain 130002 had a range of 258-904 SNPs with other strains of the ST29 complex (Supplementary Datasheet 2) and is most closely related to ST29 KL54 strain 4300STDY6470438 (accession no. ERR2397540) recovered from an unspecified human sample in Thailand in 2016 (Figure 1).

Retrieval of bla
130002 Has a bla KPC−2 and bla OXA Gene Encoding a Novel Narrow-Spectrum β-Lactamase OXA-926 Strain 130002 contains three β-lactamase-encoding genes including narrow-spectrum β-lactamase gene bla SHV−187 (Tian et al., 2020) on chromosome, carbapenemase gene bla KPC−2 on a 94.9-kb IncFII plasmid of the Y6:A-:B-type (designated pKPC2_130002), and a novel bla OXA gene on a 117.8-kb plasmid containing both IncFIA and IncFII replicons (designated pOXA926_130002; Table 3). pKPC2_130002 shows the closest similarity among the sequenced plasmids to pKPC2_020019 (accession no. CP028554), an 89.7-kb Y6:A-:Btype bla KPC−2 -carrying plasmid from a Klebsiella variicola strain recovered from a hospital of a neighboring city (Meishan, 80 km away from Chengdu) in 2017, with a 94% coverage and 99.93% identity (Figure 2). On pKPC2_130002 and pKPC2_020019, bla KPC−2 is located in a non-Tn4401 element containing a transposase-encoding tnpA gene of an unnamed transposon of the Tn3 family at upstream and another tnpA of an unnamed transposon of the TnAs1 family at downstream (Figure 2). Transconjugants containing bla KPC−2 were obtained at a frequency of 1 × 10 −4 (transconjugant per recipient), illustrating that pKPC2_130002 is readily self-transmissible. The findings above show that strain 130002 emerged as a CRKP by acquiring a self-transmissible bla KPC−2 -carrying plasmid.
The bla OXA gene encodes an OXA enzyme that shows the closest similarity to OXA-459 with a 59.1% amino acid (aa) identity (143/242 aa) and 90.3% coverage (242/268 aa) among all known OXA enzymes in the Bacterial Antimicrobial Resistance FIGURE 2 | Alignment of pKPC2_130002 with pKPC2_020019. pKPC2_130002 is a 94.9-kb IncFII plasmid of the Y6:A-:B-type and has bla KPC−2 . pKPC2_130002 is the closest to pKPC2_020019 (accession no. CP028554), an 89.7-kb Y6:A-:B-type bla KPC−2 -carrying plasmid from a K. variicola strain recovered in a hospital of a neighbor city (Meishan, 80 km away from Chengdu) in 2017, with a 94% coverage and 99.93% identity. Comparing with pKPC2_130002, pKPC2_020019 lacks several protein-encoding genes with the products being indicated. On pKPC2_130002 and pKPC2_020019, bla KPC−2 is located in a non-Tn4401 element containing a transposase-encoding tnpA gene of an unnamed transposon of the Tn3 family at upstream and another tnpA of an unnamed transposon of the TnAs1 family at downstream. This comparison was performed using BRIG (Alikhan et al., 2011) in the default settings.
Reference Gene Database 9 . OXA-459 is one of the OXA-114like enzymes intrinsic to Achromobacter spp. The findings above suggest that this OXA is a novel enzyme and is assigned OXA-926 by the Pathogen Detection group of GenBank, National Center for Biotechnology Information. OXA enzymes are very diverse in amino acid sequences and can be assigned to various subfamilies (Evans and Amyes, 2014;Yoon and Jeong, 2021). A ≥73.1% amino acid identity has been recently proposed as the cutoff to define OXA subfamilies (Yoon and Jeong, 2021) and, therefore, OXA-926 represents a novel subfamily. The secondary structure of OXA-926 contains seven α helixes, six β sheets, and four 3 10 -helixes (Figure 3).
bla OXA−926 was successfully cloned into vector pET28a, generating pET28a-OXA926. Among the β-lactams tested, only the MICs of piperacillin, piperacillin-tazobactam, and cephalothin for the transformant containing pET28a-OXA926 (BL21::pET28a-OXA926) were increased by ≥four-fold as compared with those for the transformant containing pET-28a (BL21::pET28a) ( Table 2). This suggests that OXA-926 exhibits activity against piperacillin and such activities cannot be inhibited by tazobactam, a class A (not class D) β-lactamase inhibitor. As avibactam is a non-β-lactam β-lactamase inhibitor able to inhibit classes A, C, and D β-lactamases, we, therefore, determined the MICs of piperacillin in the presence of 4 mg/L of avibactam. In the presence of avibactam, MIC of piperacillin decreased from 32 to 4 mg/L for BL21::pET28a-OXA926 but was still 8-fold of that 9 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA313047 for BL21::pET28a (0.5 mg/L). This suggests that avibactam is able to provide protection for β-lactams from the hydrolysis OXA-926 to a certain extent but cannot fully inhibit OXA-926.
pOXA926_130002 has a K4 IncFII allele and a novel IncFIA allele closest to the FIA_10 type with a 98.7% identity (378/383 nucleotides). pOXA926_130002 was the closest, with a 56% coverage and 97.1% identity (Figure 4), to pRHBSTW-00167_2 [accession no. CP058119; containing a K4 IncFII allele and a FIA_10 type IncFIA allele (K4:A10:B-) plus an IncR replicon] of Klebsiella michiganensis strain RHBSTW-00167 recovered from freshwater in the United Kingdom in 2017. By Blast, there is only one bla OXA−926 -carrying plasmid, p15WZ-82_res (accession no. CP032357), with a complete sequence available in GenBank (Table 4). p15WZ-82_res was recovered from a K. variicola clinical strain in an unspecified place in China in 2015. This plasmid contains a K4 IncFII allele and a K (pCAV1099-114 type) IncFIB allele but no IncFIA allele (K4:A-:B-; pCAV1099-114 type IncFIB was not included in the IncF pMLST scheme). pOXA926_130002 had a 53% coverage and 99.0% identity with p15WZ-82_res (Figure 4). Transconjugants containing both bla KPC−2 and bla OXA−926 were obtained but those containing bla OXA−926 alone were not, suggesting that pOXA926_130002 was not self-transmissible. Nonetheless, pOXA926_130002 was able to be transferred in the presence of pKPC2_130002 at a frequency of 1 × 10 −6 (transconjugant per recipient). On pOXA926_130002, there are no mobile genetic elements including insertion sequences and transposons present in the immediate upstream and downstream of bla OXA−926 .
FIGURE 3 | Secondary structure of OXA-926. The secondary structure was predicted using the neural network-based web service JPred4 (Drozdetskiy et al., 2015) with the default settings. Secondary structure elements, α helixes, β sheets, and 3 10 -helixes (representing by η), are indicated. β-strands are rendered as arrows, and strict αand β-turns are shown as TTT and TT letters, respectively. FIGURE 4 | Alignment of pOXA926_130002 with RHBSTW-00167_2 and p15WZ-82_res. pOXA926_130002 is a 117.8-kb IncF plasmid and has bla OXA−926 . Plasmid replication initiation-encoding genes repA of the IncFII replicon and repE of the IncFIA replicon and the conjugation module containing multiple genes (traA to traX and trbB to trbI) are indicated. A gene encoding the transpose of an IS30 family insertion sequence at upstream of bla OXA−926 and a gene encoding a tyrosine recombinase-like integrase at downstream are also shown. pOXA926_130002 is the closest (56% coverage and 97.1% identity) to pRHBSTW-00167_2 (accession no. CP058119), a 223.8-kb K4:A10:B-type IncF plasmid with an additional IncR replicon of an K. michiganensis strain recovered in United Kingdom in 2017. pRHBSTW-00167_2 has no bla OXA−926 , while p15WZ-82_res (accession no. CP032357) is another bla OXA−926 -carrying plasmid of a K. variicola clinical strain recovered from an unspecified place in China in 2015. p15WZ-82_res belongs to the K4:A-:B-IncF type and also contains a K (pCAV1099-114 type) IncFIB allele. pOXA926_130002 had a 53% coverage and 99.0% identity with p15WZ-82_res.
Nonetheless, an unnamed novel insertion sequence of the IS30 family was found 3,137 bp upstream of bla OXA−926 but no additional insertion sequences of the IS30 family to form a composite transposon were present on pOXA926_130002 (Figure 4). A tyrosine recombinase-like integrase-encoding gene was present 4,984 bp downstream of bla OXA−926 (Figure 4) but no crossover sites (recombination sites), which are required for recombination mediated by this type of integrases are found. No additional tyrosine recombinase-like integrase-encoding gene was present upstream of bla OXA−926 . Therefore, the mechanism for mobilizing bla OXA−926 remains to be elucidated and warrants further study. In GenBank, bla OXA−926 was found in one plasmid of Klebsiella variicola (GenBank accession no. CP032357) and seven Klebsiella draft genomes, including four K. pneumoniae and three K. variicola ( Table 4). Four of the eight Klebsiella strains have detailed information available, revealing that the strains were recovered from China and Japan as far back as 2008. In addition, a variant of bla OXA−926 encoding an OXA enzyme with 97.01% (260/268) aa identity with OXA-926 was found in one Klebsiella michiganensis from Malaysia and two K. variicola from Brazil ( Table 4). These findings suggest that bla OXA−926 has been circulating in Klebsiella spp. for more than a decade and has spread to multiple countries.