A Nationwide Genomic Study of Clinical Klebsiella pneumoniae Carrying blaOXA-232 and rmtF in China

ABSTRACT OXA-232 carbapenemase is becoming a threat in China due to its high prevalence, mortality, and limited treatment options. However, little information is available on the impact of OXA-232-producing Klebsiella pneumoniae in China. This study aims to characterize the clonal relationships, the genetic mechanisms of resistance, and the virulence of OXA-232-producing K. pneumoniae isolates in China. We collected 81 OXA-232-producing K. pneumoniae clinical isolates from 2017 to 2021. Antimicrobial susceptibility testing was performed using the broth microdilution method. Capsular types, multilocus sequence types, virulence genes, antimicrobial resistance (AMR) determinants, plasmid replicon types, and single-nucleotide polymorphism (SNP) phylogeny were inferred from whole-genome sequences. OXA-232-producing K. pneumoniae strains were resistant to most antimicrobial agents. These isolates showed partial differences in susceptibility to carbapenems: all strains were resistant to ertapenem, while the resistance rates to imipenem and meropenem were 67.9% and 97.5%, respectively. Sequencing and capsular diversity analysis of the 81 K. pneumoniae isolates revealed 3 sequence types (ST15, ST231, and one novel ST [ST-V]), 2 K-locus types (KL112 and KL51), and 2 O-locus types (O2V1 and O2V2). The predominant plasmid replicon types associated with the OXA-232 and rmtF genes were ColKP3 (100%) and IncFIB-like (100%). Our study summarized the genetic characteristics of OXA-232-producing K. pneumoniae circulating in China. The results demonstrate the practical applicability of genomic surveillance and its utility in providing methods to prevent transmission. It alerts us to the urgent need for longitudinal surveillance of these transmissible lineages. IMPORTANCE In recent years, the detection rate of carbapenem-resistant K. pneumoniae has increased and represents a major threat to clinical anti-infective therapy. Compared with KPC-type carbapenemases and NDM-type metallo-β-lactamases, OXA-48 family carbapenemases are another important resistance mechanism mediating bacterial resistance to carbapenems. In this study, we investigated the molecular characteristics of OXA-232 carbapenemase-producing K. pneumoniae isolated from several hospitals to clarify the epidemiological dissemination characteristics of such drug-resistant strains in China.

Aminoglycosides are used for severe infections caused by Gram-negative organisms and have shown reliable efficacy in combination with carbapenems, which can effectively reduce mortality (17). The 16S rRNA methyltransferase (16SRMTase) genes, commonly found in Enterobacterales worldwide, confer high levels of resistance to aminoglycosides, including armA, npmA, and rmtA through rmtH. The most common 16SRMTase-encoding gene in China is rmtB, followed by armA (18). While rmtF was first identified in France in 2012 (19), it has subsequently been reported sporadically in the United States, India (20), the United Kingdom (20), China (21), and other regions successively (12). In recent years, relevant reports of rmtF have emerged, always accompanied by harboring of bla OXA-232 (9,22).
In the previous studies, bla OXA-232 is mainly associated with nosocomial outbreaks in many countries, including China (14,22), the United States (23), South Korea (24), and Brunei (9). Here, a comprehensive overview of the epidemiology of 81 OXA-232-Kp isolates in China was completed to identify the genomic characterization of bla OXA-232 and rmtF, including dominant STs, clonal distribution, and virulence, and with a particular focus on the emergence and dissemination of extended-spectrum b-lactamases (ESBLs), AmpC, and outer membrane porins (Omp).
Virulence genes. In total, four virulence features documented in the virulence factor database were detected in this study, namely, the yersiniabactin receptor gene (fyuA), the siderophore genes (irp1 and irp2), the yersiniabactin siderophore cluster (ybtAEPQSTUX), and the aerobactin siderophore biosynthesis protein (IucBCD). The distribution of these virulence genes varied between strains: fyuA, irp1, and irp2 and ybtAEPQSTUX were present in all STs strains, while IucBCD was present only in ST231. In short, the ST231 isolates had the highest number of virulence operons (four). The detection of virulence factors helps to understand the pathogenesis of different strains.
Clonal distribution and plasmid repertoire. Overall, there were three sequence types (STs) among the K. pneumoniae isolates tested in this study, ST15 (n = 66), ST231 (n = 14), and ST-V (n = 1). The minimum-spanning tree indicated that the clonal transmission characteristics of the OXA-232-KP isolates are relatively dispersed. For example, clonal transmission of ST231 (n = 14) occurred in one hospital from Kunming, whereas ST15 occurred mainly in Shanghai and Zhejiang, which are geographically close to each other ( Fig. 1). Two K-locus types, KL112 (67, 82.7%) and KL51 (14, 17.3%), were identified in this study. The correlation between capsular locus type and sequence type was also clarified by the minimum-spanning tree: KL51 with ST231 and KL112 with ST15. With regard to O antigen types, all isolates belonged to O1. The O locus types also showed an ST-specific distribution. In particular, O2v1 was found in ST15, whereas O2v2 was found in ST231. The predominant plasmid replicons present in the 81 K. pneumoniae isolates were ColKP3 (100%), IncFIB-like (100%), and IncFII (K) (85.2%). Other plasmid replicons detected were ColRNAI, repB, IncHI1B(pNDM-MAR), Col440I, IncFIA, IncHI2A, and IncHI2. In this study, we observed that both ST15 and ST231 isolates carried the ColKP3 plasmid replicon.
Genetic context surrounding bla OXA-232 and rmtF. The 81 isolates in our study all shared an identical OXA-232-carrying genetic context that was located within the truncated Tn2013-like insertion sequence (IS)-based transposition, with the best identity to a ColKP3 plasmid, pKNICU5 (KY454616), of 6,141 bp (Fig. 2a). As for the 16S rRNA methyltransferase gene rmtF, which is not in the same plasmid with OXA-232, the analysis results showed that rmtF is located on the IncFIB-type plasmid and is in tandem with arr-2, aacA4'-17, and catB at transposon Tn6867b. BLAST results showed that the plasmid containing the rmtF gene had the highest identity (.99%) with pE109-1-MDR (CM012200) (Fig. 2b). The only difference was that there was a point OXA-232 and rmtF in China Microbiology Spectrum mutation in the rmtF gene in strain ET39, causing an amino acid substitution (D249E), but the antimicrobial susceptibility of ET39 did not change significantly compared to other isolates. The introduction of some functional changes may require further investigation.

DISCUSSION
Following the first description of an OXA-232-producing K. pneumoniae isolate detected in a tertiary hospital in Shanghai, China, which caused a small-scale clonal dissemination, the emergence of OXA-232-KP has been reported (22,(25)(26)(27). At present, OXA-232-producing K. pneumoniae isolates are usually isolated from vulnerable populations such as neonates and elderly patients exposed to medical facilities and have occurred extensively in neonatal intensive care units and intensive care units (25,26,28). To date, there have been no large-scale genomic studies of isolates carrying OXA-232 in China, which is of great importance in understanding their emergence and distribution in different geographical regions for public health initiatives (13). According to previous reports, most OXA-48-like carbapenemases hydrolyze carbapenems poorly, while some OXA-48-like variants (such as OXA-163, OXA-247, and OXA-405) hydrolyze only extended-spectrum cephalosporins but not carbapenems (14). However, in association with the impaired permeability or the production of extended-spectrum b-lactamases, some OXA-48-like enzymes can confer high levels of carbapenem resistance, similar to OXA-232 (14,29).
Clonal lineages of K. pneumoniae differ in their ability to acquire resistance, virulence genes, and propensity to spread within hospital and community settings (30). The high prevalence of ST15 carbapenemase-producing Enterobacteriaceae (CPE) harboring bla OXA-232 was consistent with published data from China (14,22,31). There were two novel findings in this study. ST231 K. pneumoniae harboring bla OXA-232 was reported for the first time in China, and others have been reported in India, Switzerland, and Brunei (9,12,32). In addition, the novel ST (ST-V, gapA1-infB1-mdh-1-pgi8-phoE1-rpoB1-tonB447) was a single-locus variant of ST15 with variation at the tonB gene. KL51 was reported from the United States, Sweden, the United Kingdom (33), Thailand (34), and Lebanon (35). KL112 has been reported from Russia (36). Understanding such vectors carrying the resistance genes could help improve strategies to control the OXA-232 and rmtF in China Microbiology Spectrum spread of AMR (37). The association between the ColKP3 replicon and the bla OXA-232 gene has also been observed in ST231 or ST15 isolates around the world (37,38). To the best of our knowledge, plasmid pKNICU5 was isolated from an ST15 OXA-232producing K. pneumoniae isolate in a children's hospital in Shanghai in 2017, suggesting that the plasmid carrying OXA-232 has been widely disseminated in China in recent years (22).

Conclusions.
Our findings showed that OXA-232-producing K. pneumoniae has increased and spread to different geographical areas of China through interhospital and interregional dissemination of a few high-risk K. pneumoniae clones (ST15, ST231, and ST-V). The spread of bla OXA -like genes in China was due to the spread of the plasmid groups ColKP3, IncFIB, and IncFII (K).
Whole-genome sequencing and sequence analysis. DNA was extracted using a commercial midi kit (Qiagen, Hilden, Germany) according to the manufacture's recommendations. Sequencing libraries were prepared using the Nextera XT DNA library preparation protocol (Illumina, San Diego, CA, USA), and then the genomic DNA was subjected to Illumina short-read sequencing (150-bp paired-end reads). Reads were trimmed using Sickle (GitHub). Draft genome assemblies were generated using SPAdes 3.12.0. Further assembly was performed by mapping contigs on the reference plasmid sequences (.99% identical), and the assembly was confirmed using the PCR-based gap closure method. Antimicrobial resistance genes as well as multilocus sequence typing (MLST), mobile elements, outer  (44), CARD (45), and BIGSdb (46). Genome annotation was performed using RAST (47) and BLASTp/BLASTn (48) searches for open reading frames (ORFs) and pseudogenes. BRIG (BLAST Ring Image Generator) software was used to compare genetic context sequences (49).
Kleborate software was used to analyze K and O antigen types, virulence genes, and the integrative conjugative element (ICEKp) (50). The four major virulence loci were screened, including the siderophores yersiniabactin (ybt), aerobactin (iuc), and salmochelin (iro), the genotoxin colibactin (clb), and ICEKp, a virulence-associated mobile genetic element (MGE) of K. pneumoniae (51). The plasmid types in the CRE isolates were identified using SRST2 v0.2.0 with the PlasmidFinder (44) database from the Centre for Genomic Epidemiology, v2021-01-13. A maximum-likelihood phylogenetic tree was  Data availability. Sequence data were submitted to the National Centre for Biotechnology Information (PRJNA819578).