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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., Competing Interests: The authors declare no conflict of interest.

Carbapenem-hydrolysing enzymes belonging to the OXA-48-like group are encoded by bla OXA-48 -like alleles and are abundant among Enterobacterales in the Netherlands. Therefore, the objective here was to investigate the characteristics, gene content and diversity of the bla OXA-48 -like carrying plasmids and chromosomes of Escherichia coli and Klebsiella pneumoniae collected in the Dutch national surveillance from 2014 to 2019 in comparison with genome sequences from 29 countries. A combination of short-read genome sequencing with long-read sequencing enabled the reconstruction of 47 and 132 complete bla OXA-48 -like plasmids for E. coli and K. pneumoniae , respectively. Seven distinct plasmid groups designated as pOXA-48-1 to pOXA-48-5, pOXA-181 and pOXA-232 were identified in the Netherlands which were similar to internationally reported plasmids obtained from countries from North and South America, Europe, Asia and Oceania. The seven plasmid groups varied in size, G+C content, presence of antibiotic resistance genes, replicon family and gene content. The pOXA-48-1 to pOXA-48-5 plasmids were variable, and the pOXA-181 and pOXA-232 plasmids were conserved. The pOXA-48-1, pOXA-48-2, pOXA-48-3 and pOXA-48-5 groups contained a putative conjugation system, but this was absent in the pOXA-48-4, pOXA-181 and pOXA-232 plasmid groups. pOXA-48 plasmids contained the PemI antitoxin, while the pOXA-181 and pOXA-232 plasmids did not. Furthermore, the pOXA-181 plasmids carried a virB2-virB3-virB9-virB10-virB11 type IV secretion system, while the pOXA-48 plasmids and pOXA-232 lacked this system. A group of non-related pOXA-48 plasmids from the Netherlands contained different resistance genes, non-IncL-type replicons or no replicons. Whole genome multilocus sequence typing revealed that the bla OXA-48 -like plasmids were found in a wide variety of genetic backgrounds in contrast to chromosomally encoded bla OXA-48 -like alleles. Chromosomally localized bla OXA-48 and bla OXA-244 alleles were located on genetic elements of variable sizes and comprised regions of pOXA-48 plasmids. The bla OXA-48 -like genetic element was flanked by a direct repeat upstream of IS1R, and was found at multiple locations in the chromosomes of E. coli . Lastly, K. pneumoniae isolates carrying bla OXA-48 or bla OXA-232 were mostly resistant for meropenem, whereas E. coli bla OXA-48 , bla OXA-181 and chromosomal bla OXA-48 or bla OXA-244 isolates were mostly sensitive. In conclusion, the overall bla OXA-48 -like plasmid population in the Netherlands is conserved and similar to that reported for other countries, confirming global dissemination of bla OXA-48 -like plasmids. Variations in size, presence of antibiotic resistance genes and gene content impacted pOXA-48, pOXA-181 and pOXA-232 plasmid architecture.

Bailong Hou,1,* Ying Zhou,2,* Wei Wang,1 Weifeng Shen,1 Qinlong Yu,1 Minjie Mao,1 Siheng Wang,1 Wenxiu Ai,3 Fangyou Yu,2,3 Pingyang Shao1 1Department of Clinical Laboratory Medicine, The First Hospital of Jiaxing, The Affiliated Hospital of Jiaxing University, Jiaxing, 314000, People’s Republic of China; 2Department of Clinical Laboratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200000, People’s Republic of China; 3Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Pingyang Shao, Department of Clinical Laboratory Medicine, The First Hospital of Jiaxing, The Affiliated Hospital of Jiaxing University, Jiaxing, 314000, People’s Republic of China, Email spy202001@163.com Fangyou Yu, Department of Clinical Laboratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200000, People’s Republic of China, Email wzjxyfy@163.comIntroduction: This study aimed to investigate the emergence and characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains that demonstrate resistance to multiple antibiotics, including aminoglycosides and tigecycline, in a Chinese hospital.Methods: A group of ten CRKP strains were collected from the nine patients in a Chinese hospital. Antimicrobial Susceptibility Testing (AST) and phenotypic inhibition assays precisely assess bacterial antibiotic resistance. Real-time quantitative PCR (RT-qPCR) was used to analyze the mRNA levels of efflux pump genes (acrA/acrB and oqxA/oqxB) and the regulatory gene (ramA). The core-genome tree and PFGE patterns were analyzed to assess the clonal and horizontal transfer expansion of the strains. Whole-genome sequencing was performed on a clinical isolate of K. pneumoniae named Kpn20 to identify key resistance genes and antimicrobial resistance islands (ARI).Results: The CRKP strains showed high resistance to carbapenems, aminoglycosides (CLSI, 2024), and tigecycline (EUCAST, 2024). The mRNA expression levels of efflux pump genes and regulatory genes were detected by RT-qPCR. All 10 isolates had significant differences compared to the control group of ATCC13883. The core-genome tree and PFGE patterns revealed five clusters, indicating clonal and horizontal transfer expansion. Three key resistance genes (blaoxa-232, blaCTX-M-15, and rmtF) were observed in the K. pneumoniae clinical isolate Kpn20. Mobile antibiotic resistance islands were identified containing blaCTX-M-15 and rmtF, with multiple insertion sequences and transposons present. The coexistence of blaoxa-232 and rmtF in a high-risk K. pneumoniae strain was reported. Conjugation assay was utilized to investigate the transferability of blaoxa-232-encoding plasmids horizontally.Conclusion: The study highlights the emergence of ST15-KL112 high-risk CRKP strains with multidrug resistance, including to aminoglycosides and tigecycline. The presence of mobile ARI and clonal and horizontal transfer expansion of strains indicate the threat of transmission of these strains. Future research is needed to assess the prevalence of such isolates and develop effective control measures.Keywords: blaOXA-232, rmtF, Klebsiella pneumoniae, carbapenemase, plasmid

Prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has compromised antimicrobial efficacy against severe infections worldwide. To monitor global spread, we conducted a comprehensive genomic epidemiologic study comparing sequences from 21 blaOXA-232–carrying CRKP isolates from China with K. pneumoniae sequence type (ST) 15 strains from 68 countries available in GenBank. Phylogenetic and phylogeographic analyses revealed all blaOXA-232–carrying CRKP isolates belonged to ST15 lineage and exhibited multidrug resistance. Analysis grouped 330 global blaOXA-232–carrying ST15 CRKP strains into 5 clades, indicating clonal transmission with small genetic distances among multiple strains. The lineage originated in the United States, then spread to Europe, Asia, Oceania, and Africa. Most recent common ancestor was traced back to 2000; mutations averaged ≈1.7 per year per genome. Our research helps identify key forces driving global spread of blaOXA-232–carrying CRKP ST15 lineage and emphasizes the importance of ongoing surveillance of epidemic CRKP.

The oxacillinase-48 (OXA-48)-like carbapenemases are class D β-lactamases and increasingly reported in Enterobacterial species. The detection of these carbapenemases is challenging and little information is available on the epidemiology and plasmid characteristics of OXA-48-like carbapenemase producers. We detected the presence of OXA-48-like carbapenemases in 500 clinical isolates of Escherichia coli and Klebsiella pneumoniae, followed by detection of other carbapenemases, extended spectrum β-lactamases (ESBLs) and 16S rRNA methyltransferases in OXA-48 producers. Clonal relatedness was studied using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Finally, plasmid characterisation was performed through conjugation experiment, S1-PFGE and Southern hybridisation. Around 40% of E. coli and K. pneumoniae isolates harboured OXA-48-like β-lactamases. Two OXA-48 allele variants, OXA-232 and OXA-181 were detected in our study. OXA-48 producers co-harbored diverse drug-resistant genes belonging to other classes of carbapenemases, ESBLs and 16S rRNA methyltransferases. OXA-48-like carbapenemase producers exhibited high clonal diversity. BlaOXA-48 carrying plasmids were conjugative, untypable and their size was ~ 45 kb and ~ 104.5 kb in E. coli and K. pneumoniae respectively. In conclusion, OXA-48-like carbapenemases have emerged as major cause of carbapenem resistance in Enterobacteriaceae and probably still being under reported. Strict surveillance and adequate detection methods are needed to prevent the dissemination of OXA-48-like carbapenemases. [ABSTRACT FROM AUTHOR]

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.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains and restricted therapeutic options pose a global threat to public health. Aminoglycosides are a wise choice, which can effectively reduce the mortality rate when combined with β-lactam drugs. However, in this study, we identified a ST15-KL112 CRKP FK3006 which not only exhibited resistance to carbapenems, but also exhibited high level resistance to aminoglycosides. In addition to the multidrug resistant phenotype, FK3006 also owned typical pathogenic characteristic, including hypermucoviscosity and hypervirulence phenotypes. According to the whole-genome sequencing, one pLVPK-like virulence plasmid, and three key resistant plasmids (blaOXA-232, blaCTX-M-15, and rmtF) were observed in FK3006. Compared to other typical ST15 CRKP, the presence of pLVPK-like virulence plasmid (p3006-2) endowed the FK3006 with high virulence features. High siderophore production, more cell invasive and more resistant to serum killing was observed in FK3006. The Galleria mellonella infection model also further confirmed the hypervirulent phenotype of FK3006 in vivo. Moreover, according to the conjugation assay, p3006-2 virulence plasmid also could be induced transfer with the help of conjugative IncFIIK p3006-11 plasmid (blaCTX-M-15). In addition to the transmissible plasmid, several insertion sequences and transposons were found around blaCTX-M-15, and rmtF to generate the mobile antimicrobial resistance island (ARI), which also make a significant contribution to the dissemination of resistant determinants. Overall, we reported the uncommon co-existence of blaOXA-232, rmtF-encoding plasmids, and pLVPK-like virulence plasmid in ST15-KL112 K. pneumoniae. The dissemination threatens of these high-risk elements in K. pneumoniae indicated that future studies are necessary to evaluate the prevalence of such isolates. [ABSTRACT FROM AUTHOR]

Objectives: The occurrence of OXA-232-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) has been increasing in China during the last five years. The blaOXA-232-carrying CRKP strain's clonal propagation can readily lead to nosocomial epidemics. Here, we report the genome sequence of an OXA-232 and CTX-M-15 co-producing K. pneumoniae strain isolated from a gallbladder infection in China. Methods: The genome sequence of K. pneumoniae S105 was determined using the Illumina NovaSeq 6000 platform. Antimicrobial resistance genes (ARGs), multilocus sequence typing (MLST) and plasmid replicons were identified using the BacWGSTdb server. The phylogenetic relationship between S105 and other K. pneumoniae strains was analysed using the core genome multilocus sequence typing (cgMLST) strategy. Results: The genomic sequence of K. pneumoniae S105 is made up of 111 contigs with a total length of 5 748 752 bp. According to the Pasteur MLST scheme, S105 belongs to sequence type (ST) 15. Fifteen ARGs were discovered in the genome, including the beta-lactam resistance genes blaOXA-232 and blaCTX-M-15. The blaOXA-232 gene was located in a ColKP3 plasmid. KL112 was anticipated to be the capsule and lipopolysaccharide serotype. A total of 73 phylogenetically related strains were found from 19 nations across four continents; 22 of them were from China, with 21 strains harbouring the blaOXA-232 gene, and the majority of them diverged by just 6–37 cgMLST alleles. Conclusion: In summary, we reported the genomic sequencing of a K. pneumoniae ST15 clinical strain co-carrying the blaOXA-232 and blaCTX-M-15 genes. The clonal dissemination of OXA-232-producing K. pneumoniae ST15 strains in China needs our attention.

Qingyu Shi,1,2 Renru Han,1,2 Yan Guo,1,2 Yonggui Zheng,1,2 Yang Yang,1,2 Dandan Yin,1,2 Rong Zhang,3 Fupin Hu1,2 1Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, People’s Republic of China; 2Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, People’s Republic of China; 3Department of Clinical Laboratory Medicine, Second Affiliated Hospital of Zhejiang University, Hangzhou, People’s Republic of ChinaCorrespondence: Fupin Hu Email hufupin@fudan.edu.cnBackground: RmtF, as 16S rRNA methyltransferase, leads to high-level resistance to aminoglycoside and is now barely reported.Methods and Results: Three rmtF-positive Klebsiella pneumoniae isolates, belonging to the pandemic clone sequence type 15, were isolated from children and coproduced blaOXA-232 and blaCTX-M-15. The rmtF gene was located on an IncFIB transformable plasmid of 128,536-bp and blaOXA-232 was on a 6141-bp ColKP3 plasmid, respectively.Conclusion: Plasmids with rmtF found worldwide, shared relatively low similarity, and merely matched partly in their multidrug resistance region. Notably, clinical isolates coproducing rmtF and blaOXA-232 are gradually increasing in China.Keywords: Klebsiella pneumoniae, RmtF, blaOXA-232, ST15, aminoglycoside

Background: Recently, in India, there has been a shift from NDM to OXA48-like carbapenemases. OXA-181 and OXA-232 are the frequently produced variants of OXA48-like carbapenemases. OXA48-like carbapenemases are also known to be carried on transposons such as Tn1999, Tn1999.2 and it is also associated with IS1R carried on Tn1999. In India, there are no previous reports studying the association of mobile genetic elements (MGEs) with OXA48-like carbapenemases. The present study was aimed at determining the genetic backbone of OXA48-like carbapenemases to determine the role of MGEs in its transfer and to investigate the Inc plasmid type carrying blaOXA48-like. Results: A total of 49 carbapenem resistant K. pneumoniae which included 25 isolates from South India and 24 isolates from North India, were included in the study. Whole genome sequencing using Ion Torrent PGM was performed to study the isolates. OXA-232 was present in 35 isolates (71%). In 19 isolates (39%), blaOXA48-like was associated with MGEs. Insertion sequences such as ISX4, IS1, IS3, ISKpn1, ISKpn26, ISKpn25, ISSpu2, ISKox1, IS4321R, ISEc36, and ISPa38; and transposons such as TnAs3 and Tn2, were present. Isolates from northern and southern India belonging to same sequence type (ST) had diverse genetic backbone for blaOXA48-like. ST14 isolates from north had IS5 and Tn3 families while from south they had IS1, IS5 and IS630 families. ST231 from north had IS5, IS6 and Tn3 families with blaOXA-232 while from south, IS1, IS3 and IS5 families were observed; with ISKpn26 being present among isolates from both the regions. blaOXA48-like was predominantly found on ColKP3 plasmid. ST231 was the predominant ST in 22 isolates (45%). Conclusion: OXA-232 is the predominant variant of OXA48-like carbapenemase with ST231 being the commonest ST of OXA48-like carbapenemase producing K. pneumoniae in India. Diverse MGEs have been associated with both blaOXA-232 and blaOXA-181 which contribute to their spread. The MGEs in the present study are different from those reported earlier. There is no clonal expansion of blaOXA48-like producing K. pneumoniae since diverse STs were observed. Monitoring the genetic backbone of OXA48-like carbapenemase is essential to better understand the transmission dynamics of XDR K. pneumoniae. [ABSTRACT FROM AUTHOR]