Comparative genomic analysis of plasmids harboring bla (OXA-48)-like genes in Klebsiella pneumoniae.

The emergence and spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a serious medical problem worldwide. Acquired OXA-48-like carbapenemases encoded by plasmids are important causes of carbapenem resistance in K. pneumoniae. To explore the links between plasmids and bla OXA-48-like genes in K. pneumoniae, we systematically analyzed the variants of bla OXA-48-like plasmid replicon types, phylogenetic patterns, geographic distribution, conjugative transfer regions, and the genetic environments surrounding bla OXA-48-like of 191 bla OXA-48-like-harboring plasmids, which were identified from 4451 plasmids of K. pneumoniae downloaded from GenBank. Our results showed that seven different variants of bla OXA-48-like genes were identified from the 191 bla OXA-48-like-harboring plasmids in K. pneumoniae, with bla OXA-48, bla OXA-232, and bla OXA-181 being highly prevalent. In K. pneumoniae, bla OXA-48 was mainly carried by the composite transposon Tn1999.2 located on IncL/M-type conjugative plasmids, which were mainly geographically distributed in Switzerland, Germany, and China. In K. pneumoniae, the blaOXA-232 gene was mainly carried by 6.1-kb ColKP3-type mobilizable plasmids, which were mainly isolated in India. In K. pneumoniae, bla OXA-181 was mainly carried by a group of 50-kb ColKP3-IncX3 hybrid conjugative plasmids and a group of small ColKP3-type mobilizable plasmids with lengths of 5.9-9.3 kb, the former was sporadically discovered in China, South Korea, India, and Czech Republic, while the latter was almost all isolated in India. In addition, five bla OXA-245-harboring 65.9-kb IncL plasmids of K. pneumoniae isolated in Spain were found to have the genetic context of bla OXA-245 more complicated than that of bla OXA-48-harboring IncL/M-type plasmids, with two copies of IS1R inserted both upstream and downstream of bla OXA-245-lysR. These findings enhance our understanding of the genetic diversity of bla OXA-48-like-harboring plasmids in K. pneumoniae.

Li W ，Guo H ，Gao Y ，Yang X ，Li R ，Li S ，Sun C ，Du W ，Chen S ，Xu P ，Huang W ，Shi J ，Yi X ，Li X ... -  《Frontiers in Cellular and Infection Microbiology》

Klebsiella Species and Enterobacter cloacae Isolates Harboring bla(OXA-181) and bla(OXA-48): Resistome, Fitness Cost, and Plasmid Stability.

Mahazu S ，Prah I ，Ota Y ，Hayashi T ，Nukui Y ，Suzuki M ，Hoshino Y ，Akeda Y ，Suzuki T ，Ishino T ，Ablordey A ，Saito R ... -  《Microbiology Spectrum》

In silico characterization of IncX3 plasmids carrying bla (OXA-181) in Enterobacterales.

Carbapenem-resistant Enterobacterales poses a global urgent antibiotic resistance threat because of its ability to transfer carbapenemase genes to other bacteria via horizontal gene transfer mediated by mobile genetic elements such as plasmids. Oxacillinase-181 (OXA-181) is one of the most common OXA-48-like carbapenemases, and OXA-181-producing Enterobacterales has been reported in many countries worldwide. However, systematic research concerning the overall picture of plasmids harboring bla OXA-181 in Enterobacterales is currently scarce. In this study, we aimed to determine the phylogeny and evolution of bla OXA-181-positive (gene encoding OXA-181) plasmids. To characterize the plasmids harboring bla OXA-181 in Enterobacterales, we identified 81 bla OXA-181-positive plasmids from 35,150 bacterial plasmids downloaded from the NCBI RefSeq database. Our results indicated that diverse plasmid types harbored bla OXA-181 but was predominantly carried by IncX3-type plasmids. We systematically compared the host strains, plasmid types, conjugative transfer regions, and genetic contexts of bla OXA-181 among the 66 bla OXA-181-positive IncX3 plasmids. We found that IncX3 plasmids harboring bla OXA-181 were mostly ColKP3-IncX3 hybrid plasmids with a length of 51 kb each and were mainly distributed in Escherichia coli and Klebsiella pneumoniae. Most of the IncX3 plasmids harboring bla OXA-181 were human origin. Almost all the bla OXA-181-positive IncX3 plasmids were found to carry genes coding for relaxases of the MOBP family and VirB-like type IV secretion system (T4SS) gene clusters, and all the 66 IncX3 plasmids were found to carry the genes encoding type IV coupling proteins (T4CPs) of the VirD4/TraG subfamily. Most IncX3 plasmids harbored both bla OXA-181 and qnrS1 in their genomes, and the two antibiotic resistance genes were found to a composite transposon bracketed by two copies of insertion sequence IS26 in the same orientation. Our findings provide important insights into the phylogeny and evolution of bla OXA-181-positive IncX3 plasmids and further address their role in acquiring and spreading bla OXA-181 genes in Enterobacterales.

Yu Z ，Zhang Z ，Shi L ，Hua S ，Luan T ，Lin Q ，Zheng Z ，Feng X ，Liu M ，Li X ... -  《Frontiers in Cellular and Infection Microbiology》

OXA-181-Like Carbapenemases in Klebsiella pneumoniae ST14, ST15, ST23, ST48, and ST231 from Septicemic Neonates: Coexistence with NDM-5, Resistome, Transmissibility, and Genome Diversity.

Studies on the epidemiology and genomes of isolates harboring OXA-48-like genes in septicemic neonates are rare. Here, isolates producing these carbapenemases which emerged and persisted in an Indian neonatal unit were characterized in terms of their resistome, transmissibility, and genome diversity. Antibiotic susceptibility and whole-genome sequencing were carried out. The sequence types, resistome, virulome, mobile genetic elements, and transmissibility of carbapenem-resistant plasmids were evaluated. Core genome analysis of isolates was shown in a global context with other OXA-48-like carbapenemase-harboring genomes, including those from neonatal studies. Eleven OXA-48-like carbapenemase-producing Klebsiella pneumoniae (blaOXA-181, n = 7 and blaOXA-232, n = 4) isolates belonging to diverse sequence types (ST14, ST15, ST23, ST48, and ST231) were identified. blaOXA-181/OXA-232 and blaNDM-5 were found in a high-risk clone, ST14 (n = 4). blaOXA-181/OXA-232 were in small, nonconjugative ColKP3 plasmids located on truncated Tn2013, whereas blaNDM-5 was in self-transmissible, conjugative IncFII plasmids, within truncated Tn125 Conjugal transfer of blaOXA-181/OXA-232 was observed in the presence of blaNDM-5 The study strains were diverse among themselves and showed various levels of relatedness with non-neonatal strains from different parts of the world and similarity with neonatal strains from Tanzania and Ghana when compared with a representative collection of carbapenemase-positive K. pneumoniae strains. We found that blaOXA-181/OXA-232-harboring isolates from a single neonatal unit had remarkably diverse genomes, ruling out clonal spread and emphasizing the extent of plasmid spreading across different STs. This study is probably the first to report the coexistence of blaOXA-181/232 and blaNDM-5 in neonatal isolates.IMPORTANCE Neonatal sepsis is a leading cause of neonatal mortality in low- and middle-income countries (LMICs). Treatment of sepsis in this vulnerable population is dependent on antimicrobials, and resistance to these life-saving antimicrobials is worrisome. Carbapenemases, enzymes produced by bacteria, can make these antimicrobials useless. Our study describes how OXA-48-like carbapenemases in neonatal septicemic Klebsiella pneumoniae shows remarkable diversity in the genomes of the strains and relatedness with strains from other parts of world and also to some neonatal outbreak strains. It is also the first to describe such resistance due to coproduction of dual carbapenemases, (OXA)-48 and New Delhi metallo-β-lactamase-5, in Klebsiella pneumoniae from neonatal settings. Carbapenemase genes situated on plasmids within high-risk international clones, as seen here, increase the ease and transfer of resistant genetic material. With the WHO treatment protocols not adequately poised to handle such infections, prompt attention to neonatal health care is required.

Naha S ，Sands K ，Mukherjee S ，Saha B ，Dutta S ，Basu S ... -  《mSphere》

Whole genome sequencing of OXA-232-producing wzi93-KL112-O1 carbapenem-resistant Klebsiella pneumoniae in human bloodstream infection co-harboring chromosomal ISEcp1-based bla (CTX-M-15) and one rmpA2-associated virulence plasmid.

To characterize one OXA-232-producing wzi93-KL112-O1 carbapenem-resistant Klebsiella pneumoniae (CRKP) co-harboring chromosomal bla CTX-M-15 and one rmpA2-associated virulence plasmid. Minimum inhibitory concentrations (MICs) were measured via broth microdilution method. Conjugation, chemical transformation, string test and Galleria mellonella infection model experiments were also conducted. Whole-genome sequencing (WGS) was performed on the Illumina and Nanopore platforms. Antimicrobial resistance determinants were identified using ABRicate program with ResFinder database. Insertion sequences (ISs) were identified using ISfinder. Bacterial virulence factors were identified using virulence factor database (VFDB). Wzi, capsular polysaccharide (KL) and lipoolygosaccharide (OCL) were analyzed using Kleborate with Kaptive. Phylogenetic analysis of 109 ST15 K. pneumoniae strains was performed using core genome multilocus sequence typing (cgMLST) on the Ridom SeqSphere+ server. MLST, replicons type, SNP strategies and another cgMLST analysis for 45 OXA-232-producing K. pneumoniae strains were further conducted using BacWGSTdb server. K. pneumoniae KPTCM strain belongs to ST15 with wzi93, KL112 and O1. It possessed a multidrug-resistant (MDR) profile and was resistant to carbapenems (meropenem and ertapenem), ciprofloxacin and amikacin. Virulence assays demonstrated KPTCM strain possesses a low virulence phenotype. WGS revealed it contained one circular chromosome and nine plasmids. The carbapenemase-encoding gene bla OXA-232 was located in a 6141-bp ColKP3-type non-conjugative plasmid and flanked by ΔISEcp1 and ΔlysR-ΔereA. Interestingly, bla CTX-M-15 was located in the chromosome mediated by ISEcp1-based transposon Tn2012. Importantly, it harbored a rmpA2-associated pLVPK-like virulence plasmid with iutA-iucABCD gene cluster and one IS26-mediated MDR fusion plasmid according to 8-bp (AGCTGCAC or GGCCTTTG) target site duplications (TSD). Based on the cgMLST and SNP analysis, data showed OXA-232-producing ST15 K. pneumoniae isolates were mainly isolated from China and have evolved in recent years. Early detection of CRKP strains carrying chromosomal bla CTX-M-15, OXA-232 carbapenemase and pLVPK-like virulence plasmid is recommended to avoid the extensive spread of this high-risk clone.

Tian C ，Xing M ，Zhao Y ，Fan X ，Bai Y ，Fu L ，Wang S ... -  《Frontiers in Cellular and Infection Microbiology》