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Coexistence of tmexCD-toprJ, bla_NDM-1, and bla_IMP-4 in One Plasmid Carried by Clinical Klebsiella spp.

ABSTRACT: In clinical practice, carbapenems and tigecycline are considered significant options for treating infections caused by multidrug-resistant Klebsiella spp. The continual evolution of resistance mechanisms to carbapenems and tigecycline is shattering the present condition. Meanwhile, convergence of the two resistance mechanisms in a single strain has been reported repeatedly, posing a significant threat to public health and safety. In this study, two carbapenem- and tigecycline-resistant Klebsiella species were obtained from patients and investigated using antimicrobial susceptibility testing, conjugation assay, whole-genome sequencing, and bioinformatics analysis. In Klebsiella variicola FK2020ZBJ35, an untransferable multidrug IncFIB(Mar)/IncHI1B-like plasmid carrying tmexCD2-toprJ2, bla_IMP-4, and bla_NDM-1 was discovered, as was a similar plasmid carrying tmexCD1-toprJ1 and bla_IMP-4 in Klebsiella quasipneumoniae 2019SCSN059. Genetic context analysis found that two distinct tmexCD-toprJ variants were detected in comparable mobile units with genetic array int-int-hp-hp-tnfxB-tmexCD-toprJ and integrated into separate genetic locations. bla_IMP-4 and bla_NDM-1 were carried by an integron In1377 and a truncated Tn3000, respectively. These findings revealed that the carbapenem and tigecycline resistance genes carried by the two strains were located on mobile elements and might potentially transmit horizontally to additional strains. Furthermore, our findings showed that IncFIB(Mar)/IncHI1B-like plasmids represent a significant reservoir of essential resistance genes that warrants continued monitoring. IMPORTANCE Tigecycline is an essential antibiotic that is used to treat infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). The emergence of high-level tigecycline-resistant CRKP poses a serious hazard to human health. This work screened two tigecycline-resistant CRKP strains from clinical patients and found a type of plasmid that encoded carbapenemase and TmexCD-ToprJ in Klebsiella. Importantly, one plasmid cocarried tmexCD-toprJ, bla_NDM-1, and bla_IMP-4, hinting that this plasmid could be a critical vector for superbug development. Furthermore, we discovered that the carbapenem and tigecycline resistance genes are located in mobile units by genetic structure analysis. Our research tracks the formation of clinically super-resistant Gram-negative bacteria.

SUBMITTER: Xiao T

PROVIDER: S-EPMC9241619 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Carbapenemase is the predominant enzyme in the mechanism leading to Enterobacterales resistance to carbapenems, but only a limited number of isolates harbor double classes/types of carbapenemase. Here, an IMP-4 and NDM-1 producer named Klebsiella michiganensis 7525 is reported, and the co-harboring plasmid is further characterized. K. michiganensis 7525 was positive for the blaIMP-4 and blaNDM-1 genes by the NG-Test Carba-5 method and PCR followed by sequencing, and both were located on the same plasmid (designated pKOX7525_1) according to S1-PFGE with Southern blot experiments. pKOX7525_1 was capable of transconjugation with an efficiency of 4.3 × 10-8 in a filter mating experiment. Whole-genome sequencing and bioinformatics analysis confirmed that the plasmid was novel, clustered to the incompatibility type of IncHIB/IncFIA/IncR and presented high similarity to a blaIMP-4-carrying IncHIB plasmid (pA) published with 79% coverage and 100% sequence identify. In contrast, a large-fragment insertion and inversion mediated by IS26 was observed on the plasmid, which introduced a genetic hybrid zone with multiple resistance genes, including blaNDM-1, to the plasmid. In the transconjugants, the presence of pKOX7525_1 had a negative impact on bacterial fitness. In vitro evolution experiments showed that pKOX7525_1 in the transconjugant could not be stably inherited after 10 days of passage and that blaNDM-1 could be lost during repeated laboratory passage. Our study not only reports a novel plasmid co-harboring blaIMP-4 and blaNDM-1 but also highlights the putative pathway of plasmid formation and evolution by means of genetic rearrangement through sequence insertion and homologue recombination, which may have critical value for plasmid research and increase awareness of carbapenem-resistant Enterobacteriaceae (CRE). IMPORTANCE In this study, we characterized a novel plasmid from a carbapenem-resistant K. michiganensis (CRKM) isolate, which harbors two metallo-β-lactamases (MBLs), IMP-4 and NDM-1, is capable of transconjugation and contains three replicons. Our results first expand the diversity of plasmids co-harboring carbapenemase genes in Enterobacterales, which exhibits epidemic importance in bacterial resistance. Additionally, we investigated the origin and formation of this MBL double-positive plasmid based on comparative genomics analysis, which indicated that IS26 plays a vital role through continuous genetic rearrangements. Moreover, this plasmid is unstable in transconjugants during passage at the multidrug-resistant (MDR) region of blaNDM-1, with fluctuating stability under varying antibiotic selection, highlighting auspicious considerations regarding recognition of the complexity and plasticity of plasmids in evolution and re-emphasizing clinical infection control inspired by CRE.

Project description:The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly bla NDM-1 and bla KPC-2 Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes bla NDM-1 and bla KPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the bla NDM-1 and virulence genes and a bla KPC-2-carrying plasmid.IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

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Coexistence of tmexCD-toprJ, bla_NDM-1, and bla_IMP-4 in One Plasmid Carried by Clinical Klebsiella spp.

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Coexistence of tmexCD-toprJ, blaNDM-1, and blaIMP-4 in One Plasmid Carried by Clinical Klebsiella spp.

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OmicsDI is part of the ELIXIR infrastructure

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Coexistence of tmexCD-toprJ, bla_NDM-1, and bla_IMP-4 in One Plasmid Carried by Clinical Klebsiella spp.