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Genetic Characterization and Passage Instability of a Hybrid Plasmid Co-Harboring bla_IMP-4 and bla_NDM-1 Reveal the Contribution of Insertion Sequences During Plasmid Formation and Evolution.

ABSTRACT: 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 bla_IMP-4 and bla_NDM-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 bla_IMP-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 bla_NDM-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 bla_NDM-1 could be lost during repeated laboratory passage. Our study not only reports a novel plasmid co-harboring bla_IMP-4 and bla_NDM-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 bla_NDM-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.

SUBMITTER: Li X

PROVIDER: S-EPMC8672901 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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, blaIMP-4, and blaNDM-1 was discovered, as was a similar plasmid carrying tmexCD1-toprJ1 and blaIMP-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. blaIMP-4 and blaNDM-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, blaNDM-1, and blaIMP-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.

Project description:Providencia rettgeri has recently gained increased importance owing to the New Delhi metallo-β-lactamase (NDM) and other β-lactamases produced by its clinical isolates. These enzymes reduce the efficiency of antimicrobial therapy. Herein, we reported the findings of whole-genome sequence analysis and a comprehensive pan-genome analysis performed on a multidrug-resistant P. rettgeri 18004577 clinical strain recovered from the urine of a hospitalized patient in Shandong, China, in 2018. Providencia rettgeri 18004577 was found to have a genome assembly size of 4.6 Mb with a G + C content of 41%; a circular plasmid p18004577_NDM of 273.3 Kb, harboring an accessory multidrug-resistant region; and a circular, stable IncT plasmid p18004577_Rts of 146.2 Kb. Additionally, various resistance genes were identified in its genome, including bla NDM-1, bla OXA-10, bla PER-4, aph(3')-VI, ant(2'')-Ia, ant(3')-Ia, sul1, catB8, catA1, mph(E), and tet. Conjugation experiments and whole-genome sequencing revealed that the bla NDM-1 gene could be transferred to the transconjugant via the formation of pJ18004577_NDM, a novel hybrid plasmid. Based on the genetic comparison, the main possible formation process for pJ18004577_NDM was the insertion of the [ΔISKox2-IS26-ΔISKox2]-aph(3')-VI-bla NDM-1 translocatable unit module from p18004577_NDM into plasmid p18004577_Rts in the Russian doll insertion structure (ΔISKox2-IS26-ΔISKox2), which played a role similar to that of IS26 using the "copy-in" route in the mobilization of [aph(3')-VI]-bla NDM-1. The array, multiplicity, and diversity of the resistance and virulence genes in this strain necessitate stringent infection control, antibiotic stewardship, and periodic resistance surveillance/monitoring policies to preempt further horizontal and vertical spread of the resistance genes. Roary analysis based on 30 P. rettgeri strains pan genome identified 415 core, 756 soft core, 5,744 shell, and 12,967 cloud genes, highlighting the "close" nature of P. rettgeri pan-genome. After a comprehensive pan-genome analysis, representative biological information was revealed that included phylogenetic distances, presence or absence of genes across the P. rettgeri bacteria clade, and functional distribution of proteins. Moreover, pan-genome analysis has been shown to be an effective approach to better understand P. rettgeri bacteria because it helps develop various tailored therapeutic strategies based on their biological similarities and differences.

Project description:ObjectivesWe identified a novel hybrid plasmid simultaneously carrying bla NDM-1 and bla IMP-4 in an ST20-K28 carbapenem-resistant Klebsiella pneumoniae (CRKP) strain AZS099 and reported its detailed genetic and phenotypic characterization.MethodsAntimicrobial susceptibility was characterized using broth microdilution method. Complete genome characteristics and plasmid detailed analysis were carried out by PacBio Sequel and Illumina sequencing and further bioinformatics analysis. Conjugation assay, S1-PFGE, Southern blot, plasmid stability, and fitness cost were conducted to the phenotypic characterization of this novel hybrid plasmid.ResultsAZS099 was isolated from a blood specimen obtained from a 3-month baby who presented with biliary tract infection. Susceptibility testing showed that AZS099 was resistant to almost all β-lactams examined, including cephalosporins, combinations of β-lactams and β-lactamase inhibitors, carbapenems, and aztreonam. PacBio and Illumina sequencing together with S1-PFGE and Southern blot showed that bla NDM-1 and bla IMP-4 were simultaneously located on a 296 kb IncFIB(K)/IncHI1B/IncX3 plasmid (pAZS099-NDM-IMP), which consists of four main parts that came from four different types of plasmids. The region harboring bla IMP-4 is located in a class 1 integron designated as In0, which is located in an IS6100-IS26 transposon-like structure with a total length of ~5 kb. The region harboring bla NDM-1 is located in the Tn125 transposon remnant. Conjugation and transformation assay confirmed that the plasmid pAZS099-NDM-IMP has the potential for horizontal transfer and displayed high stability (retention rate > 95%). Furthermore, growth curve assessment confirmed that the presence of pAZS099-NDM-IMP exhibits no growth pressure on bacteria.ConclusionOur research reported a hybrid plasmid coharboring bla NDM-1 and bla IMP-4 in an ST20-K28 CRKP strain. The emergence of novel hybrid plasmid could threaten the control of antimicrobial resistance and should be closely supervised.

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Genetic Characterization and Passage Instability of a Hybrid Plasmid Co-Harboring bla_IMP-4 and bla_NDM-1 Reveal the Contribution of Insertion Sequences During Plasmid Formation and Evolution.

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Genetic Characterization and Passage Instability of a Hybrid Plasmid Co-Harboring blaIMP-4 and blaNDM-1 Reveal the Contribution of Insertion Sequences During Plasmid Formation and Evolution.

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Genetic Characterization and Passage Instability of a Hybrid Plasmid Co-Harboring bla_IMP-4 and bla_NDM-1 Reveal the Contribution of Insertion Sequences During Plasmid Formation and Evolution.