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Molecular Analysis of bla_KPC-2-Harboring Plasmids: Tn4401a Interplasmid Transposition and Tn4401a-Carrying ColRNAI Plasmid Mobilization from Klebsiella pneumoniae to Citrobacter europaeus and Morganella morganii in a Single Patient.

ABSTRACT: The spread of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales is a public health concern. KPC-encoding bla_KPC is predominantly spread by strains of a particular phylogenetic lineage, clonal group 258, but can also be spread by horizontal transfer of bla_KPC-carrying plasmids. Here, we report the transfer of a bla_KPC-2-harboring plasmid via mobilization from K. pneumoniae to Citrobacter freundii complex and Morganella morganii strains in a single patient. We performed draft whole-genome sequencing to analyze 20 carbapenemase-producing Enterobacterales strains (15 of K. pneumoniae, two of C. freundii complex, and three of M. morganii) and all K. pneumoniae strains using MiSeq and/or MinION isolated from a patient who was hospitalized in New York and Montreal before returning to Japan. All strains harbored bla_KPC-2-containing Tn4401a. The 15 K. pneumoniae strains each belonged to sequence type 258 and harbored a Tn4401a-carrying multireplicon-type plasmid, IncN and IncR (IncN+R). Three of these K. pneumoniae strains also possessed a Tn4401a-carrying ColRNAI plasmid, suggesting that Tn4401a underwent interplasmid transposition. Of these three ColRNAI plasmids, two and one were identical to plasmids harbored by two Citrobacter europaeus and three M. morganii strains, respectively. The Tn4401a-carrying ColRNAI plasmids were each 23,753 bp long and incapable of conjugal transfer via their own genes alone, but they mobilized during the conjugal transfer of Tn4401a-carrying IncN+R plasmids in K. pneumoniae. Interplasmid transposition of Tn4401a from an IncN+R plasmid to a ColRNAI plasmid in K. pneumoniae and mobilization of Tn4401a-carrying ColRNAI plasmids contributed to the acquisition of bla_KPC-2 in C. europaeus and M. morganii. IMPORTANCE Plasmid transfer plays an important role in the interspecies spread of carbapenemase genes, including the Klebsiella pneumoniae carbapenemase (KPC)-coding gene, bla_KPC. We conducted whole-genome sequencing (WGS) analysis and transmission experiments to analyze bla_KPC-2-carrying mobile genetic elements (MGEs) between the bla_KPC-2-harboring K. pneumoniae, Citrobacter europaeus, and Morganella morganii strains isolated from a single patient. bla_KPC-2 was contained within an MGE, Tn4401a. WGS of bla_KPC-2-carrying K. pneumoniae, C. europaeus, and M. morganii strains isolated from one patient revealed that Tn4401a-carrying ColRNAI plasmids were generated by plasmid-to-plasmid transfer of Tn4401a from a multireplicon-type IncN and IncR (IncN+R) plasmid in K. pneumoniae strains. Tn4401a-carrying ColRNAI plasmids were incapable of conjugal transfer in C. europaeus and M. morganii but mobilized from K. pneumoniae to a recipient Escherichia coli strain during the conjugal transfer of Tn4401a-carrying IncN+R plasmid. Therefore, Tn4401a-carrying ColRNAI plasmids contributed to the acquisition of bla_KPC-2 in C. europaeus and M. morganii.

SUBMITTER: Sugita K

PROVIDER: S-EPMC8565517 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Ceftazidime-avibactam (CZA) therapy has significantly improved survival rates for patients infected by carbapenem-resistant bacteria, including KPC producers. However, resistance to CZA is a growing concern, attributed to multiple mechanisms. In this study, we characterized four clinical CZA-resistant Klebsiella pneumoniae isolates obtained between July 2019 and December 2020. These isolates expressed novel allelic variants of blaKPC-2 resulting from changes in hotspots of the mature protein, particularly in loops surrounding the active site of KPC. Notably, KPC-80 had an K269_D270insPNK mutation near the Lys270-loop, KPC-81 had a del_I173 mutation within the Ω-loop, KPC-96 showed a Y241N substitution within the Val240-loop and KPC-97 had an V277_I278insNSEAV mutation within the Lys270-loop. Three of the four isolates exhibited low-level resistance to imipenem (4 µg/mL), while all remained susceptible to meropenem. Avibactam and relebactam effectively restored carbapenem susceptibility in resistant isolates. Cloning mutant blaKPC genes into pMBLe increased imipenem MICs in recipient Escherichia coli TOP10 for blaKPC-80, blaKPC-96, and blaKPC-97 by two dilutions; again, these MICs were restored by avibactam and relebactam. Frameshift mutations disrupted ompK35 in three isolates. Additional resistance genes, including blaTEM-1, blaOXA-18 and blaOXA-1, were also identified. Interestingly, three isolates belonged to clonal complex 11 (ST258 and ST11) and one to ST629. This study highlights the emergence of CZA resistance including unique allelic variants of blaKPC-2 and impermeability. Comprehensive epidemiological surveillance and in-depth molecular studies are imperative for understanding and monitoring these complex resistance mechanisms, crucial for effective antimicrobial treatment strategies.ImportanceThe emergence of ceftazidime-avibactam (CZA) resistance poses a significant threat to the efficacy of this life-saving therapy against carbapenem-resistant bacteria, particularly Klebsiella pneumoniae-producing KPC enzymes. This study investigates four clinical isolates exhibiting resistance to CZA, revealing novel allelic variants of the key resistance gene, blaKPC-2. The mutations identified in hotspots surrounding the active site of KPC, such as K269_D270insPNK, del_I173, Y241N and V277_I278insNSEAV, prove the adaptability of these pathogens. Intriguingly, low-level resistance to imipenem and disruptions in porin genes were observed, emphasizing the complexity of the resistance mechanisms. Interestingly, three of four isolates belonged to clonal complex 11. This research not only sheds light on the clinical significance of CZA resistance but also shows the urgency for comprehensive surveillance and molecular studies to inform effective antimicrobial treatment strategies in the face of evolving bacterial resistance.

Project description:PurposeTo explore the genetic characteristics of the IMP-4, NDM-1, OXA-1, and KPC-2 co-producing multidrug-resistant (MDR) clinical isolate, Citrobacter freundii wang9.MethodsMALDI-TOF MS was used for species identification. PCR and Sanger sequencing analysis were used to identify resistance genes. In addition to agar dilution, broth microdilution was used for antimicrobial susceptibility testing (AST). We performed whole genome sequencing (WGS) of the strains and analyzed the resulting data for drug resistance genes and plasmids. Phylogenetic trees were constructed with maximum likelihood, plotted using MAGA X, and decorated by iTOL.ResultsCitrobacter freundii carrying blaKPC-2, blaIMP-4, blaOXA-1, and blaNDM-1 are resistant to most antibiotics, intermediate to tigecycline, and only sensitive to polymyxin B, amikacin, and fosfomycin. The blaIMP-4 coexists with the blaNDM-1 and the blaOXA-1 on a novel transferable plasmid variant pwang9-1, located on the integron In1337, transposon TnAS3, and integron In2054, respectively. The gene cassette sequence of integron In1337 is IntI1-blaIMP-4-qacG2-aacA4'-catB3Δ, while the gene cassette sequence of In2054 is IntI1-aacA4cr-blaOXA-1-catB3-arr3-qacEΔ1-sul1. The blaNDM-1 is located on the transposon TnAS3, and its sequence is IS91-sul-ISAba14-aph (3')-VI-IS30-blaNDM-1-ble-trpF-dsbD-IS91. The blaKPC-2 is located on the transposon Tn2 of plasmid pwang9-1, and its sequence is klcA-korC-ISkpn6-blaKPC-2-ISkpn27-tnpR-tnpA. Phylogenetic analysis showed that most of the 34\u00B0C. freundii isolates from China were divided into three clusters. Among them, wang1 and wang9 belong to the same cluster as two strains of C. freundii from environmental samples from Zhejiang.ConclusionWe found C. freundii carrying blaIMP-4, blaNDM-1, blaOXA-1, and blaKPC-2 for the first time, and conducted in-depth research on its drug resistance mechanism, molecular transfer mechanism and epidemiology. In particular, we found that blaIMP-4, blaOXA-1, and blaNDM-1 coexisted on a new transferable hybrid plasmid that carried many drug resistance genes and insertion sequences. The plasmid may capture more resistance genes, raising our concern about the emergence of new resistance strains.

Project description:Klebsiella pneumoniae carbapenemase (KPC) producers are an emerging threat to global health, and the hospital water environment is considered an important reservoir of these life-threatening bacteria. We characterized plasmids of KPC-2-producing Citrobacter freundii and Klebsiella variicola isolates recovered from hospital sewage in Japan. Antimicrobial susceptibility testing, whole-genome sequencing analysis, bacterial conjugation, and transformation experiments were performed for both KPC-2 producers. The blaKPC-2 gene was located on the Tn3 transposon-related region from an IncP-6 replicon plasmid that could not be transferred via conjugation. Compared to the blaKPC-2-encoding plasmid of the C. freundii isolate, alignment analysis of plasmids with blaKPC-2 showed that the blaKPC-2-encoding plasmid of the K. variicola isolate was a novel IncP-6/IncF-like hybrid plasmid containing a 75,218-bp insertion sequence composed of IncF-like plasmid conjugative transfer proteins. Carbapenem-resistant transformants harboring blaKPC-2 were obtained for both isolates. However, no IncF-like insertion region was found in the K. variicola donor plasmid of the transformant, suggesting that this IncF-like region is not readily functional for plasmid conjugative transfer and is maintained depending on the host cells. The findings on the KPC-2 producers and novel genetic content emphasize the key role of hospital sewage as a potential reservoir of pathogens and its linked dissemination of blaKPC-2 through the hospital water environment. Our results indicate that continuous monitoring for environmental emergence of antimicrobial-resistant bacteria might be needed to control the spread of these infectious bacteria. Moreover, it will help elucidate both the evolution and transmission pathways of these bacteria harboring antimicrobial resistance. IMPORTANCE Antimicrobial resistance is a significant problem for global health, and the hospital environment has been recognized as a reservoir of antimicrobial resistance. Here, we provide insight into the genomic features of blaKPC-2-harboring isolates of Citrobacter freundii and Klebsiella variicola obtained from hospital sewage in Japan. The findings of carbapenem-resistant bacteria containing this novel genetic context emphasize that hospital sewage could act as a potential reservoir of pathogens and cause the subsequent spread of blaKPC-2 via horizontal gene transfer in the hospital water environment. This indicates that serial monitoring for environmental bacteria possessing antimicrobial resistance may help us control the spread of infection and also lead to elucidating the evolution and transmission pathways of these bacteria.

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Molecular Analysis of bla_KPC-2-Harboring Plasmids: Tn4401a Interplasmid Transposition and Tn4401a-Carrying ColRNAI Plasmid Mobilization from Klebsiella pneumoniae to Citrobacter europaeus and Morganella morganii in a Single Patient.

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Molecular Analysis of blaKPC-2-Harboring Plasmids: Tn4401a Interplasmid Transposition and Tn4401a-Carrying ColRNAI Plasmid Mobilization from Klebsiella pneumoniae to Citrobacter europaeus and Morganella morganii in a Single Patient.

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OmicsDI is part of the ELIXIR infrastructure

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Molecular Analysis of bla_KPC-2-Harboring Plasmids: Tn4401a Interplasmid Transposition and Tn4401a-Carrying ColRNAI Plasmid Mobilization from Klebsiella pneumoniae to Citrobacter europaeus and Morganella morganii in a Single Patient.