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Dissemination of bla _NDM- ₁ Gene Among Several Klebsiella pneumoniae Sequence Types in Mexico Associated With Horizontal Transfer Mediated by IncF-Like Plasmids.

ABSTRACT: Nosocomial infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are a major health problem worldwide. The aim of this study was to describe NDM-1-producing K. pneumoniae strains causing bacteremia in a tertiary referral hospital in Mexico. MDR K. pneumoniae isolates were screened by polymerase chain reaction for the presence of resistance genes. In resistant isolates, plasmids were identified and conjugation assays were performed. Clonal diversity and the sequence types were determined by pulsed-field gel electrophoresis and multilocus sequence typing. A total of 80 K. pneumoniae isolates were collected from patients with bacteremia over a 1-year period. These isolates showed a level of resistance of 59% (47/80) to aztreonam, 56-60% (45-48/80) to cephalosporins, 54% (43/80) to colistin and 12.5% (10/80) to carbapenems. The carbapenem resistant isolates were bla _NDM- ₁ carriers and negative for bla _KPC, bla _NDM, bla _IMP, bla _VIM and bla _OXA- ₄₈ _-like carbapenemases genes. Conjugative plasmids IncFIIA and IncF group with sizes of 82-195 kbp were carriers of bla _NDM- ₁, bla _CTX-M- ₁₅, bla _TEM- ₁, aac(6')-Ib and/or aac(3')-IIa. Clonal variability and nine different multilocus sequence types were detected (ST661, ST683, ST1395, ST2706, ST252, ST1198, ST690, ST1535, and ST3368) for the first time in the isolates carrying bla _NDM- ₁ in Mexico. This study demonstrates that bla _NDM- ₁ has remained within this hospital in recent years and suggests that it is currently the most prevalent carbapenemase among K. pneumoniae MDR strains causing bacteremia in Mexico. The horizontal transfer of bla _NDM- ₁ gene through IncF-like plasmids among different clones demonstrates the dissemination pathway of antimicrobial resistance and underscore the need for strong and urgent joint measures to control the spread of NDM-1 carbapenemase in the hospital.

SUBMITTER: Toledano-Tableros JE

PROVIDER: S-EPMC8027308 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:NDM-5 carbapenemase was mainly identified in Escherichia coli, while the rapid transmission of bla NDM-5 among Enterobacteriaceae has raised serious public attention. This study identified 14 NDM-5-producing Klebsiella pneumoniae isolates from 107 carbapenem-resistant K. pneumoniae isolates, recovered from blood, urine, and normally sterile body fluids of pediatric patients from January 2016 to December 2018. All NDM-5-producing isolates were highly resistant to ?-lactams, while tigecycline and polymyxin B exhibited excellent antimicrobial activity. These 14 strains belonged to 9 different sequence types (STs) and displayed various pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they were not clonally related. S1-PFGE followed by Southern blotting showed that the bla NDM-5 gene was located on an ?46-kb IncX3 plasmid in all strains. All bla NDM-5-carrying plasmids were successfully transferred into recipient E. coli J53. PCR-based sequencing demonstrated that all of the bla NDM-5-carrying plasmids shared highly similar backbones, with nucleotide sequence identity of >99%. Moreover, this plasmid displayed high sequence similarity to the previously reported epidemic IncX3 bla NDM-5-carrying plasmids, with dynamic changes observed only in bla NDM-5-surrounding elements. Interestingly, the IncX3 bla NDM-5-carrying plasmids showed strong stability in clinical isolates when cultured in antibiotic-free medium. However, after the conjugation inhibitor linoleic acid was added, a gradual increase in the level of IncX3 plasmid loss could be observed. Clinical isolates displayed 10% to 15% bla NDM-5-carrying plasmid loss after coculture with linoleic acid for 5?days. These results showed that the IncX3 plasmid facilitated the dissemination of bla NDM-5 among multiclonal K. pneumoniae strains in children and that conjugal transfer contributed significantly to IncX3 plasmid stability within K. pneumoniae IMPORTANCE The emergence and spread of New Delhi metallo-?-lactamase (NDM)-producing Enterobacteriaceae have been a serious challenge to public health, and NDM-5 shows increased resistance to carbapenems compared with other variants. NDM-5 has been identified mostly in E. coli but has rarely been described in K. pneumoniae and other Enterobacteriaceae isolates. Here, we present the dissemination of highly similar 46-kb IncX3 bla NDM-5-carrying plasmids among multiclonal K. pneumoniae strains in children, highlighting the horizontal gene transfer of bla NDM-5 among K. pneumoniae strains via the IncX3 plasmid. Moreover, the IncX3 bla NDM-5-carrying plasmids displayed strong stability in clinical strains when cultured in antibiotic-free medium, and the plasmid maintenance was attributed partly to conjugal transfer. Plasmid conjugation is mediated by the type IV secretion system (T4SS), and T4SS is conserved among all epidemic IncX3 bla NDM-5-carrying plasmids. Therefore, combining conjugation inhibition and promotion of plasmid loss would be an effective strategy to limit the conjugation-assisted persistence of IncX3 bla NDM-5-carrying plasmids.

Project description:New Delhi metallo-β-lactamases (NDMs), including at least 28 variants, are a rapidly emerging family of β-lactamases worldwide, with a variety of infections caused by NDM-positive strains usually associated with very poor prognosis and high mortality. NDMs are the most prevalent carbapenemases in Escherichia coli (E. coli) worldwide, especially in China. The vast majority of bla NDM cases occur on plasmids, which play a vital role in the dissemination of bla NDM. To systematically explore the relationships between plasmids and bla NDM genes in E. coli and obtain an overall picture of the conjugative and mobilizable bla NDM-positive plasmids, we analyzed the variants of bla NDM, replicon types, phylogenetic patterns, conjugative transfer modules, host STs, and geographical distributions of 114 bla NDM-positive plasmids, which were selected from 3786 plasmids from 1346 complete whole genomes of E. coli from the GenBank database. We also established links among the characteristics of bla NDM-positive plasmids in E. coli. Eight variants of bla NDM were found among the 114 bla NDM-positive plasmids, with bla NDM - 5 (74 bla NDM - 5 genes in 73 plasmids), and bla NDM - 1 (31 bla NDM - 1 genes in 28 plasmids) being the most dominant. The variant bla NDM - 5 was mainly carried by the IncX3 plasmids and IncF plasmids in E. coli, the former were mainly geographically distributed in East Asia (especially in China) and the United States, and the latter were widely distributed worldwide. IncC plasmids were observed to be the predominant carriers of bla NDM - 1 genes in E. coli, which were mainly geographically distributed in the United States and China. Other bla NDM - 1-carrying plasmids also included IncM2, IncN2, and IncHI1. Moreover, the overall picture of the conjugative and mobilizable bla NDM-positive plasmids in E. coli was described in our study. Our findings enhance our understanding of the genetic diversity and characteristics of bla NDM-positive plasmids in in E. coli.

Project description:Objective:Regional dissemination is the major cause of the widespread prevalence of a plasmid-encoding NDM-1 enzyme. We investigated the drug resistance, joint efficiency, and gene environment of a Klebsiella pneumoniae strain carrying bla NDM-1 gene. Materials and Methods:Carbapenem-non-susceptible strains were analyzed using the VITEK 2 Compact. Strains carrying bla NDM-1 were identified using polymerase chain reaction and sequencing. Antimicrobial susceptibility testing and plasmid conjugation experiments were then conducted. Strains carrying bla NDM-1 were subjected to Southern blot analysis. After the gene mapping of bla NDM-1, library construction, and sequencing, plasmids were subsequently spliced and genotyped using the software Glimmer 3.0, and then analyzed using Mauve software. Results:Among 1735 carbapenem-non-susceptible strains, 54 strains of bla NDM-1-positive bacteria were identified, which consisted of 44 strains of K. pneumoniae, 8 strains of Acinetobacter baumannii and 2 strains of Escherichia coli. Strains carrying bla NDM-1 had a resistance rate of more than 50% in most antibiotics. Plasmid conjugation between strains carrying bla NDM-1 and E. coli strain J53 had a success rate of 50%. Southern blot analysis indicated that each strain had multiple plasmids containing bla NDM-1. Among the five plasmids containing bla NDM-1 in K. pneumoniae for sequencing, two plasmids with complete sequences were obtained. The findings were as follows: (i) The p11106 and p12 plasmids were highly similar to pNDM-BTR; (ii) the p11106 and p12 plasmids showed differences in the 20-30 kb region (orf00032-orf00043) from the other six plasmids; and (iii) bla NDM-1 was located at orf00037, while ble was found at orf00038. Two tnpA genes were located in the upstream region, and orf00052 (tnpA) in the 36 kb region was in the downstream sequence. Conclusion:bla NDM-1-containing bacteria exhibit multidrug resistance, which rapidly spreads and is transferred through efficient plasmid conjugation; the multidrug resistance of these bacteria may be determined by analyzing their drug-resistant plasmids. The presence of ble and tnpA genes suggests a possible hypothesis that bla NDM-1 originates from A. baumannii, which is retained in K. pneumoniae over a long period by transposition of mobile elements.

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Dissemination of bla _NDM- ₁ Gene Among Several Klebsiella pneumoniae Sequence Types in Mexico Associated With Horizontal Transfer Mediated by IncF-Like Plasmids.

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Dissemination of bla NDM- 1 Gene Among Several Klebsiella pneumoniae Sequence Types in Mexico Associated With Horizontal Transfer Mediated by IncF-Like Plasmids.

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Dissemination of bla _NDM- ₁ Gene Among Several Klebsiella pneumoniae Sequence Types in Mexico Associated With Horizontal Transfer Mediated by IncF-Like Plasmids.