Polymyxin S1

Purpose: To explore the genetic characteristics of the IMP-4 and SFO-1 co-producing multidrug-resistant (MDR) clinical isolates, Enterobacter hormaechei YQ13422hy and YQ13530hy. Methods: MALDI-TOF MS was used for species identification. Antibiotic resistance genes (ARGs) were tested by PCR and Sanger sequencing analysis. In addition to agar dilution, broth microdilution was used for antimicrobial susceptibility testing (AST). Whole-genome sequencing (WGS) analysis was conducted using the Illumina NovaSeq 6000 and Oxford Nanopore platforms. Annotation was performed by RAST on the genome. The phylogenetic tree was achieved using kSNP3.0. Plasmid characterization was conducted using S1-pulsed-field gel electrophoresis (S1-PFGE), Southern blotting, conjugation experiments, and whole genome sequencing (WGS). An in-depth study of the conjugation module was conducted using the OriTFinder website. The genetic context of bla IMP-4 and bla SFO-1 was analyzed using BLAST Ring Image Generator (BRIG) and Easyfig 2.3. Results: YQ13422hy and YQ13530hy, two MDR strains of ST51 E. hormaechei harboring bla IMP-4 and bla SFO-1, were identified. They were only sensitive to meropenem, amikacin and polymyxin B, and were resistant to cephalosporins, aztreonam, piperacillin/tazobactam and aminoglycosides, intermediate to imipenem. The genetic context surrounding bla IMP-4 was 5'CS-hin-1-IS26-IntI1-bla IMP-4-IS6100-ecoRII. The integron of bla IMP-4 is In823, which is the array of gene cassettes of 5'CS-bla IMP-4. Phylogenetic analysis demonstrated that E. hormaechei YQ13422hy and YQ13530hy belonged to the same small clusters with a high degree of homology. Conclusion: This observation revealed the dissemination of the bla IMP-4 gene in E. hormaechei in China. We found that bla IMP-4 and bla SFO-1 co-exist in MDR clinical E. hormaechei isolates. This work showed a transferable IncN-type plasmid carrying the bla IMP-4 resistance gene in E. hormaechei. We examined the potential resistance mechanisms of pYQ13422-IMP-4 and pYQ13422-SFO-1, along with their detailed genetic contexts.

The worldwide spread of carbapenem-resistant Enterobacteriaceae (CRE) has led to a major challenge to human health. In this case, colistin is often used to treat the infection caused by CRE. However, the coexistence of genes conferring resistance to carbapenem and colistin is of great concern. In this work, we reported the coexistence of bla OXA-181, bla CTX-M-55, and mcr-8 in an ST273 Klebsiella pneumoniae isolate for the first time. The species identification was performed using MALDI-TOF MS, and the presence of various antimicrobial resistance genes (ARGs) and virulence genes were detected by PCR and whole-genome sequencing. Antimicrobial susceptibility testing showed that K. pneumoniae 5589 was resistant to aztreonam, imipenem, meropenem, ceftriaxone, cefotaxime, ceftazidime, levofloxacin, ciprofloxacin, gentamicin, piperacillin-tazobactam, cefepime, and polymyxin B, but sensitive to amikacin. S1-pulsed-field gel electrophoresis (PFGE) and Southern blotting revealed the mcr-8 gene was carried on a ~ 138 kb plasmid with a conserved structure (IS903B-ymoA-inhA-mcr-8-copR-baeS-dgkA-ampC). In addition, bla OXA-181 was found on another ~51 kb plasmid with a composite transposon flanked by insertion sequence IS26. The in vitro conjugation experiments and plasmid sequence probe indicated that the plasmid p5589-OXA-181 and the p5589-mcr-8 were conjugative, which may contribute to the propagation of ARGs. Relevant detection and investigation measures should be taken to control the prevalence of pathogens coharboring bla OXA-181, bla CTX-M-55 and mcr-8.

Background: Since its first report in the country in 2013, NDM-producing Enterobacterales have been identified in all the Brazilian administrative regions. In this study, we characterized by antimicrobial susceptibility testing and by molecular typing a large collection of NDM-producing Klebsiella isolates from different hospitals in Brazil, mainly from the state of Sao Paulo, over the last decade. Methods: Bacterial isolates positive for blaNDM-genes were identified by MALDI-TOF MS and submitted to antimicrobial susceptibility testing by disk diffusion or broth microdilution (for polymyxin B). All isolates were submitted to pulsed-field gel electrophoresis, and isolates belonging to different clusters were submitted to whole genome sequencing by Illumina technology and downstream analysis. Mating out assays were performed by conjugation, plasmid sizes were determined by S1-PFGE, and plasmid content was investigated by hybrid assembly after MinIon long reads sequencing. Results: A total of 135 NDM-producing Klebsiella were identified, distributed into 107 different pulsotypes; polymyxin B was the only antimicrobial with high activity against 88.9% of the isolates. Fifty-four isolates presenting diversified pulsotypes were distributed in the species K. pneumoniae (70%), K. quasipneumoniae (20%), K. variicola (6%), K. michiganensis (a K. oxytoca Complex species, 2%), and K. aerogenes (2%); blaNDM-1 was the most frequent allele (43/54, 80%). There was a predominance of Clonal Group 258 (ST11 and ST340) encompassing 35% of K. pneumoniae isolates, but another thirty-one different sequence types (ST) were identified, including three described in this study (ST6244 and ST6245 for K. pneumoniae, and ST418 for K. michiganensis). The blaNDM-1 and blaNDM-7 were found to be located into IncF and IncX3 type transferable plasmids, respectively. Conclusions: Both clonal (mainly driven by CG258) and non-clonal expansion of NDM-producing Klebsiella have been occurring in Brazil in different species and clones, associated with different plasmids, since 2013.