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Introduction: Multi-carbapenemase-producing Enterobacterales (M-CPE) are increasingly described. We characterized the M-CPE isolates prospectively recovered in our hospital (Madrid, Spain) over two years (2021-2022)., Methods: We collected 796 carbapenem resistant Enterobacterales (CRE) from clinical and surveillance samples. Carbapenemase production was confirmed with phenotypic (immunochromatographic, disk diffusion) and molecular (PCR, WGS) techniques. Antimicrobial susceptibility was evaluated by a standard broth microdilution method. Clinical and demographic data were collected., Results: Overall, 23 M-CPE (10 Klebsiella pneumoniae, 6 Citrobacter freundii complex, 3 Escherichia coli, 2 Klebsiella oxytoca, and 2 Enterobacter hormaechei) isolates were recovered from 17 patients (3% with CPE, 0.26-0.28 cases per 1000 admissions). OXA-48 + KPC-3 (7/23) and KPC-3 + VIM-1 (5/23) were the most frequent carbapenemase combinations. All patients had prior antibiotics exposure, including carbapenems (8/17). High resistance rates to ceftazidime/avibactam (14/23), imipenem/relebactam (16/23) and meropenem/vaborbactam (7/23) were found. Ceftazidime/avibactam + aztreonam combination was synergistic in all metallo-β-lactamase producers. Clonal and non-clonal related isolates were found, particularly in K. pneumoniae (5 ST29, 3 ST147, 3 ST307) and C. freundii (3 ST8, 2 ST125, 1 ST563). NDM-1 + OXA-48 was introduced with the ST147-K. pneumoniae high-risk clone linked to the transfer of a Ukrainian patient. We identified four possible nosocomial clonal transmission events between patients of the same clone with the same combination of carbapenemases (KPC-3 + VIM-1-ST29-K. pneumoniae, NDM-1 + OXA-48-ST147-K. pneumoniae and KPC-2 + VIM-1-ST145-K. oxytoca). Carbapenemase-encoding genes were located on different plasmids, except for VIM-1 + KPC-2-ST145-K. oxytoca. Cross-species transmission and a possible acquisition overtime was found, particularly between K. pneumoniae and E. coli producing OXA-48 + KPC-3., Conclusion: M-CPE is an emerging threat in our hospital. Co-production of different carbapenemases, including metallo-β-lactamases, limits therapeutic options and depicts the need to reinforce infection control measures., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: KHM-1-metallo-β-lactamase-producing Enterobacterales strains, of which only a few have been found, were isolated from four inpatients in Osaka, Japan during 2016 to 2020. We compared whole genomes of the four KHM-1-producing isolates, including one Enterobacter hormaechei subsp. hoffmannii, one Escherichia coli, and two Citrobacter freundii., Methods: These isolates were characterized by whole-genome sequencing, comparative analysis of bla KHM-1 -encoding plasmids with earlier reported plasmids, and antimicrobial susceptibility tests., Results: Multilocus sequence typing classified the E. hormaechei subsp. hoffmannii isolate to ST78, the E. coli isolate to ST354, and the two C. freundii isolates to ST95. These isolates harboured various antimicrobial resistance genes aside from bla KHM-1 on their chromosomes and plasmids. In all four isolates, bla KHM-1 was located on 137 kbp to 213 kbp plasmids of IncC replicon type. Although there were common resistance genes such as bla KHM-1 -ISEc68, class I integron cassette, and fosG, the four bla KHM-1 -encoding plasmids were distinguishable into two lineages based on differences of the resistance gene components and their surrounding regions., Conclusion: Because no epidemiological contact was observed among the inpatients, the bla KHM-1 -encoding IncC plasmids might have spread horizontally to multiple bacterial species through repeated recombination and insertion., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: This study aimed to isolate and characterise extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) isolates from animals and wastewater in Tunisia., Methods: ESBL-E from wastewater (n=123 samples), faeces of healthy animals (poultry, sheep, goats and calves) (n=140) and raw milk from healthy cows (n=42) and goats (n=20) were investigated. Antimicrobial susceptibility was determined according to CLSI recommendations. The bla TEM , bla SHV , bla CTX-M and bla OXA-48 genes were analysed by PCR and sequencing. Phylogenetic groups were determined by PCR for Escherichia coli isolates. The clonality of E. coli and Klebsiella pneumoniae isolates was determined by XbaI-PFGE and MLST., Results: A total of 81 E. coli, 20 K. pneumoniae, 4 Enterobacter cloacae, 1 Citrobacter freundii and 1 Citrobacter braakii were isolated. The bla CTX-M-1 and bla CTX-M-15 genes were predominant in E. coli and K. pneumoniae isolates. E. cloacae and C. braakii isolates harboured the bla SHV-12 gene. The C. freundii isolated from wastewater carried bla CTX-M-15 , bla TEM-1 and bla OXA-204 . E. coli isolates belonged to phylogroups A (37), B1 (25), B2 (7) and D (12). Seventy-eight E. coli isolates were typeable by PFGE and were classified into 34 pulsotypes. The K. pneumoniae isolates belonged to 11 pulsotypes. The E. coli isolates belonged to sequence types ST131, ST224, ST162, ST845, ST5204, ST69, ST141 and ST10. The K. pneumoniae isolates belonged to ST405, ST147, ST564, ST307, ST152, ST45, ST661 and ST1564., Conclusion: This is the first report of O25b-B2 3 -CTX-M-27-ST131 E. coli isolates and of C. freundii carrying bla CTX-M-15 , bla TEM-1 and bla OXA-204 in Tunisia., (Copyright © 2019 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Objectives: Although bla NDM-1 has been widely detected in various Enterobacteriaceae clinical isolates, multiple bla NDM-1 colonisations within the same patient remain rare. The aim of the study was to describe a patient with respiratory tract colonisation with NDM-1-producing Escherichia coli and Citrobacter freundii during hospitalisation in China., Methods: Two carbapenem-resistant isolates were analysed. Antimicrobial susceptibility testing, metallo-β-lactamase production, conjugation assay, plasmid analysis and molecular typing were performed., Results: The two clinical isolates carried the bla NDM-1 gene and showed resistance to carbapenems. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) revealed that E. coli NB753 harboured a plasmid of ca. 80kb, and C. freundii NB865 harboured three plasmids (ca. 70, 80 and 135kb). Southern blot and Inc replicon typing further demonstrated that both isolates carried the bla NDM-1 gene on a self-transferrable IncX3 plasmid. Moreover, the two NDM-producing plasmids were conjugative and the transconjugants showed increased resistance to carbapenems., Conclusion: An NDM-1-encoding plasmid harboured by various clinical isolates in a single patient is worrying considering that this plasmid may be widespread in our hospital. Furthermore, the threat of carbapenemase-producing bacterial epidemics should be closely monitored. However, a limitation of this study was the extremely small sample size., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Objectives: The objective of this study was to characterise the mechanisms underlying quinolone and oxyimino-cephalosporin resistance in a Citrobacter freundii clinical isolate obtained from the ICU in a university hospital in Uruguay., Methods: Citrobacter freundii strain CF638 was isolated from a urine culture. Identification was performed using a VITEK ® 2 system, and antimicrobial susceptibility was established by MIC determination and disk diffusion assay. Resistance genes and mobile genetic elements were identified by PCR and sequencing. Plasmid transfer was assessed by conjugation and the plasmid size was estimated by S1-PFGE. Plasmid incompatibility (Inc) group and toxin-antitoxin systems were sought by PCR., Results: Strain CF638 showed a multidrug-resistant profile, including resistance to carbapenems and quinolones. Transconjugant TcCF638, harbouring an ca. 200-kb IncA/C plasmid, also showed resistance to all β-lactams (except aztreonam) and diminished susceptibility to ciprofloxacin. PCR was positive for bla NDM-1 and qnrVC in CF638 and TcCF638. Two different class 1 integrons were detected (In127 and In907). In127 featured the genetic array aadA2-ltr2. Conversely, complex In907 featured two variable regions (VRs); VR-1 consisted of aadB-bla OXA-10 -aadA1cc, whereas VR-2 featured a qnrVC6 gene 108bp downstream from ISCR1 and 45bp upstream from qacEΔ1. Expression of qnrVC6 was due to a putative promoter region, detected using the Neural Network Promoter Prediction program., Conclusion: To the best of our knowledge, this constitutes the first report of qnrVC within a complex class 1 integron, as well as the first report of the occurrence of such a gene in an NDM-1-producing enterobacterial clinical isolate., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Objectives: Non-clonal, carbapenem- and multidrug-resistant Citrobacter freundii isolates were collected from unrelated outpatients admitted to a Portuguese hospital emergency department. One patient lived in a nursing home and was never hospitalised, whereas the other patient was repeatedly hospitalised in this hospital. The aim of this study was to unveil the molecular mechanisms associated with the carbapenem resistance of these isolates and to assess its potential dissemination., Methods: Isolate identification was performed by VITEK ® 2 and was confirmed by 16S rDNA sequencing. The clonal relationship of the isolates was evaluated by repetitive element palindromic PCR (rep-PCR). Antibiotic susceptibility was determined using the automatic VITEK ® 2 AES system and disk diffusion assay. β-Lactamases, porins and mobile genetic elements were characterised by PCR and sequencing. Pulsed-field gel electrophoresis (PFGE) and Southern blot hybridisation were used to determine the genetic location of integrons, and their transferability was tested by conjugation., Results: No genetic relatedness was found, suggesting different origins of the isolates. In isolate Cf254 a VIM-2 carbapenemase integrated in In58 was detected, located in a high-frequency conjugative IncL/M plasmid that also carried CTX-M-15 and CMY-39 genes. VIM-1 in isolate Cf872 was chromosomal. This is the first description in Portugal of VIM carbapenemases in C. freundii. Loss/alteration of porins was also detected., Conclusions: Emergence of carbapenem-resistant Enterobacteria is not confined to the nosocomial environment. Community-acquired strains appear to be in circulation between inpatients and outpatients, spreading carbapenem resistance genes by horizontal gene transfer., (Copyright © 2016 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)