INTRODUCTION: In the battle against multidrug-resistant bacterial infections, ceftazidime- avibactam (CZA) stands as a pivotal defense, particularly against carbapenemresistant (CR) Gram-negative pathogens. However, the rise in resistance against this drug poses a significant threat to its effectiveness, highlighting the critical need for in-depth studies about its resistance mechanisms. METHODS: This research focuses on the genomic characterization of CR- and CZA-resistant Escherichia coli (n=26) and Klebsiella pneumoniae (n=34) strains, harboring the blaNDM and/or blaOXA-48-like genes, at a major Lebanese tertiary care medical center, using whole genome sequencing (WGS). RESULTS: Our findings revealed a notable prevalence of blaNDM in all K. pneumoniae strains isolates, with 27 of these also harboring blaOXA-48. On the other hand, E. coli strains predominantly carried the blaNDM-5 gene. Whole genome sequencing (WGS) identified a predominance of ST383 among K. pneumoniae strains, which possessed a multi-replicon IncFIB-IncHI1B plasmid harboring the blaNDM-5. Additionally, various Inc group plasmids in K. pneumoniae across multiple sequence types were found to carry the blaNDM. Similarly, diverse STs of E. coli were observed to carry blaNDM-5 on different plasmids. DISCUSSION: The study underscores NDM carbapenemases as a paramount resistance mechanism in Lebanon,jeopardizing critical last-resort treatments. It also illuminates the role of varied sequence types and mobile genetic elements in the spread of NDM resistance,stressing the urgent need for strategies to mitigate this threat, especially in nosocomial infections.

• Keywords
• Escherichia coli, Klebsiella pneumoniae, ST383, blaNDM-5, carbapenem resistance,
• MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Azabicyclo Compounds * pharmacology   MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• beta-Lactamases * genetics   metabolism   MeSH
• Ceftazidime * pharmacology   MeSH
• Tertiary Care Centers MeSH
• Carbapenem-Resistant Enterobacteriaceae genetics   drug effects   isolation & purification   MeSH
• Escherichia coli * genetics   drug effects   MeSH
• Drug Combinations * MeSH
• Genome, Bacterial MeSH
• Carbapenems * pharmacology   MeSH
• Klebsiella pneumoniae * genetics   drug effects   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Drug Resistance, Multiple, Bacterial * genetics   MeSH
• Plasmids genetics   MeSH
• Gene Transfer, Horizontal MeSH
• Whole Genome Sequencing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Lebanon MeSH
• Names of Substances
• Anti-Bacterial Agents * MeSH
• avibactam, ceftazidime drug combination MeSH Browser
• Azabicyclo Compounds * MeSH
• Bacterial Proteins MeSH
• beta lactamase NDM-5, E coli MeSH Browser
• beta-Lactamases * MeSH
• Ceftazidime * MeSH
• Drug Combinations * MeSH
• Carbapenems * MeSH

The aim of this study was to characterize four Enterobacterales co-producing NDM- and OXA-48-like carbapenemases from Czech patients with travel history or/and previous hospitalization abroad. Klebsiella pneumoniae isolates belonged to "high risk" clones ST147, ST11, and ST15, while the Escherichia coli isolate was assigned to ST167. All isolates expressed resistance against most β-lactams, including carbapenems, while retaining susceptibility to colistin. Furthermore, analysis of WGS data showed that all four isolates co-produced OXA-48- and NDM-type carbapenemases, in different combinations (Kpn47733: bla NDM- 5 + bla OXA- 181; Kpn50595: bla NDM- 1 + bla OXA- 181; Kpn51015: bla NDM- 1 + bla OXA- 244; Eco52418: bla NDM- 5 + bla OXA- 244). In Kpn51015, the bla OXA- 244 was found on plasmid p51015_OXA-244, while the respective gene was localized in the chromosomal contig of E. coli Eco52418. On the other hand, bla OXA- 181 was identified on a ColKP3 plasmid in isolate Kpn47733, while a bla OXA- 181-carrying plasmid being an IncX3-ColKP3 fusion was identified in Kpn50595. The bla NDM- 1 gene was found on two different plasmids, p51015_NDM-1 belonging to a novel IncH plasmid group and p51015_NDM-1 being an IncF K 1-FIB replicon. Furthermore, the bla NDM- 5 was found in two IncFII plasmids exhibiting limited nucleotide similarity to each other. In both plasmids, the genetic environment of bla NDM- 5 was identical. Finally, in all four carbapenemase-producing isolates, a diverse number of additional replicons, some of these associated with important resistance determinants, like bla CTX-M- 15, arr-2 and ermB, were identified. In conclusion, this study reports the first description of OXA-244-producing Enterobacterales isolated from Czech hospitals. Additionally, our findings indicated the genetic plurality involved in the acquisition and dissemination of determinants encoding OXA/NDM carbapenemases.

• Keywords
• NDM-1, NDM-5, OXA-181, OXA-244, mobile genetic elements,
• Publication type
• Journal Article MeSH

The aim of this study was to report the characterization of the first mcr-positive Enterobacterales isolated from Czech hospitals. In 2019, one Citrobacter freundii and four Enterobacter isolates were recovered from Czech hospitals. The production of carbapenemases was examined by a matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) imipenem hydrolysis assay. Additionally, bacteria were screened for the presence of carbapenemase-encoding genes and plasmid-mediated colistin resistance genes by PCR. To define the genetic units carrying mcr genes, the genomic DNAs of mcr-carrying clinical isolates were sequenced on the PacBio Sequel I platform. Results showed that all isolates carried blaVIM- and mcr-like genes. Analysis of whole-genome sequencing (WGS) data revealed that all isolates carried mcr-9-like alleles. Furthermore, the three sequence type 106 (ST106) Enterobacter hormaechei isolates harbored the blaVIM-1 gene, while the ST764 E. hormaechei and ST95 C. freundii included blaVIM-4 Analysis of plasmid sequences showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. Additionally, at least one multidrug resistance (MDR) region was identified in each mcr-9-carrying IncHI2 plasmid. The blaVIM-4 gene was found in the MDR regions of p48880_MCR_VIM and p51929_MCR_VIM. In the three remaining isolates, blaVIM-1 was localized on plasmids (∼55 kb) exhibiting repA-like sequences 99% identical to the respective gene of pKPC-CAV1193. In conclusion, to the best of our knowledge, these 5 isolates were the first mcr-9-positive bacteria of clinical origin identified in the Czech Republic. Additionally, the carriage of the blaVIM-1 on pKPC-CAV1193-like plasmids is described for the first time. Thus, our findings underline the ongoing evolution of mobile elements implicated in the dissemination of clinically important resistance determinants.IMPORTANCE Infections caused by carbapenemase-producing bacteria have led to the revival of polymyxins as the "last-resort" antibiotic. Since 2016, several reports describing the presence of plasmid-mediated colistin resistance genes, mcr, in different host species and geographic areas were published. Here, we report the first detection of Enterobacterales carrying mcr-9-like alleles isolated from Czech hospitals in 2019. Furthermore, the three ST106 Enterobacter hormaechei isolates harbored blaVIM-1, while the ST764 E. hormaechei and ST95 Citrobacter freundii isolates included blaVIM-4 Analysis of WGS data showed that, in all isolates, mcr-9 was carried on IncHI2 plasmids. blaVIM-4 was found in the MDR regions of IncHI2 plasmids, while blaVIM-1 was localized on pKPC-CAV1193-like plasmids, described here for the first time. These findings underline the ongoing evolution of mobile elements implicated in dissemination of clinically important resistance determinants. Thus, WGS characterization of MDR bacteria is crucial to unravel the mechanisms involved in dissemination of resistance mechanisms.

• Keywords
• Citrobacter freundii, Enterobacter cloacae, IncHI2, MCR-9, VIM-4,
• MeSH
• Anti-Bacterial Agents pharmacology   therapeutic use   MeSH
• Bacterial Proteins genetics   MeSH
• beta-Lactamases genetics   MeSH
• Enterobacter genetics   isolation & purification   MeSH
• Humans MeSH
• Hospitals MeSH
• Plasmids genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• beta-Lactamases MeSH

Ten Enterobacteriaceae isolates collected in a Czech hospital carried blaKPC-positive plasmids of different sizes (∼30, ∼45, and ∼80 kb). Sequencing revealed three types of plasmids (A to C) with the Tn4401a transposon. Type A plasmids comprised an IncR backbone and a KPC-2-encoding multidrug resistance (MDR) region. Type B plasmids were derivatives of type A plasmids carrying an IncN3-like segment, while type C plasmids were IncP6 plasmids sharing the same KPC-2-encoding MDR region with type A and B plasmids.

• Keywords
• Citrobacter freundii, Illumina sequencing, IncR, ST18, Tn4401a,
• MeSH
• Anti-Bacterial Agents therapeutic use   MeSH
• beta-Lactamases genetics   metabolism   MeSH
• Citrobacter freundii drug effects   enzymology   genetics   isolation & purification   MeSH
• Enterobacteriaceae Infections drug therapy   epidemiology   microbiology   MeSH
• Escherichia coli drug effects   enzymology   genetics   isolation & purification   MeSH
• Gene Expression MeSH
• Isoenzymes genetics   metabolism   MeSH
• Carbapenems therapeutic use   MeSH
• Klebsiella pneumoniae drug effects   enzymology   genetics   isolation & purification   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Drug Resistance, Multiple, Bacterial genetics   MeSH
• Morganella morganii drug effects   enzymology   genetics   isolation & purification   MeSH
• Hospitals MeSH
• Open Reading Frames MeSH
• Plasmids chemistry   classification   metabolism   MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• DNA Transposable Elements MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• beta-lactamase KPC-2 MeSH Browser
• beta-Lactamases MeSH
• Isoenzymes MeSH
• Carbapenems MeSH
• DNA Transposable Elements MeSH

VIM-39, a VIM-1-like metallo-β-lactamase variant (VIM-1 Thr33Ala His224Leu) was identified in a clinical isolate of Klebsiella pneumoniae belonging to sequence type 147. VIM-39 hydrolyzed ampicillin, cephalothin, and imipenem more efficiently than did VIM-1 and VIM-26 (a VIM-1 variant with the His224Leu substitution) because of higher turnover rates.

• MeSH
• Ampicillin metabolism   pharmacology   MeSH
• Anti-Bacterial Agents metabolism   pharmacology   MeSH
• beta-Lactamases genetics   metabolism   MeSH
• beta-Lactam Resistance genetics   MeSH
• Biotransformation MeSH
• Cephalothin metabolism   pharmacology   MeSH
• Gene Expression MeSH
• Hydrolysis MeSH
• Imipenem metabolism   pharmacology   MeSH
• Klebsiella Infections drug therapy   microbiology   MeSH
• Isoenzymes genetics   metabolism   MeSH
• Kinetics MeSH
• Klebsiella pneumoniae drug effects   enzymology   genetics   isolation & purification   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Molecular Sequence Data MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Bacterial Typing Techniques MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Greece MeSH
• Names of Substances
• Ampicillin MeSH
• Anti-Bacterial Agents MeSH
• beta-Lactamases MeSH
• Cephalothin MeSH
• Imipenem MeSH
• Isoenzymes MeSH
• VIM-1 metallo-beta-lactamase MeSH Browser

IMP-8 metallo-β-lactamase was identified in Klebsiella pneumoniae sequence type 252 (ST252), isolated in a Portuguese hospital in 2009. blaIMP-8 was the first gene cassette of a novel class 3 integron, In1144, also carrying the blaGES-5, blaBEL-1, and aacA4 cassettes. In1144 was located on a ColE1-like plasmid, pKP-M1144 (12,029 bp), with a replication region of limited nucleotide similarity to those of other RNA-priming plasmids, such as pJHCMW1. In1144 and pKP-M1144 represent an interesting case of evolution of resistance determinants in Gram-negative bacteria.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacterial Proteins genetics   MeSH
• beta-Lactamases genetics   MeSH
• DNA, Bacterial genetics   MeSH
• Klebsiella Infections microbiology   MeSH
• Carbapenems pharmacology   MeSH
• Klebsiella pneumoniae drug effects   genetics   isolation & purification   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Drug Resistance, Multiple, Bacterial genetics   MeSH
• Molecular Sequence Data MeSH
• Plasmids drug effects   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Portugal MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Bacterial Proteins MeSH
• beta-lactamase BEL-1, Klebsiella pneumoniae MeSH Browser
• beta-lactamase GES-5, Klebsiella pneumoniae MeSH Browser
• beta-Lactamases MeSH
• carbapenemase MeSH Browser
• DNA, Bacterial MeSH
• IMP-8 enzyme MeSH Browser
• Carbapenems MeSH