BACKGROUND: VIM metallo-β-lactamases are enzymes characterized by the ability to hydrolyze all β-lactams. Usually, bla VIM-like genes are carried by class 1 integrons. In the Czech Republic, only sporadic cases of VIM-producing Enterobacterales have been reported in which those isolates carried the VIM-1 carbapenemase-encoding integron In110. However, during 2019-2020, an increased number was reported. Therefore, the aim of the current study was to characterize the genetic elements involved in the increased spread of bla VIM genes. MATERIALS AND METHODS: 32 VIM-producing Enterobacterales collected between 2019 and 2020 were subjected to: antimicrobial susceptibility testing, integron analysis, and short reads sequencing. Based on the results, 19 isolates were selected as representative and sequenced using Sequel I platform. RESULTS: The 32 VIM-producing isolates exhibited variations in the MICs of carbapenems. Based on short-read data, 26 of the 32 sequenced isolates harbored the bla VIM-1 allele while six isolates carried the bla VIM-4 gene. The most prevalent was the In110 integron (n = 24) and two isolates carried the In4873 class 1 integron. The bla VIM-4 allele was identified in class 1 integrons In1174 (n = 3), In416 (n = 1), In2143 (n = 1) and In2150. Long reads sequencing revealed that the bla VIM was carried by: pKPC-CAV1193-like (n = 6), HI1 (pNDM-CIT; n = 4), HI2 (n = 3), FIB (pECLA; n = 2) and N (n = 1) incompatibility groups. Two bla VIM-carrying plasmids could not be typed by the database, while another one was integrated into the chromosome. CONCLUSION: We observed the spread of VIM-encoding integrons, mainly of In110, among Enterobacterales isolated from Czech hospitals, but also an increased number of novel elements underlining the ongoing evolution.

• Keywords
• Enterobacterales, In110, blaVIM-1, blaVIM-4, integrons, plasmids, whole-genome sequencing,
• Publication type
• Journal Article MeSH

The objective of this study was to perform molecular surveillance for assessing the spread of carbapenemase-producing Pseudomonas aeruginosa in Czech hospitals. One hundred thirty-six carbapenemase-producing isolates were recovered from 22 hospitals located throughout the country. Sequence type 357 (ST357) dominated (n = 120) among carbapenemase producers. One hundred seventeen isolates produced IMP-type (IMP-7 [n = 116] and IMP-1 [n = 1]) metallo-β-lactamases (MβLs), 15 produced the VIM-2 MβL, and the remaining isolates expressed the GES-5 enzyme. The blaIMP-like genes were located in three main integron types, with In-p110-like being the most prevalent (n = 115). The two other IMP-encoding integrons (In1392 and In1393) have not been described previously. blaVIM-2-carrying integrons included In59-like, In56, and a novel element (In1391). blaGES-5 was carried by In717. Sequencing data showed that In-p110-like was associated with a Tn4380-like transposon inserted in genomic island LESGI-3 in the P. aeruginosa chromosome. The other integrons were also integrated into the P. aeruginosa chromosome. These findings indicated the clonal spread of ST357 P. aeruginosa, carrying the IMP-7-encoding integron In-p110, in Czech hospitals. Additionally, the sporadic emergence of P. aeruginosa producing different carbapenemase types, associated with divergent or novel integrons, punctuated the ongoing evolution of these bacteria.

• Keywords
• GES, Illumina sequencing, ST111, ST235, VIM, class 1 integrons, genomic islands (GIs), integrative conjugative element (ICE),
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Chromosomes, Bacterial chemistry   MeSH
• beta-Lactamases genetics   metabolism   MeSH
• Epidemiological Monitoring MeSH
• Gene Expression MeSH
• Genomic Islands MeSH
• Genotype MeSH
• Incidence MeSH
• Integrons MeSH
• Isoenzymes genetics   metabolism   MeSH
• Carbapenems pharmacology   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Drug Resistance, Multiple, Bacterial genetics   MeSH
• Multilocus Sequence Typing MeSH
• Hospitals MeSH
• Pseudomonas Infections epidemiology   microbiology   MeSH
• Pseudomonas aeruginosa drug effects   enzymology   genetics   isolation & purification   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Multicenter Study MeSH
• Geographicals
• Czech Republic epidemiology   MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• beta-lactamase IMP-7 protein, Pseudomonas aeruginosa MeSH Browser
• beta-Lactamases MeSH
• Isoenzymes MeSH
• Carbapenems MeSH

A total of 18 Enterobacteriaceae (17 from gulls and 1 from a clinical sample) collected from Australia, carrying IncA/C plasmids with the IMP-encoding In809-like integrons, were studied. Seven plasmids, being representatives of different origins, plasmid sizes, replicon combinations, and resistance genes, were completely sequenced. Plasmid pEc158, identified in a clinical Escherichia coli ST752 isolate, showed extensive similarity to type 2 IncA/C2 plasmids. pEc158 carried none of the blaCMY-2-like region or ARI-B and ARI-A regions, while it contained a hybrid transposon structure. The six remaining plasmids, which were of wildlife origin, were highly similar to each other and probably were fusion derivatives of type 1 and type 2 A/C2 plasmids. The latter plasmids contained an ARI-B region and hybrid transposon structures. In all plasmids, hybrid transposon structures containing In809-like integrons were inserted 3,434 bp downstream of the rhs2 start codon. In all cases, the one outermost 38-bp inverted repeat (IR) of the transposon was associated with the Tn1696 tnp module, while the other outermost 38-bp IR of the transposon was associated with either a Tn6317-like module or a Tn21 mer module. However, the internal structure of the transposon and the resistance genes were different in each plasmid. These findings indicated that, for the specific periods of time and settings, different IncA/C2 plasmid types carrying In809-like elements circulated among isolates of wildlife and clinical origins. Additionally, they provided the basis for speculations regarding the reshuffling of IncA/C2 plasmids with In809-like integrons and confirmed the rapid evolution of IncA/C2 plasmid lineages.

• Keywords
• Citrobacter freundii, Escherichia coli, IMP, Klebsiella pneumoniae, PMLST, metallo-β-lactamases,
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• beta-Lactamases genetics   MeSH
• Charadriiformes microbiology   MeSH
• Citrobacter freundii genetics   isolation & purification   MeSH
• Escherichia coli genetics   isolation & purification   MeSH
• Klebsiella pneumoniae genetics   isolation & purification   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Drug Resistance, Multiple, Bacterial genetics   MeSH
• Inverted Repeat Sequences genetics   MeSH
• Plasmids genetics   MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• DNA Transposable Elements genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Australia MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• beta-Lactamases MeSH
• DNA Transposable Elements MeSH

A comparison of a matrix-assisted laser desorption ionization-time of flight mass spectrometric (MALDI-TOF MS) meropenem hydrolysis assay with the Carba NP test showed that both methods exhibited low sensitivity (approximately 76%), mainly due to the false-negative results obtained with OXA-48-type producers. The addition of NH4HCO3 to the reaction buffer for the MALDI-TOF MS assay dramatically improved its sensitivity (98%). Automatic interpretation of the MALDI-TOF MS assay, using the MBT STAR-BL software, generally agreed with the results obtained after manual analysis. For the Carba NP test, spectrophotometric analysis found six additional carbapenemase producers.

• MeSH
• Electronic Data Processing MeSH
• Bacterial Proteins analysis   MeSH
• beta-Lactamases analysis   MeSH
• Bicarbonates * MeSH
• Hydrolysis MeSH
• Automation, Laboratory MeSH
• Humans MeSH
• Meropenem MeSH
• Buffers MeSH
• Sensitivity and Specificity MeSH
• Software MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Thienamycins metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ammonium bicarbonate MeSH Browser
• Bacterial Proteins MeSH
• beta-Lactamases MeSH
• carbapenemase MeSH Browser
• Bicarbonates * MeSH
• Meropenem MeSH
• Buffers MeSH
• Thienamycins MeSH