OBJECTIVES: The beta-lactamases with extended spectrum of activity (ESBL) are medically one of the most important group of enzymes. Another group of beta-lactamases representing of Enterobacteriaceae is group of the AmpC-type cephalosporinases. The presented study provides identification and determination of the spectrum of resistance against different and clinically used antimicrobial drugs in the clinical isolates of Escherichia coli. METHODS: These isolates had origin in different departments of the L. Pasteur University Hospital in Košice. The goal was the detection of beta-lactamase production with extended-spectrum effect and testing of AmpC-type cephalosporinases by several phenotypic tests in clinical isolates. MALDI-TOF MS analysis was performed on a Microflex MALDI Biotyper. Samples were positively tested for ESBL with the use of the disc diffusion method. PCR were performed with a series of primers designed for the detection of Ambler class A, B and C beta-lactamase genes. RESULTS: For all 485 isolates, we determined the production of ESBL, which we detected in 166 E. coli isolates, which represents a 34.2% prevalence of ESBL production. It is clear from the results that the prevalence of ESBL-producing E. coli out of the total number of E. coli investigated reached 34.2%. In the monitored period, we confirmed at least one resistance gene from 485 E. coli in 188 positive isolates. CONCLUSIONS: We describe a complex ESBL epidemiology. The study revealed a high rate of ESBL-producing E. coli isolates; blaTEM and blaSHV enzymes dominated in ESBL-positive E. coli isolates in the L. Pasteur University Hospital in Košice.

• Keywords
• AmpC, E. coli, ESBL, prevalence, resistance, β-lactamase,
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Microbial MeSH
• Bacteria MeSH
• beta-Lactamases genetics   pharmacology   MeSH
• Escherichia coli * genetics   MeSH
• Escherichia coli Infections * epidemiology   microbiology   MeSH
• Humans MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• beta-Lactamases MeSH

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 aim of the present study was to characterize sporadic cases and an outbreak of NDM-like-producing Enterobacteriaceae recovered from hospital settings, in Czechia. During 2016, 18 Entrobacteriaceae isolates including 10 Enterobacter cloacae complex (9 E. xiangfangensis and 1 E. asburiae), 4 Escherichia coli, 1 Kluyvera intermedia, 1 Klebsiella pneumoniae, 1 Klebsiella oxytoca, and 1 Raoultella ornithinolytica that produced NDM-like carbapenemases were isolated from 15 patients. Three of the patients were colonized or infected by two different NDM-like producers. Moreover, an NDM-4-producing isolate of E. cloacae complex, isolated in 2012, was studied for comparative purposes. All isolates of E. cloacae complex, except the E. asburiae, recovered from the same hospital, were assigned to ST182. Additionally, two E. coli belonged to ST167, while the remaining isolates were not clonally related. Thirteen isolates carried blaNDM-4, while six isolates carried blaNDM-1 (n = 3) or blaNDM-5 (n = 3). Almost all isolates carried blaNDM-like-carrying plasmids being positive for the IncX3 allele, except ST58 E. coli and ST14 K. pneumoniae isolates producing NDM-1. Analysis of plasmid sequences revealed that all IncX3 blaNDM-like-carrying plasmids exhibited a high similarity to each other and to previously described plasmids, like pNDM-QD28, reported from worldwide. However, NDM-4-encoding plasmids differed from other IncX3 plasmids by the insertion of a Tn3-like transposon. On the other hand, the ST58 E. coli and ST14 K. pneumoniae isolates carried two novel NDM-1-encoding plasmids, pKpn-35963cz, and pEsco-36073cz. Plasmid pKpn-35963cz that was an IncFIB(K) molecule contained an acquired sequence, encoding NDM-1 metallo-β-lactamase (MβL), which exhibited high similarity to the mosaic region of pS-3002cz from an ST11 K. pneumoniae from Czechia. Finally, pEsco-36073cz was a multireplicon A/C2+R NDM-1-encoding plasmid. Similar to other type 1 A/C2 plasmids, the blaNDM-1 gene was located within the ARI-A resistance island. These findings underlined that IncX3 plasmids have played a major role in the dissemination of blaNDM-like genes in Czech hospitals. In combination with further evolvement of NDM-like-encoding MDR plasmids through reshuffling, NDM-like producers pose an important public threat.

• Keywords
• Enterobacter xiangfangensis, IncX3, NDM, ST182, metallo-β-lactamases,
• Publication type
• Journal Article 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

Two multidrug resistance (MDR) plasmids, carrying the VIM-1-encoding integron In110, were characterized. Plasmid pLec-476cz (311,758 bp), from a Leclercia adecarboxylata isolate, consisted of an IncHI1 backbone, a MDR region, and two accessory elements. Plasmid pKpn-431cz (142,876 bp), from a sequence type 323 (ST323) Klebsiella pneumoniae isolate, comprised IncFIIY-derived and pKPN3-like sequences and a mosaic region. A 40,400-bp sequence of pKpn-431cz was identical to the MDR region of pLec-476cz, indicating the en bloc acquisition of the VIM-1-encoding region from one plasmid by the other.

• Keywords
• IncFIIY, IncHI1, carbapenemases, integrative conjugative elements, metallo-β-lactamases,
• MeSH
• Bacterial Proteins genetics   MeSH
• beta-Lactamases genetics   MeSH
• beta-Lactam Resistance genetics   MeSH
• DNA, Bacterial genetics   MeSH
• Integrons genetics   MeSH
• Klebsiella pneumoniae drug effects   genetics   MeSH
• Humans MeSH
• Drug Resistance, Multiple, Bacterial genetics   MeSH
• Plasmids genetics   MeSH
• Base Sequence MeSH
• Sequence Analysis, DNA MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH
• Names of Substances
• Bacterial Proteins MeSH
• beta-Lactamases MeSH
• carbapenemase MeSH Browser
• DNA, Bacterial MeSH
• VIM-1 metallo-beta-lactamase MeSH Browser