OBJECTIVE: One of the most important threats of current medicine is the spread of multiresistant Gram-negative bacteria. We report here data from a six-month prevalence study on carbapenemase-producing K. pneumoniae and E. coli performed in Czech hospitals participating on European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE). METHODS: Ten hospitals covering all regions of the Czech Republic were selected. During the study period (1st November 2013 to 30th April 2014), first ten carbapenem non-susceptible isolates of K. pneumoniae or E. coli isolated from non-surveillance specimens (i.e., blood, lower respiratory tract secretions, urine, puncture fluids, and wound secretions) of single successive patients were collected. Successive carbapenem-susceptible isolates of the same species were also preserved as controls. Susceptibility to 15 antibiotics was determined using EUCAST recommendations. Carbapenemase activity was detected by MALDI-TOF MS meropenem hydrolysis assay. Positive isolates were subjected for molecular typing (multi-locus sequence typing, identification of carbapenemase gene). RESULTS: During the study period, thirty non-susceptible isolates (K. pneumoniae n=28, E. coli n=2) were identified in 5 hospitals. Only two of them were confirmed to be carbapenemase producers. A NDM-1-producing K. pneumoniae ST11 was recovered from a patient, transferred from Ukraine, being injured during a Maidan revolution. The second isolate, an OXA-48-producing K. pneumoniae, belonging to ST101, was recovered from a patient admitted to a hospital for an ischemic stroke. CONCLUSIONS: This study again confirmed that the Czech Republic still belongs to the countries with low prevalence of carbapenemase-producing Enterobacteriaceae (CPE). Cases of CPE are usually restricted to an import from high-prevalence countries or countries with unknown epidemiological situation.

• Klíčová slova
• carbapenemase - CPE - MALDI-TOF - susceptibility - resistance - Enterobacteriaceae.,
• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• Escherichia coli enzymologie   genetika   izolace a purifikace   MeSH
• infekce bakteriemi rodu Klebsiella epidemiologie   mikrobiologie   MeSH
• infekce vyvolané Escherichia coli epidemiologie   mikrobiologie   MeSH
• karbapenemy farmakologie   MeSH
• Klebsiella pneumoniae enzymologie   genetika   izolace a purifikace   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• multilokusová sekvenční typizace MeSH
• nemocnice MeSH
• prevalence MeSH
• průřezové studie MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• techniky typizace bakterií MeSH
• zeměpis MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Ukrajina MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• karbapenemy MeSH

Wild corvids were examined for the presence of carbapenemase-producing Gram-negative bacteria in the United States. A total of 13 isolates were detected among 590 fecal samples of American crow; 11 Providencia rettgeri isolates harboring blaIMP-27 on the chromosome as a class 2 integron gene cassette within the Tn7 transposon, 1 Klebsiella pneumoniae ST258 isolate carrying blaKPC-2 on a pKpQIL-like plasmid as a part of Tn4401a, and 1 Enterobacter bugandensis isolate with blaIMI-1 located within EcloIMEX-2.

• Klíčová slova
• IMI, IMP-27, KPC, Providencia rettgeri, carbapenemase, wild birds,
• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamasy genetika   MeSH
• Enterobacter MeSH
• infekce bakteriemi rodu Klebsiella * MeSH
• Klebsiella pneumoniae genetika   MeSH
• mikrobiální testy citlivosti MeSH
• plazmidy genetika   MeSH
• Providencia MeSH
• vrány * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Spojené státy americké MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč

Fosfomycin (FOS) has been recently reintroduced into clinical practice, but its effectiveness against multidrug-resistant (MDR) Enterobacterales is reduced due to the emergence of FOS resistance. The copresence of carbapenemases and FOS resistance could drastically limit antibiotic treatment. The aims of this study were (i) to investigate fosfomycin susceptibility profiles among carbapenem-resistant Enterobacterales (CRE) in the Czech Republic, (ii) to characterize the genetic environment of fosA genes among the collection, and (iii) to evaluate the presence of amino acid mutations in proteins involved in FOS resistance mechanisms. During the period from December 2018 to February 2022, 293 CRE isolates were collected from different hospitals in the Czech Republic. FOS MICs were assessed by the agar dilution method (ADM), FosA and FosC2 production was detected by the sodium phosphonoformate (PPF) test, and the presence of fosA-like genes was confirmed by PCR. Whole-genome sequencing was conducted with an Illumina NovaSeq 6000 system on selected strains, and the effect of point mutations in the FOS pathway was predicted using PROVEAN. Of these strains, 29% showed low susceptibility to fosfomycin (MIC, ≥16 μg/mL) by ADM. An NDM-producing Escherichia coli sequence type 648 (ST648) strain harbored a fosA10 gene on an IncK plasmid, while a VIM-producing Citrobacter freundii ST673 strain harbored a new fosA7 variant, designated fosA7.9. Analysis of mutations in the FOS pathway revealed several deleterious mutations occurring in GlpT, UhpT, UhpC, CyaA, and GlpR. Results regarding single substitutions in amino acid sequences highlighted a relationship between ST and specific mutations and an enhanced predisposition for certain STs to develop resistance. This study highlights the occurrence of several FOS resistance mechanisms in different clones spreading in the Czech Republic. IMPORTANCE Antimicrobial resistance (AMR) currently represents a concern for human health, and the reintroduction of antibiotics such as fosfomycin into clinical practice can provide further option in treatment of multidrug-resistant (MDR) bacterial infections. However, there is a global increase of fosfomycin-resistant bacteria, reducing its effectiveness. Considering this increase, it is crucial to monitor the spread of fosfomycin resistance in MDR bacteria in clinical settings and to investigate the resistance mechanism at the molecular level. Our study reports a large variety of fosfomycin resistance mechanisms among carbapenemase-producing Enterobacterales (CRE) in the Czech Republic. Our study summarizes the main achievements of our research on the use of molecular technologies, such as next-generation sequencing (NGS), to describe the heterogeneous mechanisms that reduce fosfomycin effectiveness in CRE. The results suggest that a program for widespread monitoring of fosfomycin resistance and epidemiology fosfomycin-resistant organisms can aide timely implementation of countermeasures to maintain the effectiveness of fosfomycin.

• Klíčová slova
• Enterobacterales, WGS, carbapenemase producers, drug-resistance bacteria, fosfomycin,
• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální léková rezistence genetika   MeSH
• beta-laktamasy genetika   MeSH
• Escherichia coli MeSH
• fosfomycin * farmakologie   MeSH
• karbapenemy farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• antibakteriální látky MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• fosfomycin * MeSH
• karbapenemy MeSH

Fosfomycin (FOS) is an effective antibiotic against multidrug-resistant Enterobacterales, but its effectiveness is reducing. Little is known on the current prevalence of FosA enzymes in low-risk pathogens, such as Citrobacter freundii. The aim of the study was the molecular characterization of a carbapenemase- and FosA-producing C. freundii collected in Italy. AK867, collected in 2023, showed an XDR profile, retaining susceptibility only to colistin. AK867 showed a FOS MIC >128 mg/L by ADM. Based on WGS, AK867 belonged to ST116 and owned a wide resistome, including fosA3, blaKPC-2, and blaVIM-1. fosA3 was carried by a conjugative pKPC-CAV1312 plasmid of 320,480 bp, on a novel composite transposon (12,907 bp). FosA3 transposon shared similarities with other fosA3-harboring pKPC-CAV1312 plasmids among Citrobacter spp. We report the first case of FosA3 production in clinical carbapenemase-producing C. freundii ST116. The incidence of FosA3 enzymes is increasing among Enterobacterales, affecting even low-virulence pathogens, as C. freundii.

• Klíčová slova
• Citrobacter freundii, carbapenemases, fosA3 gene, fosfomycin, fosfomycin resistance,
• MeSH
• antibakteriální látky * farmakologie   MeSH
• bakteriální proteiny * genetika   metabolismus   MeSH
• beta-laktamasy * genetika   metabolismus   MeSH
• Citrobacter freundii * genetika   enzymologie   účinky léků   MeSH
• enterobakteriální infekce * mikrobiologie   MeSH
• fosfomycin * farmakologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mnohočetná bakteriální léková rezistence genetika   MeSH
• plazmidy genetika   MeSH
• sekvenování celého genomu MeSH
• transpozibilní elementy DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Itálie epidemiologie   MeSH
• Názvy látek
• antibakteriální látky * MeSH
• bakteriální proteiny * MeSH
• beta-laktamasy * MeSH
• carbapenemase MeSH Prohlížeč
• fosfomycin * MeSH
• transpozibilní elementy DNA MeSH

Carbapenemase-producing Enterobacteriaceae and Pseudomonas spp. are increasingly reported in many countries all over the world. Due to the resistance of those bacteria to almost all antibiotics (e.g.beta-lactams, aminoglycosides, fluoroquinolones),treatment options are seriously limited. In the Czech Republic, the incidence of carbapenemase-producing Enterobacteriaceae seems to be low, restricted to only three cases detected between 2009 and 2010.Here, we describe molecular typing of 15 carbapenemase-producing Klebsiella pneumoniae isolates identified in the Czech Republic during 2011. Five VIM-1-producing isolates belonging to sequence type (ST)11 and one VIM-4-producing isolate of ST1029 have been detected. blaVIM-1 and blaVIM-4 as a part of class 1 integrons were chromosomally located or carried by a plasmid belonging to A/C replicon type (blaVIM-4). KPC-3-producing isolates of ST512, recovered from six patients, caused an outbreak. Three more isolates producing KPC-2 enzyme belonged to ST258. Both blaKPCgenes were part of the Tn4401a transposon carried on plasmids of the pKpQIL type. The isolates were resistant to all antibiotics tested except colistin and/or gentamicin.Four of these 15 strains were recovered from patients repatriated to the Czech Republic from Greece and Italy. This is the first report of outbreaks caused by carbapenemase-producing Enterobacteriaceae in the Czech Republic.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální proteiny metabolismus   MeSH
• beta-laktamasy metabolismus   MeSH
• beta-laktamová rezistence MeSH
• cestování MeSH
• incidence MeSH
• infekce bakteriemi rodu Klebsiella diagnóza   enzymologie   epidemiologie   MeSH
• integrony genetika   MeSH
• karbapenemy farmakologie   MeSH
• Klebsiella pneumoniae klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mnohočetná bakteriální léková rezistence MeSH
• molekulární typizace MeSH
• plazmidy genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Itálie MeSH
• Řecko MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• karbapenemy MeSH

ST252 Enterobacter cloacae, producing GES-5 carbapenemase, was isolated in a Czech hospital. blaGES-5 was part of a novel class 1 integron, In1406, which also included a new allele of the aadA15 gene cassette. In1406 was located on a ColE2-like plasmid, pEcl-35771cz (6953bp).

• Klíčová slova
• Class 1 integron, Class A β-lactamase, ColE2-like, Enterobacteriaceae, In1406,
• MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamasy genetika   MeSH
• Enterobacter cloacae enzymologie   genetika   MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• integrony genetika   MeSH
• lidé MeSH
• multilokusová sekvenční typizace MeSH
• nemocnice MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč

Analysis of 594 isolates of Escherichia coli sequence type (ST)131 and its single locus variants carrying carbapenemase genes from 17 European Union/European Economic Area countries revealed acquisition of 18 carbapenemase variants, mainly in ST131 clades A and C. Most frequent were bla OXA-244 (n = 230) and bla OXA-48 (n = 224), detected in 14 and 12 countries, respectively. Isolates carrying bla OXA-244 have increased rapidly since 2021. The increasing detection of carbapenemase genes in the E. coli high-risk lineage ST131 is a public health concern.

• Klíčová slova
• Carbapenem-resistant Enterobacterales, Escherichia coli, antibiotics, antimicrobial resistance, carbapenemase, cross-border spread, surveillance, whole genome sequencing,
• MeSH
• antibakteriální látky farmakologie   terapeutické užití   MeSH
• bakteriální proteiny * genetika   MeSH
• beta-laktamasy * genetika   MeSH
• Escherichia coli * genetika   izolace a purifikace   MeSH
• Evropská unie * MeSH
• infekce vyvolané Escherichia coli * epidemiologie   mikrobiologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• multilokusová sekvenční typizace MeSH
• proteiny z Escherichia coli genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Evropa epidemiologie   MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny * MeSH
• beta-laktamasy * MeSH
• carbapenemase MeSH Prohlížeč
• proteiny z Escherichia coli MeSH

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is used for the determination of molecular weights of different chemical compounds. We describe here the use of MALDI-TOF mass spectrometry to detect a carbapenem antibiotic, meropenem, and its degradation products. Buffered meropenem solution (0.1 mM Tris-HCl, pH 6.8) was mixed with an overnight culture of bacteria. After 3-h incubation, the reaction mixture was centrifuged, and the supernatant was analyzed by MALDI-TOF mass spectrometry. The presence or absence of peaks representing meropenem and its sodium salts was crucial. The average turnaround time of this test, considering the use of overnight culture, is 4 h. We validated this method for the detection of resistance to carbapenems in Enterobacteriaceae and Pseudomonas aeruginosa mediated by carbapenemase production. A total of 124 strains, including 30 carbapenemase-producing strains, were used in the study. The sensitivity of this method is 96.67%, with a specificity of 97.87%. Our results demonstrate the ability of this method to routinely detect carbapenemases in Enterobacteriaceae and Pseudomonas spp. in laboratories. This assay is comparable with a labor-intensive imipenem-hydrolyzing spectrophotometric assay that is a reference method for the detection of carbapenemase. As demonstrated here, MALDI-TOF mass spectrometry may be used in microbiological laboratories not only for microbial identification but also for other applications, such as studies of mechanisms of antibiotic resistance.

• MeSH
• antibakteriální látky analýza   MeSH
• bakteriální proteiny metabolismus   MeSH
• beta-laktamasy metabolismus   MeSH
• Enterobacteriaceae enzymologie   MeSH
• lidé MeSH
• meropenem MeSH
• Pseudomonas aeruginosa enzymologie   MeSH
• senzitivita a specificita MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• thienamyciny analýza   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• meropenem MeSH
• thienamyciny MeSH

Recently, new combinations of β-lactams and β-lactamase inhibitors became available, including ceftazidime-avibactam, and increased the ability to treat infections caused by carbapenem-resistant Enterobacterales (CRE). Despite the reduced time of clinical use, isolates expressing resistance to ceftazidime-avibactam have been reported, even during treatment or in patients with no previous contact with this drug. Here, we detailed review data on global ceftazidime-avibactam susceptibility, the mechanisms involved in resistance, and the molecular epidemiology of resistant isolates. Ceftazidime-avibactam susceptibility remains high (≥ 98.4%) among Enterobacterales worldwide, being lower among extended-spectrum β-lactamase (ESBL) producers and CRE. Alterations in class A β-lactamases are the major mechanism involved in ceftazidime-avibactam resistance, and mutations are mainly, but not exclusively, located in the Ω loop of these enzymes. Modifications in Klebsiella pneumoniae carbapenemase (KPC) 3 and KPC-2 have been observed by many authors, generating variants with different mutations, insertions, and/or deletions. Among these, the most commonly described is Asp179Tyr, both in KPC-3 (KPC-31 variant) and in KPC-2 (KPC-33 variant). Changes in membrane permeability and overexpression of efflux systems may also be associated with ceftazidime-avibactam resistance. Although several clones have been reported, ST258 with Asp179Tyr deserves special attention. Surveillance studies and rationale use are essential to retaining the activity of this and other antimicrobials against class A CRE.

• MeSH
• antibakteriální látky farmakologie   terapeutické užití   MeSH
• azabicyklické sloučeniny MeSH
• bakteriální proteiny genetika   MeSH
• beta-laktamasy genetika   MeSH
• ceftazidim farmakologie   MeSH
• fixní kombinace léků MeSH
• infekce bakteriemi rodu Klebsiella * farmakoterapie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antibakteriální látky MeSH
• avibactam, ceftazidime drug combination MeSH Prohlížeč
• azabicyklické sloučeniny MeSH
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• ceftazidim MeSH
• fixní kombinace léků MeSH

A comparison of carbapenem molecules for the detection of carbapenemase-producing bacteria by MALDI-TOF MS showed that imipenem exhibited higher sensitivity (97%) and specificity (100%) scores for Pseudomonas aeruginosa than meropenem. However, meropenem was more efficient (98% sensitivity and 100% specificity) against Enterobacteriaceae.

• Klíčová slova
• Carbapenems, Enterobacteriaceae, GES, IMP, Pseudomonas aeruginosa, VIM,
• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální proteiny analýza   metabolismus   MeSH
• bakteriologické techniky metody   MeSH
• beta-laktamasy analýza   metabolismus   MeSH
• Enterobacteriaceae účinky léků   enzymologie   MeSH
• imipenem farmakologie   MeSH
• karbapenemy farmakologie   MeSH
• meropenem MeSH
• mikrobiální testy citlivosti MeSH
• Pseudomonas aeruginosa účinky léků   enzymologie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• thienamyciny farmakokinetika   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• imipenem MeSH
• karbapenemy MeSH
• meropenem MeSH
• thienamyciny MeSH