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.
- 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
- Geografické názvy
- Česká republika MeSH
OBJECTIVES: This study examined the antimicrobial susceptibility and resistance mechanisms of Clostridium difficile recovered in Greek hospitals during 2012-2015. METHODS: C. difficile isolates (n=88) were collected from clinically-confirmed C. difficile infection from symptomatic patients in 10 Greek hospitals. Minimum inhibitory concentrations (MICs) of various antimicrobial agents were determined by Etest. Isolates were typed by multilocus sequence typing (MLST). Toxin and resistance genes were detected by PCR. Chromosomal mutations in gyrA, gyrB and rpoB were identified by PCR and sequencing. The genetic environment of resistance genes was characterised by Illumina sequencing. RESULTS: The 88 C. difficile isolates comprised 27 sequence types (STs), with ST37 (n=26) and ST11 (n=21) being the most prevalent. All isolates were susceptible to vancomycin and metronidazole, with variable resistance rates to other antimicrobials. Of the 88 isolates, 45.5% were multidrug-resistant and the majority belonged to ST11 and ST37. The presence of chromosomal mutations in gyrA, gyrB and rpoB was mainly observed in high-risk clones such as ST11 and ST37. The antimicrobial resistance genes ermB, mefA, msrA and tetM were identified at different prevalences and combinations. Additionally, cfrB and cfrC were identified for the first time in Greece and were carried by a Tn6218 transposon and a novel plasmid, respectively. CONCLUSIONS: To our knowledge, this is the first study examining the resistance profiles and respective mechanisms of C. difficile recovered in Greek hospitals. Gut commensals such as C. difficile may serve as hubs for further transfer of antimicrobial resistance genes.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální proteiny genetika MeSH
- Clostridioides difficile klasifikace účinky léků genetika MeSH
- klostridiové infekce mikrobiologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- mnohočetná bakteriální léková rezistence genetika MeSH
- multilokusová sekvenční typizace MeSH
- mutace MeSH
- nemocnice MeSH
- techniky typizace bakterií MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Řecko MeSH
BACKGROUND: The implementation of MALDI-TOF MS for microorganism identification has changed the routine of the microbiology laboratories as we knew it. Most microorganisms can now be reliably identified within minutes using this inexpensive, user-friendly methodology. However, its application in the identification of mycobacteria isolates has been hampered by the structure of their cell wall. Improvements in the sample processing method and in the available database have proved key factors for the rapid and reliable identification of non-tuberculous mycobacteria isolates using MALDI-TOF MS. AIMS: The main objective is to provide information about the proceedings for the identification of non-tuberculous isolates using MALDI-TOF MS and to review different sample processing methods, available databases, and the interpretation of the results. SOURCES: Results from relevant studies on the use of the available MALDI-TOF MS instruments, the implementation of innovative sample processing methods, or the implementation of improved databases are discussed. CONTENT: Insight about the methodology required for reliable identification of non-tuberculous mycobacteria and its implementation in the microbiology laboratory routine is provided. IMPLICATIONS: Microbiology laboratories where MALDI-TOF MS is available can benefit from its capacity to identify most clinically interesting non-tuberculous mycobacteria in a rapid, reliable, and inexpensive manner.
- MeSH
- atypické mykobakteriální infekce diagnóza MeSH
- bakteriologické techniky MeSH
- lidé MeSH
- netuberkulózní mykobakterie izolace a purifikace MeSH
- průběh práce MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Sequence type 11 Klebsiella pneumoniae, coproducing NDM-1 and VIM-1 metallo-β-lactamases, were isolated in a Greek hospital. blaNDM-1 was part of a Tn125 derivative, located on an ~90-kb plasmid similar to the NDM-1-encoding plasmid pB-3002cz. blaVIM-1 was located in an In-e541-like integron, carried on a multireplicon (IncA/C and IncR) plasmid of ~180kb.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- beta-laktamasy genetika metabolismus MeSH
- infekce bakteriemi rodu Klebsiella farmakoterapie mikrobiologie MeSH
- karbapenemy farmakologie MeSH
- Klebsiella pneumoniae klasifikace genetika izolace a purifikace MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- nemocnice MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Řecko MeSH
OBJECTIVES: An Enterococcus faecium isolate (Efa-125) carrying both the vanA and vanB genes was recovered from a patient with bacteraemia treated in a Greek hospital. Since this is the first description in Europe of E. faecium carrying both vanA and vanB genes, the isolate was further studied. METHODS: Susceptibility to several antibiotics was determined using the VITEK®2 automated system. The isolate was typed by multilocus sequence typing (MLST). To define the genetic units of the vanA and vanB genes, the plasmid content of Efa-125 was analysed by pulsed-field gel electrophoresis (PFGE) of total DNA digested with S1 nuclease followed by hybridisation with digoxigenin-labelled vanA and vanB probes. In addition, plasmids and chromosomes were sequenced using the Illumina MiSeq platform. RESULTS: E. faecium Efa-125 belonged to ST117 and expressed resistance both to vancomycin and teicoplanin, with minimum inhibitory concentrations (MICs) for both of 256mg/L. The vanA gene was carried on a 29 320-bp plasmid exhibiting high similarity to pA6981 previously characterised from Enterococcus gallinarum A6981, whereas vanB was part of a Tn1549-like transposon integrated into the chromosome. Expression of the VanA phenotype was correlated with the presence of intact vanZ and vanS genes. CONCLUSIONS: This is the first detection in Greece of vanA-vanB genotype/VanA phenotype E. faecium and indicates an evolving epidemiology of vancomycin-resistant enterococci.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální geny genetika MeSH
- bakteriální proteiny genetika MeSH
- bakteriemie mikrobiologie MeSH
- Enterococcus faecium účinky léků genetika izolace a purifikace patogenita MeSH
- enterokoky rezistentní vůči vankomycinu genetika MeSH
- fenotyp MeSH
- genotyp MeSH
- grampozitivní bakteriální infekce mikrobiologie MeSH
- lidé MeSH
- ligasy tvořící vazby C-O genetika MeSH
- mikrobiální testy citlivosti MeSH
- molekulární epidemiologie * MeSH
- multilokusová sekvenční typizace MeSH
- nemocnice MeSH
- plazmidy genetika MeSH
- proteinkinasy genetika MeSH
- pulzní gelová elektroforéza MeSH
- regulace genové exprese u bakterií MeSH
- rezistence na vankomycin MeSH
- teikoplanin farmakologie MeSH
- transkripční faktory genetika MeSH
- transpozibilní elementy DNA MeSH
- vankomycin farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Řecko MeSH
The sequence type 11 Klebsiella pneumoniae strain Kpn-3002cz was confirmed to harbor two NDM-1-encoding plasmids, pB-3002cz and pS-3002cz. pB-3002cz (97,649 bp) displayed extensive sequence similarity with the blaNDM-1-carrying plasmid pKPX-1. pS-3002cz (73,581 bp) was found to consist of an IncR-related sequence (13,535 bp) and a mosaic region (60,046 bp). A 40,233-bp sequence of pS-3002cz was identical to the mosaic region of pB-3002cz, indicating the en bloc acquisition of the NDM-1-encoding region from one plasmid by the other.
- MeSH
- beta-laktamasy genetika MeSH
- Klebsiella pneumoniae genetika MeSH
- plazmidy genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Carbapenemase-producing bacteria have now spread all over the world. Infections caused by those bacteria are difficult to treat. Therefore, there is an urgent need for accurate and fast detection of carbapenemases in diagnostic laboratories. In this review, we summarize screening methods for suspected isolates, direct assays for confirmation of carbapenemase activity (e.g. the Carba NP test and matrix-assisted laser desorption ionization time-of-flight mass spectrometry carbapenem hydrolysis assay), inhibitor-based methods for carbapenemase classification, and molecular-genetic techniques for precise identification of carbapenemase genes. We also propose a workflow for carbapenemase identification in diagnostic laboratories.
- MeSH
- bakteriální proteiny analýza genetika MeSH
- bakteriologické techniky metody MeSH
- beta-laktamasy analýza genetika MeSH
- diagnostické techniky molekulární metody MeSH
- Enterobacteriaceae enzymologie genetika MeSH
- lidé MeSH
- plošný screening metody MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
This study exploited the possibility to detect Citrobacter freundii-derived CMY-2-like cephalosporinases in Enterobacteriaceae clinical isolates using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Periplasmic proteins were prepared using a modified sucrose method and analyzed by MALDI-TOF MS. A ca. 39,850-m/z peak, confirmed to represent a C. freundii-like β-lactamase by in-gel tryptic digestion followed by MALDI-TOF/TOF MS, was observed only in CMY-producing isolates. We have also shown the potential of the assay to detect ACC- and DHA-like AmpC-type β-lactamases.
