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

BACKGROUND: During the last decade, the prevalence of carbapenem-resistant Enterobacteriaceae in human patients has increased. Carbapenemase-producing bacteria are usually multidrug resistant. Therefore, early recognition of carbapenemase producers is critical to prevent their spread. OBJECTIVES: The objective of this study was to develop the primers for single and/or multiplex PCR amplification assays for simultaneous identification of class A, class B, and class D carbapenem hydrolyzing β-lactamases in Enterobacteriaceae and then to evaluate their efficiency. MATERIALS AND METHODS: The reference sequences of all genes encoding carbapenemases were downloaded from GenBank. Primers were designed to amplify the following 11 genes: bla KPC, bla OXA, bla VIM, bla NDM, bla IMP, bla SME, bla IMI, bla GES , bla GIM, bla DIM and bla CMY . PCR conditions were tested to amplify fragments of different sizes. Two multiplex PCR sets were created for the detection of clinically important carbapenemases. The third set of primers was included for detection of all known carbapenemases in Enterobacteriaceae. They were evaluated using six reference strains and nine clinical isolates. RESULTS: Using optimized conditions, all carbapenemase-positive controls yielded predicted amplicon sizes and confirmed the specificity of the primers in single and multiplex PCR. CONCLUSIONS: We have reported here a reliable method, composed of single and multiplex PCR assays, for screening all clinically known carbapenemases. Primers tested in silico and in vitro may distinguish carbapenem-resistant Enterobacteriaceae and could assist in combating the spread of carbapenem resistance in Enterobacteriaceae.

• Publikační typ
• časopisecké články MeSH

Nalezení spolehlivé metody detekce karbapenemáz u enterobakterií a některých gramnegativních nefermentujících tyček je velkou výzvou současné klinicko-mikrobiologické diagnostiky. Tento článek shrnuje možnosti detekce karbapenemáz u enterobakterií inhibičními fenotypovými testy, pomocí MALDI-TOF hmotnostní spektrometrie a molekulárně-mikrobiologickými metodami.

Finding a reliable method for the detection of carbapenemases in enterobacteria and some Gram-negative nonfermenting rods is a big challenge for current clinical microbiological diagnosis. This article summarizes the possibilities for carbapenemase detection in enterobacteria provided by inhibitor-based phenotypic tests, MALDI- TOF mass spectrometry, and molecular microbiology techniques.

• Klíčová slova
• KPC, MBL, metropenem, DDST,
• MeSH
• beta-laktamasy analýza   izolace a purifikace   MeSH
• Enterobacteriaceae izolace a purifikace   patogenita   MeSH
• fenotyp MeSH
• financování organizované MeSH
• karbapenemy terapeutické užití   MeSH
• lidé MeSH
• mikrobiologické techniky metody   využití   MeSH
• molekulární biologie metody   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   využití   MeSH
• Check Tag
• lidé MeSH

The resistance to carbapenems is usually mediated by enzymes hydrolyzing β-lactam ring. Recently, an alternative way of the modification of the antibiotic, a β-lactone formation by OXA-48-like enzymes, in some carbapenems was identified. We focused our study on a deep analysis of OXA-48-like-producing Enterobacterales, especially strains showing poor hydrolytic activity. In this study, well characterized 74 isolates of Enterobacterales resistant to carbapenems were used. Carbapenemase activity was determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), liquid chromatography/mass spectrometry (LC-MS), Carba-NP test and modified Carbapenem Inactivation Method (mCIM). As meropenem-derived β-lactone possesses the same molecular weight as native meropenem (MW 383.46 g/mol), β-lactonization cannot be directly detected by MALDI-TOF MS. In the spectra, however, the peaks of m/z = 340.5 and 362.5 representing decarboxylated β-lactone and its sodium adduct were detected in 25 out of 35 OXA-48-like producers. In the rest 10 isolates, decarboxylated hydrolytic product (m/z = 358.5) and its sodium adduct (m/z = 380.5) have been detected. The peak of m/z = 362.5 was detected in 3 strains co-producing OXA-48-like and NDM-1 carbapenemases. The respective signal was identified in no strain producing class A or class B carbapenemase alone showing its specificity for OXA-48-like carbapenemases. Using LC-MS, we were able to identify meropenem-derived β-lactone directly according to the different retention time. All strains with a predominant β-lactone production showed negative results of Carba NP test. In this study, we have demonstrated that the strains producing OXA-48-like carbapenemases showing false-negative results using Carba NP test and MALDI-TOF MS preferentially produced meropenem-derived β-lactone. We also identified β-lactone-specific peak in MALDI-TOF MS spectra and demonstrated the ability of LC-MS to detect meropenem-derived β-lactone.

• MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální proteiny * analýza   MeSH
• beta-laktamasy analýza   MeSH
• Enterobacteriaceae * MeSH
• karbapenemy farmakologie   MeSH
• meropenem farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Publikační typ
• časopisecké články MeSH

Multirezistentní kmen Acinetobacter baumannii nesoucí gen pro metalo-ß-laktamázu NDM-1 byl letos v červenci zachycen u pacienta hospitalizovaného na oddělení intenzivní péče v Ústí nad Labem. Kmen byl importován z Egypta a je in vitro rezistentní k ß-laktamům a ostatním klinicky relevantním antibiotikům kromě kolistinu, tobramycinu, netilmicinu, doxycyklinu a tigecyklinu. Nese též gen pro karbapemázu OXA-23 (v České republice dosud nedoložený) a řadu dalších genetických determinant rezistence.

A multidrug-resistant Acinetobacter baumannii strain carrying the gene encoding metallo-ß-lactamase NDM-1 was isolated from a patient hospitalized in an intensive care unit in Ústí nad Labem. The strain was imported from Egypt and is in vitro resistant to ß-lactams and to other clinically relevant antimicrobial agents except for colistin, tobramycin, netilmicin, doxycycline and tigecycline. It also carries the gene encoding the OXA-23 carbapenemase (not yet reported from the Czech Republic) and a number of other resistance determinants.

• Klíčová slova
• genotypizace, rezistence ke karbapenemům, metalo-ß-laktamáza,
• MeSH
• Acinetobacter baumannii genetika   patogenita   účinky léků   MeSH
• bakteriální léková rezistence účinky léků   MeSH
• infekce bakteriemi rodu Acinetobacter farmakoterapie   genetika   MeSH
• karbapenemy terapeutické užití   MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Geografické názvy
• Česká republika MeSH
• Egypt MeSH

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently introduced to many diagnostic microbiological laboratories. Besides the identification of bacteria and fungi, that technique provides a potentially useful tool for the detection of antimicrobial resistance, especially of that conferred by β-lactamases. Here, we describe an assay allowing a detection of meropenem hydrolysis in clinical isolates of Enterobacteriaceae, Pseudomonas spp., and Acinetobacter baumannii using MALDI-TOF MS. This method is able to confirm carbapenemases within 3 h. The results are important for proper and fast intervention to limit the spread of carbapenemase-producing bacteria and provide information for appropriate initial therapy of the infections caused by these microbes.

• MeSH
• Acinetobacter baumannii účinky léků   enzymologie   genetika   izolace a purifikace   MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• beta-laktamová rezistence genetika   MeSH
• biotest * MeSH
• exprese genu MeSH
• hydrolýza MeSH
• infekce bakteriemi rodu Acinetobacter diagnóza   farmakoterapie   mikrobiologie   MeSH
• lidé MeSH
• pseudomonádové infekce diagnóza   farmakoterapie   mikrobiologie   MeSH
• Pseudomonas účinky léků   enzymologie   genetika   izolace a purifikace   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• thienamyciny farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a potentially useful tool for the detection of antimicrobial resistance, especially that conferred by β-lactamases. Here we describe a modification of a previously reported MALDI-TOF MS meropenem hydrolysis assay. The modified method was validated on 108 carbapenemase-producing members of the Enterobacteriaceae, two NDM-1-producing Acinetobacter baumannii isolates, and 35 carbapenem-resistant enterobacteria producing no carbapenemase. The detection of carbapenemases by MALDI-TOF MS seems to be a powerful, quick, and cost-effective method for microbiological laboratories.

• MeSH
• Acinetobacter baumannii enzymologie   MeSH
• bakteriální proteiny metabolismus   MeSH
• beta-laktamasy metabolismus   MeSH
• Enterobacteriaceae enzymologie   MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• hydrolýza MeSH
• lidé MeSH
• mikrobiální testy citlivosti metody   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• thienamyciny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH

• MeSH
• antibakteriální látky diagnostické užití   MeSH
• bakteriální léková rezistence MeSH
• bakteriální proteiny izolace a purifikace   MeSH
• beta-laktamasy izolace a purifikace   MeSH
• beta-laktamová rezistence MeSH
• Enterobacteriaceae enzymologie   MeSH
• financování organizované MeSH
• Klebsiella pneumoniae enzymologie   MeSH
• metaloproteiny izolace a purifikace   MeSH
• mikrobiální testy citlivosti metody   MeSH

• MeSH
• bakteriální léková rezistence MeSH
• beta-laktamasy škodlivé účinky   MeSH
• beta-laktamová rezistence účinky léků   MeSH
• enterobakteriální infekce epidemiologie   prevence a kontrola   MeSH
• lidé MeSH
• mezinárodní spolupráce MeSH
• Check Tag
• lidé MeSH

• MeSH
• bakteriální proteiny * genetika   MeSH
• beta-laktamasy * genetika   analýza   MeSH
• laktony metabolismus   MeSH
• lidé MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• dopisy MeSH