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.

• Klíčová slova
• Carba NP, Enterobacteriaceae, MALDI-TOF MS, carbapenemase, detection, β-Lactamase,
• 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
• Názvy látek
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč

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.

• Klíčová slova
• Carbapenems, Drug Resistance, Enterobacteriaceae, Multiplex Polymerase Chain Reaction,
• Publikační typ
• časopisecké články 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
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny * MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• karbapenemy MeSH
• meropenem 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
• meropenem 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
• Názvy látek
• antibakteriální látky MeSH
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• meropenem MeSH
• thienamyciny 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
• meropenem 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
• Názvy látek
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• meropenem MeSH
• thienamyciny MeSH

In this study, we evaluate the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories during a multicenter networking validation study. The study was divided into three different stages: "software design," "intercenter evaluation," and "clinical validation." First, a standardized procedure with an online software for data analysis was designed. Carbapenem resistance was detected by measuring imipenem hydrolysis and the results were automatically interpreted using the Clover MS data analysis software (Clover BioSoft, Spain). Second, a series of 74 genotypically characterized Enterobacterales (46 carbapenemase-producers and 28 non carbapenemase-producers) were analyzed in 8 international centers to ensure the reproducibility of the method. Finally, the methodology was evaluated independently in all centers during a 2-month period and results were compared with the reference standard for carbapenemase detection used in each center. The overall agreement rate relative to the reference method for carbapenemase resistance detection in clinical samples was 92.5%. The sensitivity was 93.9% and the specificity, 100%. Results were obtained within 60 min and accuracy ranged from 83.3 to 100% among the different centers. Further, our results demonstrate that MALDI-TOF MS is an outstanding tool for rapid detection of carbapenemase activity in Enterobacterales in clinical microbiology laboratories. The use of a simple in-house procedure with online software allows routine screening of carbapenemases in diagnostics, thereby facilitating early and appropriate antimicrobial therapy.

• Klíčová slova
• MALDI-TOF MS, carbapenemases enzymes, clinical microbiology, imipenem, resistance detection,
• Publikační typ
• časopisecké články 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

• 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
• Názvy látek
• bakteriální proteiny * MeSH
• beta-laktamasy * MeSH
• carbapenemase MeSH Prohlížeč
• laktony MeSH

To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepò Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. Enterobacteriaceae were also characterized for the Extended-Spectrum-β-Lactamases (ESBLs), carbapenemases, and mcr-1 genes presence. From December 2014 to November 2015, 246 water samples were filtered, plated on Plate Count Agar, MacConkey Agar, and MacConkey Agar with cefotaxime. Isolates were species identified using AutoSCAN-4-System and ESBLs, carbapenemases, and colistin resistance determinants were characterized by PCR, sequencing, and microarray. Plasmid conjugative transfer experiments, PCR-based Replicon typing, Pulsed-Field Gel Electrophoresis, Multi-Locus-Sequence-Typing, and in-silico plasmid characterization were performed. A total of 132 enterobacteria isolates grew on MacConkey agar with cefotaxime: 82 (62.1%) were obtained from streams, 41 (31.1%) from treatment plants, and 9 (6.8%) from wells. Thirty out of 132 (22.7%) isolates, mainly belonging to Escherichia coli (n = 15) species, showed a synergic effect with piperacillin-tazobactam. A single ESBL gene of blaCTX-M-type was identified in 19/30 isolates. In further two E. coli strains, a blaCTX-M-1 gene co-existed with a blaSHV-type ESBL determinant. A blaSHV-12 gene was detected in two isolates of E. coli (n = 1) and Klebsiella oxytoca (n = 1), while any ESBL determinant was ascertained in seven Yersinia enterocolitica strains. A blaDHA-type gene was detected in a cefoxitin resistant Y. enterocolitica from a stream. Interestingly, two Klebsiella pneumoniae strains of ST307 and ST258, collected from a well and a wastewater treatment plant, resulted KPC-2, and KPC-3 producers, respectively. Moreover, we report the first detection of mcr-1.2 ST10 E. coli on a conjugative IncX4 plasmid (33.303 bp in size) from a stream of Oltrepò Pavese (Northern Italy). Both ESBLs E. coli and ESBLs/carbapenemase-producing K. pneumoniae strains showed clonal heterogeneity by Pulsed-Field Gel Electrophoresis and Multi-Locus-Sequence-Typing. During one-year study and taking in account the whole Gram-negative bacterial population, an average percentage of cefotaxime resistance of 69, 32, and 10.3% has been obtained for the wastewater treatment plants, streams, and wells, respectively. These results, of concern for public health, highlight the need to improve hygienic measures to reduce the load of discharged bacteria with emerging resistance mechanisms.

• Klíčová slova
• Gram-negative bacteria, carbapenemases, colistin resistance, molecular characterization, water ecosystems,
• Publikační typ
• časopisecké články MeSH

Klebsiella pneumoniae, one of the most common pathogens found in hospital-acquired infections, is often resistant to multiple antibiotics. In fact, multidrug-resistant (MDR) K. pneumoniae producing KPC or OXA-48-like carbapenemases are recognized as a serious global health threat. In this sense, we evaluated the virulence of K. pneumoniae KPC(+) or OXA-48(+) aiming at potential antimicrobial therapeutics. K. pneumoniae carbapenemase (KPC) and the expanded-spectrum oxacillinase OXA-48 isolates were obtained from patients treated in medical care units in Lisbon, Portugal. The virulence potential of the K. pneumonia clinical isolates was tested using the Galleria mellonella model. For that, G. mellonella larvae were inoculated using patients KPC(+) and OXA-48(+) isolates. Using this in vivo model, the KPC(+) K. pneumoniae isolates showed to be, on average, more virulent than OXA-48(+). Virulence was found attenuated when a low bacterial inoculum (one magnitude lower) was tested. In addition, we also report the use of a synthetic polycationic oligomer (L-OEI-h) as a potential antimicrobial agent to fight infectious diseases caused by MDR bacteria. L-OEI-h has a broad-spectrum antibacterial activity and exerts a significantly bactericidal activity within the first 5-30 min treatment, causing lysis of the cytoplasmic membrane. Importantly, the polycationic oligomer showed low toxicity against in vitro models and no visible cytotoxicity (measured by survival and health index) was noted on the in vivo model (G. mellonella), thus L-OEI-h is foreseen as a promising polymer therapeutic for the treatment of MDR K. pneumoniae infections.

• Klíčová slova
• Galleria mellonella infection model, KPC and OXA-48-like carbapenemases, Klebsiella pneumoniae, linear oligoethyleneimine hydrochloride,
• Publikační typ
• časopisecké články MeSH