The objective of this study was to assess the susceptibility of cefiderocol against multidrug-resistant carbapenemase-producing and nonproducing bacteria. The panel comprised 182 isolates of the order Enterobacterales, and 40 strains of Pseudomonas aeruginosa. Antimicrobial susceptibility testing has been performed using broth microdilution method according to the European Committee on Antimicrobial Susceptibility Testing recommendations. Mass spectrometry matrix-assisted laser desorption/ionization-time of flight mass spectrometry and carbapenemase-producing test were used to verify the presence of carbapenemases in clinical isolates. The genetic expression of single carbapenemases (blaKPC, blaOXA-48, blaNDM, blaVIM, blaIMP, blaGES) was determined by real-time polymerase chain reaction. Cefiderocol exhibited a good activity against the majority of strains tested in this study. Altogether, growth of 81.9% (n = 149) strains of the order Enterobacterales and 77.5% (n = 31) of P. aeruginosa isolates were inhibited at minimal inhibitory concentration (MIC) ≤2 mg/L. Values MIC50/MIC90 were 0.5/8 mg/L for enterobacteria, and 1/8 mg/L for P. aeruginosa. One isolate (Klebsiella pneumoniae) harboring two carbapenemases (blaOXA-48, blaNDM) had cefiderocol MIC 0.5 mg/L. In enterobacteria resistant to cefiderocol, blaNDM carbapenemase prevailed (43.3%, n = 29), followed by blaOXA-48 (31.3%, n = 21) and blaKPC (4.5%, n = 3). blaIMP (n = 8) and blaVIM (n = 1) metallo-β-lactamases dominated in cefiderocol-resistant P. aeruginosa (n = 9) isolates. Very good susceptibility (100%) to this drug showed blaGES-positive strains of P. aeruginosa (n = 8) and isolates resistant to meropenem without confirmed carbapenemase gene (n = 10). In this study, cefiderocol demonstrated potent activity against important nosocomial pathogens, therefore, therapeutic options of this drug against multidrug-resistant bacteria should be considered.

• Klíčová slova
• Enterobacterales, Pseudomonas aeruginosa, carbapenemase, cefiderocol,
• MeSH
• antibakteriální látky * farmakologie   MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• cefiderokol MeSH
• Enterobacteriaceae genetika   metabolismus   MeSH
• karbapenemy * farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• Pseudomonas aeruginosa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky * MeSH
• beta-laktamasy MeSH
• karbapenemy * 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

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

Silver gulls carry phylogenetically diverse Escherichia coli, including globally dominant extraintestinal pathogenic E. coli (ExPEC) sequence types and pandemic ExPEC-ST131 clades; however, our large-scale study (504 samples) on silver gulls nesting off the coast of New South Wales identified E. coli ST457 as the most prevalent. A phylogenetic analysis of whole-genome sequences (WGS) of 138 ST457 samples comprising 42 from gulls, 2 from humans (Australia), and 14 from poultry farmed in Paraguay were compared with 80 WGS deposited in public databases from diverse sources and countries. E. coli ST457 strains are phylogenetic group F, carry fimH145, and partition into five main clades in accordance to predominant flagella H-antigen carriage. Although we identified considerable phylogenetic diversity among the 138 ST457 strains, closely related subclades (<100 SNPs) suggested zoonotic or zooanthroponosis transmission between humans, wild birds, and food-producing animals. Australian human clinical and gull strains in two of the clades were closely related (≤80 SNPs). Regarding plasmid content, country, or country/source, specific connections were observed, including I1/ST23, I1/ST314, and I1/ST315 disseminating blaCMY-2 in Australia, I1/ST113 carrying blaCTX-M-8 and mcr-5 in Paraguayan poultry, and F2:A-:B1 plasmids of Dutch origin being detected across multiple ST457 clades. We identified a high prevalence of nearly identical I1/ST23 plasmids carrying blaCMY-2 among Australian gull and clinical human strains. In summary, ST457 is a broad host range, geographically diverse E. coli lineage that can cause human extraintestinal disease, including urinary tract infection, and displays a remarkable ability to capture mobile elements that carry and transmit genes encoding resistance to critically important antibiotics.

• Klíčová slova
• AmpC, ESBL, ExPEC, I1 plasmids, ST457,
• MeSH
• antibakteriální látky farmakologie   MeSH
• beta-laktamasy genetika   MeSH
• beta-laktamy MeSH
• divoká zvířata MeSH
• Escherichia coli * genetika   MeSH
• fylogeneze MeSH
• infekce vyvolané Escherichia coli * veterinární   MeSH
• lidé MeSH
• plazmidy genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Austrálie MeSH
• Názvy látek
• antibakteriální látky MeSH
• beta-laktamasy MeSH
• beta-laktamy MeSH

In the last two decades, microbiology laboratories have radically changed by the introduction of novel technologies, like Next-Generation Sequencing (NGS) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Nevertheless, emergence of antibiotic-resistant microorganisms represents a global threat of current medicine, being responsible for increasing mortality and health-care direct and indirect costs. In addition, the identification of antibiotic-resistant microorganisms, like OXA-48 carbapenemase-producing Enterobacteriaceae, has been changeling for clinical microbiology laboratories. Even the cost of NGS technology and MALDI-TOF MS equipment is relatively high, both technologies are increasingly used in diagnostic and research protocols. Therefore, the aim of this review is to present applications of these technologies used in clinical microbiology, especially in detection of antibiotic resistance and its surveillance, and to propose a combinatory approach of MALDI-TOF MS and NGS for the investigation of microbial associated infections.

• Klíčová slova
• MLST, NGS, beta-lactamase, carbapenemase, susceptibility testing,
• MeSH
• antibakteriální látky farmakologie   MeSH
• Bacteria klasifikace   účinky léků   genetika   izolace a purifikace   MeSH
• bakteriální infekce diagnóza   mikrobiologie   MeSH
• bakteriální léková rezistence * MeSH
• hmotnostní spektrometrie metody   MeSH
• laboratoře nemocniční MeSH
• lidé MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• antibakteriální látky MeSH

A comparison of a matrix-assisted laser desorption ionization-time of flight mass spectrometric (MALDI-TOF MS) meropenem hydrolysis assay with the Carba NP test showed that both methods exhibited low sensitivity (approximately 76%), mainly due to the false-negative results obtained with OXA-48-type producers. The addition of NH4HCO3 to the reaction buffer for the MALDI-TOF MS assay dramatically improved its sensitivity (98%). Automatic interpretation of the MALDI-TOF MS assay, using the MBT STAR-BL software, generally agreed with the results obtained after manual analysis. For the Carba NP test, spectrophotometric analysis found six additional carbapenemase producers.

• MeSH
• automatizované zpracování dat MeSH
• bakteriální proteiny analýza   MeSH
• beta-laktamasy analýza   MeSH
• hydrogenuhličitany * MeSH
• hydrolýza MeSH
• laboratorní automatizace MeSH
• lidé MeSH
• meropenem MeSH
• pufry MeSH
• senzitivita a specificita MeSH
• software MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• thienamyciny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ammonium bicarbonate MeSH Prohlížeč
• bakteriální proteiny MeSH
• beta-laktamasy MeSH
• carbapenemase MeSH Prohlížeč
• hydrogenuhličitany * MeSH
• meropenem MeSH
• pufry MeSH
• thienamyciny 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.

• MeSH
• bakteriální proteiny metabolismus   MeSH
• cefalosporinasa metabolismus   MeSH
• Enterobacteriaceae enzymologie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• cefalosporinasa MeSH

• MeSH
• antibakteriální látky farmakologie   MeSH
• beta-laktamasy genetika   metabolismus   MeSH
• DNA bakterií chemie   genetika   MeSH
• Enterobacter cloacae účinky léků   enzymologie   genetika   izolace a purifikace   MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• hospitalizace MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• polymerázová řetězová reakce MeSH
• sekvenční analýza DNA MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Srí Lanka MeSH
• Názvy látek
• antibakteriální látky MeSH
• beta-laktamasy MeSH
• DNA bakterií MeSH

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been successfully applied as an identification procedure in clinical microbiology and has been widely used in routine laboratory practice because of its economical and diagnostic benefits. The range of applications of MALDI-TOF MS has been growing constantly, from rapid species identification to labor-intensive proteomic studies of bacterial physiology. The purpose of this review is to summarize the contribution of the studies already performed with MALDI-TOF MS concerning antibiotic resistance and to analyze future perspectives in this field. We believe that current research should continue in four main directions, including the detection of antibiotic modifications by degrading enzymes, the detection of resistance mechanism determinants through proteomic studies of multiresistant bacteria, and the analysis of modifications of target sites, such as ribosomal methylation. The quantification of antibiotics is suggested as a new approach to study influx and efflux in bacterial cells. The results of the presented studies demonstrate that MALDI-TOF MS is a relevant tool for the detection of antibiotic resistance and opens new avenues for both clinical and experimental microbiology.

• MeSH
• antibakteriální látky farmakologie   MeSH
• Bacteria účinky léků   MeSH
• bakteriální léková rezistence * MeSH
• biomedicínský výzkum trendy   MeSH
• mikrobiální testy citlivosti metody   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• antibakteriální látky 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