Antibiotic resistance, molecular epidemiology, mass spectrometry
128
Scopus Publications
Scopus Publications
Clonal outbreak of an extensively drug-resistant NDM-1 producing Pseudomonas aeruginosa in a local hospital in the Czech Republic Katerina Chudejova, Tsolaire Sourenian, Marc Finianos, Anna Sramkova, Costas C. Papagiannitsis, et al. Microbiology Spectrum, 2026 A clonal outbreak of 18 ST773 NDM-1 producing Pseudomonas aeruginosa strains has been detected for the first time in the Czech Republic. The strains were extremely drug-resistant (XDR) and resistant to serum killing. SNP-based phylogeny and CRISPR assay typing showed minimal genomic variations among the isolates. The results suggest a high-risk, persistent, virulent clone causing the hospital outbreak, with the possibility of a nationwide outbreak. IMPORTANCE Our research on the novel detection of the NDM-1 gene in carbapenem-resistant Pseudomonas aeruginosa ST773 in the Czech Republic is of great significance for public health and infection control. Until now, the emergence of this gene in P. aeruginosa strains was uncommon in this region, as carbapenem resistance was primarily associated with IMP and VIM types of MBLs. This nosocomial outbreak was triggered by an index case patient repatriated from areas with reported NDM-1 producing P. aeruginosa , illustrating how international travel contributes to the spread of such resistant pathogens. The results obtained in this study show that it is necessary to focus on tracing the source of infections to control and prevent nosocomial infections, helping to protect public health in the Czech Republic.
Proof-of-concept MALDI-TOF-MS assay for the detection of Toxin B enzymatic activity in Clostridioides difficile infection Josef Dvorak, Lukáš Fojtík, Ljubina Adámková, Katerina Vlkova, Vendula Studentova, et al. Microbiology Spectrum, 2025 Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometers have become an integral part of all modern clinical microbiology laboratories. They serve as the key tool for pathogen identification and antibiotic resistance determination. However, certain limiting conditions must be fulfilled. The pathogen cannot be tested directly from the sample and requires the cultivation of a pure colony, which means that the standard protocol takes additional time, workforce, and consumables. The testing protocol is also more complicated when it comes to anaerobes. In our work, we focused on the functional detection of Clostridioides difficile , an important nosocomial human pathogen that is responsible for diarrhea and can lead to life-threatening colitis, as a model diagnostic problem. The virulence of C. difficile is mainly caused by two toxins, Toxin A and Toxin B. Established diagnostic methods, including nucleic acid amplification testing methods and immunoassays, detect the presence of the microorganism or the presence and concentration of the toxins, with limited ability to gauge infection severity based on the actual biochemical activity of the toxins and thus their potency to cause harm. This work presents proof-of-concept assays that indirectly determine the toxin activity in the human stool, a very complex matrix sample, using the natural RhoA protein as substrate. The RhoA protein substrate was recombinantly prepared with biotin tag modification, which allows its attachment to the NeutrAvidin MALDI chips. In the assay, the RhoA substrate anchored on the MALDI chip undergoes enzymatic glycosylation when exposed to the Toxin B in the stool sample, and the reaction product is then detected by MALDI-TOF mass spectrometry directly from the MALDI chip. The entire assay, from sampling to final mass spectrometry detection, was performed in situ , on the NeutrAvidin MALDI chip, which was prepared by unique surface modification technology also described in this work. The assay was successfully tested for the detection of Toxin B in a cohort of patient samples as well as in cell culture of C. difficile . IMPORTANCE The diagnostics of Clostridioides difficile infection is usually based on the identification of the bacterial pathogen and/or on the detection of the Toxins A and B. Due to the variance in Toxins A and B activity across species, the toxin concentration determined by standard methods does not necessarily correlate with the severity of the disease. Assays that would target toxins’ enzymatic activity are not routinely used because the requirements are unsuitable for clinical laboratories. In this study, we demonstrate a new approach that determines the presence and potency of Toxin B indirectly by determining its enzymatic activity rather than its concentration. This is performed by detecting mass difference due to glycosylation of RhoA substrate by Toxin B, which is then determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented proof-of-concept assay thus offers the possibility to quickly determine the activity of C. difficile toxins directly in the stool samples without pathogen cultivation.
Mass spectrometric profiling of microbial polysaccharides using laser desorption/ionization – time-of-flight (LDI-TOF) and liquid chromatography-mass spectrometry (LC-MS): a novel method for structural fingerprinting and derivatization Lucia Dadovska, Veronika Paskova, Petr Novak, Jaroslav Hrabak Frontiers in Cellular and Infection Microbiology, 2025 IntroductionOver the last two decades, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been introduced into the routine diagnostic practice of microbiological laboratories for the rapid taxonomic identification of bacteria and yeasts. However, a method that effectively identifies microbes directly from clinical samples using MALDI-TOF MS has not yet been found. One of the promising targets is microbial polysaccharides, which are abundant structures in bacterial and fungal cells. Their rapid and inexpensive analysis, nevertheless, is complicated. This study focused on detecting microbial polysaccharides, such as lipopolysaccharides, using MALDI-TOF MS and liquid chromatography-tandem mass spectrometry (LC-MS). We developed a method for fingerprinting polysaccharides by acid hydrolysis and enzymatic digestion.MethodsThe mono- and oligosaccharides are then derivatized with a newly designed probe (vanillyl pararosaniline, the HD ligand), enabling efficient ionization without the use of the MALDI matrix. For precise analysis of polysaccharides, the hydroxyl groups can be esterified by formic acid.ResultsThe method was validated using several saccharides as well as Escherichia coli lipopolysaccharides (O26:B6, O55:B5, and O111:B4). Derivatization using the HD ligand also allows the detection of structures containing amines and phosphate groups in positive ion mode. We optimized the method using crude bacteria (Escherichia coli, Salmonella enterica, Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Legionella pneumophila, Staphylococcus aureus) and yeasts (Candida albicans, C. kudriavzevii, and C. tropicalis).DiscussionThis approach opens the possibility of directly detecting microbial polysaccharides from clinical specimens. Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (LDI-TOF MS) using a specific self-ionizable ligand enables direct ionization without the need for an additional matrix, allowing for the particular detection of molecules of interest while suppressing the background signal.
Genomic characterization of ST38 NDM-5-producing Escherichia coli isolates from an outbreak in the Czech Republic Katerina Chudejova, Tsolaire Sourenian, Jana Palkovicova, Katarina Stredanska, Lucie Nechutna, et al. Antimicrobial Agents and Chemotherapy, 2024 A 2-year national genomic screening in the Czech Republic identified a notable prevalence of the New Delhi metallo-β-lactamase 5 (NDM-5)-producing Escherichia coli sequence type 38 (ST38) in the city of Brno. Forty-two ST38 E. coli isolates harbored the bla NDM-5 gene on the chromosome. Virulence factors confirmed the persistence of these isolates through biofilm formation. Single Nucleotide Polymorphisms (SNPs)-based phylogeny and CRISPR assay typing showed minimal genomic variations, implying a clonally driven outbreak. Results suggest that this high-risk clone may impose a nationwide problem.
Simultaneous PCR detection of Paenibacillus larvae targeting insertion sequence IS256 and Melissococcus plutonius targeting pMP1 plasmid from hive specimens Katerina Vlkova, Tomas Erban, Martin Kamler, Dalibor Titera, Ibrahim Bitar, et al. Folia Microbiologica, 2024 Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)—American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I–IV (strain DSM 7030—ERIC I, DSM 25430—ERIC II, LMG 16252—ERIC III, DSM 3615—ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.
