Mass Spectrometric Microbial Serotyping | Lucia Ďaďovská
A Novel Method for Derivatization and Mass Spectrometric Analysis (LC/MS and LDI -TOF) of Saccharides with a Special Focus on Bacteria
Author: Lucia Ďaďovská (1), Veronika Pašková (1), Jaroslav Hrabák (1)
Supervisor: Jaroslav Hrabák
(1) Department of Microbiology
State-of-the-Art: MALDI-TOF MS shortened the turn-around time of taxonomic identification of microbes and detection of antibiotic resistance in routine diagnostic medical microbiology. However, there is no application of the technique for epidemiological typing that would allow a deeper comparison of identified bacterial isolates (serotyping) or identification of the microbe directly from clinical sample. This void could be filled with analysis of microbial cell surface structures like lipopolysaccharides and lipoteichoic acid as those molecules are abundant in the bacterial cell and stable in the body fluids. MS analysis of polysaccharides is currently complicated due to their poor ionization ability, complexity, and molecule size. Thus, polysaccharide detection in diagnostics laboratory is impossible.
Objective: The hypothesis of the study was to demonstrate whether an innovative approach to polysaccharide ionization for analyzing bacterial lipopolysaccharides and other external structures of the cell wall can facilitate bacterial identification and typing by MALDI-TOF MS.
Material and Methods: One milligram of various saccharides (e.g. D-glucose, isotopic D-glucose-1,2-13C2, glucose-6-phosphate, lactose, starch), purified lipopolysaccharides, and bacterial cultures was dissolved in 10 μL concentrated formic acid or its isotopic variant (13C) and incubated at 98 °C for 10 minutes in the thermocycler for digestion. This digestion procedure also yielded esters of saccharide’s hydroxyl groups. The reaction was filtered using Amicon® Ultra Centrifugal Filter, 3 kDa MWCO. Filtrate was derivatized with concentrated HD ligand (250 mmol/L) solution at pH 4.0. After incubation at 50 °C for 20 minutes, the derivatized saccharides were visualized using a timsTOF Pro 2 spectrometer (Bruker Daltonics, Bremen, Germany) and rapiflex MALDI-TOF MS (Bruker Daltonics, Bremen, Germany).
Results & Discussion: We developed a novel method for analyzing saccharides using mass spectrometry that is translated into detecting microbial lipopolysaccharides in LDI-TOF MS for bacterial serotyping. The first step of the method consists of acid hydrolysis of glycosidic bonds of microbial polysaccharides. In addition, hydroxyl groups of saccharides are esterified, further facilitating their identification in spectra. In the second step, aldehyde groups of digested reducing sugars react with a novel self-ionizable HD reagent we designed, synthesized, and patented. HD reagent possesses perfect ionization ability that no matrix is needed. This derivatization allows the obtaining of unique spectra for each bacterial species and purified lipopolysaccharides representing specific lipopolysaccharide fingerprints.
Conclusion: We discovered an innovative glycomic MS method for analyzing microbial polysaccharides and bacterial serotyping. The procedure can be used in diagnostics laboratories. It is also a promising tool for analyzing microbes directly from clinical specimens.
Funding: The study was financed by the Czech Health Research Council grant Nr. NW24-09-00464, the GA UK project Nr. 550225 and the NIVB (Programme EXCELES, ID Project Nr. LX22NPO5103)—funded by the EU—Next Generation EU. The method was patented (PV 2024-48), and PCT application has been submitted.
Study program: Doctoral study – Medical Microbiology | Year of Study: 3
ID: 1144
Extensively Drug-Resistant A. baumannii of ST2 | Tsolaire Sourenian
Outbreak Investigation of Extensively Drug-Resistant Acinetobacter baumannii of ST2 in the Intensive Care Unit of a Lebanese Hospital
Authors: Tsolaire Sourenian (1), Marc Finianos (1), Jaroslav Hrabak (1), Abdallah Medlij (2),
Ibrahim Bitar (1)
Supervisor: Ibrahim Bitar (1)
(1) Department of Microbiology, Faculty of Medicine in Pilsen, Charles University and University
Hospital, Pilsen (2) Sheikh Ragheb Harb University Hospital, Nabatiyeh, Lebanon
State-of-the-Art: Acinetobacter baumannii is a leading multidrug-resistant (MDR) pathogen
responsible for nosocomial infections, particularly in intensive care units (ICUs). This incidence
has been driven by unregulated antibiotic use, posing significant concerns and high mortality
rate due to its extensive drug-resistance (XDR).
Objective: The aim of the study is to investigate an XDR A. baumannii outbreak in the ICU of a
tertiary hospital in Lebanon.
Material and Methods: A total of 47 swabs were sampled from one of the ICU rooms (n=40;
environmental surfaces and n=7; clinical A. baumannii swabs from outpatients). The strains were selected by Muller Hinton (MH) agars containing 4 μl/mL of meropenem. Species identification and antibiotic susceptibility profiles were detected using MALDI-TOF MS and broth microdilution assays respectively. Both short and long reads (Illumina and Sequel I/Minion) whole genome sequencing (WGS) were performed on the positive swabs (n=21). Clonal relatedness was assessed by multilocus sequence typing (MLST) and genomes were compared through SNPbased phylogeny.
Results & Discussion: Nineteen isolates were XDR A. baumannii, of which 12 were from environmental
swabs (including side rails, monitors, air mattresses and toilet chairs). WGS revealed the presence of blaOXA-23 gene in all isolates, and it was located on the chromosome. MLST analysis showed that all strains belong to ST2 international clone. The SNP-based phylogeny suggested that the outbreak originated from multiple ST2 isolates which clustered into two main clades (A and B). Clade A confined 10 strains having only 0 to 1 SNP when compared to each other. Whereas, clade B had 2 subclades: one containing 6 strains that differed by 0 to 21 SNPs among each other, and the other one had 2 strains with 102 SNPs difference.
Conclusion: Conclusion: We reported a nosocomial ongoing four-year outbreak of XDR A. baumannii in a Lebanese ICU, intensified by the war casualties, reveals critical gaps in screening and disinfection protocols. Urgent intervention at both national and hospital levels is necessary to halt future outbreaks.
Study program: Doctoral study – Medical Microbiology | Year of study: 4
ID: 1083
Neuropeptides B/W in Diabetic Gut Dysfunction | Tomáš Chmelíř
Neuropeptides B and W as Novel Targets in Research on Gastrointestinal Dysfunction in Diabetes Mellitus
Authors: Tomáš Chmelíř (1), Dagmar Jarkovská (1), Shashank Pandey (2)
Supervisor: Magdaléna Chottová Dvořáková (1)
(1) Department of Physiology, Faculty of Medicine in Pilsen, Charles University (2) Department of Pharmacology and Toxicology, Faculty of Medicine in Pilsen, Charles University
State-of-the-Art: Neuropeptides B (NPB) and W (NPW) and their receptor (NPBWR1) are involved in the regulation of energy homeostasis, stress response, and neuroendocrine functions. Although they were identified more than two decades ago, their role in gastrointestinal physiology remains poorly understood. It has been confirmed that NPW influences gastrointestinal motility by reducing antral contractions and slowing gastric emptying. However, NPB/W function in other parts of the gastrointestinal tract has not yet been studied. In the context of diabetes mellitus (DM), it is well known that gastrointestinal motility is disrupted due to autonomic neuropathy and alterations in neuropeptide signaling. The potential NPB and NPW role in the pathophysiology of gastrointestinal dysfunction remains unclear.
Objective: Our goal was to confirm the gene expression of NPB, NPW and NPBWR1 in the cardia, corpus, pylorus, duodenum, and colon and assess the impact of type 1 and type 2 DM on the expression of these genes. Subsequently, we analyzed the effect of NPB and NPW on colonic contraction force in vitro.
Material and Methods: The study involved 12-week-old Zucker Diabetic Fatty (ZDF) rats (type 2 DM; n = 7), lean rats with streptozotocin-induced DM (type 1 DM; n = 7), and lean control rats (n = 7). Tissue samples from the cardia, corpus, pylorus, duodenum, and colon were dissected, washed, and frozen. Later, they were homogenized. The total RNA was isolated and converted into cDNA, which was analyzed by qPCR to determine the relative gene expression of the target genes (NPB, NPW, NPBWR1). For the in vitro measurements, we used a ring of colonic tissue placed into a chamber perfused with oxygenated Tyrode´s solution (37°C). Acetylcholine-induced contractions were recorded first in the control solution and then in the solution containing NPB or NPW (1 nM, 10 nM, and 100 nM gradually).
Results & Discussion: Gene expression of NPB, NPW and NPBWR1 was detected in all examined tissues. NPBWR1 expression in the corpus and pylorus was at the detection limit and, hence, not quantitatively assessed. In STZ rats, we observed a statistically significant downregulation of NPB expression in the corpus (0.56-fold, p = 0.028) and a statistically significant upregulation of NPW expression in the pylorus (2.62-fold, p = 0.034), duodenum (1.91-fold, p = 0.046), and colon (1.7-fold, p = 0.046). In the ZDF rats, we revealed a significantly upregulated NPBWR1 expression in the colon (2.19-fold, p = 0.038). The amplitude of colonic contraction significantly decreased after applying NPB and NPW during the in vitro measurements. This effect was more pronounced with increasing concentration of applied neuropeptide.
Conclusion: We observed significant changes in NPB/W signaling system gene expression in various gastrointestinal organs of DM rats. Both neuropeptides showed a relaxing effect on colonic smooth muscle, which, together with an increased NPBWR1 expression, may help to explain gut symptoms in diabetic patients.
Funding: This study was supported by the Charles University Grant Agency, project No. 10524: The effect of diabetes mellitus on the innervation of the gastrointestinal tract.
Study program: Doctoral study – Physiology and Pathological Physiology | Year of study: 2
ID: 1084