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The authors present three case reports of bacteremia with very rare microorganisms being isolated in the patients. Each brief case report is accompanied by etiological considerations related to the diagnosis and clinical condition of the patient. Routine microbiology tests were performed by examining the biochemical properties using commercial kits. Two methods were used to determine the microorganism species in the State Institute of Public Health in Prague: MALDI-TOF MS and 16S rDNA sequencing. Antibiotic susceptibility was assessed by the disk diffusion test; minimum inhibitory concentrations were determined using broth microdilution and the gradient diffusion method (E tests). This is probably the first reported case of Stenotrophomonas acidaminiphila isolated from human clinical specimens.

Early and causal administration of antibiotics in patients with a positive blood culture is an essential prerequisite for successful treatment of infection. However, isolation and subsequent identification of bacteria in a blood culture by classical (culture) methods may last several days. MALDI-TOF MS is a method allowing rapid identification of bacteria, not only cultures from culture media, but also directly in clinical specimens.The study included samples of positive blood cultures taken from patients in the University Hospital Olomouc between 2016 and 2018 and examined at the Department of Microbiology of the Faculty of Medicine, Palacký University Olomouc. Positive blood culture samples were processed using an in-house method involving the removal of blood cells by low-speed centrifugation. Subsequently, a pellet obtained by high-speed centrifugation and sample washing was tested by MALDI-TOF MS.A total of 110 positive blood cultures were examined using the method of direct identification. At a species level, more Gram-negative bacteria (88 %) than Gram-positive bacteria (79 %) were correctly identified, with higher identification score values being obtained for the former. Identification score values of 2.0 or higher were found in 62 % of blood cultures containing Gram-negative bacteria and 17 % of blood cultures containing Gram-positive bacteria. Identification score values ranging from 1.7 to 2.0 were found in 21 % of Gram-negative blood cultures and 33 % of blood cultures containing Gram-positive bacteria.Direct identification of microorganisms from positive blood cultures using MALDI-TOF MS enables more rapid diagnosis. By reducing the time required to obtain the result of pathogen identification, it may positively affect the antibiotic treatment of patients.

MALDI-TOF MS is a method enabling rapid identification of bacteria. This is also important for early initiation of adequate antibiotic therapy in patients with infections. In this review, various methods for direct detection of bacteria in clinical specimens are described. The fundamental part deals with direct identification of bacteria from positive blood cultures. Attention is also paid to identification of bacteria from urine and cerebrospinal fluid. Finally, reliability of the methods is mentioned in comparison with conventional methods.

Molecular epidemiology is a field that uses results of typing techniques to obtain information on detailed characterization of bacterial strains for determining the identity, similarity or difference in bacteria of the same genus, species or serotype. Nowadays, the most commonly used methods are based on monitoring differences in bacterial genotypes. However, most of these techniques are time-consuming and costly. A method increasingly used in routine microbiological testing is matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which is based on analysis of the bacterial proteome. It is mainly used for rapid and accurate classification of bacteria into genera and species. The aims were to assess the potential use of this method for typing of Campylobacter below the species level and to apply these results in epidemiological investigations.The study comprised 39 strains of Campylobacter jejuni isolated from food (16) and humans (23). Macrorestriction fragment profiling by pulsed-field gel electrophoresis (PFGE) and simultaneous protein profile analysis using MALDI-TOF MS were performed for all tested strains.Similar pulse profiles were found among isolates originating from the same outbreak or repeatedly collected from a single patient. The same pulse profiles were also detected in strains of unknown relationship but sharing the same place of origin and year of isolation. The comparison of dendrograms from both analyses showed that strains identified as identical by PFGE appeared in the same subgroups in dendrograms obtained by MALDI-TOF MS, the only exception being isolates repeatedly collected from a single patient.The results suggest that confirmation of the identity or similarity of strains in accordance with the established epidemiological facts has not been clearly demonstrated using MALDI-TOF MS.

To verify whether the MALDI TOF MS method can be used for rapid identification of selected zoonotic bacterial pathogens isolated from various types of materials in the real conditions of routine laboratory work.Between August 2010 and April 2015, the Bruker's MALDI TOF MS system was used for 4,174 identifications of selected zoonotic bacterial pathogens (Salmonella spp., Campylobacter jejuni, Campylobacter coli, Listeria monocytogenes, Yersinia enterocolitica, Yersinia pseudotuberculosis, Francisella tularensis, Brucella melitensis, Brucella suis and Cronobacter sakazakii). The samples were prepared for the test by simply mixing a bacterial culture with a matrix on a steel target plate. The results were evaluated with a standard protocol of the system using the MALDI Biotyper software under operating conditions.In 74.8 % of the tested isolates of the above bacterial species, the identification scores ranged between 2 and 3, which is satisfactory for result interpretation in routine practice. Acceleration of identification of Campylobacter spp. and Listeria monocytogenes by testing suspicious cultures obtained directly from selective-diagnostic media decreased the identification scores in these cases.MALDI TOF MS is a suitable and rapid method for identification of the selected zoonotic bacterial pathogens.

Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has recently been widely used in diagnostic microbiological laboratories. It is a cheap and rapid method for the identification of bacteria and micromycetes. Apart from this purpose, it is also used for the detection of antibiotic resistance mechanisms. It has the potential to be extended for other purposes in microbiology. The aim of this study was to validate MALDI-TOF MS for the identification of mycobacteria.Thirty isolates of Mycobacterium spp. isolated in the Laboratory of Mycobacteriology of the Plzeň University Hospital were included in the study. The isolates were identified to the species level using biochemical tests, gene probes, and sequencing of the gene encoding 16S rRNA. The identification by MALDI-TOF MS was performed with the use of silica beads. Strain identification by sequencing the gene encoding 16S rRNA was considered as the reference method.MALDI-TOF MS correctly identified all isolates of Mycobacterium spp. (score range 1.461 - 2.168). The species identified were Mycobacterium tuberculosis (n= 5), Mycobacterium kansasii (n=5), Mycobacterium avium (n=6), Mycobacterium intracelullare (n=3), Mycobacterium xenopi (n=3), Mycobacterium gordonae (n=1), Mycobacterium abscessus (n=1), Mycobacterium kumamotonense (n=2), Mycobacterium mantenii (n=1), Mycobacterium lentiflavum (n=1), Mycobacterium fortuitum (n=1), and Mycobacterium scrofulaceum (n=1).MALDI-TOF MS is a suitable tool for the routine identification of Mycobacterium spp. in laboratories using this method for the conventional identification of microbes.

Recently, research at genetic and molecular levels has extensively accelerated due to advances in new technologies. Since the mid-90s, a relatively new discipline--clinical proteomics, has evolved, which focuses on studying gene products--proteins. The evaluation of protein profiles may contribute to the more accurate stratification of patients in the future, in terms of both prediction of treatment results and prognosis. In pursuing this objective, proteomic approaches are currently used for the identification of new biomarkers. This is also the case with malignant melanoma, a disease without typical serum marker possessing high sensitivity and high specificity.We analyzed human blood serum samples from 25 patients with metastatic malignant melanoma treated with palliative chemotherapy at the Masaryk Memorial Cancer Institute, Brno, in 2004-2006. The analysis was performed by Surface Enhanced Laser Desorption/lonisation Time of Flight Mass Spectrometry (SELDI-TOF-MS). Our patients were divided into two subgroups: a group relatively resistant to chemotherapy--14 patients--and a group with certain clinical benefit from the treatment (complete and partial remission, stabilized disease)--11 patients. We were searching for a new biomarker or typical protein profile in the selected two subgroups. Then, we recategorized our patients into three groups according to the similarity of their protein profiles regardless of sensitivity to chemotherapy. Finally, we evaluated differences in laboratory and clinical parameters, between both the groups of chemo-resistant and chemo-sensitive patients, and newly defined subgroups with similar protein profiles.We did not identify any significant differences in protein profiles or laboratory parameters in the predefined chemo-sensitive or chemo-resistant groups of patients. However, with regard to the new groups with similar protein profiles, we identified a subgroup of patients with different laboratory and clinical parameters. The results are very interesting and merit further research.

Proteomics, a new branch of science studying proteins, only emerged in the postgenomic period. What makes so it important is that fact even the exciting recent insights into the genome have not furnished sufficient information about proteins, which are the main agents behind cellular, subcellular, and supracellular functions. The center of attention of proteomics is identification of proteins in complex protein mixtures with an emphasis on the identification of a large number of proteins at a time, search for interrelations between identified proteins, their more exact characterization, determination of quantity, and better understanding of their function. Proteomics in nephrology is still in its infancy. Although it makes use, like in other fields, of various methodological procedures, the principal techniques are two-dimensional gel electrophoresis and MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry. Nephrology-related proteomic findings made to date inform about normal protein composition of renal parenchyma, urine and plasma, and on their alterations under the influence of physiological or pathological stimuli. Search is under way for uremic toxins in uremic infiltrate, and their elimination from the body of patients with renal failure using various dialysis membranes is being investigated. Findings have made it possible to formulate hypotheses regarding renal physiology and pathology. Current nephrology-related proteomics raises more questions than it answers. Its potential for improving the understanding of physiological processes, pathological states, non-invasive diagnosis, monitoring of therapy, development of drugs and therapeutic procedures is a major promise for future.