Microflex LT Research Articles

Этиологическая структура и антибиотикорезистентность возбудителей сепсиса у больных COVID-19

The purpose — to study the etiological structure and antibiotic resistance of sepsis pathogens in patients with COVID-19. Material and methods. A retrospective observational, uncontrolled study was conducted. We analyzed the results of an examination survey of 584 patients with severe and extremely severe forms of COVID-19 hospitalized at the Republic Clinical Infectious Diseases Hospital in Kazan from April 1, 2020 to October 1, 2022. Identification of the pathogen in blood and other biological materials was carried out using a Microflex LT Bruker mass spectrometer. The antibiotic sensitivity of the isolated bacterial strains was determined by the disk diffusion method using an ADAGIO analyzer (France). Results. Various types of bacteria were isolated from the blood of 76 (13%) patients with severe and critical forms of COVID-19, including: A. baumannii (23.7%), K. pneumoniae (19.7%), S. haemolyticus (19.7%), S. aureus (7.9%), E. faecium (11.8%), E. faecalis (7.9%), E. coli (3.9%), R. planticola (2.6%), P. aeruginosa (2.6%), S. pneumoniae (2.6%), S. maltophilia (1.3%), and B. cereus (1.3%). In 15 patients (19.7%) associations of 2–3 types of bacteria were isolated from the blood. A. baumannii isolates were resistant to carbapenems (94.5%), fluoroquinolones and aminoglycosides (100%), and trimethoprim-sulfamethoxazole (83.3%) strains. K. рneumoniae strains were resistant to carbapenems (60%), cephalosporins (cefixime, ceftazidime) (73.3%), fluoroquinolones (ciprofloxacin) (80%), aminoglycosides (60%), and semisynthetic penicillins (100%). 33.3% of S. aureus strains were resistant to trimethoprim-sulfamethoxazole, 16.7% — to oxacillin, erythromycin and gentamicin. Resistance of S. haemolyticus to trimethoprim-sulfamethoxazole was detected in 86.7%, to erythromycin — in 80%, to norfloxacin — in 50% of strains. 93.3% of S. haemolyticus strains were methicillin-resistant. 88.9% of E. faecium strains were resistant to ampicillin, 22.2% to tigecycline, 11.1% to linezolid. 50% of E. faecalis strains were resistant to norfloxacin, 16.7% of strains were resistant to amoxicillin, ampicillin and piperacillin. P. aeruginosa 50% of strains were resistant to levofloxacin. R. planticola was resistant to all antibiotics tested. S. maltophilia demonstrated sensitivity to co-trimoxazole only with increased exposure. Conclusion. Bacterial sepsis was diagnosed in 13% of severe and extremely severe patients with COVID-19. The etiological structure of sepsis includes a wide range of pathogens with a predominance of multi-resistant bacteria from the group of ESKAPE pathogens. Data on the etiological structure and antibiotic resistance of isolated pathogens should be taken into account for the rational use of antimicrobial drugs in the treatment of sepsis in patients with COVID-19.

Comparison of the Bruker Microflex LT and Zybio EXS2600 MALDI TOF MS systems for the identification of clinical microorganisms

The emergence of Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI TOF MS) technology has expanded the capabilities for identifying microorganisms in clinical labs, replacing traditional biochemical testing with a proteomic approach. In the present study, we compared results between the two commercial MALDI TOF MS systems, Bruker Microflex LT Biotyper and Zybio EXS2600 Ex-Accuspec, for the identification of 1979 urinary isolates by direct extraction method. Current study found that both systems identified a high percentage of isolates to at least the genus level – Bruker 95.6 % of isolates, Zybio 92.4 %. In the case of 89.5 % of all analyzed spectra, the identification results were consistent between the used devices. The highest score values and the highest percentage of spectra identified to species were obtained for gram-negative bacteria. The results show that both systems are equally good choices in terms of analytical performance for routine microbiological diagnostic procedures.

Detection and quantification of Plasmodium falciparum in human blood by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: a proof of concept study

BackgroundMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has revolutionized identification of bacteria and is becoming available in an increasing number of laboratories in malaria-endemic countries. The purpose of this proof-of-concept study was to explore the potential of MALDI-TOF as a diagnostic tool for direct detection and quantification of Plasmodium falciparum in human blood.MethodsThree different P. falciparum strains (3D7, HB3 and IT4) were cultured and synchronized following standard protocols. Ring-stages were diluted in fresh blood group 0 blood drawn in EDTA from healthy subjects to mimic clinical samples. Samples were treated with saponin and washed in PBS to concentrate protein material. Samples were analysed using a Microflex LT MALDI-TOF and resulting mass spectra were compared using FlexAnalysis software.ResultsMore than 10 peaks specific for P. falciparum were identified. The identified peaks were consistent among the three genetically unrelated strains. Identification was possible in clinically relevant concentrations of 0.1% infected red blood cells, and a close relationship between peak intensity and the percentage of infected red blood cells was seen.ConclusionThe findings indicate that the method has the potential to detect and quantify P. falciparum at clinically relevant infection intensities and provides proof-of-concept for MALDI-TOF-based diagnosis of human malaria. Further research is needed to include other Plasmodium spp., wildtype parasite isolates and to increase sensitivity. MALDI-TOF may be a useful tool for mass-screening purposes and for diagnosis of malaria in settings where it is readily available.

Comparison of the New VITEK MS PRIME System with the Matrix-Assisted Laser Desorption Ionization Biotyper Microflex LT for the Identification of Microorganisms.

Matrix-assisted laser desorption ionization (MALDI) is the cornerstone of bacterial identification. The performance of a new MALDI time-of-flight mass spectrometry VITEK MS PRIME (VMS-P) system was compared with that of the MALDI Biotyper Microflex LT (MBT) system, which is routinely used in our laboratory. Sixteen bacterial and yeast reference strains cultured in 20 different media were analyzed over 10 consecutive rounds using both systems. Bacterial and yeast isolates from the routine workflow were processed using both systems. Microcolonies were identified after a 4-hour agar subculture from positive blood culture bottles, without extraction. To determine the repeatability based on the reference strains, 1,190 spots were processed using each system. Correct identification was achieved for 94.0% (MBT) and 98.4% (VMS-P; P<0.01) of spots. Among these, 83.0% (MBT) and 100.0% (VMS-P) were identified with a high degree of confidence. For 1,214 spots from routine isolates, species identification was achieved for 90.0% (MBT) and 91.4% (VMS-P; P=0.26) of spots. For 69.8% (MBT) and 87.4% (VMS-P) of the spots, identification was achieved with a high degree-of-confidence score. When identification was performed using both systems, the agreement between them was 97.9%. The identification of microcolonies from positive blood culture bottles was achieved for 55.5% (MBT) and 70.2% (VMS-P; P=0.01) of spots. The MBT and VMS-P systems perform similarly in routine daily practice. The new VMS-P system shows high repeatability, better confidence scores for identification, and promising ability to identify microcolonies.

Rapid and accurate detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157 : H7 by mass spectrometry directly from the isolate, using 10 potential biomarker peaks and machine learning predictive models.

Introduction. The different pathotypes of Escherichia coli can produce a large number of human diseases. Surveillance is complex since their differentiation is not easy. In particular, the detection of Shiga toxin-producing Escherichia coli (STEC) serotype O157 : H7 consists of stool culture of a diarrhoeal sample on enriched and/or selective media and identification of presumptive colonies and confirmation, which require a certain level of training and are time-consuming and expensive.Hypothesis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a quick and easy way to obtain the protein spectrum of a microorganism, identify the genus and species, and detect potential biomarker peaks of certain characteristics.Aim. To verify the usefulness of MALDI-TOF MS to rapidly identify and differentiate STEC O157 : H7 from other E. coli pathotypes.Methodology. The direct method was employed, and the information obtained using Microflex LT platform-based analysis from 60 clinical isolates (training set) was used to detect differences between the peptide fingerprints of STEC O157 : H7 and other E. coli strains. The protein profiles detected laid the foundations for the development and evaluation of machine learning predictive models in this study.Results. The detection of potential biomarkers in combination with machine learning predictive models in a new set of 142 samples, called 'test set', achieved 99.3 % (141/142) correct classification, allowing us to distinguish between the isolates of STEC O157 : H7 and the other E. coli group. Great similarity was also observed with respect to this last group and the Shigella species when applying the potential biomarkers algorithm, allowing differentiation from STEC O157 : H7Conclusion. Given that STEC O157 : H7 is the main causal agent of haemolytic uremic syndrome, and based on the performance values obtained in the present study (sensitivity=98.5 % and specificity=100.0 %), the implementation of this technique provides a proof of principle for MALDI-TOF MS and machine learning to identify biomarkers to rapidly screen or confirm STEC O157 : H7 versus other diarrhoeagenic E. coli in the future.

Performance of the MSI-2 Database for Fungal Identification by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry from Cleanroom Environments.

Accurate mold identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is dependent on robust organism representation in available databases. The Mass Spectrometry Identification (MSI) platform has proven successful for mold identification in clinical and veterinary settings but has yet to be studied with a large set of environmental isolates. Here, we performed a retrospective study using spectra collected by the Bruker MALDI Biotyper (MBT) v4.1 microflex LT instrument to evaluate the MSI-2 database alongside the combined use of the Bruker MBT (including the MBT Filamentous Fungi Library) and the National Institutes of Health (NIH) mold database (MBT/NIH databases). Analysis was performed for 462 environmental fungal isolates (representing 73 different fungi) cultured from the hospital pharmacy and cellular therapy suites as part of the current good manufacturing practices (cGMP) environmental monitoring program at the NIH. When used alone, MSI-2 identified 237 spectra (51.3%) at its higher score threshold (index A), while the MBT/NIH databases identified only 183 spectra (39.6%; P < 0.001) at the equivalent threshold of ≥2.0. The combination of all three databases at the respective high thresholds improved identification sensitivity to 327 spectra (70.8%). The combination of MSI-2 with the MBT/NIH databases at a lowered threshold (index B or ≥1.7, respectively) identified 400/462 environmental spectra (86.6%). Our results show that the MSI-2 database, in combination with existing databases, may be useful for environmental surveillance, particularly by clinical or industry laboratories involved in cGMP or current good tissue practices (cGTP) applications, such as cellular therapy manufacturing facilities and sterile compounding pharmacies.

Changes in the Protein Profile in Staphylococcal Strains from Patients Infected with the SARS-CoV-2 Virus.

Staphylococcus aureus strains are particularly often isolated from patients with SARS-CoV-2 infection. The aim of the current research was to determine whether the SARS-CoV-2 virus infection affects the protein profile of S. aureus. Bacteria were isolated from the forty swabs collected from the patients in the hospitals of the Pomeranian region. MALDI-TOF MS spectra were obtained using a Microflex LT instrument. Twenty-nine peaks were identified. The peak (2,430) is described here for the first time and was unique for the isolates from patients infected with the SARS-CoV-2 virus. These results support the hypothesis of bacterial adaptation to the conditions caused by viral infection.

SG-APSIC1053: Detection of SARS-COV-2 in nasopharyngeal swags with MALDI-TOF MS and machine learning

Objectives: The widespread distribution of SARS-CoV-2 and its high contagiousness pose a challenge for researchers seeking to develop a rapid and cost-effective screening method to identify carriers of this virus. RT-PCR is considered the gold standard for detecting viral RNA in nasopharyngeal swabs, but it is time-consuming and requires constant changes in the primer composition due to the mutation of SARS-CoV-2 strains. We propose a method for the detection of SARS-CoV-2 in nasopharyngeal swabs using MALDI-TOF MS and machine learning. Methods: Nasopharyngeal swabs from patients with PCR-confirmed COVID-19 and control participants were tested (130 and 80 swabs, respectively) with MALDI-TOF MS MicroFlex LT using the HCCA matrix. MALDI spectra were preprocessed in R version 4.1.2 software with the MALDIquant R package using the workflow: sqrt transformation, wavelet smoothing, SNIP-based base removal, and PQN intensity calibration. Peaks were detected with MAD algorithms with following Peak alignment on the following parameters: minFreq 70% and tolerance 0.005. Machine learning was performed with the rtemis r package on GLM, random forest, and XGBoost models. Results: These models were characterized by specificity, sensitivity, and F1 score. GLM models (specificity 1 and sensitivity 0.5) showed a low F1 score of 0.71. However, the random forest and XGBoost models demonstrated sensitivity, specificity, and F1 score equaling 1. Conclusions: We propose a screening method for SARS-CoV-2 detection (sensitivity 1 and specificity 1). This methodology combines the analysis of nasopharyngeal swab samples using MALDI-TOF-MS with machine learning. It is suitable for screening patients with COVID-19 at the first stages of diagnosis. Random forest and XGBoost models demonstrated sensitivity, specificity, and F1 scores equaling 1.

Oral microbiota in patients with oropharyngeal cancer with an emphasis on &lt;i&gt;Candida&lt;/i&gt; spp.

Introduction. Interactions between the 2 microbiota components – bacteria and fungi – are of interest as diagnostic and prognostic markers in selection of treatment tactics for oncological patients.Aim. To study microbiota of the oral cavity in patients with primary squamous cell carcinoma of the oropharyngeal area before and after surgical intervention to find biomarkers for rational selection of antifungal drugs.Materials and methods. At the Surgical Department of Head and Neck Tumors of the N. N. Blokhin National Research Center of Oncology, three-component study was performed: investigations of spectrum of Candida spp. isolates, Candida spp. strains’ resistance to antifungals, and oral washes in primary patients before and after surgery. mALDI-Tof microflex LT (Biotyper, Bruker Daltonics, germany) was used for strain identification; Sensititre Yeast ONE, YO10 (Trek Diagnostic System, united kingdom) plates were used for determination of minimal inhibiting concentrations of anti fungals. values of minimal inhibiting concentrations were evaluated based on the European Committee on Antimicrobial Susceptibility Testing (EuCAST) criteria (version 10.0).Results. four-year observation of patients at the surgical department of head and neck tumors of the N. N. Blokhin National Research Center of Oncology showed that the most common species of Candida is C. albicans (73.5 % of cases). Candida spp. resistance to antifungals was detected only for fluconazole (9.3 % of cases) and micafungin (8.0 % of cases), mostly among C. albicans strains. In 31.8 % of primary patients, oral washes prior to surgery showed growth of Candida spp. (probably, tissue colonization). After surgical intervention, Candida spp. growth was detected in 36.4 % of cases, only 1 of which was diagnosed as invasive mycosis. In 54.5 % of cases before and in 72.7 % of cases after surgery, gram-negative rods were detected. After surgical intervention, percentage of enterobacteria and non-fermenters significantly increased: 59.1 % versus 27.3 % (p &lt;0.05) and 63.6 % versus 27.3 % (p &lt;0.02), respectively. prior to surgery, non-fermenting gram-negative bacteria were represented only by P. aeruginosa; after surgery, the spectrum of non-fermenting gram-negative bacteria became wider but percentage of P. aeruginosa remained high: 71.4 %. ERG11 gene was identified only in 1 strain: C. albicans. FKS1 gene also was identified only in 1 strain: C. inconspicua. virulence factor genes were detected in 57.1 % of strains.Conclusion. Surgical intervention is associated with changes in bacterial microbiota but not fugal microbiota. presence of virulence factor genes and resistance genes in Candida spp. strains should be considered a biomarker allowing to differentiate between colonization and candida infection and can be used for rational selection of antifungal drugs in prevention and treatment of invasive candidiasis, especially in the absence of criteria for interpretation of measured minimal inhibiting concentrations of antifungals.

АКТУАЛЬНОСТЬ ПРИМЕНЕНИЯ ПАКЕТА ПРОГРАММНОЙ СРЕДЫ R-MALDIQUANT ДЛЯ ПРЕОБРАЗОВАНИЯ НЕОБРАБОТАННЫХ МАСС- СПЕКТРОВ ДИРОФИЛЯРИЙ

Diagnosis of human tissue helminthiasis remains a complex problem of practical medicine. Therefore, the search for alternative direct methods of their verification is an important area of laboratory diagnostics. Proteomic research methods are one of the most modern areas of laboratory practice. Updating the database of reference protein profiles of parasitic pathogens is one of the main priorities of mass spectrometry. To convert digital data of protein extract spectra, the R-MALDIquant software package for dirofilaria differentiation (Dirofilaria immitis, Dirofilaria repens) was used. For the study, two D. repens females were taken, removed from the subcutaneous fat of two patients (one from each patient), two D. immitis females obtained from the heart after autopsy of two fallen dogs. In the study, only the head ends were taken (where the stichocytes are concentrated), without reproductive organs (where the main excretory-secretory antigens of the parasite are located). The profiles of the mass spectra of the homogenate proteins were obtained using the standard Sepsityper Kit 50 and the “Formic Acid Extraction” for Microflex LT MALDI–TOF MS (Bruker Daltonics) with flexControl software (Bruker Daltonics). Visualization was performed using Flexanalysis 3.3 software (Bruker Daltonics). Preliminary processing of mass spectra was carried out using the R software environment in the MALDIquant package. The coincidence rate in the dendrogram sections for D. immitis individuals was 96-99 %, and for D. repens – 73-100 %, which is probably due to phenotypic differences in helminths isolated from different patients. The results obtained by us of processing two types of dirofilaria using the R-MALDIquant software environment are the first step in creating a library of reference protein profiles of nematodes of the genus Dirofilaria. Key words: Dirofilaria repens, Dirofilaria immitis, MALDI-TOF MS, R package, MALDIquant. erroneous identification of the causative agent of the disease D. latum, which did not affect the results of treatment

The Effectiveness of MALDI ToF Mass Spectrometry in Identification of &lt;i&gt;Francisella tularensis&lt;/i&gt; Strains

The aim of the study was to evaluate the effectiveness of MALDI‑ToF mass spectrometry in the identification of collection and newly isolated strains of tularemia pathogen using the database “Protein profiles of mass spectra of microorganisms belonging to I–II pathogenicity groups for the MALDI Biotyper software”.Materials and methods. We investigated 142 strains of Francisella tularensis, including 59 collection strains and 83 newly isolated ones. Bacteriological, molecular-genetic and proteomic research methods were used to identify them. The acquisition of mass spectra, analysis, generation and expansion of reference libraries were performed on a mass analyzer “Microflex LT” using FlexControl v. 3.3, FlexAnalysis v. 3.3, and MALDI Biotyper 3.0 software packages. The cluster analysis was performed using the BioNumerics 7.6 software.Results and discussion. The possibility of identifying tularemia pathogen has been assessed using the extended database for MALDI Biotyper 3.0 “Protein profiles of mass spectra of microorganisms belonging to I–II pathogenicity groups for the MALDI Biotyper software”. During identification to the species level, the significance of mass spectrometry results for collection strains and newly isolated ones was 91.5 % and 97.6 %, respectively. In determining the genus appurtenance, the reliability of identification was 100 %. Thus, the MALDI‑ToF mass spectrometry method allows for accurate species and genus identification of F. tularensis strains. Based on the cluster analysis of 66 F. tularensis strains in BioNumerics 7.6 software using «Pearson correlation» and the UPGMA algorithm, the possibility of subspecies differentiation has been evaluated. Due to the similarity of protein profiles of F. tularensis strains, a clear differentiation into subspecies could not be achieved. It is necessary to use other options for sample preparation, new generation devices with higher resolution, as well as apply additional approaches and analysis tools for successful subspecific differentiation.

Molecular Сharacterization of &lt;i&gt;Neisseria meningitidis&lt;/i&gt; Serogroup W in St. Petersburg

The study included 14 isolates of Neisseria meningitidis serogroup W, isolated from patients with generalized forms of meningococcal infection aged 0–17 years at the Pediatric Research and Clinical Center for Infectious Diseases during the period from 2009 to 2020, as well as from carriers. The isolates from carriers (17–18 years old) were isolated in 2016–2019. The isolates were identified by MALDI-TOF mass spectrometry using a Microflex LT mass spectrometer (Bruker Daltonics, Germany). Whole genome sequencing of N. meningitidis was carried out on the basis of the Miseq Illumina platform; it was established that they belong to ST11 (cc11) sequence type. The results of the whole genome sequencing of the studied meningococci showed that there are 2 groups of the subcluster belonging to the Hajj cluster in St. Petersburg. In addition, they show close affinity with meningococci of the Anglo-French and Swedish clusters of the Hajj line.

Antibiotic Sensitivity of &lt;i&gt;Neisseria meningitidis&lt;/i&gt; Isolated From Patients with Generalized Forms of Meningococcal Infection and From Healthy Carriers

The study included 50 isolates of Neisseria meningitidis isolated from patients with generalized forms of meningococcal infection and 48 isolates, which were isolated from carriers. The isolates were identified by MALDI-TOF mass spectrometry using a Microflex LT mass spectrometer (Bruker Daltonics, Germany). Minimum inhibitory concentrations (MICs) of benzylpenicillin, ampicillin, ceftriaxone, meropenem, ciprofloxacin, azithromycin, rifampicin, and chloramphenicol were assessed by the method of serial agar dilution with interpretation of the results according to EUCAST 2021 criteria. Clinical resistance to penicillin was detected in 7% of isolates. However, all isolates with MIC &gt;0.064 µg/ml (n=26) had mutations in the penA gene. Decreased sensitivity to rifampicin was found in four isolates isolated from patients and four isolated from carriers. Seven out of eight isolates with reduced sensitivity to rifampicin had mutations in the rpoB gene. Resistance to ceftriaxone, meropenem, ciprofloxacin was not detected.

MALDI-TOF mass spectrometry for direct KPC detection among Enterobacterales.

In this study, we evaluate a method for the KPC enzyme detection, using MALDI-TOF MS, for Enterobacterales. A total of 300 clinical Enterobacterales isolates were selected. The collection included 259 carbapenemase-producing (157 KPC and 102 non-KPC) and 41 carbapenemase non-producing isolates. Bacterial proteins were extracted from Mueller-Hinton agar plates using formic acid, isopropyl alcohol, and water (17:33:50). Samples were prepared with a double layer of synapinic acid. Analyses were performed using a Microflex LT mass spectrometer (Bruker Daltonics) and flexAnalysis 4.0 software (Bruker Daltonics). Statistical analyses were performed using SPSS Software. A distinctive peak at m/z 28,643-28,731 was found in all 157 KPC-producing isolates, and it was consistently absent in the 143 KPC non-producing group. KPC-producing peak intensities ranged from 77 to 3893. Considering an intensity cutoff value ≥ 120 for the presence of KPC, this methodology presented 98.09% and 97.90% of sensitivity and specificity, respectively.

Microflora of sputum and autopsy material of patients with COVID-19.

The rapid spread of a new coronavirus infection in the country actualizes the conduct of bacteriological studies of clinical material obtained from the respiratory tract of patients with COVID-19. During the experiments, 230 sputum samples and 260 autopsy lung samples from patients with COVID-19 were analyzed. 946 high-risk strains were isolated and identified by MALDI-ToF mass spectrometry on a Microflex LT instrument (Bruker®). According to the results of bacteriological cultures of sputum, a predominance of gram-positive ones was revealed, amounting to 50.5% (222 strains) of the total number of isolated pathogens. However, falling into this group is manifested by natural representatives of the microflora of the human mucous membranes from the genera Streptococcus, Rothia and Lactobacillus (109 strains in total), which can be manifested by the detection of improper sputum collection, causing contamination by the substance of intense salivation and nasopharyngeal discharge. In turn, the "classic" gram-positive causative agents of pneumonia were detected much less frequently: S. aureus in 5 cases, S. pneumoniae in 6 patients. The causative agents in the order Enterobacterales are represented by 42 strains, among which the most likely species are K.pneumoniae (27 strains). In the group of non-fermenting gram-negative bacteria, A. baumanii (29 strains) prevailed, and P. aeruginosa was also identified in 2 cases. When analyzing the results of a microbiological study of autopsy material (lungs) of patients with COVID-19, significant differences in the qualitative and quantitative composition of the microflora were revealed, compared with sputum. In the group of gram-positive bacteria, 15 strains of the natural microflora of the mucous membranes were identified, while sensitive species dominated among gram-negative pathogens: K. pneumoniae (102 strains), A. baumanii (75 strains), P. aeruginosa (11 strains). Regular microbiological monitoring is essential for antibiotic therapy and prevention of secondary bacterial infection. In the event of a fatal outcome, the results of microbiological analysis of autopsy material can determine the cause of death of the patient.

Pseudomonas species prevalence, protein analysis, and antibiotic resistance: an evolving public health challenge

Psychrotrophic Pseudomonas is one of the significant microbes that lead to putrefaction in chilled meat. One of the biggest problems in the detection of Pseudomonas is that several species are seemingly identical. Currently, antibiotic resistance is one of the most significant challenges facing the world's health and food security. Therefore, this study was designed to apply an accurate technique for eliminating the identification discrepancy of Pseudomonas species and to study their resistance against various antimicrobials. A total of 320 chicken meat specimens were cultivated, and the isolated bacteria’ were phenotypically recognized. Protein analysis was carried out for cultured isolates via Microflex LT. The resistance of Pseudomonas isolates was recorded through Vitek® 2 AST-GN83 cards. Overall, 69 samples were identified as Pseudomonas spp. and included 18 Pseudomonas lundensis (P. lundensis), 16 Pseudomonas fragi (P. fragi), 13 Pseudomonas oryzihabitans (P. oryzihabitans), 10 Pseudomonas stutzeri (P. stutzeri), 5 Pseudomonas fluorescens (P. fluorescens), 4 Pseudomonas putida (P. putida), and 3 Pseudomonas aeruginosa (P. aeruginosa) isolates. Microflex LT identified all Pseudomonas isolates (100%) correctly with a score value ≥ 2.00. PCA positively discriminated the identified isolates into various groups. The antimicrobial resistance levels against Pseudomonas isolates were 81.16% for nitrofurantoin, 71% for ampicillin and ampicillin/sulbactam, 65.22% for cefuroxime and ceftriaxone, 55% for aztreonam, and 49.28% for ciprofloxacin. The susceptibilities were 100% for cefotaxime, 98.55% for ceftazidime, 94.20% for each piperacillin/tazobactam and cefepime, 91.3% for cefazolin. In conclusion, chicken meat was found to be contaminated with different Pseudomonas spp., with high incidence rates of P. lundensis. Microflex LT is a potent tool for distinguishing Pseudomonads at the species level.

MASS-SPECTROMETRIC STUDIES OF HELMINTHS: INFORMATION TECHNOLOGIES FOR IDENTIFICATION OF PARASITIC INFECTION CAUSES

Mass spectrometry is currently used to identify bacteria, mycobacteria and some fungi. Unlike clinical bacteriology, MALDI-TOF MS is practically not used for the identification of human and animal parasites. Homogenate protein profiles were obtained using Microflex LT MALDI-TOF MS (Bruker Daltonics Germany) with Flex Control software, visualized using Flex analysis 3.3 software (Bruker Daltonics). Analysis of protein extracts of nematodes showed spectra with high intensity peaks in the range of 2–20 kDa. Slight differences in the mass spectrometric spectra of Ascaris lumbricoides and Ascaris suum in the same range, which characterizes nematode species, confirm the results obtained in full-genome analysis of helminth data that the causative agent of human ascariasis is an evolutionarily close relative of swine ascarids. In the high ranges of mass spectrometric analysis of Dirofilaria immitis and Dirofilaria repens, significantly frequent peaks were observed, which probably characterize the whole genus of nematodes. In the range above 6000 kDa, the spectra differed in representative peaks, which allowed to differentiate one species from another in protein profile. A comparative analysis of the mass spectrometric peaks of helminths D.repens, D. immitis using R medium (MALDIquant). On the cluster of dendrograms of averaged mass spectra, it was found that for two individuals of D. repens, as well as for two individuals of D. immitis, the sections of the dendrograms coincide with a high degree of probability. For D. immitis, the coincidence rate was 100%, for D. repens 70%. The results obtained can form the basis for the creation of a library of protein profiles with the aim of accelerated and objective dirofilaria differentiation.

ОЦЕНКА КУЛЬТУРОМА ОТДЕЛЯЕМОГО ВЕРХНИХ ДЫХАТЕЛЬНЫХ ПУТЕЙ И СОДЕРЖИМОГО ТОЛСТОЙ КИШКИ У ПАЦИЕНТОВ С АТОПИЧЕСКИМ ДЕРМАТИТОМ

Aim. The aim of the study is to study the species diversity of the microflora of the discharge of the nose, oropharynx and the contents of the colon in atopic dermatitis. Material and methods. The study included 80 patients with atopic dermatitis. Patients are divided into 3 groups depending on the severity of AD (Focusing on the SCORAD scale). The patients underwent collection of biomaterials from the oral and nasal cavities and intestines. Sowing of the material was carried out on an extended list of nutrient media. As a result of the MALDI-ToF mass spectrometry on the Microflex LT (Bruker, Germany), the resulting microorganisms were recognized by direct and extended deposition using formic acid. Results and discussion. When seeding feces patients from all groups, statistically significant differences were revealed for the following microorganisms, depending on the severity: Enterococcus faecium (61%), Streptococcus anginosus (16.7%), Parabacteroides distasonis (22.2%) were found in remission; at the stage of exacerbation with exacerbation of atopic dermatitis in the form of a limited form - Enterococcus faecalis (39.3%), Lactobacillus fermentum (16.1%), Streptococcus parasanguinis (9%);with a common form of exacerbation, Klebsiella pneumonia (50%), Klebsiella oxytoca (50%), Enterococcus mundtii (16.7%), Echerichia vulneris (16.7%), Lactobacillus salivarius (83.3%), Raoultella ornithinolytica(16.7%), Enterococcus avium (50%), Enterobacter asburie (16.7%), Citr obacter braaki (33.3%), Bacteroides vulgates (33.3%), Bifidobacterium adolescentis(16.7%), Enterococcus durans (16.7%), Lactobacillus crispatus (16.7%), Corynebacterium amycolatum (33.3%), Streptococcus sanguinis (16.7%). Inoculation of oropharyngeal discharge from patients with atopic dermatitis revealed statistically significant differences for the following microorganisms, depending on the severity: Streptococcus australis (11.1%) was detected in remission; in the stage of exacerbation with a limited form of atopic dermatitis, Rothia mucalaginosa (19.6%), Streptococcus saliverius (39.3%) were identified; in the stage of exacerbation with a widespread form of atopic dermatitis, Streptococcus anginosus (16.7%;), Candida albicans (33.3%), Streptococcus gordonii (16.7%), Staphylococcus haemolyticus (16.7%), Neisseria oralis (33.3%), Corynebacterium amycolatum (16.7%), Kocuria rhizophila (33.3%), Lactobacillus rhamnosus (16.7%). Inoculation of nasal discharge from patients with atopic dermatitis revealed statistically significant differences for the following microorganisms, depending on the severity: Staphylococcus haemolyticus (16.7%), Staphylococcus lugdunensis (11.1%), Staphylococcus copitis (11.1%) were found in remission. At the stage of exacerbation with a widespread form of atopic dermatitis, Proteus mirabilis (16.7%), Streptococcus vestibularis (16.7%), Streptococcus sobrinus (16.7%), Staphylococcus warneri (16.7%), Corynebacterium coyleae (16.7%), Lactobacillus plantarum (16.7%). Conclusion. Our results indicate enzymatic deficiency and significant changes in the qualitative composition of the microbiota in people with atopic dermatitis, which violates the body's immunomodulating function.

Carbapenemase Producing Klebsiella pneumoniae (KPC): What Is the Best MALDI-TOF MS Detection Method.

Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria is a group of highly dangerous antibiotic resistant Gram-negative Enterobacteriaceae. They cause infections associated with significant morbidity and mortality. Therefore, the rapid detection of KPC-producing bacteria plays a key role in clinical microbiology. Matrix assisted laser desorption/ionization time-of- flight (MALDI-TOF) is a rapidly evolving technology that finds application in various clinical, scientific, and industrial disciplines. In the present study, we demonstrated three different procedures of carbapenemase-producing K. pneumoniae (KPC) detection. The most basic model of MALDI-TOF instrument MS Microflex LT was used, operating in the linear ion-positive mode, commonly used in modern clinical laboratories. The first procedure was based on indirect monitoring of carbapenemase production with direct detection of hydrolyzed carbapenem antibiotic degradation products in the mass spectrum. The second procedure was based on direct detection of blaKPC accompanying peak with an 11,109 Da in the mass spectrum of carbapenemase-producing K. pneumoniae (KPC), which represents the cleaved protein (pKpQIL_p019) expressed by pKpQIL plasmid. In addition, several unique peaks were detected in the carbapenemase-producing K. pneumoniae (KPC) mass spectrum. The third procedure was the identification of carbapenemase-producing K. pneumoniae (KPC) based on the protein fingerprint using local database created from the whole mass spectra. By comparing detection procedures, we determined that the third procedure was very fast and relatively easy. However, it requires previous verification of carbapenemase-producing K. pneumoniae (KPC) using other methods as genetic blaKPC identification, detection of carbapenem degradation products, and accompanying peak with 11,109 Da, which represents cleaved pKpQIL_p019 protein expressed by pKpQIL plasmid. Detection of carbapenemase-producing K. pneumoniae using MALDI-TOF provides fast and accurate results that may help to reduce morbidity and mortality in hospital setting when applied in diagnostic situations.

Current epidemiological situation on leptospirosis in the territories adjacent to Lake Baikal

Background. In presence the epizootiological situation on leptospirosis in the territories adjacent to Lake Baikal is not studied well. The showing up of natural focus, discovering of host animal’s species composition and etiological structure of leptospirosis causative agents are essential to plan any epidemiological response and to organize the specific prophylaxis.Aim: to analyze contemporary characteristics of leptospirosis natural foci at the territories adjacent to Lake Baikal.Materials and methods. From 2011 till 2020 the territory of nine Irkutsk Region's administrative districts was investigated. Trapping has been done at four kind of sites (wetland, meadow, forest-shrub, steppificated meadow) and boroughs. 1152 small mammals which belonged to 35 species were collected. Collected samples were studied by complex of methods. Serogroup belonging was detected by micro agglutination and decomposition test, and genomic species – by Microflex LT mass analyzer.Results. A leptospirosis natural foci in the Irkutsk city boundary was newly founded and there were isolated (from small mammals) four pathogenic cultures of leptospiras belonging to Javanica serogroup. Main hosts of causative agents at the territories adjacent to Lake Baikal were defined (insect-eating mammals: tundra shrew, largetoothed Siberian shrew, even-toothed shrew, shrew-mouse, and rodents: root vole, ground vole, narrow-skulled vole, sewer rat). By micro agglutination were defined pathogenic leptospirosis serogroups (Icterohaemorrhagiae, Grippotyphosa, Javanica, Pomona, Sejroe, Autumnalis, Australis), all over 2.4 ± 0.45 % seropositive. Antibody dilution at survey animals ranged from 1 : 20 to 1 : 640. On the grounds of PCR results the mean value of infected animals in studied sample was 16.4 ± 1.14 %. Positive PCR findings were at 19 from 35 animal species.Conclusion. At the territories adjacent to Lake Baikal In the Baikal region, the proportion of animals that have had contact with pathogenic leptospira is quite high. Natural focus of this infection are highly pervasive in this region, they are evolved mainly in dewy sites.