MALDI-TOF MS Method Research Articles

Identification of cotton non-pathogenic fungal agent isolates based on morphological and MALDI-TOF mass spectrometry method

In this study, morphological and MALDI-TOF MS identification and comparison of non-pathogenic fungal species isolated from diseased root, leaf and boll tissues of cotton plants were carried out. For this purpose, surveys were conducted in Bağlar, Sur, Çınar, Bismil, Yenişehir, Ergani, Eğil, Kayapınar and Silvan districts of Diyarbakır province where cotton production is intensive between June and September 2020 and 2021. 209 samples of plants showing typical fungal disease symptoms were collected from 75 different cotton production areas. A total of 171 fungal isolates were obtained by isolation, culture and purification procedures from diseased plant tissues in the samples. The 20 isolates that were negative in the pathogenicity test in the main host were identified and compared by morphological (traditional) and MALDI-TOF MS methods. According to the results, Aspergillus niger, Aspergillus flavus, Aspergillus terreus, Aspergillus fumigatus, Aspergillus minisclerotigenes, Penicillium nalgıovense, Chaetomium globosum, Dichotomopilus funicola, Arthroderma gloria, Pseudogmnoacus pannorum, Tichophyton interdigitale, and Penicillium sp. Penicillium sp. were found to be intensively colonized in different parts of cotton such as leaves, bolls and roots. Again, it was determined that the most common species among the total saprophyte isolates was Aspergillus niger with a high similarity rate.

Successful treatment of Keratitis caused by Mycobacterium chelonae and an overview of previous cases in Europe

Introduction and purposeMycobacterium (M.) chelonae is responsible for a half of relatively rare nontuberculous mycobacteria (NTM) keratitis. We report a case of M. chelonae keratitis in a woman following sclerocorneal suture extraction after cataract surgery.ResultsA 70-year-old woman presented with a red eye and corneal infiltration of her left eye six weeks following sclerocorneal suture extraction after an elective cataract surgery in another institute. She complained of a sharp, cutting pain and photophobia. Since initial corneal scrapes and conjunctival swabs proved no pathogen using culture and PCR methods, non-specific antibiotics and antifungal agents were administered. As keratitis was complicated by an inflammation in the anterior chamber and vitreous, samples of the vitreous fluid were sent for microbiologic examination. DNA of Epstein-Barr virus (EBV) was repeatedly detected. Since the intrastromal abscess had formed, corneal re-scrapings were performed and M. chelonae was detected using culture, MALDI-TOF MS and PCR methods. Therapy was changed to a combination of oral and topical clarithromycin, intravitreal, topical and intracameral amikacin, and oral and topical moxifloxacin. The successful therapy led to stabilization. The optical penetrating keratoplasty was performed and no signs of the infection recurrence were found.ConclusionsThe diagnosis of nontuberculous mycobacterial keratitis is difficult and often delayed. An aggressive and prolonged antimicrobial therapy should include systemic and topical antibiotics. Surgical intervention in the form of corneal transplantation may be required in the active and nonresponsive infection. In the presented case this was necessary for visual rehabilitation due to scarring.

Evolving strategies in microbe identification—a comprehensive review of biochemical, MALDI-TOF MS and molecular testing methods

Abstract Detection and identification of microorganisms are the first steps to guide susceptibility testing and enable clinicians to confirm diseases and guide therapy. The faster the pathogen identification is determined, the quicker the appropriate treatment can be started. In the clinical microbiology laboratory, multiple methodologies can be used to identify organisms, such as traditional biochemical testing or more recent methods like MALDI TOF MS and nucleic acid detection/identification assays. Each of these techniques has advantages and limitations, and clinical laboratories need to determine which methodology is best suited to their particular setting in terms of clinical needs, availability of technical expertise and cost. This article presents a concise review of the history, utilization, advantages and limitations of the main methods used for identifying microorganisms in microbiology laboratories.

Occurrence, Molecular Serogroups, Antimicrobial Susceptibility and Identification by MALDI-TOF MS of Listeria monocytogenes Isolated from RTE Meat Products in Southern Poland.

L. monocytogenes is considered one of the most dangerous foodborne pathogens. This study aimed to determine the occurrence of L. monocytogenes in RTE meat products from southern Poland, including serogroups and antimicrobial susceptibility, and to assess the usefulness of MALDI-TOF MS as a tool for identifying L. monocytogenes. A total of 848 production batches of RTE meat products were analyzed for L. monocytogenes. All L. monocytogenes isolates were serotyped using the multiplex PCR method, tested for antimicrobial susceptibility using the disk diffusion method and identified using the MALDI-TOF MS method. L. monocytogenes was detected in 52/848 batches of RTE meat products (6.13%). The isolates belonged to four serogroups: 17/52 (33%) isolates to IVb; 15/52 (29%) isolates to IIa; 10/52 (19%) isolates to IIc and 10/52 (19%) isolates to IIb. All isolates (52/52) showed susceptibility to the tested antimicrobials. Using MALDI-TOF MS, 10/52 isolates (19.2%) were identified at the level of secure genus identification, probable species identification; 37/52 isolates (71.2%) were identified at the level of probable genus identification; 3/52 isolates (5.8%) were incorrectly identified as L. innocua; and 2/52 isolates (3.8%) were not identified. The occurrence of L. monocytogenes in RTE meat products was low. Almost half of the analyzed isolates were L. monocytogenes of serogroups, which are most often associated with listeriosis in humans in Poland. All isolates showed susceptibility to five commonly used antimicrobials for treating listeriosis. The use of MALDI-TOF MS as a tool for the identification of L. monocytogenes indicated its limitations related to the insufficient representation of the pathogen in the reference database.

Diversity and Antibiotic Susceptibility of Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae from the Urine of Human Population Using Bacteria-Contaminated Water in Butembo Urban Area (DR Congo, Central Africa)

Aims: This study aims to investigate the extended-spectrum beta-lactamases (ESBLs)-producing Enterobacteriaceae in the human urine samples and to understand the potential influence of the contaminated water the urine donors use. Study Design and Methodology: One hundred urine samples from females and the same number from males were collected from donors of different age groups and analysed. One hundred urine samples from females and the same number from males were collected from donors of different age groups. The enterobacterial isolation was done on CLED agar medium. Their identification was carried out first by the enzymatic technic, then using MALDI–TOF MS method. ESBLs-producing enterobacteria were identified by searching the synergy between Clavulanic acid and 3rd generation Cephalosporins. The antimicrobial susceptibility tests were carried out using the disk diffusion method. Results: In male urine samples, the identified ESBLs-producing strains belong to E. coli, E. agglomerans and K. pneumoniae. They were in 11 samples: 3 from donors aged 20 years or less, 3 also from those aged 21 to 40 years as well as in those aged 41 to 60 years, and 2 in samples from donors aged over than 60 years. In female urine samples, the identified ESBLs-producing strains belong to Escherichia coli, Enterobacter agglomerans, Yersinia frederiksenii, Salmonella typhi and Klebsiella pneumoniae. They were in 27 samples: 3 from donors aged 20 years or less, 9 from those aged 21 to 40 years, 12 from those aged 41 to 60 years, and 3 from donors aged over than 60 years. All the strains were sensitive to Imipenem and resistant to Cefotaxim, Ceftazidin, Amoxicillin+Clavulanic acid and Nitrofurantoin. The Multi-Antibiotic Resistance (MAR) index varied from 0.416 (E. coli strains, youngest male donors) to 0.916 (E. coli strains, old female donors). Most of strains react differently from one antibiotic to another (P<0.05), with the exception of E. coli strains from most samples. Most of the identified ESBLs-producing species have been reported in surface and groundwater the population use. These water resources could play a role in the human urinary tract infections. Conclusion: There is a serious health problem with great MAR indices for all bacterial species identified. Treating water before use could reduce the viability and spreading of ESBLs-producing bacteria and genes into the population.

Occurrence of Enterococci in the Process of Artisanal Cheesemaking and Their Antimicrobial Resistance.

Enterococci are a group of microorganisms that have a controversial position from some scientific points of view. The species of the greatest clinical importance are E. faecalis and E. faecium, which are common agents of nosocomial infections. However, enterococci also have important applications in the dairy industry, as they are used as non-starter lactic acid bacteria (NSLAB) in a variety of cheeses, especially artisanal cheeses. The aim of this study was to determine the presence of representatives from the Enterococcus genus using PCR and MALDI-TOF MS methods on samples of raw milk, processing environment swabs, and cheese from four different artisanal dairy plants in Slovakia. Among the 136 isolates of enterococci, 9 species of genus Enterococci (E. faecalis, E. faecium, E. durans, E. devriesi, E. hirae, E. italicus, E. casseliflavus, E. malodoratus, and E. gallinarum) were identified and were tested for their antimicrobial resistance (AMR) to 8 antibiotics (amoxicillin, penicillin, ampicillin, erythromycin, levofloxacin, vancomycin, rifampicin, and tetracycline); most of them were resistant to rifampicin (35.3%), ampicillin (22.8%), and tetracycline (19.9%). A PCR analysis of vanA (4.41%) and tetM (14.71%) revealed that antimicrobial resistance genes were present in not only phenotypic resistant isolates of enterococci but also susceptible isolates. The investigation of antimicrobial resistance in enterococci during the cheesemaking process can be a source of valuable information for public health in the concept of "One Health".

Comparison of Vaginal microbiota in HPV-negative and HPV-positive pregnant women using a culture-based approach

The purpose of this study was to conduct a comparative analysis of the composition of the dominant groups of vaginal microorganisms in healthy pregnant women and pregnant women infected with HPV using a microbiological culture-based method. The MALDI TOF MS method and 16S rRNA gene fragment sequencing were used to identify microorganisms isolated from healthy pregnant women (n=32) and pregnant women infected with HPV (n=24). It was found that vaginal secretion samples from both groups contained bacteria of 4 phyla: Bacillota, Actinomycetota, Pseudomonadota, Bacteroidota, and Ascomycota fungi. The most common microbial community in healthy pregnant women being CST I (p=0.0007), and CST V in pregnant women infected with HPV (p=0.0001). At the genus level, a total of 25 taxa were found in all samples, with Lactobacillus being the dominant genus overall. Escherichia (p<0.0001) and Prevotella (p=0.0001) concentrations were higher in HPV infected patients. When calculating the Pearson correlation coefficient for the phyla, it was found that Bacillota correlated negatively with HPV genotypes 16 and 51 (p≤0.05), but positively with HPV genotype 59 (p≤0.05), just like Actinomycetota (p≤0.05). Bacteroidota correlated positively with HPV genotype 56 (0.001<p<0.01), and Ascomycota correlated positively with HPV genotypes 39 and 51 (p≤0.05; 0.001<p<0.01). Pearson correlation coefficients between bacteria genera and HPV genotypes were statistically significant for the following genera: Lactobacillus, Streptococcus, Enterococcus, Gardnerella, Escherichia, Prevotella. The data obtained in our study indicates that the culture-based method is informative when assessing the qualitative and quantitative composition of the microbiota.

Genetic diversity and phylogenetic relationships of Streptococcus thermophilus isolates from traditional Turkish yogurt: multilocus sequence typing (MLST).

Yogurt, a globally consumed fermented dairy product, is recognized for its taste and potential health benefits attributed to probiotic bacteria, particularly Streptococcus thermophilus. In this study, we employed Multilocus Sequence Typing (MLST) to investigate the genetic diversity and phylogenetic relationships of 13 S. thermophilus isolates from traditional Turkish yogurt samples. We also assessed potential correlations between genetic traits and geographic origins. The isolates were identified as S. thermophilus using VITEK® MALDI-TOF MS, ribotyping, and 16S rRNA analysis methods. MLST analysis revealed 13 different sequence types (STs), with seven new STs for Turkey. The most prevalent STs were ST/83 (n = 3), ST/135 (n = 2), and ST/134 (n = 2). eBURST analysis showed that these isolates mainly were singletons (n = 7) defined as sequence types (STs) that cannot be assigned to any group and differ at two or more alleles from every other ST in the sample. This information suggests that the isolates under study were genetically distinct from the other isolates in the dataset, highlighting their unique genetic profiles within the population. Genetic diversity analysis of ten housekeeping genes revealed polymorphism, with some genes showing higher allelic variation than others. Tajima's D values suggested that selection pressures differed among these genes, with some being more conserved, likely due to their vital functions. Phylogenetic analysis revealed distinct genetic diversity between Turkish isolates and European and Asian counterparts. These findings demonstrate the genetic diversity of S. thermophilus isolates in Turkish yogurt and highlight their unique evolutionary patterns. This research contributes to our understanding of local microbial diversity associated with yogurt production in Turkey and holds the potential for identifyic strains with enhanced functional attributes.

Bacteriophages for Controlling Staphylococcus spp. Pathogens on Dairy Cattle Farms: In Vitro Assessment.

Pathogenic Staphylococcus spp. strains are significant agents involved in mastitis and in skin and limb infections in dairy cattle. The aim of this study was to assess the antibacterial effectiveness of bacteriophages isolated from dairy cattle housing as potential tools for maintaining environmental homeostasis. The research will contribute to the use of phages as alternatives to antibiotics. The material was 56 samples obtained from dairy cows with signs of limb and hoof injuries. Staphylococcus species were identified by phenotypic, MALDI-TOF MS and PCR methods. Antibiotic resistance was determined by the disc diffusion method. Phages were isolated from cattle housing systems. Phage activity (plaque forming units, PFU/mL) was determined on double-layer agar plates. Morphology was examined using TEM microscopy, and molecular characteristics were determined with PCR. Among 52 strains of Staphylococcus spp., 16 were used as hosts for bacteriophages. Nearly all isolates (94%, 15/16) showed resistance to neomycin, and 87% were resistant to spectinomycin. Cefuroxime and vancomycin were the most effective antibiotics. On the basis of their morphology, bacteriophages were identified as class Caudoviricetes, formerly Caudovirales, families Myoviridae-like (6), and Siphoviridae-like (9). Three bacteriophages of the family Myoviridae-like, with the broadest spectrum of activity, were used for further analysis. This study showed a wide spectrum of activity against the Staphylococcus spp. strains tested. The positive results indicate that bacteriophages can be used to improve the welfare of cattle.

Heterologous expression and fibrillary characterization of the microtubule-binding domain of tau associated with tauopathies.

Neurofibrillary tangles (NFTs) are one of the most common pathological characteristics of Alzheimer's disease. The NFTs are mainly composed of hyperphosphorylated microtubule-associated tau. Thus, recombinant tau is urgently required for the study of its fibrillogenesis and its associated cytotoxicity. Heterologous expression, purification, and fibrillation of the microtubule-binding domain (MBD) of tau (tauMBD) were performed. The tauMBD was heterologously expressed in E. coli. Ni-chelating affinity chromatography was then performed to purify the target protein. Thereafter, tauMBD was systematically identified using the SDS-PAGE, western blot and MALDI-TOF MS methods. The aggregation propensity of the tauMBD was explored by both the thioflavin T fluorescence and atomic force microscopy experiments. The final yield of the recombinant tauMBD was ~ 20 mg L-1. It is shown that TauMBD, in the absence of an inducer, self-assembled into the typical fibrils at a faster rate than wild-type tau. Finally, the in vitro cytotoxicity of tauMBD aggregates was validated using PC12 cells. The heterologously expressed tau in this study can be further used in the investigation of the biophysical and cellular cytotoxic properties of tau.

Co-colonization of different species harboring KPC or NDM carbapenemase in the same host gut: insight of resistance evolution by horizontal gene transfer.

The dissemination of carbapenem-resistant Enterobacteriales (CRE) in nosocomial settings is primarily associated with the horizontal transfer of plasmids. However, limited research has focused on the in-host transferability of carbapenem resistance. In this study, ten isolates were collected from gut specimens of five individuals, each hosting two different species, including Escherichia coli, Klebsiella pneumoniae, Klebsiella aerogenes, Enterobacter cloacae, or Citrobacter koseri. Species identification and antimicrobial susceptibility were determined by MALDI-TOF MS and broth microdilution method. Carbapenemase genes were detected and localized using PCR, S1-PFGE and southern blot. The transferability of carbapenemase genes between species was investigated through filter mating experiments, and the genetic contexts of the plasmids were analyzed using whole genome sequencing. Our results revealed that each of the ten isolates harbored a carbapenemase gene, including bla NDM-5, bla NDM-1, or bla KPC-2, on a plasmid. Five different plasmids were successfully transferred to recipient cells of E. coli, K. pneumoniae or A. baumannii by transconjugation. The genetic contexts of the carbapenemase gene were remarkably similar between the two CRE isolates from each individual. This study highlights the potential for interspecies plasmid transmission in human gut, emphasizing the colonization of CRE as a significant risk factor for the dissemination of carbapenemase genes within the host. These findings underscore the need for appropriate intestinal CRE screening and colonization prevention.

High oral carriage of multidrug resistant Gram-negative bacilli in adolescents: the SOPKARD-Junior study.

The colonization of the oral cavity by potentially pathogenic antimicrobial-resistant bacteria in adolescents and its consequences is very poorly understood. The present study focused on the occurrence of oral colonization by Gram-negative bacilli (GNB) and their multidrug resistance, including the production of extended-spectrum β-lactamases (ESBLs) and carbapenemases, among healthy adolescents and risk factors associated with GNB colonization. This study was conducted as part of "A program for the early detection of risk factors for lifestyle diseases SOPKARD-Junior" (SOPKARD-Junior). Oral samples were collected from 182 adolescents from four public elementary schools in Sopot, Poland, aged 13-14 years. Bacterial strains were identified by the MALDI-TOF MS method. Screening of antimicrobial resistance was performed using a disk diffusion method. The NG-Test® CARBA-5 was used to detect and differentiate the five most widely distributed carbapenemases. Demographic and clinical data were collected and statistical analysis of risk factors was performed. A total of 68 out of 182 (37.4%) healthy adolescents was documented oral colonization with Gram-negative bacilli, including 50/182 (27.5%) multidrug resistant (MDR-GNB) strains. Over 60% of oral carriage concerned three main genera Enterobacter spp., Pseudomonas spp., and Serratia spp., which were detected in 22.1%, 19.1%, and 19.1% of participants, respectively. Citrobacter spp., Escherichia coli, Klebsiella spp., Hafnia spp., Aeromonas spp., Acinetobacter spp., and Stenotrophomonas spp. were also isolated. The antimicrobial resistance to ampicillin (100%), ceftazidime (69.1%), meropenem (60.3%), gentamycin (60.3%), piperacillin/tazobactam (52.9%), and piperacillin (45.6%) were the most common. Among 73.5% GNB strains multidrug resistance was observed, including all Pseudomonas spp. strains. Among MDR-GNB, 30.4% were resistant to four groups of antibiotics, half of the MDR Pseudomonas spp. strains were resistant to 10 groups of antibiotics. Extended-spectrum β-lactamases were produced by Enterobacter cloacae, Klebsiella spp., and Serratia spp. (7.4%). Colonization by ESBLs-positive GNB strains was significantly associated with recurrent respiratory infections, nasal congestion, and bronchitis (p<0.05). Our study revealed high oral carriage of multi-drug resistant Gram-negative bacilli in healthy adolescents and the association of ESBL-producing strains with respiratory infections. Further studies on oral colonization with GNB are necessary due to the possibility of distinct infections and the acquisition of antibiotic resistance by resident microbiota.

Evaluating molecular epidemiology of carbapenem non-susceptible Klebsiella pneumoniae isolates with MLST, MALDI-TOF MS, PFGE

BackgroundThis study aimed to evaluate antibiotic resistance genes and virulence genes and the clonal relationship of the carbapenem-nonsusceptible Klebsiella pneumoniae strains by molecular methods which are isolated from various clinical specimens from patients treated in tertiary care hospital in Turkey.MethodsIdentification of 32 carbapenem non-susceptible K. pneumoniae were determined by VITEK-2 (BioMérieux, France) automated system. Thirteen colistin-resistant strains were tested with the broth microdilution method. Various antibiotic resistance genes and virulence genes frequently seen in carbapenem-resistant strains were screened by PCR. Immunochromatographic tests used in the rapid diagnosis of carbapenemases were compared with PCR results. In addition, PFGE, MLST and MALDI-TOF MS methods were used to determine the clonal relationship among these strains.ResultsPCR demonstrated that 31 of the strains carried at least one of the carbapenemase genes. In one strain, the coexistence of blaOXA−48+NDM was shown. The most common resistance genes were determined as blaSHV (84.3%), blaCTX−M−1 (46.8%), blaOXA−48 (40.6%), blaKPC (40.6%), blaTEM (31.2%), blaNDM (18.8%) respectively. Among the virulence genes; magA (68.7%) was the most common, followed by kpn (59.3%) and K2 (9.3%). Immunochromatographic tests were found to be 100% compatible with PCR results. All colistin-resistant isolates were also found to be resistant by colistin broth microdilution. In PFGE analysis, 25 different genotypes were determined and clustering isolates were collected in 5 different clusters and the clustering rate was 35.4%. In MLST analysis, ST101 type was determined as the most common ST type with a rate of 29%. ST101 is followed by ST16, ST307, ST14, ST147, ST309, ST377, ST395 and ST2096, respectively. The compatibility rate between MALDI-TOF MS and VITEK-2 was found 94.3%, in bacterial identification. In MALDI-TOF MS typing, the maximum similarity between the strains was less than 70% and clustering not shown.ConclusionIn addition to OXA-48, which is endemic in our country, it has been determined that KPC, which is more common in the world, is becoming increasingly common in our region. ST101 type was determined as the most common type between the strains. To the best of our knowledge, this is the first study that compares these three methods in our country. There may be differences between bacterial identifications made with VITEK-2 and MALDI-TOF MS. In this study, it was observed that MALDI-TOF MS analyses were not compatible with the typing of strains according to PFGE and MLST analysis results.

Identification of Antibody-Mediated Hydrolysis Sites of Oligopeptides Corresponding to the SARS-CoV-2 S-Protein by MALDI-TOF Mass Spectrometry.

Antibodies recognizing RBD and the S-protein have been previously demonstrated to be formed in humans after SARS-CoV-2 infection and vaccination with the Sputnik V adenovirus vaccine. These antibodies were found to be active when hydrolyzing FITC-labeled oligopeptides corresponding to linear epitopes of the S-protein. The thin-layer chromatography method allows the relative accumulation of the reaction product to be estimated but cannot identify hydrolysis sites. This study used the MALDI-TOF MS method to establish oligopeptide hydrolysis sites. Using the MALDI-TOF MS method in combination with the analysis of known hydrolysis sites characteristic of canonical proteases allowed us to establish the unique hydrolysis sites inherent only to catalytically active antibodies. We have discovered two 12-mer oligopeptides to have six hydrolysis sites equally distributed throughout the oligopeptide. The other three oligopeptides were found to have two to three closely spaced hydrolysis sites. In contrast to trypsin and chymotrypsin proteases, the catalytically active antibodies of COVID-19 patients have their peptide bond hydrolyzed mainly after proline, threonine, glycine, or serine residues. Here, we propose a new high-throughput experimental method for analyzing the proteolytic activity of natural antibodies produced in viral pathology.

Highly Efficient One-Pot Synthesis of Hexakis(m-phenyleneimine) Macrocyle Cm6 and the Thermostimulated Self-Healing Property through Dynamic Covalent Chemistry.

Highly efficient one-pot synthesis of hexakis(m-phenyleneimine) macrocycle Cm6 from acetalprotected AB-type monomer, m-aminobenzaldehyde diethylacetal, was successfully achieved based on imine dynamic covalent chemistry and precipitation-driven cyclization. The structure of Cm6 in the solid state was determined using CP/MAS NMR, X-ray single crystallographic analysis, and WAXD. Macrocycle Cm6 is composed of six phenylene and imine bonds facing the same direction, with nitrogen atoms arranged on the outside of the ring, and has a chair conformation, as predicted from DFT calculation. The macrocycle forms π-stacked columnar aggregates and hexagonally closest-packed structure. The cyclization process was investigated using MALDI-TOF MS and NMR. A mechanism of precipitation-driven cyclization based on imine dynamic covalent chemistry and π-stacked columnar aggregation is proposed. Both the nature of imine linkage and the shape anisotropy of the macrocycle played an important role in the single one-pot synthesis. The water-mediated mutual conversion between macrocycle Cm6 and linear oligomers driven by thermal stimulation was analyzed using MALDI-TOF MS and GPC methods. Macrocycle Cm6 with a dynamic covalent imine bond exhibited self-healing properties when stimulated using heat.

Improved MALDI-TOF MS Identification of Mycobacterium tuberculosis by Use of an Enhanced Cell Disruption Protocol.

Mycobacterium tuberculosis is the microorganism that causes tuberculosis, a disease affecting millions of people worldwide. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast, reliable, and cost-effective method for microorganism identification which has been used for the identification of Mycobacterium spp. isolates. However, the mycobacteria cell wall is rich in lipids, which makes it difficult to obtain proteins for MALDI-TOF MS analysis. In this study, two cell preparation protocols were compared: the MycoEx, recommended by MALDI-TOF instrument manufacturer Bruker Daltonics, and the MycoLyser protocol described herein, which used the MagNA Lyser instrument to enhance cell disruption with ethanol. Cell disruption and protein extraction steps with the two protocols were performed using the Mycobacterium tuberculosis H37Rv strain, and the MALDI-TOF MS results were compared. The MycoLyser protocol allowed for improved Biotyper identification of M. tuberculosis since the log(score) values obtained with this protocol were mostly ≥ 1.800 and significantly higher than that underwent MycoEx processing. The identification reliability was increased as well, considering the Bruker criteria. In view of these results, it is concluded that the MycoLyser protocol for mycobacterial cell disruption and protein extraction improves the MALDI-TOF MS method's efficacy for M. tuberculosis identification.

Clinical, Microbiological, and Molecular Characterization of Candia (Starmera) stellimalicola, a Rare Fungal Pathogen Causing Human Infections.

Candia (Starmera) stellimalicola is a yeast species spread worldwide and recovered from varieties of ecological reservoirs, but human infections are rarely reported. In this study, we reported an intra-abdominal infection case caused by C. stellimalicola and described its microbiological and molecular characteristics. C. stellimalicola strains were isolated from ascites fluid of an 82-year-old male patient having diffuse peritonitis with fever and elevated WBC counts. Routine biochemical and MALDI-TOF MS methods failed to identify the pathogenic strains. Phylogenetic analysis of 18S, 26S and internal transcribed space (ITS) rDNA regions, as well as whole-genome sequence identified the strains as C. stellimalicola. Compared with other Starmera species, C. stellimalicola had unique physiological characteristics including thermal tolerance (able to grow at 42°C), which may prompt its environmental adaptability and potential for opportunistic human infection. Fluconazole minimum inhibitory concentration (MIC) values of the strains identified in this case was 2mg/L, and the patient had a favorable outcome after receiving fluconazole treatment. In comparison, the majority of C. stellimalicola strains previously documented had high MIC values (≥ 16mg/L) to fluconazole. In conclusion, with the raise in human infections caused by rare fungal pathogens, molecular diagnostic remains the most efficient way for accurate species identification; and antifungal susceptibility testing is essential to guide proper patient management.

The rapid detection of procalcitonin in septic serum using immunoaffinity MALDI chips

BackgroundSepsis is a common worldwide health condition with high mortality. It is caused by a dysregulated immune response to the pathogen. Severe infections resulting in sepsis can be also determined by monitoring several bloodstream biomarkers, one of them being pro-hormone procalcitonin (PCT). PCT concentration in the bloodstream correlates well with sepsis and in severe cases increases up to a thousand times from the healthy physiological values in a short time. In this study, we developed a rapid technique for PCT detection by MALDI-TOF mass spectrometry, that uses in-situ enrichment directly on the specialized immuno MALDI chips that are utilized as MALDI plates. The method’s ability to detect PCT was confirmed by comparing the results with LC–MS bottom-up workflow. The new method detects intact PCT by its m/z and uncovers its alternations in septic serum.MethodsThe MALDI chips used for the detection of PCT were prepared by ambient ion soft landing of anti-PCT antibody on an ITO glass slide. The chips were used for the development of the rapid MALDI-TOF MS method. A parallel method based on affinity enrichment on magnetic beads followed by LC–MS/MS data-dependent peptide microsequencing was used to prove PCT presence in the sample. All samples were also tested by ELISA to determine PCT concentration prior to analyzing them by mass spectrometry methods.ResultsThe MALDI chip method was optimized using recombinant PCT spiked into the human serum. The PCT detection limit was 10 ng/mL. The optimized method was used to analyze 13 sera from patients suffering sepsis. The PCT results were confirmed by LC–MS/MS. The measurement of the intact PCT by the MALDI chip method revealed that sera of patients with severe sepsis have other forms of PCT present, which show post-processing of the primary sequence by cleavage of PCT, resulting in the formation of N and C termini fragments.ConclusionsProcalcitonin from human serum was successfully enriched and detected using immunoaffinity MALDI chips. The intact PCT was characterized in 13 septic patients. The method is more specific compared to non-MS-based immunoaffinity techniques and allows observation of different variants of PCT in septic patients.

Multiple mutations of Mycobacterium intracellulare subsp. chimaera causing false-negative reaction to the transcription-reverse transcription concerted method for pathogen detection

ObjectivesTo report an isolate of Mycobacterium intracellulare subsp. chimaera with multiple mutations in 16S ribosomal RNA (rRNA) gene, resulting in the false-negative reaction to the transcription-reverse transcription concerted (TRC) method for Mycobacterium avium-intracellulare complex. MethodsWe used TRC, polymerase chain reaction (PCR), and Matrix-assisted laser desorption/ionization Time-of-Flight/Mass Spectrometry (MALDI-TOF/MS) methods to identify a clinical isolate in 2021. Due to the discordant results between TRC and PCR or MALDI-TOF MS methods, 16S rRNA sequencing, whole-genome shotgun (WGS) sequencing, and average nucleotide identity (ANI) analysis were employed to identify the isolate. ResultsA mycobacterial isolate from a sputum sample gave negative results for the detection of Mycobacterium tuberculosis complex or M. avium-intracellulare complex by the TRC method. However, the isolate was identified as M. intracellulare by both PCR method and MALDI-TOF MS method. WGS sequencing of 16S rRNA genome revealed eight substitution mutations and one insertion mutation within the region, which could hamper the correct reaction to TRC method. Subsequent ANI analysis between the isolate and various species of nontuberculosis mycobacteria revealed that the isolate could be identified as M. intracellulare subsp. chimaera. ConclusionRare mutations within the 16S rRNA genome resulted in the false-negative identification of Mycobacterium chimaera by the TRC method. WGS sequencing and ANI analysis was necessary to identify the isolate.

The role of intestinal translocation of E.coli in the development of acute obstructive pyelonephritis in an experiment

&lt;b&gt;Aim:&lt;/b&gt; To study the role of &lt;i&gt;E. coli &lt;/i&gt;intestinal translocation in the development of acute obstructive pyelonephritis in an experiment.&lt;br /&gt; &lt;b&gt;Material and methods:&lt;/b&gt; An experimental study was conducted on 60 male rabbits weighing 3000±500 g. The animals were divided into 3 groups of 20 animals each: experimental, control and intermediate control group. The acute obstructive pyelonephritis with the ureter blocking by laparotomy and introduction of the strain into the intestine were simulated in the animals of the experimental group. In the control group, the model was performed anologically as in the experimental group, but without the ureter blocking. In the intermediate control group, laparotomy was performed, the ureter was isolated without blocking and without the introduction of a bacterial strain. 10 animals of each group were removed from the experiment on the 3&lt;sup&gt;rd&lt;/sup&gt; and 5&lt;sup&gt;th&lt;/sup&gt; days, kidney tissue and urine were intake. As a reference marker strain, the laboratory strain &lt;i&gt;E. coli&lt;/i&gt; No. 49579 was used, which was obtained from a patient with a urological infection and had resistance to cefepime, ciprofloxacin and tetracycline. Biomaterials were studied by microbiological examination and subspecific typing of strains using the MALDI-TOF MS method, antibiotic sensitivity was determined.&lt;br /&gt; &lt;b&gt;Results: &lt;/b&gt;&lt;i&gt;E. coli&lt;/i&gt; strain was isolated in all animals of the experimental group and in 2 animals of the control group on the 5&lt;sup&gt;th&lt;/sup&gt; day. During subspecific typing by the MALDI-TOF MS method, the isolated strains were identical in ribosomal proteins, and also had the same sensitivity to the said antibiotics. When analyzing the amount of lg CFU &lt;i&gt;E.coli&lt;/i&gt; in urine after the experiment between the experimental and control group, we found that, on day 3, there were statistically significant differences between the groups (p=0.005), and on day 5, the amount of lg CFU &lt;i&gt;E.coli&lt;/i&gt; was 13 times greater (p=0.004).A comparative analysis of the lg CFU &lt;i&gt;E.coli&lt;/i&gt; index in kidney tissue on 3 (p=0.004) and 5 (p=0.003) days revealed statistically significant differences between the experimental group and the control group.&lt;br /&gt; &lt;b&gt;Conclusion:&lt;/b&gt; The results of identification and subspecific typing of isolated microorganisms confirmed that the strains isolated from the urinary tract were identical to the reference strain introduced into the gastrointestinal tract during the experiment, which confirms the role of translocation of intestinal microorganisms in the development of acute obstructive pyelonephritis.