Potential of MALDI-TOF MS biotyping to detect deltamethrin resistance in the dengue vector Aedes aegypti.

Insecticide resistance in mosquitoes is spreading worldwide and represents a growing threat to vector control. Insecticide resistance is caused by different mechanisms including higher metabolic detoxication, target-site modification, reduced penetration and behavioral changes that are not easily detectable with simple diagnostic methods. Indeed, most molecular resistance diagnostic tools are costly and labor intensive and then difficult to use for routine monitoring of insecticide resistance. The present study aims to determine whether mosquito susceptibility status against the pyrethroid insecticides (mostly used for mosquito control) could be established by the protein signatures of legs and/or thoraxes submitted to MALDI-TOF Mass Spectrometry (MS). The quality of MS spectra for both body parts was controlled to avoid any bias due to unconformity protein profiling. The comparison of MS profiles from three inbreeds Ae. aegypti lines from French Guiana (IRF, IR03, IR13), with distinct deltamethrin resistance genotype / phenotype and the susceptible reference laboratory line BORA (French Polynesia), showed different protein signatures. On both body parts, the analysis of whole protein profiles revealed a singularity of BORA line compared to the three inbreeding lines from French Guiana origin, suggesting that the first criteria of differentiation is the geographical origin and/or the breeding history rather than the insecticide susceptibility profile. However, a deeper analysis of the protein profiles allowed to identify 10 and 11 discriminating peaks from leg and thorax spectra, respectively. Among them, a specific peak around 4870 Da was detected in legs and thoraxes of pyrethroid resistant lines compared to the susceptible counterparts hence suggesting that MS profiling may be promising to rapidly distinguish resistant and susceptible phenotypes. Further work is needed to confirm the nature of this peak as a deltamethrin resistant marker and to validate the routine use of MS profiling to track insecticide resistance in Ae. aegypti field populations.

Predicting the age of field Anopheles mosquitoes using mass spectrometry and deep learning.

Mosquito-borne diseases like malaria are rising globally, and improved mosquito vector surveillance is needed. Survival of Anopheles mosquitoes is key for epidemiological monitoring of malaria transmission and evaluation of vector control strategies targeting mosquito longevity, as the risk of pathogen transmission increases with mosquito age. However, the available tools to estimate field mosquito age are often approximate and time-consuming. Here, we show a rapid method that combines matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry with deep learning for mosquito age prediction. Using 2763 mass spectra from the head, legs, and thorax of 251 field-collected Anopheles arabiensis mosquitoes, we developed deep learning models that achieved a best mean absolute error of 1.74 days. We also demonstrate consistent performance at two ecological sites in Senegal, supported by age-related protein changes. Our approach is promising for malaria control and the field of vector biology, benefiting other disease vectors like Aedes mosquitoes.

Elucidating Gender-Specific Distribution of Imipramine, Chloroquine, and Their Metabolites in Mice Kidney Tissues through AP-MALDI-MSI.

Knowledge of gender-specific drug distributions in different organs are of great importance for personalized medicine and reducing toxicity. However, such drug distributions have not been well studied. In this study, we investigated potential differences in the distribution of imipramine and chloroquine, as well as their metabolites, between male and female kidneys. Kidneys were collected from mice treated with imipramine or chloroquine and then subjected to atmospheric pressure matrix-assisted laser desorption ionization-mass spectrometry imaging (AP-MALDI-MSI). We observed differential distributions of the drugs and their metabolites between male and female kidneys. Imipramine showed prominent distributions in the cortex and medulla in male and female kidneys, respectively. Desipramine, one of the metabolites of imipramine, showed significantly higher (*** p < 0.001) distributions in the medulla of the male kidney compared to that of the female kidney. Chloroquine and its metabolites were accumulated in the pelvis of both male and female kidneys. Interestingly, they showed a characteristic distribution in the medulla of the female kidney, while almost no distributions were observed in the same areas of the male kidney. For the first time, our study revealed that the distributions of imipramine, chloroquine, and their metabolites were different in male and female kidneys.

Kodamaea ohmeri: A rare yeast causing invasive infections in immunocompromised patients.

INTRODUCTION: Kodamaea ohmeri is a rare, recognized pathogen that has previously been isolated from environmental sources. The patients commonly affected by this yeast include immunocompromised as well as immunocompetent patients having several associated risk factors. METHODOLOGY: We report three cases in which K. ohmeri was isolated from blood using Bact T/ALERT. Identification was carried out by MALDI-TOF MS (Vitek-MS, BioMérieux, Marcy-l'Etoile, France) in addition to color characteristics on chromogenic media. The patients had diminished immune response on account of a multitude of comorbidities. RESULTS: K. ohmeri can be misidentified as Candida tropicalis, Candida albicans, or Candida hemolounii by conventional methods; correct and timely identification can be achieved by MALDI-TOF MS. Antifungal susceptibility breakpoints for K. ohmeri are currently not defined. An Echinocandin was added to the treatment regimen of all three of the cases. CONCLUSIONS: Identification of K. ohmeri using conventional methods is difficult and unusual yeasts should be carefully observed, especially upon prolonged incubation.

Exploring the fragmentation efficiency of proteins analyzed by MALDI-TOF-TOF tandem mass spectrometry using computational and statistical analyses.

Matrix-assisted laser desorption/ionization time-of-flight-time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry (MS/MS) is a rapid technique for identifying intact proteins from unfractionated mixtures by top-down proteomic analysis. MS/MS allows isolation of specific intact protein ions prior to fragmentation, allowing fragment ion attribution to a specific precursor ion. However, the fragmentation efficiency of mature, intact protein ions by MS/MS post-source decay (PSD) varies widely, and the biochemical and structural factors of the protein that contribute to it are poorly understood. With the advent of protein structure prediction algorithms such as Alphafold2, we have wider access to protein structures for which no crystal structure exists. In this work, we use a statistical approach to explore the properties of bacterial proteins that can affect their gas phase dissociation via PSD. We extract various protein properties from Alphafold2 predictions and analyze their effect on fragmentation efficiency. Our results show that the fragmentation efficiency from cleavage of the polypeptide backbone on the C-terminal side of glutamic acid (E) and asparagine (N) residues were nearly equal. In addition, we found that the rearrangement and cleavage on the C-terminal side of aspartic acid (D) residues that result from the aspartic acid effect (AAE) were higher than for E- and N-residues. From residue interaction network analysis, we identified several local centrality measures and discussed their implications regarding the AAE. We also confirmed the selective cleavage of the backbone at D-proline bonds in proteins and further extend it to N-proline bonds. Finally, we note an enhancement of the AAE mechanism when the residue on the C-terminal side of D-, E- and N-residues is glycine. To the best of our knowledge, this is the first report of this phenomenon. Our study demonstrates the value of using statistical analyses of protein sequences and their predicted structures to better understand the fragmentation of the intact protein ions in the gas phase.

A comparison between full-length 16S rRNA Oxford nanopore sequencing and Illumina V3-V4 16S rRNA sequencing in head and neck cancer tissues.

Describing the microbial community within the tumour has been a key aspect in understanding the pathophysiology of the tumour microenvironment. In head and neck cancer (HNC), most studies on tissue samples have only performed 16S rRNA short-read sequencing (SRS) on V3-V5 region. SRS is mostly limited to genus level identification. In this study, we compared full-length 16S rRNA long-read sequencing (FL-ONT) from Oxford Nanopore Technology (ONT) to V3-V4 Illumina SRS (V3V4-Illumina) in 26 HNC tumour tissues. Further validation was also performed using culture-based methods in 16 bacterial isolates obtained from 4 patients using MALDI-TOF MS. We observed similar alpha diversity indexes between FL-ONT and V3V4-Illumina. However, beta-diversity was significantly different between techniques (PERMANOVA - R2 = 0.131, p < 0.0001). At higher taxonomic levels (Phylum to Family), all metrics were more similar among sequencing techniques, while lower taxonomy displayed more discrepancies. At higher taxonomic levels, correlation in relative abundance from FL-ONT and V3V4-Illumina were higher, while this correlation decreased at lower levels. Finally, FL-ONT was able to identify more isolates at the species level that were identified using MALDI-TOF MS (75% vs. 18.8%). FL-ONT was able to identify lower taxonomic levels at a better resolution as compared to V3V4-Illumina 16S rRNA sequencing.

Mass spectrometric analysis of Odonthobuthus Doriae scorpion venom and its non-neutralized fractions after interaction with commercial antivenom.

It is believed that antivenoms play a crucial role in neutralizing venoms. However, uncontrolled clinical effects appear in patients stung by scorpions after the injection of antivenom. In this research, non-neutralized components of the venom of the Iranian scorpion Odonthobuthus doriae were analyzed after interacting with the commercial antivenom available in the market. The venom and antivenom interaction was performed, then centrifuged, and the supernatant was analyzed by high-performance liquid chromatography (HPLC). Two peaks of Odonthobuthus doriae venom were observed in the chromatogram of the supernatant. Two components were isolated by HPLC and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) instruments. Peptide sequencing was done by Liquid Chromatography Quadrupole Time-of-Flight Tandem Mass Spectrometry (LC-Q-TOF MS/MS). Results indicate that the components of scorpion venom mainly have a molecular weight below 10 kDa, consisting of toxic peptides that disrupt the function of sodium and potassium channels. The MALDI-TOF MS results show that two toxic peptides with molecular masses of 6941 Da and 6396 Da were not neutralized by the antivenom. According to the MS/MS sequencing data, the components have been related to peptides A0A5P8U2Q6_MESEU and A0A0U4FP89_ODODO, which belong to the sodium and potassium channels toxins family, respectively.

Mannheimia indoligenes sp. nov., proposed for clade V organisms of Mannheimia.

A set of 25 strains belonging to clade V of Mannheimia mainly isolated from cattle was investigated and is proposed to represent Mannheimia indoligenes sp. nov. The species can be separated from the other validly published species of the genus by pheno- and genotype. Only indole separates M. indoligenes and Mannheimia varigena while two to seven characters separate M. indoligenes from other species of Mannheimia. Thirteen strains belonging to biogroups 6, 7, 8C, 9, 10, 12 and UG5 formed a monophyletic group based on 16S rRNA gene sequence comparisons with 98-100 % similarity. Eight of these strains were further included in the whole genome comparison. Digital DNA-DNA hybridization showed that the similarities between the suggested type strain M14.4T and the other strains of M. indoligenes were 62.9 % or higher. The average nucleotide identity was 95.5 % or higher between M14.4T and the other strains of the species. The rpoB gene sequence similarity was 95-100 % within M. indoligenes. MALDI-TOF allowed a clear separation from other Mannheimia species further supporting classification as a novel species and making it the diagnostic identification tool of choice for M. indoligenes. The type strain is M14.4T (=CCUG 77347T=DSM 116804T) isolated from a cattle tongue in Scotland.

Matrix-free laser desorption/ionization mass spectrometry imaging for rapid evaluation of wood biomass conversion.

RATIONALE: This study overcomes traditional biomass analysis limitations by introducing a pioneering matrix-free laser desorption/ionization (LDI) approach in mass spectrometry imaging (MSI) for efficient lignin evaluation in wood. The innovative acetic acid-peracetic acid (APA) treatment significantly enhances lignin detection, enabling high-throughput, on-site analysis. METHODS: Wood slices, softwood from a conifer tree (Japanese cypress) and hardwood from a broadleaf tree (Japanese beech), were analyzed using MSI with a Fourier transform ion cyclotron resonance mass spectrometer. The developed APA treatment demonstrated effectiveness for MSI analysis of biomass. RESULTS: Our imaging technique successfully distinguishes between earlywood and latewood and enables the distinct visualization of lignin in these and other wood tissues, such as the radial parenchyma. This approach reveals significant contrasts in MSI. It has identified intense ions from ß-O-4-type lignin, specifically in the radial parenchyma of hardwood, highlighting the method's precision and utility in wood tissue analysis. CONCLUSIONS: The benefits of matrix-free LDI include reduced peak overlap, consistent sample quality, preservation of natural sample properties, enhanced analytical accuracy, and reduced operational costs. This innovative approach is poised to become a standard method for rapid and precise biomass evaluation and has important applications in environmental research and sustainable resource management and is crucial for the effective management of diverse biomass, paving the way towards a sustainable, circular society.

Delay in the diagnosis of Brucella abortus bacteremia in a nonendemic country: a case report.

BACKGROUND: It is challenging to diagnose brucellosis in nonendemic regions because it is a nonspecific febrile disease. The accurate identification of Brucella spp. in clinical microbiology laboratories (CMLs) continues to pose difficulties. Most reports of misidentification are for B. melitensis, and we report a rare case of misidentified B. abortus. CASE PRESENTATION: A 67-year-old man visited an outpatient clinic complaining of fatigue, fever, and weight loss. The patient had a history of slaughtering cows with brucellosis one year prior, and his Brucella antibody tests were negative twice. After blood culture, the administration of doxycycline and rifampin was initiated. The patient was hospitalized due to a positive blood culture. Gram-negative coccobacilli were detected in aerobic blood culture bottles, but the CML's lack of experience with Brucella prevented appropriate further testing. Inaccurate identification results were obtained for a GN ID card of VITEK 2 (bioMérieux, USA) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using a MALDI Biotyper (Bruker, Germany). The strain showed 100.0% identity with Brucella spp. according to 16S rRNA sequencing. MALDI-TOF MS peaks were reanalyzed using the CDC MicrobeNet database to determine Brucella spp. (score value: 2.023). The patient was discharged after nine days of hospitalization and improved after maintaining only doxycycline for six weeks. The isolate was also identified as Brucella abortus by genomic evidence. CONCLUSION: Automated identification instruments and MALDI-TOF MS are widely used to identify bacteria in CMLs, but there are limitations in accurately identifying Brucella spp. It is important for CMLs to be aware of the possibility of brucellosis through communication with clinicians. Performing an analysis with an additional well-curated MALDI-TOF MS database such as Bruker security-relevant (SR) database or CDC MicrobeNet database is helpful for quickly identifying the genus Brucella.

Impact of the rearing environment on the metabolism of shrimps and tracing the origins and species of shrimps using specific metabolites.

Herein, the link between rearing environmental condition and metabolism was explored. Metabolite fingerprint datasets of black tiger shrimp (Penaeus monodon) from three production sites were collected and studied using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and HPLC-MS/MS. Two compounds, benzisothiazolinone and hippuric acid, were identified to be potentially related to pollution in the rearing environment and showed different abundances in the analysed shrimp samples with different origins. Furthermore, metabolomic analysis on three shrimp species, black tiger shrimp, kuruma shrimp (Penaeus japonicus) and sword shrimp (Parapenaeopsis hardwickii), under an identical rearing environment was also conducted. Two compounds, diethanolamine and benzisothiazolinone, potentially linked with pollution in the rearing environment were identified. The present protocol holds promise to be extended to the studies of exploring the relationship between rearing environmental conditions and metabolism. Furthermore, the analysis of single-blind samples was conducted. The results show that specific metabolites can be utilized as markers for tracing the origins of shrimp samples. The present protocol holds potential for application in tracing the origin and species of certain seafoods.

Distinguishing methicillin-resistant Staphylococcus aureus from methicillin-sensitive strains by combining Fe3O4 magnetic nanoparticle-based affinity mass spectrometry with a machine learning strategy.

Pathogenic bacteria, including drug-resistant variants such as methicillin-resistant Staphylococcus aureus (MRSA), can cause severe infections in the human body. Early detection of MRSA is essential for clinical diagnosis and proper treatment, considering the distinct therapeutic strategies for methicillin-sensitive S. aureus (MSSA) and MRSA infections. However, the similarities between MRSA and MSSA properties present a challenge in promptly and accurately distinguishing between them. This work introduces an approach to differentiate MRSA from MSSA utilizing matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in conjunction with a neural network-based classification model. Four distinct strains of S. aureus were utilized, comprising three MSSA strains and one MRSA strain. The classification accuracy of our model ranges from ~ 92 to ~ 97% for each strain. We used deep SHapley Additive exPlanations to reveal the unique feature peaks for each bacterial strain. Furthermore, Fe3O4 MNPs were used as affinity probes for sample enrichment to eliminate the overnight culture and reduce the time in sample preparation. The limit of detection of the MNP-based affinity approach toward S. aureus combined with our machine learning strategy was as low as ~ 8 × 103 CFU mL-1. The feasibility of using the current approach for the identification of S. aureus in juice samples was also demonstrated.

MazF-rolling circle amplification combined MALDI-TOF MS for site-specific detection of N6-methyladenosine RNA.

N6-methyladenosine (m6A) is one of the most abundant chemical modifications in RNA and has vital significance in cellular processes and tumor development. However, the accurate analysis of site-specific m6A modification remains a challenge. In this work, a MazF endoribonuclease activated rolling circle amplification (MazF-RCA) combined MALDI-TOF MS assay is developed for the detection of site-specific m6A-RNA. MazF endoribonuclease can specifically cleave the ACA motif, leaving methylated (m6A)CA motif intact. The intact methylated RNA can then be amplified through rolling circle amplification, and the generated reporter oligonucleotides are detected by MALDI-TOF MS. The assay exhibits good quantification ability, presenting a wide linear range (100 fM to 10 nM) with the limit-of-detection lower than 100 fM. Additionally, the assay can accurately detect methylated RNA in the presence of large amount of non-methylated RNA with a relative abundance of methylated RNA down to 0.5%. The developed assay was further applied to detect m6A-RNA spiked in MCF-7 cell RNA extracts, with the recovery rates in the range of 90.64-106.93%. The present assay provides a novel platform for the analysis of site-specific m6A-RNA at high specificity and sensitivity, which can promote the study of RNA methylation in clinical and biomedical research.

A blood test to monitor bee health across a European network of agricultural sites of different land-use by MALDI BeeTyping mass spectrometry.

There are substantial concerns about impaired honey bee health and colony losses due to several poorly understood factors. We used MALDI profiling (MALDI BeeTyping®) analysis to investigate how some environmental and management factors under field conditions across Europe affected the honey bee haemolymph peptidome (all peptides in the circulatory fluid), as a profile of molecular markers representing the immune status of Apis mellifera. Honey bees were exposed to a range of environmental stressors in 128 agricultural sites across eight European countries in four biogeographic zones, with each country contributing eight sites each for two different cropping systems: oilseed rape (OSR) and apple (APP). The full haemolymph peptide profiles, including the presence and levels of three key immunity markers, namely the antimicrobial peptides (AMPs) Apidaecin, Abaecin and Defensin-1, allowed the honey bee responses to environmental variables to be discriminated by country, crop type and site. When considering just the AMPs, it was not possible to distinguish between countries by the prevalence of each AMP in the samples. However, it was possible to discriminate between countries on the amounts of the AMPs, with the Swedish samples in particular expressing high amounts of all AMPs. A machine learning model was developed to discriminate the haemolymphs of bees from APP and OSR sites. The model was 90.6 % accurate in identifying the crop type from the samples used to build the model. Overall, MALDI BeeTyping® of bee haemolymph represents a promising and cost-effective "blood test" for simultaneously monitoring dozens of peptide markers affected by environmental stressors at the landscape scale, thus providing policymakers with new diagnostic and regulatory tools for monitoring bee health.

Hepatic Topology of Glycosphingolipids in Schistosoma mansoni-Infected Hamsters.

Schistosomiasis is a neglected tropical disease caused by worm parasites of the genus Schistosoma. Upon infection, parasite eggs can lodge inside of host organs like the liver. This leads to granuloma formation, which is the main cause of the pathology of schistosomiasis. To better understand the different levels of host-pathogen interaction and pathology, our study focused on the characterization of glycosphingolipids (GSLs). For this purpose, GSLs in livers of infected and noninfected hamsters were studied by combining high-spatial-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) with nanoscale hydrophilic interaction liquid chromatography tandem mass spectrometry (nano-HILIC MS/MS). Nano-HILIC MS/MS revealed 60 GSL species with a distinct saccharide and ceramide composition. AP-SMALDI MSI measurements were conducted in positive- and negative-ion mode for the visualization of neutral and acidic GSLs. Based on nano-HILIC MS/MS results, we discovered no downregulated but 50 significantly upregulated GSLs in liver samples of infected hamsters. AP-SMALDI MSI showed that 44 of these GSL species were associated with the granulomas in the liver tissue. Our findings suggest an important role of GSLs during granuloma formation.

Analysis and Characterization of the Extracellular Vesicles Released in Non-Cancer Diseases Using Matrix-Assisted Laser Desorption Ionization/Mass Spectrometry.

The extracellular vesicles (EVs) released by cells play a crucial role in intercellular communications and interactions. The direct shedding of EVs from the plasma membrane represents a fundamental pathway for the transfer of properties and information between cells. These vesicles are classified based on their origin, biogenesis, size, content, surface markers, and functional features, encompassing a variety of bioactive molecules that reflect the physiological state and cell type of origin. Such molecules include lipids, nucleic acids, and proteins. Research efforts aimed at comprehending EVs, including the development of strategies for their isolation, purification, and characterization, have led to the discovery of new biomarkers. These biomarkers are proving invaluable for diagnosing diseases, monitoring disease progression, understanding treatment responses, especially in oncology, and addressing metabolic, neurological, infectious disorders, as well as advancing vaccine development. Matrix-Assisted Laser Desorption Ionization (MALDI)/Mass Spectrometry (MS) stands out as a leading tool for the analysis and characterization of EVs and their cargo. This technique offers inherent advantages such as a high throughput, minimal sample consumption, rapid and cost-effective analysis, and user-friendly operation. This review is mainly focused on the primary applications of MALDI-time-of-flight (TOF)/MS in the analysis and characterization of extracellular vesicles associated with non-cancerous diseases and pathogens that infect humans, animals, and plants.

LC-MS and MALDI-MSI-based metabolomic approaches provide insights into the spatial-temporal metabolite profiles of Tartary buckwheat achene development.

Tartary buckwheat, celebrated as the "king of grains" for its flavonoid and phenolic acid richness, has health-promoting properties. Despite significant morphological and metabolic variations in mature achenes, research on their developmental process is limited. Utilizing Liquid chromatography-mass spectrometry and atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging, we conducted spatial-temporal metabolomics on two cultivars during achene development. Metabolic profiles including 17 phenolic acids and 83 flavonoids are influenced by both varietal distinctions and developmental intricacies. Notably, flavonols, as major flavonoids, accumulated with achene ripening and showed a tissue-specific distribution. Specifically, flavonol glycosides and aglycones concentrated in the embryo, while methylated flavonols and procyanidins in the hull. Black achenes at the green achene stage have higher bioactive compounds and enhanced antioxidant capacity. These findings provide insights into spatial and temporal characteristics of metabolites in Tartary buckwheat achenes and serve as a theoretical guide for selecting optimal resources for food production.

Discriminatory power of MALDI-TOF MS protein profiling analysis of pork meat and meat products.

Forty different sample preparation methods were tested to obtain the most informative MALDI-TOF MS protein profiles of pork meat. Extraction by 25% formic acid with the assistance of zirconia-silica beads followed by defatting by methanol:chloroform mixture (1:1, v/v) and deposition by using the layer-by-layer method was determined as the optimum sample preparation protocol. The discriminatory power of the method was then examined on samples of pork meat and meat products. The method was able to discriminate between selected salami based on the production method and brand and was able to monitor the ripening process in salami. However, it was not able to differentiate between different brands of pork ham or closely located parts of pork meat. In the latter case, a more comprehensive analysis using LC-MS/MS was used to assess the differences in protein abundance and their relation to the outputs of MALDI - TOF MS profiling.

Nitrospirillum viridazoti sp. nov., an Efficient Nitrogen-Fixing Species Isolated from Grasses.

A group of Gram-negative plant-associated diazotrophic bacteria belonging to the genus Nitrospirillum was investigated, including both previously characterized and newly isolated strains from diverse regions and biomes, predominantly in Brazil. Phylogenetic analysis of 16S rRNA and recA genes revealed the formation of a distinct clade consisting of thirteen strains, separate from the formally recognized species N. amazonense (the closest species) and N. iridis. Comprehensive taxonomic analyses using the whole genomes of four strains (BR 11140T = AM 18T = Y-2T = DSM 2788T = ATCC 35120T, BR 11142T = AM 14T = Y-1T = DSM 2787T = ATCC 35119T, BR 11145 = CBAmC, and BR 12005) supported the division of these strains into two species: N. amazonense (BR 11142 T and BR 12005) and a newly proposed species (BR 11140 T and BR 11145), distinct from N. iridis. The phylogenomic analysis further confirmed the presence of the new Nitrospirillum species. Additionally, MALDI-TOF MS analysis of whole-cell mass spectra provided further evidence for the differentiation of the proposed Nitrospirillum species, separate from N. amazonense. Analysis of chemotaxonomy markers (i.e., genes involved in fatty acid synthesis, metabolism and elongation, phospholipid synthesis, and quinone synthesis) revealed that the new species highlights high similarity and evolutionary convergence with other Nitrospirillum species. This new species exhibited nitrogen fixation ability in vitro, it has similar NifHDK protein phylogeny position with the closest species, lacked denitrification capability, but possessed the nosZ gene, enabling N2O reduction, distinguishing it from the closest species. Despite being isolated from diverse geographic regions, soil types, and ecological niches, no significant phenotypic or physiological differences were observed between the proposed new species and N. amazonense. Based on these findings, a new species, Nitrospirillum viridazoti sp. nov., was classified, with the strain BR 11140T (DSM 2788T, ATCC 35120T) designated as the type strain.

[Detection of Virulence Genes in Pseudomonas aeruginosa Isolates by Polymerase Chain Reaction and Investigation of High-Risk Clones by Matrix Assisted Laser Desorption/ Ionization Time-of-Flight Mass Spectrometer Method]. / Pseudomonas aeruginosa Izolatlarinda Virülans Genlerinin Polimeraz Zincir Reaksiyonuyla Tespiti ve Yüksek Riskli Klonlarin Matriksle Desteklenmis Lazer Desorpsiyon/Iyonizasyon Uçus Zamani Kütle Spektrometresi Yöntemi ile Arastirilmasi.

Pseudomonas aeruginosa is a non-fermentative gram-negative bacillus. Many virulence factors play a role in the pathogenesis of P.aeruginosa. The aim of this study was to early detection of ST111, ST175, ST235, ST253, ST395 which are named high-risk clones with increased epidemic potential due to multidrug resistance in P.aeruginosa isolates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method and to evaluate the relationship between high-risk clones and the presence of P.aeruginosa virulence factors and carbapenemase production genes.P.aeruginosa isolates (n= 100) found to be resistant to at least imipenem or meropenem antibiotics isolated from the various clinical samples in the medical microbiology laboratory between 01.01.2021 and 07.06.2022 were included in the study. For the detection of virulence genes uniplex polymerase chain reaction (PCR) for toxA and multiplex PCR for algD, plcN, lasB, plcH were performed in P.aeruginosa isolates. In the detection of carbapenemase genes, two separate multiplex PCRs used for blaKPC , blaNDM , blaVIM , blaOXA-48 and for blaIMP , blaSPM , blaSIM , blaGIM , blaGES . Investigation of the peaks specific to high-risk clones was performed by using VITEK®-MS (bioMérieux, France) system. P.aeruginosa isolates were mostly isolated from intensive care units (45%) and respiratory tract samples (46%). The antibiotic to which the isolates were found to be most susceptible was amikacin, while highest resistance was detected for piperacillin. In PCR results, toxA, lasB, plcH, plcN and algD were detected as 89%, 99%, 98%, 100%, 100%, respectively. When the presence of characteristic peaks belonging to high-risk clones was evaluated with MALDI-TOF MS, ST253 (7%) and ST175 (2%) were detected. The peaks specific to ST235 and ST395 clones were not detected in our study. blaVIM was detected in two isolates and blaGES-5 carbapenemase was detected in two isolates. Virulence factors were detected at high rates in both high-risk clones and other strains and no significant relationship was found between high-risk clones and virulence factors. Early detection of high-risk clones, identification of antimicrobial resistance mechanisms will help to develop strategic treatment options and prevent their worldwide spread.