MALDI-TOF MS Research Articles

The tracement of origins of aquatic products by characterization of their gut or gill microbiome via MALDI-TOF MS analysis

Tracement of the origin of seafoods and aquatic products is an important issue for avoiding the falsification of production regions of aquatic products. Herein, by analyzing black tiger shrimp (Penaeusmonodon) as a model of aquatic products, we demonstrated a method to trace the origin of aquatic products by analyzing the gut microbiome of black tiger shrimp (Penaeusmonodon) collected from three commercial farms in China, via matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. Multivariate analysis was executed on the obtained mass spectrometric datasets of bacteria extracted and collected from shrimp samples with different production regions. The gut microorganisms of black tiger shrimps can be identified by scoring the gut microbiota of black tiger shrimps collected from three producing areas. The black tiger shrimp from three production regions were found with distinguished characteristics of bacterial prevalence in their digestive gland microbiota. The dominant bacteria genus in digestive gland microbiota from black tiger shrimp samples from the three production regions are completely different. Single-blind shrimp samples with unknown production region was successfully screened by comparing the detected MS data in different ways. This strategy can trace the production region of the analyzed shrimp samples in a quick and efficient mode. The application of the present protocol was further demonstrated by analyzing the biometric species from gill tissue of Chinese mitten crab (Eriocheir sinensis) samples collected from crab-producing areas in Jiangsu Province (China), that is, Taihu Lake, Gaoyou Lake, and Hongze Lake for tracing their production area. It is expected to extend the presently developed method to provide clues for the origin traceability of other aquatic products.

Novel Ralstonia species from human infections: improved matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification and analysis of antimicrobial resistance patterns.

A collection of 161 Ralstonia isolates, including 90 isolates from persons with cystic fibrosis, 27 isolates from other human clinical samples, 8 isolates from the hospital environment, 7 isolates from industrial samples, and 19 environmental isolates, was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identification and yielded confident species level identification scores for only 62 (39%) of the isolates, including four that proved misidentified subsequently. Whole-genome sequence analysis of 32 representative isolates for which no confident MALDI-TOF MS species level identification was obtained revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively, and provided the basis for updating an in-house MALDI-TOF MS database. A reanalysis of all mass spectra with the updated MALDI-TOF MS database increased the percentage of isolates with confident species level identification up to 77%. The antimicrobial susceptibility of 30 isolates mainly representing novel human clinical and environmental Ralstonia species was tested toward 17 antimicrobial agents and demonstrated that the novel Ralstonia species were generally multi-resistant, yet susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline. An analysis of genomic antimicrobial resistance genes in 32 novel and publicly available genome sequences revealed broadly distributed beta-lactam resistance determinants.IMPORTANCEThe present study demonstrated that a commercial matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification database can be tailored to improve the identification of Ralstonia species. It also revealed the presence of seven novel Ralstonia species, including three and four that were isolated from cystic fibrosis or other human clinical samples, respectively. An analysis of minimum inhibitory concentration values demonstrated that the novel Ralstonia species were generally multi-resistant but susceptible to trimethoprim/sulfamethoxazole, ciprofloxacin, and tigecycline.

Evaluation of the first Candida auris isolates reported from Türkiye in terms of identification by various methods and susceptibility to antifungal drugs

PurposeCandida auris is increasingly being isolated from patients all over the world. It has five clades. In this study, it was aimed to compare the results of biochemical tests obtained using different methods and the antifungal susceptibility profiles of C. auris strains isolated from the first seven cases reported in Türkiye, and evaluate whether this information could be useful as preliminary data in determining the clade of strains in centers that lack the opportunity to apply molecular methods. MethodsIdentification test results obtained using API ID 32 C, API 20 C AUX, VITEK-2 YST, and MALDI-TOF MS; colony color and morphology on Chromagar Candida, CHROMagar Candida Plus media, and cornmeal-Tween 80 agar; susceptibility to antifungals were tested and compared. Antifungal susceptibility test was studied using microdilution method according to the recommendations of EUCAST. Additionally, a pilot study was conducted to investigate the value of CHROMagar Candida Plus. ResultsAll seven strains were identified as Lachancea kluyveri with API ID 32 C, Rhodotorula glutinis; Cryptococcus neoformans with API 20 C AUX, and C. auris with both VITEK-2 YST and MALDI-TOF MS. MIC values for fluconazole were very high (≥64 mg/L) for all seven strains. It was observed that 11 (37.9%) of 29 Candida parapsilosis strains formed colonies with morphology similar to C. auris on CHROMagar Candida Plus medium, leading to false positivity. ConclusionsAlthough there have been many isolations of C. auris in our country in recent years, clade distribution of only a small number of strains is known yet. In this study, when the biochemical properties and antifungal susceptibility profiles of the seven strains were evaluated, it was concluded that they exhibited some characteristics compatible with clade I. It was also observed that strains 1 and 2 may belong to a different clade.

[Comparison of Three Different Methods in Investigating the Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates].

The aim of the study was to evaluate the relationship between carbapenem-resistant Acinetobacter baumannii isolates carrying oxacillinase-type carbapenemase genes with "international high-risk clones" (IC I, II, and III) by different molecular epidemiological methods and to statistically compare the concordance and discrimination power of the methods. Carbapenem-resistant and moderately susceptible A.baumannii isolates from non-repeating blood cultures of 72 patients were included in the study. The presence of "blaOXA-23 , blaOXA-24 , blaOXA-51 ve blaOXA-58 " genes within OXA-type carbapenemases was detected by polymerase chain reaction (PCR) method and confirmed by DNA sequence analysis. Pulsed f ield gel electrophoresis (PFGE), multilocus sequence typing (MLST) and matrix-assisted laser desorption/ ionization time- of-flight mass spectrometry (MALDI-TOF MS) analyses were performed to evaluate the clonal relations of IC I, II and III clones together with clinical isolates. In the statistical comparison of the methods, discrimination power was evaluated by Simpson index of diversity (SID) and concordance by "Wallace coefficient". All of the isolates were found to carry blaOXA-23 and blaOXA-51 genes. As a result of the bioinformatic analysis of the four isolates selected for sequence analysis; blaOXA-23 and blaOXA-51 genes were detected in the selected isolates, and the analysis of two isolates carrying blaOXA-51 gene showed 99% similarity with blaOXA-92 gene. The isolates were clustered into five pulsotypes (A, B, C, D and E) according to ≥ 85% similarity coefficient by PFGE. The isolates and RUH 875, RUH 134, LUH 5875 strains belonging to high-risk clones ICI, ICII and ICIII, respectively, were divided into five main groups [A (n= 58), B (n= 8), C (n= 4), D (n= 4) and E (n= 1)] and 10 subgroups (A1, A2, A4, A5, A6, A9, B1, B4, C3, D1) by PFGE. IC clone III (E1) and seven strains showed singleton PFGE profiles (A3, A7, A8, B2, B3, C1, C2). ICII was found in A5 subtype, ICI in C1 subtype and ICIII in E1 subtype. By PFGE subtype groups, 18 pulsotypes were determined and ST1, ST2, ST81, ST157 and ST604 sequence types were found in 20 isolates randomly selected from pulsotypes according to MLST Pasteur scheme (cpn60, fusA, gltA, pyrG, recA, rplB, rpoB). Principal component analysis (PCA) of the spectra of 72 A. baumannii isolates and ICI, ICII and ICIII clones was performed by MALDI-TOF MS. In PCA analysis, the cluster distance level was defined as 1.5 and the isolates were divided into three clusters. IC clone I, II and III together with 70 clinical isolates were grouped in one cluster, while two clinical isolates (AB083 and AB0115) formed singleton clusters. There was no significant agreement between MALDI-TOF MS; MLST and PFGE data according to Wallace coefficient. It was found that PFGE method gave significant results in terms of discrimination power with SID coefficient, MALDI-TOF MS PCA analysis had the lowest discrimination power value, and the Wallace coefficient result of PFGE and MLST was concordant. In conclusion, MALDI-TOF MS may not function as a gold standard method like PFGE and MLST for epidemiological analysis in A.baumannii species and the epidemiological typing protocols used for MALDI-TOF MS need to be improved and developed.

[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 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.

Effects of Speleotherapy on Aerobiota: A Case Study from the Sežana Hospital Cave, Slovenia

Speleotherapy is one of the non-pharmacological methods for the treatment and rehabilitation of patients with chronic respiratory diseases, especially those with chronic obstructive pulmonary disease (COPD) and asthma. On the one hand, one of the alleged main advantages of speleotherapeutic caves is the low microbial load in the air and the absence of other aeroallergens, but on the other hand, due to the lack of comprehensive air monitoring, there is little information on the pristine and human-influenced aerobiota in such environments. The aim of this study was to assess the anthropogenic effects of speleotherapy on the air microbiota and to investigate its potential impact on human health in Sežana Hospital Cave (Slovenia). From May 2020 to January 2023, air samples were collected in the cave before and after speleotherapeutic activities using two different volumetric air sampling methods—impaction and impingement—to isolate airborne microbiota. Along with sampling, environmental data were measured (CO2, humidity, wind, and temperature) to explore the anthropogenic effects on the aerobiota. While the presence of patients increased microbial concentrations by at least 83.3%, other parameters exhibited a lower impact or were attributed to seasonal changes. The structure and dynamics of the airborne microbiota are similar to those in show caves, indicating anthropization of the cave. Locally, concentrations of culturable microorganisms above 1000 CFU/m3 were detected, which could have negative or unpredictable effects on the autochthonous microbiota and possibly on human health. A mixture of bacteria and fungi typically associated with human microbiota was found in the air and identified by MALDI-TOF MS with a 90.9% identification success rate. Micrococcus luteus, Kocuria rosea, Staphylococcus hominis, and Staphylococcus capitis were identified as reliable indicators of cave anthropization.

Bacterial discrimination by Fourier transform infrared spectroscopy, MALDI-mass spectrometry and whole-genome sequencing.

Aim: Proof-of-concept study, highlighting the clinical diagnostic ability of FT-IR compared with MALDI-TOF MS, combined with WGS. Materials & methods: 104 pathogenic isolates of Neisseriameningitidis, Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus were analyzed. Results: Overall prediction accuracy was 99.6% in FT-IR and 95.8% in MALDI-TOF-MS. Analysis of N. meningitidis serogroups was superior in FT-IR compared with MALDI-TOF-MS. Phylogenetic relationship of S. pyogenes was similar by FT-IR and WGS, but not S. aureus or S. pneumoniae. Clinical severity was associated with the zinc ABC transporter and DNA repair genes in S. pneumoniae and cell wall proteins (biofilm formation, antibiotic and complement permeability) in S. aureus via WGS. Conclusion: FT-IR warrants further clinical evaluation as a promising diagnostic tool.

Epidemiology and management of tinea capitis in France: a 6-year nationwide retrospective survey.

Tinea capitis (TC) is still a frequent dermatophytosis in France, both autochthonous and imported. A nationwide retrospective survey was performed and a total of 4,395 TC cases were recorded within 36 French mycology laboratories during a 6-year period. TC is a disease that occurs in childhood with 85% of the cases occurring before 10 years old and 94% before the age of 15. Anthropophilic origin was predominant with 779 cases of Trichophyton tonsurans (32.6%), 738 cases of Trichophyton soudanense/T. violaceum (31%), and 445 cases of Microsporum audouinii (19.2%). Of note, T. tonsurans represents more than 80% of the cases in the French West Indies (Martinique and Guadeloupe). By contrast, zoophilic species were less prevalent with mainly M. canis (10.3%) confirming the shift from zoophilic to anthropophilic species observed in many centers during the last decades. During this survey, diagnosis methods were also collected. Most labs had a classical process for the diagnosis: microscopic direct examination associated to cultures on Sabouraud and Sabouraud-cycloheximide media (incubated between 25±5°C for 2 to 3 weeks) in all laboratories. Identification of the causal dermatophyte was performed by microscopic and macroscopic examination of the cultures in 100% of the labs, with various specific culture media available when fructification was insufficient (mainly malt or potato-dextrose agar, or Borelli medium). New techniques were also implemented with the introduction of MALDI-TOF mass spectrometry identification in more than two third of the labs, and molecular identification available if necessary in half of the labs.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry: An Innovative Tool for Rapid Identification of Hylurgus ligniperda, an Invasive Pest

Hylurgus ligniperda is an imported quarantine plant pest in China. Its identification is usually based on morphological characteristics; therefore, species identification needs high professional requirements of staff and professionals with high experience accumulated through long-term training. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid identification technology, which is based on protein profiles of species. It has been widely used for the identification of pathogenic microorganisms. Many studies have reported the identification of mosquitoes, ticks, and other arthropods. The application of MALDI-TOF MS in the identification of H. ligniperda can improve the identification efficiency of H. ligniperda, preventing and control its harm and further spread. To construct a spectra database for H. ligniperda, we analyzed the effect of different factors, such as different body parts, developmental stages, populations, and preservation conditions, on its protein spectrum. We collected protein spectrum profiles from 19 specimens of H. ligniperda and its related species, obtaining 211 protein spectra to construct a reference database and validate identification. The protein spectrum from the chest specimens of H. ligniperda showed many peaks, high intensity, and a stable signal, indicating a successful data establishment. The difference in protein spectra between different regions of the same species was less, but did not affect the identification results. Clear differences were observed in the protein spectrum across many developmental stages. The database established by the adult specimens protein spectrum can accurately identify Dendroctonus valens, Tomicus piniperda, and H. ligniperda. MALDI-TOF MS technology can be used for the rapid identification of H. ligniperda. This method is rapid and direct, and the identification results are robust. It does not require specialized entomological expertise and can be used for customs interception and field investigations.

Intact cell mass spectrometry coupled with machine learning reveals minute changes induced by single gene silencing

Intact (whole) cell MALDI TOF mass spectrometry is a commonly used tool in clinical microbiology for several decades. Recently it was introduced to analysis of eukaryotic cells, including cancer and stem cells. Besides targeted metabolomic and proteomic applications, the intact cell MALDI TOF mass spectrometry provides a sufficient sensitivity and specificity to discriminate cell types, isogenous cell lines or even the metabolic states. This makes the intact cell MALDI TOF mass spectrometry a promising tool for quality control in advanced cell cultures with a potential to reveal batch-to-batch variation, aberrant clones, or unwanted shifts in cell phenotype. However, cellular alterations induced by change in expression of a single gene has not been addressed by intact cell mass spectrometry yet. In this work we used a well-characterized human ovarian cancer cell line SKOV3 with silenced expression of a tumor suppressor candidate 3 gene (TUSC3). TUSC3 is involved in co-translational N-glycosylation of proteins with well-known global impact on cell phenotype. Altogether, this experimental design represents a highly suitable model for optimization of intact cell mass spectrometry and analysis of spectral data. Here we investigated five machine learning algorithms (k-nearest neighbors, decision tree, random forest, partial least squares discrimination, and artificial neural network) and optimized their performance either in pure populations or in two-component mixtures composed of cells with normal or silenced expression of TUSC3. All five algorithms reached accuracy over 90 % and were able to reveal even subtle changes in mass spectra corresponding to alterations of TUSC3 expression. In summary, we demonstrate that spectral fingerprints generated by intact cell MALDI-TOF mass spectrometry coupled to a machine learning classifier can reveal minute changes induced by alteration of a single gene, and therefore contribute to the portfolio of quality control applications in routine cell and tissue cultures.

Experimental Validation of Comprehensive Calculation for High-Resolution Linear MALDI-TOF Mass Spectrometry.

This work discusses the effectiveness of the previously developed comprehensive calculation model to optimize linear MALDI-TOF mass spectrometers. The model couples space- and velocity-focusing to precisely analyze the flight-time distribution of ions and predict optimal experimental parameters for the highest mass resolving power. Experimental validation was conducted using a laboratory-made instrument to analyze CsI3 and angiotensin I ions in low to medium m/z range. The results indicate that the predicted optimal extraction voltage and delay were reasonably accurate and effective. In the low m/z range, the peak width obtained using optimal parameters reached the sub nanosecond range, corresponding to a mass resolving power of 10 000-17 000, or 20 000-34 000 if shot-to-shot random fluctuations were minimized by the dynamic data correction method. The observed optimal mass resolving power in the current experiment is 4.8-7.8 times that of commercial instruments. Practical limitations resulting in the gap between the observed and theoretical ultimate mass resolving power are discussed.

Purification and characterization of 3-O-methyl-D-glucose from the seed coat of Vigna mungo (L.) Hepper

Vigna mungo. L (Black gram) belonging to the Leguminosae family is a most abundant source of protein for human and animal diets. Over 8.5 million tonnes of black gram are being produced worldwide, and after confiscating the endosperm for the protein source, the seed coat is dumped as agro-waste and used as feed for the cattle. This study aims to investigate and profile the metabolites in three distinct parts of V. mungo seeds: coat, cotyledon, and intact seed. Also, it has been hypothesized that sugar methylation plays a pivotal role in facilitating black gram seed germination by serving as an energy source during metabolic processes. In our study, A total of forty-nine compounds belonging to the class such as fatty acids, saccharides, triterpenes, sterols, and vitamins were detected in various parts of V. mungo seed by gas chromatography-mass spectrometry (GC-MS). Most interestingly, 3-O-methyl-D-glucose (3-OMG) was commonly detected in methanolic extract of coat, intact seed, and cotyledon with peak area percentages of 3.076, 1.157, and 1.100 %, respectively. Based on the highest peak area percentage in the coat, this compound was purified by using a silica gel column with a mesh size of 60–120 followed by Biotage SNAP cartridge KP-C18-HS (12 g). The yield and purity of the purified fractions were found to be 11.78 & 94.19 %, respectively by high-performance liquid chromatography (HPLC) analysis. Further, the mass confirmation of purified fraction containing 3-OMG (m/z - 217.06 g/mol) was done by liquid chromatography-mass spectrometry (LC-MS/MS) and reconfirmed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The MS/MS analysis of the fragment showed m/z 206.91 for 3-OMG which corresponds to the association of the sodium adduct. The concentration of 3-OMG was quantified as 332.65 µg/g of dry weight in the coat of V. mungo by HPLC analysis. Also, among the antioxidant assays conducted, such as DPPH, ABTS, FRAP, and NOS, it was observed that the methanolic extract of coat exhibited the most significant antioxidant activity. The 3-OMG is commonly used as a non-metabolized analogue of glucose by regulating glucose transporter, especially GLUT-4 that regulates the absorption of glucose by insulin into muscle and fat cells. This study supports the black gram seed with coat as an alternative medicine to regulate blood glucose.

Structural characterization of strawberry pomace

Strawberries are a nutrient dense food rich in vitamins, minerals, non-nutrient antioxidant phenolics, and fibers. Strawberry fiber bioactive structures are not well characterized and limited information is available about the interaction between strawberry fiber and phenolics. Therefore, we analyzed commercial strawberry pomace in order to provide a detailed carbohydrate structural characterization, and to associate structures with functions. The pomace fraction, which remained after strawberry commercial juice extraction, contained mostly insoluble (49.1 % vs. 5.6 % soluble dietary fiber) dietary fiber, with pectin, xyloglucan, xylan, β-glucan and glucomannan polysaccharides; glucose, fructose, xylose, arabinose, galactose, fucose and galacturonic acid free carbohydrates; protein (15.6 %), fat (8.34 %), and pelargonidin 3-glucoside (562 μg/g). Oligosaccharides from fucogalacto-xyloglucan, methyl-esterified rhamnogalacturonan I with branched arabinogalacto-side chains, rhamnogalacturonan II, homogalacturonan and β-glucan were detected by MALDI-TOF MS, NMR and glycosyl-linkage analysis. Previous reports suggest that these oligosaccharide and polysaccharide structures have prebiotic, bacterial pathogen anti-adhesion, and cholesterol-lowering activity, while anthocyanins are well-known antioxidants. A strawberry pomace microwave acid-extracted (10 min, 80 °C) fraction had high molar mass (2376 kDa) and viscosity (3.75 dL/g), with an extended rod shape. A random coil shape, that was reported previously to bind to phenolic compounds, was observed for other strawberry microwave-extracted fractions. These strawberry fiber structural details suggest that they can thicken foods, while the polysaccharide and polyphenol interaction indicates great potential as a multiple-function bioactive food ingredient important for gut and metabolic health.

A Day Saved is a Life Saved: Direct Antimicrobial Susceptibility Testing from Positively Flagged Blood Culture Bottles and their Concordance with the Routine Method.

Sepsis is a major health problem worldwide and is associated with high morbidity and mortality with every hour delay in initiation of therapy. A conventional method of blood culture and Antimicrobial Susceptibility Testing (AST) takes around 48-72 hours. Empirical antibiotics need to be administered until the sensitivity report is made available. It has been estimated that 20-50% of the empirical antibiotics are inappropriate, resulting in prolonged hospital stays, adverse effects, and emergence of drug resistance. Additionally, this also puts an extra financial burden on both the patients and healthcare settings. Performing direct Antimicrobial Sensitivity Testing (dAST) is an important tool to reduce turn-around time (TAT) by at least 18-24 hours, thus reducing morbidity and mortality among critically ill patients. Direct AST (dAST) was performed from the positively flagged blood culture bottles received between December, 2021 to May, 2022 from Intensive Care Units (ICUs) on MuellerHinton Agar (MHA) using four drops of withdrawn blood. dAST was performed for six drugs: Ceftriaxone-30 µg (CTR), Piperacillin/Tazobactam-100/10 µg (PIT), Meropenem-10 µg (MRP), Ciprofloxacin-5 µg (CIP), Aztreonam-30 µg (AT), and Colistin (CL). The zone of inhibition was interpreted as per CLSI M100 ed32, 2022 guidelines. A parallel conventional method was also performed to examine for categorical agreement and disagreement. Identification was carried out using MALDI-TOF MS from the colonies that appeared on the dAST plate on the subsequent day. A total of 162 positively flagged blood culture bottles were included in the study. The majority of the Gram-negative organisms were from Enterobacterales (n=109), followed by Acinetobacter spp. (n=28) and Pseudomonas aeruginosa (n=25). Out of the 972 isolate-antimicrobial combinations, overall Categorical Agreement (CA) was seen in 936 (96.3%), whereas disagreement was observed in 36 with minor error (mE) in 21 (2.2%), major error (ME) in 7 (0.7%), and very major error (VME) in 8 (0.8%) when compared to the routine method. Categorical agreement (CA) of > 99% was seen in ceftriaxone (CTR) and ciprofloxacin (CIP). In comparison, the lowest CA was observed with meropenem (MRP) at 92%. Colistin dAST was performed using the E-strip method, and the result obtained was highly convincing, with an overall disagreement of only 1.2%. Rapid dAST from positively flagged blood culture bottles proved to significantly reduce the TAT from the time of sample collection to the first availability of antimicrobial susceptibility report with excellent categorical agreement of > 95% using the conventional disc diffusion method. Results obtained were within the acceptance criteria set by U. S. Food and Drug Administration (FDA) guidelines of > 90% categorical agreement for a new method. We were able to obtain excellent concordance for colistin using the E-strip method. Performing dAST not only saves a "day", but its proper implementation would save a "life".

Evidence and antibiotic resistance profiles of clinical Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) and non-ACB complex members in companion animals: A 2020–2022 retrospective study

To evaluate the frequency of Acinetobacter spp., belonging to both Acinetobacter calcoaceticus-baumannii (ACB) and non-ACB complex, and their antibiotic resistance profiles in veterinary medicine, a three-year (2020–2022) retrospective study was carried out on sick companion animals. Epidemiological data from different clinical canine, feline, and equine samples, were acquired. For each strain, MALDI-TOF MS identification and susceptibility to a panel of 11 antibiotics, by Kirby-Bauer and E-test methods, were performed. Out of 628 bacteriological examinations, 2.5% resulted positive for strains belonging to Acinetobacter genus. Frequencies of 2.3%, 1.9%, and 3% were obtained from both in-visiting and hospitalized dogs, cats, and horses, respectively. Members of ACB-complex accounted for 50% of isolates. Since all strains resulted susceptible to aminoglycosides and polymyxins, no pandrug-resistant (PDR) species were recorded. While 12.5% A. baumannii resulted extensively-drug resistant (XDR), a higher percentage of multidrug-resistant strains was recorded among non-ACB strains (35.5%) than ACB strains (25%). Susceptibility was observed in the same percentage in both groups (62.5%). All ACB strains confirmed their intrinsic resistances. Non-ACB species showed lower resistances against antipseudomonal penicillins plus beta-lactamase inhibitors (P=0.1306), III generation cephalosporins (P=0.0547), and tetracyclines (P=0.0209) than ACB species. Carbapenem-resistance was observed for XDR A. baumannii (12.5%) and, in particular for MDR non-ACB complex members (25%). To our knowledge, A. lactucae represents the first description in two sick dogs in Italy. Furthermore, our results emphasize the role of non-ACB-complex species as important zoonotic pathogens, which could be reservoirs of clinically relevant resistance profiles.

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 rRNAand 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 11142T and BR 12005) and a newly proposed species (BR 11140T 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.

Enrichment and MALDI-TOF MS Analysis of Phosphoinositides in Brain Tissue.

Triazolium cyclodextrin click cluster (+CCC) is an ideal scaffold to specifically bind phosphoinositides (PIPs) via multivalent electrostatic interaction. A new enrichment material, triazolium cyclodextrin click cluster-magnetic agarose bead conjugate (+CCC-MAB), was synthesized and applied to the PIP enrichment of brain tissue. The enriched sample was analyzed using MALDI-TOF MS in negative ion mode without any derivatization. The PIP extract of brain tissue is known to contain abundant lipid interferences. By employing magnetic pull-down separation using +CCC-MAB, we effectively removed the weak-binding interferences in the PIP extract, thereby improving the signal-to-noise ratio (S/N) of the PIPs. Our +CCC-MAB-based PIP enrichment enabled us to analyze 16 PIP species in brain tissue. Six species with high S/N were assigned by MS/MS, while the remaining 10 species with low S/N were characterized by an empirical selection guide based on the biological relevance of PIPs. We conclude that +CCC-MAB-based PIP enrichment is a promising MALDI sample preparation method for specific PIP analysis in brain tissue.

Burkholderia cepacia: Understanding Pathogenicity, Virulence Factors, and Therapeutic Strategies

Burkholderia cepacia, formerly described as Pseudomonas cepacia, belongs to the Burkholderia cepacia complex (Bcc), a collection of closely related gram-negative bacteria that is considered a leading organism in causing pulmonary infections in humans. In healthcare settings, Burkholderia species have the ability to colonise medical equipment and surfaces, posing a threat of nosocomial infections. Moreover, they exhibit inherently resistance to various classes of antibiotics, thus being nominated as multi-drug resistant pathogens (MDR), that is why treatment of infections caused by these organisms is challenging. Enzymes such as metalloproteases, serine proteases, metalloproteases, and other extracellular lipases play a major role in its pathogenesis. In addition to this, different components, such as pili, flagella, and lipopolysaccharide (LPS), have a role in both the motility of the bacterial cell as well as its adherence to host cells. Identification is usually done with different biochemical reactions such as late oxidase activity, oxidation of glucose, variable lysine decarboxylation, maltose, mannitol, and lactose, and hydrogen sulphide gas production. Advanced techniques like semi-nested PCR, real-time PCR, or MALDI-TOF mass spectrometry enable quick and precise identification.
 Key words: Multi-drug resistant, Polymerase chain reaction, Burkholderia cepacia complex, matrix assisted laser desorption ionisation, time of flight mass spectrometry

New type paraben decorated mono- and dispirocyclotriphosphazene compounds: Synthesis, characterization and DNA binding studies

Parabens are a group of chemical preservatives that are widely used in the cosmetics, pharmaceutical, and food industries. Although some studies have raised concerns about potential health risks associated with parabens, it is important to note that the presence of parabens in tumor tissue does not provide conclusive evidence that parabens cause cancer. This is an area that requires further research. For this purpose, six new mono- and dispirocyclotriphosphazene compounds decorated with paraben were synthesized and designed for the first time. Their structures were identified by MALDI-TOF MS, 1H, 31P NMR and FT-IR spectroscopy. Moreover, the molecular structure of Compound 8 was determined by X-Ray crystallography. The DNA interaction properties of the targeted molecules were investigated via agarose gel electrophoresis.

The Detection of Crown Gall Disease in Kahramanmaraş and Surrounding Provinces

Aims: The aim of the study is to isolate the bacterial agent causing crown gall disease in Kahramanmaraş and its surrounding provinces, determine its prevalence in the region, and conduct its identification. 
 Material and methods: Between June and September of 2017-2019, strains obtained from the shoots and galls of vines showing symptoms of galls in vineyards in Kahramanmaraş, Adıyaman, Kilis, Gaziantep, and Şanlıurfa provinces were subjected to pathogenicity tests, classical, molecular, and MALDI-TOF MS identification.
 Results and Discussion: In this study, the prevalence of crown gall disease in 46 vineyards showing disease symptoms was determined as follows: Kahramanmaraş 76.47%, Adıyaman 75%, Gaziantep 50%, Kilis 33.33%, and Şanlıurfa 20%. From the 46 contaminated vineyards in the study area, 24 pathogenic of R. vitis strains were obtained. In the pathogenicity test conducted with R. vitis strains on the Hatun Parmağı grape variety under greenhouse conditions, the most virulent strains were determined based on the weights of the galls formed, and they were identified as CU52-3/2, CU39-11/5, and CU39-8/1. According to classical tests, 13 strains showed 100% similarity to R. vitis, while molecular characterization with R. vitis-specific primers revealed that 17 strains had the virA gene, 13 strains had the pehA gene, 10 strsains had the virF gene, and 6 strains had the octopine-type plasmid. Ten strains, representing the sample, were identicated as R. vitis through MALDI-TOF MS analysis. This study revealed the impact of pathogenic strains with different virulences on the same grape variety. In line with this, of control methods against highly virulent bacterial agents in reducing losses in plants can be investigated for their applicability in field and orchard areas in subsequent studies.
 Conclusions: Research on different grape varieties is essential to develop varieties that are resistant or tolerant to R. vitis, the pathogen causing this prevalent disease in Turkish viticulture.