Ionization Time Of Flight Mass Spectrometry Research Articles

The Public Health Risks of β-Hemolytic Bacillus pumilus Bacteria Resistant to Gastrointestinal Conditions from Medicinal Plant.

In numerous countries, the utilization of plants for both nutritional and therapeutic purposes is a common practice. However, the inadvertent use of these plants can pose risks due to their active molecules or microbiota. The traditional use of Herniaria glabra L. (H. glabra) plant in treating various diseases is well-known; however, its application in yogurt production raises concerns. In this study, Bacillus pumilus isolated from H. glabra was identified through 16s rRNA sequencing and MALDI-TOF MS (Matriks Assisted Lazer Desorption Ionization Time of Flight Massspectrometry). The bacterium's resistance under simulated gastrointestinal tract (GIT) conditions was assessed, followed by investigations into its aggregation ability, antibiotic resistance, hemolytic activity, and antagonistic potential through in vitro tests. The study revealed that B. pumilus exhibited 100% resistance to GIT conditions. Notably, the bacterium demonstrated strong autoaggregation (34.48%) and coaggregation abilities (49.82% for Escherichia coli, 49.13% for Listeria monocytogenes), signifying a potent aggregative potential. Sensitivity to most tested antibiotics was observed, while no antagonistic activity against tested bacteria was evident. Furthermore, the bacterium exhibited β-hemolytic activity, indicative of potential virulence. The findings suggest that this resistant yet virulent bacterium, with its hemolytic activity, could disrupt the GIT balance, posing serious health risks. The study underscores the need for caution and awareness regarding the potential dangers posed by bacteria in plant microbiota in herbal therapies.

Impact of boiling on the allergens in fish bone samples identified by microfluidic chips and MALDI-TOF MS

Fish bones hold significant potential in the food industry. Investigating the allergenic characteristics of fish bone food products can enhance our understanding of fish allergies. In this study, the allergenic proteins in aqueous extracts from large yellow croaker (Larimichthys crocea) bones boiled for various durations were analyzed and identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and protein database searches. Tryptic products of potential allergens were detected using MALDI-TOF MS and further identified through analysis with Bruker's FlexAnalysis software and the FindMod tool available on the ExPASy proteomics server of the SIB (http://expasy.org/). 25 proteins related to allergy were identified. Two proteins reported as allergens were identified, along with twenty-three (23) proteins that, to the best knowledge of the authors, have not been reported as allergens. At least one aligned tryptic peptide were detected for 17 of the 25 proteins. The 17 potentially allergenic proteins exhibit peptide coverage ranging from 6.72 % to as high as 80 %. The results indicate that boiling the bones for 10 min releases many potentially allergenic proteins. However, the sensitization of most proteins diminishes when the bones are boiled for 30 to 120 min. Despite this, boiling does not completely eliminate the allergenicity of proteins in large yellow croaker bone samples. It is recommended to boil large yellow croaker fish bones for 30 min or longer to reduce most of the protein allergenicity when processing fish bones. Boiling may affect the allergenicity of proteins in fish bones by modifying their structure. These findings provide valuable guidance for monitoring allergens in aquatic food by-products, promisingly assisting to ensure the safety of allergy sufferers. Additionally, this research offers a reference for allergy management and the development of diagnostic reagents derived from aquatic food by-products.

Evaluation of the Activity of Triazoles Against Non-fumigatus Aspergillus and Cryptic Aspergillus Species Causing Invasive Infections Tested in the SENTRY Program.

The activity of isavuconazole and other triazoles against non-fumigatus (non-AFM) Aspergillus causing invasive aspergillosis was evaluated. A total of 390 non-AFM isolates were collected (1/patient) in 2017-2021 from 41 hospitals. Isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry and/or internal spacer region/β-tubulin sequencing and tested by Clinical and Laboratory Standards Institute (CLSI) broth microdilution. CLSI epidemiological cutoff values were applied, where available. Isavuconazole showed activity against Aspergillus sections Flavi (n = 122; minimum inhibitory concentration [MIC]50/90, 0.5/1 mg/L), Terrei (n = 57; MIC50/90, 0.5/0.5 mg/L), Nidulantes (n = 34; MIC50/90, 0.12/0.25 mg/L), Versicolores (n = 7; MIC50, 1 mg/L), and Circumdati (n = 2; MIC range, 0.12-2 mg/L). Similar activity was displayed by other triazoles against those Aspergillus sections. Most of the isolates from Aspergillus sections Fumigati (n = 9), Nigri (n = 146), and Usti (n = 12) exhibited elevated MIC values to isavuconazole (MIC50/90, 2/-, 2/4, and 2/8 mg/L), voriconazole (MIC50/90, 2/-, 1/2, and 4/8 mg/L), itraconazole (MIC50/90, 2/-, 2/4, and 8/>8 mg/L), and posaconazole (MIC50/90, 0.5/-, 0.5/1, and >8/>8 mg/L), respectively. Isavuconazole was active (MIC values, ≤1 mg/L) against Aspergillus parasiticus, Aspergillus tamarii, Aspergillus nomius, Aspergillus nidulans, Aspergillus unguis, Aspergillus terreus, Aspergillus alabamensis, and Aspergillus hortai, while isavuconazole MIC values between 2 and 8 mg/L were observed against cryptic isolates from Aspergillus section Fumigati. Isavuconazole inhibited 96.1% of Aspergillus niger and 80.0% of Aspergillus tubingensis at ≤4 mg/L, the CLSI wild-type cutoff value for A niger. Voriconazole, itraconazole, and posaconazole showed similar activity to isavuconazole against most cryptic species. Isavuconazole exhibited potent in vitro activity against non-AFM; however, the activity of triazoles varies among and within cryptic species.

Accuracy of various matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based rapid identification methods—As a tool to augment diagnostic stewardship in blood culture laboratory, South India

BackgroundBlood stream infection is a medical emergency associated with high morbidity and mortality. Prompt identification of bloodstream infection-causing microorganisms directly from positive blood culture will significantly enhance patient care by reducing the turnaround time of pathogen recognition. MethodsA total of 256 freshly flagged positive blood culture bottles were subjected to Gram staining. Direct MALDI-TOF MS analysis was performed following sample preparation techniques such as lysis centrifugation, lysis filtration and VITEK® MS BC kit to directly identify microorganisms from positive blood cultures. Along with these short-term incubation methods of Choco spot and minute colony(8–10h) were also performed. All those positive bottles were identified by the routine (reference) laboratory method. Results177 isolates (69.14 %) were correctly identified by Lysis centrifugation, 163 isolates (63.67 %) were correctly identified by Lysis filtration, 206 isolates (80.47 %) were correctly identified by Choco spot,250 isolates (97.65 %) were correctly identified from minute colony (8–10h) of incubation. Of 162 isolates,115 isolates (70.99 %) were correctly identified by VITEK® MS Blood culture kit, (BioMérieux). VITEK® MS BC kit method revealed higher agreement with the kappa value of 0.697 than lysis centrifugation (0.672) followed by lysis filtration (0.611). ConclusionsIn house method of lysis centrifugation is found to be equivalent to VITEK® MS BC kit method and superior to lysis filtration method in correct direct identification of bacteria from positive blood cultures by MALDI-TOF MS analysis. As lysis centrifugation requires only 10 min of processing time as compared to overnight incubation, thus it offers a less expensive substitute for the VITEK® MS BC kit in the clinical laboratory. As a consequence of this study, we have implemented direct MALDI-TOF-based identification from positive BCs in our daily routine diagnostic management.

Multi-year comparison of VITEK MS performance for identification of rarely encountered pathogenic Gram-negative organisms (GNOs) in a large integrated Canadian healthcare region.

This multi-year study (2014-2019) compared identification of rare and unusual Gram-negative organisms (GNOs) by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (VITEK MS, bioMérieux, Laval Que.) to 16S rRNA gene sequencing (16S) according to our laboratories routine workflow; 16S is done if initial MALDI-TOF MS gave discordant, wrong, or no results. GNB isolates were first analyzed by standard phenotypic methods and MALDI-TOF MS using direct deposit-full formic acid extraction; proteomics was repeated if no result occurred. Medically approved 16S analyses were done using fast protocols. Isolate sequences were analyzed using the Integrated Database Network System (IDNS3) bacterial database (SmartGene, Lausanne, Switzerland). Three hundred thirty-one GNOs including 251 (76%) aerobic Gram-negative bacilli (GNB), 63 (19%) fastidious Gram-negative coccobacilli (fGNCBs), and 17 (5%) Campylobacterales (CAMPB) isolates were recovered from 304 specimens; >1 isolate was recovered from 19 (6%). GNOs were mainly recovered from blood cultures (31.6%) and lower respiratory specimens (43%) (one-half were isolated from cystic fibrosis patients). Accurate genus vs species identities were obtained for 67.7% and 32.5% aerobic GNBs, 73% and 60% fGNCBs, and 23.5% CAMPB (with no discrepant species), respectively. Wrong or no results were obtained for 81 (32.3%) aerobic GNBs, 17 (27%) fGNCBs, and 13 (72.2%) CAMPB. No results or misidentifications occurred for 33% of aerobic GNBs, 26% of fGNCBs, and 76.5% of CAMPB due to absence of species in the instrument's database. VITEK MS performance remained stable for aerobic GNBs and fGNCBs but improved for CAMPB with addition of Campylobacter rectus and Campylobacter curvus to the database. 16S remains important for identification of GNOs when proteomics fails.IMPORTANCEMatrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has transformed the identification of commonly encountered Gram-negative organisms (GNOs) in the clinical laboratory, but rare and unusual organisms continue to challenge the technology. This study verified performance of VITEK MS for identification of a broad range of rare and unusual clinical GNO isolates by our large reference laboratory workflow over a multi-year period. Although most GNOs were accurately identified by MALDI-TOF MS, a small number of clinical isolates (~1%-6%) required 16S sequencing for identification depending on the GNO category. Approximately one-third of aerobic Gram-negative bacilli (GNBs) and two-thirds of Campylobacterales could not be accurately identified by proteomics due to lack of an organism in the instrument's database. MALDI-TOF MS databases should be continuously updated and validated, and laboratories should have a workflow for identification of unusual or rarely encountered aerobic, fastidious, and Campylobacterales GNOs that includes 16S rRNA gene sequencing whenever proteomics cannot give a definitive identification.

Synthesis of a dehydrodieugenol B derivative as a lead compound for visceral leishmaniasis-mechanism of action and in vivo pharmacokinetic studies.

Leishmaniasis is a parasitic neglected tropical disease, affecting 12 million people. Available treatments present several limitations, with an increasing number of resistance cases. In the search for new chemotherapies, the natural product dehydrodieugenol B was used as a scaffold for the synthesis of a series of derivatives, resulting in the discovery of the promising analog [4-(4-(5-allyl-3-methoxy-2-((4-methoxybenzyl)oxy)phenoxy)-3-methoxybenzyl)morpholine, 1]. In this work, we investigated the effect of compound 1 on cell signaling in Leishmania (L.) infantum, culminating in cell death, as well as its immunomodulatory effect in the host cell. Additionally, we performed a pharmacokinetic profile study in an animal model. After treatment, compound 1 induced the alkalinization of acidocalcisomes and concomitant Ca2+ release in the parasite. These events may induce depolarization of the mitochondrial potential, with successive collapse of the bioenergetic system, leading to a reduction of ATP and reactive oxygen species (ROS) levels. The analysis of total proteins and protein profile by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) demonstrated that compound 1 also altered the parasite proteins after treatment. Transmission electron microscopy studies revealed ultrastructural damage to mitochondria; together, these data suggest that compound 1 may promote autophagic cell death. Additionally, compound 1 also induced an immunomodulatory effect in host cells, with a reduction of Th1 and Th2 cytokine response, characterizing an anti-inflammatory compound. The obtained pharmacokinetic profile in rats enhances the potential of the compound, with a mean plasma half-life (T1/2) of 21 h. These data reinforce the potential of compound 1 as a new lead for future efficacy studies.

Multi-Locus Sequence Typing-Based Genetic Analysis, Antifungal Resistance, and Clinical Prognosis of Cryptococcus neoformans Infections in HIV-Infected Patients in Northern China.

This study aimed to investigate the molecular epidemiological characteristics and drug sensitivity of Cryptococcus from HIV-infected patients and their relationship with patients' prognosis. Seventy-six strains were collected and identified to the species level by matrix-assisted laser desorption ionization-time of flight mass spectrometry, confirmed by internal transcribed spacer sequencing. Multi-locus sequence typing was used for the typing of Cryptococcus, and its antifungal susceptibility was tested using FUNGUS 3. The clinical outcomes of the patients were reviewed at 3-, 6-, 9-, and 12-month follow-ups. All strains were Cryptococcus neoformans var. grubii classified into seven sequence types (STs) dominated by ST5, ST31, and a new ST702 strain. The 6- and 9-month survival rates were highest for patients infected with ST31, ST32, and ST174. The antifungal resistant rates were 13.2%, 2.6%, and 1.4% for fluconazole, amphotericin B, and 5-fluorocytosine. Except itraconazole, the minimum inhibitory concentration (MIC) values and wild type (WT)/non-wild type (NWT) of Cryptococcus for antifungal drugs were not related to the clinical prognosis of HIV-infected patients with cryptococcal infection. ST5 was the main ST type, and the new ST702 type was found in a patient who died in a short period of time. Cryptococcus neoformans var. grubii had a relatively high antifungal drug resistance rate to fluconazole. The WT strain accounted for the highest proportions for 5-fluorocytosine, amphotericin B, fluconazole, voriconazole, and itraconazole. The MIC values of Cryptococcus for first-line antifungal drugs showed no relationship with clinical prognosis, implying that MIC values cannot be used to predict the clinical outcome of these patients.

Characterization of neurologic disease-associated Streptococcus suis strains within the United States swine herd and use of diagnostic tools.

Streptococcus suis negatively impacts swine health, posing diagnostic and preventative challenges. S. suis can induce disease and also quietly reside on mucosal surfaces. The limited use of diagnostic tools to identify disease-associated strains and rule out differential diagnoses, alongside the complex ecology of S. suis, poses significant challenges in comprehending this important pathogen and defining pathotypes. This study evaluated 2,379 S. suis central nervous system (CNS) isolates from diagnostic submissions between 2015 and 2019. Isolates originating from submissions with histologic evidence of CNS infection (n = 1,032) were further characterized by standard and advanced diagnostic techniques. We identified 29 S. suis serotypes and 4 reclassified serotypes as putative causes of CNS disease. Among these, serotypes 1 and 7 emerged as the predominant putative causes of CNS infection (32% of submissions). Furthermore, 51 sequence types (STs), of which 15 were novel, were detected with ST1 predominating. Through whole-genome sequencing of 145 isolates, we observed that five commonly used virulence-associated genes (VAGs; epf, mrp, sly, ofs, and srtF) were not present in most disease-associated isolates, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) yielded false-positive results in 7% of isolates. These data indicate that (i) clinical signs and site of isolation alone are insufficient for defining a pathotype, (ii) S. suis serotypes and STs associated with CNS infection are more diverse than previously reported, (iii) MALDI-TOF MS may need to be supplemented with additional diagnostic tools for precise S. suis identification, and (iv) VAGs remain an unreliable means for identifying isolates associated with CNS disease.IMPORTANCEStreptococcus suis is an important and complex systemic bacterial pathogen of swine. Characterization of S. suis strains originating from pigs with histologic confirmation of neurologic disease is limited. Review of swine diagnostic submissions revealed that fewer than half of cases from which S. suis was isolated from the brain had histologic evidence of neurologic disease. This finding demonstrates that clinical signs and site of isolation alone are not sufficient for identifying a neurologic disease-associated strain. Characterization of strains originating from cases with evidence of disease using classic and advanced diagnostic techniques revealed that neurologic disease-associated strains are diverse and commonly lack genes previously associated with virulence.

Genomic Epidemiology of Extrapulmonary Nontuberculous Mycobacteria Isolates at Emerging Infections Program Sites - United States, 2019-2020.

Nontuberculous mycobacteria (NTM) cause pulmonary and extrapulmonary infections. Although isolation of NTM from clinical specimens has increased nationally, few studies delineated the molecular characteristics of extrapulmonary NTM. Extrapulmonary isolates were collected by four Emerging Infections Program sites from October 2019 to March 2020 and underwent laboratory characterization, including matrix-assisted laser desorption ionization-time of flight mass spectrometry, Sanger DNA sequencing, and whole genome sequencing. Bioinformatics analyses were employed to identify species, sequence types (STs), antimicrobial resistance (AR), and virulence genes; isolates were further characterized by phylogenetic analyses. Among 45 isolates, the predominant species were Mycobacterium avium (n=20, 44%), Mycobacterium chelonae (n=7, 16%), and Mycobacterium fortuitum (n=6, 13%). The collection represented 31 STs across 10 species; the most common ST was ST11 (M. avium, n=7). Mycobacterium fortuitum and Mycobacterium abscessus isolates harbored multiple genes conferring resistance to aminoglycosides, beta-lactams, and macrolides. No known AR mutations were detected in rpoB, 16S, or 23S rRNAs. Slow-growing NTM species harbored multiple virulence genes including type-VII secretion components, adhesion factors, and phospholipase C. Continued active laboratory- and population-based surveillance will further inform the prevalence of NTM species and STs, monitor emerging clones, and allow AR characterization.

Robust online monitoring system for PCDD/Fs in a full-scale MSWI by Deans switch: Efficiently separation and purification

Existing online monitoring system for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), technically named Thermal Desorption-Gas Chromatography-Tunable Laser Ionization-Time of Flight Mass Spectrometry (TD-GC-TLI-TOFMS), has been applied in several incinerators in China. TD-GC-TLI-TOFMS can realize rapid detection of PCDD/Fs emissions from incineration sources. However, the long-term measurement of unclean flue gas will pollute the instruments in TD-GC-TLI-TOFMS, and interfere with the peak output of the target 1,2,4-trichlorobenzene (1,2,4-TrCBz). In this study, Deans switch (DS) was utilized for the first time in an online monitoring system for PCDD/Fs to separate 1,2,4-TrCBz signal from impurity signals, which improved the anti-interference capability of the system. Laboratory standard gas experiments showed that after adding a DS device between GC and TLI pulse valve, when the pressure set in DS was 4 psi and switched before or near the peak output of 1,2,4-TrCBz, the change of 1,2,4-TrCBz signal intensity was minimal. The impurities near the target peak were removed, and TLI-TOFMS was highly stable during continuous measurement. Moreover, the maximum intensity peak time of 1,2,4-TrCBz was stable after using DS in different switching time intervals. When connecting DS to TD-GC-TLI-TOFMS for field validation on the tail flue gas of a municipal solid waste incinerator (MSWI), results showed that a better 1,2,4-TrCBz signal could be obtained with a 69.52 % reduction of impurity peaks at the moments closer to the target peak. Furthermore, DS improved the sensitivity of the system to low concentration variations of 1,2,4-TrCBz in the flue gas. The robust system developed in this study can be better applied to incineration factories with poor combustion or suboptimal purification technology, facilitating online PCDD/Fs monitoring.

Antifungal susceptibility and biofilm production of Candida species- causative agents of female genital tract infections.

Recurrent vulvovaginal candidosis (RVVC) is a chronic infection affecting 8-10% of women worldwide. Biofilm production of the infecting species and reduced sensitivity to antimycotics could contribute to the recurrence of this infection. This study aimed to examine the biofilm production ability and antifungal susceptibility of genital yeast isolates to determine their virulence potential. Matrix-assisted laser desorption in ionization-time of flight mass spectrometry (MALDI-TOF MS) was used to identify 300 Candida species. Using crystal violet method, strains were categorized into non-producers, weak, moderate, and strong biofilm producers (BFP). Antifungal susceptibility testing was performed using commercial Integral System YEASTS Plus test (ISYPT) and broth microdilution method (BMM). MALDI-TOF MS identified 150 Candida albicans, 124 non-albicans Candida (NAC), and 26 Saccharomyces cerevisiae strains. Within 138 (46.0%) BFP, 23 (16.7%) were strong, 44 (31.9%) moderate, and 71 (51.4%) weak. BMM was done for 43 BFP selected isolates with nystatin MIC ˃1.25 μl, fluconazole MIC ˃64 μl, and clotrimazole MIC ˃1.0 μl determined by ISYPT. Compared to all examined isolates, BMM confirmed that: i) C. albicans and NAC BFP showed low sensitivity to fluconazole (12% and 4%, respectively); ii) all BFP showed low sensitivity to nystatin (12.7% C. albicans, 14.5% NAC, and 23.1% S. cerevisiae); iii) clotrimazole in vitro was the most efficient regarding C. albicans and S. cerevisiae strains, but in 4.0% NAC BFP for this antimycotic higher MIC was established. Novel antimycotics or possible combinations of antifungal agents and natural products could be a new treatment option for RVVC.

Feasibility of qualitative identification of some metal elements in rice grains using laser desorption ionization-time of flight mass spectrometry

Rice is one of the popular foods in the world, and the identification and measurement of the concentration of metal elements in therein is necessary for food safety. To this end, this work investigated the feasibility of using laser desorption ionization time-of-flight mass spectrometry (LDI-TOF-MS) for the qualitative identification of metal elements present in rice. The effect of different sample preparation methods (solvation of rice powder in water, acid digestion, and rice tablet) and laser wavelength on the mass spectral pattern of rice was investigated. Based on the experimental results, LDI-TOF-MS proves to be a reliable method for the qualitative identification of the metal elements in rice. It was determined that the rice sample prepared as a tablet and using visible laser radiation, are the most suitable choice for the identification of metallic elements using LDI-TOF-MS. The mass spectrum of rice was experimentally modeled using starch and theoretically simulated.

A novel barcoded nanopore sequencing workflow of high-quality, full-length bacterial 16S amplicons for taxonomic annotation of bacterial isolates and complex microbial communities.

Due to recent improvements, Nanopore sequencing has become a promising method for experiments relying on amplicon sequencing. We describe a flexible workflow to generate and annotate high-quality, full-length 16S rDNA amplicons. We evaluated it for two applications, namely, (i) identification of bacterial isolates and (ii) species-level profiling of microbial communities. We assessed the identification of single bacterial isolates by sequencing, using a set of barcoded full-length 16S rRNA gene primer pairs (pair A), on 47 isolates encompassing multiple genera and compared those results with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS)-based identification. Species-level community profiling was tested with two sets of barcoded full-length 16S primer pairs (A and B) and compared to the results obtained with shotgun Illumina sequencing using 27 stool samples. We developed a Nextflow pipeline to retain high-quality reads and taxonomically annotate them. We found high agreement between our workflow and MALDI-TOF data for isolate identification (positive predictive value = 0.90, Cramér's V = 0.857, and Theil's U = 0.316). For species-level community profiling, we found strong correlations (rs > 0.6) of alpha diversity indices between the two primer sets and Illumina sequencing. At the community level, we found significant but small differences when comparing sequencing techniques. Finally, we found a moderate to strong correlation when comparing the relative abundances of individual species (average rs = 0.6 and 0.533 for primers A and B). Despite identified shortcomings, the proposed workflow enabled accurate identification of single bacterial isolates and prominent features in microbial communities, making it a worthwhile alternative to MALDI-TOF MS and Illumina sequencing.IMPORTANCEA quick, robust, simple, and cost-effective method to identify bacterial isolates and communities in each sample is indispensable in the fields of microbiology and infection biology. Recent technological advances in Oxford Nanopore Technologies sequencing make this technique an attractive option considering the adaptability, portability, and cost-effectiveness of the platform, even with small sequencing batches. Here, we validated a flexible workflow to identify bacterial isolates and characterize bacterial communities using the Oxford Nanopore Technologies sequencing platform combined with the most recent v14 chemistry kits. For bacterial isolates, we compared our nanopore-based approach to matrix-assisted laser desorption ionization-time of flight mass spectrometry-based identification. For species-level profiling of complex bacterial communities, we compared our nanopore-based approach to Illumina shotgun sequencing. For reproducibility purposes, we wrapped the code used to process the sequencing data into a ready-to-use and self-contained Nextflow pipeline.

MALDI-TOF mass spectrometry from nucleic acid: development and evaluation of a novel platform for identification of mycobacteria and detection of genetic markers of resistance.

Complete identification methods are critical for evaluating nontuberculous mycobacteria (NTM). Here, we describe a novel diagnostic method for identification of eight NTM, Mycobacterium tuberculosis complex, and three drug resistance markers using PCR/matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) from cultured organisms. With this technology, a multiplex end-point PCR is performed for targets of interest. Detection probes that are extended in the presence of a target are added. The extended probes have greater molecular weight and can be detected by MALDI-TOF MS. An AFB Primary Panel was designed to differentiate Mycobacterium avium; Mycobacterium intracellulare subsp. chimaera; Mycobacterium avium complex (other); Mycobacterium abscessus subsp. abscessus, bolletii, and massiliense; Mycobacterium kansasii, and M. tuberculosis complex. This design should cover 90% (3,483/3,691) of mycobacteria seen onsite. A development set of unblinded isolates (n = 217) was used to develop PCR primers, detection probes, and probe barcodes. It demonstrated 99.1% (215/217) agreement with reference methods. An evaluation set using blinded isolates (n = 320) showed an overall sensitivity of 94.3% (range by target: 90.0-100%). Overall specificity from negative media, non-target mycobacteria, and bacteria was 99.1% (108/109; range by target: 94.4-100%). Three drug resistance markers erm (41), rrl, and rrs demonstrated 100%, 91%, and 100% sensitivity, respectively, and >99% specificity. Limit of detection per target ranged from 2.2 × 103 to 9.9 × 106 CFU/mL. The AFB Primary Panel allows for mycobacterial speciation, subspeciation, and resistance mutation detection, which is essential for diagnosis, appropriate therapy, identifying outbreaks, and managing treatment-refractory disease. It can perform with high-throughput and high specificity and sensitivity from isolates.IMPORTANCEEven closely related mycobacteria can have unique treatment patterns, but differentiating these organisms is a challenge. Here, we tested an innovative platform that combines two commonly used technologies and creates something new: matrix-assisted, laser-desorption ionization time-of flight mass spectrometry was performed on PCR amplicons instead of on proteins. This created a robust system with the advantages of PCR (high discriminatory power, high throughput, detection of resistance) with the advantages of mass spectrometry (more targets, lower operational cost) in order to identify closely related mycobacterial organisms.

Rapid species differentiation and typing of Acinetobacter baumannii

Acinetobacter (A.) baumannii has emerged as a difficult-to-treat nosocomial bacterial human pathogen. A. baumannii is to be dealt with under the “One Health” approach, and its surveillance in human, animal, and environmental settings assumes paramount importance in understanding its plausible transmission dynamics. Accurate identification of A. baumannii, its clonal complexes, and sequence types is important for understanding the epidemiological distribution, evolutionary relationships, and transmission dynamics. A wide range of genotyping techniques are applied for the differentiation of the Acinetobacter calcoaceticus-baumannii (ACB) complex. However, there is no single straight-forward genotype method applied for rapid assays. Currently, two multilocus sequence typing (MLST) Oxford and Pasture schemes exist; though considered a gold standard for sequence typing, harmonizing the schemes is not a straightforward process. The whole genome sequencing-based core-genome multilocus sequence typing (cgMLST) and core single nucleotide polymorphism (cgSNP) are robust and precise sequence typing; however, they are expensive, depending on the quality of sequencing and demand a higher level of computational skills. In the past decade, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) based species identification has been successfully employed for rapid discrimination of the ACB complex. MALDI typing is rapid, easier, cheaper, and as reliable as molecular methods. Strain level A. baumannii identification confidence improved upon augmentation of existing databases with in-house reference spectra of well-defined isolates. The application of artificial intelligence and machine learning might be useful in clonal sequence types (ST)-level identification. The genus Acinetobacter’s taxonomic classification is evolving, and newer STs are being described; hence, the establishment of a central repository of A. baumannii reference spectra will help in harmonizing across the laboratories and help in the global level surveillance program on A. baumannii in “One Health” perspective. This review sheds light on the challenges related to techniques employed for the identification of Acinetobacter and the potential application and future perspectives of MALDI-TOF MS.

Genetic Characterization of Antibiotic-Resistant Staphylococcus spp. and Mammaliicoccus sciuri from Healthy Humans and Poultry in Nigeria.

Staphylococcus spp. poses a significant threat to human and animal health due to their capacity to cause a wide range of infections in both. In this study, resistance genes conferring antibiotic resistance in Staphylococcus spp. and Mammaliicoccus sciuri isolates from humans and poultry in Edo state, Nigeria, were investigated. In April 2017, 61 Staphylococcus spp. isolates were obtained from urine, wounds, nasal and chicken fecal samples. Species identification was carried out by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Antimicrobial susceptibility testing was performed using the Kirby-Bauer method for 16 antibiotics. Whole-genome sequencing was used for characterization of the isolates. The 61 investigated isolates included Staphylococcus aureus, S. arlettae, M. sciuri, S. haemolyticus, and S. epidermidis. A total of 47 isolates (77%) belonged to human samples and 14 (23%) isolates were collected from poultry samples. All were phenotypically resistant to at least three antimicrobial(s). Multiple resistance determinants were detected in the human and poultry isolates analyzed. Phylogenetic analysis revealed close relatedness among the isolates within each species for S. arlettae, M. sciuri, and S. haemolyticus, respectively. This study delivered comprehensive genomic insights into antibiotic-resistant Staphylococcus species and M. sciuri isolates from human and poultry sources in Edo state, Nigeria, from a One Health perspective.

Staphylococcus spp. in Salad Vegetables: Biodiversity, Antimicrobial Resistance, and First Identification of Methicillin-Resistant Strains in the United Arab Emirates Food Supply.

Contamination of leafy greens with Staphylococcus spp. can occur at various supply chain stages, from farm to table. This study comprehensively analyzes the species diversity, antimicrobial resistance, and virulence factors of Staphylococci in salad vegetables from markets in the United Arab Emirates (UAE). A total of 343 salad items were sampled from three major cities in the UAE from May 2022 to February 2023 and tested for the presence of Staphylococcus spp. using standard culture-based methods. Species-level identification was achieved using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Antimicrobial susceptibility testing was conducted using the VITEK-2 system with AST-P592 cards. Additionally, whole genome sequencing (WGS) of ten selected isolates was performed to characterize antimicrobial resistance determinants and toxin-related virulence factors. Nine Staphylococcus species were identified in 37.6% (129/343) of the tested salad items, with coagulase-negative staphylococci (CoNS) dominating (87.6% [113/129]) and S. xylosus being the most prevalent (89.4% [101/113]). S. aureus was found in 4.6% (14/343) of the salad samples, averaging 1.7 log10 CFU/g. One isolate was confirmed as methicillin-resistant S. aureus, harboring the mecA gene. It belonged to multi-locus sequence type ST-672 and spa type t384 and was isolated from imported fresh dill. Among the characterized S. xylosus (n = 45), 13.3% tested positive in the cefoxitin screen test, and 6.6% were non-susceptible to oxacillin. WGS analysis revealed that the cytolysin gene (cylR2) was the only toxin-associated factor found in S. xylosus, while a methicillin-sensitive S. aureus isolate harbored the Panton-Valentine Leukocidin (LukSF/PVL) gene. This research is the first to document the presence of methicillin-resistant S. aureus in the UAE food chain. Furthermore, S. xylosus (a coagulase-negative staphylococcus not commonly screened in food) has demonstrated phenotypic resistance to clinically relevant antimicrobials. This underscores the need for vigilant monitoring of antimicrobial resistance in bacterial contaminants, whether pathogenic or commensal, at the human-food interface.

Molecular analysis of recombinant collagenase from Bacillus siamensis strain Z1: Gene Cloning, expression and in-silico characterization

This study focuses on gene cloning, expression, biochemical and analytical characterization along with structural and functional characterization of collagenase followed with molecular docking, dynamics study and Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA). The collagenase gene identified from the genome of the novel collagenase Bacillus siamensis strain Z1 is introduced into E.coli DH5α, subsequently expressed in E.coli BL21 (DE3) withisopropyl β −d – 1 − thiogalactopyranoside(IPTG) induction and further affinity purified yielding in ∼ 89.4 kDa recombinant collagenase which demonstrated alkali characteristics and thermostability determined by thermodynamic parameters. Recombinant collagenase revealed good stability when exposed to diverse biochemical components. The recombinant collagenase identity was confirmed through matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showing specific mass peaks and via N-terminal sequence analysis as MTAVNQTISK. Moreover, the concluded N-terminal amino acid sequence from Edman degradation displayed significant resemblance. The structural and functional analysisof recombinant collagenase was analysed by Circular dichroism (CD), Proton nuclear magnetic resonance (1H NMR) spectrometry and Thermogravimetric analysis (TGA). The recombinant collagenase also showed gelatin liquefaction ability. The collagenase gene sequence is also assessed for structural and functional characterizations by using various computational tools and revealed its classification in U32 family peptidase. A Grand average of hydropathicity index (GRAVY) score of −0.295 and instability index of 37.22 was obtained. Homology model of collagenase gene was generated by employing SWISS-MODEL and structure analysis by Ramachandran plot. Molecular docking of modelled collagenase with four different substrates was carried out by PyRx and Autodocking. Highest docking score of −12.7 kcal/mol was obtained for Alaska pollock hydroxyproline containing marine collagen peptide (APHCP). Subsequently, Molecular dynamics and simulations for highest score docked complex was assessed using GROningenMAchine for Chemical Simulations (GROMACS).

Characteristics of Umami Taste of Soy Sauce Using Electronic Tongue, Amino Acid Analyzer, and MALDI-TOF MS.

The objective of this study was to investigate the umami characteristics of soy sauce using electronic tongue evaluation and amino acid composition and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. The soy sauce peptides were isolated from soy sauce using XAD-16 macroporous resin combined with ethanol solution. The results showed that the soy sauce peptide fraction eluted by 60% ethanol (SS-60%) exhibited a prominent umami taste, and the umami scores were highly positively correlated with the amino acid nitrogen contents of soy sauces. The umami scores of SS-60% were significantly positively correlated with the contents of free amino acids. Especially, Phe showed the highest positive correlation with the umami scores. In addition, five characteristic ion peaks with m/z at 499, 561, 643, 649, and 855 were identified in the peptide mass fingerprinting. Therefore, this study provides new insights into the umami characteristics for the taste evaluation and reality identification of soy sauce.

Characterisation of glucose-induced protein fragments among the order Enterobacterales using matrix-assisted laser desorption ionization-time of flight mass spectrometry

To characterise the glucose-induced protein fragments by MALDI-TOF MS analysis, we compared data for samples from Escherichia coli cultured in media with or without glucose. Characteristic peaks were observed in the presence of glucose, and MS/MS revealed Asr-specific fragments. The amino acid sequences of the fragments suggested sequence-specific proteolysis. Blast-analysis revealed that numerous Enterobacterales harbored genes encoding Asr as well as E. coli. Here, we analysed 32 strains from 20 genera and 25 species of seven Enterobacterales families. We did not detect changes in the mass spectra of four strains of Morganellaceae lacking asr, whereas peaks of Asr-specific fragments were detected in the other 28 strains. We therefore concluded that the induction of Asr production in the presence of glucose is common among the Enterobacterales, except for certain Morganellaceae species. In members of family Budviciaceae, unfragmented Asr was detected. Molecular genetic information suggested that the amino acid sequences of Asr homologs are diverse, with fragments varying in number and size, indicating that Asr may serve as a discriminative biomarker for identifying Enterobacterales species.