Integrating MALDI-TOF Mass Spectrometry with Machine Learning Techniques for Rapid Antimicrobial Resistance Screening of Foodborne Bacterial Pathogens.

Although MALDI-TOF mass spectrometry (MS) is considered as the gold standard for rapid and cost-effective identification of microorganisms in routine laboratory practices, its capability for antimicrobial resistance (AMR) detection has received limited focus. Nevertheless, recent studies explored the predictive performance of MALDI-TOF MS for detecting AMR in clinical pathogens when machine learning techniques are applied. This chapter describes a routine MALDI-TOF MS workflow for the rapid screening of AMR in foodborne pathogens, with Campylobacter spp. as a study model.

Integration of MALDI-TOF MS and machine learning to classify enterococci: A comparative analysis of supervised learning algorithms for species prediction.

This research focused on distinguishing distinct matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectral signatures of three Enterococcus species. We evaluated and compared the predictive performance of four supervised machine learning algorithms, K-nearest neighbor (KNN), support vector machine (SVM), and random forest (RF), to accurately classify Enterococcus species. This study involved a comprehensive dataset of 410 strains, generating 1640 individual spectra through on-plate and off-plate protein extraction methods. Although the commercial database correctly identified 76.9% of the strains, machine learning classifiers demonstrated superior performance (accuracy 0.991). In the RF model, top informative peaks played a significant role in the classification. Whole-genome sequencing showed that the most informative peaks are biomarkers connected to proteins, which are essential for understanding bacterial classification and evolution. The integration of MALDI-TOF MS and machine learning provides a rapid and accurate method for identifying Enterococcus species, improving healthcare and food safety.

Diversity of culturable bacterial isolates and their potential as antimicrobial against human pathogens from Afar region, Ethiopia.

Antimicrobial resistance is a growing global concern exacerbated by the scarcity of new medications and resistance to current antibiotics. Microbes from unexplored habitats are promising sources of natural products to combat this challenge. This study aimed to isolate bacteria producing secondary metabolites and assess their antimicrobial efficacy against human pathogens. Soil and liquid samples were collected from Afar region, Ethiopia. Bacterial isolates were obtained using standard serial dilution techniques. Antimicrobial activity was evaluated using agar plug and well diffusion methods. matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF MS) and whole-genome sequencing (WGS) were conducted for the isolate exhibiting the highest antimicrobial activity. Secondary metabolites were extracted and analyzed using gas chromatography-mass spectra (GC-MS). In this study, 301 bacteria isolates were identified, of which 68 (22.6%) demonstrated antagonistic activity against at least one reference pathogen. Whole-genome sequencing revealed that Sl00103 belongs to the genus Bacillus, designated as Bacillus sp. Sl00103. The extract of Sl00103 showed zones of inhibition ranging between 17.17 ± 0.43 and 26.2 ± 0.4 mm against bacterial pathogens and 19.5 ± 0.44 to 21.0 ± 1.01 mm against Candida albicans. GC-MS analysis of ethyl acetate and n-hexane extracts identified major compounds including (R,R)-butane-2,3-diol; 3-isobutylhexahydropyrrolo[1,2a] pyrazine-1,4-dione; cyclo(L-prolyl-L-valine); and tetradecanoic acid, 12-methyl-, methyl ester; hexadecanoic acid, methyl ester among other. In conclusion, this study isolated several promising bacterial strains from the Afar region in Ethiopia, with strain Sl00103 (Bacillus sp. Sl00103) demonstrating notable antimicrobial and antioxidant activities and warranting further studies. IMPORTANCE: Antimicrobial resistance (AMR) is an escalating global health threat affecting humans, animals, and the environment, underscoring the urgent need for alternative pathogen control methods. Natural products, particularly secondary metabolites from bacteria, continue to be a vital source of antibiotics. However, microbial habitats and metabolites in Africa remain largely unexplored. In this study, we isolated and screened bacteria from Ethiopia's Afar region, characterized by extreme conditions like high temperatures, volcanic activity, high salinity, and hot springs to identify potential bioactive compounds. We discovered diverse bacterial isolates with antimicrobial activity against various pathogens, including strain Sl00103 (Bacillus sp. Sl00103), which demonstrated significant antimicrobial and antioxidant activities. GC-MS analysis identified several antimicrobial compounds, highlighting strain Sl00103 as a promising source of secondary metabolites with potential pharmaceutical applications and warranting further investigation.

Bacterial Pesticides: Mechanism of Action, Possibility of Food Contamination, and Residue Analysis Using MS.

As Sustainable Development Goals (SDGs) and the realities of climate change become widely accepted around the world, the next-generation of integrated pest management will become even more important for establishing a sustainable food production system. To meet the current challenge of food security and climate change, biological control has been developed as one sustainable crop protection technology. However, most registered bacteria are ubiquitous soil-borne bacteria that are closely related to food poisoning and spoilage bacteria. Therefore, this review outlined (1) the mechanism of action of bacterial pesticides, (2) potential concerns about secondary contamination sources associated with past food contamination, and, as a prospective solution, focused on (3) principles and methods of bacterial identification, and (4) the possibility of identifying residual bacteria based on mass spectrometry.

Human identification of single hair shaft using a mass spectrometry mRNA typing system.

Hair is one of the most common forms of forensic biological material at various crime scenes. So far, human identification cannot be effectively accomplished with a single telogen hair encountered in forensic casework due to the detection limit. Emerging studies have revealed RNA as a promising biomarker in hair shafts, while the single telogen hair could not be successfully genotyped even after being examined with the recently developed mRNA typing system. MALDI-TOF MS, the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enables sensitive and accurate measurement of DNA products. To address this problem and further develop the analysis technology of hairs, we established a mass spectrometry system for human identification based on a single hair shaft using 25 polymorphic SNPs located on 18 mRNA molecules (KRT31, RFK, KRT86, KRT35, PABPC1, KMT2D, LEMD2, TBC1D4, CTC1, PPP1R15A, RBM33, LRRC15, KRT33A, KRTAP12-2, KRT81, AHNAK, KRTAP4-8, FLG2). The forensic application of the detection system was evaluated, and all hair samples used were collected from individuals in Shanxi province. Firstly, we demonstrated that the RNA typing results of a single hair shaft were in perfect concordance with DNA typing results and confirmed the consistency between hairs from different body parts. To assess the potential influence of positions along the hair shaft, 6 cm long hair shafts from the distal end were examined by the MALDI-TOF MS system, whose genotype could be successfully detected. The system was capable of detecting aged samples stored for 390 days and could also be employed on various types of hair samples, such as white hair and permed or dyed hair. Finally, 50 unrelated individuals from Shanxi province were genotyped for the population study, and the CDP of the system in the Shanxi population is 0.998928. In this study, we established a mass spectrometry system for human identification based on a single hair shaft. We used a single hair shaft, rather than multiple hair shafts reported in our previous report, to get a full typing profile. The system sensitivity was substantially enhanced, which provided a valuable strategy for forensic practice to perform human identification using hairs.

Evaluation of protein extraction protocols for MALDI-TOF Biotyper analysis of mycobacteria.

Infections caused by Mycobacterium tuberculosis and nontuberculous mycobacteria represent a significant global threat and medical concern. Therefore, accurate and reliable methods must be employed to identify mycobacteria rapidly. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a technique that compares the cellular protein profiles of unknown isolates with reference mass spectra in a database to identify microorganisms. However, the thick and waxy lipid layer, which is rich in mycolic acids and is present in mycobacterial cells, makes protein extraction challenging. To identify the optimal protocol for correctly identifying bacilli using MALDI-TOF mass spectrometry, this study compared four different cellular protein extraction methods. Four strains of M. bovis BCG were selected as representatives of slow-growing mycobacteria, while three strains of fast-growing mycobacteria were also included: M. peregrinum, M. smegmatis, and M. farcinogenes. The extraction method that proved most effective was the extraction of inactivated cells with chloroform and methanol, which partially delipidates the cells. These cells were then extracted with formic acid, as is standard practice for protein extraction. The advantage of this method is that it allows the parallel analysis of cellular lipids and proteins from a single sample. It is therefore important to optimize mycobacterial protein extraction for MALDI-TOF MS analysis in clinical microbiology laboratories.

Utilization of MALDI-TOF MS in the etiological diagnosis of deep-seated anaerobic bacterial infections.

PURPOSE: Deep-seated abscesses can be caused by a wide array of bacteria in various anatomical sites, the precise identification of which is crucial for implementing organism-specific treatments which can reduce morbidity and mortality. MALDI-TOF MS is a powerful proteomic method for the swift and accurate identification of anaerobic organisms. The aim of this study was to investigate deep-seated infections by MALDI-TOF MS (in comparison to VITEK®2 ANC ID card and phenotypic biochemical tests) and to determine the susceptibility pattern of identified microorganisms. MATERIALS AND METHODS: A total of 104 samples from patients suspected of deep-seated infections were aseptically collected and subjected to microscopy, aerobic/anaerobic cultures and subsequent identification via MALDI-TOF MS followed by antimicrobial susceptibility testing. Anaerobic bacteria were also identified using the VITEK-2 system and phenotypic biochemical tests. RESULTS: Out of the 104 samples tested, 41.3 % (43/104) showed positive results, predominantly in pus specimens (88 %). Mixed infections were found in 21 % of the positive cases. Of the 52 organisms identified from positive specimens, 19.2 % (10/52) were obligate anaerobes, with Bacteroides fragilis group being the most prevalent, followed by both Clostridium perfringens and Clostridium sporogenes respectively. Escherichia coli was observed to be the most common facultative anaerobic isolate. All obligate anaerobes were successfully identified to the species level via MALDI-TOF MS. In contrast, the VITEK®2 ANC ID card identified only 40 % (4/10) anaerobic bacteria to the species level. All obligate anaerobic organisms showed 100 % susceptibility to metronidazole, vancomycin and ertapenem. 25 % of the Bacteroides spp. and 50 % of Clostridium perfringens isolates were found to be resistant to clindamycin. CONCLUSION: MALDI-TOF MS proves as a beneficial diagnostic tool for bacterial identification, eliminating the labour-intensive and time consuming conventional microbiological methods. Its accuracy of bacterial detection further helps in combating antibiotic resistance and improving patient outcomes in deep-seated infections.

Characteristic of new Phaffia rhodozyma yeast strains isolated from birch slime fluxes in Poland.

Three new strains of Phaffia rhodozyma yeast have recently been isolated in Poland. The aim of this study was to phenotypically characterize these strains and to compare them with the properties of the reference strain. The potential for carotenoid biosynthesis in these strains was also determined, depending on temperature, carbon, and nitrogen sources in the medium. Phaffia rhodozyma yeasts were also identified by MALDI-TOF MS. There were minor differences in cell morphology among the strains. All strains reproduced asexually by budding and formed spherical chlamydospores. No ability for sexual reproduction was observed. Physiological tests showed minor variations between the reference strain and the isolates, likely due to the geographical specificity of the habitat from which they were originally isolated. Analysis of protein spectra showed that the tested yeast isolates had seven common peaks of different intensities, with masses at 2200, 2369, 3213, 3628, 3776, 3921, and 4710 m/z. Moreover, additional strain-dependent spectra were found. The amount of synthesized carotenoids varied with the carbon and nitrogen sources used, as well as the temperature. The best producer of carotenoids was the P. rhodozyma CMIFS 102 isolate.

Repurposing MALDI-TOF MS for effective antibiotic resistance screening in Staphylococcus epidermidis using machine learning.

The emergence of Staphylococcus epidermidis as a significant nosocomial pathogen necessitates advancements in more efficient antimicrobial resistance profiling. However, existing culture-based and PCR-based antimicrobial susceptibility testing methods are far too slow or costly. This study combines machine learning with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to develop predictive models for various antibiotics using a comprehensive dataset containing thousands of S. epidermidis isolates. Optimized machine learning models utilized feature selection and achieved high AUROC scores ranging from 0.80 to 0.95 while maintaining AUPRC scores up to 0.97. Shapley Additive exPlanations were employed to analyze relevant features and assess the significance of corresponding protein biomarkers while also verifying that predictive power was derived from the detection of proteins rather than noise. Antimicrobial resistance models were validated externally to evaluate model performance outside the original data collection site. The approaches and findings in this study demonstrate a significant advancement in rapid, cost-effective antimicrobial resistance profiling, offering a promising solution for improving treatments for nosocomial infections and being potentially applicable to other microbial pathogens in the future.

A Case of Pulmonary Infection Due to Magnusiomyces capitatus in a Non-Immunocompromised Patient with Cerebral Palsy.

Background: Magnusiomyces capitatus (M. capitatus) is a dimorphic opportunistic fungus that is a rare yeast and rarely reported in Asia. Owing to the absence of established clinical breakpoints, the treatment of this fungus poses challenges. Case Presentation: We report a rare case of a young, non-immunocompromised man with cerebral palsy, spinal deformity, and pulmonary M. capitatus infection. The patient's condition improved after treatment with voriconazole and became stable. Conclusion: M. capitatus infection is commonly associated with hematologic tumors and compromised immunity. Reports of M. capitatus infection in patients with non-immunocompromised host are uncommon. Insufficient understanding of these fungi may lead to underdiagnosis of fungal infection and clinical misdiagnosis, potentially resulting in delayed treatment and increased mortality.

Comparison of Nucleotide MALDI-TOF MS with Xpert MTB/RIF for Rifampicin Susceptibility Identification and Associated Risk Factors of Rifampicin Resistance Among Drug Resistant Mycobacterium tuberculosis.

Purpose: Nucleotide-based matrix-assisted laser desorption ionization time-of-flight mass spectrometry (nucleotide MALDI-TOF MS) is an emerging molecular technology used for the diagnosis of tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB)and its drug resistance. This study aimed to compare the ability of nucleotide MALDI-TOF MS to detect rifampicin (RIF) resistance in drug-resistant TB (DR-TB) patients with Xpert MTB/RIF and to analyze the disparate results individually. Additionally, potential factors associated with rifampicin resistance among DR-TB patients in Qingdao were investigated. Patients and Methods: A retrospective study was conducted at Qingdao Chest Hospital, and patients with DR-TB were enrolled. Corresponding frozen isolates were recovered and subjected to nucleotide MALDI-TOF MS, Xpert MTB/RIF, and phenotypic drug susceptibility testing (pDST). Sanger sequencing was performed for the discordant results of nucleotide MALDI-TOF MS and Xpert MTB/RIF. Univariate and multivariate logistic regression analyses were used to identify potential factors associated with rifampicin resistance among patients with DR-TB. Results: A total of 125 patients with DR-TB (18.8%, 125/668) were enrolled in this study from May 1 to July 31, 2023. Rifampicin-resistant (DR-TB/RR, 29) and rifampicin-sensitive (DR-TB/RS, 96) groups were divided according to the pDST results. Nucleotide MALDI-TOF MS performed better than Xpert MTB/RIF in terms of sensitivity, specificity, accuracy, and agreement with pDST. Only six cases had inconsistent results, and the sequencing results of five cases were identical to nucleotide MALDI-TOF MS. Furthermore, chest pain (aOR=12.84, 95% CI, 2.29-91.97, p=0.005), isoniazid sensitivity (aOR=0.14, 0.02-0.59, p=0.013), and ethambutol sensitivity (aOR=0.02, 0.00-0.10, p=0.000) were potential factors associated with rifampicin resistance among DR-TB patients in Qingdao. Conclusion: The overall concordance between nucleotide MALDI-TOF MS and Xpert MTB/RIF was 95.2%, with the former performing better in determining rifampicin susceptibility among DR-TB cases in Qingdao. Chest pain, isoniazid, and ethambutol resistance might be factors associated with RIF resistance among patients with DR-TB in Qingdao.

Comparison of Zybio Kit and saponin in-house method in rapid identification of bacteria from positive blood cultures by EXS2600 matrix-assisted laser desorption ionization time-of-flight mass spectrometry system.

We evaluated the performance of Zybio EXS2600 matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Zybio Inc., Chongqing, China) for the identification of bacteria from positive blood culture (BC) bottles using Blood Culture Positive Sample Pretreatment Kit (Zybio Inc., Chongqing, China) in comparison to an in-house saponin method. Following a positive signal by the BACTEC™ FX system, confirmation of identification was achieved using subcultured growing biomass used for MALDI-TOF MS analysis. A total of 94 positive BC bottles with 97 bacterial isolates were analyzed. The overall identification rates at the genus and species levels for the saponin method were 89.7% (87/97) and 74.2% (72/97), respectively. With the Zybio Kit, 88.7% (86/97) and 80.4% (78/97) of microorganisms were correctly identified to the genus and species levels, respectively. The saponin method identified 65.3% (32/49) of Gram-positive bacteria at the species level, whereas the Zybio Kit achieved a higher species-level identification rate of 79.6% (39/49) (p = 0.1153). The saponin method with additional on-plate formic acid extraction showed a significantly higher overall identification rate in comparison to the saponin method without that step for both genus (87.6% [85/97] vs. 70.1% [68/97], p = 0.0029) and species level (70.1% [68/97] vs. 46.4% [45/97], p = 0.0008). Identification rates of Gram-negative bacteria showed a higher identification rate, however, not statistically significant with additional Zybio Kit protocol step on both genus (85.4% [41/48] vs. 81.3% [39/48], p = 0.5858) and species level (77.1% [37/48] vs. 75% [36/48], p = 0.8120). Zybio Kit could offer an advantage in species-level identification, particularly for Gram-positive bacteria. The inclusion of on-plate formic acid extraction in the saponin method notably enhanced identification at both genus and species levels for Gram-positive bacteria. The extended protocol provided by the Zybio Kit could potentially offer an advantage in the identification of Gram-negative bacteria at both genus and species levels. Enhancements to the Zybio EXS2600 MALDI-TOF instrument software database are necessary.

Development of a rapid mass spectrometric method for the analysis of ten-eleven translocation enzymes.

In DNA, methylation at the fifth position of cytosine (5mC) by DNA methyltransferases is essential for eukaryotic gene regulation. Methylation patterns are dynamically controlled by epigenetic machinery. Erasure of 5mC by Fe2+ and 2-ketoglutarate (2KG) dependent dioxygenases in the ten-eleven translocation family (TET1-3), plays a key role in nuclear processes. Through the event of active demethylation, TET proteins iteratively oxidize 5mC to 5-hydroxymethyl cytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC), each of which has been implicated in numerous diseases when aberrantly generated. A wide range of biochemical assays have been developed to characterize TET activity, many of which require multi-step processing to detect and quantify the 5mC oxidized products. Herein, we describe the development and optimization of a sensitive MALDI mass spectrometry-based technique that directly measures TET activity and eliminates tedious processing steps. Employing optimized assay conditions, we report the steady-state activity of wild type TET2 enzymes to furnish 5hmC, 5fC and 5caC. We next determine IC50 values of several small-molecule inhibitors of TETs. The utility of this assay is further demonstrated by analyzing the activity of V1395A which is an activating mutant of TET2 that primarily generates 5caC. Lastly, we describe the development of a secondary assay that utilizes bisulfite chemistry to further examine the activity of wildtype TET2 and V1395A in a base-resolution manner. The combined results demonstrate that the activity of TET proteins can be gauged, and their products accurately quantified using our methods.

Identification of N-linked glycans recognized by WGA in saliva from patients with non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths. Saliva diagnosis is an essential approach for clinical applications owing to its noninvasive and material-rich features. The purpose of this study was to investigate differences in wheat germ agglutinin (WGA)-based recognition of salivary protein N-linked glycan profiles to distinguish non-small cell lung cancer (NSCLC) patients from controls. We used WGA-magnetic particle conjugates to isolate glycoproteins in the pooled saliva of healthy volunteers (HV, n = 35), patients with benign pulmonary disease (BPD, n = 35), lung adenocarcinoma (ADC, n = 35), and squamous cell carcinoma (SCC, n = 35), following to release the N-linked glycans from the isolated proteins with PNGase F, and further identified and annotated the released glycans by MALDI-TOF/TOF-MS, respectively. The results showed that 34, 35, 39, and 44 N-glycans recognized by WGA were identified and annotated from pooled saliva samples of HV, BPD, ADC, and SCC, respectively. Furthermore, the proportion of N-glycans recognized by WGA in BPD (81.2 %), ADC (90.1 %), and SCC (88.7 %), increased compared to HV (71.9 %). Two N-glycan peaks (m/z 2286.799, and 3399.211) specifically recognized by WGA were present only in NSCLC. These findings suggest that altered salivary glycopatterns such as sialic acids and GlcNAc containing N-glycans recognized by WGA might serve as potential personalized biomarkers for the diagnosis of NSCLC patients.

Artificial intelligence-clinical decision support system for enhanced infectious disease management: Accelerating ceftazidime-avibactam resistance detection in Klebsiella pneumoniae.

BACKGROUND: Effective and rapid diagnostic strategies are required to manage antibiotic resistance in Klebsiella pneumonia (KP). This study aimed to design an artificial intelligence-clinical decision support system (AI-CDSS) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and machine learning for the rapid detection of ceftazidime-avibactam (CZA) resistance in KP to improve clinical decision-making processes. METHODS: Out of 107,721 bacterial samples, 675 specimens of KP with suspected multi-drug resistance were selected. These specimens were collected from a tertiary hospital and four secondary hospitals between 2022 and 2023 to evaluate CZA resistance. We used MALDI-TOF MS and machine learning to develop an AI-CDSS with enhanced speed of resistance detection. RESULTS: Machine learning models, especially light gradient boosting machines (LGBM), exhibited an area under the curve (AUC) of 0.95, indicating high accuracy. The predictive models formed the core of our newly developed AI-CDSS, enabling clinical decisions quicker than traditional methods using culture and antibiotic susceptibility testing by a day. CONCLUSIONS: The study confirms that MALDI-TOF MS, integrated with machine learning, can swiftly detect CZA resistance. Incorporating this insight into an AI-CDSS could transform clinical workflows, giving healthcare professionals immediate, crucial insights for shaping treatment plans. This approach promises to be a template for future anti-resistance strategies, emphasizing the vital importance of advanced diagnostics in enhancing public health outcomes.

Co-Infection of Chickens with Staphylococcus lentus and Staphylococcus aureus from an Outbreak of Arthritis, Synovitis, and Osteomyelitis Argues for Detailed Characterisation of Isolates.

Staphylococcus species are widespread in poultry environments and can cause various infections, often when the host's defences are compromised. This manuscript reports on a co-infection of chickens with Staphylococcus lentus and Staphylococcus aureus associated with an outbreak of arthritis, synovitis, and osteomyelitis in an organic broiler breeder flock in Austria. Clinically, the affected flock showed weakness, lethargy, lameness, and increased mortality. Post-mortem examinations identified purulent arthritis and femoral head necrosis. Bacteriological analysis using MALDI-TOF MS identified both S. aureus and S. lentus in the affected joints. Antibiotic resistance testing revealed significant resistance, particularly in S. lentus. Histological analysis showed severe inflammation and bacterial colonies in the joints. While S. aureus is a common pathogen in poultry, S. lentus is less frequently reported. This study emphasises the need for detailed bacterial characterisation in outbreaks to better understand the role of less common pathogens like S. lentus. Further research is necessary to elucidate the impact of S. lentus on poultry health and its role in causing arthritis and synovitis, highlighting the importance of comprehensive investigation in such outbreaks.

Murine hepatic proteome adaptation to high-fat diets with different contents of saturated fatty acids and linoleic acid : α-linolenic acid polyunsaturated fatty acid ratios.

Introduction: Some health disorders, such as obesity and type 2 diabetes, are associated with a poor diet and low quality of the fat in it. The type and duration of the diet have an impact on the liver. This investigation uses the proteomic approach to identify changes in the mouse liver protein profile in adaptation to high-fat diets with different saturated fatty acid contents and linoleic acid (18:2n-6) to α-linolenic acid (18:3n-3) fatty acid ratios. Material and Methods: Four groups of male mice were fed different diets: one standard diet and three high-fat diets were investigated. After six months on these diets, the animals were sacrificed for liver dissection. Two-dimensional electrophoresis was used to separate the complex liver protein mixture, which enabled the separation of proteins against a wide, 3-10 range of pH and molecular weights of 15-250 kDa. Protein profiles were analysed in the PDQuest Advanced 8.0.1 program. Differentially expressed spots were identified using matrix-assisted laser desorption/ionisation-time-of-flight tandem mass spectrometry and peptide mass fingerprinting. The levels of identified proteins were validated using Western blotting. Transcript levels were evaluated using a real-time quantitative PCR. Results: The analysis of mouse liver protein profiles enabled the identification of 32 protein spots differing between nutritional groups. Conclusion: A diet high in polyunsaturated fatty acids modulated the levels of liver proteins involved in critical metabolic pathways, including amino acid metabolism, carbohydrate metabolism and cellular response to oxidative stress.

Metabolite profiling and molecular characterization of NBAIR BSWG1: A potential strain of Bacillus subtilis against Fusarium oxysporium f. sp. udum.

To address the fungal wilt of pigeon pea caused by Fusarium oxysporium f. sp. udum, farmers currently rely on chemical fungicides, despite their harmful effects. However, there is a growing need for safer alternatives like green pesticides. Bacterial biocontrol agents and their derivatives serve as potential green pesticides in the management of plant pathogens. In the present study, we aimed to identify indigenous Bacillus subtilis strains effective against F. oxysporium f. sp. udum. We used PCR and MALDI-TOF analysis to identify the active components responsible for the efficiency of efficient strain. Biochemical studies of cell-free extracts extracted from B. subtilis strains demonstrated the highest biosurfactant activity in NBAIR BSWG1, with an oil displacement of 2 cm and an emulsification index of 60 %. Molecular characterization confirmed the presence of surfactin, fengycin, and iturin coding genes in the B. subtilis strains, among them, NBAIR BSWG1 showed the highest number of lipopeptide-producing genes. Meanwhile, NBAIR BSWG1 showed inhibition of 79.84 % against F. oxysporium f. sp. udum using cell-free extract. Further metabolite profiling of NBAIR BSWG1 using MALDI-TOF analysis further confirmed surfactin, fengycin, and iturin in the purified cell-free extract of NBAIR BSWG1. Two peaks with m/z of 923.77 and 1149.92 were identified as novel lipopeptide compounds which need further characterization. The present study identified NBAIR BSWG1 as an efficient bacterial strain for the inhibition of F. oxysporium f. sp. udum and its antifungal properties are mainly due to the production of cyclic lipopeptides.

Application of MALDI-TOF mass spectrometry for identification of Nocardia species.

BACKGROUND: Nocardiosis, despite its rarity and underreporting, is significant due to its severe impact, characterized by high morbidity and mortality rates. The development of a precise, reliable, rapid, and straightforward technique for identifying the pathogenic agent in clinical specimens is crucial to reduce fatality rates and facilitate timely antimicrobial treatment. In this study, we aimed to identify Nocardia spp. in clinical isolates, using MALDI-TOF MS as the primary method, with molecular methods as the gold standard. Clinical Nocardia isolates were identified using 16S rRNA/hsp65/gyrB/secA1/rpoB gene sequencing. Identification performance of the Bruker MALDI Biotyper 3.1 (V09.0.0.0_8468) and MBT Compass 4.1 (V11.0.0.0_10833) for Nocardia identification was evaluated. RESULTS: Seventy-six Nocardia isolates were classified into 12 species through gene sequencing. The MALDI Biotyper 3.1 (V09.0.0.0_8468) achieved 100% genus-level accuracy and 84.2% species accuracy (64/76). The MBT Compass 4.1 with the BDAL Database (V11.0.0.0_10833) improved species identification to 98.7% (75/76). The updated database enhanced species level identification with scores > 1.7, increasing from 77.6% (59/76) to 94.7% (72/76), a significant improvement (P = 0.001). The new and simplified extraction increased the proportion of strains identified to the species level with scores > 1.7 from 62.0% (18/29) to 86.2% (25/29) (P = 0.016). An in-house library construction ensured accurate species identification for all isolates. CONCLUSIONS: The Bruker mass spectrometer can accurately identify Nocardia species, albeit with some variations observed between different database versions. The MALDI Biotyper 3.1 (V09.0.0.0_8468) has limitations in identifying Nocardia brasiliensis, with some strains only identifiable to the genus level. MBT Compass 4.1 (V11.0.0.0_10833) effectively addresses this shortfall, improving species identification accuracy to 98.7%, and offering quick and reliable identification of Nocardia. Both database versions incorrectly identified the clinically less common Nocardia sputorum as Nocardia araoensis. For laboratories that have not upgraded their databases and are unable to achieve satisfactory identification results for Nocardia, employing the new and simplified extraction method can provide a degree of improvement in identification outcomes.

Epidemiological trends and clinical relevance of nontuberculous mycobacterial pulmonary disease in a referral hospital in Japan, 2017-2021.

BACKGROUND: Epidemiological trends and clinical relevance of NTM species in Japan following the adoption of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry remain unclear. METHODS: We analyzed the results of mycobacterial culture tests of respiratory specimens collected between January 2017 and December 2021. We assessed the clinical relevance of NTM species by analyzing the proportion of patients diagnosed with NTM pulmonary infection (NTM-PI). We illustrated the incidence and clinical relevance of each NTM species using a two-dimensional scatter plot. Medical chart review and radiological analysis were also performed for less common species. RESULTS: Among 65,368 respiratory specimens tested for acid-fast bacilli, NTM were identified in 12,802 specimens from 3177 patients. The number of incident cases with NTM-PI has continued to increase. Notably, the number of incident cases with M. abscessus species (MABS) was continuously increasing and accounted for 10.6% of all incident cases with NTM-PI. The clinical relevance of the common NTM species, M. avium complex, MABS and M. kansasii, ranged from 57 to 72%. Seven other species exhibited a higher clinical relevance than these common NTM species, with M. shinjukuense (100%) having the highest clinical relevance. On the other hand, 11 species, including M. fortuitum (32.4%), M. xenopi (20.0%), and M. gordonae (22.9%), showed clinical relevance below 50%. CONCLUSIONS: The present study clarified the incidence and clinical relevance of NTM species using a two-dimensional scatter plot, which could serve as a useful tool for clinical decision-making and future epidemiological research.