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Cyclotides are small disulfide-rich peptides (28–37 amino acids) with a wide range of biological activities. The Rubiaceae family is a good source for cyclotide production, but the quantity produced is not sufficient. Therefore, we aimed to enhance cyclotide production in Oldenlandia umbellata (Rubiaceae family) cell cultures using a fungal elicitation process. Aspergillus niger was used as an elicitor to enhance the cyclotides in this plant. After elicitation, proteins were extracted and analyzed using 2-D gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The results of this study showed a 3.4-fold increase in protein spot intensity in fungal elicitor-treated cultures compared to untreated cultures. Mass spectrometric data showed two intense peaks in the monoisotopic protonated mass. Peptide sequencing of these peaks yielded two sequence hits that matched known cyclotide sequences in the CyBase database. The present study identified Varv-A and cycloviolacin O23-like peptides with antimicrobial properties, low cell permeability and solubility, and a lack of hemolytic, allergenic, toxic, and aggregation properties. Varv-A-like and cycloviolacin O23-like peptides are predicted to exhibit anticancer and antiviral properties, respectively. In conclusion, the cyclotides identified in this study may improve drug design and protein engineering, highlighting their therapeutic potential in the pharmaceutical industry.Key message: The production of antimicrobial varv-A and cycloviolacin O23-like peptides in O. umbellata cell cultures was significantly increased by 3.4 times using A. niger elicitation. [ABSTRACT FROM AUTHOR]

Cyclotides are small disulfide-rich peptides (28–37 amino acids) with a wide range of biological activities. The Rubiaceae family is a good source for cyclotide production, but the quantity produced is not sufficient. Therefore, we aimed to enhance cyclotide production in Oldenlandia umbellata (Rubiaceae family) cell cultures using a fungal elicitation process. Aspergillus niger was used as an elicitor to enhance the cyclotides in this plant. After elicitation, proteins were extracted and analyzed using 2-D gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The results of this study showed a 3.4-fold increase in protein spot intensity in fungal elicitor-treated cultures compared to untreated cultures. Mass spectrometric data showed two intense peaks in the monoisotopic protonated mass. Peptide sequencing of these peaks yielded two sequence hits that matched known cyclotide sequences in the CyBase database. The present study identified Varv-A and cycloviolacin O23-like peptides with antimicrobial properties, low cell permeability and solubility, and a lack of hemolytic, allergenic, toxic, and aggregation properties. Varv-A-like and cycloviolacin O23-like peptides are predicted to exhibit anticancer and antiviral properties, respectively. In conclusion, the cyclotides identified in this study may improve drug design and protein engineering, highlighting their therapeutic potential in the pharmaceutical industry.Key message: The production of antimicrobial varv-A and cycloviolacin O23-like peptides in O. umbellata cell cultures was significantly increased by 3.4 times using A. niger elicitation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

During the last few decades, the main focus of numerous studies has been on the human breast milk microbiota and its influence on the infant intestinal microbiota and overall health. The presence of lactic acid bacteria in breast milk affects both the quantitative and qualitative composition of the infant gut microbiota. The aim of this study was to assess the most frequently detected cultivable rod-shaped lactobacilli, specific for breast milk of healthy Bulgarian women and fecal samples of their infants over the first month of life, in 14 mother–infant tandem pairs. Additionally, we evaluated the strain diversity among the most common isolated species. A total of 68 Gram-positive and catalase-negative strains were subjected to identification using the MALDI-TOF technique. Predominant cultivable populations belonging to the rod-shaped lactic acid bacteria have been identified as Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Lacticaseibacillus paracasei, and Limosilactobacillus reuteri. Also, we confirmed the presence of Lactiplantibacillus plantarum and Lactobacillus gasseri. Up to 26 isolates were selected as representatives and analyzed by 16S rRNA sequencing for strain identity confirmation and a phylogenetic tree based on 16S rRNA gene sequence was constructed. Comparative analysis by four RAPD primers revealed genetic differences between newly isolated predominant L. rhamnosus strains. This pilot study provides data for the current first report concerning the investigation of the characteristic cultivable lactobacilli isolated from human breast milk and infant feces in Bulgaria. [ABSTRACT FROM AUTHOR]

The currently dominant SARS-CoV-2 omicron variant, while causing mild respiratory symptoms, exhibits high transmissibility, drug resistance, and immune evasion. We investigated whether the presence of the SARS-CoV-2 affected the dynamics of fecal microbial composition isolated in culture in moderate COVID-19 patients. Blood, stool, and medical records were collected from 50 patients with confirmed SARS-CoV-2 infection. Two samples were taken per patient, at disease onset (within 5 days) and after symptom resolution (30–35 days). The part of the gut microbiota identifiable using MALDI-TOF MS was analyzed, and inflammatory cytokines and blood markers were measured in serum. The analysis identified 566 isolates at the species level, including 83 bacterial and 9 fungal species. Our findings indicate a change in the gut microbiota composition isolated in culture during the initial phase of infection, characterized by the proliferation of opportunistic bacteria such as Enterococcus spp. and Citrobacter spp., at the expense of beneficial commensal bacteria from the genus Bacillus and Lactobacillus. Additionally, the enrichment of fungal pathogens in fecal samples collected 30 days after the cessation of disease symptoms might suggest a prolonged disruption of the gut microbiota even after the resolution of COVID-19 symptoms. This study contributes to a growing body of evidence on the systemic effects of SARS-CoV-2 and highlights the importance of considering gastrointestinal involvement in the management and treatment of COVID-19. [ABSTRACT FROM AUTHOR]

Fishes are bio-concentrators for heavy metals, but cannot thrive in excessively polluted water. Heavy metals can accumulate in Arius arius, and cadmium (Cd) activates its metal-binding protein. Presently, Cd-binding metallothionein (MT) in A. arius is subjected to sub-lethal cadmium chloride (CdCl2 at 20 mg/L for 72 h). Separation of MT was achieved from liver homogenate supernatants via affinity chromatography and Sephadex G-25, assessed by 15% SDS-PAGE, verified by Western blotting, and quantified by MALDI-TOF. Using mass fingerprinting, peptide masses were searched in Swiss-Prot. A. arius bounded MT amino acid sequences matched Oreochromis mossambicus with 100%. The 60 amino acids of MT and cysteine content were identified (20 residues). MT contains 6021 daltons based on the MALDI-TOF–MS. The MT protein was modeled using modular9v8 software, confirmed using Ramachandran plot, and utilized as a biomarker for Cd toxicity in fish. The 3D structures might help to identify the binding sites and may lead to innovative treatments for stress and cadmium toxicity. [ABSTRACT FROM AUTHOR]

Background: In November 2023, our hospital confirmed a case of Mycolicibacterium mucogenicum infection in the right back. The patient sought medical attention at our hospital due to "a right back lump for 2 months, ruptured for 1 month." Two months ago, the patient's back collided with a fire hose and developed a local walnut sized lump. The lump was soft, painless when pressed, and there was no obvious redness or swelling around it. The patient went to a private hospital for incision and drainage treatment on their own. One month later, the condition did not improve and there was yellow pus exuding from the incision site. For additional diagnosis and treatment, the patient went to our hospital for treatment. Methods: Back MRI (Magnetic Resonance Imaging) and abdominal ultrasound. Right back abscess incision and drainage surgery, pus pathogen examination: pus bacterial smear, bacterial culture, and identification. Auxiliary examinations: urine routine, blood routine, liver function, kidney function, blood lipids, and blood sugar. Results: Back MR: Abnormal signal shadow of subcutaneous soft tissue in the lower back, suggesting inflammatory lesions with abscess formation. Acid fast staining of pus: positive. Culture and identification of pus bacteria (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS): Mycolicibacterium mucogenicum. Clinical treatment plan: Clarithromycin 500 mg bid po+ minocycline 100 mg bid po, abscess incision and drainage, 5-aminolevulinic acid photodynamic therapy (ALA-PDT) qd. After 14 days of treatment, the patient's incision base tissue showed excellent activity, no obvious bleeding or exudation, slight redness and swelling around the wound, and normal skin temperature. The patient improved and was discharged. Conclusions: This article reports a case of Mycolicibacterium mucogenicum infection in the right back. The Mycolicibacterium mucogenicum was quickly and accurately identified by MALDI-TOF MS, and clinical treatment with antibiotics combined with 5-aminolevulinic acid photodynamic therapy (ALA-PDT) was used. The patient improved and was discharged. I hope that in the future, this study can provide assistance for the clinical diagnosis and treatment of Mycolicibacterium mucogenicum. [ABSTRACT FROM AUTHOR]

Simple Summary: This manuscript reports on an outbreak of joint infections in an organic broiler breeder flock in Austria, associated with two types of bacteria: Staphylococcus aureus and Staphylococcus lentus. The clinical picture of the affected chickens included weakness, lethargy, and difficulty walking, with some birds succumbing to the infection. Detailed examinations revealed severe joint inflammation and damage. Laboratory analyses confirmed the presence of both bacteria, with tests showing resistance to many antibiotics. This study underscores the importance of recognising and understanding less common bacteria like S. lentus, which are not frequently reported but can seriously impact poultry health. The findings highlight the need for thorough bacterial identification in outbreaks to improve disease management and prevention strategies. 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. [ABSTRACT FROM AUTHOR]

This study investigated the occurrence and dynamics of oral Staphylococcus species in patients with orofacial clefts undergoing surgical rehabilitation treatment. Patients (n = 59) were statistically stratified and analyzed (age, gender, types of orofacial clefts, surgical history, and types of previous surgical rehabilitation). Salivary samples were obtained between hospitalization and the return to the specialized medical center. Microbiological diagnosis was performed by classical methods, and MALDI-TOF MS. MRSA strains (SCCmec type II, III, and IV) were characterized by the Decision Tree method. A total of 33 (55.9%) patients showed oral staphylococcal colonization in one, two, or three sampling steps. A high prevalence has been reported for S. aureus (including HA-, MRSA and CA-MRSA), followed by S. saprophyticus, S. epidermidis, S. sciuri, S. haemolyticus, S. lentus, S. arlettae, and S. warneri. The dynamics of oral colonization throughout surgical treatment and medical follow-up may be influenced by (i) imbalances in staphylococcal maintenance, (ii) efficiency of surgical asepsis or break of the aseptic chain, (iii) staphylococcal neocolonization in newly rehabilitated anatomical oral sites, and (iv) total or partial maintenance of staphylococcal species. The highly frequent clinical periodicity in specialized medical and dental centers may contribute to the acquisition of MRSA in these patients. [ABSTRACT FROM AUTHOR]

Helicobacter pylori (H. pylori) infection is a widespread global health issue with a varying prevalence influenced by geography, socioeconomic status, and demographics. In the U.S., the prevalence is lower, though certain groups, such as older adults and immigrants from high-prevalence regions, show higher rates. The decrease in infection rates in developed countries is due to improved sanitation, antibiotics, and healthcare, whereas developing countries continue to experience high rates due to poor living conditions. H. pylori infection can be asymptomatic or cause symptoms like dyspepsia, abdominal pain, bloating, nausea, and loss of appetite. Pathophysiologically, H. pylori contribute to conditions such as gastritis, peptic ulcers, and gastric cancer through mechanisms including urease production and the release of virulence factors, leading to chronic inflammation and an increased cancer risk. Diagnostic methods for H. pylori have progressed significantly. Non-invasive techniques, such as serological assays, stool antigen tests, and urea breath tests, are practical and sensitive. Invasive methods, including endoscopic biopsy and molecular diagnostics, are more definitive but resource intensive. Recent advancements in diagnostic technology, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), biosensor technology, and next-generation sequencing (NGS), promise improved speed, accuracy, and accessibility. These innovations are expected to enhance the detection and management of H. pylori, potentially reducing the global disease burden. This review aims to discuss these diagnostic modalities with a focus on further advances under investigation. [ABSTRACT FROM AUTHOR]

Background: The increased resistance rate of Salmonella to third-generation cephalosporins represented by ceftriaxone (CRO) may result in the failure of the empirical use of third-generation cephalosporins for the treatment of Salmonella infection in children. The present study was conducted to evaluate a novel method for the rapid detection of CRO-resistant Salmonella (CRS). Methods: We introduced the concept of the ratio of optical density (ROD) with and without CRO and combined it with matrix-assisted laser desorption–ionization time-of-flight mass spectrometry (MALDI-TOF MS) to establish a new protocol for the rapid detection of CRS. Results: The optimal incubation time and CRO concentration determined by the model strain test were 2 h and 8 µg/ml, respectively. We then conducted confirmatory tests on 120 clinical strains. According to the receiver operating characteristic curve analysis, the ROD cutoff value for distinguishing CRS and non-CRS strains was 0.818 [area under the curve: 1.000; 95% confidence interval: 0.970–1.000; sensitivity: 100.00%; specificity: 100%; P < 10− 3]. Conclusions: In conclusion, the protocol for the combined ROD and MALDI-TOF MS represents a rapid, accurate, and economical method for the detection of CRS. [ABSTRACT FROM AUTHOR]

Bacillus cereus sensu stricto is a foodborne pathogen that causes food poisoning. Their spore and biofilm-forming abilities persist in various environments and foods. This study investigated the prevalence, virulence, antibiotic resistance, and genetic diversity of B. cereus s. s. strains isolated from various food samples. Of 179 samples, 22.34% were positive for B. cereus s. s., with significantly high detection rates in milk products and raw chicken meat. Forty strains were isolated from positive samples. Matrix-assisted laser desorption ionization/time of flight mass spectrometry analysis revealed nine distinct clusters and multi-locus sequence typing revealed 34 sequence types including 23 novel sequences, demonstrating high genetic diversity among the isolates. PCR analysis revealed that all the strains contained at least one toxin gene, but none contained the cytK gene. Antibiotic resistance tests revealed that all isolates were classified as multidrug-resistant, with high resistance levels, particularly to β-lactam antibiotics and vancomycin, but were susceptible to gentamicin. All isolates showed variations in biofilm formation. This study highlights the significant public health risk due to B. cereus s. s. and underscores the need for stringent monitoring and control measures in food production to manage antimicrobial resistance and ensure food safety. [ABSTRACT FROM AUTHOR]

The rhizosphere are site of plant-microorganism interaction, and have critical impotance in events like nutrient supply, the production of compounds that support plant growth and phytopathogen protection. This study reports 52 bacteria isolated from 5 different plant rhizospheres (Hordeum vulgare, Avena sativa, Cicer arietinum, Helianthus annuus, and Phaseolus vulgaris) of two agricultural districts of Kırşehir (Akçakent and Çicekdağı regions) in Türkiye. Morphological, biochemical, plant growth-promoting characteristics (including production of indole-3-acetic acid, siderophore, nitrogen fixation, and phosphate solubilization) using MALDI-TOF-MS techniques were used to identify the isolates. Among all isolates, 33 isolate were Gram positive and 19 were Gram negative. Furthermore 40% of them were classified as Bacillus species, 10% belonged to Pseudomonas species, 7% were Acinetobacter species, 4% were Pantoeae species, and 39% other species (Blastomonas, Lysinibacillus, Comamonas, Alcaligenes, Bifidobacterium, Aromatoleum). It was seen that 12 isolates fixed nitrogen, 8 isolates produced dissolved inorganic phosphate, 12 isolates produced siderophores, 9 isolates were produced by IAA, and 14 isolates produced HCN during the screening of plant growth promoting properties. It is concluded that the culture of bacteria living in the rhizosphere are essential to promote soil fertility and sustainable agriculture. Therefore, there is a need to identify and use these strains with plant growth promoting properties in agriculture. [ABSTRACT FROM AUTHOR]

The fisheries and aquaculture sectors are growing rapidly, reflecting their importance in meeting the ever-increasing human population's demands for animal protein. Production progress in this sector, however, is challenging as a result of increased deaths from epidemics caused by bacterial infectious diseases in aquaculturally important species. In order to minimize the impact of such diseases, quick and reliable diagnosis of pathogens, timely intervention, and control of the disease are essential to ensure the health of aquaculture and fisheries stocks. Thus, high-throughput proteomics-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used as a rapid, accurate, and species-specific tool for the identification of bacterial fish pathogens and antibiotic resistance. The aim of this article is to review and evaluate the results of nearly three hundred papers published from 2010 to 2023 on the application of MALDI-TOF MS in aquaculture, showing that this method has been increasingly used over the years for the species-level identification and antibiotic resistance of fifty different gram-positive and negative bacterial aquatic pathogens encountered in wild or cultured fish, shellfish, invertebrates, and crustaceans. In addition, the history and general principles of the MALDI-TOF MS method are also mentioned in the article so that fish disease researchers interested in the use of this technology can see all aspects of the topic. [ABSTRACT FROM AUTHOR]

Background: Mediterraneibacter gnavus is a Gram positive, non-sporulated, obligate anaerobe diplococci. It was first described in 1974 by Moore et al. (under the name Ruminococcus gnavus) from faeces and contents of the gastrointestinal tract of humans. It is a relatively common member of the human gut microbiota, nevertheless its role as a pathogenic bacterium has not been completely elucidated yet and it seems to depend on numerous factors, including those of the host. Here we present a case of prosthetic joint infection following total knee arthroplasty by M. gnavus. Case presentation: A 74 years old patient was admitted to the emergency department presenting with acute onset of left knee pain and swelling 20 days after total left knee arthroplasty. Follow-up revealed erythema and oedema without signs of fluctuation or purulent discharge from the surgical wound and elevated inflammatory reactants. Synovial fluid was taken for bacterial culture and antibiotic treatment with ceftazidime and daptomycin was established. Examination of the synovial fluid revealed abundant polymorphonuclear leucocytes, without visualizing bacteria. After four days of incubation, anaerobic culture exhibit growth of small, grey, umbilicated colonies in pure culture on Schaedler agar. The microorganism was identified as R. gnavus by MALDI-TOF (Bruker Daltonics) and M. gnavus by 16S ribosomal bacterial sequencing. The isolated showed susceptibility to the most commonly used anaerobicidal antibiotics except for clindamycin. Surgical treatment and infection source control included DAIR (debridement, antibiotics, and implant retention) and vacuum assisted therapy. The patient was discharged after six weeks with a 3-month course of oral amoxicillin as consolidation therapy. Subsequent follow-up revealed adequate wound healing with no signs of infection. Conclusions: Mediterraneibacter gnavus have been reported as the causal microorganism in a range of human infections, nevertheless its identification remains challenging. Infection of prosthetic joints by anaerobic microorganisms is uncommon and is not considered in its empirical antibiotic treatment, thus, correct and swift identification of anaerobic bacteria in these cases is paramount. [ABSTRACT FROM AUTHOR]

Objectives: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is extensively employed for the identification of filamentous fungi on MALDI Biotyper (Bruker Daltonics) and Vitek MS (biomerieux), but the performance of fungi identification on new EXS2600 (Zybio) is still unknow. Our study aims to evaluate the new EXS2600 system's (Zybio) ability to rapidly identify filamentous fungi and determine its effect on turnaround time (TAT) in our laboratory. Methods: We tested 117 filamentous fungi using two pretreatment methods: the formic acid sandwich (FA-sandwich) and a commercial mold extraction kit (MEK, Zybio). All isolates were confirmed via sequence analysis. Laboratory data were extracted from our laboratory information system over two 9-month periods: pre-EXS (April to December 2022) and post-EXS (April to December 2023), respectively. Results: The total correct identification (at the species, genus, or complex/group level) rate of fungi was high, FA-sandwich (95.73%, 112/117), followed by MEK (94.02%, 110/117). Excluding 6 isolates not in the database, species-level identification accuracy was 92.79% (103/111) for FA-sandwich and 91.89% (102/111) for MEK; genus-level accuracy was 97.29% (108/111) and 96.39% (107/111), respectively. Both methods attained a 100% correct identification rate for Aspergillus, Lichtheimia, Rhizopus Mucor and Talaromyces species, and were able to differentiate between Fusarium verticillioides and Fusarium proliferatum within the Fusarium fujikuroi species complex. Notably, high confidence was observed in the species-level identification of uncommon fungi such as Trichothecium roseum and Geotrichum candidum. The TAT for all positive cultures decreased from pre EXS2600 to post (108.379 VS 102.438, P < 0.05), and the TAT for tissue decreased most (451.538 VS 222.304, P < 0.001). Conclusions: The FA-sandwich method is more efficient and accurate for identifying filamentous fungi with EXS2600 than the MEK. Our study firstly evaluated the performance of fungi identification on EXS2600 and showed it is suitable for clinical microbiology laboratories use. Highlights: (1) The EXS2600 platform demonstrated superior capability in distinguishing between Fusarium species. (2) The data demonstrated that EXS2600 platform provided comparable results and was suitable for filamentous identification. (3) The EXS2600 platform significantly reduced the TAT for the processing of positive cultures in tissue specimens. [ABSTRACT FROM AUTHOR]

The effective identification of bacterial and fungal isolates is essential for microbiological monitoring in environments like speleotherapeutic caves. This study compares MALDI-TOF MS and the OmniLog ID System, two high-throughput culture-based identification methods. MALDI-TOF MS identified 80.0% of bacterial isolates to the species level, while the OmniLog ID System identified 92.9%. However, species-level matches between the methods were only 48.8%, revealing considerable discrepancies. For discrepant results, MALDI-TOF MS matched molecular identification at the genus level in 90.5% of cases, while the OmniLog ID System matched only in 28.6%, demonstrating MALDI-TOF MS's superiority. The OmniLog ID System had difficulties identifying genera from the order Micrococcales. Fungal identification success with MALDI-TOF MS was 30.6% at the species level, potentially improvable with a customised spectral library, compared to the OmniLog ID System's 16.7%. Metagenomic approaches detected around 100 times more microbial taxa than culture-based methods, highlighting human-associated microorganisms, especially Staphylococcus spp. In addition to Staphylococcus spp. and Micrococcus spp. as indicators of cave anthropisation, metagenomics revealed another indicator, Cutibacterium acnes. This study advocates a multi-method approach combining MALDI-TOF MS, the OmniLog ID System, culture-based, and metagenomic analyses for comprehensive microbial identification. Metagenomic sampling on nitrocellulose filters provided superior read quality and microbial representation over liquid sampling, making it preferable for cave air sample collection. [ABSTRACT FROM AUTHOR]

Streptococcus uberis is a common bovine mastitis pathogen in dairy cattle. The rapid identification and characterization of antimicrobial resistance (AMR) in S. uberis plays an important role in its diagnosis, treatment, and prevention. In this study, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify S. uberis and screen for potential AMR biomarkers. Streptococcus uberis strains (n = 220) associated with bovine mastitis in northern Thailand were identified using the conventional microbiological methods and compared with the results obtained from MALDI-TOF MS. Streptococcus uberis isolates were also examined for antimicrobial susceptibility using a microdilution method. Principal component analysis (PCA) and the Mann–Whitney U test were used to analyze the MALDI-TOF mass spectrum of S. uberis and determine the difference between antimicrobial-resistant and -susceptible strains. Using MALDI-TOF MS, 73.18% (161/220) of the sampled isolates were identified as S. uberis, which conformed to the identifications obtained using conventional microbiological methods and PCR. Using PCR, antimicrobial-resistant strains could not be distinguished from antimicrobial-susceptible strains for all three antimicrobial agents, i.e., tetracycline, ceftiofur, and erythromycin. The detection of spectral peaks at 7531.20 m/z and 6804.74 m/z was statistically different between tetracycline- and erythromycin-resistant and susceptible strains, respectively. This study demonstrates a proteomic approach for the diagnosis of bovine mastitis and potentially for the surveillance of AMR among bovine mastitis pathogens. [ABSTRACT FROM AUTHOR]

We used inter-delta typing (IDT) and MALDI-TOF profiling to characterize the genetic and phenotypic diversity of 45 commercially available winemaking Saccharomyces cerevisiae strains and 60 isolates from an organic winemaker from Waipara, New Zealand, as a stratified approach for predicting the commercial potential of indigenous isolates. A total of 35 IDTs were identified from the commercial strains, with another 17 novel types defined among the Waipara isolates. IDT 3 was a common type among strains associated with champagne production, and the only type in commercial strains also observed in indigenous isolates. MALDI-TOF MS also demonstrated its potential in S. cerevisiae typing, particularly when the high-mass region (m/z 2000–20,000) was used, with most indigenous strains from each of two fermentation systems distinguished. Furthermore, the comparison between commercial strains and indigenous isolates assigned to IDT 3 revealed a correlation between the low-mass data (m/z 500–4000) analysis and the recommended use of commercial winemaking strains. Both IDT and MALDI-TOF analyses offer useful insights into the genotypic and phenotypic diversity of S. cerevisiae, with MALDI-TOF offering potential advantages for the prediction of applications for novel, locally isolated strains that may be valuable for product development and diversification. [ABSTRACT FROM AUTHOR]

This case report describes the case of a female infant hospitalized for severe pneumonia. During the treatment process, various antibiotics are used to treat and prevent further infection. The child had a weak physical condition, combined with neuroblastoma, paraneoplastic syndrome, and low immune function, leading to Tsukamurella tyrosinosolvens infection. The treatment was eventually abandoned owing to poor prognosis. This study aims to through the medium, dyeing, electron microscope observation, 16s rRNA and high-throughput sequencing investigated the morphological characteristics, staining properties, electron microscope morphology, antibiotic resistance, and genomic characteristics of Tsukamurella tyrosinosolvens. The aim of the study is to provide data reference for clinical laboratory staff in bacteria identification research, and to provide relevant help for clinicians in diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Purpose: The World Health Organization has identified Klebsiella pneumoniae (KP) as a significant threat to global public health. The rising threat of carbapenem-resistant Klebsiella pneumoniae (CRKP) leads to prolonged hospital stays and higher medical costs, necessitating faster diagnostic methods. Traditional antibiotic susceptibility testing (AST) methods demand at least 4 days, requiring 3 days on average for culturing and isolating the bacteria and identifying the species using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), plus an extra day for interpreting AST results. This lengthy process makes traditional methods too slow for urgent clinical situations requiring rapid decision-making, potentially hindering prompt treatment decisions, especially for fast-spreading infections such as those caused by CRKP. This research leverages a cutting-edge diagnostic method that utilizes an artificial intelligence-clinical decision support system (AI-CDSS). It incorporates machine learning algorithms for the swift and precise detection of carbapenem-resistant and colistin-resistant strains. Patients and Methods: We selected 4307 KP samples out of a total of 52,827 bacterial samples due to concerns about multi-drug resistance using MALDI-TOF MS and Vitek-2 systems for AST. It involved thorough data preprocessing, feature extraction, and machine learning model training fine-tuned with GridSearchCV and 5-fold cross-validation, resulting in high predictive accuracy, as demonstrated by the receiver operating characteristic and area under the curve (AUC) scores, laying the groundwork for our AI-CDSS. Results: MALDI-TOF MS analysis revealed distinct intensity profiles differentiating CRKP and susceptible strains, as well as colistin-resistant Klebsiella pneumoniae (CoRKP) and susceptible strains. The Random Forest Classifier demonstrated superior discriminatory power, with an AUC of 0.96 for detecting CRKP and 0.98 for detecting CoRKP. Conclusion: Integrating MALDI-TOF MS with machine learning in an AI-CDSS has greatly expedited the detection of KP resistance by approximately 1 day. This system offers timely guidance, potentially enhancing clinical decision-making and improving treatment outcomes for KP infections. [ABSTRACT FROM AUTHOR]

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

In this study, we report the first isolation of Hanseniaspora opuntiae obtained from four pregnant women in Brazil. Clinical isolates were obtained from four samples taken between 35 and 37 gestational weeks, as part of the routine antenatal care for maternal colonization screening for Streptococcus agalactiae group B. The patients were immunocompetent, with two of them diagnosed with gestational diabetes mellitus. Species identification was performed by MALDI-TOF MS and rDNA sequencing. While Hanseniaspora species have not traditionally been considered a typical opportunist pathogen, our findings emphasize the importance of investigating and screening for Hanseniaspora in pregnant populations, highlighting H. opuntiae as a potential agent of human infections. [ABSTRACT FROM AUTHOR]

The elucidation of disease pathogenesis can be achieved by analyzing the low-abundance phosphopeptides in organisms. Herein, we developed a novel and easy-to-prepare polymer-coated nanomaterial. By improving the hydrophilicity and spatial conformation of the material, we effectively enhanced the adsorption of phosphopeptides and demonstrated excellent enrichment properties. The material was able to successfully enrich the phosphopeptides in only 1 min. Meanwhile, the material has high selectivity (1:2000), good loading capacity (100 μg/mg), excellent sensitivity (0.5 fmol), and great acid and alkali resistance. In addition, the material was applied to real samples, and 70 phosphopeptides were enriched from the serum of Parkinson's disease (PD) patients and 67 phosphopeptides were enriched from the serum of normal controls. Sequences Logo showed that PD is probably associated with threonine, glutamate, serine, and glutamine. Finally, gene ontology (GO) analysis was performed on phosphopeptides enriched in PD patients' serum. The results showed that PD patients expressed abnormal expression of the cholesterol metabolic process and cell–matrix adhesion in the biological process (BP), endoplasmic reticulum and lipoprotein in the cellular component (CC), and heparin-binding, lipid-binding, and receptor-binding in the molecular function (MF) as compared with normal individuals. All the experiments indicate that the nanomaterials have great potential in proteomics studies. [ABSTRACT FROM AUTHOR]

Healthy cattle, sheep, and goats can be reservoirs for gastrointestinal pathogenic fecal enterococci, some of which could be multidrug-resistant to antimicrobials. The objective of this study was to determine the prevalence and diversity of Enterococcus species in healthy sheep, goat, and cattle carcasses, as well as to analyze the antimicrobial resistance phenotype/genotype and the virulence gene content. During 2019–2020, carcass surface samples were collected from 150 ruminants in a slaughterhouse. A total of 90 enterococci, comprising five species, were obtained. The overall prevalence of enterococci was found to be 60%, out of which 37.7% were identified as Enterococcus (E.) hirae, 33.3% as E. casseliflavus, 15.5% as E. faecium, 12.2% as E. faecalis, and 1.1% as E. gallinarum. Virulence-associated genes of efaA (12.2%) were commonly observed in the Enterococcus isolates, followed by gelE (3.3%), asaI (3.3%), and ace (2.2%). High resistance to quinupristin-dalfopristin (28.8%), tetracycline (21.1%), ampicillin (20%), and rifampin (15.5%) was found in two, four, four, and five of the Enterococcus species group, respectively. The resistance of Enterococcus isolates to 11 antibiotic groups was determined and multidrug resistant (MDR) strains were found in 18.8% of Enterococcus isolates. Characteristic resistance genes were identified by PCR with an incidence of 6.6%, 2.2%, 1.1%, 1.1%, 1.1%, and 1.1% for the tetM, ermB, ermA, aac(6ʹ)Ie-aph(2")-la, VanC1, and VanC2 genes in Enterococcus isolates, respectively. Efflux pump genes causing multidrug resistance were detected in Enterococcus isolates (34.4%). The results showed that there were enterococci in the slaughterhouse with a number of genes linked to virulence that could be harmful to human health. [ABSTRACT FROM AUTHOR]

Cervical cancer screening is an effective mean of preventing cervical cancer, however, there are limitations of the current screening methods. In this study, an automated high throughput paired box-1 (PAX1) methylation detection method was designed by employing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which analyzed 88 PAX1 methylation sites. In total, 188 subjects were enrolled in the study. The diagnostic efficacy of various PAX1 methylation sites for detecting cervical intraepithelial neoplasia II or more severe lesions was analyzed and compared with current cervical cancer screening methods. A predictive model was constructed using logistic regression. Five sites with the largest area under receiver operating characteristic curve values were GRCh38/hg38, chr20: X21705687, X21705946, X21706427, X21706285 and X21706637. PAX1 methylation method had higher diagnostic efficacy than high risk human papillomavirus (hr-HPV) and ThinPrep cytology tests. A predictive model combining hr-HPV and PAX1 methylation sites was constructed as follows: prediction index = -5.993 + 15.211 × X21705687 + 7.890 × X21706427 + 1.846 × HPV16/18 (1,0) + 1.821 × other hr-HPV (1,0). Area under the curve was 0.868 and sensitivity and specificity were 0.863 and 0.756, respectively. Analysis of PAX1 promoter methylation alone or in combination with hr-HPV was a promising approach for cervical cancer screening. The study was registered in Chinese Clinical Trial Registry (registration number: ChiCTR2000029231, http://www.chictr.org.cn/index.aspx). [ABSTRACT FROM AUTHOR]

In the early 2000s, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) emerged as a performant and relevant tool for identifying micro-organisms. Since then, it has become practically essential for identifying bacteria in microbiological diagnostic laboratories. In the last decade, it was successfully applied for arthropod identification, allowing researchers to distinguish vectors from non-vectors of infectious diseases. However, identification failures are not rare, hampering its wide use. Failure is generally attributed either to the absence of respective counter species MS spectra in the database or to the insufficient quality of query MS spectra (i.e., lower intensity and diversity of MS peaks detected). To avoid matching errors due to non-compliant spectra, the development of a strategy for detecting and excluding outlier MS profiles became compulsory. To this end, we created MSProfileR, an R package leading to a bioinformatics tool through a simple installation, integrating a control quality system of MS spectra and an analysis pipeline including peak detection and MS spectra comparisons. MSProfileR can also add metadata concerning the sample that the spectra are derived from. MSProfileR has been developed in the R environment and offers a user-friendly web interface using the R Shiny framework. It is available on Microsoft Windows as a web browser application by simple navigation using the link of the package on Github v.3.10.0. MSProfileR is therefore accessible to non-computer specialists and is freely available to the scientific community. We evaluated MSProfileR using two datasets including exclusively MS spectra from arthropods. In addition to coherent sample classification, outlier MS spectra were detected in each dataset confirming the value of MSProfileR. [ABSTRACT FROM AUTHOR]

Plant growth-promoting rhizobacteria (PGPR) support plant growth through direct and indirect mechanisms. To investigate PGPR strains that support plant growth, 21 bacterial isolates, mostly Bacillus ssp. and Pseudomonas ssp., were isolated from different rhizospheric soils of sunflowers in Kırşehir districts in 2020. All isolates were characterized morphologically, biochemically by screening under in vitro conditions for plant growth-promoting properties such as nitrogen fixation, IAA (indoleacetic acid) production, siderophore production, HCN (hydrogen cyanide) production, inorganic phosphate solubility. It was also screened for extracellular enzyme production and antifungal activity against Fusarium oxysporum. Among the 21 isolates, 3 isolates (MH-35-4, MH-49-4, MH-64-3) fixed nitrogen, 2 isolates (MH-59-6, MH-64-3), produced siderophores, 8 isolates (MH-35-4, MH-35-6, MH-54-3, MH-54-4, MH-59-1, MH-59-2, MH-59-4, MH-59-8) produced HCN, 6 isolates (MH-35-6, MH-54-4, MH-59-1, MH- 59-2, MH-59-4, MH-59-8) produced IAA, and 7 isolates (MH-35-4, MH-35-6, MH-59-1, MH-59-2, MH-59-4, MH-59-8, MH-64-3) solubilized inorganic phosphate. Additionally, only 2 isolates (MH-54-3, MH-54-4) were positive amylase tests, 8 isolates (MH-35-6, MH-54-4, MH-59-1, MH-59-2, MH-59-4, MH-59-6, MH-59-7, MH-59-8) were positive citrate tests, 8 isolates (MH-35-1, MH-35-4, MH-35-7, MH-49-4, MH-54-4, MH-59-6, MH-59-7, MH-64-3) were positive protease tests, and 6 isolates (MH-35-1, MH-35-3, MH-35-7, MH-54-3, MH-54-4, MH-59-7) were positive gelatin hydrolysis tests. Among 21 isolates, 38% were determined as hydrogen cyanide producers, 10% as siderophore producers, 29% IAA producers, 33% as phosphate solubilizers and 14% as nitrogen fixers. Isolate MH-35-6 showed the highest antifungal activity against Fusarium oxysporum with an inhibition rate of 53.57%. This was followed by isolates MH-54-1 (51.19%), MH-54-3 (47.61%) and MH-59-2 (38.09%), respectively. Therefore, our study reveals that bacteria that promote plant growth in sunflowers can be used to increase crop yield and as a biocontrol agent. [ABSTRACT FROM AUTHOR]

Kars Kashar cheese is an artisanal pasta-filata type cheese and geographically marked in Eastern Anatolia of Turkey. The aims of this research were to determine for the first time thermophilic lactic acid bacteria (LAB) of Kars Kashar cheese and characterize the technological properties of obtained isolates. In our research, a number of 15 samples of whey were collected from the different villages in Kars. These samples were incubated at 45 °C and used as the source material for isolating thermophilic LAB. A total of 250 colonies were isolated from thermophilic whey, and 217 of them were determined to be presumptive LAB based on their Gram staining and catalase test. A total of 170 isolates were characterized by their phenotypic properties and identified using the MALDI-TOF mass spectrometry method. Phenotypic identification of isolates displayed that Enterococcus and Lactobacillus were the predominant microbiota. According to MALDI-TOF MS identification, 89 isolates were identified as Enterococcus (52.35%), 57 isolates as Lactobacillus (33.53%), 23 isolates as Streptococcus (13.53%), and one isolate as Lactococcus (0.59%). All thermophilic LAB isolates were successfully identified to the species level and it has been observed that MALDI-TOF MS can be successfully used for the identification of selected LAB. The acidification and proteolytic activities of the isolated thermophilic LAB were examined, and the isolates designated for use as starter cultures were also genotypically defined. [ABSTRACT FROM AUTHOR]

In order to obtain the best mass spectrometry identification results for using the most appropriate methods in clinical practice, we explore the optimal pretreatment methods for different species and morphologies of filamentous fungi. 98 fungal strains were treated with formic acid sandwich method, dispersion method, extraction method, and other methods using a medium element mass spectrometer (EXS3000) as a platform. Each strain had three targets, and the identification rates and confidence differences under different pre-treatment methods were compared to evaluate the identification effects of these methods. The mass spectrometry identification rates of 98 filamentous fungi obtained after pre-treatment with formic acid sandwich method, dispersion method, and extraction method were 85.71%, 82.65%, and 75.51%, respectively. The identification rate of the formic acid sandwich method was significantly higher than the other two methods (P < 0 005) has the best identification ability and the obtained confidence is also higher than the other two methods. The use of formic acid sandwich method for mass spectrometry identification of filamentous fungi can achieve ideal identification results, which is suitable for mass spectrometry identification of filamentous fungi in conventional laboratories. [ABSTRACT FROM AUTHOR]

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

Our study aimed to identify markers of enterococci's virulence potential by evaluating the properties of strains of different sites of isolation. Enterococcal strains were isolated as commensals from faeces and as invasive strains from the urine and blood of patients from the University Clinical Centre, Gdańsk, Poland. Changes in monocytes' susceptibility to the cytotoxic activity of isolates of different origins and their adherence to biofilm were evaluated using a flow cytometer. The bacterial protein profile was estimated by matrix assisted laser desorption ionization-time of flight mass spectrometer. The cytotoxicity of biofilm and monocytes' adherence to it were the most accurate factors in predicting the prevalence of the strain in the specific niche. Additionally, a bacterial protein with mass-to-charge ratio (m / z) 5000 was found to be responsible for the increased bacterial cytotoxicity, while monocytes' decreased adherence to biofilm was linked with the presence of proteins either with m / z 3330 or 2435. The results illustrate that monocytes' reaction when exposed to the bacterial biofilm can be used as an estimator of pathogens' virulence potential. The observed differences in monocytes' response are explainable by the bacterial proteins' profile. Additionally, the results indicate that the features of both bacteria and monocytes impact the outcome of the infection. [ABSTRACT FROM AUTHOR]

Periprosthetic joint infection (PJI) is a rare but most vital complication after joint arthroplasty and requires a revision surgery. Synovial fluid analysis is essential in diagnosis of the PJI, and conventional and molecular microbiologic investigations may help in determining the cause of the infection. With this unusual case, we aimed to present the second instance in the literature of PJI of the knee caused by Streptococcus dysgalactiae subspecies dysgalactiae (SDSD). S. dysgalactiae PJI in the literature are commonly Streptococcus dysgalactiae subspecies equisimilis (SDSE), and SDSD mostly infects animals. A farmer with comorbid illnesses who works with cattle and sheep experienced periprosthetic knee joint infection caused by SDSD. Surgical excisional debridement with open washing, decompression, and liner exchange were performed. The identification of the bacteria was done with VITEK MS as SDSD. After 1-year follow-up, the patient has fully recovered without recurrence. [ABSTRACT FROM AUTHOR]

Rationale: The gut microbiota may play an important role in the development and functioning of the mammalian central nervous system. The assumption of the experiment was to prove that the use of probiotic bacterial strains in the diet of mice modifies the expression of brain proteins involved in metabolic and immunological processes. Objectives and results: Albino Swiss mice were administered with Bifidobacterium longum Rosell®-175 or Lactobacillus rhamnosus JB-1 every 24 h for 28 days. Protein maps were prepared from hippocampal homogenates of euthanized mice. Selected proteins that were statistically significant were purified and concentrated and identified using MALDI-TOF mass spectrometry. Among the analysed samples, 13 proteins were identified. The mean volumes of calcyon, secreted frizzled-associated protein 3, and catalase in the hippocampus of mice from both experimental groups were statistically significantly higher than in the control group. In mice supplemented with Lactobacillus rhamnosus JB-1, a lower mean volume of fragrance binding protein 2, shadow of prion protein, and glycine receptor α4 subunit was observed compared to the control. Conclusion: The psychobiotics Bifidobacterium longum Rosell®-175 and Lactobacillus rhamnosus JB-1enhances expression of proteins involved in the activation and maturation of nerve cells, as well as myelination and homeostatic regulation of neurogenesis in mice. The tested psychobiotics cause a decrease in the expression of proteins associated with CNS development and in synaptic transmission, thereby reducing the capacity for communication between nerve cells. The results of the study indicate that psychobiotic bacteria can be used in auxiliary treatment of neurological disorders. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) in pathogens important to aquatic animal health is of increasing concern but vastly understudied. Antimicrobial therapy is used to both treat and prevent bacterial disease in fish and is critical for a viable aquaculture industry and for maintenance of wild fish populations. Unfortunately, phenotypic antimicrobial susceptibility testing is technically difficult for bacteria recovered from aquatic animal hosts resulting in challenges in resistance monitoring using traditional methods. Whole-genome sequencing provides an appealing methodology for investigation of putative resistance. As part of the ongoing efforts of the FDA CVM Vet-LIRN to monitor AMR, source laboratories cultured and preliminarily identified pathogenic bacteria isolated from various fish species collected in 2019 from across the United States. Sixty-one bacterial isolates were evaluated using whole-genome sequencing. We present here the assembled draft genomes, AMR genes, predicted resistance phenotypes, and virulence factors of the 61 isolates and discuss concurrence of the identifications made by source laboratories using matrix-assisted laser desorption/time-of-flight mass spectrometry. [ABSTRACT FROM AUTHOR]

The emerging pathogen Trichophyton indotineae, often resistant to terbinafine (TRB), is known to cause severe dermatophytoses such as tinea corporis and tinea cruris. In order to achieve successful treatment for these infections, insight in the resistance profile of T. indotineae strains and rapid, reliable identification is necessary. In this research, a screening medium was tested on T. indotineae strains (n = 20) as an indication tool of TRB resistance. The obtained results were confirmed by antifungal susceptibility testing (AST) for TRB following the in vitro broth microdilution reference method. Additionally, AST was performed for eight other antifungal drugs: fluconazole, itraconazole, voriconazole, ketoconazole, griseofulvin, ciclopirox olamine, naftifine and amorolfine. Forty-five percent of the strains were confirmed to be resistant to terbinafine. The TRB resistant strains showed elevated minimal inhibitory concentration values for naftifine and amorolfine as well. DNA sequencing of the squalene epoxidase-encoding gene showed that TRB resistance was a consequence of missense point mutations in this gene, which led to amino acid substitutions F397L or L393F. MALDI-TOF MS was used as a quick, accurate identification tool for T. indotineae, as it can be challenging to distinguish it from closely related species such as Trichophyton mentagrophytes or Trichophyton interdigitale using morphological characteristics. While MALDI-TOF MS could reliably identify ≥ 95% of the T. indotineae strains (depending on the spectral library), it could not be used to successfully distinguish TRB susceptible from TRB resistant strains. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria living in marine waters have evolved peculiar adaptation strategies to deal with the numerous stress conditions that characterize aquatic environments. Among the multiple mechanisms for efficient adaptation, these bacteria typically exhibit chemical modifications in the structure of the lipopolysaccharide (LPS), which is a fundamental component of their outer membrane. In particular, the glycolipid anchor to the membrane of marine bacteria LPSs, i.e. the lipid A, frequently shows unusual chemical structures, which are reflected in equally singular immunological properties with potential applications as immune adjuvants or anti-sepsis drugs. In this work, we determined the chemical structure of the lipid A from Cellulophaga pacifica KMM 3664T isolated from the Sea of Japan. This bacterium showed to produce a heterogeneous mixture of lipid A molecules that mainly display five acyl chains and carry a single phosphate and a D-mannose disaccharide on the glucosamine backbone. Furthermore, we proved that C. pacifica KMM 3664T LPS acts as a weaker activator of Toll-like receptor 4 (TLR4) compared to the prototypical enterobacterial Salmonella typhimurium LPS. Our results are relevant to the future development of novel vaccine adjuvants and immunomodulators inspired by marine LPS chemistry. [ABSTRACT FROM AUTHOR]

The frequent mutations in SARS-CoV-2 significantly increase the virus's pathogenicity and transmissibility while also diminishing the effectiveness of vaccines. Consequently, assays capable of rapidly and simultaneously identifying multiple SARS-CoV-2 variants are essential for large-scale applications that aim to monitor the evolution of the virus. In this work, we propose a method combining duplex-specific nuclease (DSN)-assisted cyclic amplification with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) detection, enabling the simultaneous identification of multiple SARS-CoV-2 variants at high-throughput. Due to the high specificity of DSN, single-base mutations can be resolved by the method. With ultra-sensitive detection by MALDI-TOF MS, a limit of detection of 100 pM viral RNA fragment was demonstrated. The assay was used for simultaneous identification and typing of SARS-CoV-2 Alpha, Beta, and Delta variants. The whole assay can be accomplished within 3 h, and the amplification is performed under constant temperature, making the technique simple in operation and efficient. It is also feasible to extend the technique to the detection of many other variants of the virus. We expect that the method can add value to the rapid screening of viral variants and can play an important role in pandemic control. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is routinely used as a rapid and cost-effective method for pathogen identification in clinical settings. In comparison, its performance in other microbiological fields, such as environmental microbiology, is still being tested, although isolates of environmental microbes are essential for in-depth in vivo studies of their biology, including biotechnological applications. We investigated the applicability of MALDI-TOF MS for the identification of bacterial isolates from a highly oligotrophic environment — Dinaric Karst caves, which likely harbor specific microorganisms. We cultured bacteria from the shell surface of the endemic mussel Congeria jalzici, one of the three known cave mussels in the world that lives in the Dinaric karst underground. The bacterial isolates were obtained by swabbing the shell surface of mussels living in microhabitats with different amounts of water: 10 air-exposed mussels, 10 submerged mussels, and 10 mussels in the hygropetric zone. A collection of 87 pure culture isolates was obtained, mostly belonging to the phylum Bacillota (72%), followed by Pseudomonadota (16%), Actinomycetota (11%), and Bacteroidota (1%). We compared the results of MALDI-TOF MS identification (Bruker databases DB-5989 and version 11, v11) with the results of 16S rDNA-based phylogenetic analysis, a standard procedure for bacterial identification. Identification to the genus level based on 16S rDNA was possible for all isolates and clearly outperformed the results from MALDI-TOF MS, although the updated MALDI-TOF MS database v11 gave better results than the DB-5989 version (85% versus 62%). However, identification to the species-level by 16S rDNA sequencing was achieved for only 17% of isolates, compared with 14% and 40% for the MALDI-TOF MS databases DB-5989 and v11 database, respectively. In conclusion, our results suggest that continued enrichment of MALDI-TOF MS libraries will result with this method soon becoming a rapid, accurate, and efficient tool for assessing the diversity of culturable bacteria from different environmental niches. [ABSTRACT FROM AUTHOR]

This study investigates the biodegradation of Reactive Red 141 (RR 141), an azo dye prevalent in the textile industry, by bacteria isolated from activated sludge in a textile effluent treatment plant. RR 141, characterized by nitrogen–nitrogen double bonds (-N=N-), contributes to environmental issues when improperly disposed of in textile effluents, leading to reduced oxygen levels in water bodies, diminished sunlight penetration, and the formation of potentially carcinogenic and mutagenic aromatic amines. This research focuses on identifying bacteria from activated sludge with the potential to decolorize RR 141. Microbiological identification employs MALDI-TOF-MS, known for its precision and rapid identification of environmental bacteria, enhancing treatment efficiency. Results highlight Bacillus thuringiensis and Kosakonia radicincitans as the most promising strains for RR 141 decolorization. Analysis of micro-organisms in activated sludge and database exploration suggests a correlation between these strains and the decolorization process. It is worth noting that this is the first report on the potential use of K. radicincitans for azo dye decolorization. Three distinct culture media—BHI, MSG, and MS—were assessed to investigate their impact on RR 141 decolorization. Notably, BHI and MSG media, incorporating a carbon source, facilitated the bacterial growth of both tested species (B. thuringiensis and K. radicincitans), a phenomenon absent in the MS medium. This observation suggests that the bacteria exhibit limited capability to utilize RR 141 dye as a carbon source, pointing towards the influence of the culture medium on the discoloration process. The study evaluates performance kinetics, decolorization capacity through UV-VIS spectrophotometry, potential degradation pathways via HPLC-MS analysis, phytotoxicity, and enzymatic activity identification. B. thuringiensis and K. radicincitans exhibit potential in decolorizing RR141, with 38% and 26% removal individually in 120 h. As a consortium, they achieved 36% removal in 12 h, primarily through biosorption rather than biodegradation, as indicated by HPLC-MS analyses. In conclusion, the research emphasizes the importance of exploring bacteria from activated sludge to optimize azo dye degradation in textile effluents. B. thuringiensis and K. radicincitans emerge as promising candidates for bioremediation, and the application of MALDI-TOF-MS proves invaluable for rapid and precise bacteria identification. [ABSTRACT FROM AUTHOR]

Vagococcus fluvialis infection is rare in humans, and there is limited research on the clinical manifestations and antimicrobial susceptibility testing of Vagococcus fluvialis infection. Here, We isolated Vagococcus fluvialis from the urine samples of bladder cancer patients at Hunan Provincial People's Hospital, and it is the first reported case of Vagococcus fluvialis isolated from the urine. The fully automated microbial identification system and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified the bacterium as Vagococcus fluvialis with a confidence level of 99.9%. The VITEK-2Compact fully automated microbial susceptibility analysis system indicated that it was most sensitive to tigecycline, vancomycin, quinupristin/dalfopristin, linezolid, and showed moderate sensitivity to erythromycin, levofloxacin, ciprofloxacin, ampicillin/sulbactam, and tetracycline. Additionally, it exhibited synergy when combined with high-level gentamicin and vancomycin, showing sensitivity. However, it displayed poor activity against penicillin and furanth. According to our knowledge, this is the first study to isolate and identify Vagococcus fluvialis from the urine of bladder cancer patients and the systematically reviewed other reported Vagococcus infections on human, which provide an experimental basis for guiding the rational use of drugs in the clinical treatment and diagnose of Vagococcus fluvialis infection and related pathogenic mechanism research. Meanwhile, we have systematically reviewed other reported. [ABSTRACT FROM AUTHOR]

Objective: A comprehensive strategy for microbial identification and contamination investigation during sterile drug manufacturing was innovatively established in this study, mainly based on MALDI-TOF MS for the identification and complemented by sequencing technology on strain typing. Methods: It was implemented to monitor the bacterial contamination of a sterile drug manufacturing facility, including its bacterial distribution features and patterns. In three months, two hundred ninety-two samples were collected covering multiple critical components of raw materials, personnel, environment, and production water. Results: Based on our strategy, the bacterial profile across the production process was determined: 241/292 bacterial identities were obtained, and Staphylococcus spp. (40.25%), Micrococcus spp.(11.20%), Bacillus spp. (8.30%), Actinobacteria (5.81%), and Paenibacillus spp. (4.56%) are shown to be the most dominant microbial contaminants. With 75.8% species-level and 95.4% genus-level identification capability, MALDI-TOF MS was promising to be a first-line tool for environmental monitoring routine. Furthermore, to determine the source of the most frequently occurring Staphylococcus cohnii, which evidenced a widespread presence in the entire process, a more discriminating S. cohnii whole-genome SNP typing method was developed to track the transmission routes. Phylogenetic analysis based on SNP results indicated critical environment contamination is highly relevant to personnel flow in this case. The strain typing results provide robust and accurate information for the following risk assessment step and support effective preventive and corrective measures. Conclusion: In general, the strategy presented in this research will facilitate the development of improved production and environmental control processes for the pharmaceutical industry, and give insights about how to provide more sound and reliable evidence for the optimization of its control program. [ABSTRACT FROM AUTHOR]

Objectives: Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a potent tool for detecting drug resistance in tuberculosis (TB); however, concerns about its reliability have been raised. In this study, we assessed the reliability of MassARRAY (Sequenom, Inc.), which is a MALDI-TOF MS-based method, by comparing it to the wellestablished GeneXpert assay (Cepheid) as a reference method. Methods: A retrospective study was conducted using laboratory data retrieved from Henan Chest Hospital (Zhengzhou, China). To ensure a rigorous evaluation, we adopted a comprehensive assessment approach by integrating multiple outcomes of the Xpert assay across various specimen types. Results: Among the 170 enrolled TB cases, MassARRAY demonstrated significantly higher sensitivity (85.88%, 146 of 170) compared to the Xpert assay (76.62%, 118 of 154) in TB diagnosis (p < 0.05). The concordance in detecting rifampicin resistance between MassARRAY and the combined outcomes of the Xpert assay was 90%, while it was 97.37% (37 of 38) among smear-positive cases and 89.06% (57 of 64) among culture-positive cases. When compared to the phenotypic susceptibility outcomes of the 12 included drugs, consistency rates of 81.8 to 93.9% were obtained, with 87.9% for multiple drug resistance (MDR) identification. Conclusion: MassARRAY demonstrates high reliability in detecting rifampicin resistance, and these findings may offer a reasonable basis for extrapolation to other drugs included in the test panel. [ABSTRACT FROM AUTHOR]

Background: Pseudomonas nitroreducens is a non-fermenting, gram-negative, rod-shaped bacterium commonly inhabiting soil, particularly soil contaminated with oil brine. To our knowledge, no cases of human infection with P. nitroreducens have been previously reported. Here, we present the first documented case of cholangitis caused by P. nitroreducens in a patient with bacteremia. Case presentation: A 46-year-old Japanese man with an advanced pancreatic neuroendocrine tumor was hospitalized with fever and chills. Four days before admission, the patient developed right upper abdominal pain. Two days later, he also experienced fever and chills. Endoscopic retrograde cholangiopancreatography was performed on the day of admission, and the patient was diagnosed as having cholangitis associated with stent dysfunction. Gram-negative rods were isolated from blood cultures, but attempts to identify the bacteria using VITEK2 and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with VITEK MS ver. 4.7.1 (bioMérieux Japan Co. Ltd., Tokyo, Japan) were unsuccessful. Finally, the organism was identified as P. nitroreducens using MALDI-TOF MS with a MALDI Biotyper (Bruker Daltonics Co., Ltd., Billerica, MA, USA) and 16 S ribosomal RNA sequencing. Despite thorough interviews with the patient, he denied any exposure to contaminated soil. The patient was treated with intravenous cefepime and oral ciprofloxacin for 16 days based on susceptibility results, achieving a good therapeutic outcome. At the outpatient follow-up on day 28, the patient was in good general condition. Conclusions: This is the first reported human case of cholangitis with bloodstream infection caused by P. nitroreducens. This report provides clinicians with novel insights into the clinical manifestations and diagnostic methods necessary for the accurate diagnosis of P. nitroreducens, along with guidance on treatment. [ABSTRACT FROM AUTHOR]

The identification of microorganisms at the species level has always constituted a diagnostic challenge for clinical microbiology laboratories. The aim of the present study has been the evaluation in a real-time assay of the performance of Autobio in comparison with the Bruker mass spectrometry system for the identification of bacteria and yeasts. A total of 535 bacteria and yeast were tested in parallel with the two systems by direct smear or fast formic acid extraction for bacteria and yeasts, respectively. Discordant results were verified by 16S, ITS rRNA or specific gene sequencing. Beyond giving comparable results for bacteria with respect to the MBT smart system, Autof MS2600 mass spectrometer provided excellent accuracy for the identification of yeast species of clinical interest. [ABSTRACT FROM AUTHOR]

Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in the last decade has revealed itself as a valid support in the workflow in the clinical microbiology laboratory for the identification of bacteria and fungi, demonstrating high reliability and effectiveness in this application. Its use has reduced, by 24 h, the time to obtain a microbiological diagnosis compared to conventional biochemical automatic systems. MALDI-TOF MS application to the detection of pathogens directly in clinical samples was proposed but requires a deeper investigation, whereas its application to positive blood cultures for the identification of microorganisms and the detection of antimicrobial resistance are now the most useful applications. Thanks to its rapidity, accuracy, and low price in reagents and consumables, MALDI-TOF MS has also been applied to different fields of clinical microbiology, such as the detection of antibiotic susceptibility/resistance biomarkers, the identification of aminoacidic sequences and the chemical structure of protein terminal groups, and as an emerging method in microbial typing. Some of these applications are waiting for an extensive evaluation before confirming a transfer to the routine. MALDI-TOF MS has not yet been used for the routine identification of parasites; nevertheless, studies have been reported in the last few years on its use in the identification of intestinal protozoa, Plasmodium falciparum, or ectoparasites. Innovative applications of MALDI-TOF MS to viruses' identification were also reported, seeking further studies before adapting this tool to the virus's diagnostic. This mini-review is focused on the MALDI-TOF MS application in the real life of the diagnostic microbiology laboratory. [ABSTRACT FROM AUTHOR]

Non-aureus staphylococci and mammaliicocci (NASM) are associated with bovine mastitis and increased milk somatic cell count (SCC) but their relationships with mammary gland health at the species level are not clearly defined. Regional differences have also been reported in their specific prevalence. The implementation of MALDI-TOF MS in milk microbiology is generating large and dependable datasets with the potential of providing useful epidemiological information. We present the retrospective analysis of 17,213 milk samples sent to our laboratory in 2021–2022, including 13,146 quarter samples from cows with subclinical (SCM) or clinical mastitis (CM) from 104 farms, and 4,067 composite herd survey (HS) samples from 21 farms. NASM were isolated from 21.12% of SCM, 11.49% of CM, and 15.59% of HS milk samples. The three most frequently identified NASM in SCM milk were Staphylococcus chromogenes (33.33%), S. haemolyticus (26.07%), and S. epidermidis (10.65%); together with S. microti and S. hyicus, these species were significantly more prevalent in quarters with SCM (p < 0.05). The three most frequently identified NASM in CM milk were S. chromogenes (31.69%), S. haemolyticus (21.42%), and Mammaliicoccus sciuri (18.38%), although no significant associations were found between these NASM species and CM. The three most frequently identified NASM in HS milk were S. chromogenes (44.49%), S. epidermidis (17.84%), and S. haemolyticus (17.23%), with S. chromogenes being isolated in all the farms sending HS milk (100%). In conclusion, this retrospective study provides the first information on the NASM species isolated from cow milk in Italy, expanding our knowledge on the epidemiology of NASM at the species level and providing further insights into their relationships with mammary gland health in modern dairy farms. [ABSTRACT FROM AUTHOR]

Semen samples contain bacteria originating from the animal urogenital tract, environment, and/or contamination during semen processing, negatively affecting sperm quality by producing toxins and/or competing for nutrients in extenders. The aims of this study were to evaluate two methods of Single-layer centrifuges (SLC), high and low density colloid, as a method for bacterial removal from bull semen, and to evaluate sperm quality after treatment. In total, semen samples from 20 bulls (3 ejaculates per bull) were used in this study. Bacterial reduction was evaluated by bacterial quantification (colony forming unit - CFU/mL) while bacterial identification was performed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) after culturing bacteria on blood agar. Sperm motility parameters were evaluated by Computer Assisted Sperm Analyses (CASA), and sperm chromatin structure assay (SCSA) by Flow cytometry. Both, High and Low density SLC reduced number of bacteria significantly (p < 0.001) compared with control. The difference in bacterial count between High and Low SLC was also significant (p < 0.001). Furthermore, High density SLC was successful in removing almost all Bacillus and Proteus spp. Most CASA parameters were significantly improved after both treatments (p < 0.001, p < 0.01, p < 0.05). The Deoxyribonucleic acid (DNA) fragmentation index evaluated by SCSA in High (p < 0.01) and Low (p < 0.05) SLC group differed significantly compared with control. Single-layer centrifugation (SLC) with either a high or a low density colloid is a suitable method for bacterial removal in bull semen. [ABSTRACT FROM AUTHOR]

The protein profiles of bacteria are useful markers in their identification. In this study, we examined the influence of environmental settings on the protein profiles of culturable bacteria using matrix‐assisted laser desorption ionization‐time of flight mass spectrometry (MALDI–TOF MS). We compared the protein profiles of bacteria collected from geographically distinct port environments (Kolkata, a freshwater port, and Kandla, a marine port) from India. The results indicated that bacterial species were distinctly different between the two ports, except for a few bacteria, that were common, such as Bacillus cereus, Enterobacter cloacae, Micrococcus luteus, Pseudomonas aeruginosa, P. putida, P. stutzeri, P. mosselii and Vibrio parahaemolyticus. Mass spectra of these common bacterial species, a few pathogenic among them, differed in their protein profiles and relative intensity of peaks. These variations point out that environmental settings/factors have a significant role in the protein expression and can result in differentiated virulence of pathogens. Identifying these risk factors is crucial in developing appropriate environmental port management practices. [ABSTRACT FROM AUTHOR]