MALDI-TOF MS Research Articles

Xanthomonas rydalmerensis sp. nov., a non-pathogenic member of Group 1 Xanthomonas.

Five bacterial isolates were isolated from Fragaria × ananassa in 1976 in Rydalmere, Australia, during routine biosecurity surveillance. Initially, the results of biochemical characterisation indicated that these isolates represented members of the genus Xanthomonas. To determine their species, further analysis was conducted using both phenotypic and genotypic approaches. Phenotypic analysis involved using MALDI-TOF MS and BIOLOG GEN III microplates, which confirmed that the isolates represented members of the genus Xanthomonas but did not allow them to be classified with respect to species. Genome relatedness indices and the results of extensive phylogenetic analysis confirmed that the isolates were members of the genus Xanthomonas and represented a novel species. On the basis the minimal presence of virulence-associated factors typically found in genomes of members of the genus Xanthomonas, we suggest that these isolates are non-pathogenic. This conclusion was supported by the results of a pathogenicity assay. On the basis of these findings, we propose the name Xanthomonas rydalmerensis, with DAR 34855T = ICMP 24941 as the type strain.

Structure dependent fermentation kinetics of dietary carrot rhamnogalacturonan-I in an in vitro gut model

Plant derived dietary polysaccharides are important for gut health and have the potential to modulate the gut microbial community. Dietary rhamnogalacturonan-I obtained by enzymatic treatment of carrot pomace has shown prebiotic properties. In the present study, fermentability of carrot rhamnogalacturonan-I (cRG-I) by faecal microbiota of four donors was studied in an adapted M-SHIME® intestinal model. Despite its complex structure, cRG-I was degraded rapidly in the proximal colon compartment and fermentation became quicker and more complete during three weeks of repeated supplementation. Tracking the change in the molecular weight distribution pattern of cRG-I during the supplementation showed two main donor-dependent gut microbial fermentation strategies designated as either the general or preferential pathway. In the general fermentation pathway, different cRG-I structures were hydrolysed concomitantly, while in the preferential pathway discrete structures were sequentially fermented in a selective manner. Especially arabinan sidechains were utilized before the RG-I backbone, which correlated with an increase in Bifidobacterium longum absolute abundance over the three weeks period. MALDI-TOF MS confirmed that arabinan-, galactan- and arabinogalactan-sidechains were first to be released and degraded. Donor specific production of all SCFA increased over time with a general trend of higher levels of acetate and propionate than butyrate. Strikingly, although the host's baseline gut microbiota composition led to distinct cRG-I hydrolysis routes, the final RG-I consumption was almost complete for both routes, leading to similar metabolic profiles at the end of the three weeks treatment period.

Antifungal susceptibility profile of Candida species isolated from women with vulvovaginal candidiasis

Objectives: The objective of this study was to study the occurrence of vulvovaginal candidiasis (VVC) among women with vaginitis and to determine the antifungal susceptibility testing (AFST) pattern of Candida spp. isolated. Materials and Methods: A prospective study was conducted in a tertiary care setting. The two high vaginal swabs were collected and subjected to Gram’s stain, wet mount examination, and cultured on blood agar and sabouraud dextrose agar with chloramphenicol. The cultures were incubated at 37°C for 18–24 h. The Candida spp. was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS), and AFST was performed according to the Clinical and Laboratory Standards Institute guidelines M27-A3. Statistical Analysis: The data was collected in excel sheet. The qualitative variables are presented as frequencies and percentages. The quantitative variables are presented as mean with SD and median. p value of less than 0.05 was considered statistically significant. Analysis will be done by using Epi InfoTM statistical software. Results: A total of 961 women with symptoms of vaginitis were screened. Candida spp. was isolated in 108 (11.2%) patients. The median age of affected women was 29 years, and the majority belonged to 20–30 years (60.2%). Age <40 years was the significant risk factor (P = 0.016). Vaginal discharge was the most common complaint (82.4%),followed by itching (55.6%), vulvar edema, dyspareunia, and dysuria. Candida albicans was the predominant species (46.3%),followed by Candida glabrata (36.1%), Candida krusei (6.5%), and Candida tropicalis (6.5%). Ninety-six percentages of C. albicans were sensitive to fluconazole, while only 22.4% of non-albicans Candida spp (NAC) were susceptible. C. albicans showed 8% resistance to itraconazole and 8% intermediate resistance to caspofungin, whereas C. glabrata showed resistance to fluconazole, itraconazole, and caspofungin. Conclusions: The study showed a higher incidence of NAC and higher antifungal resistance leading to treatment failure. It is, hence, crucial to send fungal cultures, speciate, and perform AFST for all symptomatic patients of vulvo-vaginal candidiasis (VVC).

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

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

Multidrug-Resistant Staphylococcus sp. and Enterococcus sp. in Municipal and Hospital Wastewater: A Longitudinal Study.

The objective of the study was to detect multidrug-resistant Staphylococcus sp. and Enterococcus sp. isolates in municipal and hospital wastewater and to determine their elimination or persistence after wastewater treatment. Between August 2021 and September 2022, raw and treated wastewater samples were collected at two hospital and two community wastewater treatment plants (WWTPs). In each season of the year, two treated and two raw wastewater samples were collected in duplicate at each of the WWTPs studied. Screening and presumptive identification of staphylococci and enterococci was performed using chromoagars, and identification was performed with the Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-TOF MS®). Antimicrobial susceptibility was performed using VITEK 2® automated system. There were 56 wastewater samples obtained during the study period. A total of 182 Staphylococcus sp. and 248 Enterococcus sp. were identified. The highest frequency of Staphylococcus sp. isolation was in spring and summer (n = 129, 70.8%), and for Enterococcus sp. it was in autumn and winter (n = 143, 57.7%). Sixteen isolates of Staphylococcus sp. and sixty-three of Enterococcus sp. persisted during WWTP treatments. Thirteen species of staphylococci and seven species of enterococci were identified. Thirty-one isolates of Staphylococcus sp. and ninety-four of Enterococcus sp. were multidrug-resistant. Resistance to vancomycin (1.1%), linezolid (2.7%), and daptomycin (8.2%/10.9%%), and a lower susceptibility to tigecycline (2.7%), was observed. This study evidences the presence of Staphylococcus sp. and Enterococcus sp. resistant to antibiotics of last choice of clinical treatment, in community and hospital wastewater and their ability to survive WWTP treatment systems.

Molecular characterization of non-aureus staphylococci and Mammaliicoccus from Hipposideros bats in Southwest Nigeria

Bats are not only ecologically valuable mammals but also reservoirs of zoonotic pathogens. Their vast population, ability to fly, and inhabit diverse ecological niches could play some role in the spread of antibiotic resistance. This study investigated non-aureus staphylococci and Mammaliicoccus colonization in the Hipposideros bats at Obafemi Awolowo University, Ile-Ife, Nigeria. Pharyngeal samples (n = 23) of the insectivorous bats were analyzed, and the presumptive non-aureus staphylococcal and Mammaliicoccus isolates were confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The isolates were characterized based on antibiotic susceptibility testing and whole-genome sequencing (WGS). Six bacterial genomes were assembled, and three species were identified, including Mammaliicoccus sciuri (n = 4), Staphylococcus gallinarum (n = 1), and Staphylococcus nepalensis (n = 1). All the isolates were resistant to clindamycin, while the M. sciuri and S. gallinarum isolates were also resistant to fusidic acid. WGS analysis revealed that the M. sciuri and S. gallinarum isolates were mecA-positive. In addition, the M. sciuri isolates possessed some virulence (icaA, icaB, icaC, and sspA) genes. Multi-locus sequence typing identified two new M. sciuri sequence types (STs) 233 and ST234. The identification of these new STs in a migratory mammal deserves close monitoring because previously known ST57, ST60, and ST65 sharing ack (8), ftsZ (13), glpK (14), gmk (6), and tpiA (10) alleles with ST233 and ST234 have been linked to mastitis in animals. Moreover, the broad host range of M. sciuri could facilitate the dispersal of antibiotic resistance genes. This study provides evidence of the importance of including migratory animals in monitoring the development and spread of antibiotic resistance.

Fungemia by Wickerhamomyces anomalus-A Narrative Review.

Wickerhamomyces anomalus has been previously classified as Hansenula anomala, Pichia anomala, and Candida pelliculosa and was recently reclassified in the genus Wickerhamomyces after phylogenetic analysis of its genetic sequence. An increasing number of reports of human infections by W. anomalus have emerged, suggesting that this microorganism is an emerging pathogen. The present review aimed to provide data on the epidemiology, antifungal resistance, clinical characteristics, treatment, and outcomes of fungemia by W. anomalus by extracting all the available information from published original reports in the literature. PubMed/Medline, Cochrane Library, and Scopus databases were searched for eligible articles reporting data on patients with this disease. In total, 36 studies involving 170 patients were included. The age of patients with fungemia by W. anomalus ranged from 0 to 89 years; the mean age was 22.8 years, the median age was 2.2 years, with more than 37 patients being less than one month old, and 54% (88 out of 163 patients) were male. Regarding patients' history, 70.4% had a central venous catheter use (CVC), 28.7% were on total parenteral nutrition (TPN), 97% of neonates were hospitalized in the neonatal ICU (NICU), and 39.4% of the rest of the patients were hospitalized in the intensive care unit (ICU). Previous antimicrobial use was noted in 65.9% of patients. The most common identification method was the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in 34.1%, VITEK and VITEK 2 in 20.6%, and ID32 C in 15.3%. W. anomalus had minimal antifungal resistance to fluconazole, echinocandins, and amphotericin B, the most commonly used antifungals for treatment. Fever and sepsis were the most common clinical presentation noted in 95.8% and 86%, respectively. Overall mortality was 20% and was slightly higher in patients older than one year. Due to the rarity of this disease, future multicenter studies should be performed to adequately characterize patients' characteristics, treatment, and outcomes, which will increase our understanding and allow drawing safer conclusions regarding optimal management.

A promoted MALDI-TOF-MS platform towards rapid and accurate identifications of bacteria

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is emerging as the most powerful tool for the identification of bacteria. Herein, we demonstrated that the capability of MALDI-TOF-MS towards the discrimination of clinical bacteria can be further dramatically improved. A cooperative matrix consisting of (E)-propyl α-cyano-4-hydroxylcinnamylate (CHCA-C3) and α-cyano-4-hydroxycinnamic acid (CHCA) was used to replace the commonly used matrix CHCA. As a result, 1.4–3.8 times more fingerprint peaks have been identified by the cooperative matrix in comparison with CHCA when six different bacteria were studied. The peaks distribute in the wider m/z range, and the spectral quality has also been significantly improved. As an example of practical applications, the direct and more reliable discrimination between Escherichia coli (E. coli) and Shigella sonnei (S. sonnei) was achieved based on their protein fingerprints, which was previously claimed to be impossible by CHCA. Thus, a promoted MALDI-TOF-MS platform towards bacterial identification has been established by utilizing the new cooperative matrix, which will exhibit great potential for many applications.

Vibrio infection in freshwater fish in Poland.

Vibrio species are common inhabitants of aquatic environments and have been described in connection with fish and human diseases. Six Vibrio species were isolated from diseased freshwater and ornamental fish in Poland. The strains were identified based on morphological and biochemical characteristics and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as V. albensis (n=3) from Gymnocephalus cernua, Sander lucioperca, Paracheirodon innesi, and Xiphophorus hellerii; V. mimicus (n=1) from Xiphophorus maculatus; and V. vulnificus (n=1) from Nematobrycon palmeri. This is the first time that Vibrio species have been isolated and described from ornamental fish in Poland. The isolates were resistant to ampicillin (83.3%), gentamicin (16.6%), ciprofloxacin (16.6%), sulfamethoxazole-trimethoprim (16.6%), and chloramphenicol (16.6%). The multiple antibiotic resistance (MAR) index was 0.00-0.08 for V. albensis, 0.17 for V. mimicus, and 0.33 for V. vulnificus. Our study confirmed the presence of potentially pathogenic Vibrio species in freshwater and ornamental fish. Therefore, further monitoring of the presence of Vibrio species, mainly in ornamental fish, is necessary.

Performance evaluation of machine-assisted interpretation of Gram stains from positive blood cultures.

Manual microscopy of Gram stains from positive blood cultures (PBCs) is crucial for diagnosing bloodstream infections but remains labor intensive, time consuming, and subjective. This study aimed to evaluate a scan and analysis system that combines fully automated digital microscopy with deep convolutional neural networks (CNNs) to assist the interpretation of Gram stains from PBCs for routine laboratory use. The CNN was trained to classify images of Gram stains based on staining and morphology into seven different classes: background/false-positive, Gram-positive cocci in clusters (GPCCL), Gram-positive cocci in pairs (GPCP), Gram-positive cocci in chains (GPCC), rod-shaped bacilli (RSB), yeasts, and polymicrobial specimens. A total of 1,555 Gram-stained slides of PBCs were scanned, pre-classified, and reviewed by medical professionals. The results of assisted Gram stain interpretation were compared to those of manual microscopy and cultural species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The comparison of assisted Gram stain interpretation and manual microscopy yielded positive/negative percent agreement values of 95.8%/98.0% (GPCCL), 87.6%/99.3% (GPCP/GPCC), 97.4%/97.8% (RSB), 83.3%/99.3% (yeasts), and 87.0%/98.5% (negative/false positive). The assisted Gram stain interpretation, when compared to MALDI-TOF MS species identification, also yielded similar results. During the analytical performance study, assisted interpretation showed excellent reproducibility and repeatability. Any microorganism in PBCs should be detectable at the determined limit of detection of 105 CFU/mL. Although the CNN-based interpretation of Gram stains from PBCs is not yet ready for clinical implementation, it has potential for future integration and advancement.

Creatine kinase elevation in chronic hepatitis B patients with telbivudine therapy: influence of telbivudine plasma concentration and single nucleotide polymorphisms of TK2, RRM2B, and NME4.

To study the correlations of genetic variants of telbivudine phosphorylase kinases and telbivudine plasma concentration with creatine kinase elevation in chronic hepatitis B patients who received telbivudine. An observational study was performed in China chronic hepatitis B patients receiving telbivudine therapy at 600mg once daily. Plasma concentration was measured 12h after taking telbivudine using ultra-performance liquid chromatography-tandem mass spectrometry and SNPs located in RRM2B, TK2, and NME4 was detected by MALDI-TOF mass spectrometry. All statistical analyses were performed with R 4.3.1 and all graphs were drawn by Origin 2023b and P value < 0.05 was considered statistically significant. A total of 140 patients receiving telbivudine therapy were recruited with a median plasma concentration of 952.49 (781.07-1238.98) ng/mL. The value of plasma concentration was proportional to the grade of creatine kinase elevation and the best telbivudine plasma concentration threshold to discriminate the grade 3/4 CK elevation was 1336.61ng/mL. Multivariate analysis revealed that plasma concentration and rs3826160 were the independent risk factor of telbivudine-induced creatine kinase elevation. Patients with TC and CC genotype in rs3826160 not only had a higher incidence of creatine kinase elevation but also a higher plasma concentration than TT genotype carriers. Chronic hepatitis B patients with TC and CC genotype in rs3826160 have high telbivudine plasma concentration are at risk of elevated creatine kinase.

The Home Environment Is a Reservoir for Methicillin-Resistant Coagulase-Negative Staphylococci and Mammaliicocci.

Coagulase-negative staphylococci (CoNS) and mammaliicocci are opportunistic human and animal pathogens, often resistant to multiple antimicrobials, including methicillin. Methicillin-resistant CoNS (MRCoNS) have traditionally been linked to hospitals and healthcare facilities, where they are significant contributors to nosocomial infections. However, screenings of non-hospital environments have linked MRCoNS and methicillin-resistant mammaliicocci (MRM) to other ecological niches. The aim of this study was to explore the home environment as a reservoir for MRCoNS and MRM. A total of 33 households, including households with a dog with a methicillin-resistant staphylococcal infection, households with healthy dogs or cats and households without pets, were screened for MRCoNS and MRM by sampling one human, one pet (if present) and the environment. Samples were analyzed by a selective culture-based method, and bacterial species were identified by MALDI-TOF MS and tested for antibiotic susceptibility by the agar disk diffusion method. Following whole-genome sequencing, a large diversity of SCCmec elements and sequence types was revealed, which did not indicate any clonal dissemination of specific strains. Virulome and mobilome analyses indicated a high degree of species specificity. Altogether, this study documents that the home environment is a reservoir for a variety of MRCoNS and MRM regardless of the type of household.

Application of MALDI-TOF MS and machine learning for the detection of SARS-CoV-2 and non-SARS-CoV-2 respiratory infections.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) could aid the diagnosis of acute respiratory infections (ARIs) owing to its affordability and high-throughput capacity. MALDI-TOF MS has been proposed for use on commonly available respiratory samples, without specialized sample preparation, making this technology especially attractive for implementation in low-resource regions. Here, we assessed the utility of MALDI-TOF MS in differentiating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vs non-COVID acute respiratory infections (NCARIs) in a clinical lab setting in Kazakhstan. Nasopharyngeal swabs were collected from inpatients and outpatients with respiratory symptoms and from asymptomatic controls (ACs) in 2020-2022. PCR was used to differentiate SARS-CoV-2+ and NCARI cases. MALDI-TOF MS spectra were obtained for a total of 252 samples (115 SARS-CoV-2+, 98 NCARIs, and 39 ACs) without specialized sample preparation. In our first sub-analysis, we followed a published protocol for peak preprocessing and machine learning (ML), trained on publicly available spectra from South American SARS-CoV-2+ and NCARI samples. In our second sub-analysis, we trained ML models on a peak intensity matrix representative of both South American (SA) and Kazakhstan (Kaz) samples. Applying the established MALDI-TOF MS pipeline "as is" resulted in a high detection rate for SARS-CoV-2+ samples (91.0%), but low accuracy for NCARIs (48.0%) and ACs (67.0%) by the top-performing random forest model. After re-training of the ML algorithms on the SA-Kaz peak intensity matrix, the accuracy of detection by the top-performing support vector machine with radial basis function kernel model was at 88.0%, 95.0%, and 78% for the Kazakhstan SARS-CoV-2+, NCARI, and AC subjects, respectively, with a SARS-CoV-2 vs rest receiver operating characteristic area under the curve of 0.983 [0.958, 0.987]; a high differentiation accuracy was maintained for the South American SARS-CoV-2 and NCARIs. MALDI-TOF MS/ML is a feasible approach for the differentiation of ARI without specialized sample preparation. The implementation of MALDI-TOF MS/ML in a real clinical lab setting will necessitate continuous optimization to keep up with the rapidly evolving landscape of ARI.IMPORTANCEIn this proof-of-concept study, the authors used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and machine learning (ML) to identify and distinguish acute respiratory infections (ARI) caused by SARS-CoV-2 versus other pathogens in low-resource clinical settings, without the need for specialized sample preparation. The ML models were trained on a varied collection of MALDI-TOF MS spectra from studies conducted in Kazakhstan and South America. Initially, the MALDI-TOF MS/ML pipeline, trained exclusively on South American samples, exhibited diminished effectiveness in recognizing non-SARS-CoV-2 infections from Kazakhstan. Incorporation of spectral signatures from Kazakhstan substantially increased the accuracy of detection. These results underscore the potential of employing MALDI-TOF MS/ML in resource-constrained settings to augment current approaches for detecting and differentiating ARI.

Functional Melanin Nanoparticles-Assisted Laser Desorption Ionization Mass Spectrometry for High-Sensitivity Detection of TBBPA and TBBPS Contaminations in Animal-Derived Foodstuffs.

Tetrabromobisphenol A (TBBPA) and tetrabromobisphenol S (TBBPS) have been widely used as additives in various products; however, their residues damage human health mainly via dietary ingestion. The current detection techniques remain challenging in directly and sensitively identifying TBBPA and TBBPS from food samples. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has great potential as an alternative tool for the analysis of low-mass environmental pollution. Herein, we successfully screened and optimized COOH-MNP-COOH as a novel MALDI matrix to enhance deprotonation for the analysis of TBBPA and TBBPS from animal-derived food samples in negative-ion mode. Notably, COOH-MNP-COOH was synthesized by a facile self-assembly strategy and characterized by TEM, FT-IR, UV-vis, and zeta potential analysis. Compared with conventional and control matrices, the COOH-MNP-COOH matrix exhibited excellent performance of TBBPA and TBBPS with high chemical stability, favorable reproducibility, remarkable salt and protein tolerance, and high sensitivity owing to abundant active groups, stronger UV-vis absorption at 355 nm, and better hydrophilicity and biocompatibility. TBBPA and TBBPS were detected with the assistance of an internal standard with limits of detection (LODs) of 300 and 200 pg/mL, respectively. Moreover, this method was applied to directly identify the residues of TBBPA and TBBPS in milk products, followed by basa catfish and meat. This research may provide a promising approach for the analysis of environmental pollutants in foodstuffs.

Comparison of MBT biotargets with MBT steel targets for matrix-assisted laser desorption/ionization time of flight mass spectrometry identifications of microorganisms in a clinical microbiology laboratory

MALDI-TOF MS identifications of microorganisms in a clinical laboratory were investigated, comparing steel targets with MBT Biotargets. By using MBT Biotargets, the score values of yeast identifications increased, whereas the score values of Gram-negative bacteria decreased. Switching to MBT Biotargets did not negatively impact overall frequencies of high confidence identifications.

Chromogenic, Biochemical and Proteomic Identification of Yeast and Yeast-like Microorganisms Isolated from Clinical Samples from Animals of Costa Rica.

Yeast infections are challenging human and animal medicine due to low rates of detection and the emergence of unknown ecology isolates. The aim of this study was to verify the biochemical identification of yeasts and yeast-like microorganisms obtained from animals comparing the results with chromogenic media and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS). Between January and August 2023, yeast and yeast-like isolates from samples of animals with suspicion of mycosis were identified using Vitek® 2 Compact, Brilliance® Candida Agar and MALDI Biotyper® MSP. A total of 39 cases were included, and 45 isolations were obtained. Cryptococcus neoformans (15.5%, 7/45), Meyerozyma guilliermondii (13.3%, 6/45), Candida parapsilosis (11.1%, 5/45), Candida albicans and Candida tropicalis (8.9%, each one 4/45) were the most identified organisms. There was full agreement with the three identification methods in 71.1% (32/45) of the isolates, disagreement on species in 17.8% (8/45), disagreement on genus and species in 6.7% (3/45) and, in 4.4% (2/45), there was no matched pattern in MALDI-TOF to compare the results. Biochemical methods are a good option in laboratories where proteomics are not available, and chromogenic media enhances diagnostics by detecting mixed infections. Surveillance must be implemented to improve the detection of agents shared between humans and animals.

The usefulness of the MALDI-TOF MS technique in the determination of dairy samples' microbial composition: comparison of the new EXS 2600 system with MALDI Biotyper platform.

This study compared the EXS 2600 system with the MALDI Biotyper for identifying microorganisms in dairy samples. Of the 196 bacterial isolates from milk, whey, buttermilk, cream, and dairy wastewater, the species and genus consistent identification between two systems showed 74% and 99%, respectively. However, the level of species identification rate exhibited a difference, which was higher in Zybio than in Bruker-76.0% and 66.8%, respectively. Notably, the EXS 2600 system performed better with certain yeast species and H. alvei, while the Biotyper excelled with Pseudomonas bacteria. Unique microbial compositions were found in 85% of dairy samples, with whey and buttermilk having the highest diversity. This research highlights the EXS 2600's potential as a reliable dairy microbial identification tool and underscores the need for a more diverse and comprehensive spectral database, despite the database's focus on clinical applications (as announced).

Resistance Profile, Terbinafine Resistance Screening and MALDI-TOF MS Identification of the Emerging Pathogen Trichophyton indotineae

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.

Next-Generation Sequencing-Based Identification of Enterobacter hormaechei as Causative Agent of High Mortality Disease in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) Mice.

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research.

Stereochemical Heterogeneity Analysis of Polylactides by Multidimensional Liquid Chromatography.

A new and robust high-performance liquid chromatography (HPLC) method that separates poly(lactic acid) (PLA) according to its stereochemical composition is presented. Using this method, poly(l-lactide) incorporating trace amounts of meso-lactide resulting from the racemization is separated from the pristine polymer. To prove this aspect in more detail, a representative poly(l-lactic acid) standard, assumed to be highly homogeneous, was separated using this method. The result showed that this was not the case as a fraction incorporating meso-lactide due to racemization occurring during the synthesis is separated. Employing two-dimensional liquid chromatography (2D-LC), the molar mass differences of the separated species were investigated, and fractions with similar molecular sizes were detected, confirming that the LC separation is solely based on stereochemical heterogeneity. The sample was further fractionated by preparative HPLC, followed by an in-depth analysis of the fractions using homonuclear decoupling in proton nuclear magnetic resonance (1H NMR). Convincing results that unveiled significant differences in the stereochemistry of the isolated PLA fractions were obtained. Subsequent analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) also confirmed oligomer series with different end group structures, indicating that the applied HPLC method is very sensitive to minor variations in stereochemistry and end groups. This integrated approach offers detailed insight into the structural characteristics of PLA polymers, contributing to a better understanding of their composition and potential applications.