The biological functions of microcystins.

Microcystins are potent hepatotoxins predominantly produced by bloom-forming freshwater cyanobacteria (e.g., Microcystis, Planktothrix, Dolichospermum). Microcystin biosynthesis involves large multienzyme complexes and tailoring enzymes encoded by the mcy gene cluster. Mutation, recombination, and deletion events have shaped the mcy gene cluster in the course of evolution, resulting in a large diversity of microcystin congeners and the natural coexistence of toxic and non-toxic strains. The biological functions of microcystins and their association with algal bloom formation have been extensively investigated over the past decades. This review synthesizes recent advances in decoding the biological role of microcystins in carbon/nitrogen metabolism, antioxidation, colony formation, and cell-to-cell communication. Microcystins appear to adopt multifunctional roles in cyanobacteria that reflect the adaptive plasticity of toxic cyanobacteria to changing environments.

Development and validation of LC-MS/MS for quantifying omadacycline from stool for gut microbiome studies.

This study developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify omadacycline and its epimerization in stool to facilitate microbiome studies. Omadacycline was extracted in a methanol-water-ethylenediaminetetraacetic acid (ETDA) solvent containing deuterated omadacycline as internal standard, followed by dilution. In an optimal gradient elution mode, omadacycline and its C4 epimer were separated within 5 min on reversed-phase C18 column. The method showed a broad working range of 0.1-200 ng/ml with a limitation of detection (LOD) of 0.03 ng/ml, little fecal matrix effect, good intra-day and inter-day accuracy (90-101 %), precision (2-15 %), and recovery rate (99-105 %). The method was sufficiently sensitive to quantify omadacycline in human fecal samples (n = 82) collected during a 10-day therapy course and at follow-up (day 13 and day 30) that ranged from 1 to 4785 µg/g. Further analysis revealed that ∼9 % of omadacycline was epimerized in fecal matrix control while, on average, 37.4 % was epimerized in human fecal samples. This study developed and validated a novel, simple, sensitive, and accurate method utilizing LC-MS/MS to quantify omadacycline its epimerization in the human gut. This has important implications for future studies of omadacycline and other tetracycline-class antibiotics as part of gut microbiome studies.

Microscopy methods for Clostridioides difficile.

Microscopic technologies including light and fluorescent, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and cryo-electron microscopy have been widely utilized to visualize Clostridioides difficile at the molecular, cellular, community, and structural biology level. This comprehensive review summarizes the microscopy tools (fluorescent and reporter system) in their use to study different aspects of C. difficile life cycle and virulence (sporulation, germination) or applications (detection of C. difficile or use of antimicrobials). With these developing techniques, microscopy tools will be able to find broader applications and address more challenging questions to study C. difficile and C. difficile infection.

Fecal Pharmacokinetics and Gut Microbiome Effects of Oral Omadacycline Versus Vancomycin in Healthy Volunteers.

BACKGROUND: Clostridioides difficile infection (CDI) is a common healthcare-associated infection with limited treatment options. Omadacycline, an aminomethylcycline tetracycline, has potent in vitro activity against C difficile and a low propensity to cause CDI in clinical trials. We aimed to assess fecal pharmacokinetics and gut microbiome effects of oral omadacycline compared to oral vancomycin in healthy adults. METHODS: This was a phase 1, nonblinded, randomized clinical trial conducted in healthy volunteers aged 18-40 years. Subjects received a 10-day course of omadacycline or vancomycin. Stool samples were collected at baseline, daily during therapy, and at follow-up visits. Omadacycline and vancomycin stool concentrations were assessed, and microbiome changes were compared. RESULTS: Sixteen healthy volunteers with a mean age of 26 (standard deviation [SD], 5) years were enrolled; 62.5% were male, and participants' mean body mass index was 23.5 (SD, 4.0) kg/m2. Omadacycline was well tolerated with no safety signal differences between the 2 antibiotics. A rapid initial increase in fecal concentrations of omadacycline was observed compared to vancomycin, with maximum concentrations achieved within 48 hours. A significant difference in alpha diversity was observed following therapy in both the omadacycline and vancomycin groups (P < .05). Bacterial abundance and beta diversity analysis showed differing microbiome changes in subjects who received omadacycline versus vancomycin. CONCLUSIONS: Subjects given omadacycline had high fecal concentrations with a distinct microbiome profile compared to vancomycin. CLINICAL TRIALS REGISTRATION: NCT06030219.

Beyond cyanotoxins: increased Legionella, antibiotic resistance genes in western Lake Erie water and disinfection-byproducts in their finished water.

Background: Western Lake Erie is suffering from harmful cyanobacterial blooms, primarily toxic Microcystis spp., affecting the ecosystem, water safety, and the regional economy. Continued bloom occurrence has raised concerns about public health implications. However, there has been no investigation regarding the potential increase of Legionella and antibiotic resistance genes in source water, and disinfection byproducts in municipal treated drinking water caused by these bloom events. Methods: Over 2 years, source water (total n = 118) and finished water (total n = 118) samples were collected from drinking water plants situated in western Lake Erie (bloom site) and central Lake Erie (control site). Bloom-related parameters were determined, such as microcystin (MC), toxic Microcystis, total organic carbon, N, and P. Disinfection byproducts (DBPs) [total trihalomethanes (THMs) and haloacetic acids (HAAs)] were assessed in finished water. Genetic markers for Legionella, antibiotic resistance genes, and mobile genetic elements were quantified in source and finished waters. Results: Significantly higher levels of MC-producing Microcystis were observed in the western Lake Erie site compared to the control site. Analysis of DBPs revealed significantly elevated THMs concentrations at the bloom site, while HAAs concentrations remained similar between the two sites. Legionella spp. levels were significantly higher in the bloom site, showing a significant relationship with total cyanobacteria. Abundance of ARGs (tetQ and sul1) and mobile genetic elements (MGEs) were also significantly higher at the bloom site. Discussion: Although overall abundance decreased in finished water, relative abundance of ARGs and MGE among total bacteria increased after treatment, particularly at the bloom site. The findings underscore the need for ongoing efforts to mitigate bloom frequency and intensity in the lake. Moreover, optimizing water treatment processes during bloom episodes is crucial to maintain water quality. The associations observed between bloom conditions, ARGs, and Legionella, necessitate future investigations into the potential enhancement of antibiotic-resistant bacteria and Legionella spp. due to blooms, both in lake environments and drinking water distribution systems.

The Novel DNA Binding Mechanism of Ridinilazole, a Precision Clostridiodes difficile Antibiotic.

Clostridioides difficile infection (CDI) causes substantial morbidity and mortality worldwide with limited antibiotic treatment options. Ridinilazole is a precision bisbenzimidazole antibiotic being developed to treat CDI and reduce unacceptably high rates of infection recurrence in patients. Although in late clinical development, the precise mechanism of action by which ridinilazole elicits its bactericidal activity has remained elusive. Here, we present conclusive biochemical and structural data to demonstrate that ridinilazole has a primary DNA binding mechanism, with a co-complex structure confirming binding to the DNA minor groove. Additional RNA-seq data indicated early pleiotropic changes to transcription, with broad effects on multiple C. difficile compartments and significant effects on energy generation pathways particularly. DNA binding and genomic localization was confirmed through confocal microscopy utilizing the intrinsic fluorescence of ridinilazole upon DNA binding. As such, ridinilazole has the potential to be the first antibiotic approved with a DNA minor groove binding mechanism of action.

Nonmammalian models to study Clostridioides difficile infection; a systematic review.

Clostridioide difficile is the leading cause of diarrhea disease worldwide and is a CDC-designated urgent threat level pathogen. Mammalian models are commonly utilized as gold standard to study the pathogenesis of C. difficile infection (CDI); however, alternatives are needed due to cost, higher throughput ability, and mammalian animal ethics. Nonmammalian models such as great wax worm, nematode, fruit fly, and zebrafish have been used as CDI models. This review provides a comprehensive summary of nonmammalian models used to study CDI. Multiple studies were identified using these models to study C. difficile infection, pathogenicity, colonization, host immunity, and therapy. Translational outcomes and strength and weakness of each nonmammalian model are discussed.

Heterogeneity and lyophilization comparison of stool processing for gastrointestinal bile acid measurement by LC-MS/MS.

Fecal bile acid (BA) analysis is an emerging area of gut microbiome research. However, sample preparation procedures for fecal BA analysis are not standardized. Current fecal BA analysis often utilizes either original or lyophilized aliquot, and fecal BA result difference between these two processing steps remains not systematically investigated. Moreover, the distribution pattern of fecal BA in the collected stool sample also remains unclear but affects interpretation of fecal BA for downstream experiments. To address these two questions regarding effect of lyophilization on fecal BA and fecal heterogeneity, fourteen separate BAs were quantified from 60 aliquots obtained from 10 clinical fecal samples using liquid chromatography-tandem mass spectrometry (LC-MS/MS). BA concentrations in the lyophilized sample were typically 2-4 folds higher than those in the original sample, but were almost identical using a water-adjusted lyophilized BA concentration. The fecal BA compositional profile and four BA ratios were similar utilizing either the original or lyophilized samples. BA concentrations were similar among different aliquots of differing starting mass except for the relatively trace-level BA. Therefore, it is suggested that fecal BA concentrations should be presented as the original sample concentration or water-adjusted lyophilization concentration to allow comparisons between studies. A single aliquot (20-100 mg) of stool can be used to reflect the concentrations in the entire sample. These results help to standardize analyses in this emerging field.

COVID-19 Vaccine Booster Strategies for Omicron SARS-CoV-2 Variant: Effectiveness and Future Prospects.

In the light of the lack of authorized COVID-19 vaccines adapted to the Omicron variant lineage, the administration of the first and second booster dose is recommended. It remains important to monitor the efficacy of such an approach in order to inform future preventive strategies. The present paper summarizes the research progress on the effectiveness of the first and second booster doses of COVID-19. It also discusses the potential approach in vaccination strategies that could be undertaken to maintain high levels of protection during the waves of SARS-CoV-2 infections. Although this approach can be based, with some shortcomings, on the first-generation vaccines, other vaccination strategies should be explored, including developing multiple antigen-based (multivariant-adapted) booster doses with enhanced durability of immune protection, e.g., through optimization of the half-life of generated antibodies.