ObjectivesAlthough Clostridium perfringens sporulation is a key event in the pathogenesis of the food-borne illness, the molecules and underlying mechanisms responsible for regulating sporulation are incompletely understood. The present study sought to identify amino acids that affect sporulation in C. perfringens strain SM101. MethodsThe bacterial strain was cultured in the chemically defined medium deficient in an amino acid. The bacterial growth was determined by spectrophotometrically measuring culture turbidity and by calculating colony-forming unit. Morphological characteristics were assessed by phase-contrast microscopy with fluorescent staining and by electron microscopy. ResultsThe amino acids Arg, Cys, Gly, His, Ile, Leu, Met, Phe, Thr, Trp, Tyr, and Val were important for sporulation, and furthermore, Ser reduced sporulation. The mechanism underlying Ser-induced prevention of sporulation was assessed morphologically. The numbers of bacterial cells in sporulation stage II were significantly higher in the presence than in the absence of Ser. In the presence of Ser, almost all cells were in stage II−III, characterized by polar septation−early engulfment, and did not proceed to late engulfment. ConclusionsThese results suggest that Ser accelerated the early stage of sporulation of C. perfringens strain SM101, but disturbed the engulfment process, resulting in reduction of sporulation. To the best of our knowledge, this is the first study reporting that an amino acid affects engulfment during the C. perfringens sporulation process.

We investigated the effects of Clostridioides difficile toxin B (TcdB), a major virulence factor in C. difficile infection (CDI), on human neutrophils. TcdB inhibits neutrophil migration via loss of polarity of F-actin polymerization in response to interleukin-8. TcdB facilitates CDI by allowing C. difficile to avert the host immune system.

ObjectivesFructo-oligosaccharide (FOS) and citrus pectin (CP) are soluble fibers with different chemical composition. However, their fermentation pattern in large intestine remains unclear. MethodsAn in vitro batch fermentation using colonic digesta from pigs as inoculum was employed to investigate the fermentation dynamics of FOS and CP. The monosaccharides and SCFAs contents were assayed by High-Performance Liquid Chromatography and Gas Chromatography, respectively. And the microbiota community was assessed by 16S rRNA gene high-throughput sequencing. ResultsThe decline of monosaccharides in both substrates after 6 h, especially to a neglected level in FOS. FOS showed higher abundances of butyrate-producing bacteria such as Eubacterium rectale, Roseburia faecis and Coprococcus comes and butyrate compared to CP. CP stimulated the growth of pectinolytic microbe Lachnospira pectinoschiza, succinate-producing bacteria Succinivibrio dextrinosolvens, succinate-utilizing bacteria Phascolarctobacterium succinatutens and the production of acetate and propionate compared to FOS. Moreover, the relative abundances of key enzymes (e.g. butyrate kinase) involving in butyrate formation via the butyrate kinase route were upregulated in the FOS group. And the key enzymes (e.g. acetyl-CoA synthetase) associated with propionate production through the succinate pathway were upregulated in the CP group. ConclusionsFOS was preferred to ferment by butyrate-producing bacteria to yield a higher level of butyrate via the butyrate kinase pathway, while CP enhanced the cross-feeding of succinate-producing and succinate-utilizing bacteria to form propionate through the succinate pathway. These findings deepen our understanding on the fermentation characteristics of the soluble fibers, and also provide guidelines for fiber choice in precisely modulating the microbial composition and metabolism in large intestine.

ObjectivesThe central challenge in third-generation sequencing lies in meeting the requirements for DNA quality (integrity and purity) and quantity. Therefore, novel improvements in DNA extraction methods are needed to satisfy these requirements. We reasoned that in anaerobic microbial communities, the presence of certain strict anaerobes containing oxygen-activated DNase activity might contribute substantially to the poor integrity of extracted metagenomic DNA (or genomic DNA from some pure cultures) if exposed to air. MethodsTo test this hypothesis, we developed an enhanced genomic and metagenomic DNA isolation technique that we applied to a specifically chosen set of both strict and aerotolerant anaerobes, as well as to the hindgut microbiota of an herbivorous marine fish. ResultsConsidering the quality (or degradation) of extracted DNA obtained under anaerobic versus aerobic conditions, we found that DNA extracted aerobically from cells of some strict anaerobes showed more degradation of high molecular weight DNA than analogous preparations under anaerobic conditions. In contrast, with the selected aerotolerant anaerobes, no discernable difference was found between the molecular sizes of DNA extracted aerobically and anaerobically. Metagenomic DNA extracted from the fish hindgut microbiota showed higher yields and better quality under anaerobic conditions compared to aerobic conditions. ConclusionOur study effectively demonstrates the advantages of our improved extraction protocol in anaerobic conditions. This is evident through the improved quality of extracted DNA. Such findings may be valuable for studies, especially metagenomic studies, where the quality and quantity of DNA are crucial for downstream analysis.

We present the first described case of bacteremia due to Butyricimonas paravirosa, a commensal gram-negative anaerobic bacterium identified by whole-genome sequencing in an elderly patient with acute terminal ileitis, who was successfully treated with ceftriaxone and metronidazole. We reviewed eleven previous cases of infection due to other Butyricimonas spp, which can cause a range of diseases but may be treated conservatively with a short antimicrobial course in the appropriate clinical setting. Additionally, while most Butyricimonas spp are susceptible to empiric anaerobic therapy, drug resistance has been reported in some cases.

ObjectivesTo comprehensively characterize a new species, named Finegoldia dalianensis sp. nov., isolated from the pus of a skin abscess from a patient and genomic analysis of the strains belonging to Finegoldia genus. MethodsStrain LY240594T was definitively characterized through phylogenetic, genomic, and biochemical approach. Extensive genomic comparisons, involving the genome of LY240594T and those of 82 Finegoldia strains from GenBank, were instrumental in revealing genetic relationships within the Finegoldia genus. ResultsStrain LY240594 was initially identified as F. magna based on MALDI-TOF MS analysis, showing 99.7% 16S rRNA gene sequence similarity with the type strain of F. magna CCUG 17636T. However, there were 68.5% similarity with dDDH method and 90.9% similarity by ANI analysis respectively, between LY240594T and the selected type strain, F. magna DSM 20470T.Biochemical differences were also found between two strains. The ANI and genomic analysis of 82 Finegoldia sp. strains and Strain LY240594 revealed that those strains could be categorized into at least three groups using a 95% ANI threshold. ConclusionComprehensive characterization supported the proposal of a new species within the genus Finegoldia, named Finegoldia dalianensis sp. nov. The type strain, LY240594T (=GDMCC 1.4375T =KCTC 25838T), features 1,938 genes and a G+C content of 31.8 mol%. Genomic comparisons and ANI studies elucidated substantial heterogeneity within the Finegoldia genus.

Clostridium perfingens bloodstream infections (BSIs) can be associated with high mortality rates. We performed a subanalysis of all C. perfringens BSIs enrolled during a multicentric retrospective observational study (ITANAEROBY). Data were collected from January 2016 to December 2020. C. perfringens BSIs were 134 (134/1960, 6.8 %). The highest resistance rate was observed for clindamycin (26/120, 21.6 %), penicillin (11/71, 15.4 %) and metronidazole (14/131, 10.7 %). In conclusion, C. perfringens reduced susceptibility phenotype to first-line therapy.

ObjectivesThis study investigated the codigestion of corn straw (CS) with cow manure (CM), cow digestion solution (CD), and a strain consortium (SC) for enhanced volatile fatty acid (VFA) production. The aims of this study were to develop a sustainable technique to increase VFA yields, examine how combining microbial reagents with CS affects VFA production by functional microorganisms, and assess the feasibility of improving microbial diversity through codigestion. MethodsBatch experiments evaluated VFA production dynamics and microbial community changes with different combinations of CS substrates with CM, CD, and SC. Analytical methods included measuring VFAs by GC, ammonia and chemical oxygen demand (COD) by standard methods and microbial community analysis by 16S rRNA gene sequencing. ResultsCodigesting CS with the strain consortium yielded initial VFA concentrations ranging from 0.6 to 1.0 g/L, which were greater than those of the other combinations (0.05–0.3 g/L). Including CM, and CD further increased VFA production to 1.0–2.0 g/L, with the highest value of 2.0 g/L occurring when all four substrates were codigested. Significant ammonium reduction (194–241 mg/L to 29–37 mg/L) and COD reduction (3310–5250 mg/L to 730–1210 mg/L) were observed. Codigestion with CM and CD had greater Shannon diversity indices (3.19–3.24) than did codigestion with the other consortia (2.26). Bacillota dominated (96.5–99.6 %), with Clostridiales playing key roles in organic matter breakdown. ConclusionsThis study demonstrated the feasibility of improving VFA yields and harnessing microbial diversity through anaerobic codigestion of lignocellulosic and animal waste streams. Codigestion substantially enhanced VFA production, which was dominated by butyrate, reduced ammonium and COD, and enriched fiber-degrading and fermentative bacteria. These findings can help optimize codigestion for sustainable waste management and high-value chemical production.

Oxygen tolerance of anaerobes is a virulence factor, but can also be a beneficial property. Many species have evolved to tolerate or take advantage of the presence of low, especially nanaerobic (≤0.14 %) oxygen concentrations. Oxygen tolerance is genus-, species- and strain-dependent according to their protective mechanisms. It was better expressed in some pathogenic species such as Bacteroides fragilis, Clostridioides difficile, and Clostridium perfringens, as well as in Akkermansia muciniphila than in other potential probiotics such as Alistipes, Blautia and Roseburia spp. Different degrees of oxygen sensitivity were found between the strains of Anaerostipes, Faecalibacterium, and Bifidobacterium spp. Importantly, clostridial spores and anaerobes in biofilms are protected from oxidation. Rubrerythrins and flavodiiron proteins and two regulators (sigma factor B and PerR) contribute to C. difficile protection from reactive oxygen species (ROS). The frequent pathogen, B. fragilis, has numerous protective factors such as enzymes (catalase, superoxide dismutase, alkyl hydroperoxidase, thioredoxin peroxidase, and aerobic-type NrdAB ribonucleotide reductase), and nanaerobic respiration. Seven proteins confer strain-specific oxygen adaptation of Faecalibacterium prausnitzii. Oxygen tolerance protects anaerobes from ROS, shields their DNA and modulates gene expression. Furthermore, oxygen can induce mutations leading to antibiotic resistance as shown in Prevotella melaninogenica. Some Faecalibacterium, Anaerostipes, Bifidobacterium, and Akkermansia strains from the intestinal microbiota exhibiting oxygen tolerance may become next-generation probiotic candidates. Further studies are needed to reveal oxygen effects on more anaerobic species and strains, and the influence of oxygen on antibiotic resistance. More studies on oxygen-tolerant probiotic strains can be useful to optimize biotechnological methods.

ObjectiveFlagellin protein, an integral component of flagella, provides motility to several bacterial species and also acts as a candidate antigen in diagnostics and subunit vaccines. The bulk production of flagellin with retention of all conformational epitopes using recombinant protein technology is of paramount importance in the development of pathogen-specific immuno-assays and vaccines. We describe the production of highly soluble and immuno-reactive rFliA(C) protein of Clostridium chauvoei, a causative agent of blackleg or black quarter (BQ) affecting cattle and small ruminants worldwide. The bacterium is known to possess peritrichous flagella that provide motility and also act as a virulence factor with high protective antigenicity. MethodsUpon sequence and structural analysis, a partial fliA(C) gene from Clostridium chauvoei was cloned and the recombinant mature protein with N- and C- terminal truncation was over-expressed as a His-tagged fusion protein (∼25 kDa) in Escherichia coli. Subsequently, rFliA(C) protein was purified by single-step affinity chromatography and characterized for its immuno-reactivity in laboratory animals, Western blot, and indirect-ELISA format. ResultsrFliA(C) was highly soluble and was purified in high quantity and quality. rFliA(C) elicited antigen-specific conformational polyclonal antibodies in rabbit and guinea pig models, as well as anti-Clostridium chauvoei-specific antibodies being specifically detected in BQ-vaccinated and convalescent sera of bovines in Western blot and in indirect-ELISA format. Further, no cross reactivity was noted with antibodies against major bovine diseases (e.g., foot-and-mouth disease, IBR, LSDV, hemorrhagic septicaemia, brucellosis, and leptospirosis). ConclusionThe study indicated the production of conformational recombinant flagellin—rFliA(C)—antigen and its potential utility in development of diagnostics for detection of Clostridium chauvoei-specific antibodies in BQ-recovered and/or vaccinated animals.