Toxin genotypes, antibiotic resistance and their correlations in Clostridioides difficile isolated from hospitals in Xi'an, China.

BACKGROUND: Clostridioides difficile is the main pathogen of antimicrobial-associated diarrhoea and health care facility-associated infectious diarrhoea. This study aimed to investigate the prevalence, toxin genotypes, and antibiotic resistance of C. difficile among hospitalized patients in Xi'an, China. RESULTS: We isolated and cultured 156 strains of C. difficile, representing 12.67% of the 1231 inpatient stool samples collected. Among the isolates, tcdA + B + strains were predominant, accounting for 78.2% (122/156), followed by 27 tcdA-B + strains (27/156, 17.3%) and 6 binary toxin gene-positive strains. The positive rates of three regulatory genes, tcdC, tcdR, and tcdE, were 89.1% (139/156), 96.8% (151/156), and 100%, respectively. All isolates were sensitive to metronidazole, and the resistance rates to clindamycin and cephalosporins were also high. Six strains were found to be resistant to vancomycin. CONCLUSION: Currently, the prevalence rate of C. difficile infection (CDI) in Xi'an is 12.67% (156/1231), with the major toxin genotype of the isolates being tcdA + tcdB + cdtA-/B-. Metronidazole and vancomycin were still effective drugs for the treatment of CDI, but we should pay attention to antibiotic management and epidemiological surveillance of CDI.

Genomic and phenotypic studies among Clostridioides difficile isolates show a high prevalence of clade 2 and great diversity in clinical isolates from Mexican adults and children with healthcare-associated diarrhea.

Clostridioides difficile (C. difficile) is widely distributed in the intestinal tract of humans, animals, and in the environment. It is the most common cause of diarrhea associated with the use of antimicrobials in humans and among the most common healthcare-associated infections worldwide. Its pathogenesis is mainly due to the production of toxin A (TcdA), toxin B (TcdB), and a binary toxin (CDT), whose genetic variants may be associated with disease severity. We studied genetic diversity in 39 C. difficile isolates from adults and children attended at two Mexican hospitals, using different gene and genome typing methods and investigated their association with in vitro expression of toxins. Whole-genome sequencing in 39 toxigenic C. difficile isolates were used for multilocus sequence typing, tcdA, and tcdB typing sequence type, and phylogenetic analysis. Strains were grown in broth media, and expression of toxin genes was measured by real-time PCR and cytotoxicity in cell-culture assays. Clustering of strains by genome-wide phylogeny matched clade classification, forming different subclusters within each clade. The toxin profile tcdA+/tcdB+/cdt+ and clade 2/ST1 were the most prevalent among isolates from children and adults. Isolates presented two TcdA and three TcdB subtypes, of which TcdA2 and TcdB2 were more prevalent. Prevalent clades and toxin subtypes in strains from children differed from those in adult strains. Toxin gene expression or cytotoxicity was not associated with genotyping or toxin subtypes. In conclusion, genomic and phenotypic analysis shows high diversity among C. difficile isolates from patients with healthcare-associated diarrhea. IMPORTANCE: Clostridioides difficile is a toxin-producing bacterial pathogen recognized as the most common cause of diarrhea acquired primarily in healthcare settings. This bacterial species is diverse; its global population has been divided into five different clades using multilocus sequence typing, and strains may express different toxin subtypes that may be related to the clades and, importantly, to the severity and progression of disease. Genotyping of children strains differed from adults suggesting toxins might present a reduced toxicity. We studied extensively cytotoxicity, expression of toxins, whole genome phylogeny, and toxin typing in clinical C. difficile isolates. Most isolates presented a tcdA+/ tcdB+/cdt+ pattern, with high diversity in cytotoxicity and clade 2/ST1 was the most prevalent. However, they all had the same TcdA2/TcdB2 toxin subtype. Advances in genomics and bioinformatics tools offer the opportunity to understand the virulence of C. difficile better and find markers for better clinical use.

Unveiling the effect of interacting forecasted abiotic factors on growth and aflatoxin B1 production kinetics by Aspergillus flavus.

The aim was to decipher the temporal impact of key interacting climate change (CC) abiotic factors of temperature (30 vs 37 °C), water activity (aw; 0.985 vs 0.930) and CO2 exposure (400 vs 1000 ppm) on (a) growth of Aspergillus flavus and effects on (b) gene expression of a structural (aflD) and key regulatory gene (aflR) involved in aflatoxin B1 (AFB1) biosynthesis and (c) AFB1 production on a yeast extract sucrose medium over a period of 10 days. A. flavus grew and produced AFB1 very early with toxin detected after only 48 h. Both growth and toxin production were significantly impacted by the interacting abiotic factors. The relative expression of the aflD gene was significantly influenced by temperature; aflR gene expression was mainly modulated by time. However, no clear relationship was observed for both genes with AFB1 production over the experimental time frame. The optimum temperature for AFB1 production was 30 °C. Maximum AFB1 production occurred between days 4-8. Exposure to higher CO2 conditions simulating forecasted CC conditions resulted in the amount of AFB1 produced in elevated temperature (37 °C) being higher than with the optimum temperature (30 °C) showing a potential for increased risk for human/animal health due to higher accumulation of this toxin.

Capsaicin inhibitory effects on Vibrio cholerae toxin genes expression.

OBJECTIVE: Cholera is an acute secretory diarrhea caused by the Gram-negative bacterium, Vibrio cholerae mostly through production of cholera toxin (CT) and zonula occludens toxin (Zot). Isolates of V. cholerae have acquired resistance elements during the last decade. One of the most promising ways to treat resistant strains is to use antivirulence agents instead of killing the causative agent with conventional antibiotics. In this study, we examined whether different concentrations of capsaicin - the pungent fraction of red chili- can act as an antivirulence agent and inhibit V. cholerae toxin production. MATERIALS AND METHODS: Two standard strains namely, V. cholerae ATCC 14035 and V. cholerae PTCC 1611 were used in this study. Minimum Inhibitory Concentration (MIC) of capsaicin was determined by broth microdilution method. Based on MIC results, the bacteria were cultured in the presence of sub-MIC concentrations of capsaicin and a negative control without capsaicin. Real-time PCR (RT-PCR) was carried out to determine the expression level of V. cholerae toxin genes at each concentration. RESULTS: MIC test showed that 200 mg/mL of capsaicin in 2% dimethyl sulfoxide (DMSO) could inhibit the growth of the two standard strains of V. cholerae. The expression of V. cholerae toxin genes was significantly reduced following treatment with sub-MIC concentrations of capsaicin as assessed by RT-PCR. CONCLUSION: Capsaicin showed great inhibitory effect against cholera toxin and reduced Zot production in the tested strains of V. cholerae. The results showed promising insights into antivirulence effects of capsaicin.

Equistatin and equinatoxin gene expression is influenced by environmental temperature in the sea anemone Actinia equina.

We examined the gene expression levels of equinatoxin and equistatin in the sea anemone Actinia equina, when reared at varying environmental temperatures for five months. Both genes were significantly downregulated at 10 °C compared to 16 °C but showed no significant change at 22 °C. This provides the first evidence of an effect of temperature on gene expression, but with no effect of increasing temperatures such as those predicted due to climate change.

Small and Smaller-sRNAs and MicroRNAs in the Regulation of Toxin Gene Expression in Prokaryotic Cells: A Mini-Review.

Non-coding small RNAs (sRNAs) have been identified in the wide range of bacteria (also pathogenic species) and found to play an important role in the regulation of many processes, including toxin gene expression. The best characterized prokaryotic sRNAs regulate gene expression by base pairing with mRNA targets and fall into two broad classes: cis-encoded sRNAs (also called antisense RNA) and trans-acting sRNAs. Molecules from the second class are frequently considered as the most related to eukaryotic microRNAs. Interestingly, typical microRNA-size RNA molecules have also been reported in prokaryotic cells, although they have received little attention up to now. In this work we have collected information about all three types of small prokaryotic RNAs in the context of the regulation of toxin gene expression.

MicroRNA-like RNAs in plant pathogenic fungus Fusarium oxysporum f. sp. niveum are involved in toxin gene expression fine tuning.

MicroRNA-like RNAs (milRNAs) are short non-coding regulatory sRNAs which play an important role in regulating gene expression at the post-transcriptional level by targeting mRNAs for degradation or inhibiting protein translation. To explore the presence of milRNAs in Fusarium oxysporum f. sp. niveum (Fon) and analyze their expression at different propagules, two categories of sRNAs were identified from Fon hyphae and microconidia using illumina sequencing. A total of 650,960 and 561,114 unique sRNAs were obtained from the hyphae and microconidia samples. With a previously constructed pipeline to search for microRNAs, 74 and 56 milRNA candidates were identified in hyphae and microconidia, respectively, based on the short hairpin structure analysis. Global expression analysis showed an extensively differential expression of sRNAs between the two propagules. Altogether, 78 significantly differently expressed milRNAs were identified in two libraries. Target prediction revealed two interesting genes involved in trichothecene production, necrosis and ethylene-inducing peptide 1 (NEP1) biosynthesis and in silico analysis indicated that they were down-regulated by Fon-miR7696a-3p and Fon-miR6108a. The expression levels of these two milRNAs were further validated by qRT-PCR and the results were consistent. The negative correlation of the expression levels between these two milRNAs and their potential target genes imply that they play a role in trichothecene and NEP1 biosynthesis. And this negative regulation for toxin-related gene expression is more specific in microconidia. The present study provides the first large-scale characterization of milRNAs in Fon and the comparison between hyphae and microconidia propagules gives an insight into how milRNAs are involve in toxin biosynthesis.