Transcriptomic analysis of the ΔPaLoc mutant of Clostridioides difficile and verification of its toxicity / 中华预防医学杂志

Objective: Comparative analyses of wild-type Clostridioides difficile 630 (Cd630) strain and pathogenicity locus (PaLoc) knockout mutant (ΔPaLoc) by using RNA-seq technology. Analysis of differential expression of Cd630 wild-type strain and ΔPaLoc mutant strain and measurement of its cellular virulence changes. Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioides difficile. Methods: Analysis of Cd630 and ΔPaLoc mutant strains using high-throughput sequencing (RNA-seq). Clustering differentially expressed genes and screening differentially expressed genes by DESeq software. Further analysis of differential genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, cytotoxicity assays of ΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell (Vero) and the human colonic cell (Caco-2) lines. Results: The transcriptome data showed that the ΔPaLoc mutant toxin genes tcdA and tcdB were not transcribed. Compared to the wild-type strain, CD630_36010, CD630_020910,CD630_02080 and cel genes upregulated 17.92,11.40,8.93 and 7.55 fold, respectively. Whereas the hom2 (high serine dehydrogenase), the CD630_15810 (spore-forming protein), CD630_23230 (zinc-binding dehydrogenase) and CD630_23240 (galactitol 1-phosphate 5-dehydrogenase) genes were down-regulated by 0.06, 0.075, 0.133 and 0.183 fold, respectively. The GO and KEGG enrichment analyses showed that the differentially transcribed genes in ΔPaLoc were enriched in the density-sensing system, ABC transport system, two-component system, phosphotransferase (PTS) system, and sugar metabolism pathway, as well as vancomycin resistance-related pathways. Cytotoxicity assays showed that the ΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain. Conclusion: Transcriptional sequencing analysis of the Cd630 and ΔPaLoc mutant strains showed that the toxin genes were not transcribed. Those other differential genes could provide a reference for further studies on the physiological and biochemical properties of the ΔPaLoc mutant strain. Cytotoxicity assays confirmed that the ΔPaLoc mutant lost virulence to Vero and Caco-2 cells, thus laying the foundation for constructing an toxin-attenuated vaccine strain against C. difficile.

New Biotransformation Mode of Zearalenone Identified in Bacillus subtilis Y816 Revealing a Novel ZEN Conjugate.

An increasing number of Bacillus strains have been identified, and the removal capacity of zearalenone (ZEN) was determined; however, they failed to reveal the detoxification mechanism and transformation product. Here, Bacillus subtilis Y816, which could transform 40 mg/L of ZEN within 7 h of fermentation, was identified and studied. First, the biotransformation products of ZEN and 17-ß-estradiol (E2) were identified as ZEN-14-phosphate and E2-3-phosphate by HPLC-TOF-MS and NMR, respectively. An intracellular zearalenone phosphotransferase (ZPH) was found through transcriptome sequencing analysis of B. subtilis Y816. The phosphorylated reaction conditions of ZEN by ZPH were further revealed in this work. Furthermore, the phosphorylated conjugates showed reduced estrogenic toxicity compared with their original substances (ZEN and α/ß-zearalenol) using an engineered yeast biosensor system. The first report on the phosphorylated conjugated mode of ZEN in B. subtilis Y816 will inspire new perspectives on the biotransformation of ZEN in Bacillus strains.

Application and evaluation of multiple cross displacement amplification in detection of Listeria monocytogenes / 中国人兽共患病学报

Listeria monocytogenes is a significant food-borne pathogen that is capable of causing severe listeriosis in both humans and animals.Traditional techniques such as culture-based approaches have good specificity and sensitivity,while they are time-consuming and tend to be tedious.Hence,we presented a simple and rapid molecular technique using multiple cross displacement amplification (MCDA) targeting L.monocytogenes-specific gene lmo0733 for sensitive and specific detection of the pathogen.L.monocytogenes-MCDA assay used a set of ten primers spanning ten distinct regions of target sequence and was carried out at a constant temperature 61 ℃ to evaluate the specificity,sensitivity and feasibility.A total of three methods were used to confirm MCDA products,including color change of Loopamp Fluorescent Detection Reagent,real-time measurement of turbidity and agarose gel electrophoresis.The limit of detection (LoD) of the MCDA was 10 fg DNA,which was 25-fold and 250-fold more sensitive than that of the LAMP and CPA assay for detecting L.monocytogenes DNA,respectively.The detection ability of MCDA method was identical to the culture-biotechnical method for 153 rat intestinal feces samples,and was better than LAMP,CPA and conventional PCR.The results in this study indicated that the MCDA assay could be used as a rapid,sensitive and effective tool for the detection of L.monocytogenes in the food industry,medical institutions and field.

Structure and sequence analysis-based engineering of pullulanase from Anoxybacillus sp. LM18-11 for improved thermostability.

Pullulanase (EC 3.2.1.41) is a well-known starch-debranching enzyme. Enhancing the thermostability of Pullulanase is required for industrial application. In this study, we used two methods to improve the thermostability of the pullulanase from Anoxybacillus sp. LM18-11; these methods were the modified amino acid consensus method combined with the analyses of the residue water-exposed surface (ACC) and the deletion of flexible domains. Four mutants (Y477A, Y175C, L215C and R473E) were obtained via the modified consensus method exhibited varying degrees of improvements in terms of thermostability. One deletion mutant termed D3 (residues(686-688)) was obtained and exhibited enhanced thermostability due to deletion of the flexible region at the C-terminus. The combination of the two strategies yielded the mutant M18 (Y477A/D3/Y175C/L215P/R473E). It retained 66% of its initial activity after incubation at 60 °C for 72 hrs, whereas that of the wild-type enzyme was only 35%. After incubation at 65 °C for 4 h, M18 retained 50.6% of its initial activity, whereas that of the wild-type was only 16.8%, respectively. Additionally, kinetic studies revealed that the Km of M17 (Y477A/D3/Y175C/L215P) was decreased by 33.9% and that the Kcat/Km value of M17 increased by 50%, while M18 exhibited Km and Kcat/Km values that were similar to those of the wild-type enzyme. The attractive improved thermostability and the high catalytic efficiency made M17 and M18 more suitable for industrial application.

Direct cloning, expression of a thermostable xylanase gene from the metagenomic DNA of cow dung compost and enzymatic production of xylooligosaccharides from corncob.

OBJECTIVES: To acquire a thermostable xylanase, that is suitable for xylooligosaccharide production from pretreated corncobs, the metagenomic method was used to obtain the gene from an uncultured environmental microorganism. RESULTS: A thermostable xylanase-encoding gene (xyn10CD18) was cloned directly from the metagenomic DNA of cow dung compost. When xyn10CD18 was expressed in Bacillus megaterium MS941, extracellular xylansae activity at 106 IU/ml was achieved. The purified recombinant Xyn10CD18 was optimally active at pH 7 and 75 °C as measured over 10 min. It retained over 55% of its initial activity at 70 °C and pH 7 after 24 h. Its action on birchwood xylan for 18 h liberated xylooligosaccharides with 2°-4° of polymerization, with xylobiose and xylotetraose as the main products. When pretreated corncobs were hydrolyzed by Xyn10CD18 for 18 h, the xylooligosaccharides (DP 2-4) products increased to 80% and the xylose was just increased by 3%. CONCLUSION: Xyn10CD18 is a thermostable endoxylanase and is a promising candidate for biomass conversion and xylooligosaccharide production.

Purification and characterization of a thermostable xylanase from Paenibacillus sp. NF1 and its application in xylooligosaccharides production.

High levels of extracellular xylanase activity (211.79 IU/mg) produced by Paenibacillus sp. NF1 were detected when it was submerged-cultured. After three consecutive purification steps using Octyl-Sepharose, Sephadex G75, and Q-Sepharose columns, a thermostable xylanase (XynNF) was purified to homogeneity and showed a molecular mass of 37 kDa according to SDS-PAGE. The specific activity of the purified XynNF was up to 3,081.05 IU/mg with a 14.55-fold purification. The activity of XynNF was stimulated by Ca(2+), Ba(2+), DTT, and ß-mercaptoethanol, but was inhibited by Fe(3+), Zn(2+), Fe(2+), Cu(2+), SDS, and EDTA. The purified XynNF displayed a greater affinity for oat spelt xylan with the maximal enzymatic activity at 60°C and pH 6.0. XynNF, which was shown to be cellulose-free, with high stability at high temperature (70°C-80°C) and low pH range (pH 4.0-7.0), is potentially valuable for various industrial applications. The end products of high efficient oat spelt xylan hydrolysis by XynNF (an endoxylanase) containing 95.8% xylooligosaccharides of 2-4 degree of polymerization (DP2-4) with the enrichment of xylobiose (61.5%) indicated that XynNF is a promising candidate for xylooligosaccharides production.

Penetrability of amikacin into prostate tissues in rat models of chronic bacterial prostatitis / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish the rat model of chronic bacterial prostatitis and investigate the penetrability of amikacin to chronic inflammatory prostatic tissues.</p><p><b>METHODS</b>A total of 180 male rats were randomly divided into a normal control group (NC, n=48), a chronic bacterial prostatitis group (CBP, n = 84) and a CBP treatment group (CBPT, n = 48). The prostates of the animals were injected with Xiaozhiling and E. coli respectively to make CBP and CBPT models. The animals of the CBPT group were treated with amikacin by intramuscular injection, their prostate tissues and sera isolated at 1-150 min after the treatment and detected for antibiotic activities, bacteria counts and pathological changes.</p><p><b>RESULTS</b>Obvious chronic inflammatory pathological changes including leukocyte invasion and fibre hyperplasia were observed and E. coli isolated from the prostate tissues of the rats in the CBP and CBPT groups, but no pathological changes, antibiotic activity and bacteria were detected in the the NC group. The numbers of E. coli in the prostate tissues markedly decreased with the time in the two model groups, 30 CFU/mg in the CBP rats at 28 days and 0 CFU/mg in the CBPT group at 10 days after the treatment. Obvious antibiotic activities were found in both the prostate tissues and sera at 2-150 min after the injection. No antimicrobial activities were detected at 12 hours after the treatment either in the sera or in the prostate samples. With the increase of the treatment time and decrease of the bacteria counts, the chronic inflammatory pathological changes were obviously alleviated in the CBPT group.</p><p><b>CONCLUSION</b>Rat models of CBP with chronic inflammatory pathological changes can be successfully established by direct injection of Xiaozhiling and E. coli into the prostate. Amikacin, given by intramuscular injection, can penetrate into the prostate of the CBP rat and produce an antibiotic activity equal to or higher than that of the sera, which may kill sensitive bacteria in the prostate and help to reduce the inflammatory pathological changes and repair the damage to the prostate tissues.</p>

Penetrability and therapeutic effect of vancomycin to the prostates of rats with bacterial prostatitis (BP) or BPH-BP / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the penetrability and therapeutic effect of vancomycin to the prostates of rats with bacterial prostatitis (BP) or benign prostate hyperplasia (BPH)-BP.</p><p><b>METHODS</b>The experimental rats with BP or BPH-BP were injected with vancomycin through the tail vein. The prostate tissues and sera were isolated respectively from the rats at 10 min to approximately 24 h after treatment and the antibiotic activities of the samples were detected by serial dilution test and agar diffusion test. The rats with BP or BPH-BP were treated with vancomycin by intravenous injection daily for 5 days. The prostates were collected the second day after injection and bacteria were isolated and determined. One to five weeks after treatment, the prostates of the animals were isolated and pathologic tests were done.</p><p><b>RESULTS</b>No bacteria could be isolated from the prostates of the normal rats, but positive isolation was achieved from the prostates of the infected animals 28th day after infection. In the first 4 days after treatment, a decrease of bacteria could be detected in the prostate samples of the rats treated with BP or BPH-BP. After 5th day, no bacteria could be detected from 91.7% prostates of the treated groups. Obvious antibiotic activity in both sera and prostates could be detected 10 to approximately 150 min after the antibiotic injection. Antibiotic activity of the prostate tissues could be lower or higher than or equal to that of the sera in the same period. Pathologic tests detected obvious exudation and leukocyte invasion in the prostate tissues of the BP rats and gland proliferation in the BPH rats. Vancomycin treatment and the consequent reduction of bacteria obviously alleviated the inflammatory pathological changes in the prostates of the BP rats.</p><p><b>CONCLUSION</b>Vancomycin given intravenously has more penetrability to the prostates of either BP or BPH-BP rats. The antibiotic concentration in the prostate tissues may be equal to or higher than that of the sera, so that the susceptive bacteria in the prostates will be killed and the alleviation of the inflammation and repair of the tissues accelerated effectively.</p>