转座子Tn916对固氮螺菌Azospirillum brasilense的接合诱变及氨分泌菌株的筛选

以携带质粒pAM120(Ap~r,Tc~r/Tn916)的大肠杆菌(E.coli CG120)为供体菌株,与受体菌巴西固氮螺菌(Azospirillum brasilense)采用滤膜接合法进行接合转移,在选择平板上得到具较高频率的接合子(10~(-5)/每个供体菌,选择四环素抗性)。从846株四环素抗性接合子中进一步用奈氏法筛选得到氨分泌突变株3株。在无氮培养基上,其氨分泌量可达7.5～14.0mmol/L。用乙炔还原法分析氨分泌突变株在不同浓度氮源上的固氮活力,发现20mmol/L NH Cl的存在不抑制其固氮活性,固氮活力与无氮条件下野生株的活力相差不多。无选择压力下细胞分裂50代后的稳定性实验证明,转座子Tn916在氨分泌突变株中的稳定性在50％～80％之间。以固氮螺菌氨分泌突变株为供体菌株,对E.coli HBX1进行反向接合转移实验,证实Tn916确实存在于氨分泌固氮螺菌接合子中。     

Demonstration and preliminary characterization of a regular array in the cell wall of Clostridium difficile

Abstract The presence of a regular array (RA) was demonstrated on the outer layer of the cell wall in Clostridium difficile GAI0714 by electron microscopy. The RA was composed of squarely arranged subunits with a center-to-center spacing of about 8.2 nm. The outer wall layer carrying the RA was isolated from the wall fragments of early log-phase cells by autolysis. The outer wall layer was composed of two main proteins with apparent M _rs of about 45 000 and 32 000 upon sodiumdodecylsul-fate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar RAs were also present in the cell walls of the other 9 strains of C. difficile . These strains were divided into two groups on the basis of the wall protein composition: one containing M _r 45 000–47 000 and 32 000 proteins and the other containing M _r 42 000 and 38 000 proteins.     

益生菌预防成人艰难梭菌相关性腹泻的Meta分析

目的 系统评价益生菌预防成人艰难梭菌相关性腹泻（Clostridium difficile associated diarrhea,CDAD）的临床疗效。方法 计算机检索PubMed、EMBASE、Cochrane、Web of Science、CBM、万方数据库、维普资讯网和中国知网全文数据库,纳入益生菌预防成人CDAD的随机对照试验（RCT）,采用随机效应模型进行Meta分析。结果 共有13项符合纳入标准的随机对照试验,包括5 179名患者,其中干预组2 730例,对照组2 449例。Meta分析结果显示,预防性使用益生菌可降低CDAD的风险（RR=0.49,95％CI 0.33‒0.75,I2=16.4％）。亚组分析发现益生菌高剂量（RR=0.41,95%CI 0.22‒0.77,P=0.043）、混合菌种（RR=0.43,95%CI 0.24‒0.75,P=0.037）与对照组相比,差异均有统计学意义。结论 辅助性益生菌的应用能够有效预防成人艰难梭菌相关性腹泻的发生。但受纳入研究数量较少与研究质量的参差不齐的影响,本结论尚需要开展更多高质量、大样本的研究予以证实。     

Comparison of methods for the production and purification of toxin A from Clostridium difficile

Abstract The production and purification of toxin A from Clostridium difficile were studied. When the toxin was produced in dialysis culture it preicipitated quantitatively at pH 5.5 and after purification it appeard homogeneous in polyacrylamide gel electrophoresis (PAGE). The toxin probably consists of two noncovalently bound peptides, each with a molecular mass of about 250 dDa. It is resistant to trypsin but sensitive to papain and chymotrypsin. In contrast, toxin A produced in anaerbic chamber culture precipitated poorly at pH 5.5 (yield 14%) and easily formed aggregates as observed in gel filtration and PAGE Accordingly, dialysis culture seems to be a better method for producing and purifying toxin A.     

ICU患者艰难梭菌分离培养与流行病学特征研究

目的 了解ICU患者艰难梭菌的定植和感染情况,为预防艰难梭菌的流行提供参考。方法 收集2016年9月至2017年6月福建医科大学附属第一医院ICU中139例住院时间＞7 d的患者的粪便样本,对其进行选择性厌氧培养和质谱鉴定。对艰难梭菌培养阳性标本进行毒素基因（tcdA、tcdB、cdtA、cdtB）的PCR检测以及毒素A、B表型检测。收集所有患者的临床资料,并对艰难梭菌培养阳性患者的临床特征和实验室检查结果进行单因素分析和多因素回归分析。结果 艰难梭菌检出率为17.27%（24/139）。其中,14株艰难梭菌的tcdA和tcdB基因检测阳性,占58.3%（14/24）;10株为tcdA和tcdB基因检测阴性,占41.7%（10/24）。所有菌株二元毒素基因（ctdA/ctdB）均未检出。单因素分析提示,高龄、长时间住院、高淋巴细胞数、使用β-内酰胺类抗生素是艰难梭菌定植的高危因素;多因素回归分析提示,使用β-内酰胺类抗生素是艰难梭菌定植的独立危险因素（OR=3.881,P=0.039）。结论 我院ICU可能存在艰难梭菌感染和传播的风险,对具有高龄、长期住院以及使用抗生素等高危因素的患者应进行艰难梭菌的监测,以防艰难梭菌的传播、感染和艰难梭菌相关性腹泻的发生。     

A novel multi‐strain probiotic and synbiotic supplement for prevention of Clostridium difficile infection in a murine model

The protective effect of a multi‐strain probiotic and synbiotic formulation was evaluated in C57BL/6 mice infected with Clostridium difficile (CD) NAP1/027. Antibiotic‐treated mice were divided into the following four groups: Group 1, fed with a synbiotic formulation consisting of Lactobacillus plantarum F44, L. paracasei F8, Bifidobacterium breve 46, B. lactis 8:8, galacto‐oligosaccharides, isomalto‐oligosaccharides, and resistant starch; Group 2, fed with the same four probiotic strains as Group 1; Group 3, fed with the same prebiotic supplements as Group 1 for 7 days before CD infection; and Group 4 (control group) antibiotic treated and infected with NAP1/027 strain. Feces and cecal contents were collected for microbial cell viability, quantitative PCR (qPCR), toxin analyses and histopathology. Synbiotics‐ and probiotics‐fed mice showed a significant increase in total bifidobacteria (P < 0.05). The total lactobacilli count was increased in Group 1. Tests for cecal toxins were negative in Group 2 mice, whereas one sample each from Group 1 and 3 was positive. qPCR of cecal contents showed significant reduction in NAP1/027 DNA copies in Groups 1 and 2 and significantly higher numbers of B. breve 46, L. plantarum F44, and L. paracasei F8 in Groups 1 and 2 (P < 0.05); these changes were much less pronounced in Groups 3 and 4. Our findings indicate that the newly developed synbiotic or multi‐strain probiotic formulation confers protection against NAP1/027 infection in C57BL/6 mice. This holds promise for performing human studies.     

Detection of the ADP-ribosyltransferase toxin gene (cdtA) and its activity in Clostridium difficile isolates from Equidae

Clostridium difficile is an antibiotic-associated emerging pathogen of humans and animals. Thus far three toxins of C. difficile have been described: an enterotoxin (ToxA), a cytotoxin (ToxB) and an ADP-ribosyltransferase (CDT). In the present work we describe the first isolation of CDT producing C. difficile from Equidae with gastro-intestinal disease. Out of 17 C. difficile strains isolated from Equidae, 11 were positive for the genes tcdA and tcdB encoding ToxA and ToxB. In addition four of these 11 isolates were positive for the cdtA gene encoding the catalytic subunit of the ADP-ribosyltransferase CDT. Interestingly none of the isolates derived from canines (41 isolates) and felines (4 isolates) harboured the cdtA gene. In C. difficile field isolates which contained the cdtA gene, ADP-ribosyltransferase activity could also be detected in culture supernatants indicating expression and secretion of CDT. All strains were associated with intestinal disorders, but no association was found for the occurrence of toxins with a specific clinical diagnosis.     

新鲜粪菌移植在治疗艰难梭菌感染性腹泻方面的疗效及安全性的Meta 分析

目的 应用循证研究方法评价新鲜粪菌移植在治疗艰难梭菌感染相关性腹泻方面的效果及安全性。 方法 计算机检索中国生物医学文献数据库、中国学术期刊全文数据库、万方数据库、 PubMed、Embase、Web of Science、The Cochrane Library数据库,搜集关于新鲜粪菌移植治疗艰难梭菌感染的随机对照实验,检索时限均为建库至2019年7月30日。由2名研究者按照纳入和排除标准进行文献筛选、数据提取和质量评价,采用RevMan 5.3软件进行Meta分析。 结果 最终纳入7个RCT,共543名患者。Meta分析结果显示:新鲜FMT比传统方法在治疗CDI引起的腹泻方面更有效(OR=6.70,95%CI:1.64～27.43,P=0.040),敏感性分析显示:此结果敏感性较低,结果较为稳定可信。多次输注方式比起单次输注在治愈率方面更显优势(OR=-0.09,95%CI:0.04～3.13,P结论 新鲜粪菌移植能明显提高艰难梭菌感染性腹泻的治愈率,且在不同输注方式方面,多次输注更能提高治愈率,且不良反应较少,与FTM无直接关系,有一定的临床推广价值。     

Effects of Gibberellic Acid on Primary Terpenoids and Δ9-Tetrahydrocannabinol in Cannabis sativa at Flowering Stage

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. However, little is known about the effects of plant hormones on the regulation of these pathways. In the present study we investigated the effect of gibberellic acid (GA₃) on changes in the amounts of many produced terpenoids and the activity of the key enzymes, 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), in these pathways. Our results showed GA₃ caused a decrease in DXS activity in both sexes that it was accompanied by a decrease in chlorophylls, carotenoids and Δ⁹-tetrahydrocannabinol (THC) contents and an increase in α-tocopherol content. The treated plants with GA₃ showed an increase in HMGR activity. This increase in HMGR activity was followed by accumulation of stigmasterol and β-sitosterol in male and female plants and campestrol in male plants. The pattern of the changes in the amounts of sterols was exactly similar to the changes in the HMGR activity. These data suggest that GA₃ can probably influence the MEP and MVA pathways oppositely, with stimulatory and inhibitory effects on the produced primary terpenoids in MVA and DXS pathways, respectively.     

Cloning and Functional Expression of cDNA Encoding Pheromone Δ9 Acyl-CoA Desaturase of the Tobacco Cutworm, Spodoptera litura (Lepidoptera: Noctuidae)

ABSTRACT A cDNA encoding pheromone Δ9 acyl-CoA desaturase, Slit KPSE was isolated from sex pheromone gland of the tobacco cutworm, Spodoptera litura which uses a diene unsaturated fatty acid (UFA) derivative, Z9E11-14 : 2 as a major pheromone component. The fulllength open reading frame coding region of Slit KPSE was inserted in a yeast shuttle vector, YEpOLEX, and two kinds of yeast ( Saccharomyces cerevisiae ) mutant strains were transformed with the recombinant vector. In the desaturase-deficient ole 1 strain, Slit KPSE expressed a complementary enzyme producing two kinds of diene UFAs, more 9–16 : 1 and less 9–18 : 1 at a ratio of 1 : 0.74 exhibiting a typical functional characteristics as one of the pheromone Δ9 acyl-CoA desaturase lineage group, KPSE, but no Δ9 14C monoene was detectable because of too small amount of 14C saturated fatty acid precursor to be reliably used by Slit KPSE in the transformed cells. However, the another transformed yeast strain elo 1 which is deficient of elongase 1, an enzyme converting 14C to 16C hydrocarbon substrate, was supplemented with some myristic acid (14 : 0) in the medium, and produced a significant amount of 9–14 : 1 in due to a much enhanced level of the 14C substrate suggesting that Slit KPSE may be responsible for making the Δ9 double bond on the diene pheromone component.     

Interaction of the chromosomal Tn557 with two thermosensitive derivatives, pS1 and p ΔD, of the plasmid pI9789 in Staphylococcus aureus

Abstract The plasmid pI9789::Tn552 carries genes conferring resistance to penicillins and to cadmium, mercury and arsenate ions. The presence of Tn551 at one location in the chromosome of Staphylococcus aureus enhances the frequency of suppression of thermosensitivity of replication of the plasmids pSl and pΔD which are derivatives of pI9789::Tn552. Bacteriophage propagated on the bacteria in which thermosensitivity of replication had been suppressed was used to transduce cadmium resistance to S. aureus PS80N. The cadmium-resistant transductants obtained carried plasmid pS1 or p ΔD with a copy of Tn551 inserted into a specific site on pSl but into several different sites on p ΔD. The possible mechanisms of the suppression are discussed.     

Inhibition of glucose transport by phenylglyoxal: Both dissipation of ΔpH and essential arginyl residues are involved

The effects of the arginine modifying reagent phenylglyoxal (PGO) on solute transport was studied in two cellular systems: protoplasts isolated from the mesophyll of Vicia faba L. and XD cell suspension culture of Nicotiana tabacum L. cv. Xanthi. The solutes in the case of the protoplasts were the non‐metabolizable glucose analog 3‐O‐methyl‐D‐glucose (MeG), and a non‐metabolizable amino acid analog α‐aminoisobutyric acid (AIB), whereas the solutes for the cell suspension were AIB and nitrate. Solute transport in both systems was rapidly inhibited by PGO. Exposure of the protoplasts to light enhanced the initial rate of MeG uptake. PGO rapidly inhibited MeG uptake in both the light and the dark, the half‐time for inactivation being less than 3 min. Flux analysis of double‐labeled MeG showed that initial MeG uptake was mediated mainly by the plasma membrane transport system and that it was inhibited by PGO. Maximal inhibition of initial MeG uptake rate was observed at PGO concentrations of 1 m M and above. PGO treatment altered rapidly the equilibrium distribution of the ΔpH probe dimethyloxazolidine (DMO) in both cellular systems, indicating dissipation of ΔpH between cell and medium. In the protoplasts, PGO inhibited both DMO and MeG uptake at pH 5.5; however, at pH 7.0, where ΔpH is minimal, only MeG uptake was inhibited. Our results suggest that PGO has two effects on glucose uptake: an indirect effect through ΔpH dissipation and a direct effect through interaction with essential arginyl residues in the glucose transporter.     

Cloning and expression of the thermostable α-amylase gene from Clostridium thermosulfurogenes (DSM 3896) in Escherichia coli

Abstract The gene coding for a thermostable α-amylase from Clostridium thermosulfurogenes (DSM 3896) was cloned in Escherichia coli using pUC18 as a vector. The recombinant plasmid pCT2 of an amylolytic positive transformant of E. coli contained a 2.9 kbp fragment of chromosomal DNA of C. thermosulforogenes carrying the α-amylase gene. In E. coli the gene was apparently transcribed by its own promoter. Comparative studies showed no difference between the original and the heterologously in E. coli expressed enzyme. The latter was not secreted into the medium.     

A nonsense mutation abrogates production of a functional enterotoxin A in Clostridium difficile toxinotype VIII strains of serogroups F and X

Clostridium difficile strains of toxinotype VIII from serogroups F and X are described as toxin B-positive, toxin A-negative (TcdB+ A-), although they harbour almost the entire tcdA gene. To identify the reason for the lack of TcdA detection, we analyzed catalytic and ligand domains of TcdA-1470 of the type strain of serogroup F, strain 1470. Using recombinant fragments, the C-terminal immunodominant ligand domain TcdA3-1470, spanning amino acid residues 1694-2711 (corresponding to VPI 10463 sequence), was detected in Western blots. Similar experiments using the recombinant N-terminal catalytic fragment TcdAc1-2-1470 (amino acid positions 1-544) failed. In addition, this fragment showed no glucosylation activity. We determined the size and the position of alterations in the ligand domain tcdA3-1470 by DNA sequencing. Within the N-terminal fragment tcdAc1-2-1470, a nonsense mutation was identified introducing a stop codon at amino acid position 47. Identical mutations were found in the two serogroup X strains 17663 and 10355. The mutation might explain the lack of TcdA production observed in strains of serotypes F and X.     

Expression and regulation of the lactose transposon Tn951 in Rhizobium spp.

Abstract The expression of β-galactosidase by the lac transposon Tn 951 , in Escherichia coli , was found to be cAMP-dependent. This finding provided the basis for an investigation of the effect of cAMP on Tn 951 lac expression in Rhizobium , with the ultimate aim of using the Tn 951 system as a specific probe for cAMP mediated catabolite repression. When introduced into Rhizobium , Tn 951 directed the synthesis of β-galactosidase, which was inducible by isopropyl-β- d -thiogalactopyranoside (IPTG). Marked quantitative and qualitative differences in β-galactosidase expression were found between R. meliloti and R. japonicum during the growth cycle, with expression being higher in the former. β-Galactosidase levels were, however, unaffected by exogenous cAMP under catabolite repressing conditions.