艰难梭菌相关性腹泻的治疗进展及对我国的启示

艰难梭菌相关性腹泻（Clostridium difficile associated diarrhea,CDAD）是艰难梭菌感染（Clostridium difficile Infection,CDI）引起的一种机体严重疾病。随着艰难梭菌感染率的增加及高毒力株027／NAP1／BI的出现,导致该病在全球特别是北美、及欧洲等地区爆发性流行,对于该病的控制引起全球研究者的高度重视。本文就其治疗进展进行综述,并结合我国实际分析该病防治中存在的问题并提出建议。     

Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway

Solventogenic clostridia are well-known since almost a century due to their unique capability to biosynthesize the solvents acetone and butanol. Based on recently developed genetic engineering tools, a targeted 3-hydroxybutyryl-CoA dehydrogenase (Hbd)-negative mutant of Clostridium acetobutylicum was generated. Interestingly, the entire butyrate/butanol (C₄) metabolic pathway of C. acetobutylicum could be inactivated without a severe growth limitation and indicated the general feasibility to manipulate the central fermentative metabolism for product pattern alteration. Cell extracts of the mutant C. acetobutylicum hbd::int(69) revealed clearly reduced thiolase, Hbd and crotonase but increased NADH-dependent alcohol dehydrogenase enzyme activities as compared to the wildtype strain. Neither butyrate nor butanol were detected in cultures of C. acetobutylicum hbd::int(69), and the formation of molecular hydrogen was significantly reduced. Instead up to 16 and 20 g/l ethanol were produced in glucose and xylose batch cultures, respectively. Further sugar addition in glucose fed-batch fermentations increased the ethanol production to a final titer of 33 g/l, resulting in an ethanol to glucose yield of 0.38 g/g.     

住院腹泻患者艰难梭菌快速检测结果分析

目的 了解住院腹泻患者艰难梭菌带菌情况,为抗生素相关腹泻患者的诊断和治疗提供参考。方法 收集2015年9月至2016年8月住院腹泻患者粪便标本163份,用艰难梭菌快速检测试剂盒检测艰难梭菌。结果 艰难梭菌检出总阳性率为20.86%（34/163）,其中春季16.67%、夏季15.79%、秋季17.86%、冬季27.87%,经比较差异无统计学意义（2=2.943,P＞0.05）;成人粪便标本阳性率为22.52%（25/111）,儿童粪便标本阳性率为17.31%（9/52）,差异无统计学意义（2=0.583,P＞0.05）;男性粪便标本阳性率为21.69%（18/83）,女性粪便标本阳性率为20.0%（16/80）,差异无统计学意义（2=0.202,P＞0.05）;艰难梭菌阳性患者抗菌药物平均使用天数为12.74 d,阴性患者为8.21 d,差异有统计学意义（t=－7.81,P＜0.01）。结论 住院腹泻患者艰难梭菌阳性率高低与抗菌药物使用时间长短有关。     

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.     

Variation in the surface layer proteins of Clostridium difficile

Surface layers (S-layers) form regular crystalline structures on the outermost surface of many bacteria. Clostridium difficile possesses such an S-layer consisting of two protein subunits. Treatment of whole cells of C. difficile with 5 M guanidine hydrochloride revealed two major proteins of different molecular masses characteristic of the S-layer on SDS-PAGE. In this study 25 isolates were investigated. A high degree of variability in the molecular mass of the two S-layer proteins was evident. Molecular masses ranged from 48 to 56 kDa for the heavier protein and from 37 to 45 kDa for the lighter protein. A further protein component of 70 kDa was detectable in all isolates. No cross-reaction was seen between the two major proteins from isolates that produced different S-layer patterns, and most S-layer proteins from isolates with the same or similar banding patterns did not cross-react. The S-layer proteins, when detected by a combination of Coomassie blue staining and immunoblotting, are a useful marker for phenotyping.     

婴幼儿艰难梭菌的无症状定植与防治措施研究进展

艰难梭菌是一种肠道条件致病菌,能形成芽胞有效抵抗抗生素的杀灭.由于临床上抗生素的不规范使用,导致人体肠道有益菌被杀死.艰难梭菌因抵抗力强而大量繁殖,破坏人体肠道菌群平衡,引起抗生素相关性腹泻.艰难梭菌同时也是院内感染的主要病原菌.艰难梭菌可在人体肠道无症状定植,不同年龄阶段艰难梭菌定植率有较大差异,相比于成人,婴幼儿时...     

Evaluation of the proposed interaction of nucleic acid with Clostridium difficile toxins A and B and the effects of nucleases on cytotoxicity

Both DNA and RNA were found to co-purify with Clostridium difficile toxin B but not toxin A. DNAase treatment greatly reduced the cytotoxicity of toxin B but not of toxin A. RNAase had no effect on either toxin. The effects on toxin B were shown to be due to a contaminating protease and could be inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride.     

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.     

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。     

Effect of Arginine on Toxin Production by Clostridium difficile in Defined Medium

Twenty strains of Clostridium difficile were examined for the effect of arginine on toxin production in a defined medium. In three strains, the production of toxins A and B was greatly enhanced in the absence of arginine. These strains showed distinctively poorer growth in the absence of arginine in comparison with the remaining 17 strains, indicating that the presence of arginine is required for good growth among the three strains. From the present results, test strains were divided into two groups: a group in which arginine insufficiency caused distinctly poor growth and enhanced toxin production, and another group in which there was neither distinctly poor growth nor enhanced toxin production. The phenomenon is discussed in relation to the biosynthesis and catabolism of arginine.     

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.     

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.     

Transfer of Tn916 and Tn916ΔE into Clostridium difficile: demonstration of a hot-spot for these elements in the C. difficile genome

The conjugative transposon Tn916 and a derivative Tn916 delta E was transferred from Bacillus subtilis into Clostridium difficile CD37 by filter mating. All the C. difficile transconjugants appeared to contain one copy of the transposon integrated into the same position in the genome. Transposition from the original site of integration was not observed. Like Tn916 the transferable tetracycline resistance determinant (Tc-CD) of C. difficile has a preferred site of integration in C. difficile and is homologous with Tn916 along the whole length of Tn916. However comparisons of the distribution of TaqI and Sau3AI sites in the homologous regions of the two elements did not demonstrate any hybridizing fragments in common.     

Genes encoding peroxisomal enzymes are not necessarily assigned on the same chromosome of an n-alkane-utilizable yeast Candida tropicalis

We have resolved eight chromosomal bands from an n-alkane-assimilating yeast, Candida tropicalis pK 233, by using contour-clamped homogeneous electric field gel electrophoresis (CHEF). From the results of hybridization of DNA probes of yeast peroxisomal enzymes--catalase, acyl-CoA oxidase, carnitine acetyltransferase, isocitrate lyase, malate synthase, acetoacetyl-CoA thiolase, and 3-ketoacyl-CoA thiolase--to Southern transfers of CHEF gels, these genes were proven not necessarily to be located on the same chromosome. This fact shows that the genes encoding the enzymes tested were not distributed to be cistronic, although simultaneous and inducible synthesis of peroxisomal enzymes occurred in harmony with the proliferation of peroxisomes, suggesting that their co-ordinated expression might be mainly regulated by certain trans-acting factors.     

The internal pH of Clostridium acetobutylicum and its effect on the shift from acid to solvent formation

Clostridium acetobutylicum was unable to keep a constant pH inside the cells when grown on a phosphatelimited synthetic medium which allowed production of organic acids in a first phase and of solvents in a second phase. At external pH values between 5.9 and 4.3, the cells kept a constant pH of 0.9 to 1.3. A similar pH was measured in continuous culture under solventproducing conditions. The pH was abolished by protonovorous uncouplers, such as tetrachlorosalicylanilide (TCS) or carbonyl-p-trifluormethoxyphenylhydrazone (FCCP). n-Butanol at concentration of 150 mM and above led also to a complete abolition of the pH gradient.The internal pH stayed above 5.5 in cultures that shifted from acid to solvent formation. It is concluded that this is a prerequisite for the shift. The possible function of high internal concentrations of butyrate, butyryl phosphate and butyryl coenzyme A in the triggering mechanisms of the shift is discussed.Abbreviations TCS Tetrachlorosalicylanilide - FCCP carbonyl-p-trifluormethyoxyphenylhydrazone