Case of Clostridium perfringens bacteremia after routine colonoscopy and polypectomy

Bacteremia is an uncommon complication after polypectomy and colonoscopy. We report one of the first cases of Clostridium perfringens bacteremia after polypectomy. Our patient was a four years old boy with congenital polyposis, who underwent colonoscopy and polypectomy without complication. Approximately 12 h later he developed a fever and tachycardia with no other clinical symptoms. His blood cultures grew out penicillin susceptible C. perfringens and Enterococcus faecalis. He responded to antibiotic therapy and remained clinically asymptomatic for the duration of his course. There are a few reports of bacteremia after routine polypectomy, but no reported cases of C. perfringens bacteremia in the pediatric population. Clostridial sp. bacteremia can be fatal with devastating consequences if appropriate antibiotics and/or surgical debridement are delayed. Polymicrobial infection, as illustrated in our patient, is also common and can be a poor prognostic risk factor. Therefore, for patients with a history of polypectomy and new onset fever, anaerobic infections should be considered and empiric antibiotic therapy should include coverage for these organisms.     

Characterization of an ATP-binding cassette from Clostridium perfringens with homology to an ABC transporter from Clostridium hathewayi

A ciprofloxacin-resistant mutant of Clostridium perfringens, strain VPI-C, which had stable mutations in the topoisomerase genes, accumulated less norfloxacin and ethidium bromide than the wild type, strain VPI. Efflux pump inhibitors both increased the accumulation of ethidium bromide by cells of the mutant and enhanced their sensitivity to this toxic dye. Cloning a gene, which codes for a putative ABC transporter protein (NP_562422) of 527 amino acids, from the mutant strain VPI-C into the wild-type strain VPI not only reduced the accumulation of ethidium bromide by the recombinant strain but also reduced its sensitivity to norfloxacin and ciprofloxacin. Efflux pump inhibitors decreased the rate at which ethidium bromide was removed from the cells of the recombinant strain. It appears that the putative ABC transporter protein (NP_562422) may contribute to extrusion of drugs from C. perfringens.     

Characterization of two putative fibronectin-binding proteins of Clostridium perfringens

Clostridium perfringens is a Gram-positive anaerobic pathogen that causes gas gangrene and food poisoning in humans and animals. Genomic analysis of C. perfringens strain 13 revealed that this bacterium contains two genes (CPE0737 and CPE1847) that encode putative fibronectin (Fn)-binding proteins (Fbps). These genes, named fbpA and fbpB, were found to be constitutively expressed in all three strains (13, NCTC8237, CPN50) of C. perfringens, isolated from gas gangrene of human, that were tested. Both fbpA and fbpB were cloned and His-tagged, recombinant FbpA (rFbpA) and recombinant FbpB (rFbpB) were purified by Ni²⁺-Sepharose column chromatography from transformed Escherichia coli. These recombinant Fbps were shown to bind to Fn, purified from human serum, in a ligand blotting assay. Additionally, Fn bound to these rFbps in a dose-dependent manner in an enzyme-linked immunosorbent assay. Furthermore, it was found that pre-incubation of Fn with either rFbpA or rFbpB inhibited the binding of Fn to C. perfringens cells.     

一株抑制产气荚膜梭菌的枯草芽孢杆菌BS-2特性

【背景】感染产气荚膜梭菌会引起动物坏死性肠炎,通常使用抗生素进行预防和治疗。随着我国饲料禁抗、养殖减抗的实施,寻找绿色微生态制剂及其代谢产物成为当前研究的热点。【目的】旨在研究前期筛选的一株抑制产气荚膜梭菌的枯草芽孢杆菌BS-2特性。【方法】检测了菌株生长曲线、代谢物质的抑菌特性及细菌素基因簇mRNA表达。【结果】枯草芽孢杆菌BS-2代谢物质对革兰氏阴性菌无抑制作用,而对革兰氏阳性菌具有较强的抑菌性能,并且对产气荚膜梭菌的抑菌性能在2-12 h内迅速增长,在12-24 h内抑菌性能较稳定;该抑菌性能不受胃蛋白酶、胰蛋白酶、蛋白酶K的影响,具有良好的热稳定性;进一步分析抑菌物质基因簇mRNA表达,发现枯草芽孢杆菌BS-2抑制产气荚膜梭菌的活性可能与表面活性素(surfactin)和美杀菌素(mersacidin)表达有关。【结论】枯草芽孢杆菌BS-2对产气荚膜梭菌具有较强的抑制作用,可能通过抑菌物质surfactin和mersacidin表达发挥作用。     

产气荚膜梭菌前噬菌体的分布特点及遗传进化分析

【目的】分析和研究产气荚膜梭菌中前噬菌体的分布情况、基因组特点及遗传进化关系。【方法】利用PHASTER (phage search tool enhanced release)软件预测产气荚膜梭菌携带的前噬菌体,基于ANI (average nucleotide identity)值对前噬菌体进行分群,利用CARD (comprehensive antibiotic research database)、Res Finder 4.1、VFDB (virulence factors database)和Bac Met(antibacterial biocide&metal resistance genes database)分析前噬菌体携带的耐药基因、毒力基因、抗菌剂/金属离子抗性基因,利用CRISPRCas Finder分析产气荚膜梭菌的CRISPR-Cas系统,利用MEGA 7.0进行前噬菌体的遗传进化关系分析。【结果】产气荚膜梭菌平均携带前噬菌体2.67条,其长度呈双峰分布,平均占基因组2.23%;前噬菌体不携带耐药基因,但携带了α毒素、唾液酶和溶血素等毒力基因以及重金属...     

Sequence Heterogeneity of the Ten rRNA Operons in Clostridium perfringens

We have cloned and sequenced rRNA operons of Clostridium perfringens strain 13 and analyzed the sequence structure in view of the phylogenesis. The organism had ten copies of rRNA operons all of that comprised of 16S, 23S and 5S rDNAs except for one operon. The operons clustered around the origin of replication, ranging within one-third of the whole genome sequence as it is arranged in a circle. Seven operons were transcribed in clockwise direction, and the remaining three were transcribed in counter clockwise direction assuming that the gyrA was transcribed in clockwise direction. Two of the counter clockwise operons contained tRNA^Ile genes between the 16S and 23S rDNAs, and the other had a tRNA^Ile genes between the 16S and 23S rDNAs and a tRNA^Asn gene in the place of the 5S rDNA. Microheterogeneity was found within the rRNA structural genes and spacer regions. The length of each 16S, 23S and 5S rDNA were almost identical among the ten operons, however, the intergenic spacer region of 16S-23S and 23S-5S were variable in the length depending on loci of the rRNA operons on the chromosome. Nucleotide sequences of the helix 19, helix 19a, helix 20 and helix 21 of 23S rDNA were divergent and the diversity appeared to be correlated with the loci of the rRNA operons on the chromosome.     

Induction of pCW3-Encoded Tetracycline Resistance in Clostridium perfringens Involves a Host-Encoded Factor

The tetracycline resistance determinant Tet P, which is encoded by the conjugative plasmid pCW3 from Clostridium perfringens, is induced by subinhibitory concentrations of tetracycline. In this study we have shown that the inducible phenotype is strain dependent. When pCW3 is present in derivatives of the wild-type strains CW234 and CW362 resistance is inducible. However, transfer to derivatives of strain 13 leads to a constitutive phenotype that is only observed in this strain background. Based on these results it is proposed that induction of the pCW3-encoded tet(P) genes in C. perfringens requires a host-encoded factor that is either absent or nonfunctional in strain 13 derivatives.     

Effect of Different Carbon and Nitrogen Sources on Clostridium argentinense Toxigenicity in Coculture withPseudomonas mendocina

The effect of different carbon and nitrogen sources on the production of toxin by Clostridium argentinense was examined. The toxin production by C. argentinense in coculture with Pseudomonas mendocina increased in all the cases in relation to that produced by monocultures independent of the nature of the source. Using dextrin as carbon source C. argentinense produced the highest levels of toxin both in monocultures (300 LD₅₀/mL) and in cocultures with P. mendocina (5000 LD₅₀/mL). Experiments run in a microfermenter showed that the slow growth of cocultures associated with the assimilation of dextrin and the pH and Eh profiles favoured the production of toxin. Of the nitrogen sources assayed, corn steep liquor sustained the highest levels of toxin in both monocultures and cocultures with 3 and 2.8 fold increases with respect to that obtained using proteose peptone. The toxin production by C. argentinense cultures and C. argentinense–P. mendocina cocultures was highly dependent on the nature of the carbon and nitrogen sources used in the culture media. Growth of C. argentinense on substrates slowly assimilated stimulated the production of toxin.     

艰难梭菌研究现状

艰难梭菌(Clostridium difficile,CD)是产芽胞革兰阳性厌氧菌,产毒素艰难梭菌可导致艰难梭菌感染(Clostridium difficile infection,CDI)。产毒素艰难梭菌已被公认是抗菌药物相关性腹泻最常见的病原体,也是发达国家最常见的导致住院患者腹泻感染的原因之一。高毒力菌株导致全世界范围内CDI的发病率和死亡率增加。如何快速而准确地检测CD一直是备受关注的问题,国内外多推荐两步法和三步法进行检测。艰难梭菌治疗的一线用药是甲硝唑、万古霉素以及非达霉素,应用粪便微生物移植(faecal microbiota transplantation,FMT)也可作为治疗轻度或严重复发CDI(recurrence Clostridium difficile infection,rCDI)的可选方法。艰难梭菌的芽胞能抵抗不良环境,但抗菌药物的使用,医院环境的复杂性和医务工作者的手等因素会导致艰难梭菌易于传播,因此医疗机构应加强艰难梭菌的预防。本文主要对艰难梭菌的流行病学、致病机制、检测及治疗进行综述。     

克氏梭菌硫解酶的鉴定及其功能研究

【目的】硫解酶是梭菌属微生物合成短中链脂肪酸的关键酶。克氏梭菌(Clostridium kluyveri)具有3个高度同源的硫解酶编码基因,对这3个基因的功能鉴定是解析克氏梭菌高己酸合成能力的关键。【方法】通过发酵动力学分析确定克氏梭菌的己酸和丁酸生成动力学特征;转录组测序结合反转录-荧光定量RCR分析克氏梭菌3个硫解酶编码基因的表达水平和时序表达特征;在大肠杆菌中异源表达这3个硫解酶,并对其硫解酶动力学参数进行测定。【结果】克氏梭菌生成丁酸、己酸、辛酸,其中己酸为主要代谢产物;转录组数据显示,在乙酸消耗完全之前,thlA1基因维持恒定表达,thlA2基因表达时序上调,thlA3基因表达时序下调,转录组测序表明3个硫解酶编码基因均具有较高水平的转录活性,thlA2和thlA3的最高表达量分别约为thlA1的29%和43%;硫解酶动力学参数测定结果表明,克氏梭菌3个硫解酶对于四碳底物均显示出相似的底物亲和力(K_m),但ThlA1对四碳底物的催化效率(k_(cat)/K_m)略低于ThlA2和ThlA3。【结论】克氏梭菌的3个硫解酶均具有催化活性,在克氏梭菌体内均呈活跃表达,表明克氏梭菌拥有3个具有催化活性的硫解酶,这为后续深入研究克氏梭菌己酸合成机理奠定了基础。     

丙酮丁醇梭菌的基因编辑工具及代谢工程改造

丙酮丁醇梭菌作为极具潜力的新型生物燃料丁醇的生产菌,受到各国研究学者的广泛关注。通过丙酮丁醇梭菌(ABE)发酵生产丁醇,由于生产成本高,限制了其工业化应用。随着基因组学和分子生物学的快速发展,适用于丙酮丁醇的基因编辑工具不断发展并应用于提高菌株的发酵性能。本文对丙酮丁醇梭菌基因编辑工具和代谢工程改造取得的进展进行综述。     

丁酸梭菌影响肠炎沙门氏菌对SPF小鼠致病性的作用

【目的】旨在探究丁酸梭菌影响肠炎沙门氏菌(CVCC3377)对SPF小鼠的致病性及对小鼠的保护作用。【方法】选用72只6周龄SPF级雌性C57BL/6小鼠,按照完全随机区组设计分为阴性对照(CON)、饲喂丁酸梭菌组(CB)、沙门氏菌感染组(SE)和丁酸梭菌保护组(CB+SE)4组,每组18只。CON组和SE组饲喂基础日粮和饮水,CB组和CB+SE组饲喂基础日粮和每天更换添加丁酸梭菌(1×10⁷CFU/mL)的饮水,连续饲喂一周;第8天进行肠炎沙门氏菌感染实验,分别对SE组和CB+SE组灌服0.2mL 1.1×10⁴CFU/只的肠炎沙门氏菌,同时对阴性对照组和丁酸梭菌组灌服0.2mL生理盐水。在感染后第6天处死小鼠并采集肝脏、脾脏和盲肠及内容物样品。采用H.E染色检测肝脏和脾脏组织的病理学变化,免疫组化检测脾脏组织中TLR4、MyD88蛋白的表达水平,荧光定量PCR检测盲肠组织中TNF-α和IL-6的表达水平,并通过高通量测序方法分析其对肠道菌群的影响。【结果】结果显示,丁酸梭菌能够缓解肠炎沙门氏菌感染引起的小鼠体重下降;H.E染色结果显示,丁...     

解纤维梭菌脱水四环素诱导的ClosTron基因打靶系统构建

【背景】解纤维梭菌是发酵木质纤维素生产乙醇的中温模式菌株,构建可控诱导的基因打靶技术是研究解纤维梭菌遗传调控的重要手段。【目的】在解纤维梭菌中构建脱水四环素诱导的ClosTron基因打靶系统。【方法】首先,分析解纤维梭菌对脱水四环素的敏感性,筛选合适的诱导剂浓度,并以β-葡萄糖苷酶为报告基因,检测其在解纤维梭菌中的诱导效果。然后,将脱水四环素诱导型操纵子与II型内含子元件组合,构建脱水四环素诱导的ClosTron质粒。最后,以解纤维梭菌mspI、ldh和ack为例,检测其在解纤维梭菌中的打靶效率。【结果】脱水四环素诱导系统在解纤维梭菌中能够诱导ClosTron元件表达,在mspI、ldh和ack这3个基因位点的打靶效率分别为29.48%±15.51%、23.61%±7.08%和28.09%±6.97%。【结论】在解纤维梭菌中成功构建脱水四环素诱导的ClosTron基因打靶系统,为梭菌基因工程改造奠定了基础。     

A Protease-Resistant Novel Hemagglutinin Purified from Type A Clostridium botulinum

Botulinum neurotoxin is a food poisoning agent produced by Clostridium botulinum. The neurotoxin is a 150-kDa protein that causes flaccid muscle paralysis by blocking neurotransmitter release at neuromuscular junctions. The neurotoxin is produced along with a group of neurotoxin associated proteins (NAPs), which protect it from the low pH and proteases of the gastrointestinal (GI) tract. We have isolated, for the first time, one of the major components of NAPs in a pure form. The isolated protein is a 33-kDa single polypeptide (Hn-33) that exhibits hemagglutination activity. Specific polyclonal antibodies against the Hn-33 are able to block the hemagglutination activity of the neurotoxin complex, which indicates that perhaps Hn-33 is the only strong hemagglutinating protein in the complex. The Hn-33 was found be resistant to trypsin and other protease digestion, a feature that could play a role in the protection of the neurotoxin in the GI tract during its toxicoinfection.     

Improvement of xylose utilization in Clostridium acetobutylicum via expression of the talA gene encoding transaldolase from Escherichia coli

Clostridium acetobutylicum ATCC 824 was metabolically engineered for improved xylose utilization. The gene talA, which encodes transaldolase from Escherichia coli K-12, was cloned and overexpressed in C. acetobutylicum ATCC 824. Compared with C. acetobutylicum ATCC 824 (824-WT), the transformant bearing the E. coli talA gene (824-TAL) showed improved ability on xylose utilization and solvents production using xylose as the sole carbon source. During the fermentation of xylose and glucose mixtures with three xylose/glucose ratios (approximately 1:2, 1:1 and 2:1), the rate of xylose consumption and final solvents titers of 824-TAL were all higher than those of 824-WT, despite glucose repression on xylose uptake still existing. These results suggest that the insufficiency of transaldolase in the pentose phosphate pathway (PPP) of C. acetobutylicum is one of the bottlenecks for xylose metabolism and therefore, overexpressing the gene encoding transaldolase is able to improve xylose utilization and solvent production.     

丙酮丁醇梭菌硫氧还蛋白系统在溶剂生产过程中的功能

[目的]探究丙酮丁醇梭菌硫氧还蛋白系统在生长和代谢过程中的功能.[方法]使用ClosTron系统对硫氧还蛋白系统中的硫氧还蛋白还原酶基因(trxB)进行插入失活,得到突变株,通过Southern杂交方法验证插入内含子的拷贝数;在基本培养基中进行分批发酵,比较并分析突变株的生长特点;通过pH控制,利用限磷的连续发酵方法使...     

一株产丁酸羊源拜氏梭菌的筛选及其培养条件优化

【背景】人类和动物消化道内栖息着极其复杂和多样化的微生物群落,这些微生物群落分布在肠道的不同位置并执行着特定的功能。近年来,产丁酸菌逐渐成为微生物领域的研究热点,产丁酸菌主要为产芽孢革兰氏阳性厌氧菌,对肠道健康有重要意义。【目的】从反刍动物瘤胃中筛选出产丁酸菌株并研究其生长特性,进一步优化其培养条件,从而提高产丁酸菌的丁酸产量。【方法】以绵羊瘤胃内容物为样品,运用稀释涂布法进行产丁酸菌的筛选,通过形态学观察和16S rRNA基因序列分析等方法对菌株进行鉴定。通过单因素试验与Box-Behnken design试验相结合,对培养条件进行优化,确定筛选菌株在梭菌增殖培养基(reinforced clostridium medium,RCM)中的最佳产酸培养条件。【结果】经过筛选鉴定得到的菌株为梭菌属的拜氏梭菌(clostridium beijerinckii,CB),命名为拜氏梭菌R8(CB.R8)。对拜氏梭菌R8的培养条件进行优化,得出该菌株在接种量为1.22%、温度为38.45℃、pH6.08和培养时间为64.67h的条件下丁酸产量为2.48g/L。【结论】筛选到1株拜氏梭菌R8,该菌能够在RCM培养基中生长并代谢产生丁酸,具备较高的应用价值。