牛源致病性蜡样芽孢杆菌溶血素BL亚基的原核表达及其生物学活性分析

蜡样芽孢杆菌属于革兰氏阳性菌,分布广泛,具有一定的致病性。不同的蜡样芽孢杆菌携带有不同的毒力因子,这直接决定了蜡样芽孢杆菌株致病性的差异。研究和探讨毒力因子分布以及具体毒素的生物活性有助于对蜡样芽孢杆菌病采取更科学的防控。本研究通过原核表达系统将溶血素BL三亚基重组表达,并对表达蛋白进行了纯化和部分生物学活性的检测。研究结果表明,牛源致病性蜡样芽孢杆菌溶血素BL可以在原核表达系统中成功表达和纯化,牛源致病性蜡样芽孢杆菌溶血素BL拥有溶血性、细胞毒性、很好的免疫原性以及对小鼠具有一定的免疫保护性。本研究表达了溶血素BL三亚基,并探究了牛源致病性蜡样芽孢杆溶血素BL的生物学活性。本研究为进一步揭示蜡样芽孢杆菌溶血素BL的致病作用机制和建立针对牛源致病性蜡样芽孢杆菌病的检测方法奠定了理论基础。     

空肠弯曲杆菌感染小鼠模型的研究进展

空肠弯曲杆菌主要导致食源性胃肠疾病,对人类健康有重要影响,然而相关小鼠感染模型的建立是该致病菌研究的难点。本文综述了影响空肠弯曲杆菌感染模型建立的关键因素,包括菌株的毒力因子、小鼠的品系、免疫功能、肠道菌群、饮食组成、宿主适应等因素,以期为成功建立合适的小鼠感染模型提供参考。     

食品中蜡样芽孢杆菌耐药性及其机制研究进展

蜡样芽孢杆菌是常见的食源性致病菌之一,其产生的毒素会引起食物中毒。蜡样芽孢杆菌主要引起2种类型的食物中毒,即呕吐和腹泻综合征,并可造成各种局部和全身感染。随着抗生素的广泛、大量使用,蜡样芽孢杆菌的耐药性不断增强,现已有报道出现多重耐药性。本文对蜡样芽孢杆菌的耐药现状及耐药性机制进行了综述,以期正确理解蜡样芽孢杆菌耐药性的特点及其规律,从而为防治蜡样芽孢杆菌耐药性的产生及合理用药提供理论依据。     

Sigma B因子活性的调节及其在几种革兰氏阳性食源性致病菌中的作用

Sigma B(-B)是许多革兰氏阳性菌对环境胁迫产生应答反应的主要调控因子.不仅在芽孢形成中具有重要作用,而且-B也直接或间接地调控包括蜡样芽孢杆菌(Bacillus cereus)、单核细胞增生李斯特菌(Listeria monocytogenes)和金黄色葡萄球菌(Staphylococcus aureus)在内的一些革兰氏阳性食源性致病菌毒力及毒力相关基因的表达.本文结合作者在国内外的相关工作,综述了-B活性的调节方式及其在上述革兰氏阳性食源性致病菌中相关作用的最新研究进展,为深入研究革兰氏阳性食源性致病菌的致病机理、预防和治疗细菌感染提供新的思路和理论依据.     

中药91—4对抗生素相关性腹泻小鼠肠道膜菌群及腔菌群的影响

本实验以肠道菌检测与电镜观察法,初步研究了中药９１－４对抗生素相关性腹泻小鼠肠道菌群的影响。结果表明,盐酸林可霉素对小鼠肠道膜菌群、腔菌群干扰显著、乳酸杆菌、双歧杆菌及肠杆菌等比正常对照组明显减少,致小鼠腹泻或肠炎;中药９１－４能够促进肠道乳酸杆菌、双歧杆菌等生长繁殖,对肠道膜菌群、腔菌群均具有调整作用,加速肠粘膜病变的愈合及修复,可有效地控制ＡＡＤ模型小鼠的腹泻症状。     

中药91－4对抗生素相关性腹泻小鼠肠道膜菌群及腔菌群的影响

本实验以肠道菌群检测与电镜观察法,初步研究了中药９１－４对抗生素相关性腹泻（ＡＡＤ）小鼠肠道菌群的影响。结果表明,盐酸林可霉素对小鼠肠道膜菌群、腔菌群干扰显著,乳酸杆菌、双歧杆菌及肠杆菌等比正常对照组明显减少,致小鼠腹泻或肠炎;中药９１－４能够促进肠道乳酸杆菌、双歧杆菌等生长繁殖,对肠道膜菌群、腔菌群均具有调整作用,加速肠粘膜病变的愈合及修复,可有效地控制ＡＡＤ模型小鼠的腹泻症状。     

致病性大肠杆菌和蜡样芽胞杆菌的分离鉴定

【背景】猪只消化道疾病是养猪业上一大重要疾病,给养猪业带来一定的经济损失。大肠杆菌是引起猪腹泻的一种常见病原菌,可以引起不同日龄的猪腹泻,但主要以幼龄猪为主。【目的】旨在分离鉴定引起四川省眉山市一规模化养猪场病猪大规模腹泻的病原菌。【方法】采用常规细菌分离方法结合16S rRNA基因序列的分析方法从发病猪肝脏、胃以及污染的饲料分离鉴定细菌,并对分离株进行小鼠致病性试验、16S rRNA基因遗传进化树分析、毒力基因的检测、药物敏感试验。【结果】从腹泻猪肝脏中分离到一株致病性大肠杆菌,胃中分离到一株蜡样芽胞杆菌,并且追溯到传染源是该猪场饲料。通过检测这两株菌相应的毒力基因发现大肠杆菌不属于肠外致病性型,蜡样芽胞杆菌检测到了nheA、nheB、nheC、bceT、entFM 5种毒力基因;药敏试验表明常规的氨基糖苷类和头孢类抗生素对大肠杆菌抑菌效果较好,红霉素、氟苯尼考、头孢氨苄、头孢哌酮对蜡样芽胞杆菌抑菌效果较好,而蜡样芽胞杆菌对青霉素、阿莫西林等常规药物不敏感。【结论】饲料存在大肠杆菌和蜡样芽胞杆菌的混合污染。     

蜡样芽孢杆菌ATCC14579毒力基因plcR缺失株的构建及其一般特性

蜡样芽孢杆菌是土壤中的优势菌,具有作为益生菌的潜在能力,同时它也是条件致病菌,能引起食物中毒等。蜡样芽孢杆菌的多种毒力因子受到多效性调控子(pleiotropic regulator,plcR)的调控,在其条件致病性作用中起着重要作用。真养产碱杆菌JMP34(Alcaligenes eutrophus)质粒上的2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid,2,4-D)单加氧酶(tfdA)基因可以降解2,4-D。本研究利用同源重组技术使tfdA基因置换掉蜡样芽孢杆菌的plcR基因,构建了蜡样芽孢杆菌ATCC14579突变株B.cereus△plcRΩtf-dA,并对其毒性、一般生理生化特性进行分析。研究结果表明,突变株B.cereus△plcRΩtfdA的毒性显著减弱;生理生化实验结果显示突变株与野生株并没有明显区别,且突变株并没有表现出tfdA酶活性。所有的结果表明plcR控制着蜡样芽孢杆菌ATCC14579的致病性,同时剔除plcR并不破坏其酶系统。本研究为今后构建蜡样芽孢杆菌工程菌提供了新的思路和依据。     

益生菌地衣芽孢杆菌治疗鸡腹泻机理初探

目的：探讨益生菌地衣芽孢杆菌饲喂鸡腹泻的治疗效果以及分子机理。方法：从养鸡场土壤中选择性分离芽孢杆菌,通过优 化条件提高产生芽孢的比例;利用芽孢和大米混合后饲喂腹泻鸡,通过克隆测序分析腹泻鸡肠道微生物群落在饲喂前后的差异。 结果：我们分离到一株地衣芽孢杆菌,通过优化条件使产芽孢的比例提高到60 %。利用芽孢饲喂一周后能够有效地治疗鸡腹泻。 分析鸡肠道微生物群落发现：鸡腹泻后其肠道细菌群落的多样性和均匀度降低。造成腹泻鸡肠道主要菌群为动胶杆菌（Zoogloea sp.）;经芽孢饲喂后,主要菌群转变为拟杆菌（Bacteroides sp.）,与健康鸡的肠道微生物主要菌群相似。结论：我们筛选到一株可能 治疗鸡腹泻的芽孢杆菌,通过优化培养条件能够提高其产芽孢比例;腹泻鸡经芽孢饲喂后,其肠道中致病菌被益生菌所代替,形 成正常的肠微生物菌群而恢复健康。     

抗生素所致腹泻大鼠肠屏障功能损伤及乳酸杆菌保护机制的研究

目的探讨抗生素对所致腹泻大鼠肠道屏障功能、肠道菌群结构和肠道细菌移位的影响及乳酸杆菌制剂的保护机制。方法采用细菌培养法动态测定抗生素所致腹泻大鼠肠道菌群变化及肠系膜淋巴结、肝脏、脾脏和结肠组织的移位细菌量;应用光镜和电子显微镜观察肠黏膜组织超微结构变化。结果应用抗生素可致大鼠腹泻,肠道菌群失调,肠黏膜组织受损,发生肠道细菌移位。大肠埃希菌攻击可加重肠道菌群失调和肠黏膜损伤程度,促发细菌移位发生。乳酸杆菌可扶正肠菌群结构,修复损伤的肠黏膜,抑制肠细菌移位发生。结论阐明了抗生素、肠黏膜屏障功能、肠道菌群结构和肠道细菌移位间的互为因果,相互影响的关系。微生态制剂在维持机体微生态平衡、修复肠黏膜方面具有保护作用。     

Ecological consequences of ingestion of Bacillus cereus on Bacillus thuringiensis infections and on the gut flora of a lepidopteran host

The Bacillus cereus group comprises a diverse array of non-pathogenic bacteria as well as pathogens such as Bacillus thuringiensis. Their spores are found together in soil and leaves and are therefore likely to commonly interact within hosts. Mixed infections of pathogenic B. thuringiensis and non-pathogenic strains have been little studied, despite their potential impact on biological control and the evolutionary ecology of virulence. Antibiotic secreting strains of B. cereus have been shown to be able to synergize B. thuringiensis (Bt) infections. We explored the ecology of these mixed infections more broadly in the diamondback moth (DBM). We tested whether antibiotic-expressing B. cereus can synergize Bt infections initiated with spores, investigated whether ingestion of antibiotic-expressing B. cereus had any consequences for the larval gut flora and whether synergistic interactions with B. cereus increase Bt reproduction. Ingestion of high-antibiotic secreting B. cereus synergized infections of B. thuringiensis in diamondback moth larvae, but at a lower level than previously reported. Coinfection also increased slightly the number of Bt spores found in cadavers. Culture independent analysis of gut homogenates indicated that ingestion of an antibiotic-expressing strain of B. cereus reduced the abundance of the gut flora and led to gut communities being dominated bacteria with DGGE profiles very similar to pure B. cereus cultures. Ingestion of B. cereus, regardless of genotype, reduced densities of an enteric isolate of Enterobacter sp. These findings support the hypothesis that antibiotic secretion in the gut synergizes B. thuringiensis infections by reducing the abundance of the commensal gut flora and facilitating invasion by bacteria in the B. cereus group.     

马蜂肠道菌的分离鉴定及产消化酶功能菌株的筛选

【背景】马蜂(Vespa mandarinia Smith)可以防治多种田间害虫,还具有药用价值,其肠道菌群结构和功能还有待研究。【目的】获得马蜂肠道可培养细菌并筛选出具有产消化酶功能的菌株,为理解肠道菌对宿主的影响机理及功能菌株的利用提供科学依据和研究材料。【方法】采用传统细菌分离培养法获得马蜂肠道菌,结合形态学以及16S rRNA基因序列分析进行鉴定;利用水解圈法分别筛选产蛋白酶、脂肪酶、淀粉酶和纤维素酶菌株;通过测量水解圈D与菌落直径d的比值,比较不同细菌的产酶能力。【结果】在马蜂肠道中共分离出6属10种细菌,其中芽孢杆菌属5种,肠球菌属、葡萄球菌属、明串珠菌属、乳球菌属和不动杆菌属各1种。从获得的61个菌株中筛选出6个具有产消化酶功能的菌株。其中,苏云金芽孢杆菌V44具有产蛋白酶、淀粉酶、脂肪酶和纤维素酶4种消化酶的能力;粪肠球菌V6具有产淀粉酶、蛋白酶和脂肪酶3种消化酶的能力;蜡样芽孢杆菌V43具有产蛋白酶、淀粉酶和纤维素酶3种消化酶的能力;粪肠球菌V20、蜡样芽孢杆菌V19和维德曼氏芽孢杆菌V22均具有产蛋白酶的能力。【结论】马蜂肠道细菌资源较丰富,部分有产消化酶的功能,可帮助马蜂消化食物,对宿主健康有一定影响。本研究筛选的6个菌株都能产蛋白酶,其中菌株V43和V44分别具有最强产淀粉酶和脂肪酶的能力,是可进一步开发利用的肠道功能菌株资源。     

Characterization of a broad range antibacterial substance from a new <Emphasis Type="Italic">Bacillus</Emphasis> species isolated from Amazon basin

A Bacillus sp. strain producing a bacteriocin-like substance was characterized by biochemical profiling and 16S rDNA sequencing. The phylogenetic analysis indicated that this strain has low sequence similarity with most Bacillus spp., suggesting a new species was isolated. The antimicrobial activity was detected starting at the exponential growth phase, and maximum activity was observed at stationary phase. The substance was inhibitory to a broad range of indicator strains, incluing pathogenic and food spoilage bacteria such as Listeria monocytogenes, B. cereus, Aeromonas hydrophila, Erwinia carotovora, Pasteurella haemolytica, Salmonella Gallinarum, among other. The antibacterial substance was stable over a wide pH range, but it was sensitive to pronase E and lipase. The antibacterial substance was bactericidal and bacteriolytic to L. monocytogenes and B. cereus at 160 AU ml⁻¹. The identification of a broad range bacteriocin-like inhibitory substance active against L. monocytogenes addresses an important aspect of food protection against pathogens and spoilage microorganisms.     

百里酚和香芹酚改变肠道微生物组成抑制羔羊腹泻的作用研究

【目的】本试验研究百里酚和香芹酚对滩羊羔羊腹泻、肠道短链脂肪酸和微生物组成的影响,旨在为利用植物提取物手段治疗羔羊腹泻提供可用的添加剂和理论基础。【方法】本研究选择腹泻发病时间为2-3 d的10只腹泻羔羊,随机分为2组,分为腹泻羔羊组(CON组)和百里酚和香芹酚处理组(SCAT组),试验期间SCAT组羔羊补充200 mg/只/d的百里酚和香芹酚1:1混合品,处理时间为7 d。【结果】百里酚和香芹酚处理能够显著提高腹泻羔羊日增重,降低羔羊腹泻指数和腹泻率(P<0.05),降低血清促炎因子表达水平(P<0.05),提高肠道短链脂肪酸(short-chain fatty acids,SCFA)丁酸浓度(P<0.05);同时百里酚和香芹酚能够改变腹泻羔羊肠道微生物组成,提高肠道中双歧杆菌(Bifidobacteriaceae)和阿克曼氏菌(Akkermansiaceae)的相对丰度,降低肠杆菌科(Enterobacteriaceae)相关菌的相对丰度。【结论】百里酚和香芹酚能够提高肠道有益菌相对丰度和有益代谢物丁酸的浓度,降低致病菌相对丰度,从而有效抑制羔羊腹泻,可作为新型替...     

Determination of Toxigenic Potentials of <Emphasis Type="Italic">Bacillus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Okpehe</Emphasis>, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.     

Enteric bacteria isolated from acute diarrheal patients in the Republic of Korea between the year 2004 and 2006

In an epidemiological survey of human enterobacterial infections in the Republic of Korea during three years from 2004 to 2006, we isolated 1,784 (6.2%, isolation rate of enteropathogens from stool samples) in 2004, 2,547 (9.5%) in 2005 and 3,506 bacteria (12.3%) from people who visited clinics. Among the isolated bacteria, pathogenic Escherichia coli, especially, EAEC was the most frequently identified pathogen in both urban and rural regions followed by Staphylococcus aureus, Salmonella species, Bacillus cereus, Vibrio parahaemolyticus, Campylobacter jejuni, Clostridium perfringens, and Shigella species. Distinct seasonality was found in V. parahaemolyticus species, while this pathogen showed no age-specific patterns. However, other bacteria, i.e., pathogenic E. coli, S. aureus, Salmonella spp., and B. cereus showed similar seasonality throughout the year, showing a slight increase in the infection rate during the summer months and high prevalence among children under 10 years of age and elder-age people. The antibiotic susceptibility patterns of pathogenic E. coli, Salmonella spp., and S. aureus showed high resistance to penicillins. However, both pathogenic E. coli and Salmonella spp. were susceptible to several cephems, imipenem, and amikacin. Moreover, S. aureus strains resistant to vancomycin were not found. In conclusion, these surveillances can play an important role for the control and prevention to the diseases originated by enteritis bacteria.     

Biosorption of cadmium by a Cd2+-hyperresistant Bacillus cereus strain HQ-1 newly isolated from a lead and zinc mine

A Cd²⁺-hyperresistant bacterial strain HQ-1 was isolated from a lead–zinc mine. The strain was characterized and identified as Bacillus cereus based on morphology, physiological tests and 16S rRNA gene analysis. The minimal inhibitory concentration of Cd²⁺ for the bacterium was 0.012 mol/l. Isotherms for cadmium (Cd) biosorption by cells of B. cereus strain HQ-1 were investigated. The equilibrium data could be fitted by a Langmuir isotherm equation. The possible functional sites that might be influenced by the sorption were determined. The results indicate that this B. cereus strain has excellent potential for biosorption of Cd. Physiological characterization of the isolate also indicates possible application of this strain for bioremediation of sites with Cd contamination.     

Cereulide-producing strains of <Emphasis Type="Italic">Bacillus cereus</Emphasis> show diversity

Producers of cereulide, the emetic toxin of Bacillus cereus, are known to constitute a specific subset within this species. We investigated physiological and genetic properties of 24 strains of B. cereus including two high cereulide producers (600–1,800 ng cereulide mg⁻¹ wet weight biomass), seven average producers (180–600 ng cereulide mg⁻¹ wet weight biomass), four low cereulide producers (20–160 ng cereulide mg⁻¹ wet weight biomass) and 11 non-producers representing isolates from food, food poisoning, human gut and environment. The 13 cereulide producers possessed 16S rRNA gene sequences identical to each other and identical to that of B. anthracis strains Ames, Sterne from GenBank and strain NC 08234–02, but showed diversity in the adk gene (two sequence types), in ribopatterns obtained with EcoRI and PvuII (three types of patterns), in tyrosin decomposition, haemolysis and lecithin hydrolysis (two phenotypes). The cereulide-producing isolates from the human gut represented two ribopatterns of which one was novel to cereulide-producing B. cereus and two phenotypes. We conclude that the cereulide-producing B. cereus are genetically and biochemically more diverse than hitherto thought.     

凡纳滨对虾肠道红杆菌科细菌富集碳源筛选及其定向分离

【目的】红杆菌科(Rhodobacteraceae)细菌为凡纳滨对虾肠道微生物的优势类群,在健康对虾肠道中具有较高的相对丰度,是指示对虾健康的关键类群,探究对虾肠道红杆菌科细菌定向富集和分离方法,可为对虾养殖益生菌菌剂的研发提供基础。【方法】利用16S rRNA基因高通量测序技术研究不同碳源添加对凡纳滨对虾肠道中红杆菌科细菌的富集作用,筛选对红杆菌科细菌有显著富集作用的碳源;利用纯培养技术从红杆菌科细菌富集的样品中定向分离红杆菌科细菌,并对其进行鉴定和遗传多样性分析。【结果】添加短链脂肪酸(乙酸、丙酸、丁酸、戊酸)和碳酸氢钠对红杆菌科细菌有显著富集作用,主要富集到Cribrihabitans、Tritonibacter、Rhodovulum、Ruegeria、Sagittula和Thalassobius属相关菌株;对红杆菌科细菌相对丰度最高的样品进行稀释涂布培养,共分离纯化出303株细菌,分属于2门12科,其中红杆菌科细菌为主导类群共119株,主要包括Tritonibacter (90株)、Phaeobacter (25株)、Sulfitobacter (1株)、Ruegeria (1...