Characterization of Bacillus Species Used for Oral Bacteriotherapy and Bacterioprophylaxis of Gastrointestinal Disorders

Bacillus subtilis spores are being used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders in both humans and animals. Since B. subtilis is an aerobic saprophyte, how spores may benefit the gut microbiota is an intriguing question, since other probiotics such as Lactobacillus spp. which colonize the gut are anerobes. As a first step in understanding the potential effects of ingesting spores, we have characterized five commercial products. An extensive biochemical, physiological, and phylogenetic analysis has revealed that four of these products are mislabeled. Moreover, four of these products showed high levels of antibiotic resistance.     

Molecular characterization and identification of Bacillus clausii Strains marketed for use in oral bacteriotherapy

A substantial number of Bacillus species have been marketed for use in oral bacteriotherapy because of their purported ability to prevent or treat various gastrointestinal disorders. Recently, some of the Bacillus strains in Enterogermina, which is made up of aqueous suspensions of viable Bacillus spores, have been partially characterized and aligned with members of the Bacillus alcalophilus subgroup rather than with Bacillus subtilis, as previously reported. With a view toward verifying the original taxonomic position of the Enterogermina strains, we catalogued both phenotypic and genotypic traits exhibited by the four Bacillus strains isolated from the spore mixtures found in original commercial preparations dated 1975 and 1984 and commercial preparations now being propagated industrially. Analyses of physiological and biochemical traits, complete 16S rRNA gene sequences, DNA-DNA reassociation, tRNA intergenic spacer length polymorphism, single-strand conformation polymorphism of PCR-amplified spacer regions of tRNA genes, and randomly amplified polymorphic DNA led to the finding that all of the Enterogermina strains belong to a unique genospecies, which is unequivocally identified as the alkalitolerant species Bacillus clausii. Moreover, we provide evidence that in contrast to several reference strains of B. clausii, the strains constituting Enterogermina are characterized by a notable low level of intraspecific genome diversity and that each strain has remained the same for the last 25 years.     

Characterization of a New erm-Related Macrolide Resistance Gene Present in Probiotic Strains of Bacillus clausii

The mechanism of resistance to macrolides, lincosamides, and streptogramins B was studied in four Bacillus clausii strains that are mixed in a probiotic administered to humans for prevention of gastrointestinal side effects due to oral antibiotic chemotherapy and in three reference strains of B. clausii, DSM8716, ATCC 21536, and ATCC 21537. An 846-bp gene called erm(34), which is related to the erm genes conferring resistance to these antibiotics by ribosomal methylation, was cloned from total DNA of B. clausii DSM8716 into Escherichia coli. The deduced amino acid sequence presented 61% identity with that of Erm(D) from B. licheniformis, B. halodurans, and B. anthracis. Pulsed-field gel electrophoresis of total DNA digested by I-CeuI, followed by hybridization with an erm(34)-specific probe, indicated a chromosomal location of the gene in all B. clausii strains. Repeated attempts to transfer resistance to macrolides by conjugation from B. clausii strains to Enterococcus faecalis JH2-2, E. faecium HM1070, and B. subtilis UCN19 were unsuccessful.     

Strategy for Identification of Bacillus cereus and Bacillus thuringiensis Strains Closely Related to Bacillus anthracis

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these “near neighbors” are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.     

Characterization of a new erm-related macrolide resistance gene present in probiotic strains of Bacillus clausii

The mechanism of resistance to macrolides, lincosamides, and streptogramins B was studied in four Bacillus clausii strains that are mixed in a probiotic administered to humans for prevention of gastrointestinal side effects due to oral antibiotic chemotherapy and in three reference strains of B. clausii, DSM8716, ATCC 21536, and ATCC 21537. An 846-bp gene called erm(34), which is related to the erm genes conferring resistance to these antibiotics by ribosomal methylation, was cloned from total DNA of B. clausii DSM8716 into Escherichia coli. The deduced amino acid sequence presented 61% identity with that of Erm(D) from B. licheniformis, B. halodurans, and B. anthracis. Pulsed-field gel electrophoresis of total DNA digested by I-CeuI, followed by hybridization with an erm(34)-specific probe, indicated a chromosomal location of the gene in all B. clausii strains. Repeated attempts to transfer resistance to macrolides by conjugation from B. clausii strains to Enterococcus faecalis JH2-2, E. faecium HM1070, and B. subtilis UCN19 were unsuccessful.     

Degradation of Toluene Hydrocarbon by Isolated Yeast Strains: Molecular Genetic Approaches for Identification and Characterization

Removal of petroleum benzene, toluene, and xylene compounds from the environment is necessary to ensure quality life. In this research, 41 yeasts were isolated from oily soils. Among them, nine yeasts named KKUs (A5, A6, A12, A20, A23, A24, A26, A29, and A38) were selected based on their use of benzene, toluene, and xylene as a sole carbon and energy source. Based on their growth rates, all selected yeasts displayed a high efficiency for toluene degradation, but had no ability to degrade benzene and a low ability to degrade xylene, except A29 and A38, which could not degrade xylene. HPLC analysis for toluene removal indicated that A6, A12, A20, A23, A24, and A26 almost completely removed the toluene compound after 3 days of incubation (92.74, 94.61, 95.05, 91.74, 91.85, and 97.29%, respectively). In addition, strains A29 and A38 showed moderate degradation (88.29 and 85.30%, respectively), while the ability of A5 was low (39.00%). The isolates were identified based on amplifying and sequencing the D1/D2 domain of the 26S rRNA gene. Alignments and comparisons of the 26S rRNA gene sequences of the isolates with those available in GenBank, plus phylogenetic analysis, proved isolates as Rhodotorula lactose KKU-A5, Rhodotorula nymphaeae KKU-A6, Rhodotorula graminis KKU-A12, Rhodotorula minuta KKU-A20, Exophiala dermatitidis KKU-A23, Candida davisiana KKU-A24, Rhodotorula slooffiae KKU-A26, Rhodotorula mucilaginosa KKU-A29, and Rhodosporidium diobovatum KKU-A38. Random amplified polymorphic DNA-PCR fingerprinting was accomplished within seven toluene-degrading red yeasts (A5, A6, A12, A20, A26, A29, and A38). The results indicated no correlation between the random amplified polymorphic DNA profile and the geographic origin of the isolates.     

Identification of Bacillus Strains for Biological Control of Catfish Pathogens

Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10⁷ CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05). A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture.     

Characterization of Bacillus Probiotics Available for Human Use

Bacillus species (Bacillus cereus, Bacillus clausii, Bacillus pumilus) carried in five commercial probiotic products consisting of bacterial spores were characterized for potential attributes (colonization, immunostimulation, and antimicrobial activity) that could account for their claimed probiotic properties. Three B. cereus strains were shown to persist in the mouse gastrointestinal tract for up to 18 days postadministration, demonstrating that these organisms have some ability to colonize. Spores of one B. cereus strain were extremely sensitive to simulated gastric conditions and simulated intestinal fluids. Spores of all strains were immunogenic when they were given orally to mice, but the B. pumilus strain was found to generate particularly high anti-spore immunoglobulin G titers. Spores of B. pumilus and of a laboratory strain of B. subtilis were found to induce the proinflammatory cytokine interleukin-6 in a cultured macrophage cell line, and in vivo, spores of B. pumilus and B. subtilis induced the proinflammatory cytokine tumor necrosis factor alpha and the Th1 cytokine gamma interferon. The B. pumilus strain and one B. cereus strain (B. cereus var. vietnami) were found to produce a bacteriocin-like activity against other Bacillus species. The results that provided evidence of colonization, immunostimulation, and antimicrobial activity support the hypothesis that the organisms have a potential probiotic effect. However, the three B. cereus strains were also found to produce the Hbl and Nhe enterotoxins, which makes them unsafe for human use.     

水产养殖中枯草芽孢杆菌的分子鉴定

微生物种类繁多,以形态学和生理生化指标为基础的细菌鉴定较为复杂,是一项繁琐、费时的工作,对一种未知细菌的鉴定和分类,不仅需要分析多个生化指标,有时还要取决于工作人员的经验。因此迫切需要建立一些简单、方便、易于操作的分类鉴定方法对微生物进行分析,使人们在一定程度上更科学、更精确、更快速地找到分离物的分类地位。     

低温条件下诱变菌株的营养动力学研究及分子鉴定

以实验室保存的经过低温混合诱导的乳酸菌菌株Q1-4-6为研究对象,通过生长及产酸情况,研究其在低温条件下对于各种营养素的需求,以期为工业大规模生产提供数据参考。低温条件下通过研究碳源对菌株生长及产酸的影响发现,麦芽糖和乳糖对于菌株的生长及产酸有非常好的促进作用,菌株在以蔗糖为碳源的培养基中生长缓慢,而在以乳糖为碳源的培养基中生长最好。3种氮源对于菌株生长的差异不显著,以酵母膏为氮源的菌株生长略好于其他2种。不同浓度金属离子对于菌株生长有不同影响,Zn2+的促生长作用略高于Fe2+,Zn2+浓度越低,菌株生长越好,高浓度的Zn2+则对菌株生长有抑制作用。Fe2+浓度为0.5 g/L时,菌株生长最好,Fe2+浓度过高或过低对于菌株的生长都有抑制作用。根据16S rDNA序列分析结果,同时结合形态学特征、生理生化特性,将菌株鉴定为棉籽糖肠球菌(Enterococcus raffinosus)。     

Isolation and Characterization of Bacillus thuringiensis Strains from Aquatic Environments in Spain

Samples collected from aquatic environments from Spain were analyzed for the occurrence and dipteran toxicity of Bacillus thuringiensis. From a total of 41 samples, 122 isolates were obtained, yielding a B. thuringiensis index of 0.22. Isolates were assigned to 13 different serovars, with serovar thuringiensis (serotype H1) the most frequently found. Toxicity tests carried out revealed that eight isolates (6.6% out of the total) were active against Tipula oleracea larvae. Serological tests assigned these toxic isolates to serovar thuringiensis. The toxicity found in these isolates against the tipulid was approximately seven times lower than that shown by the standard strain B. thuringiensis ser. israelensis IPS-82. Implication of Cry2A protein in toxic activity is hypothesized. Received: 3 December 1999 / Accepted: 5 January 2000     

New Strategy for Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.     

Isolation and Characterization of Antagonistic Bacillus Strains Capable to Degrade Ethylenethiourea

In this study, more than 150 bacteria showing antagonistic properties against bacterial and fungal pathogens of the tomato plant were isolated and characterized. The most efficient agents against these phytopathogenic microorganisms belong to the genus Bacillus: the best biocontrol isolates were representatives of Bacillus subtilis, B. mojavensis and B. amyloliquefaciens species. They intensively produced fengycin or/and surfactin depsipeptide antibiotics and also proved to be excellent protease secretors. It was proved, that the selected strains were able to use ethylenethiourea (ETU) as sole nitrogen source. These antagonistic and ETU-degrading Bacillus strains can be applied as biocontrol and also as bioremediation agents.     

Isolation and Characterization of Thymineless Mutants from Ultraviolet-Sensitive Bacillus subtilis Strains

Thymineless mutants of Bacillus subtilis 168 ind, hcr-9 were isolated by using trimethoprim. These and other Thy⁻ strains differed drastically from Thy⁺ ones in their patterns of [³H]thymidine uptake and growth in trimethoprim-containing medium. Transformation was negligible between most mutants derived from the ultraviolet-sensitive strain 168 ind, hcr-9 but significant between 168 ind, thy and these mutants. The latter and these new mutants all grow in the presence of trimethoprim plus thymidine or thymine and fail to grow if thymine or thymidine is omitted.     

Identification of cry1I-Type Genes from Bacillus thuringiensis Strains and Characterization of a Novel cry1I-Type Gene

A PCR-restriction fragment length polymorphism method for identification of cry1I-type genes from Bacillus thuringiensis was established by designing a pair of universal primers based on the conserved regions of the genes to amplify 1,548-bp cry1I-type gene fragments. Amplification products were digested with the Bsp119I and BanI enzymes, and four kinds of known cry1I-type genes were successfully identified. The results showed that cry1I-type genes appeared in 95 of 115 B. thuringiensis isolates and 7 of 13 standard strains. A novel cry1I-type gene was found in one standard strain and six isolates. The novel cry1I gene was cloned from B. thuringiensis isolate Btc007 and subcloned into vector pET-21b. Then it was overexpressed in Escherichia coli BL21(DE3). The expressed product was shown to be toxic to the diamondback moth (Plutella xylostella), Asian corn borer (Ostrinia furnacalis), and soybean pod borer (Leguminivora glycinivorella). However, it was not toxic to the cotton bollworm (Helicoverpa armigera), beet armyworm (Spodoptera exigua), or elm leaf beetle (Pyrrhalta aenescens) in bioassays. Subsequently, the Cry protein encoded by this novel cry gene was designated Cry1Ie1 by the B. thuringiensis δ-endotoxin nomenclature committee.     

Characterization of Fluorescent and Nonfluorescent Peptide Siderophores Produced by Pseudomonas syringae Strains and Their Potential Use in Strain Identification

Nonfluorescent highly virulent strains of Pseudomonas syringae pv. aptata isolated in different European countries and in Uruguay produce a nonfluorescent peptide siderophore, the production of which is iron repressed and specific to these strains. The amino acid composition of this siderophore is identical to that of the dominant fluorescent peptide siderophore produced by fluorescent P. syringae strains, and the molecular masses of the respective Fe(III) chelates are 1,177 and 1,175 atomic mass units. The unchelated nonfluorescent siderophore is converted into the fluorescent siderophore at pH 10, and colors and spectral characteristics of the unchelated siderophores and of the Fe(III)-chelates in acidic conditions are similar to those of dihydropyoverdins and pyoverdins, respectively. The nonfluorescent siderophore is used by fluorescent and nonfluorescent P. syringae strains. These results and additional mass spectrometry data strongly suggest the presence of a pyoverdin chromophore in the fluorescent siderophore and a dihydropyoverdin chromophore in the nonfluorescent siderophore, which are both ligated to a succinamide residue. When chelated, the siderophores behave differently from typical pyoverdins and dihydropyoverdins in neutral and alkaline conditions, apparently because of the ionization occurring around pH 4.5 of carboxylic acids present in β-hydroxyaspartic acid residues of the peptide chains. These differences can be detected visually by pH-dependent changes of the chelate colors and spectrophotochemically. These characteristics and the electrophoretic behavior of the unchelated and chelated siderophores offer new tools to discriminate between saprophytic fluorescent Pseudomonas species and fluorescent P. syringae and P. viridiflava strains and to distinguish between the two siderovars in P. syringae pv. aptata.     

Molecular Characterization of the Flagellar Hook in Bacillus subtilis

The structure of the Gram-positive flagellum is poorly understood, and Bacillus subtilis encodes three proteins homologous to the flagellar hook protein from Salmonella enterica. Here we generated a modified B. subtilis hook protein that could be fluorescently stained using a cysteine-reactive dye. We used the fluorescently labeled hook to demonstrate that FlgE is the hook structural protein and that FliK regulated hook length. We further demonstrate that two proteins of unknown function, FlhO and FlhP, and the putative hook cap, FlgD, were required for hook assembly, such that when flhO, flhP, or flgD was mutated, hook protein was secreted into the supernatant. All mutants defective in hook completion resulted in homogeneously reduced σ(D)-dependent gene expression due to the action of the anti-sigma factor FlgM.     

Production and Use of Monoclonal Antibodies for Identification of Strains of Rhizobium trifolii

We produced a monoclonal antibody against Rhizobium trifolii 162×95. This antibody in cell culture supernatant was used in an indirect enzyme-linked immunosorbent assay to differentiate strain 162×95 from naturalized strains in the Appalachian region. Nodules crushed in 0.1 to 0.2 ml of phosphate-buffered saline and used to charge enzyme-linked immunosorbent assay plates gave strong absorbance readings. Heat-inactivated and noninactivated portions of 162×95 cultures were strongly reactive, indicating that the antigen is probably a carbohydrate. Of 10 strains from California, where 162×95 was isolated, 6 strongly cross-reacted with the antibody. The cellular protein patterns in a sodium dodecyl sulfate-polyacrylamide gradient gel of cross-reactive strains were essentially identical. A Western blot analysis indicated that the antibody was against a 19.8-kilodalton band. The Western blot analysis also revealed that the polyvalent antiserum contained other strongly reacting antibodies with molecular weights of approximately 20,000, indicating the possibility that other monoclonal antibodies to detect strain 162×95 may be produced. However, the available antibody has been shown to be useful for short-term experiments. Based upon protein profiles and immunological reactions, there are 4 or 5 California strains rather than 10.     

Quantitation of Bacillus clausii in biological samples by real-time polymerase chain reaction

A real-time PCR assay targeting the highly specific erm34 sequence of Bacillus clausii DNA was developed and optimized. The quantitative assay showed a sensitivity level of 10(2) CFU/microl of sample. The method may represent a useful tool for monitoring the role of B. clausii as probiotic in vivo.     

抗动物病原菌芽孢杆菌的筛选、初步鉴定和抗菌活性

从鸡肠道分离、挑选的18株芽孢杆菌经培养特征、形态观察、生理生化实验,初步被鉴定为枯草芽孢杆菌(Bacillus subtilus)、地衣芽孢杆菌(Bacillus licheniformis)、蜡状芽孢杆菌(Bacillus cereus)、巨大芽孢杆菌(Bacillus megaterium)和凝结芽孢杆菌(Bacillus coagulans)。同时测定了它们对5种常见动物病原菌大肠杆菌(E.coli)、金黄色葡萄球菌(Staphylococcus aureus)、肠炎沙门氏菌(Salmonella typhimurium)、猪链球菌(Streptococcus suis)、奇异变形杆菌(Proteus mirabilis)的抑菌活性,其中有4株芽孢杆菌对5种病原菌都有抑制作用。还分别测定了它们产木聚糖酶(从0.895 U1到3.239 U1)和纤维素酶活性(分别为0.391 U2和0.465 U2)。结果表明,芽孢杆菌分离株BC17、BC32、BC106、BC228、BC247和BC261具有作为益生菌的开发潜力。