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PubMed2001

Rapid identification of Bacillus cereus based on the detection of a 28.5-kilodalton cell surface antigen.

Conventional procedures for the identification of suspect Bacillus cereus isolated on mannitol-egg yolk-polymyxin (MYP) agar may need several days. To facilitate the identification of the bacterium, an enzyme-linked immunosorbent assay (ELISA) was developed. The assay was based on the detection of a 28.5-kDa cell surface antigen of B. cereus. Bacterial colonies grown on MYP agar or nutrient agar were suspended in phosphate-buffered saline (pH 7.2) containing 0.1% Teepol. The cell suspensions were heated at 100 degrees C for 5 min and added to the microtiter plates coated with antibodies against the 28.5-kDa antigen. After washing, the same antibodies labeled with horseradish peroxidase were used as secondary antibodies to reveal the signal of antigen-antibody reaction. For 38 strains of B. cereus and 127 strains of non-B. cereus bacteria (including 79 isolates of Bacillus spp.) tested, the sensitivity and specificity of the ELISA were 100 and 88.2%, respectively. Strains producing false-positive results were members of the B. cereus group (i.e., Bacillus anthracis, Bacillus mycoides, and Bacillus thuringiensis), which are genetically and biochemically similar to B. cereus. Similar ELISA results were obtained by using antibodies against another cell surface antigen with a molecular mass of 20 kDa. If members of the B. cereus group were recognized as a single species, the sensitivity and specificity of the ELISA were 100 and 99.1%, respectively. The ELISA could be used as a rapid method for presumptive identification of B. cereus grown on MYP agar.

Antibodies, Bacterial↗
PubMed1982

An enrichment isolation procedure for minor Bacillus populations.

In general, spores of B. cereus, B. megaterium, B. Sphaericus and B. subtilis strains germinated uniformly within a short time of incubation in a germination medium. In contrast, spores of B, circulans, B. brevis, B. laterosporus, B. pulvifaciens, B. polymyxa, B. pumilus, B. licheniformis and B. coagulans strains were usually slow and/or uneven germinators under the same conditions of incubation. The former group of Bacillus strains occur frequently in soils as the predominant population and the latter group of Bacillus species are found in many cases as minor populations. The minor populations of Bacillus were isolated with difficulty by the standard dilution-plate technique, but could easily be enriched by treating the soil sample in a germination medium for 2 to 3 hours at 30 to 35 degrees C, followed by heating it at 65 degrees C for 10 minutes ("minor-shifted isolation"). Using this technique, the minor Bacillus strains could be isolated from samples containing 100- to 1,000-fold more of the rapid germinators of Bacillus.

Bacillus↗
PubMed1999

Changes in fecal microflora induced by intubation of mice with Bacillus subtilis (natto) spores are dependent upon dietary components.

We examined changes in mouse fecal microflora after various dietary components and Bacillus subtilis (natto) spores were delivered by intubation. The administration of intact spores of Bacillus subtilis (natto) did not affect fecal Enterobacteriaceae and Enterococcus spp. in all three diet groups; on the other hand, it did affect fecal Bacteroidaceae and Lactobacillus spp., depending upon the diets fed. The administration of autoclaved spores did not alter fecal microflora. In vitro cultures of Lactobacillus murinus obtained from mouse feces, together with Bacillus subtilis (natto) under aerobic conditions as a mixed culture, revealed that the growth of L. murinus was enhanced by the addition of intact spores of Bacillus subtilis (natto). This enhancement of growth was displayed only in media containing either sucrose, glucose, maltose, or fructose but not in media containing cornstarch, soluble starch, or microcrystalline cellulose. From these results it was evident that some metabolites of Bacillus subtilis (natto) produced during germination and (or) outgrowth of spores of this strain, requiring monosaccharides or oligosaccharides, participated in the enhancement of the growth of L. murinus.

Animals↗
PubMedDate not supplied

[The monosaccharide composition of the glycocalyx in Mollicutes and phylogenetically related bacteria in the genus Bacillus].

A number of monosaccharides composing the glycocalix carbohydrates of some Mollicutes (Mycoplasma pneumoniae FH, M. hominis PG21, M. fermentans PG18, Acholeplasma laidlawii PG8, A. laidlawii var. granulum st. 118) and phylogenetically related bacteria from the genus Bacillus (Bacillus cereus 89, B. cereus DM423, B. subtilis 1/2, B. licheniformis 31, B. licheniformis 49) were detected using plant lectins of definite carbohydrate specificity labelled by colloid gold. The structure of extracellular glycopolymers of microorganisms was discussed concerning their role in the adhesion process and their specializations in colonization of the appropriate organs and tissues of macroorganisms as well as the action of probiotics on the basis of bacteria from the genus Bacillus. It was marked, that the mollicute cells typically interacted with all vegetable lectins tested while bacilli studied bind certain lectins only. The surface carbohydrates prevailing in monosaccharides from tested one for superficial structures of Bacillus pathogenic strains were: sialic acid, beta-D-galactose and alpha-N-acetyl-D-galactosamine, while L-fucose and alpha-D-glucose were observed in insignificant quantity or were absent. The nonpathogenic Bacillus strains being a basis of biopreparations were distinguished by the insignificant amount of sialic acid and other monosaccharides in the composition of carbohydrates of their glycocalix. Thus, as a result of research it was confirmed, that the superficial glycopolymers of Mollicutes and some related bacilli were alike as to presence of some monosaccharides that depended on taxonomic position of microorganisms and their biological activity.

Bacillus↗
PubMed2000

Management of suspected nosocomial infection: an audit of 19 hospitalized patients with septicemia caused by Bacillus species.

From April to August of 2000, Bacillus spp. were detected in the blood culture of 29 patients in a hospital in Japan. Of these patients, 19 had clinical signs of septicemia; positive culture in the remaining 10 patients was attributed to contamination with skin flora at the site of puncture. Of the 18 strains evaluated, 15 were Bacillus cereus, 2 were Bacillus subtilis, and one was Bacillus licheniformis. The only hospital death observed was that of a patient who had no clinical signs of septicemia at the time of blood sampling. That death is now considered attributable to the underlying neoplasm. The hospital committee for prevention of nosocomial infection concluded after a critical review of the patient records that the cause of septicemia in most cases had been contaminated intravenous lines. To control the situation, the committee recommended the use of a new skin disinfectant, and medical personnel were advised to avoid infusion pauses with interruption of intravenous lines and to replace the caps for the stopcocks with new ones each time the caps were removed. These measures were rigorously observed in addition to the conventional measures for preventing catheter sepsis, and the incidence of septicemia due to the Bacillus spp. declined dramatically thereafter.

Adult↗
PubMed2001

Study on the effect of Bacillus thuringiensis (B.t. serotype H-14) and its toxins against Cephalopina titillator (Clark), the Egyptian camel myiasis producing dipterous fly.

Bacillus t. israelensis (B.t. serotype H-14) and its toxins have a marked lethal effect on both the eggs and the newly moulted 3rd stage larvae of Cephalopina titillator. The Bacillus was less effective than Bacillus and its toxins on both the eggs and larvae. On the other hand, the larvae were more affected by both Bacillus and Bacillus and its toxins than the eggs. Prolonged exposure time showed more lethal effect.

Animals↗
PubMed2003

Genetic polymorphism by RAPD-PCR and phenotypic characteristics of isolated thermotolerant Bacillus strains from hot spring sources.

The polymerase chain reaction (PCR) based random amplified polymorphic DNA (RAPD) assay, morphological, physiological, biochemical and antimicrobial susceptibility test methods have been evaluated for use in the taxonomy of isolated thermotolerant Bacillus from Jordanian hot springs, with specific reference to strains Geobacillus stearothermophilus (ATCC 12980), Bacillus circulans (ATCC 4513) and Bacillus sphaericus (ATCC 14577). A RAPD assay has been optimized and is able to discriminate between numerous thermotolerant Bacillus strains. RAPD-PCR was found to give reproducible thermotolerant Bacillus strains classification of DNA fingerprints for 14 strains including 3 reference strains. A study of 14 isolates and 3 reference strains, analyzing 53 phenotypic characters, resulted in their allocation to five major clusters at 60% similarity. Whereas at 80% similarity, twelve taxonomically distinct groups were evident.

Bacillus↗
PubMed2005

Bacillus clausii exerts immuno-modulatory activity in allergic subjects: a pilot study.

UNLABELLED: Bacillus clausii has been demonstrated to exert some immunomodulatory activities and to be safe. Allergic subjetcs show Th2 polarization and defect of T regulatory cells. Aim of the study was to investigate whether Bacillus clausii administration in allergic subjects might modulate cytokine pattern. Ten subjects (mean age 22.3 years) suffering from allergic rhinitis were enrolled. Bacillus clausii spores (Enterogermina: 2 billions spores/vial) were administered at the dosage schedule of 3 vials a day for 4 weeks. Nasal lavage was performed in all subjects before and after the treatment. A panel of cytokines, including IL4, IL10, IFNgamma, and TGFbeta, was measured by immunoassay in the fluid recovered from nasal lavage, before and after the treatment. RESULTS: Bacillus clausii treatment showed a significant decrease of IL4 levels (p=0.004) and a significant increase of IFNgamma (p=0.038), TGFbeta (p=0.039), and IL10 (p=0.009) levels. In conclusion, this study shows that the Bacillus clausii may exert immuno-modulating activity by affecting cytokine pattern in allergic subjects and confirms previous study conducted in allergic children.

Adolescent↗
PubMed2005

Partial purification and characterization of protease enzyme from Bacillus subtilis megatherium.

Bacteria of genus Bacillus are active producers of extracellular proteases, and characteristics of enzyme production by Bacillus species have been well studied. The aim of this experimental study is isolation and partial purification of protease enzyme from the Bacillus subtilis megatherium bacteria species. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species on suitable media. The partial purification was realized by applying successively ammonium sulfate precipitation, dialysis, DEAE-cellulose ion exchange chromatography to the supernatant. In this study, the effect of substrate concentration, reaction time, the effect of inhibitor and activator on the optimum pH, optimum temperature, pH stability, and temperature stability was determined. Molecular weight of the obtained enzyme was investigated by SDS-PAGE. In this study, the specific activity of the supernatant, which was partially purified from Bacillus subtilis megatherium bacteria, was 10.4 U/mg, specific activity of supernatant was 13.5 U/mg after 80% ammonium sulfate fractionation. The final enzyme preparation was 1.1-fold purer than the crude homogenate. Molecular weight of the protease was determined, and it was found that the weight of enzyme was 45 kDa by using SDS-PAGE.

Bacillus subtilis↗
PubMed1990

Inhibition of Bordetella pertussis and Bacillus anthracis adenylyl cyclases by polyadenylate and "P"-site agonists.

Inhibition of adenylyl cyclases from Bacillus anthrasis and Bordetella pertussis by polyadenylate and by the most potent "P"-site agonists was investigated. These bacterial adenylyl cyclases differed in their sensitivity to inhibition by nominal "P"-site agents and in the effect of divalent cations on this inhibition. The enzyme from Bordetella pertussis was relatively insensitive to inhibition by "P"-site agonists, exhibiting a rank order of potency of 2'd3'AMP greater than 3'-AMP greater than 2',5'-ddAdo approximately Ado approximately 2'-dAdo, with IC50 values for 2'd3'AMP and 3'-AMP of 1-3 mM. Inhibition by 2'd3'AMP, however, was not affected by divalent cation, making it distinct from "P"-site-mediated inhibition of most mammalian adenylyl cyclases. The sensitivity to these nucleosides was comparable with potency for inhibition of bovine sperm adenylyl cyclase but was 3 orders of magnitude less potent than for activated enzyme from bovine or rat brain. The Bordetella pertussis enzyme was similarly insensitive to inhibition by polyadenylate, with 16 microM inhibiting less than 20%. By comparison, Bacillus anthrasis adenylyl cyclase was more potently inhibited by 2'd3'AMP (IC50 approximately 85 microM) but not by the other nucleosides (less than 15% inhibition at 1 mM), and inhibition by 2'd3'AMP was optimally enhanced by 5-10 mM Mg2+ or Mn2+, as is typical for inhibition by "P"-site agonists. The Bacillus anthrasis enzyme was potently inhibited by polyadenylate (IC50 approximately 0.3 microM), comparable to inhibition of brain adenylyl cyclases. Sensitivity of Bacillus anthrasis adenylyl cyclase to poly(A) was diminished somewhat by Ca2+/calmodulin (to IC50 approximately 1 microM) although Ca2+/calmodulin was without effect on inhibition by 2'd3'AMP. In contrast to inhibition of mammalian adenylyl cyclases via the "P"-site, inhibition of both bacterial adenylyl cyclases by 2'd3'AMP was competitive with respect to substrate MgATP. The data indicate basic differences in susceptibilities of these bacterial adenylyl cyclases to inhibition by poly(A), by adenosine analogs, and the effects of divalent cations. Although the potency of 2'd3'AMP and the metal-dependent nature of inhibition of Bacillus anthrasis adenylyl cyclase shared characteristics of "P"-site-mediated inhibition, the fact that inhibition of both bacterial adenylyl cyclases was competitive with respect to substrate strongly suggests that this inhibition was at the catalytic site and that these bacterial enzymes do not contain a distinct "P"-site.

Adenosine↗
PubMed2005

Genomics of the Bacillus cereus group of organisms.

Members of the Bacillus cereus group of organisms include Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis. Collectively, these organisms represent microbes of high economic, medical and biodefense importance. Given this significance, this group contains the highest number of closely related fully sequenced genomes, giving the unique opportunity for thorough comparative genomic analyses. Much of the disease and host specificity of members of this group can be attributed to their plasmids, which vary in size and number. Chromosomes exhibit a high level of synteny and protein similarity, with limited differences in gene content, questioning the speciation of the group members. Genomic data have spurred functional studies that combined microarrays and proteomics. Recent advances are reviewed in this article and highlight the advantages of genomic approaches to the study of this important group of bacteria.

Bacillus anthracis↗
PubMed1997

Sequencing and phylogenetic analysis of the spoIIA operon from diverse Bacillus and Paenibacillus species.

In order to clone the spoIIA operon from three different Bacillus and Paenibacillus species, we designed two sets of PCR primers based on three previously published Bacillus spoIIA sequences. One set of primers corresponded to the C-terminal region of SpoIIAB and a region near the middle of SpoIIAC. These primers were used to amplify the corresponding region of spoIIA from Bacillus stearothermophilus and Paenibacillus polymyxa (previously called Bacillus polymyxa [see Ash, C., Priest, F.G., Collins, M.D., 1993. Molecular identification of ribosomal-RNA group 3 bacilli using a PCR probe test - proposal for the creation of a new genus Paenibacillus. Antonie van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 64, 253-260]. The other set of primers, corresponding to an N-terminal and a C-terminal region of SpoIIAC, was used for B. sphaericus. The PCR products were used as probes for Southern blotting of homologous chromosomal DNA. DNA corresponding to spoIIA from the three organisms was identified by screening chromosomal DNA libraries, and cloned. Sequence analysis showed that all spoIIA sequences were conserved, but conservation was strongest in SpoIIAC and least strong in SpoIIAA. In the promoter the -35 region was conserved well but the -10 region rather poorly. Within the proteins, certain regions were particularly strongly conserved, suggesting that they are essential to the function of the protein. Phylogenetic analysis of spoIIA suggested that B. stearothermophilus is close to B. subtilis and B. licheniformis, but that P. polymyxa and B. sphaericus are remote from B. subtilis.

Amino Acid Sequence↗
PubMed1999

PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis.

Members of the Bacillus cereus group (B. anthracis, B. cereus, B. mycoides and B. thuringiensis) are well-known pathogens of mammals (B. anthracis and B. cereus) and insects (B. thuringiensis). The specific diseases they cause depend on their capacity to produce specific virulence factors, such as the lethal toxin of B. anthracis and the Cry toxins of B. thuringiensis. However, these Bacillus spp. also produce a variety of proteins, such as phospholipases C, which are known to act as virulence factors in various pathogenic bacteria. Few genes encoding these virulence factors have been characterized in pathogenic Bacillus spp. and little is known about the regulation of their expression. We had previously reported that in B. thuringiensis expression of the phosphatidylinositol-specific phospholipase C gene is regulated by the transcriptional activator PlcR. Here we report the identification of several extracellular virulence factor genes by the virtue of their PlcR-regulated expression. These PlcR-regulated genes encode degradative enzymes, cell-surface proteins and enterotoxins. The PlcR-regulated genes are widely dispersed on the chromosome and therefore do not constitute a pathogenic island. Analysis of the promoter region of the PlcR-regulated genes revealed the presence of a highly conserved palindromic region (TATGNAN4TNCATA), which is presumably the specific recognition target for PlcR activation. We found that the plcR gene is also present in and probably restricted to all the members of the B. cereus group. However, although the polypeptide encoded by the B. cereus PlcR gene is functionally equivalent to the B. thuringiensis regulator, the polypeptide encoded by the B. anthracis gene is truncated and not active as a transcriptional activator. PlcR is the first example described of a pleiotropic regulator involved in the control of extracellular virulence factor expression in pathogenic Bacillus spp. These results have implications for the taxonomic relationships among members of the B. cereus group, the virulence properties of these bacteria and the safety of B. thuringiensis-based biopesticides.

Amino Acid Sequence↗
PubMed2001

Distribution of S-layers on the surface of Bacillus cereus strains: phylogenetic origin and ecological pressure.

Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis have been described as members of the Bacillus cereus group but are, in fact, one species. B. anthracis is a mammal pathogen, B. thuringiensis an entomopathogen and B. cereus a ubiquitous soil bacterium and an occasional human pathogen. In two clinical isolates of B. cereus, in some B. thuringiensis strains and in B. anthracis, an S-layer has been described. We investigated how the S-layer is distributed in B. cereus, and whether phylogeny or ecology could explain its presence on the surface of some but not all strains. We first developed a simple biochemical assay to test for the presence of the S-layer. We then used the assay with 51 strains of known genetic relationship: 26 genetically diverse B. cereus and 25 non-B. anthracis of the B. anthracis cluster. When present, the genetic organization of the S-layer locus was analysed further. It was identical in B. cereus and B. anthracis. Nineteen strains harboured an S-layer, 16 of which belonged to the B. anthracis cluster. All 19 were B. cereus clinical isolates or B. thuringiensis, except for one soil and one dairy strain. These findings suggest a common phylogenetic origin for the S-layer at the surface of B. cereus strains and, presumably, ecological pressure on its maintenance.

Bacillus anthracis↗
PubMed1975

Characteristics of a Bacillus megaterium bacteriophage.

A bacteriophage which infects and lyses Bacillus megaterium ATCC 19213 was isolated from the soil. The phage produces lysis on nine strains of B, megaterium tested but did not lyse a Bacillus cereus or Bacillus licheniformis strain, nor any of eight Bacillus subtilis strains tested. Physical characteristics of the phage including morphology, size, thermal and pH stability, and buoyant density were examined. The nucleic acid is double-stranded DNA of mol. wt. 41.7 times 10 and 36 to 38.5 mol percent guanine plus cytosine (G+C).

Bacillus cereus↗
PubMed1986

Numerical taxonomy of Bacillus isolated from orally administered drugs.

Numerical taxonomy procedures were used to study 118 strains of Bacillus isolated from non-sterile drugs prepared for oral administration. Similarities between pairs of strains were calculated by the simple matching coefficient of Sokal and Michener (SSM). Each strain was tested for 60 unit characters and three clusters were defined. The strains in each cluster presented a similarity level of at least 60%. Cluster A comprised the strains identified as Bacillus cereus (SSM = 93.13%), cluster B contained three subgroups corresponding to the species B. pumilus, B. subtilis and B. licheniformis (SSM = 84.35%) and cluster C also included three subgroups that belonged to the species B. firmus, B. lentus and B. badius (SSM = 80.14%). The most discriminating tests were selected to differentiate the clusters from the subgroups. The feature with the highest discriminating power between clusters A and B was the lack of acid production from arabinose and mannitol. The Voges-Proskauer, methyl red tests and sensitivity to polymyxin B clearly distinguished cluster A from C. The Voges-Proskauer test and acid production from arabinose were the best to differentiate between B and C. Bacillus pumilus and B. subtilis differed in starch hydrolysis and B. licheniformis in growing anaerobically. To discriminate B. firmus from B. lentus the most important tests were the acid production from glucose and sucrose; intermediate strains were found. Bacillus badius was differentiated from B. firmus by 10 tests, and from B. lentus by the production of urease.

Administration, Oral↗
PubMed2005

Plasmid-encoded regulator of extracellular proteases in Bacillus anthracis.

Bacillus anthracis Sterne cured of the pXO1 plasmid had enhanced secreted protease activity during the postexponential phase but no change in hemolytic or lecithinase activities. A zymogen profile revealed at least six proteases, including serine, metal, and perhaps cysteine types. There were similar amounts of protease secreted by the closely related species Bacillus cereus and Bacillus thuringiensis, but the patterns differed. Among the pXO1 plasmid-encoded proteins, there is a tetratricopeptide protein designated Cot43 that is related to the Rap proteins of Bacillus subtilis and the PlcR pleiotropic regulator of secreted enzymes and toxins in B. thuringiensis. A disruption of the cot43 gene resulted in overproduction of several proteases to a somewhat greater extent than in the plasmid-cured strain. Transformation of either of these strains with a clone of the cot43 gene resulted in the inhibition of accumulation of some of the proteases and induction of at least one. On the basis of lacZ fusions, transcription of the cot43 gene increased in late exponential cells at the time of protease accumulation. The expression of lacZ fusions to the upstream regions of two B. anthracis extracellular protease genes was greater in the strain with the disruption of cot43 than in the Sterne strain, indicating regulation at the level of transcription. In B. anthracis, a pXO1 plasmid-encoded protein directly modulates or indirectly regulates the transcription of genes for several chromosomally encoded extracellular proteases.

Bacillus anthracis↗
PubMed1965

CONSERVATION OF RIBOSOMAL AND MESSENGER RIBONUCLEIC ACID CISTRONS IN BACILLUS SPECIES.

Doi, Roy H. (Syracuse University, Syracuse, N.Y.), and Richard T. Igarashi. Conservation of ribosomal and messenger ribonucleic acid cistrons in Bacillus species. J. Bacteriol. 90:384-390. 1965.-Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) fractions from various Bacillus species were tested for interspecies DNA-RNA hybrid formation. DNA preparations from B. subtilis, B. cereus, B. megaterium, B. stearothermophilus, and B. macerans, whose base composition varied from 33 to 50% guanylate + cytidylate content, were used in the hybrid annealing mixtures with pulse-labeled RNA from sporulating cells and from log-phase cells of B. subtilis and B. cereus. Efficient hybridization in these cases was obtained only in homologous annealing situations. When heterologous DNA and RNA preparations were tested for hybrid formation, only 1 to 6% of the homologous hybridization was obtained. Although the efficiency of hybrid formation was low, the results were reproducible. No difference in efficiency of hybrid formation was observed between the messenger RNA from sporulating cells and that from log-phase cells. When B. subtilis ribosomal RNA was placed in annealing mixtures with heterologous DNA, 47.5 to 62.9% of the homologous hybridization was obtained. These results suggest that a small number of identical sequences are present among the Bacillus species. Furthermore, the ribosomal RNA cistrons appear to be more highly conserved relative to other genetic sequences.

Bacillus↗