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PubMed1999

Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay.

An improved qualitative cell cytotoxicity assay for the detection of Bacillus cereus emetic and enterotoxin is described. The presence of toxin in culture supernatant fluids was detected by measurement with the tetrazolium salt MTT, as it adversely affects the metabolic status of cultured CHO cells. Psychrotrophic B. cereus isolates (65) were assessed for toxin production using the cytotoxicity assay, and 91% of culture supernatant fluids were cytotoxic. Toxin assessment using BCET-RPLA and ELISA immunoassays indicated that 51% and 85% of the cultures, respectively, were toxigenic. There were pronounced strain differences in the amount of toxin produced by the B. cereus isolates. Some isolates of B. circulans, B. laterosporus/cereus, B. lentus, B. licheniformis, B. mycoides, B. subtilis and B. thuringiensis were also toxigenic.

Animals↗
PubMed2003

Genetic relationship in the 'Bacillus cereus group' by rep-PCR fingerprinting and sequencing of a Bacillus anthracis-specific rep-PCR fragment.

AIMS: To evaluate the genetic relationship in the Bacillus cereus group by rep-PCR fingerprinting. METHODS AND RESULTS: A collection of 112 strains of the six species of the B. cereus group was analysed by rep-PCR fingerprinting using the BOX-A1R primer. A relative genetic distinctness was found among the species. Cluster analysis of the rep-PCR patterns showed clusters of B. thuringiensis strains quite separate from those of B. cereus strains. The B. anthracis strains represented an independent lineage in a B. cereus cluster. The B. mycoides, B. pseudomycoides and B. weihenstephanensis strains were clustered into three groups at some distance from the other species. Comparison of sequences of AC-390, a typical B. anthracis rep-PCR fragment, from 27 strains of B. anthracis, B. cereus, B. thuringiensis and B. weihenstephanensis, representative of different clusters identified by rep-PCR fingerprinting, confirmed that B. anthracis diverges from its related species. CONCLUSIONS: The genetic relationship deduced from the rep-PCR patterns indicates a relatively clear separation of the six species, suggesting that they can indeed be considered as separate units. SIGNIFICANCE AND IMPACT OF THE STUDY: rep-PCR fingerprinting can make a contribution in the clarification of the genetic relationships between the species of the B. cereus group.

Bacillus anthracis↗
PubMed2003

Efficacy and efficiency of new Bacillus thuringiensis var israelensis and Bacillus sphaericus formulations against Afrotropical anophelines in Western Kenya.

We evaluated the efficacy of new water-dispersible granular (WDG) formulations of Bacillus thuringienis var. israelensis (Bti; VectoBac) and B. sphaericus (Bs; VectoLex), Valent BioScience Corp., Illinois, USA) for the control of larval Anopheles gambiae sensu lato Giles mosquitoes in a malaria-endemic area around Lake Victoria, Western Kenya. WDG and powder formulations were compared in laboratory bioassays and followed by efficiency and residual effect assessments of both WDG formulations in open field experiments. LC50 and LC95 values for the Bti/Bs strains and their formulations show high susceptibility of A. gambiae sensu stricto under laboratory conditions. The larvae proved more susceptible to Bs than to Bti and the WDG formulations were slightly superior to the powder formulations. High efficiency was also shown in the open field trials, and a minimum dosage of 200 g/ha Bti WDG, representing the LC95 of the laboratory tests, was sufficient to fully suppress emergence of mosquitoes when applied at weekly intervals. Bti WDG did not show a residual effect, irrespective of the concentration applied. The Bs WDG formulation, however, showed significant larval reductions up to 11 days post-treatment at application doses of either 1 or 5 kg/ha. We conclude that the main malaria vector in our study area is highly susceptible to these microbial control agents. Minimum effective dosages to achieve elimination of the larval population in a given habitat are extremely low and environmental impact is negligible. Microbial products for larval control have therefore great potential within Integrated Vector Management programmes and may augment control efforts against adult vector stages, such as the use of insecticide-treated bednets, in many parts of Africa.

Animals↗
PubMed1992

Mass production of sphingomyelinase of Bacillus cereus by a protein-hyperproducing strain, Bacillus brevis 47, and its purification.

Sphingomyelinase (sphingomyelin cholinephosphohydrolase) [EC 3.1.4.12] of Bacillus cereus was overproduced in a protein-hyperproducing strain, B. brevis 47, by cloning the gene into an expression vector pNU211, which has been developed to express a foreign gene utilizing a promoter and a signal sequence of an outer cell wall protein gene. From 1 liter of culture, about 10 mg of protein was purified to near-homogeneity by two steps of column chromatography; this is almost 500 times higher production compared to the conventional preparation from the original strain, B. cereus IAM 1208. The N-terminal amino acid sequence of the secreted enzyme was identical to that of the authentic enzyme, indicating that the signal sequence for secretion of B. cereus was processed properly in B. brevis 47.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the Bacillus licheniformis homologue of the sporulation locus spoIIA of Bacillus subtilis.

The spoIIA locus of Bacillus licheniformis was cloned into the phage vector phi 105J9; selection was based on the ability of the clone to complement mutations in both the first and the last of the spoIIA genes of B. subtilis. The B. licheniformis DNA was subcloned into M13 and sequenced; it includes three genes of identical lengths to those of B. subtilis. The average interspecies difference in nucleotide sequence is 24%; C occurs at the third position of codons 55% more often in the B. licheniformis than in the B. subtilis sequence. The average difference in predicted amino acid sequence is 11%, but the distribution of these differences is far from random, and there are several long stretches of amino acid sequence that are identical in the two organisms. The distribution of non-conservative amino acid changes between the species is also strikingly non-random; no such changes are found in those regions in which missense mutations are known in B. subtilis. Each species has an open reading frame 5' of the spoIIAA gene, but the predicted amino acid sequence, and the distribution of differences between the two species in both nucleotides and predicted amino acids, suggest that these open reading frames are not expressed. Both species have regions 3' of the open reading frames which resemble rho-independent terminators of transcription, but that in B. licheniformis is longer and could form a more elaborate secondary structure than that in B. subtilis.

Amino Acid Sequence↗
PubMed1987

Comparative antigenicity of spore coat proteins from Bacillus species using antibody to spore coat proteins of Bacillus megaterium.

Spore coat proteins obtained by extraction with sodium dodecylsulfate/dithiothreitol from six Bacillus spores were compared by immunoblot analysis using antibodies to spore coat proteins from two strains of B. megaterium. Although the extract from spores of each strain had heterogenous proteins with various molecular weights, there were some bands which cross-reacted with specific antibodies from B. megaterium spores. Specific antibody to 48K protein from B. megaterium ATCC 12872 cross-reacted with 17K protein from B. megaterium ATCC 19213, 13K protein from B. cereus and 50K protein from B. subtilis 60015 and B. subtilis NRRL B558. Also, specific antibody to 22K protein from the same strain cross-reacted with 22K and 17K proteins from B. megaterium ATCC 19213 and 13K protein from B. cereus T. Specific antibody to 17K protein from B. megaterium ATCC 19213 reacted with 22K and 19K proteins in addition to 17K protein of own strain, and it was cross-reactive with 16K protein from B. megaterium ATCC 12872, 19K and 27K proteins from B. thiaminolyticus, 13K protein from B. cereus.

Antibodies, Bacterial↗
PubMed1994

A small (2.4 Mb) Bacillus cereus chromosome corresponds to a conserved region of a larger (5.3 Mb) Bacillus cereus chromosome.

We have determined the sizes of the chromosomes of six Bacillus cereus strains (range 2.4-4.3 Mb) and constructed a physical map of the smallest B. cereus chromosome (2.4 Mb). This map was compared to those of the chromosomes of four B. cereus strains and one B. thuringiensis strain previously determined to be 5.4-6.3 Mb. Of more than 50 probes, 30 were localized to the same half of the larger B. cereus and B. thuringiensis chromosomes. All 30 were also present on the small chromosome. Twenty of the probes present on the other half of the larger chromosomes were either present on extrachromosomal DNA, or absent from the B. cereus strain carrying the small chromosome. We propose that the genome of B. cereus/B. thuringiensis has one constant part and another less stable part which is more easily mobilized into other genetic elements. This part of the genome is localized to one region of the chromosome and may be subject to deletions or more frequent relocations between the chromosome and episomal elements of varying sizes up to the order of megabases.

Bacillus cereus↗
PubMed2004

Field evaluation of the effect of a probiotic-containing Bacillus licheniformis and Bacillus subtilis spores on the health status, performance, and carcass quality of grower and finisher pigs.

The aim of the study was to assess the efficacy of BioPlus 2B, a probiotic containing Bacillus licheniformis and B. subtilis spores, on the health status and productivity of pigs, during weaning, growing and finishing stages of growth. On a commercial farrow-to-finish farm, five experimental groups were formed, each of 54 weaned piglets. The pigs of the first group (double controls) received normal feed with no probiotic and the pigs of the second group (untreated controls) received BioPlus 2B only during the weaning stage. The pigs of the third, the fourth and the fifth group received the same as the second group feed but, at the growing and at a part of the finishing stages, supplemented with three different doses of Bioplus 2B, a low, medium and high dose, respectively. The results have shown that, compared with the double controls, BioPlus 2B-treated pigs had a lower morbidity and mortality during the whole trial period, compared with the double controls (range from 9.26 to 14.81% versus 25.93% and from 0.00 to 3.70% versus 11.1%, respectively), as a result of the lower incidence of post-weaning diarrhoea due mainly to Escherichia coli. Weight gain, feed conversion ratio and carcass quality of the BioPlus 2B-treated pigs were significantly improved compared with the double controls, whilst the beneficial effects of the probiotic were more pronounced when the medium and high doses were used.

Animal Husbandry↗
PubMed1993

An operator binding-negative mutation of Xyl repressor from Bacillus subtilis is trans dominant in Bacillus megaterium.

We have selected a Bacillus subtilis 168-borne xylR Ser to Leu mutation at position 41 of the encoded amino acid sequence showing a constitutive expression phenotype for the xyl operon. When cloned on a multi-copy plasmid in a B. megaterium strain harbouring a single-copy xylA-lacZ fusion it leads to derepression of beta-galactosidase expression. Thus, it is trans dominant over the endogenous xylR, indicating that Xyl repressor functions as a multimer. This result also supports the assumption that the mutation is in a putative alpha-helix-turn-alpha-helix operator binding motif of Xyl repressor.

Amino Acid Sequence↗
PubMed1995

Differentiation of Bacillus anthracis and other 'Bacillus cereus group' bacteria using IS231-derived sequences.

Sequences based on the conserved 20 bp inverted repeat of IS231 variants were used as polymerase chain reaction-based fingerprinting primers of the member species of the Bacillus cereus group (B. anthracis, B. cereus, B. thuringiensis and B. mycoides), because of their close association with transposons, principally Tn4430 in B. thuringiensis. Fingerprints of B. anthracis were simple, and specifically allowed its identification and sub-differentiation from other members of the group. Fingerprints for B. cereus were strain-specific; those for B. thuringensis gave a 1650 bp product, characteristic of IS231 variants A-F. The same reaction conditions gave one or two bands for both B. anthracis and B. cereus that differed by restriction endonuclease mapping from the B. thuringiensis PCR product and established IS231 restriction maps; this does not preclude some kind of relationship between these products and IS231.

Bacillus anthracis↗
PubMed1996

The chromosome map of Bacillus thuringiensis subsp. canadensis HD224 is highly similar to that of the Bacillus cereus type strain ATCC 14579.

A physical map of the Bacillus thuringiensis subsp. canadensis HD224 chromosome based on AscI, NotI, and SfiI restriction sites has been established. The chromosome map of 4.3 Mb was similar to a revised map of the chromosome of the B. cereus type strain ATCC 14579, except that the B. thuringiensis subsp. canadensis HD224 chromosome lacked a NotI site and had two additional AscI sites. The positions of 27 probes were identical in the common macromap. A probe for the insecticidal toxin gene, cryIA, hybridized only to the B. thuringiensis subsp. canadensis HD224 chromosome. The BssHII ribotype patterns were almost identical confirming the similarity between the two strains.

Bacillus cereus↗
PubMed1997

Discrimination between Bacillus cereus and Bacillus thuringiensis using specific DNA probes based on variable regions of 16S rRNA.

Identification of Bacillus cereus and differentiation between B. cereus and closely related species are currently based on biochemical tests. The main problem is to discriminate between B. cereus and B. thuringiensis. Sequencing part of the 16S rRNA showed that several B. cereus isolates present in food and involved in food poisoning, confirmed according to the classical biochemical methods, were in fact B. thuringiensis. As this organism is the most commonly used microbial insecticide worldwide, the results of this study emphasize the need for accurate identification methods and for careful screening of strains for use as insecticides. Therefore, specific DNA probes based on the variable region VI of 16S rRNA of B. cereus and B. thuringiensis were designed. The probes were used in hybridization experiments with the variable region amplified using the polymerase chain reaction. In this way, a rapid and sensitive method was developed to distinguish B. cereus and B. thuringiensis.

Bacillus cereus↗
PubMed2002

Molecular analysis of rifampin resistance in Bacillus anthracis and Bacillus cereus.

Rifampin-resistant mutants were selected from UV-light-treated Bacillus cereus (20 mutants) and attenuated B. anthracis (23 mutants). In addition, spontaneous rifampin-resistant mutants were also isolated in attenuated B. anthracis (22 mutants). The rifampin resistance clusters of the rpoB gene were sequenced for all 65 mutants. Mutations associated with resistance were consistent with those from other bacteria, though two novel changes were observed. The spontaneous rate of resistance was estimated at 1.57 x 10(-9) mutations/generation by a Luria-Delbrück fluctuation test.

Amino Acid Sequence↗
PubMed1989

Enzyme activities and antibiotic susceptibility of colonial variants of Bacillus subtilis and Bacillus licheniformis.

A nonmucoid colonial variant of a mucoid Bacillus subtilis strain produced less amylase activity and a transparent colonial variant of a B. licheniformis strain produced less protease activity compared with their parents. Antibiotic susceptibility patterns of the colonial variants differed, and increased resistance to beta-lactam antibiotics was correlated with increased production of extracellular beta-lactamase.

Amylases↗
PubMed2001

Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis.

Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereus and B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceT gene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes in B. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.

Bacillus cereus↗
PubMed2001

Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates.

We examined 154 Norwegian B. cereus and B. thuringiensis soil isolates (collected from five different locations), 8 B. cereus and 2 B. thuringiensis reference strains, and 2 Bacillus anthracis strains by using fluorescent amplified fragment length polymorphism (AFLP). We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria. Both B. anthracis strains analyzed were closely related and affiliated with a B. cereus milk isolate (ATCC 4342) and a B. cereus human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including B. anthracis, were more closely related to one another than to the environmental isolates. Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups. This analysis was consistent with the AFLP analysis, although of much lower resolution. The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future.

Bacillus cereus↗
PubMed2003

Bacillus anthracis diverges from related clades of the Bacillus cereus group in 16S-23S ribosomal DNA intergenic transcribed spacers containing tRNA genes.

Mung bean nuclease treatment of 16S-23S ribosomal DNA intergenic transcribed spacers (ITS) amplified from several strains of the six species of the Bacillus cereus group showed that B. anthracis Davis TE702 and B. mycoides G2 have other intermediate fragments in addition to the 220- and 550-bp homoduplex fragments typical of the B. cereus group. Long and intermediate homoduplex ITS fragments from strains Davis TE702 and G2 and from another 19 strains of the six species were sequenced. Two main types of ITS were found, either with two tRNA genes (tRNA(Ile) and tRNA(Ala)) or without any at all. Strain Davis TE702 harbors an additional ITS with a single tRNA gene, a hybrid between the tRNA(Ile) and tRNA(Ala) genes, suggesting that a recombination event rather than a deletion generated the single tDNA-containing ITS. Strain G2 showed an additional ITS of intermediate length with no tDNA and no similarity to other known sequences. Neighbor-joining analysis of tDNA-containing long ITS indicated that B. cereus and B. thuringiensis represent a single clade. Three signature sequences discriminated B. anthracis from B. cereus and B. thuringiensis, indicating that the anthrax agent started evolving separately from the related clades of the B. cereus group. B. mycoides and B. weienstephanensis were very closely related, while B. pseudomycoides appeared the most distant species.

Bacillus anthracis↗
PubMed2005

Fingerprinting of Bacillus thuringiensis type strains and isolates by using Bacillus cereus group-specific repetitive extragenic palindromic sequence-based PCR analysis.

A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native beta-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.

Bacillus cereus↗