Bacillus and relatives in foodborne illness.

Species of Bacillus and related genera have long been troublesome to food producers on account of their resistant endospores. These organisms have undergone huge taxonomic changes in the last 30 years, with numbers of genera and species now standing at 56 and over 545, respectively. Despite this expansion, relatively few new species have been isolated from infections, few are associated with food and no important new agents of foodborne illness have been reported. What has changed is our knowledge of the established agents. Bacillus cereus is well known as a cause of food poisoning, and much more is now understood about its toxins and their involvement in infections and intoxications. Also, although B. licheniformis, B. subtilis and B. pumilus have occasionally been isolated from cases of food-associated illness, their roles were usually uncertain. Much more is now known about the toxins that strains of these species may produce, so that their significances in such episodes are clearer; however, it is still unclear why such cases are so rarely reported. Another important development is the use of aerobic endosporeformers as probiotics, as the potentials of such organisms to cause illness or to be sources of antibiotic resistance need to be borne in mind.

Evaluation of the VITEK2 BCL card for identification of Bacillus species and other aerobic endosporeformers.

AIMS: To evaluate the performance of the VITEK2 Bacillus identification card (BCL) for the identification of aerobic endospore-forming bacteria, using fresh isolates and reference strains. METHODS AND RESULTS: One hundred and nine industrial, environmental and clinical isolates were tested using the BCL card. The card contained 46 substrates for measuring carbon source utilization, enzymatic activities, inhibition by 6.5% NaCl and resistance to the antibiotics kanamycin, oleandomycin and polymyxin B. Identifications were made after 14 h incubation, using a database allowing identification of 42 species of the genera Aneurinibacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus and Virgibacillus. The reference identities of all isolates were authenticated by phenotypic methods, with 16S rRNA gene sequencing used to resolve discrepancies. CONCLUSIONS: One hundred and one strains (93%) were identified correctly to species level, seven strains (6%) were incorrectly identified, and one strain (1%) remained unidentified. SIGNIFICANCE AND IMPACT OF THE STUDY: The VITEK2 BCL card provides a major advance in the reliable identification of Bacillus species and members of related genera.

Changes in the properties of Bacillus thuringiensis after prolonged culture in a rich medium.

AIM: To investigate the effect of repeated culture in a rich medium on certain genetic, metabolic, pathogenic and structural characteristics of fresh isolates of Bacillus thuringiensis. METHODS AND RESULTS: Four strains of B. thuringiensis, which had been isolated in vegetative form from leaf surfaces, were grown for 500 generations in batch culture in a rich medium. One of the strains, S4g, differed from the parent in the following respects: greater cell width; changed plasmid profile; complete loss of ability to produce delta-endotoxins; loss of ability to produce beta-exotoxin and disruption of vip3 gene; radically different fatty acid composition; and altered metabolic activity. Two of the other evolved strains (S1g and S6g) showed differences in fatty acid profiles compared with the parents. Genetic finger-printing showed that there were also mutations in the cry genes of two of the evolved strains (S1g and S2g). The delta-endotoxins of strain S6g were significantly less toxic to the larvae of Pieris brassica compared with those of the parent and it also differed in the plasmid content. CONCLUSION: Radical and unpredictable changes can occur in fresh isolates of B. thuringiensis when subjected to growth in the laboratory. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first analysis of a Gram positive and biotechnologically significant bacterium after repeated laboratory culture. It is of great relevance to the biotechnological exploitation of B. thuringiensis that prolonged growth of environmental isolates on laboratory culture media can have profound effects on their structure, genome and virulence determinants.

Identification of Bacillus anthracis by API tests.

API and morphological tests were examined for their ability to distinguish between 37 Bacillus anthracis strains (virulent and avirulent) and 194 strains of closely related Bacillus species (B. cereus, B. mycoides and B. thuringiensis). In addition, 34 strains of B. anthracis and four of B. cereus were tested by several other methods that included capsule formation, ability to grow on a selective medium, and sensitivity to phage. It was found that virulent strains of B. anthracis were easily separated from the closely related Bacillus species by most of the test methods; but separation of slightly virulent and avirulent strains of B. anthracis from the closely related species could be done only by API and phage-sensitivity tests.

Production of biologically active equine interleukin 12 through expression of p35, p40 and single chain IL-12 in mammalian and baculovirus expression systems.

Interleukin-12 (IL-12) is a key cytokine in the development of cell-mediated immune responses. Bioactive IL-12 is a heterodimeric cytokine composed of disulphide linked p35 and p40 subunits. The aim of this study was to verify biologically activity of the products expressed from equine interleukin-12 (IL-12) p35 and p40 cDNAs and to establish whether equine IL-12 could be expressed as a p35/p40 fusion polypeptide, as has been reported for IL-12a of several mammalian species. We report production of equine IL-12 through expression of p35 and p40 subunits in mammalian and insect cells and of a p35:p40 fusion polypeptide in mammalian cells. Conditioned medium recovered from cultures transiently transfected with constructs encoding equine p35 and p40 subunits or single chain IL-12 enhanced IFN-gamma production in cells derived from equine lymph nodes. Preincubation of IFN-gamma inducing preparations with anti-p40 monoclonal antibody resulted in a significant decrease in IFN-gamma induction capacity. Medium recovered from p35 and p40-expressing baculovirus infected cultures enhanced target cell IFN-gamma production and proliferation. Experimental studies in mice and other animals have revealed a therapeutic benefit of IL-12 in cancer, inflammatory and infectious disease and an adjuvant effect in prophylactic regimes. Production of a bioactive species-specific IL-12 is a first step towards an investigation of its potential application in equine species.

Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria.

Minimal standards for describing new taxa within the aerobic endospore-forming bacteria are proposed, following Recommendation 30b of the Bacteriological Code (1990 Revision). These minimal standards are recommended as guidelines to assist authors in the preparation of descriptions for novel taxa. They encourage broad polyphasic characterization and the construction of descriptions that are practically useful in routine diagnostic laboratories. The proposals have been endorsed by the Subcommittee on the Taxonomy of the Genus Bacillus and Related Organisms of the International Committee on Systematics of Prokaryotes.

Brevibacillus levickii sp. nov. and Aneurinibacillus terranovensis sp. nov., two novel thermoacidophiles isolated from geothermal soils of northern Victoria Land, Antarctica.

Thirteen strains of endospore-forming bacteria were isolated from geothermal soils at Cryptogam Ridge, the north-west slope of Mt Melbourne, and at the vents and summit of Mt Rittmann in northern Victoria Land, Antarctica. 16S rRNA gene sequencing, SDS-PAGE and routine phenotypic characterization tests indicated that the seven isolates from the north-west slope of Mt Melbourne represent a novel species of Brevibacillus and that the six isolates from Cryptogam Ridge and the vents and summit of Mt Rittmann represent a novel species of Aneurinibacillus. Brevibacillus strains were not isolated from the sites at Mt Rittmann or Cryptogam Ridge and Aneurinibacillus strains were not isolated from the north-west slope of Mt Melbourne. Preliminary metabolic studies revealed that L-glutamic acid, although not essential for growth, was utilized by both species. The Brevibacillus species possessed an uptake system specific for L-glutamic acid, whereas the Aneurinibacillus species possessed a more general uptake system capable of transporting other related amino acids. Both species utilized a K(+) antiport system and similar energy systems for the uptake of l-glutamic acid. The rate of uptake by the Brevibacillus species type strain was 20-fold greater than that shown by the Aneurinibacillus species type strain. The names Brevibacillus levickii sp. nov. and Aneurinibacillus terranovensis sp. nov. are proposed for the novel taxa; the type strains are Logan B-1657(T) (= LMG 22481(T) = CIP 108307(T)) and Logan B-1599(T) (LMG 22483(T) = CIP 108308(T)), respectively.

Identification of Bacillus strains using the API system.

A system is described for the rapid and accurate identification of Bacillus isolates using a matrix of results from tests in the API 20E and API 50CHB strips and from supplementary tests. API System tests have been shown to be more reproducible than the classical tests. A taxonomy based upon API tests is in good agreement with those obtained by other methods. The results matrix can also be used in computer assisted identification.

Isolation of Bacillus thuringiensis from Northern Victoria Land, Antarctica.

Sixty-two samples of Antarctic soils, mosses, penguin guano, algae and lichens were examined for the presence of aerobic endospore-forming bacteria; 36 samples (58%) yielded such organisms, and two samples from Edmonson Point (74 degrees 21'S 165 degrees 08'E) and one sample from Apostrophe Island (73 degrees 32'S 167 degrees 24'E), northern Victoria Land, yielded strains of Bacillus thuringiensis. Further isolations from two of the samples, appreciable variation in biotypes among the strains, failure of the strains to grow on routine B. thuringiensis media, and the fact that one of the sampling sites is very rarely visited by humans, suggest that the organisms were living in these soils rather than being chance contaminants. A representative strain, from Apostrophe Island, was identified as serovar pirenaica (H57).

Improved cytotoxicity assay for Bacillus cereus diarrhoeal enterotoxin.

An improved McCoy cell cytotoxicity assay for Bacillus cereus diarrhoeal toxin, which includes a staining procedure in addition to visual examination, was developed and the method was compared with two commercially available kits (Oxoid BCET-RPLA and Tecra BDE-VIA). A total of 71 strains of 15 different Bacillus, Brevibacillus and Paenibacillus species, including 16 strains of B. cereus from outbreaks of food-borne illness, were evaluated for toxin production. Eleven of the outbreak strains exhibited cytotoxicity, including all six B. cereus strains associated with diarrhoeal illness. Several other isolates of B. cereus, and its relatives B. anthracis, B. mycoides and B. thuringiensis, exhibited similar cytotoxicity. The other species showed no cytotoxicity, with the exception of certain B. subtilis strains. The cytotoxicity assay was more sensitive than the Oxoid kit and unlike the Tecra kit, did not give false positive results with supernatant fluids heat-treated to destroy the toxin.

Bacillus cereus produces most emetic toxin at lower temperatures.

Seven emetic toxin-producing strains of Bacillus cereus were examined for toxin production in Skim Milk Medium at incubation temperatures ranging from 10 to 50 degrees C. Minimum and maximum growth temperatures were found to be 12 and 46 degrees C, respectively. At 12 and 15 degrees C, levels of toxin production were significantly higher (P < 0.01) than that observed at 30 degrees C, while no toxin was produced above 37 degrees C. Increased levels of sporulation were observed at increased temperatures, and no correlation was found between levels of sporulation and toxin production (R(2) = 0.086).

Studies on the Bacillus flora of milk and milk products.

Bacillus licheniformis and B. cereus were the most commonly isolated species of Bacillus found in milk at all stages of processing. Bacillus licheniformis was ubiquitous in the farm environment and counts in raw milks heat-treated in the laboratory were higher during the winter months, whilst B. cereus was associated with cattle feed throughout the year, and tended to be more common in raw milks during the summer months. Although B. licheniformis was usually isolated in larger numbers than B. cereus, this pattern changed after raw and pasteurized milks and reconstituted milk powders were pre-incubated at ambient temperatures, and B. cereus came to dominate the Bacillus population, reaching levels associated with enterotoxin production. Investigation of the growth kinetics of strains of both species showed that B. cereus grew faster than B. licheniformis at ambient temperatures. It is suggested that post-pasteurization contamination, which is commonly blamed for spoilage of milk and milk products by B. cereus, is not necessarily the most important source of this organism.

Semiautomated metabolic staining assay for Bacillus cereus emetic toxin.

This paper describes a specific, sensitive, semiautomated, and quantitative Hep-2 cell culture-based 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay for Bacillus cereus emetic toxin. Of nine Bacillus, Brevibacillus, and Paenibacillus species assessed for emetic toxin production, only B. cereus was cytotoxic.

Observation of soft contact lens disinfection with fluorescent metabolic stains.

A rapid fluorescent staining method using a tetrazolium dye and propidium iodide for the in-use assessment of disinfection of Pseudomonas aeruginosa biofilms on soft contact lenses showed that 11 to 13% of cells on lenses remained actively respiring and recoverable by culture methods after 30 min of exposure to 3% hydrogen peroxide.

Detection of the dipicolinic acid biomarker in Bacillus spores using Curie-point pyrolysis mass spectrometry and Fourier transform infrared spectroscopy.

Thirty-six strains of aerobic endospore-forming bacteria confirmed by polyphasic taxonomic methods to belong to Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis (including Bacillus niger and Bacillus globigii), Bacillus sphaericus, and Brevi laterosporus were grown axenically on nutrient agar, and vegetative and sporulated biomasses were analyzed by Curie-point pyrolysis mass spectrometry (PyMS) and diffuse reflectance-absorbance Fourier-transform infrared spectroscopy (FT-IR). Chemometric methods based on rule induction and genetic programming were used to determine the physiological state (vegetative cells or spores) correctly, and these methods produced mathematical rules which could be simply interpreted in biochemical terms. For PyMS it was found that m/z 105 was characteristic and is a pyridine ketonium ion (C6H3ON+) obtained from the pyrolysis of dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA), a substance found in spores but not in vegetative cells; this was confirmed using pyrolysis-gas chromatography/mass spectrometry. In addition, a pyridine ring vibration at 1447-1439 cm-1 from DPA was found to be highly characteristic of spores in FT-IR analysis. Thus, although the original data sets recorded hundreds of spectral variables from whole cells simultaneously, a simple biomarker can be used for the rapid and unequivocal detection of spores of these organisms.

Aerobic endospore-forming bacteria from geothermal environments in northern Victoria Land, Antarctica, and Candlemas Island, South Sandwich archipelago, with the proposal of Bacillus fumarioli sp. nov.

Aerobic endospore-forming bacteria were isolated from soils taken from active fumaroles on Mount Rittmann and Mount Melbourne in northern Victoria Land, Antarctica, and from active and inactive fumaroles on Candlemas Island, South Sandwich archipelago. The Mt Rittmann and Mt Melbourne soils yielded a dominant, moderately thermophilic and acidophilic, aerobic endospore-former growing at pH 5.5 and 50 degrees C, and further strains of the same organism were isolated from a cold, dead fumarole at Clinker Gulch, Candlemas Island. Amplified rDNA restriction analysis, SDS-PAGE and routine phenotypic tests show that the Candlemas Island isolates are not distinguishable from the Mt Rittmann strains, although the two sites are 5600 km apart, and 16S rDNA sequence comparisons and DNA relatedness data support the proposal of a new species, Bacillus fumarioli, the type strain of which is LMG 17489T.

A polyphasic reassessment of the genus Paenibacillus, reclassification of Bacillus lautus (Nakamura 1984) as Paenibacillus lautus comb. nov. and of Bacillus peoriae (Montefusco et al. 1993) as Paenibacillus peoriae comb. nov., and emended descriptions of P. lautus and of P. peoriae.

Seventy-seven strains representing 10 species in the Paenibacillus polymyxa 16S rRNA group and 3 other species that exhibit phenetic relatedness to members of this group, Bacillus lautus, "Bacillus longisporus," and Bacillus peoriae, were characterized genotypically and phenotypically by performing an amplified ribosomal DNA restriction analysis, a randomly amplified polymorphic DNA analysis, a fatty acid methyl ester analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, pyrolysis mass spectrometry, and API and other routine phenotypic tests. These analyses revealed distinct clusters representing Paenibacillus alvei, Paenibacillus amylolyticus, Paenibacillus azotofixans, Paenibacillus durum, Paenibacillus larvae subsp. larvae, Paenibacillus larvae subsp. pulvifaciens, B. lautus, Paenibacillus macerans, Paenibacillus macquariensis, B. peoriae, P. polymyxa, and Paenibacillus validus, which confirmed the distinctness of these species, but appreciable within-species heterogeneity was observed in P. alvei, B. lautus, P. macerans, P. polymyxa, and P. validus. The type strain of Paenibacillus pabuli did not cluster with other strains of this species, and in several analyses a relationship between strains of P. pabuli and "B. longisporus" was observed. As the analyses showed that B. lautus and B. peoriae are closely related to the genus Paenibacillus, these species are reclassified as members of this genus.