Pyrolysis mass spectrometry studies on Bacillus anthracis, Bacillus cereus and their close relatives.

Pyrolysis mass spectrometry was used to examine strains of B. anthracis, of B. cereus, of B.cereus either proven to cause emetic illness or connected with outbreaks of emetic food poisoning and of B.thuringiensis. Analysis of the data-set for all strains allowed differentiation between B.anthracis, the emetic B.cereus and B.thuringiensis but B.cereus strains could not be clearly discriminated. Removal of data for the B.thuringiensis and the emetic B.cereus strains, followed by re-analysis, allowed clear separation of the B. anthracis and B. cereus groups. Furthermore, PyMS was found to be capable of discriminating between some strains of B.anthracis, and demonstrating sub-groupings of others. This work provides further evidence of the ability of PyMS to distinguish rapidly between very closely related organisms and indicates its potential in epidemiology.

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.

Reclassification of Paenibacillus (formerly Bacillus) pulvifaciens (Nakamura 1984) Ash et al. 1994, a later subjective synonym of Paenibacillus (formerly Bacillus) larvae (White 1906) Ash et al. 1994, as a subspecies of P. larvae, with emended descriptions of P. larvae as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens.

A polyphasic taxonomic study of four strains of Paenibacillus larvae and four strains of Paenibacillus pulvifaciens (including duplicates of both type strains) supported the reclassification of both former Bacillus species into one species, P. larvae. Our conclusions were based on morphological and Analytab Products (API) tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins, gas chromatography of methylated fatty acids, pyrolysis mass spectrometry, DNA-DNA binding, and the following genomic fingerprinting methods: amplified ribosomal DNA restriction analysis, random amplified polymorphic DNA analysis, and AFLP analysis. The last method is a novel high-resolution DNA fingerprinting technique based on the selective amplification of restriction fragments. Despite more than 90% DNA relatedness between the strains studied, SDS-PAGE of whole-cell proteins, biochemical tests, and DNA fingerprinting (AFLP) distinguished between the P. larvae and P. pulvifaciens strains at the subspecies level. Taking this evidence along with differences in pathogenicity, we propose to reclassify the honeybee pathogens P. larvae and P. pulvifaciens as P. larvae subsp. larvae and P. larvae subsp. pulvifaciens. An emended description of the species and descriptions of the subspecies are given. The type strains are P. larvae subsp. larvae ATCC 9545 (LMG 9820) and P. larvae subsp. pulvifaciens NRRL B-3685 (LMG 6911 and ATCC 13537).

Paenibacillus (formerly Bacillus) gordonae (Pichinoty et al. 1986) Ash et al. 1994 is a later subjective synonym of Paenibacillus (formerly Bacillus) validus (Nakamura 1984) Ash et al. 1994: emended description of P. validus.

A polyphasic study in which we performed an amplified ribosomal DNA restriction analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, a gas chromatographic analysis of methylated fatty acids, pyrolysis mass spectrometry, a random amplified polymorphic DNA analysis, a phenotypic analysis, and an analysis of the levels of DNA binding of Paenibacillus gordonae and Paenibacillus validus strains (including both type strains) showed that these organisms form a homogeneous group and that the names P. gordonae and P. validus are therefore synonyms. P. validus has nomenclatural priority, and an emended description of this species is given; the type strain is strain LMG 11161 (= ATCC 43897).

Characterization of exopolymers of aquatic bacteria by pyrolysis-mass spectrometry.

Exopolymers from a diverse collection of marine and freshwater bacteria were characterized by pyrolysis-mass spectrometry (Py-MS). Py-MS provides spectra of pyrolysis fragments that are characteristic of the original material. Analysis of the spectra by multivariate statistical techniques (principal component and canonical variate analysis) separated these exopolymers into distinct groups. Py-MS clearly distinguished characteristic fragments, which may be derived from components responsible for functional differences between polymers. The importance of these distinctions and the relevance of pyrolysis information to exopolysaccharide function in aquatic bacteria is discussed.

Detection of small genotypic changes in Escherichia coli by pyrolysis mass spectrometry.

Pyrolysis mass spectrometry (Py-MS) has been used to discriminate between four very closely related strains of Escherichia coli; a parent strain UB5021 and three derivatives each containing one of the antibiotic resistance plasmids, pBR322, pACYC184 or R388.

Reproducibility of pyrolysis mass spectrometry: effect of growth medium and instrument stability on the differentiation of selected Bacillus species.

The discrimination of a set of 53 strains, taken from four closely related Bacillus species (Bacillus subtilis, B. pumilus, B. licheniformis and B. amyloliquefaciens), was examined using pyrolysis mass spectrometry. Strains were grown on six different media to examine the effect of media variation, especially batch-to-batch variation of a single medium, on the pyrolysis mass spectra and strain discrimination achieved. Long-term reproducibility over a period of 14 months was also examined. It was shown that batch-to-batch media variation is insufficient to affect spectra and strain discrimination significantly, but different media types do affect this. It was shown that species groups could still be recovered from the data, however, with an appropriate data-handling system. It was not possible to directly compare spectra produced 14 months apart, but the strain and species discrimination achieved using each data-set were highly comparable.

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.

Microcomputer assisted identification of Bacillus species.

A microcomputer based system for the identification of unknown isolates of Bacillus species is described. The identification matrix includes 78 test probabilities for 38 recognised species and other groups in the genus Bacillus and it is based on the work of Logan and Berkeley (1984). Morphological characters together with the results of tests using API 20E and API 50CHB, read after 24 and 48 h incubation, are used to obtain a probabilistic identification of an unknown aerobic endospore forming rod. Any differences between the observed and expected results for any identified organism are listed. Identification can be attempted on the basis of a limited set of test results, although this is rarely if ever done with this largely API based system, and if the unknown cannot be successfully identified then a set of additional tests can be selected which should permit identification. The computer system can store and recall test results entered for any isolate. This feature allows the accumulation of data on isolates which could be used to update the identification matrix in future taxonomic studies.

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.

Curie-point pyrolysis mass spectrometry applied to characterization and identification of selected Bacillus species.

The use of pyrolysis mass spectrometry in the characterization and identification of Bacillus species was studied. Fifty-three strains of four closely related groups, Bacillus subtilis, B. pumilus, B. licheniformis and 'B. amyloliquefaciens', were used in a study of both sporulated and nonsporulated cultures. Pyrolysis was carried out using a Pyromass 8-80, a novel pyrolysis mass spectrometer specifically designed for fingerprinting complex samples. The pyrolysis data obtained were analysed using multivariate statistical techniques. All four groups could be differentiated using data from non-sporulated cultures but the data from sporulated cultures did not separate B. subtilis from 'B. amyloliquefaciens' or B. pumilus. In contrast, B. licheniformis was more clearly differentiated from the other three species using these data. Culture maturity affected the mass spectra obtained from non-sporulated cultures.

The influence of ionic strength, pH and a protein layer on the interaction between Streptococcus mutans and glass surfaces.

The initial interaction between Streptococcus mutans and hard surfaces has been investigated using a rotating disc technique. The deposition to clean and BSA-coated glass of two strains of S. mutans, FA-1 (serotype b) and KPSK2 (serotype c), which exhibit different surface properties, was studied. Organisms were harvested from cultures grown in a chemostat at a dilution rate of 0.06 h-1 and suspended in NaCl solutions of defined ionic strengths and pH values. The deposition of both strains showed a strong dependence on electrolyte concentration, particularly at low ionic strengths, which was inversely related to the zeta potentials of the organisms. Similarly, the ionic strength at which maximum deposition was first noted (critical coagulation concentration) for the two strains correlated with their relative potentials. Deposition was insensitive to changes in pH at an electrolyte concentration of 0.05 M. The maximum observed deposition did not approach values predicted by theory, suggesting that a further barrier to deposition, other than electrostatic repulsion, might exist. Under all experimental conditions, some of the deposited bacteria were observed to be oscillating, suggesting that they were held at a distance from the collector surface. The cells did not, however, appear to be deposited in a secondary minimum predicted by DLVO theory hence it may be that long-range polymer interactions are also involved in the deposition of these organisms.

Export of extracellular levansucrase by Bacillus subtilis: inhibition by cerulenin and quinacrine.

Bacillus subtilis B secretes an inducible, extracellular enzyme, levansucrase. Inhibition studies were undertaken to investigate the possible mechanism of release of this enzyme. The antibiotic cerulenin, at a concentration of 10 micrograms/ml, totally inhibited de novo lipid synthesis in B. subtilis B for at least 1 h, while only slightly reducing protein and RNA synthesis. At this concentration cerulenin, added concomitantly with the inducer sucrose, prevented the release of levansucrase for at least 150 min. This was not due to the prevention of inducer uptake by the cells. The release of the enzyme was also independent of cell division. In B. subtilis 1007 the induction of beta-galactosidase by 5 mM lactose was not prevented by cerulenin. Preliminary evidence indicated the association of a lipid moiety with the enzyme as it passes through the cytoplasmic membrane. Quinacrine (0.2 mM), which inhibits the penicillinase-releasing protease of Bacillus licheniformis, inhibited levansucrase release from B. subtilis B, but had no effect on lipid synthesis.

Exo-beta-N-acetylmuramidase--a novel hexosaminidase. Production by Bacillus subtilis B, purification and characterization.

Exo-beta-N-acetylmuramidase, or beta-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-D-glucoside acetamidodeoxyglucohydrolase, is produced by Bacillus subtilis B, growing in a succinate/peptone/salts medium, at the end of exponential growth and occurs partly in the medium and partly bound to the cells. A lysozyme digest of Micrococcus lysodeikticus cell walls, O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 leads to 4)-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-D-glucose and O-[2-acetamide-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl]-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose in decreasing order of efficiency, induce the enzyme but O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose does not do so. The enzyme was purified from the growth medium, after removal of the cells by continuous centrifugation, by ammonium sulphate precipitation, continuous filtration through XM-300 membranes (to remove the high-molecular-weight material which renders the enzyme sedimentable in low-ionic-strength solutions), diafiltration through PM-30 membranes and ion-exchange chromatography on DEAE-Sephadex and CM-Sephadex. Two peaks of activity were obtained. Peak A was purified 1800-fold and was homogenous on polyacrylamide disc gel electrophoresis. A second heterogeneous fraction (peak B) was also collected. Exo-beta-N-acetylmuramidase is most stable at pH 8.0 and has a molecular weight of about 90000. The results of studies on its ability to attack several synthetic and natural substrates are given. The Km and V values for 4-methylumbelliferyl-2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucose and O-[2-acetamido-3-O-(D-1-carboxyethyl)-2-deoxy-beta-D-glucopyranosyl]-(1 leads to 4)-2-acetamido-2-deoxy-D-glucose are respectively 0.19 and 0.65 mM and 1.50 and 16.29 mumol min(-1) mg(-1). From these results and those of inhibition studies it is concluded that the enzyme is specific for substrates with non-reducing N-acetylmuramic acid end groups. Possible roles for this enzyme are discussed.