Photoreactivation in the genus Bacillus

Photoreactivation of ultraviolet radiation-induced DNA damage was examined in exponential-phase cells of six mesophilic species of the genus Bacillus. Under the experimental conditions used, it was observed that the laboratory strains B. cereus strain T and B. thuringiensis var. thuringiensis strain NRRL-B4039 exhibited strong photoreactivation (86-fold and 70-fold respectively). Bacillus licheniformis strain ATCC 8480 exhibited moderate (15-fold) photoreactivation. Weak photoreactivation was observed in B. subtilis strain 168 (4-fold) and B. megaterium strain QM B1551 (3.4-fold). Bacillus amyloliquefaciens strain H demonstrated no detectable photoreactivation.     

Conjugal transfer of transposon Tn916 from Enterococcus faecalis to Bacillus licheniformis

Transposon Tn916 was conjugally transferred from Enterococcus faecalis to Bacillus licheniformis bacterial strains ATCC 10716 and ATCC 9945A by filter and broth matings. The Tn916 transfer frequencies to B. licheniformis ranged from 10(-7) to 10(-5) selecting for tetracycline-resistant (Tcr) transconjugants depending on broth or filter mating. Movement of Tn916 was demonstrated when Tcr B. licheniformis transconjugants were mated with Bacillus subtilis strain W23. Tn916 insertion caused several auxotrophic and bacitracin deficient mutants. Southern blot analyses of HindIII chromosomal digests extracted from Tcr transconjugants showed that the transposon inserted at different sites in the B. licheniformis chromosome and the copy number of Tn916 varied. This approach should be useful for genetic studies of B. licheniformis.     

Some pharmacokinetic parameters of doxycycline in east African goats after intramuscular administration of a long-acting formulation

A compartmental and non-compartmental study was carried out on five adult goats following intramuscular administration of doxycycline at 20 mg/kg bodyweight. The concentration of the drug in serum was determined by a microbiological assay employing Bacillus cereus var mycoides (ATCC 11778) as the test organism. The mean serum concentration (Cmax) and the time of maximum concentration (Tmax) were 1.87 micrograms/ml and 0.85 h, respectively. Using compartmental analysis, the plasma concentration-time curve of doxycycline best fitted a three-compartment open model with first-order absorption. A three-phase disposition of doxycycline was found, the terminal elimination half-life being approximately 40 h. The statistical moment theory was mainly used for non-compartmental analysis. The value obtained for the mean residence time (MRT) was 16.4 h. The mean values for the volume of distribution at steady state (Vdss), determined by compartmental and non-compartmental analyses, were 8.73 and 13.19 L/kg, respectively. There were no statistically significant differences when the major pharmacokinetic parameters were compared. It was concluded that the pharmacokinetic behaviour of doxycycline in goats after intramuscular administration is characterized by a three-compartment model with a slow terminal elimination phase. Based on current knowledge, this could be due to enterohepatic recycling and/or flip-flop kinetics. The study indicated that a single intramuscular administration of 20 mg/kg of doxycycline may only provide therapeutic concentrations for up to 24 h owing to slow absorption at the injection site.     

Testing of raw milk for tetracycline residues

A newly improved Bacillus calidolactis tube diffusion test and two postscreening test systems--a receptor assay (Charm HVS; Charm Sciences, Inc., Malden, MA) and a newly developed Bacillus cereus ATCC 11778 mycoides test system--were evaluated for the detection and identification of tetracycline residues using 973 samples of bulk milk taken at random in The Netherlands. All milk samples were assayed with the B. calidolactis tube and the receptor test. The milk samples testing as suspect or positive with one or both of the test systems were analyzed with HPLC (limit of detection, 10 ng/ml) and the recently developed B. cereus test system. The B. calidolactis tube diffusion test detected tetracycline residues > 45 ng/ml in milk. With the B. cereus test plate, residues of oxytetracycline and tetracycline of > 30 ng/ml milk were detected; for chlortetracycline and doxycycline, the detection limit was 10 ng/ml. Raw milk exhibiting inhibition diameters of < 20 mm on the B. cereus test plate fulfilled the European Union criterion for maximum residue level for tetracyclines of < 100 ng/ml (including their 4-epimer derivatives). The detection limits of the receptor assay depended on the type of milk used. The scintillation counts that were obtained for control samples of bulk milk were considerably lower than for the milks obtained from Charm Sciences, Inc. or processed using UHT pasteurization. One of 973 milk samples was suspect for tetracycline residues by means of the B. calidolactis tube test as well as by the receptor assay; 8 other samples were also considered to be positive using the receptor assay alone. The presence of tetracycline residues could not be proved for these 9 samples (residue concentration, < 10 ng/ml) with HPLC. We concluded that the receptor assay was not reliable to detect tetracycline residues in raw milk at < 150 ng/ml. The B. cereus test plate was determined to be an inexpensive, reliable alternative for the receptor assay for detection of tetracycline residues.     

Application of a diagnostic DNA probe for the differentiation of the two types of Mycoplasma mycoides subspecies mycoides

Contagious bovine pleuropneumonia (CBPP), which is caused by Mycoplasma mycoides subspecies mycoides, is still a serious disease in some parts of the world. There is also a commonly occurring mycoplasma which is sufficiently related to the CBPP organism to bear the same name, even though this organism does not cause CBPP. Thus it is very important to be able to distinguish between these organisms and identify either with certainty. Fragments derived from M mycoides subspecies capri by restriction enzyme digestion of genomic DNA were cloned into the vector M13mp8. One resulting clone CAP-21, with a 1.5 kb insert was used as a probe in Southern hybridisation assays where genomic DNA was digested with the restriction endonuclease TaqI. This probe could differentiate a strain of M mycoides subspecies mycoides which does not cause CBPP. Subsequent tests on 14 other strains from cattle and goats showed that although they were isolated from diverse geographical areas, CAP-21 could clearly differentiate between these two types of M mycoides subspecies mycoides.     

On N2-fixing clostridia and bacilli from soils (author's transl)

In total 30 nitrogen-fixing, saccharolytic Clostridia and 4 nitrogen-fixing bacilli, all freshly isolated from gleyed soils, were screened for sensitivity to 20 antibiotics. The isolates were compared in their sensitivity with 5 type cultures representing the species Clostridium butyricum, C. saccharobutyricum (2 strains) C. multifermentans and C. sporogenes. Generally speaking, both clostridia and bacilli are sensitive to the same antibiotics (Table 2). In addition, the nitrogen-fixing bacilli belonging to Bacillus polymyxa and B. macerans showed sensitivity to neomycin and kanamycin. Except for the species C. tyrobutyricum, none of the various saccharolytic Clostridium species could be distinguished by differences in sensitivity to antibiotics. Differential methods are given in Table 3.     

Alkaline-fermented foods: a review with emphasis on pidan fermentation

Alkaline-fermented foods constitute a group of less-known food products that are widely consumed in Southeast Asia and African countries. They can be made from different raw ingredients. For instance, Japanese natto, Thai thua-nao, and kinema are made from cooked soybeans, dawadawa from African locust beans, ogiri from melon seeds, ugba from African oil beans, kawal from fresh legale leaves, owoh from cotton seeds, and pidan from fresh poultry eggs. In alkaline-fermented foods, the protein of the raw materials is broken down into amino acids and peptides; ammonia is released during the fermentation, raising the pH of the final products and giving the food a strong ammoniacal smell. Most alkaline fermentations are achieved spontaneously by mixed bacteria cultures, principally dominated by Bacillus subtilis. In other cases, pure cultures can be used. For example, Japanese natto is inoculated with a pure culture of B. subtilis var natto. Pidan is a special example of alkaline fermentation. Instead of using microorganisms, pidan is made using an alkali-treated fermentation. Sodium hydroxide (NaOH) is produced from the reaction of sodium carbonate (Na2CO3), water (H2O), and calcium oxide (CaO) of pickle or coating mud. NaOH penetrates into the eggs, causing the physicochemical changes, color changes, and gelation. The appearance of pidan differs from fresh eggs in that the white becomes a semitransparent tea-brown color, and the yolk is solid or semisolid with a dark-green color. The nutritional value of pidan is slightly decreased compared with fresh eggs, but pidan has an extremely long shelf life and a pleasant, fragrant taste that is preferred by most people in Southeast Asian countries. In a small-scale laboratory study conducted by the authors, B. subtilis was not found in pidan. Four Staphylococcus spp. (S. cohnii, S. epidermidis, S. haemolyticus, and S. warneri) and two strains of Bacillus spp. (B. cereus and B. macerans) were isolated from pidan. Staphylococcus spp. did not contribute to the fermentation and were considered contaminants.     

Lipoprotein profile determinants in children and adolescents from a lipid consultation clinic. The impact of diet, body composition and physical activity]

The production of extracellular beta-amylase by some Bacillus cereus, Bacillus megaterium and Bacillus polymyxa [corrected] strains was investigated, and the maximal yields of the enzyme were 3.6; 9.3 and 20.4 U/mL of the culture fluid, respectively (U, 1 mumol of maltose equivalent per min at 30 degrees C). Several cultivation media were used for beta-amylase production. Bacillus cereus and some strains of Bacillus megaterium gave good yields of beta-amylase only in medium with the addition of nutrient broth. However, beta-amylase produced during growth in protein rich medium (nutrient broth) was highly unstable, probably due to inactivation by proteolytic enzymes co-existing in the culture fluid. Bacillus polymyxa [corrected] strains can produce good yields of beta-amylase on a semi-synthetic medium consisting of inorganic salts, potato starch and inexpensive soybean extract instead of costly peptone and meat extract. The most potential beta-amylase producer was the strain Bacillus polymyxa [corrected] NCIB 8524. The tested Bacillus megaterium and Bacillus polymyxa [corrected] strains were apparently differentiated by temperature cultivation (30 and 37 degrees C) suitable for beta-amylase amylase yield.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Identification of Bacillus cereus by Fourier transform infrared spectroscopy (FTIR)

The objective of this study was to evaluate the potential of Fourier transform infrared spectroscopy (FTIR) for rapid identification of Bacillus cereus isolates. Ten B. cereus group isolates (comprising B. cereus, Bacillus mycoides, and Bacillus thuringiensis strains), five other Bacillus spp., and five non-Bacillus spp. were used. Two types of media, brain heart infusion (BHI) and Trypticase soy agar (TSA), were tested. The results indicated that all B. cereus group isolates produced characteristic absorbance peaks at wave numbers between 1738 and 1740 cm-1. These peaks were not affected by the growth medium. None of the other bacteria tested showed a similar peak after growth on BHI or TSA. Absorbance peaks between 1800 and 1500 cm-1 of members of the B. cereus group had different shapes and sizes, suggesting that FTIR may be useful for rapid identification of species within the B. cereus group.     

Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues

To identify the critical sites for protein thermostabilization by proline substitution, the gene for oligo-1,6- glucosidase from a thermophilic Bacillus coagulans strain, ATCC 7050, was cloned as a 2.4-kb DNA fragment and sequenced. In spite of a big difference in their thermostabilities, B. coagulans oligo-1,6-glucosidase had a large number of points in its primary structure identical to respective points in the same enzymes from a mesophilic Bacillus cereus strain, ATCC 7064 (57%), and an obligately thermophilic Bacillus thermoglucosidasius strain, KP1006 (59%). The number of prolines (19 for B. cereus oligo-1,6-glucosidase, 24 for B. coagulans enzyme, and 32 for B. thermoglucosidasius enzyme) was observed to increase with the rise in thermostabilities of the oligo-1,6-glucosidases. Classification of proline residues in light of the amino acid sequence alignment and the protein structure revealed by X-ray crystallographic analysis also supported this tendency. Judging from proline residues occurring in B. coagulans oligo-1,6-glucosidase and the structural requirement for proline substitution (second site of the beta turn and first turn of the alpha helix) (K. Watanabe, T. Masuda, H. Ohashi, H. Mihara, and Y. Suzuki, Eur. J. Biochem. 226:277-283, 1994), the critical sites for thermostabilization were found to be Lys-121, Glu-290, Lys-457, and Glu-487 in B. cereus oligo-1,6-glucosidase. With regard to protein evolution, the oligo-1,6-glucosidases very likely follow the neutral theory. The adaptive mutations of the oligo-1,6-glucosidases that appear to increase thermostability are consistent with the substitution of proline residues for neutrally occurring residues. It is concluded that proline substitution is an important factor for the selection of thermostability in oligo-1,6-glucosidases.     

Possible functions of peptide antibiotics during growth of producer organisms: bacitracin and metal (II) ion transport

The inhibitory effect of bacitracin upon growth of the producer strain Bacillus licheniformis ATCC 10716 was dependent upon the presence of several different metal (II) ions, particularly Mn (II), Co (II), or Zn (II) ions. This supports our previous suggestion that the normal function of bacitracin during growth of the producer organism may be to promote the uptake of several divalent metal ions. Due to the striking similarity between the antimicrobial effect of bacitracin towards susceptible organisms and the effect of bacitracin towards the producer organisms B. licheniformis ATCC 10716, the possibility that the antimicrobial effect of bacitracin may be an induction of uptake of toxic amounts of metal ions is discussed. The possibility that peptide antibiotics may normally participate in ion transport during growth of producer organisms is also discussed.     

Isolation of penicillin-resistant Streptococcus pneumoniae in Saga Medical School Hospital

A recent nationwide increase in beta-lactams-resistant Streptococcus pneumoniae has attracted a great deal of attention. We studied the drug sensitivity of S. pneumoniae isolated from various clinical specimens in Saga Medical School Hospital between April 1988 and December 1991. To determine the drug sensitivity of the strains, we used a micro-dilution method and determined the MIC. Drug resistance was evaluated using MIC of ampicillin (ABPC) as a reference MIC, and the results were roughly classified into the following three groups: sensitive (< or = 0.1 microgram/ml), moderately resistant (0.2-3.13 micrograms/ml) and highly resistant (> or = 6.25 micrograms/ml). The isolation frequency was calculated on the basis of one strain from one patient. No strain of S. pneumoniae with high resistance against ABPC was found in 1988 (94 strains of S. pneumoniae were isolated) and 1990 (115 strains isolated), but one such strain (0.8%) was found among 129 strains isolated in 1989, and 2 such strains (2.4%) among 84 strains isolated in 1991. Moderately resistant strains were isolated at the frequencies of 12.8%, 15.5%, 22.6%, and 21.4% respectively, in 1988, 1989, 1990, and 1991. A sum of the frequencies of "moderately resistant" and "highly resistant" (2.4%) strains was 23.8% in 1991. The frequency of resistant strains is increasing and the intensity of resistance is also being elevated.     

Discrimination of psychrotrophic and mesophilic strains of the Bacillus cereus group by PCR targeting of major cold shock protein genes

Detection of psychrotrophic strains (those able to grow at or below 7 degreesC) of the Bacillus cereus group (Bacillus cereus, Bacillus thuringiensis, and Bacillus mycoides) in food products is at present extremely slow with conventional microbiology. This is due to an inability to discriminate these cold-adapted strains from their mesophilic counterparts (those able to grow only above 7 degreesC) by means other than growth at low temperature, which takes 5 to 10 days for detection. Here we report the development of a single PCR assay that, using major cold shock protein-specific primers and appropriate annealing temperatures, is capable of both rapidly identifying bacteria of the B. cereus group and discriminating between psychrotrophic and mesophilic strains. It is intended that this development help to more accurately predict the shelf life of refrigerated pasteurized food and dairy products and to reduce the incidence of food poisoning by psychrotrophic strains of the B. cereus group.     

A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination

The Bacillus subtilis sleB gene, which codes for the enzyme homologous to the germination-specific amidase from Bacillus cereus, was cloned and its nucleotide sequence was determined. Sequence analysis showed that it had an open reading frame of 918 bp, coding for a polypeptide of 305 amino acids with a putative signal sequence of 29 residues. Enzyme activity was not found in germination exudate of B. subtilis spores, which differs from the case of B. cereus enzyme. A B. subtilis mutant with an insertionally inactivated sleB gene revealed normal behavior in growth and sporulation. However, the sleB mutant was unable to complete germination mediated by L-alanine.     

Bacillus species: the dominant bacteria of the rhizosphere of established tea bushes

A number of species belonging to the genus Bacillus were found to be well adapted to the rhizoplane and rhizosphere of established tea bushes. Amongst the species, Bacillus subtilis and B. mycoides appeared to be closely associated with tea roots. The two species comprised a major part of the bacterial population, even during unfavourable periods. In extreme winter months the population of B. subtilis and B. mycoides were recorded upto 3.9 X 10(6) and 10(7) cells/g rhizosphere soil, respectively. The soil temperature during this period was in the range of 0 to 5 degrees C. Under laboratory conditions pure cultures of these Bacillus species did not grow upto 14 degrees C. While the pH of tea rhizosphere soil samples ranged from 4.3 to 6.3, these two species were able to grow at 28 degrees C in a much wider range of pH (4 to 12.0-12.5) under laboratory conditions. Survival of these bacterial species under adverse environmental conditions was probably due to their spore forming property. Various species of Bacillus behaved antagonistically amongst themselves, indicating perhaps to their bacteriocinogenic property. The observations also indicate that the tea bushes tend to make the soil acidic.     

Inactivation of Bacillus Spores by Ultraviolet or Gamma Radiation

Bacillus anthracis spores represent an important bioterrorism agent that can be dispersed in air or water. Existing decontamination practices based on these spores have focused on chemical disinfectants; however, the basic characteristics of radiation-based disinfectants suggest potential advantages in their application for control of Bacillus spores. Experiments were conducted to examine the effectiveness of ultraviolet (UV254) radiation and γ radiation for inactivation of Bacillus spores. Spores of Bacillus cereus were used for most experiments because of their similarity to B. anthracis. A limited number of experiments were also conducted using B. anthracis Sterne spores. In aqueous suspension, B. anthracis Sterne spores were observed to be slightly more resistant to UV254 than the spores of B. cereus. For the conditions of culture and assay used in these experiments, both spore types were more sensitive to UV254 radiation in aqueous suspension than the spores of B. subtilis, which are commonly used to characterize the performance of UV disinfection systems for water. Dried spores on surfaces were observed to be more resistant to UV254 than the same spores in aqueous suspension; it is likely that the increased resistance to UV of the dried spores was attributable to surface characteristics (porosity and texture) of the solid materials. γ radiation was shown to accomplish similar rates of inactivation for spores in aqueous suspension and for dried spores on surfaces. Collectively, these results suggest that the application of UV or ionizing radiation may hold promise for decontamination following bioterrorism events.     

Introduction and expression of the cry1Ac gene of Bacillus thuringiensis in a cereal-associated bacterium, Bacillus polymyxa

The abilities of Bacillus polymyxa and Bacillus thuringiensis to survive on the rice phyllospere were compared; it was found that B. polymyxa colonizes the crop better. This study also showed that B. polymyxa inoculation to rice plants increased the shoot and the root growth of the crop. Efforts were made to introduce the cry1Ac gene of B. thuringiensis subsp. kurstaki into B. polymyxa so that the application of such transgenic B. polymyxa strains would prove to be dually beneficial to rice crops both as a biopesticide and as a biofertilizer. Immunoblot analysis of the recombinant organism containing the cry1Ac gene, strain BP113, indicated efficient expression of this gene in the heterologous host. Bioassays with the first instar larvae of the yellow stem borer of rice (Scirpophaga incertulas) revealed that the protein preparations from BP113 were toxic.     

HIV testing for HIV prevention: a comparative analysis of policies in Britain, Hungary and Sweden

Growth of vegetative cells and outgrowth of spores of enterotoxigenic psychrotrophic Bacillus cereus in refrigerated minimally processed food products is a public health concern. A study was undertaken to determine the combined effects of pH, nisin, and temperature on growth and survival of 20 strains of B. cereus. The minimum growth temperatures in tryptic soy broth (pH 7.3) and brain heart infusion broth (BHI broth, pH 7.4) were 5 degrees C for two strains and 8 degrees C for five other strains. Vegetative cells of four of eight strains grew at 8 degrees C in BHI broth (pH 6.01 and 6.57) containing 10 micrograms of nisin per ml. At 15 degrees C, all strains grew at pH 5.53 to 6.57; three strains tolerated nisin at 50 micrograms/ml (pH 6.57), whereas two other strains had a maximum tolerance of 10 micrograms of nisin per ml. Tolerance of vegetative cells of B. cereus to nisin increased as the pH of the broth was increased from 5.53 to 6.01 and again to pH 6.57. Outgrowth of spores (six of six strains tested) was inhibited by 5 and 50 micrograms of nisin per ml at 8 and 15 degrees C, respectively. At 15 degrees C, outgrowth of spores of two strains occurred at pH 6.52 in BHI broth containing 10 micrograms of nisin per ml. The effectiveness of nisin in controlling the growth of psychrotrophic strains of B. cereus capable of causing human illness was more pronounced at 8 degrees C than at 15 degrees C and as the pH was decreased from 6.57 to 5.53. Studies to determine the effectiveness of nisin in controlling growth of psychrotrophic B. cereus in nonpasteurized foods held at refrigeration temperatures are warranted.     

Study of the antagonistic action of actinomycetes on anthrax bacilli

Capacity for the growth inhibition of the highly virulent causative agent of Siberia plague was studied with respect to actinomycetous strains from soil samples of the Ukraine. It was found that on nutrient media 88.4, 88.9, 88.1 and 93.4% of the isolates inhibited the growth of Bac. anthracis, Bac. cereus, Bac. mycoides and Tsenkovsky vaccine strain respectively. Representatives of very different taxonomic groups were found among the antagonistic actinomycetes. Clearance of the soil from Siberia plague bacilli stable to the outer effect with the help of the active strain L-721 of Act. chromofuscus was observed. The effect was chown in sterilized soil, not sterile soil and soil monolith.