Intestinal bacteria affect growth of Bacillus thuringiensis in larvae of the oriental tea tortrix, Homona magnanima diakonoff (Lepidoptera: tortricidae)

Spores and parasporal crystals of a Bacillus thuringiensis serovar aizawai were fed to fifth instar larvae of the oriental tea tortrix, Homona magnanima, that had been reared aseptically or that had been reared normally. Viable cell numbers of B. thuringiensis and other bacteria in H. magnanima larvae were estimated by homogenization of samples and dilution plating on peptone-polymyxin agar medium for B. thuringiensis cells and on nutrient agar medium for the other bacterial cells. B. thuringiensis did not grow in the larval cadavers of normally reared H. magnanima while bacteria other than B. thuringiensis grew rapidly. In contrast, B. thuringiensis within the larval cadavers of aseptically reared H. magnanima grew and increased 20 times. The bacteria other than B. thuringiensis from the sample homogenates of normally reared larvae that were fed on B. thuringiensis-treated diets had the same characteristics as the bacteria isolated from the guts of healthy H. magnanima larvae, which were putatively identified as Streptococcus spp. and Staphylococcus spp., typical intestinal bacteria of insects. The results strongly suggest that intestinal bacteria influence the growth of B. thuringiensis in the larvae.     

Urea-mercaptoethanol-soluble protein from spores of Bacillus thuringiensis and other species

Treatment with urea-mercaptoethanol of purified spores of Bacillus thuringiensis, other Bacillus species, and Clostridium roseum solubilizes a protein fraction between 5 and 12% of the dry weight of the spores. This fraction behaves identically to the crystal protein of B. thuringiensis on acrylamide-gel electrophoresis. The protein from all of the Bacillus species shows partial homology with crystal protein, using the Ouchterlony immunodiffusion technique. A further fraction, similar in amount, can be removed from spores of B. thuringiensis by the addition of sodium lauryl sulfate to the urea-mercaptoethanol. Spores of B. thuringiensis extracted in these ways show no difference when compared to untreated spores with respect to viability or resistance to heat and ultraviolet-irradiation. The extracted spores do show differences in their germination requirements and their susceptibility to phase-darkening by lysozyme. It is concluded that an urea-mercaptoethanol-soluble protein or class of protein is a widespread component of bacterial spores, possibly located in the spore coat, and that this protein may be related to the crystal protein of B. thuringiensis.     

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light.

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.     

Virulent spores of Bacillus anthracis and other Bacillus species deposited on solid surfaces have similar sensitivity to chemical decontaminants

AIMS: To compare the relative sensitivity of Bacillus anthracis and spores of other Bacillus spp. deposited on different solid surfaces to inactivation by liquid chemical disinfecting agents. METHODS AND RESULTS: We prepared under similar conditions spores from five different virulent and three attenuated strains of B. anthracis, as well as spores of Bacillus subtilis, Bacillus atrophaeus (previously known as Bacillus globigii), Bacillus cereus, Bacillus thuringiensis and Bacillus megaterium. As spore-surface interactions may bias inactivation experiments, we evaluated the relative binding of different spores to carrier materials. The survival of spores deposited on glass, metallic or polymeric surfaces were quantitatively measured by ASTM standard method E-2414-05 which recovers spores from surfaces by increasing stringency. The number of spores inactivated by each decontaminant was similar and generally within 1 log among the 12 different Bacillus strains tested. This similarity among Bacillus strains and species was observed through a range of sporicidal efficacy on spores deposited on painted metal, polymeric rubber or glass. CONCLUSIONS: The data obtained indicate that the sensitivity of common simulants (B. atrophaeus and B. subtilis), as well as spores of B. cereus, B. thuringiensis, and B. megaterium, to inactivation by products that contain either: peroxide, chlorine or oxidants is similar to that shown by spores from all eight B. anthracis strains studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The comparative results of the present study suggest that decontamination and sterilization data obtained with simulants can be safely extrapolated to virulent spores of B. anthracis. Thus, valid conclusions on sporicidal efficacy could be drawn from safer and less costly experiments employing non-pathogenic spore simulants.     

Pathogenicity of intrathoracically administrated Bacillus thuringiensis spores in Blatta orientalis

The ability of Bacillus thuringiensis to produce septicaemia in Periplaneta americana and Blatta orientalis has been investigated. Spores and crystals from several wild-type strains as well as spores of a B. thuringiensis crystal-deficient mutant, were first orally administrated at high doses, and no significant mortality was recorded. Intrathoracic injection of spore suspensions in P. americana revealed that this species is not very susceptible to B. thuringiensis spores. B. orientalis, by contrast, was found to be very susceptible to B. thuringiensis, with a LD(50) of about 35,000 spores, that is similar to that reported on Lepidoptera challenged with parenterally injected B. thuringiensis.     

Rapid characterization of spores of Bacillus cereus group bacteria by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption-ionization (MALDI) time-of-flight mass spectrometry was used to characterize the spores of 14 microorganisms of the Bacillus cereus group. This group includes the four Bacillus species B. anthracis, B. cereus, B. mycoides, and B. thuringiensis. MALDI mass spectra obtained from whole bacterial spores showed many similarities between the species, except for B. mycoides. At the same time, unique mass spectra could be obtained for the different B. cereus and B. thuringiensis strains, allowing for differentiation at the strain level. To increase the number of detectable biomarkers in the usually peak-poor MALDI spectra of spores, the spores were treated by corona plasma discharge (CPD) or sonicated prior to MALDI analysis. Spectra of sonicated or CPD-treated spores displayed an ensemble of biomarkers common for B. cereus group bacteria. Based on the spectra available, these biomarkers differentiate B. cereus group spores from those of Bacillus subtilis and Bacillus globigii. The effect of growth medium on MALDI spectra of spores was also explored.     

Identification of Bacillus anthracis by polyclonal antibodies against extracted vegetative cell antigens

The extractable protein antigens EA1 and EA2 of Bacillus anthracis were prepared from electrophoresis transblots of SDS extracts of vegetative bacteria of the Sterne strain. Hyperimmune guinea-pig antiserum against EA2 failed to react with B. anthracis cells in immunofluorescence (IF) tests. Guinea-pig antiserum against EA1 (anti-EA1) reacted strongly in IF tests with non-encapsulated vegetative cell of 10 of 12 strains of B. anthracis and with cells of strains of B. cereus and B. thuringiensis. The unreactive B. anthracis strains were delta-Vollum-1B-1 and Texas. Encapsulated cells of B. anthracis stained poorly except for small bright regions. Absorption of anti-EA1 with cells of B. cereus NCTC 8035 and NCTC 9946 removed activity towards all B. cereus strains tested, but only partly reduced cross-reaction with B. thuringiensis strains. Absorption of anti-EA1 with B. thuringiensis 4041 removed activity towards this strain and B. cereus strains. Evidence is produced that B. thuringiensis cells grown on nutrient agar possess more cross-reacting antigens than cells grown in nutrient broth. The reaction of anti-EA1 with Bacillus spores immobilized in clumps on microscope slides was attributed to contaminating vegetative debris because well-separated individual spores failed to react. A rapid IF test was developed allowing identification of B. anthracis sampled from overnight cultures on blood plates. When sodium dodecyl sulphate extracts of B. anthracis vegetative cells were analysed on immunoblots (Western blots) by reaction with anti-EA1, a number of bands were visualized in addition to the expected 91 kiloDalton EA1 band. Prior absorption of anti-EA1 with B. cereus or B. thuringiensis cells resulted in the disappearance of most or all of the brands in blots of these species, but had less effect on blots of the B. anthracis strains. All six B. anthracis strains that were blotted including delta-Vollum-1B-1 and Texas, could thus be distinguished from B. cereus and B. thuringiensis by their differential reaction with unabsorbed and absorbed anti-EA1.     

Estimation of the spectrum of the aerial dispersion of Bacillus thuringiensis

The present paper estimates the number of viable spores of Bacillus thuringiensis per droplet and reveals the importance of this data to improve B. thuringiensis treatments. For a given diameter, droplets from Futura formula contained two times more spores than droplets from the formulas used to date. It is preferable to have B. thuringiensis droplets larger than those of chemical insecticides. A Grumman AgCat aircraft calibrated to give the desired larger droplets resulted in successful B. thuringiensis treatments.     

Fate of Bacillus thuringiensis strains in different insect larvae

In favorable conditions Bacillus thuringiensis spores germinate and vegetative cells multiply, whereas in unfavorable conditions Bacillus thuringiensis sporulates and produces insecticidal crystal proteins. The development of B. thuringiensis strains was investigated in the larvae of insects belonging to the orders Lepidoptera and Diptera. Bacillus thuringiensis strains able to kill the insects did not always multiply in cadavers. Strains with no specificity to kill the insect sometimes multiplied when the insects were killed mechanically. These results indicate that some insect larvae represent an environment that favors the germination of B. thuringiensis spores and the multiplication of vegetative cells; however, there was no correlation between the toxin specificity and the specificity of the host.     

Immigration of Bacillus thuringiensis to bean leaves from soil inoculum or distal plant parts

AIMS: We addressed the process of immigration of Bacillus thuringiensis from soil to leaves and its capacity to grow on bean diffusate medium (BDM), a medium designed to simulate the nutrient composition of the phylloplane. METHODS AND RESULTS: Two different B. thuringiensis strains were inoculated into soils, onto seeds or onto lower leaves of bean plants to determine if they were able to disperse to upper leaves under controlled conditions. While B. thuringiensis isolates were commonly recovered from leaves exposed to such inocula, populations were very low (<10 CFU cm(-2) of leaf). In addition, the number of cells of B. thuringiensis recovered decreased with increasing distance from the soil or from the inoculated leaves. Moreover, B. thuringiensis colonies did not grow well on BDM. CONCLUSIONS: This indicates that B. thuringiensis disperses poorly from the soil or the seed to the leaves or between leaves of the same plant under controlled conditions. Bacillus thuringiensis apparently has greater nutrient requirements than other bacterial species that are prominent inhabitants of the phylloplane. SIGNIFICANCE AND IMPACT OF THE STUDY: Finding the mechanisms that favour bacteria colonization on leaves will in turn help to improve the efficacy of biocontrol agents against the target pests.     

Toxicity of chitinase-producing Bacillus thuringiensis ssp. kurstaki HD-1 (G) toward Plutella xylostella

One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium. Of 14 strains that produced chitinase, B. thuringiensis ssp. kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study. This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30 degrees C for 2 days. The toxicities of B. thuringiensis ssp. kurstaki HD-1(G) and B. thuringiensis ssp. kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC(50)'s of 4.93 x 10(4) and 1.32 x 10(5) spores/ml, respectively. If the culture broth from B. thuringiensis ssp. kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC(50)'s were reduced to 6.23 x 10(3) and 7.60 x 10(4) spores/ml, respectively. The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B. thuringiensis ssp. kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy. The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae. A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B. thuringiensis ssp. kurstaki HD-1(G) supplemented with supernatant containing chitinase. Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.     

Subspecies-specific haemagglutination patterns of fimbriated Bacillus thuringiensis spores

Electron microscopy revealed fimbrial appendages (pili) of three types on the surface of Bacillus thuringiensis spores. Fimbriated spores induced mannose-resistant agglutination of rat, guinea pig and/or chicken red blood cells. Each haemagglutination pattern correlated with the subspecies of a given B. thuringiensis strain, rather than with its flagellar serotype. Fimbriation and haemagglutination patterns of B. thuringiensis spores are considered to be of possible taxonomic value.     

Induced release of Bacillus spores from sporangia by sodium sulphate

Incubation of sporulating cultures of Bacillus anthracis, B. cereus, B. subtilis and B. thuringiensis in 1°0 mol/l sodium sulphate markedly increased the release of free spores from sporangia. It is postulated that the release of spores is due to activation of latent autolysins which hydrolyse sporangial cell walls. Sodium sulphate-induced lysis of sporangia represents a novel and highly effective method for the recovery of spores from cultures of Bacillus species.     

Induced release of Bacillus spores from sporangia by sodium sulphate

Incubation of sporulating cultures of Bacillus anthracis, B. cereus, B. subtilis and B. thuringiensis in 1.0 mol/l sodium sulphate markedly increased the release of free spores from sporangia. It is postulated that the release of spores is due to activation of latent autolysins which hydrolyse sporangial cell walls. Sodium sulphate-induced lysis of sporangia represents a novel and highly effective method for the recovery of spores from cultures of Bacillus species.     

Germination of Bacillus thuringiensis var. israelensis spores in the gut of Aedes larvae (Diptera: Culicidae)

In laboratory experiments, germination and growth of Bacillus thuringiensis israelensis in the gut of Aedes aegypti and A. vexans larvae (Culicidae: Diptera) was observed. The number of spores and vegetative cells in the gut of living larvae and in cadavers was estimated by plaing homogenized larvae on selective agar plates. The number of spores per gut increased in the first 40–140 min of exposure to a maximum, and decreased in the subsequent time, demonstrating spore germination in living larvae, moribunds, and in cadavers. Twenty-four hours after the death of the larvae, a minimal amount of spores, but an increased number of vegetative cells, was found in cadavers. In A. aegypti larvae, germination and growth of B. thuringiensis israelensis in the larval gut was photographically documented.     

Difference between the spore sizes of Bacillus anthracis and other Bacillus species

AIMS: To determine the size distribution of the spores of Bacillus anthracis, and compare its size with other Bacillus species grown and sporulated under similar conditions. METHODS AND RESULTS: Spores from several Bacillus species, including seven strains of B. anthracis and six close neighbours, were prepared and studied using identical media, protocols and instruments. Here, we report the spore length and diameter distributions, as determined by transmission electron microscopy (TEM). We calculated the aspect ratio and volume of each spore. All the studied strains of B. anthracis had similar diameter (mean range between 0.81 +/- 0.08 microm and 0.86 +/- 0.08 microm). The mean lengths of the spores from different B. anthracis strains fell into two significantly different groups: one with mean spore lengths 1.26 +/- 0.13 microm or shorter, and another group of strains with mean spore lengths between 1.49 and 1.67 microm. The strains of B. anthracis that were significantly shorter also sporulated with higher yield at relatively lower temperature. The grouping of B. anthracis strains by size and sporulation temperature did not correlate with their respective virulence. CONCLUSIONS: The spores of Bacillus subtilis and Bacillus atrophaeus (previously named Bacillus globigii), two commonly used simulants of B. anthracis, were considerably smaller in length, diameter and volume than all the B. anthracis spores studied. Although rarely used as simulants, the spores of Bacillus cereus and Bacillus thuringiensis had dimensions similar to those of B. anthracis. SIGNIFICANCE AND IMPACT OF THE STUDY: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefence against B. anthracis. The data presented here should help in the selection of simulants that better resemble the properties of B. anthracis, and thus, more accurately represent the performance of collectors, detectors and other countermeasures against this threat agent.     

Effect of bovicin HC5 on growth and spore germination of Bacillus cereus and Bacillus thuringiensis isolated from spoiled mango pulp

AIMS: To use bovicin HC5 to inhibit predominant bacteria isolated from spoiled mango pulp. METHODS AND RESULTS: Bovicin HC5 and nisin were added to brain heart infusion (BHI) medium (40-160 AU ml(-1)) or mango pulp (100 AU ml(-1)) and the growth of Bacillus cereus and Bacillus thuringiensis was monitored. Cultures treated with bovicin HC5 or nisin showed longer lag phases and grew slower in BHI medium. Bovicin HC5 and nisin were bactericidal and showed higher activity in mango pulp at acidic pH values. To determine the effect on spore germination and D values, mango pulp containing bovicin HC5 was inoculated with 10(6) and 10(9) spores per ml(-1), respectively, from each strain tested. Bovicin HC5 reduced the outgrowth of spores from B. cereus and B. thuringiensis, but thermal sensitivity was not affected. CONCLUSIONS: Bovicin HC5 was bactericidal against B. cereus and B. thuringiensis isolated from spoiled mango pulp. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus cereus and B. thuringiensis had not been previously isolated from spoiled mango pulp and bovicin HC5 has the potential to inhibit such bacteria in fruit pulps.     

Activation and germination of Bacillus thuringiensis spores in Manduca sexta larval gut fluid

Incubation of Bacillus thuringiensis HD-1 spores in the larval gut fluid of Manduca sexta (tobacco hornworm) resulted in increased viable counts, conversion to phase-dark spores, and a loss of absorbance in spore suspensions, indicative of spore germination. Heat-activated and untreated spores incubated in water did not exhibit these changes. Only when spores were heat activated and incubated in germinants L-alanine and adenosine did changes in the spores approximate those observed in gut fluid. These data suggest that M. sexta larval gut fluid induces the activation and germination of B. thuringiensis spores.     

Digestion of Bacillus thuringiensis var. israelensis spores by larvae of Aedes aegypti.

The larvicidal activity of Bacillus thuringiensis var. israelensis against mosquitoes and the blackfly is included in parasporal crystalline bodies which are produced during sporulation. Following ingestion, the crystals are solubilized in the larval midgut and induce death within a short time; the spores germinate in the dead larvae and complete a growth cycle. The fate of the spores in surviving live larvae was elucidated by using a nonlarvicidal B. thuringiensis var. israelensis mutant. When introduced as the only food source, spores of this mutant support development to the adult stage of newly hatched Aedes aegypti larvae at a rate directly related to spore concentration. The conclusion that spores of B. thuringiensis var. israelensis are digested in the larval gut was substantiated by following the incorporation of [35S]methionine-labeled spores into larval tissues.