Aims: Oryctes rhinoceros beetle is one of the most damaging pests of oil palm and cause high oil palm mortality. The empty fruit bunch mulch and rotten old trunk of oil palm in the field provide the organic matter for the breeding sites and increases the number of O. rhinoceros larvae. Bacillus thuringiensis as bioinsecticide can synthesize crystal proteins toxic to the larvae. The present study was aimed to find effective B. thuringiensis isolates as biopesticide against O. rhinoceros larvae. Methodology and results: Screening process was carried out through heating of soil sample suspension at 80 °C to eliminate the non-spore formers and plated onto T3 medium. Colony morphology was observed, followed by Gram and endospore staining. The crystal protein was observed by Coomassie Brilliant Blue (CBB) staining. Bioassay test was conducted by force-feed method followed by food contamination method. The results showed isolates SBB33 and SBB35 were able to infect and caused high mortality to the O. rhinoceros larvae. Isolates SBB33 and SBB35 showed the highest mortality against 1st instar larvae (94.44% and 75% respectively) and 3rd instar larvae (64.8% and 60% respectively) compared to control treatments. The 16S rRNA gene sequencing showed SBB33 has high similarity with B. thuringiensis strain 3S2-3, while SBB35 has high similarity with B. thuringiensis strain GCU_BTi10. Protein separation of the spore-crystal mixture by SDS-PAGE showed the prominence of 66 kDa protein band that was predicted to be Cry toxins which is specific to coleopterans insect. Conclusion, significance and impact of study Bacillus thuringiensis isolates SBB33 and SBB35 have high potential as biopesticides against O. rhinoceros larvae and could be used to control major pests in oil palm plantation.
Bacillus thuringiensis--isolation & ; purification ; Coleoptera

Aims: Potatoes are considered one of the most strategic vegetable crops all over the world. Alternaria alternata has recently contaminated certain potatoes farms in different regions in Egypt. Among thirteen samples from fifteen regions were studied in a precedent study. Our study was aimed to investigate the effect of Bacillus thuringiensis subsp. Kurosaki suspension on inhibiting the growth of the three tested isolates of A. alternata and minimizing their mycotoxins production in vitro using three isolates with three levels of highly, moderate and low pathogenicity with unequal amounts of dual mycotoxins production. Methodology and results: Three isolates of A. alternata from three regions, Kom Hamada (KH3), Alamin (Alam1) and Nobaria (NO3), which were determined as a producer of tenuazonic acid (TeA) and alternariol monomethyl ether (AME) toxins. Bacillus thuringiensis (Bt) use as commercial fungicide was applied with three suspension concentrations (75, 150 and 300 μg/mL) as inhibitor for the two mycotoxins. Our results illustrated that the three tested isolates recorded high TeA and AME inhibition efficacies by increasing the Bt suspension concentration. The highest inhibitory concentration of Bt was at concentration 75 μg/mL for isolated from Nobaria third region (NO3) and Alam1 it was (99.83 and 99.74%) for mycotoxin (AME) while, TeA mycotoxin had the most inhibition percentage (99.58%) at concentration 150 μg/mL for the isolate (NO3). Conclusion, significance and impact of study The preliminary results of the study suggest that B. thuringiensis spores’ suspension with different concentrations can be used as anti-mycotoxigenic agents to inhibit the (TeA) and (AME) mycotoxins produced by Alternaria alternata.
Bacillus thuringiensis ; Alternaria--isolation & ; purification ; Solanum tuberosum

We isolated the strain MBL13 with high collagenase productivity from the soil of piled up animal bones. It was identified as Bacillus cereus. We purified and characterized Bacillus cereus collagenase (BCC). The molecular weight of BCC was 38.0 kDa and the optimum temperature and pH for the enzyme activity were 40 degrees C and 8.0 respectively. The enzyme was stable when the temperature was below 50 degrees C, but only retained 10% activity when kept at 60 degrees C for 1 h. The enzyme activity was stable between pH 7.0-8.5. Some metal ions such as Ca2+, Zn2+, Mg2+ enhanced the enzyme activity, and Cu2+ brought the obvious inhibition. In addition, EDTA and EGTA could inhibit the enzyme activity. We suggested that the purified enzyme was a member of the metalloproteases. Based on the experiment of substrate specificity, we found that the purified enzyme was bone collagenolytic protease, and had a much stronger capacity of hydrolysis for type I collagen than that for type II collagen and type III collagen. By BCC hydrolyzing bone collagen, we obtained polypeptides with different chain lengths. The comparative test indicated that the hydrolysis capacity of BBC was higher than that of standard type I collagenase. The results introduced a new strain and a novel collagenolytic protease for industrial enzyme.
Animals ; Bacillus cereus ; enzymology ; isolation & purification ; Bone and Bones ; microbiology ; Collagenases ; biosynthesis ; isolation & purification ; Hydrolysis ; Soil Microbiology

Cockroaches are abundant in Nigeria and are seen to harbour an array of pathogens. Environmental and sanitary conditions associated with demographic/socio-economic settings of an area could contribute to the prevalence of disease pathogens in cockroaches. A total of 246 cockroaches (Periplaneta americana) in urban (Benin, n=91), semi-urban (Ekpoma, n=75) and rural (Emuhi, n=70) settings in Edo State, Nigeria were collected within and around households. The external body surfaces and alimentary canal of these cockroaches were screened for bacterial, fungal, and parasitological infections. Bacillus sp. and Escherichia coli were the most common bacteria in cockroaches. However, Enterococcus faecalis could not be isolated in cockroaches trapped from Ekpoma and Emuhi. Aspergillus niger was the most prevalent fungus in Benin and Ekpoma, while Mucor sp. was predominant in Emuhi. Parasitological investigations revealed the preponderance of Ascaris lumbricoides in Benin and Emuhi, while Trichuris trichura was the most predominant in Ekpoma. The prevalence and burden of infection in cockroaches is likely to be a reflection of the sanitary conditions of these areas. Also, cockroaches in these areas making incursions in homes may increase the risk of human infections with these disease agents.
Animals ; Ascaris lumbricoides/isolation & purification ; Aspergillus niger/isolation & purification ; Bacillus/isolation & purification ; Cockroaches/*microbiology/*parasitology ; Escherichia coli/isolation & purification ; Nigeria ; Sanitation ; Trichuris/isolation & purification

In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 µg/mL lysozyme) produced sharp bands on the gel. Restriction enzyme NotI was chosen as the most suitable. Twenty-two isolates were analyzed by NotI digestion, using three electrophoretic parameters (EPs). The EP-a was optimal for distinguishing between isolates. The optimized protocol could be completed within 40 h which is a significant improvement over the previous methods.
Bacillus cereus ; genetics ; isolation & purification ; Bacteriological Techniques ; DNA, Bacterial ; chemistry ; genetics ; Electrophoresis, Gel, Pulsed-Field ; methods

OBJECTIVETo evaluate the significance of traditional index on the identification of Bacillus anthracis and its correlation with pathogenic strains.

METHODSRoutine bacteriologic methods and PCR of virulence genes were used to determine the difference on traditional identification index between pathogenic and nonpathogenic Bacillus.

RESULTSThe characteristics of colony, with hemolysis and mobility both negative are the important interrelated characters of pathogenic strains of Bacillus anthracis.

CONCLUSIONTo judge the risk of anthrax, virulence of genes must be first defined. Some traditional methods for identification are still useful when molecular biological methods are not available.

Animals ; Bacillus anthracis ; genetics ; isolation & purification ; pathogenicity ; Hemolysis ; Sheep ; Virulence ; genetics

Laser scanning confocal microscope (LSCM) is currently the only equipment to observe fluorescence. However, this technique has disadvantages such as high cost and long test process. In this study, we developed a new system of laser-induced fluorescence (LIF) for microfluidic chip applied to detecting the expression of green fluorescent protein (GFP) in Bacillus subtilis. This novel system was comprised of laser device, optics unit, microfluidic chip, photomultiplier and computer treatment unit. The tests indicated that microfluidic chip could detect the expression of GFP as sensitively as LSCM in Bacillus subtilis. Moreover, this LIF detection system could instead of PCR to identify the positive clone in this special case. Nevertheless, the LIF system only was suitable to detect the fluorescent strength of GFP, and could not meet the request of some cases for example protein location. Therefore, this system will be applied in environmental detection with microbe, drug discovery and other cases.
Bacillus subtilis ; isolation & purification ; metabolism ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Microfluidic Analytical Techniques ; methods

The beta-glucosidase encoding gene bglA was cloned from Bacillus polymyxa 1.794. The bglA gene was inserted in expression vector pET28a(+) and transformed into Escherichia coli BL21 (DE3), finally the recombinant strain BL1979 was obtained. Induced by IPTG, the expression P-glucosidase activity reached to 24.7 IU/mL. The optimum temperature and optimum pH of the recombinant expression P-glucosidase in BL1979 were 37 degrees C and 7.0 respectively,the purity can reach to 92.7%. Analysis of the fusion protein by nondenaturing gradient gel electrophoresis, we found the fusion protein exists in dimmer, tetramer,hexamer and octamer, they all have hydrolase activity.
Bacillus ; enzymology ; Escherichia coli ; genetics ; Plasmids ; Recombinant Proteins ; biosynthesis ; isolation & purification ; beta-Glucosidase ; genetics ; isolation & purification ; metabolism

An alkaline catalase has been purified and characterized from a slightly halophilic and alkaliphilic bacterium Bacillus sp. F26. The purification was performed with a four step procedure consisting of ammonium sulfate precipitation, ion exchange, gel filtration and hydrophobic interaction chromatography, and finally achieved a 58.5-fold-purifying over the crude extract. The purified catalase was composed of two identical subunits with a native molecular mass of 140 kD. The native enzyme showed the typical Soret band appearing at 408 nm. The pyridine hemochrome spectrum indicated the presence of protoheme IX as the prosthetic group. The apparent Km value for enzyme activity on H2O2 was calculated to be 32.5 mmol/L. The activity of this catalase was not reduced by dithionite but was strongly inhibited by cyanide, azide, and 3-amino-1,2,4-triazole (the specific inhibitor of monofunctional catalase). No peroxidase activity of this enzyme was detected when using o-dianisidine, diaminobenzidine (DAB) and p-phenylenediamine as electron donor. Moreover, the N-terminal sequence of this catalase exhibited substantial similarity to the monofunctional catalase subgroup rather than catalase-peroxidase or Mn-catalase one. Therefore, we characterize the purified catalase as a monofunctional catalase. Besides, this monofunctional catalase was thermosensitive and its activity exhibited pH-independent over pH 5-9 but showed a sharp maximum at pH 11. An activity half-life of approximately 49 h was measured when the enzyme was incubated at 20 degrees C and pH 11. To our knowledge, pH 11 is the most alkaline condition for optimum catalysis and enzyme stability among the catalases reported up to now. Furthermore, this monofunctional catalase also showed excellent halo-alkali-stability with a half-life of approximately 90 h at 0.5 mol/L NaCl and pH 10.5. On the other hand, so far as we know, the characterized catalase is the first dimeric monofunctional catalase from alkaliphiles and is also the first monofunctional catalase derived from a natural soda lake, which could partially reflect the oxidative stress response in the corresponding environment.
Bacillus ; enzymology ; Bacterial Proteins ; chemistry ; isolation & purification ; Catalase ; chemistry ; isolation & purification ; Enzyme Stability ; Hydrogen-Ion Concentration ; Temperature

OBJECTIVETo establish a testing and evaluating method for filtration efficiency of the canister against microbial aerosol.

METHODSSerratia marcescens aerosol served as model of bacterial aerosol, Bacillus subtilis var niger aerosol as model of spores aerosol, bacteriophage f(2) aerosol as model of viral aerosol. Employing the microbial aerosol testing platform was established in lab, models of microbial aerosol generated artificially were sampled quantitatively by air samplers before and after filtrating by canisters, respectively. Filtration efficiency was determined by the concentration of microbial aerosol in the air sample before and after filtrating. The four canisters of 1-1, 1-2, 1-3, 1-4 were tested for the filtration efficiency against Serratia marcescens, Bacillus subtilis var niger and phage f(2) aerosol. The two canisters of 543 and 544 canisters equipped with active carbon were tested for the filtration efficiencies against Serratia marcescens aerosol.

RESULTSThe filtration efficiency of 1-1, 1-2, 1-3 canisters against Serratia marcescens, Bacillus subtilis var niger and phage f(2) aerosol was 100.000%. The filtration efficiency of 1-4 canister filtration efficiency against Bacillus subtilis var niger spores aerosol was 99.997% and efficiency of the other two aerosol was 100.000%. The filtration efficiency of the two canisters of 543 and 544 to those attached with active carbon against Serratia marcescens aerosol was 100.000%.

CONCLUSIONThe testing method might be used to evaluate the protective performance of the canister against microbiological aerosol. The effect of the canisters (including those equipped with active carbon) against microbiological aerosol should be reliable.

Aerosols ; Air Microbiology ; Bacillus subtilis ; isolation & purification ; Filtration ; methods ; Levivirus ; isolation & purification ; Respiratory Protective Devices ; standards ; Serratia marcescens ; isolation & purification ; Spores, Bacterial ; isolation & purification