Antimicrobial activity of Bacillus subtilis and Bacillus pumilus during the fermentation of African locust bean (Parkia biglobosa) for Soumbala production

Aims:  To examine predominant isolates of Bacillus subtilis and B. pumilus isolated from Soumbala for their antimicrobial activity against indicator microorganisms as Micrococcus luteus , Staphyloccocus aureus , Bacillus cereus , Enterococus facium , Listeria monocytogenes , Escherichia coli , Salmonella typhimurium , Shigella dysenteriae , Yersinia enterocolitica , Aspergillus ochraceus and Penicillium roqueforti .
Methods and Results:  Growth inhibition of indicator microorganisms by cells and supernatants of three B. subtilis and two B. pumilus strains was investigated using agar diffusion tests. Inactivation of indicator microorganisms was investigated in laboratory broth and during the fermentation of African locust bean for Soumbala production. The Bacillus isolates showed variable ability of inhibition and inactivation according to the indicator microorganism. The supernatants of pure cultures of B. subtilis inhibited one strain of B. cereus , one of Staph. aureus and E. coli and caused abnormal germination of Aspergillus ochraceus . The supernatant of mixed cultures of B. subtilis and indicators inhibited all the indicators. A treatment with protease eliminated the inhibitions. Isolates of B. subtilis inactivated all the indicators organisms during the fermentation of African locust bean as well as in laboratory broth with about five to eight decimal reduction.
Conclusion:  Bacillus isolates from Soumbala inhibit and inactivate Gram-positive and Gram-negative bacteria as well as ochratoxin A producing fungi during both laboratory cultivation and natural fermentation.
Significance and Impact of the Study:  Selection of starter cultures of Bacillus spp. for controlled production of Soumbala.     

Volatile compounds of Soumbala, a fermented African locust bean (Parkia biglobosa) food condiment

AIMS: To determine the profile of volatile compounds responsible for the aroma of Soumbala produced spontaneously and with pure and mixed cultures of Bacillus subtilis and Bacillus pumilus. METHODS AND RESULTS: Traditional and controlled fermentation trials of African locust bean with pure and mixed starter cultures of B. subtilis (B7, B9 and B15) and B. pumilus (B10) were performed. Aroma volatiles were analysed using Likens-Nikerson method coupled with gas chromatography and mass spectrophotometry. Sensory analysis of Soumbala as well as rice dishes prepared with each type of Soumbala were carried out by 10 panellists. In total 116 compounds were identified. They included pyrazines, aldehydes, ketones, esters, alcohols, acids, alkanes, alkenes, amines, pyridines, benzenes, phenols, sulphurs, furans and other compounds. Using principal component analysis for comparison, the aroma profiles of the Soumbala samples could be separated into three groups. The sensory evaluation showed variable acceptability. However, it was noticed that Soumbala samples produced with starter cultures were scored higher than traditionally prepared Soumbala. CONCLUSIONS: Aroma volatiles and organoleptic properties of Soumbala vary according to the Bacillus isolates involved in the fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus starter cultures for controlled production of Soumbala.     

Degradation of African locust bean oil by Bacillus subtilis and Bacillus pumilus isolated from soumbala,a fermented African locust bean condiment

AIMS: To investigate predominant isolates of Bacillus subtilis and B. pumilus in soumbala, a fermented African locust bean condiment, for their ability to degrade African locust bean oil (ALBO). METHODS AND RESULTS: Agar diffusion test in tributyrin and ALBO agar was used for screening of the isolates for esterase and lipase activity, respectively. The quantity and the profile of free fatty acids (FFA) during 72 h of degradation of ALBO by the Bacillus isolates were studied by titration and gas chromatography. The degradation of tributyrin and ALBO was variable among the isolates. Two strains of B. subtilis and two strains of B. pumilus showed significantly higher esterase and lipolytic activities than the others. The degradation ALBO was most pronounced in enriched nutrient agar except for one isolate of B. pumilus degrading ALBO to the same extent regardless of the enrichment. The quantity of FFA released from ALBO by the most lipolytic strains of Bacillus increased mainly between 0 and 24 h and differed among the isolates. The profile of FFA was similar for the Bacillus isolates with oleic acid (C18:2) occurring as the major FFA in all the samples except in samples incubated with B. subtilis B9 where stearic acid (C18) was dominant. CONCLUSION: Bacillus isolates from soumbala showed high strain dependent lipolytic activity against ALBO. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the selection of Bacillus strains to be used as starter cultures for controlled production of soumbala.     

Characterization of Bacillus thuringiensis soil isolates from Cuba, with insecticidal activity against mosquitoes

Chemical insecticides may be toxic and cause environmental degradation. Consequently, biological control for insects represents an alternative with low ecological impact. In this work, three soil isolates (A21, A51 and C17) from different regions of the Cuban archipelago were identified, characterized and evaluated against Aedes aegypti and Culex quinquefasciatus. The new isolates were compared with reference IPS82 strain and two strains isolated from biolarvicides Bactivec and Bactoculicida, respectively. The differentiation was done by morphological, biochemical, bioassays activity and molecular methods (SDS-PAGE, plasmid profile and random amplified polymorphic analysis). All isolates were identified as Bacillus thuringiensis. The A21, A51 and C17 isolates showed higher larvicide activity than Bactivec's isolated reference strain, against both A. aegypti and C. quinquefasciatus. A21 isolate had a protein profile similar to IPS82 and Bactivec strain. A51 and C17 isolates produced a characteristic proteins pattern. A21 and A51 isolates had plasmid patterns similar to IPS82 standard strain, while C17 isolate had different both plasmid profile and protein bands. All the studied isolates showed a diverse RAPD patterns and were different from the strains previously used in biological control in Cuba.     

Occurrence of Bacillus sporothermodurans and other aerobic spore-forming species in feed concentrate for dairy cattle

AIMS: To determine the aerobic spore composition and presence of Bacillus sporothermodurans spores in feed concentrate for dairy cattle. METHODS AND RESULTS: Six feed concentrate samples from five different farms were analysed. High levels of spores (up to 10(6) spores g(-1)) were found. Identification of 100 selected isolates was obtained by a combination of fatty acid methyl esters analysis, amplified ribosomal DNA restriction analysis and 16S rDNA sequencing. Ninety-seven isolates could be identified to the species level or assigned to a phylogenetic species group. Most of the isolates obtained after a heat treatment of 10 min at 80 degrees C were identified as members of the B. subtilis group (32 isolates), B. pumilus (25 isolates), B. clausii (eight isolates) and B. licheniformis (eight isolates). The isolates with very heat-resistant spores, obtained after a heat treatment of 30 min at 100 degrees C, were identified as members of the B. subtilis group (five isolates), B. sporothermodurans (three isolates), B. amyloliquefaciens (one isolate), B. oleronius (one isolate) and B. pallidus (one isolate). Bacillus cereus was present in each feed concentrate sample and was isolated using a selective mannitol egg yolk polymyxin agar medium. CONCLUSIONS: Feed concentrate for dairy cattle contains known as well as as yet unknown species of Bacillus and related genera with properties relevant to the dairy sector. SIGNIFICANCE AND IMPACT OF THE STUDY: The results formulate the hypothesis that feed concentrate can be a contamination source of spores, including those of B. sporothermodurans, for raw milk at the farm level.     

Production of poly-beta-hydroxyalkanoates from soy molasses oligosaccharides by new, rapidly growing Bacillus species

AIMS: To isolate and characterize bacteria from nature capable of producing poly-beta-hydroxyalkanoates in high yields from soy molasses oligosaccharides. METHODS AND RESULTS: Several strains of bacteria were obtained from enrichment cultures employing raffinose as major carbon source and inoculated with soybean field soil, lake sediment, or lake water. Many of the isolates were Bacillus species and produced polyhydroxyalkanoates (PHAs) to high yield. The raffinose-degrading isolates produced endospores, were highly saccharolytic, and both respired and fermented a variety of mono-, di-, tri- and tetrasaccharides. Strain CL1 produced 90% of cell dry mass as PHA from various sugars, including raffinose, and did so without requiring a nutrient limitation. CONCLUSIONS: Strain CL1 could be the catalyst for an industrial fermentation converting soy molasses and other waste carbohydrates to PHAs. The properties of this organism that make it ideally suited for such a fermentation include (i) its ability to use a wide variety of plant-associated carbohydrates as PHA feedstocks; (ii) its rapid growth; (iii) its ability to grow under anoxic conditions; and (iv) its ability to produce spores. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of bacteria capable of making biodegradable plastics to high yield from soy molasses oligosaccharides.     

Assessment of toxic potential of local Jordanian Bacillus thuringiensis strains on Drosophila melanogaster and Culex sp. (Diptera)

AIMS: To assess the toxic potential of different local Jordanian Bacillus thuringiensis isolates on larvae of Drosophila melanogaster and Culex sp. METHODS AND RESULTS: Scanning electron microscopy revealed the presence of spherical, bi-pyramidal, and bi-pyramidal and cuboidal parasporal bodies produced by the toxic isolates. Spherical inclusions dominated. The toxicity of the isolates to the two insects, determined using 24-well plates or vials, indicated that the 50% lethal concentration (LC50) of the bacterial suspension for D. melanogaster and Culex sp. larvae varied from 4.60 to 8.65, and from 5.30 to 6.74, respectively. CONCLUSION: Comparison of the LC50 values of isolate 82 with those of the reference strain B. t. israelensis showed that this isolate has a higher toxicity potential. SIGNIFICANCE AND IMPACT OF THE STUDY: Some local Jordanian B. thuringiensis isolates exhibit toxic potential that could be used to control some important pests, and could replace chemical pesticides.     

Isolation and selection of Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish

AIMS: To isolate, select and evaluate Bacillus spp. as potential biological agents for enhancement of water quality in culture of ornamental fish. METHODS AND RESULTS: Natural isolates obtained from mud sediment and Cyprinus carpio were purified and assessed in vitro for efficacy based on the inhibition of growth of pathogenic Aeromonas hydrophila and the decrease in concentrations of ammonium, nitrite, nitrate and phosphate ions. Based on suitability to predefined characteristics, the isolates B001, B002 and B003 were selected and evaluated in vitro in the presence of Aer. hydrophila and in a preliminary in vivo trial with C. carpio. The inhibitory effect on pathogen growth and the decrease in concentrations of waste ions was demonstrated. Based on 16S RNA sequence homology, the isolates were identified as Bacillus subtilis, Bacillus cereus and Bacillus licheniformis, respectively. Isolate B002 did not contain the anthrax virulence plasmids pOX1, pOX2 or the B. cereus enterotoxin. CONCLUSIONS: Selected isolates effected synergistic reduction in pathogen load and the concentrations of waste ions in vitro and in vivo and are safe for use in ornamental aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: A new approach for assessment of biological agents was demonstrated and has yielded putative isolates for development into aquaculture products.     

Application of actinomycetes to soil to ameliorate water repellency

AIMS: The aim of this study was to develop a novel isolation technique using a mixture of Bacillus and Streptomyces phages to selectively isolate wax-utilizing non-streptomycete actinomycetes effective in ameliorating water repellency in a problem soil. METHODS AND RESULTS: Phages added to a soil suspension reduced the dominance of Bacillus and Streptomyces isolates and significantly increased the number of non-streptomycete actinomycetes on isolation plates. Promising isolates, grown on a medium containing beeswax as sole carbon source, were selected for application to water repellent soil. Their addition significantly reduced water repellency. CONCLUSIONS: Phage application significantly increased the isolation of non-streptomycete actinomycetes. Wax-utilizing isolates were found to significantly reduce water repellency in a problem soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The phage technique can be used for the routine isolation of non-streptomycete actinomycetes. Beeswax medium can be used to selectively isolate wax-utilizing micro-organisms with the potential to ameliorate water repellency in soil.     

Adaptation of a neutrophilic dairy-associated Bacillus cereus isolate to alkaline pH

AIMS: This study identified and studied the response of five Bacillus strains, isolated from alkaline cleaning in place (CIP) solutions, to alkaline conditions. METHODS AND RESULTS: Isolates were identified as B. cereus by 16S rDNA sequencing. External and internal cell pH and buffering capacity data of a representative strain, Bacillus DL5, were compared to B. cereus ATCC 10702. Results indicated that a buffering system was induced when the pH of the growth medium increased to above pH 10, which was effective up to pH 12 and presumably cell wall associated. Volume measurements and confocal scanning laser microscope images of Bacillus DL5 cells showed that cells exhibited more pronounced stress symptoms when exposed to pH 10 than at pHs above 10. Long-term exposure of Bacillus DL5 to pH 10 or 10.5 indicated that cells grew in planktonic form and formed biofilms at both pHs. CONCLUSIONS: Bacillus DL5 was a neutrophile with a growth pH range similar to B. cereus ATCC 10702, but tolerated alkaline pH. This may be a general trait of the B. cereus species rather than a specific phenomenon of isolates from alkaline ecosystems. SIGNIFICANCE AND IMPACT OF THE STUDY: Other neutrophilic B. cereus isolates may exhibit similar responses to alkaline conditions as the isolates studied here. These results may have important implications for dairy manufacturers.     

The role of Bacillus species in the fermentation of cassava

Cassava dough inoculum is added to grated cassava in order to achieve a modification of texture during fermentation into the fermented cassava meal, agbelima. The microflora of two different types of inocula and subsequently inoculated cassava mash at 0, 24 and 48 h of fermentation were examined in order to determine the mechanism responsible for the breakdown of cassava tissue. Bacillus spp. occurred in high numbers, 10₇–10₈ cfu g_-1, in both types of inocula and persisted throughout the cassava dough fermentation. Bacillus spp. found were B. subtilis , B. mycoides , B. pumilus , B. cereus , B. amyloliquefaciens and B. licheniformis , with B. subtilis accounting for more than half of Bacillus isolates. All Bacillus isolates produced a wide spectrum of enzymes and showed similar enzymatic activities but only B. pumilus , B. licheniformis and B. amyloliquefaciens produced linamarase. Some isolates produced the tissue degrading enzymes polygalacturonase and pectin esterase and nearly all isolates hydrolysed starch. All isolates showed cellulase activity and were able to disintegrate cassava tissue. When cassava pieces were incubated in amylase, cellulase, pectin esterase and polygalacturonase solutions, only pieces in cellulase solution were dissolved revealing that the breakdown of cassava dough texture during fermentation with the inocula examined is brought about by Bacillus spp. through cellulase activity.     

Isolation and identification of nitrogen-fixing bacilli from plant rhizospheres in Beijing region

AIMS: To isolate and identify nitrogen-fixing bacilli from the plant rhizospheres in Beijing region of China. METHODS AND RESULTS: A total of 29 isolates were selectively obtained from the rhizospheres of wheat, maize, ryegrass and willow based on their growth on nitrogen-free medium and their resistance to 100 degrees C for 10 min. Of the 29 isolates, seven had nifH gene determined by PCR amplification. The seven isolates were found to belong to the genera Bacillus and Paenibacillus based on phenotypic characterization, 16S rDNA sequence, G+C content and DNA-DNA hybridization. Isolates T1 and W5 were identified as Bacillus cereus and Bacillus marisflavi respectively. Isolates G1, C4 and C5 were identified as Bacillus megaterium. Isolate G2 was identified as Paenibacillus polymyxa and isolate T7 as Paenibacillus massiliensis. CONCLUSIONS: This study suggests that nifH gene could be detected in the both genera Bacillus and Paenibacillus. These degenerate primers for nifH gene fragment used in this study were shown to be useful for identifying nitrogen-fixing bacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first demonstration that nitrogen fixation exists in B. marisflavi and P. massiliensis and the first report of the sequences of the nifH gene from B. megaterium and B. cereus. The nitrogen-fixing bacilli obtained in this study will be used in our future research for investigating the mechanisms of nitrogen fixation in bacilli.     

Relative toxicity of native Chilean Bacillus thuringiensis strains against Scrobipalpuloides absoluta (Lepidoptera: Gelechiidae)

The larva of Scrobipalpuloides absoluta , a South American moth, is the most devastating insect pest of tomato production in Chile. The potential for using bacterial insecticides was studied analysing the relative toxicity of native Bacillus thuringiensis (BT) isolates belonging to the Chilean collection. The polymerase chain reaction (PCR) technique was used in order to facilitate the prescreening. Mixtures of homologous specific primers to regions within genes encoding CryI , CryIII and CryIV crystal proteins were employed to generate a PCR product profile of each BT isolate. Four isolates were selected and further characterized by means of SDS-PAGE, Western blot and bioassays on fourth-instar S. absoluta larvae. Relative toxicities were evaluated by LD₅₀ determinations. The entomocidal activity of isolate 121e, an autoagglutinating strain, was threefold higher than toxin synthesized by B. thuringiensis var. kurstaki . This native strain was also active against Culex pipiens larvae, although much less than towards S. absoluta .     

Biodegradation of feather waste by keratinase produced from newly isolated Bacillus licheniformis ALW1

Keratinase are proteolytic enzymes which have gained much attention to convert keratinous wastes that cause huge environmental pollution problems. Ten microbial isolates were screened for their keratinase production. The most potent isolate produce 25.2?U/ml under static condition and was primarily identified by partial 16s rRNA gene sequence as Bacillus licheniformis ALW1. Optimization studies for the fermentation conditions increased the keratinase biosynthesis to 72.2?U/ml (2.9-fold). The crude extracellular keratinase was optimally active at pH 8.0 and temperature 65?°C with 0.7% soluble keratin as substrate. The produced B. licheniformis ALW1 keratinase exhibited a good stability over pH range from 7 to 9 and over a temperature range 50–60?°C for almost 90?min. The crude enzyme solution was able to degrade native feather up to 63% in redox free system.     

Inactivation of Bacillus spores in reconstituted skim milk by combined high pressure and heat treatment

AIMS: To determine the resistance of a variety of Bacillus species spores to a combined high pressure and heat treatment; and to determine the affect of varying sporulation and treatment conditions on the level of inactivation achieved. METHODS AND RESULTS: Spores from eight Bacillus species (40 isolates) were high pressure-heat treated at 600 MPa, 1 min, initial temperature 72 degrees C. The level of inactivation was broad (no inactivation to 6 log10 spores ml(-1) reduction) and it varied within species. Different sporulation agar, high pressure equipment and pressure-transmitting fluid significantly affected the response of some isolates. Varying the initial treatment temperature (75, 85 or 95 degrees C) shifted the relative order of isolate high pressure-heat resistance. CONCLUSIONS: The response of Bacillus spores to combined high pressure-heat treatment is variable and can be attributed to both intrinsic and extrinsic factors. The combined process resulted in a high level of spore inactivation for several Bacillus species and is a potential alternative treatment to traditional heat-only processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Sporulation conditions, processing conditions and treatment temperature all affect the response of Bacillus spores to the combined treatment of high pressure and heat. High levels of spore inactivation can be achieved but the response is variable both within and between species.     

Fluorescent amplified fragment length polymorphism analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis isolates

DNA from over 300 Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis isolates was analyzed by fluorescent amplified fragment length polymorphism (AFLP). B. thuringiensis and B. cereus isolates were from diverse sources and locations, including soil, clinical isolates and food products causing diarrheal and emetic outbreaks, and type strains from the American Type Culture Collection, and over 200 B. thuringiensis isolates representing 36 serovars or subspecies were from the U.S. Department of Agriculture collection. Twenty-four diverse B. anthracis isolates were also included. Phylogenetic analysis of AFLP data revealed extensive diversity within B. thuringiensis and B. cereus compared to the monomorphic nature of B. anthracis. All of the B. anthracis strains were more closely related to each other than to any other Bacillus isolate, while B. cereus and B. thuringiensis strains populated the entire tree. Ten distinct branches were defined, with many branches containing both B. cereus and B. thuringiensis isolates. A single branch contained all the B. anthracis isolates plus an unusual B. thuringiensis isolate that is pathogenic in mice. In contrast, B. thuringiensis subsp. kurstaki (ATCC 33679) and other isolates used to prepare insecticides mapped distal to the B. anthracis isolates. The interspersion of B. cereus and B. thuringiensis isolates within the phylogenetic tree suggests that phenotypic traits used to distinguish between these two species do not reflect the genomic content of the different isolates and that horizontal gene transfer plays an important role in establishing the phenotype of each of these microbes. B. thuringiensis isolates of a particular subspecies tended to cluster together.     

Properties of Bacillus thuringiensis isolated from bank voles

AIMS: To assess the properties of B. thuringiensis naturally occurring in the intestines of bank voles. METHODS AND RESULTS: Seventeen Bacillus thuringiensis strains, exhibiting typical growth on selective medium for the B. cereus group and characterized by the ability to produce parasporal crystals, were isolated from bank voles trapped in the ?omza Landscape Park of the Narew River Valley (north-east Poland). All isolates were characterized by pulsed field gel electrophoresis (PFGE) of chromosomal DNA and SDS polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins. Six pulsotypes were found with PFGE typing, using SmaI or NotI as restriction enzymes. Significant differences in chromosome size, ranging from 2.4 to 4.2 Mb for the B. thuringiensis strains studied, were noted. Strain heterogeneity in pulsotypes was also reflected by the similarity of whole-cell protein profiles of the strains. Environmental isolates and reference strains grouped at 71% similarity according to SDS-PAGE data and at 84% on the basis of biochemical tests. CONCLUSIONS: B. thuringiensis from intestines of bank voles demonstrated an important level of heterogeneity. The comparison of PFGE profiles and SDS-PAGE of whole-cell protein patterns may be useful to evaluate the relationship between B. thuringiensis isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented in this paper may help to explain the diversity of B. thuringiensis.     

Fate of Escherichia coli O157 and detection of stx phage during fermentation of maize, an animal feedstuff

AIMS: The fate of inoculated Escherichia coli O157, stx phages and the physico-chemical properties of maize were studied during laboratory-scale fermentation by naturally occurring lactic acid bacteria. METHODS AND RESULTS: Before fermentation, chopped maize was inoculated with 6.2 log(10) CFU g(-1) of a five-isolate mixture of E. coli O157. After fermentation, the silage contained 70.6 g kg(-1) dry matter (DM) lactic acid and 12.8 g kg(-1) DM acetic acid and was pH 4.0. Levels of E. coli O157 fell rapidly, and none of the five isolates could be recovered from the fermenting maize after 8 days. Using a resuscitation step did not consistently enhance recovery of E. coli O157. Stx phages were not isolated from the fermenting maize at any time. Pulsed-field gel electrophoresis (PFGE) genotyping showed that E. coli O157 2975 and 64a/01 survived better than the other three isolates studied. Escherichia coli O157 isolate 1474/00 was particularly sensitive to the laboratory procedures used to harvest the inocula and contaminate the maize. Some colonies recovered during the fermentation had one to three band alterations compared with the initial PFGE genotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: None of the five different E. coli O157 genotypes survived maize fermentation. Fermentation of maize produces an animal feedstuff that is unlikely to contain E. coli O157 or stx phages.     

Isolation and characterization of Bacillus sp. BP-6 LipA,a ubiquitous lipase among mesophilic Bacillus species

AIMS: The aim of this study was to perform the isolation, cloning and characterization of a lipase from Bacillus sp. BP-6 bearing the features of a biotechnologically important group of enzymes. METHODS AND RESULTS: Strain Bacillus sp. BP-6, showing activity on tributyrin plates, was used for isolation of lipase-coding gene lipA by means of inverse and direct PCR. The complete 633 nucleotide ORF isolated was cloned in Escherichia coli for further characterization. The amino acid sequence of the cloned protein was 98% identical to B. subtilis and B. megaterium lipases, the enzyme also showing similar molecular and biochemical features. CONCLUSIONS: The gene coding for Bacillus sp. BP-6 LipA was found in all mesophilic Bacillus species assayed, indicating its ubiquity in the genus. The cloned enzyme displayed the same properties as those of homologous lipases. SIGNIFICANCE AND IMPACT OF THE STUDY: The overall profile of Bacillus sp. BP-6 LipA was found to be that of a ubiquitous and highly conserved subfamily I.4 bacterial lipase. Previously described lipases within this family have shown to be well suited for biotechnological applications, suggesting that the cloned enzyme could be used accordingly.     

A simple and sensitive detection system for Bacillus anthracis in meat and tissue

AIMS: To detect and isolate Bacillus anthracis from meat and tissue by rapid and simple procedures. METHODS AND RESULTS: Bacillus anthracis Pasteur II cells were added to 1 g lymph node and pig meat, which were then cut into small pieces and suspended in PBS. Aliquots were spread on Bacillus cereus selective agar (BCA) plates to isolate B. anthracis cells, and incubated in trypticase soy broth. The enrichment culture was used for nested PCR with B. anthracis specific primers, which were to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. CONCLUSION: One cell of B. anthracis was detected by nested PCR from 1 g of the samples, and was also isolated on BCA plates according to colony morphology within two days. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be useful for detecting animals with latent anthrax, and meat contaminated with B. anthracis, rapidly and simply.