Cloning and nucleotide sequence analysis of gyrB of Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and their application to the detection of B. cereus in rice

As 16S rRNA sequence analysis has proven inadequate for the differentiation of Bacillus cereus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic marker. The gyrB genes of B. cereus JCM 2152(T), Bacillus thuringiensis IAM 12077(T), Bacillus mycoides ATCC 6462(T), and Bacillus anthracis Pasteur #2H were cloned and sequenced. Oligonucleotide PCR primer sets were designed from within gyrB sequences of the respective bacteria for the specific amplification and differentiation of B. cereus, B. thuringiensis, and B. anthracis. The results from the amplification of gyrB sequences correlated well with results obtained with the 16S rDNA-based hybridization study but not with the results of their phenotypic characterization. Some of the reference strains of both B. cereus (three serovars) and B. thuringiensis (two serovars) were not positive in PCR amplification assays with gyrB primers. However, complete sequencing of 1.2-kb gyrB fragments of these reference strains showed that these serovars had, in fact, lower homology than their originally designated species. We developed and tested a procedure for the specific detection of the target organism in boiled rice that entailed 15 h of preenrichment followed by PCR amplification of the B. cereus-specific fragment. This method enabled us to detect an initial inoculum of 0.24 CFU of B. cereus cells per g of boiled rice food homogenate without extracting DNA. However, a simple two-step filtration step is required to remove PCR inhibitory substances.     

Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis

Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereus and B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceT gene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes in B. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.     

Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis.

A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.     

Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray

Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect differences in both the genotypes and phenotypes of the B. cereus group organisms.     

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms

AIMS: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them. METHODS AND RESULTS: Using 16S rRNA gene restriction fragment length polymorphisms generated by HindIII and EcoRI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensis serovars and five more strains, kurstaki HD-1, subtoxicus, dendrolimus, tenebrionis and sandiego, to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis. All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia, at the 92.5% DNA relatedness rate. CONCLUSION: 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains. SIGNIFICANCE AND IMPACT OF THE STUDY: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.     

gyrB as a phylogenetic discriminator for members of the Bacillus anthracis-cereus-thuringiensis group

Bacillus anthracis, the causative agent of the human disease anthrax, Bacillus cereus, a food-borne pathogen capable of causing human illness, and Bacillus thuringiensis, a well-characterized insecticidal toxin producer, all cluster together within a very tight clade (B. cereus group) phylogenetically and are indistinguishable from one another via 16S rDNA sequence analysis. As new pathogens are continually emerging, it is imperative to devise a system capable of rapidly and accurately differentiating closely related, yet phenotypically distinct species. Although the gyrB gene has proven useful in discriminating closely related species, its sequence analysis has not yet been validated by DNA:DNA hybridization, the taxonomically accepted "gold standard". We phylogenetically characterized the gyrB sequences of various species and serotypes encompassed in the "B. cereus group," including lab strains and environmental isolates. Results were compared to those obtained from analyses of phenotypic characteristics, 16S rDNA sequence, DNA:DNA hybridization, and virulence factors. The gyrB gene proved more highly differential than 16S, while, at the same time, as analytical as costly and laborious DNA:DNA hybridization techniques in differentiating species within the B. cereus group.     

Differentiation between Bacillus thuringiensis strains by gyrB PCR-Sau3Al fingerprinting

gyrB DNA fragments of seven Bacillus thuringiensis local collection family representatives were amplified by PCR and sequenced. Several differences in their corresponding sequences were evidenced. Both in silico and in vitro restriction maps of gyrB sequences and fragments respectively confirmed that EcoRI and Sau3AI could be used to differentiate between B. thuringiensis strains. However, the phylogeny analysis showed that only the gyrB PCR-Sau3AI allows a strains classification that correlates very well with that obtained on the basis of the sequences analysis. Thus, these finds show that gyrB PCR- Sau3AI digestion could be considered as an efficient, rapid, and easy method to make a distinction, not only between strains belonging to the Bacillus cereus group, but also between those belonging to B. thuringiensis.     

Diversity of cry genes and genetic characterization of Bacillus thuringiensis isolated from Brazil

Two hundred and eighteen Bacillus thuringiensis isolates from Brazil were characterized by the presence of crystal protein genes by PCR with primers specific to different cry and cyt genes. Among these isolates, 95 were selected according to their geographic origin for genetic characterization with the 16S rRNA gene, RAPD, and plasmid profile. Isolates containing cry1 genes were the most abundant (48%) followed by the cry11 and cyt (7%) and cry8 genes (2%). Finally, 40.3% of the isolates did not produce any PCR product. The plasmid profile and RAPD analysis showed a remarkable diversity among the isolates of B. thuringiensis not observed in the 16S rRNA gene. These results suggest that the genetic diversity of B. thuringiensis species results from the influence of different ecological factors and spatial separation between strains generated by the conquest of different habitats.     

A phylogenetic analysis of Bacillus thuringiensis serovars by RFLP-based ribotyping

AIMS: To determine the 23S and 5S rRNA gene fingerprints in order to reveal phylogenetic relationships among Bacillus thuringiensis strains. METHODS AND RESULTS: Eighty-six B. thuringiensis strains which include 80 serovar type strains, five intraserovar strains and a non-serotypeable strain, wuhanensis, were tested. Total DNA was digested with EcoRI and HindIII. The 23S and 5S rRNA gene restriction fragment length polymorphisms showed 82 distinctive ribopatterns. The dendrogram generated by numerical analysis showed 10 phylogenetic groups and six ungrouped serovars at the 95.5% DNA relatedness rate. A second dendrogram was constructed using a combination of the data from this study and from a previous study on 16S rRNA gene fingerprinting. It revealed eight distinct phylogenetic groups and three ungrouped serovars at the 94% DNA relatedness rate. CONCLUSION: This method permitted the classification and positioning of a wide variety of B. thuringiensis strains on a phylogenetic tree. Bacillus thuringiensis strains appear to be relatively homogeneous and to share a high degree of DNA relatedness. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes a further step to the definition of valid taxonomic sublevels for the B. thuringiensis species.     

Phylogenetic analysis of Bacillus thuringiensis serovars based on 16S rRNA gene restriction fragment length polymorphisms

K.-B. JOUNG AND J.-C. CÔTÉ. 2001 .
Aims: To determine the 16S rRNA gene fingerprints of Bacillus thuringiensis strains to reveal phylogenetic relationships among them.
Methods and Results: Using 16S rRNA gene restriction fragment length polymorphisms generated by Hin dIII and Eco RI, 86 Bacillus thuringiensis strains were classified. This includes 80 B. thuringiensi s serovars and five more strains, kurstaki HD-1, subtoxicus , dendrolimus , tenebrionis and sandiego , to assess not only interserovar DNA relatedness but also intraserovar DNA relatedness, and the non-motile strain, hence non-serotypeable, B. thuringiensis var. wuhanensis . All 86 B. thuringiensis strains tested showed distinct ribotypes. The dendrogram resulting from the numerical analysis of the distance matrix shows four distinct phylogenetic groups and two ungrouped serovars, finitimus and bolivia , at the 92·5% DNA relatedness rate.
Conclusions: 16S rRNA gene fingerprinting cannot only be used for the classification of B. thuringiensis strains amenable or not to serotyping, but can also reveal phylogenetic relationships between strains.
Significance and Impact of the Study: In future screening programmes, 16S rRNA gene restriction pattern analysis could be determined for novel B. thuringiensis strains, allowing them not only to be grouped but also to be positioned on the phylogenetic tree.     

Comparative analysis of the 16S to 23S ribosomal intergenic spacer sequences of Bacillus thuringiensis strains and subspecies and of closely related species.

Bacillus thuringiensis spacer regions between the 16S and 23S rRNAs were amplified with conserved primers, designated 19-mer and 23-mer primers. A spacer region of 144 bp was determined for all of 6 B. thuringiensis strains, 7 B. thuringiensis subspecies, and 11 B. thuringiensis field isolates, as well as for the closely related species Bacillus cereus and Bacillus anthracis. Computer analysis and alignment of nucleotide sequences identified three mutations and one deletion in the intergenic spacer region (ISR) of B. thuringiensis subsp. kurstaki HD-1 when compared with ISR sequences from other subspecies. The same differences were identified between the ISR of B. thuringiensis strains and the ISR of B. cereus and B. anthracis. These minor differences do not seem to be sufficient to allow the design of a species-specific oligonucleotide probe.     

Markers for species-specific detection of bacteria from Bacillus cereus group

rpoB and gyr genes (and their fragments) of chromosomal DNA of bacteria from Bacillus cereus group - B. anthracis, B. cereus, and B. thuringiensis - which are the potential markers for their genotyping were sequenced and phylogenetic trees were constructed. Sets of primers for species-specific detection of B. anthracis, B. cereus, and B. thuringiensis by multiplex polymerase chain reaction were designed. Also primers sets, which allow to differentiate strains of B. anthracis with various plasmid profiles (containing both plasmids (pXO1+, pXO2+), and without one (pXO1+, pXO2- or pXO1-, pXO2+) or both plasmids (pXO1-, pXO2-), determining pathogenic characteristics of the strains, were developed. For multiplex PCR primer sets were optimized on the annealing temperature of primers and amplicon length. Itwas shown that phylogenetic tree can be applied as an indicator of reliability and accuracy of taxonomical classification of microorganisms' species and subspecies. Comparison of pXO1 and pXO2 plasmid sequences of B. anthracis showed that these plasmids contain 18 and 4 palindrome sequences respectively which can potentially form thermodynamically stable hairpin-loop structures.     

A single phylogenetic analysis of Bacillus thuringiensis strains and bacilli species inferred from 16S rRNA gene restriction fragment length polymorphism is congruent with two independent phylogenetic analyses

AIMS: To assess the congruence between two earlier independent phylogenetic studies on Bacillus thuringiensis strains and on Bacillus-related species and the single, all-encompassing, phylogenetic tree presented here inferred from the combination of the two earlier datasets. METHODS AND RESULTS: A dendrogram was constructed using a combination of the data from our previous studies on 16S rRNA gene fingerprinting of 86 B. thuringiensis strains and of 77 species of Bacillus and related taxa. It revealed that all B. thuringiensis strains were clustered together in four distinct groups at a DNA similarity rate of 93%, except two serovars, bolivia and finitimus. Four bacilli species, Paenibacillus alvei, P. azotofixans, B. lentus and B. licheniformis, share a DNA similarity rate of 92% with Bt Group IV. CONCLUSIONS: Most, but not all, B. thuringiensis strains could be grouped together based on the DNA similarity rate. They were also very close to some other bacilli species. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined phylogenetic study presented here, inferred from 16S rRNA gene restriction fragment length polymorphism, is congruent with two earlier independent phylogenetic studies, one on B. thuringiensis and the other on bacilli species.     

Simultaneous detection and identification of Bacillus cereus group bacteria using multiplex PCR

Bacillus cereus group bacteria share a significant degree of genetic similarity. Thus, to differentiate and identify the Bacillus cereus group efficiently, a multiplex PCR method using the gyrB and groEL genes as diagnostic markers is suggested for simultaneous detection. The assay yielded a 400 bp amplicon for the groEL gene from all the B. cereus group bacteria, and a 253 bp amplicon from B. anthracis, 475 bp amplicon from B. cereus, 299 bp amplicon from B. thuringiensis, and 604 bp amplicon from B. mycoides for the gyrB gene. No nonspecific amplicons were observed with the DNA from 29 other pathogenic bacteria. The specificity and sensitivity of the B. cereus group identification using this multiplex PCR assay were evaluated with different kinds of food samples. In conclusion, the proposed multiplex PCR is a reliable, simple, rapid, and efficient method for the simultaneous identification of B. cereus group bacteria from food samples in a single tube.     

Isolation and characterization of a novel Bacillus strain from coffee phyllosphere showing antifungal activity

AIMS: The isolation and characterization of a novel coffee-associated Bacillus mojavensis strain, designated as strain AB1, and its survival on the coffee phyllosphere. METHODS AND RESULTS: A pair of 16S rDNA primers was designed to amplify a highly variable region within the 16S rDNA gene of Bacillus spp., with the purpose of identifying the AB1 isolate through PCR and sequence analysis. By this method, AB1 was identified as a strain of B. mojavensis. Bioassays were carried out to characterize the broad spectrum antifungal activity of AB1. Plant colonization studies revealed that AB1 could colonize the coffee phyllosphere better than Bacillus thuringiensis. CONCLUSIONS: These studies suggest that AB1 could be a new strain of B. mojavensis. AB1 is also shown to have antifungal activity against a wide spectrum of pathogenic fungi. The antifungal metabolite of AB1 has been partially characterized as a thermostable, protease- and alkali-resistant substance that is secreted into the surrounding medium. SIGNIFICANCE AND IMPACT OF THE STUDY: As far as is known, this is the first strain of B. mojavensis which has been identified as inhabiting the coffee phyllosphere. The study highlights the potential use of AB1 as an antifungal agent in the coffee crop and as a delivery agent of the insecticidal toxin of B. thuringiensis to the coffee phyllosphere. The 16S rRNA identification strategy discussed could also be used in the identification of other new Bacillus strains.     

Development of a PCR assay for identification of the Bacillus cereus group species

Aims:  A PCR technique was developed as a reliable and rapid identification method for the Bacillus cereus group species, based on a unique conserved sequence of the motB gene (encoding flagellar motor protein) from B. cereus , Bacillus thuringiensis and Bacillus anthracis .
Methods and Results:  Primer locations were identified against eight strains of the B. cereus group spp. from nucleotide sequences available in the National Centre for Biotechnology Information database. The PCR assay was applied for the identification of 117 strains of the B. cereus group spp. and 19 strains from other microbial species, with special emphasis on foodborne pathogens.
Conclusion:  The designed cross-species primers are group specific and did not react with DNA from other Bacillus and non- Bacillus species either motile or not. The primers system enabled us to detect 10³ CFU of B. cereus cells per millilitre of sample.
Significance and Impact of the Study:  Bacillus cereus group spp. belongs to one of the most prevalent foodborne pathogens. Bacterial growth results in production of different toxins; therefore, consumption of food containing >10⁶ bacteria per gram may result in emetic and diarrhoeal syndromes. A rapid and sensitive bacterial detection method is significant for food safety.     

Polymerase chain reaction identification of Bacillus sporothermodurans from dairy sources

AIMS: A new polymerase chain reaction (PCR) method for the identification of Bacillus sporothermodurans strains from sterilized or ultrahigh temperature-treated milk and milk products and from other non-milk sources and environments, including the dairy farm. METHODS AND RESULTS: Two strains from raw milk and feed concentrate could be allocated to B. sporothermodurans based on 16S rDNA sequencing and DNA-DNA hybridization results. Two specific PCR primers were derived from the 16S rRNA gene of B. sporothermodurans. CONCLUSIONS: The PCR identification method was validated using a collection of B. sporothermodurans strains from different sources and on a large collection of dairy and non-dairy Bacillus spp. and other relevant taxa. SIGNIFICANCE AND IMPACT OF THE STUDY: This PCR method was used as a screening method for strains with very heat-resistant endospores, isolated at the dairy farm level after heat treatment for 30 min at 100 degrees C. Seventeen strains isolated at the dairy farm were identified as B. sporothermodurans. They originated mainly from feed concentrate and also from soy, pulp and silage. The PCR identification method described here can, therefore, contribute to a better understanding of the route by which B. sporothermodurans contaminates raw and/or heat-treated milk.     

Cloning and partial characterization of zwittermicin A resistance gene cluster from Bacillus thuringiensis subsp. kurstaki strain HD1

AIM: The study seeks to shed light on the aminopolyol, broad-spectrum antibiotic zwittermicin A gene cluster of Bacillus thuringiensis subsp. kurstaki HD1 and to identify any new uncharacterized genes with an eventual goal to establish a better understanding of the resistance gene cluster. METHODS AND RESULTS: We screened 51 serovars of B. thuringiensis by PCR and identified 12 zmaR-positive strains. The zmaR-positive B. thuringiensis subsp. kurstaki HD1 strain displayed inhibition zones against indicator fungal strain Phytophthora meadii and bacterial strain Erwinia herbicola as well as against Rhizopus sp., Xanthomonas campestris and B. thuringiensis subsp. finitimus. The zmaR gene cluster of strain HD1 was partially cloned using a lambda library and was extensively characterized based on the information available from a study performed on a similar group of genes in Bacillus cereus. CONCLUSIONS: Three of the five genes in the zwittermicin gene cluster, including the zmaR gene, had counterparts in B. cereus, and the other two were new members of the B. thuringiensis zmaR gene cluster. SIGNIFICANCE AND IMPACT OF THE STUDY: The two new genes were extensively analysed and the data is presented. Understanding antifungal activity of B. thuringiensis may help us to design suitable Cry toxin delivery agents with antifungal activity as well as enhanced insecticidal activity.     

Genetic differentiation between sympatric populations of Bacillus cereus and Bacillus thuringiensis

Little is known about genetic exchanges in natural populations of bacteria of the spore-forming Bacillus cereus group, because no population genetics studies have been performed with local sympatric populations. We isolated strains of Bacillus thuringiensis and B. cereus from small samples of soil collected at the same time from two separate geographical sites, one within the forest and the other at the edge of the forest. A total of 100 B. cereus and 98 B. thuringiensis strains were isolated and characterized by electrophoresis to determine allelic composition at nine enzymatic loci. We observed genetic differentiation between populations of B. cereus and B. thuringiensis. Populations of a given Bacillus species--B. thuringiensis or B. cereus--were genetically more similar to each other than to populations of the other Bacillus species. Hemolytic activity provided further evidence of this genetic divergence, which remained evident even if putative clones were removed from the data set. Our results suggest that the rate of gene flow was higher between strains of the same species, but that exchanges between B. cereus and B. thuringiensis were nonetheless possible. Linkage disequilibrium analysis revealed sufficient recombination for B. cereus populations to be considered panmictic units. In B. thuringiensis, the balance between clonal proliferation and recombination seemed to depend on location. Overall, our data indicate that it is not important for risk assessment purposes to determine whether B. cereus and B. thuringiensis belong to a single or two species. Assessment of the biosafety of pest control based on B. thuringiensis requires evaluation of the extent of genetic exchange between strains in realistic natural conditions.