Fingerprinting of Bacillus thuringiensis Type Strains and Isolates by Using Bacillus cereus Group-Specific Repetitive Extragenic Palindromic Sequence-Based PCR Analysis

A total of 119 Bacillus thuringiensis strains (83 type strains and 26 native isolates), as well as five B. cereus group species, were analyzed by repetitive extragenic palindromic sequence-based PCR analysis (Rep-PCR) fingerprinting. Primers Bc-REP-1 and Bc-REP-2 were specifically designed according to an extragenic 26-bp repeated sequence found in the six B. cereus group genomes reported. A total of 47 polymorphic bands were detected, and the patterns varied from 5 to 13 bands in number and from 0.2 to 3.8 kb in size. Virtually each type strain showed a distinctive B. cereus (Bc)-Rep-PCR pattern, except for B. thuringiensis serovars dakota (H serotype 15 [H15]) and sotto (H4a,4b), as well as serovars amagiensis (H29) and seoulensis (H35), which shared the same patterns. As expected, serovar entomocidus (H6) and its biovar subtoxicus showed an identical pattern; similarly, serovars sumiyoshiensis (H3a,3d) and fukuokaensis (H3a,3d,3e), which share two antigenic determinants, also showed identical Bc-Rep-PCR patterns. Interestingly, serovars israelensis (H14) and malaysiensis (H36), which share several phenotypic attributes, also showed identical Bc-Rep-PCR patterns. Native, coleopteran-active strains, including the self-agglutinated LBIT-74 strain, showed Bc-Rep-PCR patterns identical or very similar to that of the tenebrionis strain. Likewise, native mosquitocidal strains (including some self-agglutinated strains) also showed patterns identical or very similar to that of the serovar israelensis IPS-82 strain. Additionally, native β-exotoxin-producing strains from serovar thuringiensis showed patterns identical to that of the B. thuringiensis type strain. The B. cereus group-specific Bc-Rep-PCR fingerprinting technique was shown to be highly discriminative, fast, easy, and able to identify B. thuringiensis serotypes, including nonflagellar and self-agglutinated strains.     

Influence of Lysogeny of Tectiviruses GIL01 and GIL16 on Bacillus thuringiensis Growth,Biofilm Formation,and Swarming Motility

Bacillus thuringiensis is an entomopathogenic bacterium that has been used as an efficient biopesticide worldwide. Despite the fact that this bacterium is usually described as an insect pathogen, its life cycle in the environment is still largely unknown. B. thuringiensis belongs to the Bacillus cereus group of bacteria, which has been associated with many mobile genetic elements, such as species-specific temperate or virulent bacteriophages (phages). Temperate (lysogenic) phages are able to establish a long-term relationship with their host, providing, in some cases, novel ecological traits to the bacterial lysogens. Therefore, this work focuses on evaluating the potential influence of temperate tectiviruses GIL01 and GIL16 on the development of different life traits of B. thuringiensis. For this purpose, a B. thuringiensis serovar israelensis plasmid-cured (nonlysogenic) strain was used to establish bacterial lysogens for phages GIL01 and GIL16, and, subsequently, the following life traits were compared among the strains: kinetics of growth, metabolic profiles, antibiotics susceptibility, biofilm formation, swarming motility, and sporulation. The results revealed that GIL01 and GIL16 lysogeny has a significant influence on the bacterial growth, sporulation rate, biofilm formation, and swarming motility of B. thuringiensis. No changes in metabolic profiles or antibiotic susceptibilities were detected. These findings provide evidence that tectiviruses have a putative role in the B. thuringiensis life cycle as adapters of life traits with ecological advantages.     

Phenotypic characterization of Bacillus thuringiensis and Bacillus cereus

One hundred and thirty-seven strains of Bacillus thuringiensis and 35 strains of Bacillus cereus were tested for the presence or absence of 99 traits. An analysis of these data indicated that strains of B. thuringiensis were indistinguishable from B. cereus, except for their ability to produce parasporal crystals. This conclusion was based on a comparison of the phenotypic properties of B. thuringiensis and B. cereus, as well as on the results of numerical analyses of the data which grouped strains into clusters on the basis of phenotypic similarity. In the resulting dendrograms, strains of B. thuringiensis and B. cereus were interspersed, exhibiting no tendency to segregate. In addition, with the exception of serovar israelensis, strains on B. thuringiensis belonging to the same flagellar serovar showed little or no tendency to group in different clusters. A comparison of the phenotypic differences between serovars indicated that the greater the number of strains in the serovars, the fewer, if any, phenotypic traits separating them. This suggests that the properties reported to differentiate serovars can be attributed to the internal phenotypic diversity of the species. Characterization of 10 mosquitocidal strains of Bacillus sphaericus indicated that the traits employed in this study readily distinguished these highly related organisms from strains of B. thuringiensis and B. cereus.     

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.     

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.     

Esterase electrophoretic polymorphism ofBacillus thuringiensis andBacillus cereus reference strains

The genomic diversity and relationship among 61Bacillus thuringiensis andBacillus cereus reference strains were investigated by electrophoretic analysis of esterase enzymes on native polyacrylamide gel. Polymorphism of the esterolytic bands revealed seven esterases, designed as Est A to Est G in order of decreasing anodal migration. Each esterase showed two to three mobility variants that assigned the analysed strains into 35 electrophoretic types (ETs). This high diversity allowed the identification of several serovar or strain-specific ETs. Cluster analysis of ETs showed three major groups in which the strains belonging to the serovartolworthi were the most distant. The ETs distribution showed thatB. thuringiensis andB. cereus are intermingled in the dendrogram with the resolution of some common serovars ofB. thuringiensis in tight phylogenetic lineages. These results indicate that the esterase enzyme electrophoresis, applied as a sole typing method for the closely related speciesB. thuringiensis andB. cereus is suitable to highlight the intraspecific genetic diversity.     

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—One Species on the Basis of Genetic Evidence

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Development of a Fluorogenic Probe-Based PCR Assay for Detection of Bacillus cereus in Nonfat Dry Milk

A fluorogenic probe-based PCR assay was developed and evaluated for its utility in detecting Bacillus cereus in nonfat dry milk. Regions of the hemolysin and cereolysin AB genes from an initial group of two B. cereus isolates and two Bacillus thuringiensis isolates were cloned and sequenced. Three single-base differences in two B. cereus strains were identified in the cereolysin AB gene at nucleotides 866, 875, and 1287, while there were no species-consistent differences found in the hemolysin gene. A fluorogenic probe-based PCR assay was developed which utilizes the 5′-to-3′ exonuclease of Taq polymerase, and two fluorogenic probes were evaluated. One fluorogenic probe (cerTAQ-1) was designed to be specific for the nucleotide differences at bases 866 and 875 found in B. cereus. A total of 51 out of 72 B. cereus strains tested positive with the cerTAQ-1 probe, while only 1 out of 5 B. thuringiensis strains tested positive. Sequence analysis of the negative B. cereus strains revealed additional polymorphism found in the cereolysin probe target. A second probe (cerTAQ-2) was designed to account for additional polymorphic sequences found in the cerTAQ-1-negative B. cereus strains. A total of 35 out of 39 B. cereus strains tested positive (including 10 of 14 previously negative strains) with cerTAQ-2, although the assay readout was uniformly lower with this probe than with cerTAQ-1. A PCR assay using cerTAQ-1 was able to detect approximately 58 B. cereus CFU in 1 g of artificially contaminated nonfat dry milk. Forty-three nonfat dry milk samples were tested for the presence of B. cereus with the most-probable-number technique and the fluorogenic PCR assay. Twelve of the 43 samples were contaminated with B. cereus at levels greater than or equal to 43 CFU/g, and all 12 of these samples tested positive with the fluorogenic PCR assay. Of the remaining 31 samples, 12 were B. cereus negative and 19 were contaminated with B. cereus at levels ranging from 3 to 9 CFU/g. All 31 of these samples were negative in the fluorogenic PCR assay. Although not totally inclusive, the PCR-based assay with cerTAQ-1 is able to specifically detect B. cereus in nonfat dry milk.     

Pathogenomic sequence analysis of Bacillus cereus and Bacillus thuringiensis isolates closely related to Bacillus anthracis

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are closely related gram-positive, spore-forming bacteria of the B. cereus sensu lato group. While independently derived strains of B. anthracis reveal conspicuous sequence homogeneity, environmental isolates of B. cereus and B. thuringiensis exhibit extensive genetic diversity. Here we report the sequencing and comparative analysis of the genomes of two members of the B. cereus group, B. thuringiensis 97-27 subsp. konkukian serotype H34, isolated from a necrotic human wound, and B. cereus E33L, which was isolated from a swab of a zebra carcass in Namibia. These two strains, when analyzed by amplified fragment length polymorphism within a collection of over 300 of B. cereus, B. thuringiensis, and B. anthracis isolates, appear closely related to B. anthracis. The B. cereus E33L isolate appears to be the nearest relative to B. anthracis identified thus far. Whole-genome sequencing of B. thuringiensis 97-27and B. cereus E33L was undertaken to identify shared and unique genes among these isolates in comparison to the genomes of pathogenic strains B. anthracis Ames and B. cereus G9241 and nonpathogenic strains B. cereus ATCC 10987 and B. cereus ATCC 14579. Comparison of these genomes revealed differences in terms of virulence, metabolic competence, structural components, and regulatory mechanisms.     

Conjugal transfer between Bacillus thuringiensis and Bacillus cereus strains is not directly correlated with growth of recipient strains

Bacillus thuringiensis and Bacillus cereus belong to the B. cereus species group. The two species share substantial chromosomal similarity and differ mostly in their plasmid content. The phylogenetic relationship between these species remains a matter of debate. There is genetic exchange both within and between these species, and current evidence indicates that insects are a particularly suitable environment for the growth of and genetic exchange between these species. We investigated the conjugation efficiency of B. thuringiensis var. kurstaki KT0 (pHT73-Em^R) as a donor and a B. thuringiensis and several B. cereus strains as recipients; we used one-recipient and two-recipient conjugal transfer systems in vitro (broth and filter) and in Bombyx mori larvae, and assessed multiplication following conjugation between Bacillus strains. The B. thuringiensis KT0 strain did not show preference for genetic exchange with the B. thuringiensis recipient strain over that with the B. cereus recipient strains. However, B. thuringiensis strains germinated and multiplied more efficiently than B. cereus strains in insect larvae and only B. thuringiensis maintained complete spore germination for at least 24 h in B. mori larvae. These findings show that there is no positive association between bacterial multiplication efficiency and conjugation ability in infected insects for the used strains.     

Distribution of Genes Encoding Putative Virulence Factors and Fragment Length Polymorphisms in the vrrA Gene among Brazilian Isolates of Bacillus cereus and Bacillus thuringiensis

One hundred twenty-one strains of the Bacillus cereus complex, of which 80 were isolated from a variety of sources in Brazil, were screened by PCR for the presence of sequences (bceT, hblA, nheBC, plc, sph, and vip3A) encoding putative virulence factors and for polymorphisms in variable-number tandem repeats (VNTR), using a variable region of the vrrA open reading frame as the target. Amplicons were generated from isolates of B. cereus and Bacillus thuringiensis for each of the sequences encoding factors suggested to play a role in infections of mammals. Intriguingly, the majority of these sequences were detected more frequently in Bacillus thuringiensis than in B. cereus. The vip3A sequence, which encodes an insecticidal toxin, was detected exclusively in B. thuringiensis. VNTR analysis demonstrated the presence of five different fragment length categories in both species, with two of these being widely distributed throughout both taxa. In common with data generated from previous studies examining European, Asian, or North American populations, our investigation of Brazilian isolates supports the notion that B. cereus and B. thuringiensis should be considered to represent a single species.     

Influence of Tomato Genotype on Growth of Inoculated and Indigenous Bacteria in the Spermosphere

We previously demonstrated a genetic basis in tomato for support of the growth of a biological control agent, Bacillus cereus UW85, in the spermosphere after seed inoculation (K. P. Smith, J. Handelsman, and R. M. Goodman, Proc. Natl. Acad. Sci. USA 96:4786–4790, 1999). Here we report results of studies examining the host effect on the support of growth of Bacillus and Pseudomonas strains, both inoculated on seeds and recruited from soil, using selected inbred tomato lines from the recombinant inbred line (RIL) population used in our previous study. Two tomato lines, one previously found to support high and the other low growth of B. cereus UW85 in the spermosphere, had similar effects on growth of each of a diverse, worldwide collection of 24 B. cereus strains that were inoculated on seeds and planted in sterilized vermiculite. In contrast, among RILs that differed for support of B. cereus UW85 growth in the spermosphere, we found no difference for support of growth of the biocontrol strains Pseudomonas fluorescens 2-79 or Pseudomonas aureofaciens AB254. Thus, while the host effect on growth extended to all strains of B. cereus examined, it was not exerted on other bacterial species tested. When seeds were inoculated with a marked mutant of B. cereus UW85 and planted in soil, RIL-dependent high and low support of bacterial growth was observed that was similar to results from experiments conducted in sterilized vermiculite. When uninoculated seeds from two of these RILs were planted in soil, changes in population levels of indigenous Bacillus and fluorescent Pseudomonas bacteria differed, as measured over time by culturing and direct microscopy, from growth patterns observed in the inoculation experiments. Neither RIL supported detectable levels of growth of indigenous Bacillus soil bacteria, while the line that supported growth of inoculated B. cereus UW85 supported higher growth of indigenous fluorescent pseudomonads and total bacteria. The vermiculite system used in these experiments was predictive for growth of B. cereus UW85 inoculated on seeds and grown in soil, but the patterns of growth of inoculated strains—both Bacillus and Pseudomonas spp.—did not reflect host genotype effects on indigenous microflora recruited from soil to the spermosphere.     

Isolation and Characterization of Bacillus thuringiensis Strains from Aquatic Environments in Spain

Samples collected from aquatic environments from Spain were analyzed for the occurrence and dipteran toxicity of Bacillus thuringiensis. From a total of 41 samples, 122 isolates were obtained, yielding a B. thuringiensis index of 0.22. Isolates were assigned to 13 different serovars, with serovar thuringiensis (serotype H1) the most frequently found. Toxicity tests carried out revealed that eight isolates (6.6% out of the total) were active against Tipula oleracea larvae. Serological tests assigned these toxic isolates to serovar thuringiensis. The toxicity found in these isolates against the tipulid was approximately seven times lower than that shown by the standard strain B. thuringiensis ser. israelensis IPS-82. Implication of Cry2A protein in toxic activity is hypothesized. Received: 3 December 1999 / Accepted: 5 January 2000     

Comparison of Minisatellite Polymorphisms in the Bacillus cereus Complex: a Simple Assay for Large-Scale Screening and Identification of Strains Most Closely Related to Bacillus anthracis

Polymorphism of five tandem repeats that are monomorphic in Bacillus anthracis was investigated in 230 isolates of the B. cereus group and in 5 sequenced B. cereus genomes in search for markers allowing identification of B. cereus and B. thuringiensis strains most closely related to B. anthracis. Using this multiple-locus variable number of tandem repeat analysis (MLVA), a cluster of 30 strains was selected for further characterization. Eventually, six of these were characterized by multilocus sequence type analysis. One of the strains is only six point mutations (of almost 3,000 bp) away from B. anthracis and was also proposed to be closest to B. anthracis by MLVA analysis. However, this strain remains separated from B. anthracis by a number of significant genetic events observed in B. anthracis, including the loss of the hemolysin activity, the presence of four prophages, and the presence of the two virulence plasmids, pXO1 and pXO2. One particular minisatellite marker provides an efficient assay to identify the subset of B. cereus and B. thuringiensis strains closely related to B. anthracis. Based on these results, a very simple assay is proposed that allows the screening of hundreds of strains from the B. cereus complex, with modest equipment and at a low cost, to eventually fill the gap with B. anthracis and better understand the origin and making of this dangerous pathogen.     

Differentiation of Bacillus anthracis, B. cereus, and B. thuringiensis by Using Pulsed-Field Gel Electrophoresis

A pulsed-field gel electrophoresis (PFGE) method was developed for discriminating Bacillus anthracis from B. cereus and B. thuringiensis. A worldwide collection of 25 B. anthracis isolates showed high-profile homology, and these isolates were unambiguously distinguished from B. cereus and B. thuringiensis isolates by cluster analysis of the whole-genome macrorestriction enzyme digestion patterns generated by NotI.     

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.     

Effect of incubation in natural and autoclaved soil upon potency and viability of Bacillus thuringiensis

Spores and parasporal crystals of a Bacillus thuringiensis var. aizawai H-serotype 7, strain HD137, streptomycin-resistant mutant, were added to normal and autoclaved aliquots of pH 5 soil incubated at 25°C and ?0.10 MPa water availability. Viable B. thuringiensis in soil samples were estimated by dilution-plating on a streptomycin-based medium, and combined spore and crystal insecticidal activity was bioassayed with larvae of Galleria mellonella. Populations of B. thuringiensis in both soil treatments suffered exponential rates of mortality, which were represented by segmented linear regression. Mortality was far greater in natural than autoclaved soil. Potency also fell in both soil treatments. This loss of potency was greater in natural soil, although the rates of potency loss in either soil treatment correlated poorly with the respective mortality rates of the B. thuringiensis populations, as potency losses were not exponential functions. The results suggest that the presence of indigenous microorganisms in natural soil accelerated the rate of mortality and loss of potency of B. thuringiensis.     

Biofilm Formation and Cell Surface Properties among Pathogenic and Nonpathogenic Strains of the Bacillus cereus Group

Biofilm formation by 102 Bacillus cereus and B. thuringiensis strains was determined. Strains isolated from soil or involved in digestive tract infections were efficient biofilm formers, whereas strains isolated from other diseases were poor biofilm formers. Cell surface hydrophobicity, the presence of an S layer, and adhesion to epithelial cells were also examined.The Bacillus cereus group includes B. cereus sensu stricto, B. anthracis, and B. thuringiensis, three genetically close pathogenic species. Based on genetic evidence, it has been suggested that they could represent one species (7). B. cereus sensu stricto is itself an opportunistic human pathogen occasionally found to cause various diseases such as endophthalmitis or periodontitis but is more frequently involved in gastrointestinal diseases with diarrheal or emetic syndromes (4, 12). Emetic syndromes result from the presence of cereulide, a heat-stable toxin produced in food before ingestion, whereas diarrheal syndromes require survival of the bacterium in the host digestive tract. B. thuringiensis is an insect pathogen, and B. anthracis causes anthrax, a lethal human disease.The persistent contamination of industrial food processing systems by B. cereus (12) may facilitate its involvement in gastroenteritis. This persistence is due to spores, which may survive pasteurization, heating, and gamma-ray irradiation (9, 13), and to biofilms, which have been shown to be highly resistant to cleaning procedures (18). Biofilms are also suspected to be involved in bacterial pathogenicity, as they may form on host epithelia (15).In this study, we wanted to test whether biofilm formation by species of the B. cereus group could be connected to the pathogenicity of the bacterium. For this purpose, we screened a collection of 102 pathogenic (diarrheal, emetic, and oral diseases) and nonpathogenic strains of B. cereus and B. thuringiensis for their capability to form biofilms. As adhesion to inert or living surfaces is a prerequisite for biofilm formation, we have investigated relationships within our collection of strains between biofilm formation and cell surface hydrophobicity, the presence of an S-layer, or adhesion to epithelial cells.     

Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereus and Bacillus thuringiensis Soil Isolates

We examined 154 Norwegian B. cereus and B. thuringiensis soil isolates (collected from five different locations), 8 B. cereus and 2 B. thuringiensis reference strains, and 2 Bacillus anthracis strains by using fluorescent amplified fragment length polymorphism (AFLP). We employed a novel fragment identification approach based on a hierarchical agglomerative clustering routine that identifies fragments in an automated fashion. No method is free of error, and we identified the major sources so that experiments can be designed to minimize its effect. Phylogenetic analysis of the fluorescent AFLP results reveals five genetic groups in these group 1 bacilli. The ATCC reference strains were restricted to two of the genetic groups, clearly not representative of the diversity in these bacteria. Both B. anthracis strains analyzed were closely related and affiliated with a B. cereus milk isolate (ATCC 4342) and a B. cereus human pathogenic strain (periodontitis). Across the entire study, pathogenic strains, including B. anthracis, were more closely related to one another than to the environmental isolates. Eight strains representing the five distinct phylogenetic clusters were further analyzed by comparison of their 16S rRNA gene sequences to confirm the phylogenetic status of these groups. This analysis was consistent with the AFLP analysis, although of much lower resolution. The innovation of automated genotype analysis by using a replicated and statistical approach to fragment identification will allow very large sample analyses in the future.