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.     

Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites

AIMS: To evaluate the possibility for discrimination of Bacillus cereus and B. thuringiensis using 16S rRNA and gyrB gene based PCR methods, and to obtain the sequences of the primer annealing sites so that the PCR results may be explained. METHODS AND RESULTS: Based on the sequence difference in the variable region (V1) of 16S rRNA and in the gyrB gene between B. cereus and B. thuringiensis, PCR primers specific to these Bacillus spp. were designed. When these primers were used to discriminate B. cereus and B. thuringiensis, six of 82 B. cereus strains were identified as B. thuringiensis while 67 of 73 B. thuringiensis strains were identified as B. cereus. Sequence analysis of the primer annealing sites showed that there is no clear-cut difference in the V1 region of 16S rRNA, and in the gyrB gene, between B. cereus and B. thuringiensis strains. CONCLUSIONS: Although 16S rDNA based probes and gyrB gene based PCR primers have been suggested for the discrimination of B. cereus and B. thuringiensis strains, when a large number of Bacillus strains was tested, results showed that discrimination between B. cereus and B. thuringiensis is difficult. Therefore, to distinguish B. thuringiensis from B. cereus, a single feature, such as the presence of a parasporal crystal protein or cry gene, may sometimes be reliable. SIGNIFICANCE AND IMPACT OF THE STUDY: Discrimination between B. cereus and B. thuringiensis is a challenging debate to which this paper makes a contribution.     

1株产聚-β-羟基丁酸芽胞杆菌的分离、筛选与鉴定

从浙江省金华市花生地土样中筛选、分离纯化得到1株产聚-β-羟基丁酸编号为PX-95的芽胞杆菌,PHB产量为135.81 mg/L。根据形态特征、生理生化特征初步鉴定为芽胞杆菌属蜡样芽胞杆菌群,16S rDNA序列分析显示PX-95菌株与苏云金芽胞杆菌以及蜡样芽胞杆菌均具有高度同源性,最后采用扩增gyrB（DNA促旋酶B亚单位）基因的方法将其鉴定为苏云金芽胞杆菌（Bacillus thuringiensis）。     

A DNA microarray facilitates the diagnosis of Bacillus anthracis in environmental samples

Aims:  In order to improve the diagnosis of Bacillus anthracis in environmental samples, we established a DNA microarray based on the ArrayTube technology of Clondiag.
Methods and Results:  Total DNA of a bacterial colony is randomly biotinylated and hybridized to the array. The probes on the array target the virulence genes, the genomic marker gene rpoB , as well as the selective 16S rDNA sequence regions of B. anthracis , of the Bacillus cereus group and of Bacillus subtilis . Eight B. anthracis reference strains were tested and correctly identified. Among the analysed environmental Bacillus isolates, no virulent B. anthracis strain was detected.
Conclusions:  This array clearly differentiates B. anthracis from members of the B. cereus group and other Bacillus species in environmental samples by chromosomal ( rpoB ) and plasmid markers. Additionally, recognition of B. cereus strains harbouring the toxin genes or atypical B. anthracis strains that have lost the virulence plasmids is feasible.
Significance and Impact of the Study:  The array is applicable to the complex diagnostics for B. anthracis detection in environmental samples. Because of low costs, high security and easy handling, the microarray is applicable to routine diagnostics.     

Polymerase chain reaction assay for the detection of Bacillus cereus group cells

Recent investigations have shown that members of the Bacillus cereus group carry genes which have the potential to cause gastrointestinal and somatic diseases. Although most cases of diseases caused by the B. cereus group bacteria are relatively mild, it is desirable to be able to detect members of the B. cereus group in food and in the environment. Using 16S rDNA as target, a PCR assay for the detection of B. cereus group cells has been developed. Primers specific for the 16S rDNA of the B. cereus group bacteria were selected and used in combination with consensus primers for 16S rDNA as internal PCR procedure control. The PCR procedure was optimized with respect to annealing temperature. When DNA from the B. cereus group bacteria was present, the PCR assay yielded a B. cereus specific fragment, while when non-B. cereus prokaryotic DNA was present, the consensus 16S rDNA primers directed synthesis of the PCR products. The PCR analyses with DNA from a number of non-B. cereus confirmed the specificity of the PCR assay.     

MALDI-TOFMS compared with other polyphasic taxonomy approaches for the identification and classification of Bacillus pumilus spores

To verify the efficacy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) protein profiling for identifying and differentiating bacterial species, several strains of Bacillus pumilus were examined in a thorough taxonomic study incorporating a polyphasic approach. Sixteen isolates of putative B. pumilus isolated from spacecraft assembly facilities, the Mars Odyssey spacecraft, and the International Space Station, were characterized for their biochemical and molecular profiles using the Biolog system, DNA techniques, and MALDI-TOFMS protein profiling. MALDI-TOFMS protein profiling was more accurate than Biolog metabolic profiling, more discriminating than 16S rDNA sequence analysis, and complemented the results of gyrB sequence analysis and DNA-DNA hybridization for the identification of the B. pumilus spores. This is the first report whereby MALDI-TOFMS generated protein profiles from a set of microbes is compared directly with DNA-DNA hybridization yielding a positive correlation. Unique, cluster-specific biomarker peaks have been identified in the spores of the B. pumilus examined in this study. MALDI-TOFMS protein profiling is a rapid and simple analysis and has been demonstrated as a useful taxonomic tool for differentiating spores of the genus Bacillus. For practical purposes, it would be ideal (and necessary) to have a publicly available, standardized MALDI profile database, to facilitate the use of the technique as a diagnostic method to differentiate bacterial species.     

Correlation of 16S ribosomal DNA signature sequences with temperature-dependent growth rates of mesophilic and psychrotolerant strains of the Bacillus cereus group

Sequences of the 16S ribosomal DNA (rDNA) from psychrotolerant and mesophilic strains of the Bacillus cereus group revealed signatures which were specific for these two thermal groups of bacteria. Further analysis of the genomic DNA from a wide range of food and soil isolates showed that B. cereus group strains have between 6 and 10 copies of 16S rDNA. Moreover, a number of these environmental strains have both rDNA operons with psychrotolerant signatures and rDNA operons with mesophilic signatures. The ability of these isolates to grow at low temperatures correlates with the prevalence of rDNA operons with psychrotolerant signatures, indicating specific nucleotides within the 16S rRNA to play a role in psychrotolerance.     

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.     

具细菌群体感应抑制活性海洋细菌的筛选鉴定

目的:从海洋环境中筛选对细菌群体感应有抑制作用的活性菌株,为以致病菌群体感应系统为靶点的新型疗法提供新的药用资源。方法:从海水中分离纯化细菌菌株,采用根癌农杆菌平板筛选模型筛选细菌群体感应抑制活性细菌,对筛选出的海洋细菌进行生理生化和16S rDNA序列测定,根据《伯杰氏手册》进行菌种分类鉴定。结果:从217株海洋细菌中筛选出1株能显著抑制细菌群体感应效应的海洋细菌Y2,该海洋细菌具有蜡样芽孢杆菌(Bacillus cereus)的典型特征,其16S rDNA序列与GenBank中蜡样芽孢杆菌16S rDNA的部分序列有100%的同源性。结论:海洋环境中也存在具有抑制细菌群体感应活性的微生物。     

Development of PCR primers specific for the amplification and direct sequencing of gyrB genes from microbacteria, order Actinomycetales

PCR primer sets were developed for the specific amplification and sequence analyses encoding the gyrase subunit B (gyrB) of members of the family Microbacteriaceae, class Actinobacteria. The family contains species highly related by 16S rRNA gene sequence analyses. In order to test if the gene sequence analysis of gyrB is appropriate to discriminate between closely related species, we evaluate the 16S rRNA gene phylogeny of its members. As the published universal primer set for gyrB failed to amplify the responding gene of the majority of the 80 type strains of the family, three new primer sets were identified that generated fragments with a composite sequence length of about 900 nt. However, the amplification of all three fragments was successful only in 25% of the 80 type strains. In this study, the substitution frequencies in genes encoding gyrase and 16S rDNA were compared for 10 strains of nine genera. The frequency of gyrB nucleotide substitution is significantly higher than that of the 16S rDNA, and no linear correlation exists between the similarities of both molecules among members of the Microbacteriaceae. The phylogenetic analyses using the gyrB sequences provide higher resolution than using 16S rDNA sequences and seem able to discriminate between closely related 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.     

Fluorescent heteroduplex assay for monitoring Bacillus anthracis and close relatives in environmental samples

A fluorescent heteroduplex method was developed to assess the presence of 16S rRNA gene (rDNA) sequences from Bacillus anthracis and close relatives in PCR-amplified 16S rDNA sequence mixtures from environmental samples. The method uses a single-stranded, fluorescent DNA probe, 464 nucleotides in length, derived from a B. anthracis 16S rRNA gene. The probe contains a unique, engineered deletion such that all probe-target duplexes are heteroduplexes with an unpaired G at position 343 (deltaG343). Heteroduplex profiles of sequences >/=85% similar to the probe were produced using an ABI 377 sequencer in less than 3 h. The method divides strains of the Bacillus cereus-Bacillus thuringiensis-B. anthracis group into two subgroups. Each subgroup is defined by a specific 16S rRNA gene sequence type. Sequence type A, containing one mismatch with the probe, occurs in B. anthracis and a small number of closely related clonal lineages represented mostly by food-borne pathogenic isolates of B. cereus and B. thuringiensis. Sequence type B, containing two mismatches with the probe, is found in the majority of B. cereus and B. thuringiensis strains examined to date. Sequence types A and B, when hybridized to the probe, generate two easily differentiated heteroduplexes. Thus, from heteroduplex profiles, the presence of B. cereus-B. thuringiensis-B. anthracis subgroups in environmental samples can be inferred unambiguously. The results show that fluorescent heteroduplex analysis is an effective profiling technique for detection and differentiation of sequences representing small phylogenetic or functional groups in environmental samples.     

The phylogenetic diversity of thermophilic members of the genus Bacillus as revealed by 16S rDNA analysis

Abstract Sixteen thermophilic strains of the genus Bacillus , representing eight validly described and six invalidly described species, as well as one unassigned strain, were investigated by comparative 16S rDNA analyses and the sequences compared to the existing database for the genera Bacillus and Alicyclobacillus . The majority of strains were found to cluster in two groups represented by B. stearothermophilus and B. pallidus. Bacillus smithii, B. thermocloacae , and B. thermoruber are phylogenetically well separated and cluster within the radiation of mesophilic bacilli. The as yet undescribed taxon 'B. flavothermus' warrants species status. B. schlegelii and B. tusciae group peripherally with members of Alicyclobacillus and may be reclassified when more phenotypic data support their phylogenetic position.     

Homoduplex and heteroduplex polymorphisms of the amplified ribosomal 16S-23S internal transcribed spacers describe genetic relationships in the "Bacillus cereus group"

Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis, and Bacillus weihenstephanensis are closely related in phenotype and genotype, and their genetic relationship is still open to debate. The present work uses amplified 16S-23S internal transcribed spacers (ITS) to discriminate between the strains and species and to describe the genetic relationships within the "B. cereus group," advantage being taken of homoduplex-heteroduplex polymorphisms (HHP) resolved by polyacrylamide gel electrophoresis and silver staining. One hundred forty-one strains belonging to the six species were investigated, and 73 ITS-HHP pattern types were distinguished by MDE, a polyacrylamide matrix specifically designed to resolve heteroduplex and single-strand conformation polymorphisms. The discriminating bands were confirmed as ITS by Southern hybridization, and the homoduplex or heteroduplex nature was identified by single-stranded DNA mung bean nuclease digestion. Several of the ITS-HHP types corresponded to specific phenotypes such as B. anthracis or serotypes of B. thuringiensis. Unweighted pair group method arithmetic average cluster analysis revealed two main groups. One included B. mycoides, B. weihenstephanensis, and B. pseudomycoides. The second included B. cereus and B. thuringiensis, B. anthracis appeared as a lineage of B. cereus.     

黑水虻幼虫肠道和体表产酶细菌的分离和鉴定

从野外收集和室内饲养的黑水虻Hermetia illucens L.幼虫体表和肠道分离出同时具蛋白质和有机磷分解能力的细菌10株,其中编号为T2、T4、T6、T7和T8的菌株分离自体表,编号为S14、S15、S16、S19和S20的菌株分离自肠道。克隆细菌的16S rDNA和DNA促旋酶B亚基编码基因gyrB对10株细菌进行了鉴定。通过16S rDNA序列确定10株菌为芽孢杆菌属,根据gyrB基因以及BLAST结果构建系统发育树,将10株细菌鉴定为枯草芽孢杆菌。然后将10株菌的gyrB基因以B.subtilis subsp.subtilisstr.168和模式菌株B.subtilis subsp.subtilis BCRC10255相应基因序列为参照构建系统发育树,分析10株菌在种的水平上的亲缘关系,发现10株菌存在亲缘关系差异,没有重复菌株。     

Phylogenetic Analysis of the Genus Bifidobacterium and Related Genera Based on 16S rDNA Sequences

The 16S rRNA gene sequences were determined for type strains of 21 Bifidobacterium species. A phylogenetic tree was constructed using the determined sequences and sequences from DNA databases, which contain the sequences of 11 type strains of Bifidobacterium species and 11 strains of related genera. All species of the genus Bifidobacterium and Gardnerella vaginalis ATCC 14018 belonged to a cluster phylogenetically distinct from the other genera. The cluster was divided into two subclusters: subcluster 1 composed of most species of Bifidobacterium and G. vaginalis, and subcluster 2 consisting of two species, B. denticolens and B. inopinatum; both of which were isolated from human dental caries. In the genus Bifidobacterium, four groups of species are known to be moderately to highly related by DNA-DNA hybridization. The four groups of species exhibited more than 99% similarity among their 16S rDNA sequences within each group. These results indicated that species with around 99% or more similarity in their 16S rDNA sequences should be confirmed for species identities.     

Phylogenetic study and identification of Vibrio splendidus-related strains based on gyrB gene sequences

Different strains related to Vibrio splendidus have been associated with infection of aquatic animals. An epidemiological study of V. splendidus strains associated with Crassostrea gigas mortalities demonstrated genetic diversity within this group and suggested its polyphyletic nature. Recently 4 species, V. lentus, V. chagasii, V. pomeroyi and V. kanaloae, phenotypically related to V. splendidus, have been described, although biochemical methods do not clearly discriminate species within this group. Here, we propose a polyphasic approach to investigate their taxonomic relationships. Phylogenetic analysis of V. splendidus-related strains was carried out using the nucleotide sequences of 16S ribosomal DNA (16S rDNA) and gyrase B subunit (gyrB) genes. Species delineation based on 16S rDNA-sequencing is limited because of divergence between cistrons, roughly equivalent to divergence between strains. Despite a high level of sequence similarity, strains were separated into 2 clades. In the phylogenetic tree constructed on the basis of gyrB gene sequences, strains were separated into 5 independent clusters containing V. splendidus, V. lentus, V. chagasii-type strains and a putative new genomic species. This phylogenetic grouping was almost congruent with that based on DNA-DNA hybridisation analysis. V. pomeroyi, V. kanaloae and V. tasmaniensis-type strains clustered together in a fifth clade. The gyrB gene-sequencing approach is discussed as an alternative for investigating the taxonomy of Vibrio species.     

A cloned 23S rRNA gene fragment of Bacillus subtilis and its use as a hybridization probe of conserved character

Abstract A subclone of plasmid p14B8 containing the major part of a 23S rRNA gene of Bacillus subtilis was constructed and designated pJK1. Labeled plasmid pJK1 could be used as a DNA probe with conserved gene sequences. DNA-DNA hybridization experiments between filter-bound DNA from various bacteria and labeled pJK1 showed a good correlation between oligonucleotide sequence analysis of 16S rRNA and DNA homology values. Application of suboptimal or stringent hybridization conditions and an additional short incubation under the same conditions following hybridization yielded the best data for differentiating organisms related to B. subtilis from less or non-related bacteria.     

Climatic influence on mesophilic Bacillus cereus and psychrotolerant Bacillus weihenstephanensis populations in tropical, temperate and alpine soil

Bacillus weihenstephanensis strains are psychrotolerant and grow from below 7°C to 38°C. Closely related mesophilic Bacillus cereus strains can grow from above 7°C to 46°C. We classified 1060 B. cereus group isolates from different soil samples with respect to their psychrotolerant and mesophilic genotypes by polymerase chain reaction (PCR) targeting of specific 16S rDNA and cold shock protein A gene signatures. In parallel, growth tests at 7°C were carried out to determine the thermal phenotype. The geographic distribution of psychrotolerant and mesophilic isolates was found to depend significantly on the prevalent annual average temperature. In one tropical, one temperate and two alpine habitats, the proportion of psychrotolerant cspA genotypes was found to be 0%, 45% and 86% and 98%, respectively, with the corresponding annual average temperatures being 28°C, 7°C, 4°C and 1°C. In the tropical habitat, only the mesophilic B. cereus was found, characterized by correspondence of thermal genotype and phenotype. In the alpine habitat, almost only the psychrotolerant B. weihenstephanensis was isolated. In the temperate habitat, mesophilic B. cereus and psychrotolerant B. weihenstephanensis as well as 'intermediate thermal types' occurred, the latter having opposite thermal genotypes and phenotypes or opposing sets of thermal DNA signatures, characterized by the coexistence of mesophilic and psychrotolerant 16S rDNA operon copies within a single isolate. Both sugar utilization and DNA fingerprinting patterns revealed a high, probably non-clonal microsite diversity within the population of the temperate habitat. We interpret our observations in terms of a temperature-dependent selection regime, acting on recombining B. cereus / B. weihenstephanensis populations in soil.