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ABSTRACT: 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.
Annals of Microbiology 04/2012; 57(1):21-27. · 0.69 Impact Factor
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ABSTRACT: This study aimed to evaluate the plant growth promoting (PGP) potential ofBacillus thuringiensis. In this context, several genetic determinants of factors implicated in PGP potential were investigated by polymerase chain
reaction (PCR) in 16B. thuringiensis strains of different origin and belonging to different subspecies. PCR screening was performed on acid phosphatase, phytase,
siderophore biosynthesis protein, 1-aminocyclopropane-1-carboxylate (ACC) deaminase and indolpyruvate decarboxylase (ipdC). Production of indol acetic acid (IAA)-like compounds and of ACC deaminase, and capability of solubilising mineral phosphate
were investigated by phenotypic tests. All the strains were PCR positive for the presence of the siderophore biosynthesis
protein, ACC deaminase and acid phosphatase genes. Five and seven strains gave an amplicon with the expected length for the
phytase andipdC genes respectively. All the strains produced IAA compounds and seven had a high capacity to solubilise inorganic phosphorous.
Qualitative phenotypic test for ACC deaminase activity showed that seven strains are able to grow on salt minimal medium containing
ACC as sole nitrogen source, indicating the expression of theaccd genes. Our screening results in thirteen strains having more than one PGP trait and showed thatB. thuringiensis harbours and expresses several PGP determinants that could be very interesting in field application to enhance the plant
growth. To our knowledge, this is the first report on the multiple plant growth promoting potential ofB. thuringiensis.
Key words
Bacillus thuringiensis
-PGPR-IAA-ACCD
Annals of Microbiology 04/2012; 58(1):47-52. · 0.69 Impact Factor
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ABSTRACT: The entomopathogenic bacteriumBacillus thuringiensis is widely used for the control of many agricultural insect pests and vectors of human diseases. Several studies reported
also on its antibacterial and antifungal activities. However, to our knowledge there were no studies dealing with its capacity
to act as a plant growth promoting bacterium. This review surveys the potential ofB. thuringiensis as a polyvalent biocontrol agent, a biostimulator and biofertiliser bacterium that could promote the plant growth. Also,
discussed is the safety ofB. thuringiensis as a bacterium phylogenetically related toBacillus cereus the opportunistic human pathogen andBacillus anthracis, the etiological agent of anthrax.
Annals of Microbiology 04/2012; 57(4):481-494. · 0.69 Impact Factor
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ABSTRACT: Horizontal gene transfer through natural transformation of members of the microbiota of the lower gastrointestinal tract (GIT) of mammals has not yet been described. Insufficient DNA sequence similarity for homologous recombination to occur has been identified as the major barrier to interspecies transfer of chromosomal DNA in bacteria. In this study we determined if regions of high DNA similarity between the genomes of the indigenous bacteria in the GIT of rats and feed introduced DNA could lead to homologous recombination and acquisition of antibiotic resistance genes.
Plasmid DNA with two resistance genes (nptI and aadA) and regions of high DNA similarity to 16S rRNA and 23S rRNA genes present in a broad range of bacterial species present in the GIT, were constructed and added to standard rat feed. Six rats, with a normal microbiota, were fed DNA containing pellets daily over four days before sampling of the microbiota from the different GI compartments (stomach, small intestine, cecum and colon). In addition, two rats were included as negative controls. Antibiotic resistant colonies growing on selective media were screened for recombination with feed introduced DNA by PCR targeting unique sites in the putatively recombined regions. No transformants were identified among 441 tested isolates.
The analyses showed that extensive ingestion of DNA (100 μg plasmid) per day did not lead to increased proportions of kanamycin resistant bacteria, nor did it produce detectable transformants among the aerobic microbiota examined for 6 rats (detection limit < 1 transformant per 1,1 × 10(8) cultured bacteria). The key methodological challenges to HGT detection in animal feedings trials are identified and discussed. This study is consistent with other studies suggesting natural transformation is not detectable in the GIT of mammals.
BMC Research Notes 04/2012; 5:170.
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ABSTRACT: The fate of dietary DNA in the gastrointestinal tract (GIT) of animals has gained renewed interest after the commercial introduction of genetically modified organisms (GMO). Among the concerns regarding GM food, are the possible consequences of horizontal gene transfer (HGT) of recombinant dietary DNA to bacteria or animal cells. The exposure of the GIT to dietary DNA is related to the extent of food processing, food composition, and to the level of intake. Animal feeding studies have demonstrated that a minor amount of fragmented dietary DNA may resist the digestive process. Mammals have been shown to take up dietary DNA from the GIT, but stable integration and expression of internalized DNA has not been demonstrated. Despite the ability of several bacterial species to acquire external DNA by natural transformation, in vivo transfer of dietary DNA to bacteria in the intestine has not been detected in the few experimental studies conducted so far. However, major methodological limitations and knowledge gaps of the mechanistic aspects of HGT calls for methodological improvements and further studies to understand the fate of various types of dietary DNA in the GIT.
Critical reviews in food science and nutrition 02/2012; 52(2):142-61. · 3.73 Impact Factor
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ABSTRACT: 'Candidatus Liberibacter spp.' cause serious plant diseases. 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus' and 'Ca. L. africanus' are the aetiological agents of citrus greening (Huanglongbing) in Asia, America and Africa. 'Candidatus Liberibacter solanacearum' causes diseases in Solanaceae in America and New Zealand. All four species are vectored by psyllid insects of different genera. Here, we show that the pear psyllid pest Cacopsylla pyri (L.) hosts a novel liberibacter species that we named 'Ca. Liberibacter europaeus'. It can bloom to high titres in the psyllid host, with more than 10(9) 16S rRNA gene copies per individual. Fluorescent in situ hybridization experiments showed that 'Ca. L. europaeus' is present in the host midgut lumen, salivary glands and Malpighian tubules. 'Candidatus L. europaeus' has a relatively high prevalence (> 51%) in C. pyri from different areas in the Piedmont and Valle d'Aosta regions in Italy and can be transmitted to pear plants in experimental transmission trials. However, even though high titres of the bacterium (more than 10(8) 16S rRNA gene copies g(-1) of pear plant tissue) could be detected, in the pear tissues no specific disease symptoms could be observed in the infected plants over a 6-month period. Despite liberibacters representing potential quarantine organisms, 'Ca. L. europaeus', first described in Italy and Europe, apparently behaves as an endophyte rather than a pathogen.
Environmental Microbiology 10/2010; 13(2):414-26. · 5.84 Impact Factor
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Elena Crotti,
Claudia Damiani,
Massimo Pajoro,
Elena Gonella,
Aurora Rizzi,
Irene Ricci,
Ilaria Negri,
Patrizia Scuppa,
Paolo Rossi,
Patrizia Ballarini, Noura Raddadi,
Massimo Marzorati,
Luciano Sacchi,
Emanuela Clementi,
Marco Genchi,
Mauro Mandrioli,
Claudio Bandi,
Guido Favia,
Alberto Alma,
Daniele Daffonchio
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ABSTRACT: Bacterial symbionts of insects have been proposed for blocking transmission of vector-borne pathogens. However, in many vector models the ecology of symbionts and their capability of cross-colonizing different hosts, an important feature in the symbiotic control approach, is poorly known. Here we show that the acetic acid bacterium Asaia, previously found in the malaria mosquito vector Anopheles stephensi, is also present in, and capable of cross-colonizing other sugar-feeding insects of phylogenetically distant genera and orders. PCR, real-time PCR and in situ hybridization experiments showed Asaia in the body of the mosquito Aedes aegypti and the leafhopper Scaphoideus titanus, vectors of human viruses and a grapevine phytoplasma respectively. Cross-colonization patterns of the body of Ae. aegypti, An. stephensi and S. titanus have been documented with Asaia strains isolated from An. stephensi or Ae. aegypti, and labelled with plasmid- or chromosome-encoded fluorescent proteins (Gfp and DsRed respectively). Fluorescence and confocal microscopy showed that Asaia, administered with the sugar meal, efficiently colonized guts, male and female reproductive systems and the salivary glands. The ability in cross-colonizing insects of phylogenetically distant orders indicated that Asaia adopts body invasion mechanisms independent from host-specific biological characteristics. This versatility is an important property for the development of symbiont-based control of different vector-borne diseases.
Environmental Microbiology 10/2009; 11(12):3252-64. · 5.84 Impact Factor
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ABSTRACT: Members of the genus Bacillus and related genera are ubiquitous in nature. However, Bacillus species isolated from marine sediments have attracted less interest respect to their terrestrial relatives. Here, we report the phylogenetic diversity of a collection of 96 Bacilli, isolated from 17 distinct stations of 5 oceanographic campaigns. The diversity was analysed by phenotypic and molecular approaches based on the amplified rDNA restriction analysis (ARDRA), amplification of the internal transcribed spacers (ITS-PCR) and on 16S rRNA sequencing. Intra-specific polymorphism was efficiently detected by biochemical analysis and ARDRA while results of ITS-PCR were in agreement with 16S rRNA sequencing. The identification results assigned 68% of the isolates to the species B. subtilis, B. licheniformis, B. pumilus and B. cereus. Phylogenetic analysis allowed the separation of 9 isolates in a clade that may represent a group of obligate marine Bacillus since they clustered with B. firmus, B. foraminis and marine isolates with metal oxidation and bioaccumulation capabilities. The remaining isolates showed a close affiliation to the genera Virgibacillus, Gracilibacillus and Paenibacillus. The widespread of Bacilli and their high diversity level observed in this work point out the need of more extensive studies to understand their distribution and ecology in deep-sea environments.
Journal of Basic Microbiology 04/2009; 49 Suppl 1:S13-23. · 1.27 Impact Factor
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ABSTRACT: Sixteen Bacillus thuringiensis (Bt) strains were screened for their anti-insect, antibacterial and antifungal determinants by phenotypic tests and PCR targeting major insecticidal proteins and complements, chitinases, lactonases, beta-1,3-glucanases and zwittermicinA. Six strains had genes of at least two major insecticidal toxins and of insecticidal complements. With regard to fungal biocontrol, all the strains inhibited Fusarium oxysporum and Aspergillus flavus growth and four strains had all or most of the antifungal determinants examined, with strain Bt HD932 showing the widest antifungal activity spectrum. Autolysins, bacteriocin and AHL-lactonases were produced by all or most of the tested strains with different activity spectra including pathogens like Listeria monocytogenes. Safety evaluation was carried out via PCR by screening the B. cereus psychrotolerance-related genes, toxin genes and the virulence pleiotropic regulator plcR. Diarrheal enterotoxins and other toxin genes were widespread among the collection with strains Bt HD9 and H45 lacking psychrotolerance-related genes, while five strains were positive. Only three strains (BMG1.7, H172, H156) resulted positive with primer sets targeting partial or complete plcR gene. By Vero Cell Assays, Bt HD868 followed by Bt HD9 were shown to be the safest strains. These polyvalent and safe Bt strains could be very promising in field application.
Journal of Basic Microbiology 12/2008; 49(3):293-303. · 1.27 Impact Factor
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ABSTRACT: Forty three strains were isolated from knots induced by Pseudomonas savastanoi in different olive cultivars. All the selected bacteria were shown to produce variable amounts of the plant growth hormone indole-3-acetic acid (IAA). Amplification of the intergenic transcribed spacers (ITS) between 16S and 23S rDNA genes, allowed the clustering of the isolates into seven distinct groups. All isolates from ITS group 1 were positive to the Pseudomonas savastanoi pv. savastanoi specific iaa L gene as shown by PCR. Partial sequencing of 16S rDNA gene confirmed the identity of these isolates to Pseudomonas savastanoi strains and allowed to tentatively assign the other isolates from the remaining ITS groups to Pantoea oleae/agglomerans, Burkholderia cepacia, Pseudomonas putida, Stenotrophomonas maltophilia and Hafnia alvei. Identification of endophytic knot-derived isolates revealed association of various saprophytic and putative human pathogenic bacteria with P. savastanoi pv. savastanoi in knot environment of olive infected trees.
Journal of Basic Microbiology 09/2008; 48(5):370-7. · 1.27 Impact Factor
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ABSTRACT: A new bacteriocin produced by Bacillus thuringiensis subsp. entomocidus was identified. The antibacterial activity termed entomocin 110 was produced starting at mid-logarithmic growth phase, reaching its maximum at the early and during stationary phase. The bacteriocin obtained from culture supernatant was inhibitory to several Gram-positive bacteria including Listeria monocytogenes, Paenibacillus larvae and other Bacillus species. Entomocin 110 was shown to be heat stable and resistant to pH variation and to organic solvents. The inhibitory activity was totally lost after proteinase K treatment, thereby revealing its proteinaceous nature. The mode of action of entomocin 110 was bactericidal and bacteriolytic. Upon partial purification with ammonium sulphate precipitation followed by butanol extraction, an active peptide with an apparent molecular weight of 4.8 kDa was identified. Cross inhibition tests with bacteriocin producer strains and plasmid profiles indicated that entomocin 110 is a new bacteriocin, which genetic determinants are probably harbored by the chromosome.
Microbiological Research 02/2008; 163(6):684-92. · 2.31 Impact Factor
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Elena CROTTI,
Massimo PAJORO,
Claudia DAMIANI,
Irene RICCI,
Ilaria NEGRI,
Aurora RIZZI,
Ema- nuela CLEMENTI, Noura RADDADI,
Patrizia SCUPPA,
Massimo MARZORATI,
Luciano PASQUALINI,
Claudio BANDI,
Luciano SACCHI,
Guido FAVIA,
Alberto ALMA,
Daniele DAFFONCHIO
01/2008;
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ABSTRACT: A RSI-PCR assay was developed for the detection of a Bacillus anthracis-specific nonsense mutation in the plcR gene. The assay specificity was tested using 170 Bacillus spp. strains including 47 strains of B. anthracis. The plcR RSI-PCR distinguished Bacillus cereus group strains closely related to B. anthracis from the anthrax agent. The assay was found to be a robust, simple and cost effective tool for B. anthracis identification. In contrast to previously developed real time PCR-based methods, the RSI-PCR needs basic molecular biology equipment only, and thus may be easily introduced in developing countries, where anthrax is endemic.
FEMS Microbiology Letters 08/2007; 272(1):55-9. · 2.04 Impact Factor
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Guido Favia,
Irene Ricci,
Claudia Damiani, Noura Raddadi,
Elena Crotti,
Massimo Marzorati,
Aurora Rizzi,
Roberta Urso,
Lorenzo Brusetti,
Sara Borin, [......],
Marco Genchi,
Silvia Corona,
Ilaria Negri,
Giulio Grandi,
Alberto Alma,
Laura Kramer,
Fulvio Esposito,
Claudio Bandi,
Luciano Sacchi,
Daniele Daffonchio
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ABSTRACT: Here, we show that an alpha-proteobacterium of the genus Asaia is stably associated with larvae and adults of Anopheles stephensi, an important mosquito vector of Plasmodium vivax, a main malaria agent in Asia. Asaia bacteria dominate mosquito-associated microbiota, as shown by 16S rRNA gene abundance, quantitative PCR, transmission electron microscopy and in situ-hybridization of 16S rRNA genes. In adult mosquitoes, Asaia sp. is present in high population density in the female gut and in the male reproductive tract. Asaia sp. from An. stephensi has been cultured in cell-free media and then transformed with foreign DNA. A green fluorescent protein-tagged Asaia sp. strain effectively lodged in the female gut and salivary glands, sites that are crucial for Plasmodium sp. development and transmission. The larval gut and the male reproductive system were also colonized by the transformed Asaia sp. strain. As an efficient inducible colonizer of mosquitoes that transmit Plasmodium sp., Asaia sp. may be a candidate for malaria control.
Proceedings of the National Academy of Sciences 06/2007; 104(21):9047-51. · 9.68 Impact Factor
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ABSTRACT: The genomic diversity and relationship among 56 Bacillus thuringiensis and Bacillus cereus type strains were investigated by multi-REP-PCR fingerprinting consisting of three PCR reactions targeting the enterobacterial ERIC1 and ERIC2 and the streptococcal BOXA1R consensus sequences. A total of 113 polymorphic bands were generated in the REP-PCR profiles that allowed tracing of a single dendrogram with three major groups. Bacillus cereus strains clustered together in the A and B groups. Most of the B. thuringiensis strains clustered in group C, which included groups of serovars with a within-group similarity higher than 40% as follows: darmstadiensis, israelensis, and morrisoni; aizawai, kenyae, pakistani, and thompsoni; canadensis, entomocidus, galleriae, kurstaki, and tolworthi; alesti, dendrolimus, and kurstaki; and finitimus, sotto, and thuringiensis. Multi-REP-PCR fingerprinting clustered B. thuringiensis serovars in agreement with previously developed multilocus sequence typing schemes, indicating that it represents a rapid shortcut for addressing the genetic relationship of unknown strains with the major known serovars.
Canadian Journal of Microbiology 04/2007; 53(3):343-50. · 1.36 Impact Factor
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Maya Merabishvili,
Merab Natidze,
Sergo Rigvava,
Lorenzo Brusetti, Noura Raddadi,
Sara Borin,
Nina Chanishvili,
Marina Tediashvili,
Richard Sharp,
Maurizio Barbeschi,
Paolo Visca,
Daniele Daffonchio
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ABSTRACT: Despite the increased number of anthrax outbreaks in Georgia and the other Caucasian republics of the former Soviet Union, no data are available on the diversity of the Bacillus anthracis strains involved. There is also little data available on strains from the former Soviet Union, including the strains previously used for vaccine preparation. In this study we used eight-locus variable-number tandem repeat analyses to genotype 18 strains isolated from infected animals and humans at different sites across Georgia, where anthrax outbreaks have occurred in the last 10 years, and 5 strains widely used for preparation of human and veterinary vaccines in the former Soviet Union. Three different genotypes affiliated with the A3.a cluster were detected for the Georgian isolates. Two genotypes were previously shown to include Turkish isolates, indicating that there is a regional strain pattern in the South Caucasian-Turkish region. Four of the vaccine strains were polymorphic, exhibiting three different patterns of the cluster A1.a genotype and the cluster A3.b genotype. The genotype of vaccine strain 71/12, which is considered an attenuated strain in spite of the presence of both of the virulence pXO plasmids, appeared to be a novel genotype in the A1.a cluster.
Applied and Environmental Microbiology 09/2006; 72(8):5631-6. · 3.83 Impact Factor
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Journal of Clinical Microbiology 05/2006; 44(4):1606-7. · 4.15 Impact Factor
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ABSTRACT: The use of length-heterogeneity PCR was explored to monitor lactic acid bacteria succession during ensiling of maize. Bacterial diversity was studied during the fermentation of 30-day-old maize in optimal and spoilage-simulating conditions. A length heterogeneity PCR profile database of lactic acid bacteria isolated from the silage and identified by 16S rRNA gene sequencing was established. Although interoperonic 16S rRNA gene length polymorphisms were detected in some isolates, strain analysis showed that most of the lactic acid bacteria species thriving in silage could be discriminated by this method. The length heterogeneity PCR profiles of bacterial communities during maize fermentation were compared with those on a database. Under optimal fermentation conditions all the ecological indices of bacterial diversity, richness and evenness, deduced from community profiles, increased until day thirteen of fermentation and then decreased to the initial values. Pediococcus and Weissella dominated, especially in the first days of fermentation. Lactococcus lactis ssp. lactis and Lactobacillus brevis were mainly found after six days of fermentation. A peak corresponding to Lactobacillus plantarum was present in all the fermentation phases, but was only a minor fraction of the population. Unsuitable fermentation conditions and withered maize leaves in the presence of oxygen and water excess caused an enrichment of Enterococcus sp. and Enterobacter sp.
FEMS Microbiology Ecology 05/2006; 56(1):154-64. · 3.41 Impact Factor
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Massimo Marzorati,
Alberto Alma,
Luciano Sacchi,
Massimo Pajoro,
Simona Palermo,
Lorenzo Brusetti, Noura Raddadi,
Annalisa Balloi,
Rosemarie Tedeschi,
Emanuela Clementi,
Silvia Corona,
Fabio Quaglino,
Piero Attilio Bianco,
Tiziana Beninati,
Claudio Bandi,
Daniele Daffonchio
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ABSTRACT: Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, "Candidatus Phytoplasma vitis," which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the "Candidatus Cardinium hertigii" group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of "Ca. Cardinium hertigii." This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of "Ca. Phytoplasma vitis" were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and "Ca. Phytoplasma vitis" have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.
Applied and Environmental Microbiology 03/2006; 72(2):1467-75. · 3.83 Impact Factor
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Daniele Daffonchio, Noura Raddadi,
Maya Merabishvili,
Ameur Cherif,
Lorenzo Carmagnola,
Lorenzo Brusetti,
Aurora Rizzi,
Nina Chanishvili,
Paolo Visca,
Richard Sharp,
Sara Borin
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ABSTRACT: Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these "near neighbors" are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.
Applied and Environmental Microbiology 03/2006; 72(2):1295-301. · 3.83 Impact Factor
