Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains

Streptococci from different collections and dairy materials were characterized by conventional and molecular methods. After amplification of the 16S-23S rDNA spacer region, all the strains referable to the genus Streptococcus exhibited a single polymerase chain reaction (PCR) product, allowing their differentiation from enterococci. Cleaving this PCR product with Hae III, two different restriction patterns could be observed, allowing Streptococcus salivarius DSM 20560T, Strep. thermophilus NCDO 822 and two strains of Streptococcus spp. to be gathered in one group and all the other strains in another. In order to achieve strain typing, all the cultures were investigated by random amplified polymorphic DNA (RAPD)-PCR analysis employing two selected primers. The results were treated by cluster analysis, appearing significantly consistent with both the taxonomic position and the origin of the strains. Pulsed-field gel electrophoresis (PFGE) of Sma I digests of the genomic DNA from 11 representative strains with decreasing levels of RAPD similarity allowed their diversity to be confirmed, even though RAPD-PCR proved to be less discriminating than PFGE analysis. The results are discussed with reference to the capability of the analytical procedures used to aid both identification and strain typing of streptococci, as well as the taxonomic structure of the species Strep. thermophilus.

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“…(Molnár et al 1995), Bacillus spp. (Brousseau et al 1993 ;Stephan et al 1994) and Lactobacillus spp. (Du Plessis and Dicks 1995).…”

Section: Introductionmentioning

confidence: 99%

Random amplified polymorphic DNA and amplified ribosomal DNA spacer polymorphism : powerful methods to differentiate Streptococcus thermophilus strains

Moschetti

Blaiotta

Aponte

et al. 1998

Journal of Applied Microbiology

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“…Any non-mucoid colonies from a population initially containing pX02 were stained with polychrome methylene Table 1 Strains used in this study blue (M'Fadyean 1903) and examined by light microscopy for the presence of capsular material (Welkos 1991). The pX01 ¦ /pX02 − and pX01 − /pX02 − derivatives appeared non-mucoid under both sets of growth conditions and the presence of pX01 was determined using the polymerase chain reaction (PCR) (Turnbull et al 1992;Brousseau et al 1993;Beyer et al 1996). Colonies were tested periodically for the production of Protective Antigen by growth in Ristroph and Ivins medium Leppla 1991) and by using a rapid immunochromatographic assay kindly supplied by Dr J.…”

Section: Bacterial Strains Growth Media and Antibioticsmentioning

confidence: 99%

The native virulence plasmid combination affects the segregational stability of a theta-replicating shuttle vector in Bacillus anthracis var. New Hampshire

Bowen¹,

Quinn²

1999

J Appl Microbiol

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The segregational stability of a small, θ‐replicating, non‐mobilizable shuttle plasmid (pAEX‐5E) was determined in fully virulent (pX01+/pX02+), partially cured (pX01+/pX02− and pX01−/pX02+) and fully cured (pX01−/pX02−) derivatives of Bacillus anthracis var. New Hampshire. Under the growth conditions used (L‐broth, 37 °C, aerobic, batch culture), pAEX‐5E remained segregationally stable in the pX01−/pX02+ and pX01−/pX02− derivatives for in excess of 100 culture generations, but was expelled from the pX01+/pX02+ and pX01+/pX02− derivatives (100% loss occurred after 101±3·8 and 54±6·0 culture generations, respectively). In the presence of antibiotic selection pressure to maintain pAEX‐5E (5 μg erythromycin ml−1) no comparable loss of pX01 or pX02 was observed over 100 generations of growth in any of the derivatives of B. anthracis. Under these conditions the pX01+/pX02− derivative had an extended culture doubling time (td±S.E. of the mean) of 75·3 ± 1·4 min compared with 47·3 ± 1·1, 46·2 ± 0·86 and 43·2 ± 1·2 min for the pX01+/pX02+, pX01−/pX02+ and pX01−/pX02− derivatives, respectively. That antibiotic resistance was pAEX‐5E‐mediated was confirmed using a second antibiotic marker (kanamycin). After100 generations of growth in the presence of erythromycin, colonies were shown to have retained kanamycin resistance. Southern blot analysis, in conjunction with plasmid rescue to Escherichia coli confirmed that, after 100 culture generations in the presence of antibiotic selection pressure, pAEX‐5E had remained structurally stable and had not integrated into the B. anthracis genome.

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“…No genetic relatedness among B. thuringiensis serovars and among intraserovar strains can be revealed, and no phylogenetic relationships can be established. Several different alternative or complementary classi®cation methods have been developed (Lereclus et al 1982;Lynch and Baumann 1985;Visser 1989;Miteva et al 1991;Brousseau et al 1993;Nakamura 1994;Priest et al 1994;Bourque et al 1995;Akhurst et al 1997;Hansen et al 1998). However, because of the limitations of some these methods and the limited number of strains tested, the genetic relatedness among B. thuringiensis serovars is still unclear.…”

Section: Introductionmentioning

confidence: 99%

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

Joung

Côté

2001

J Appl Microbiol

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Aims: To determine the 23S and 5S rRNA gene ®ngerprints in order to reveal phylogenetic relationships among Bacillus thuringiensis strains. Methods and Results: Eighty-six B. thuringiensis strains which include 80 serovar type strains, ®ve 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 95á5% 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 ®ngerprinting. It revealed eight distinct phylogenetic groups and three ungrouped serovars at the 94% DNA relatedness rate. Conclusions: This method permitted the classi®cation 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. Signi®cance and Impact of the Study: This study contributes a further step to the de®nition of valid taxonomic sublevels for the B. thuringiensis species.

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Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains

Cited by 120 publications

References 21 publications

Random amplified polymorphic DNA and amplified ribosomal DNA spacer polymorphism : powerful methods to differentiate Streptococcus thermophilus strains

Random amplified polymorphic DNA and amplified ribosomal DNA spacer polymorphism : powerful methods to differentiate Streptococcus thermophilus strains

The native virulence plasmid combination affects the segregational stability of a theta-replicating shuttle vector in Bacillus anthracis var. New Hampshire

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

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