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Applied and Environmental Microbiology, March 2005, p. 1346-1355, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1346-1355.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Arturo Reyes-Ramirez and Jorge E. Ibarra^*

Departamento de Biotecnología y Bioquímica, CINVESTAV, Irapuato, Guanajuato, México

Received 6 September 2004/ Accepted 19 October 2004

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 ß-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.

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* Corresponding author. Mailing address: CINVESTAV, Apartado Postal 629, 36500 Irapuato, Guanajuato, Mexico. Phone: 52-462-623-9643. Fax: 52-462-624-5996. E-mail: jibarra{at}ira.cinvestav.mx.

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Applied and Environmental Microbiology, March 2005, p. 1346-1355, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1346-1355.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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