Journal of Microbiological Methods 2009-12-01

Improvement of a selective media for the isolation of B. anthracis from soils.

Vicki A Luna, Jenny Gulledge, Andrew C Cannons, Philip T Amuso

Index: J. Microbiol. Methods 79(3) , 301-6, (2009)

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Abstract

To prove linkage between an environmental sample and an anthrax case, there must be isolates obtained from both that can be compared. Although Bacillus anthracis is easily isolated from powder samples, isolating it from soil is difficult because of the high bacterial count in it. Formulations of PLET were prepared, inoculated with B. anthracis, B. cereus and B. thuringiensis and examined for growth. Two hundred eighty-three isolates including 23 B. anthracis were placed onto one formulation while MICs against trimethoprim-sulfamethoxazole were determined. The media supported B. anthracis growth at 30 degrees C and inhibited almost all other bacterial growth, including closely-related species. Sensitivity for B. anthracis and selectivity against other Bacillus and against non-Bacillus were 96.8%, 100% and 97.2% respectively. Isolates that grew had MICs >4 and >76 microg mL(-1) against trimethoprim and sulfamethoxazole, respectively. Soils spiked with 10(2)B. anthracis spores and suspended in PLET broth yielded a 6-7 log(10) increase in B. anthracis. Other growth was inhibited. PLET supplemented with sulfamethoxazole (38 microg mL(-1)), trimethoprim (2 microg mL(-1)), polymyxin B (15,000 U L(-1)), and lysozyme (150,000 U L(-1)) can successfully select for B. anthracis and will facilitate agricultural, environmental and forensic investigations of B. anthracis isolates.