Isolation of glucosidase and phospholipase positive Bacillus circulans on ALOA medium

Abstract

To report the growth of glucosidase and phospholipase positive bacteria on agar Listeria according to Ottaviani and Agosti (ALOA) different from Listeria monocytogenes, Listeria ivanovii and Bacillus cereus. Raw water-buffalo milk was analysed according to EN ISO 11290. Streaking of Fraser broth on ALOA resulted in green colonies with an opaque halo after 48 h at 30°C. Colonies were transferred onto Tryptone soya yeast extract agar and showed cultural characteristics atypical for L. monocytogenes. Results of confirmation tests according to EN ISO 11290 method: negative haemolysis test, weak positive camp test in correspondence with Staphylococcus aureus, no fermentation of rhamnose, fermentation of xylose. Gram staining showed tapered, curved, Gram-positive rods with subterminal to terminal ellipsoidal spores, 0.5-0.7 μm diameter 4-5 μm. API 50CH CHB kit (99.9% percentage of identification) and the sequence of the 833 bp PCR product (portion of 16S rRNA, generic primers 1492-r; p27-f) showed 97% identity with Bacillus circulans ATCC 4513 (GenBank AY724690). Some B. circulans strains can grow on ALOA, according to ISO 11290, confirmation tests readily differentiate B. circulans from L. monocytogenes. The different morphology of the colonies must be kept in mind to select true L. monocytogenes for confirmation test and to avoid overestimation of L. monocytogenes count.