Isolation of glucosidase and phospholipase positive Bacillus circulans on ALOA medium

Aim: To report the growth of glucosidase and phospholipase positive bacteria on agar Listeria according to Ottaviani and Agosti (ALOA) different from Liste- ria monocytogenes, Listeria ivanovii and Bacillus cereus. Methods and Results: 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. mono- cytogenes. Results of confirmation tests according to EN ISO 11290 method: negative haemolysis test, weak positive camp test in correspondence with Sta- phylococcus aureus, no fermentation of rhamnose, fermentation of xylose. Gram staining showed tapered, curved, Gram-positive rods with subterminal to ter- minal ellipsoidal spores, 0Æ5–0Æ7 lm diameter 4–5 lm. API 50CH CHB kit (99Æ9% percentage of identification) and the sequence of the 833 bp PCR prod- uct (portion of 16S rRNA, generic primers 1492-r; p27-f) showed 97% identity with Bacillus circulans ATCC 4513 (GenBank AY724690). Conclusions: Some B. circulans strains can grow on ALOA, according to ISO 11290, confirmation tests readily differentiate B. circulans from L. monocytogenes. Significance and Impact of the Study: The different morphology of the colo- nies must be kept in mind to select true L. monocytogenes for confirmation test and to avoid overestimation of L. monocytogenes count.