Traceability of potential enterotoxigenic Bacillus cereus in bee-pollen samples from Argentina throughout the production process.

Bee-pollen is a functional food sold for human and animal consumption but also is a favorable microhabitat for many spore-forming bacteria. Among them, Bacillus cereus can produce several toxins and other virulence factors, causing an emetic or diarrheal syndrome after ingestion. The study involved 36 bee-pollen samples obtained from different sampling points throughout the production process (collecting, freezing, drying, and cleaning) in Argentina. Fifty isolates of B. cereus yielded 24 different fingerprint patterns with BOX and ERIC primers. Only three fingerprint patterns were maintained throughout the production process. In contrast, others were lost or incorporated during the different steps, suggesting that cross-contamination occurred as shown by differences in fingerprint patterns after freezing, drying, and cleaning steps compared to the initial collection step. Genes encoding for cereulide (ces), cytotoxin K (cytK), sphingomyelinase (sph), the components of hemolysin BL (hblA, hblB, hblC, hblD) and non-hemolytic complex (nheAB) were studied. All the isolates displayed one or more enterotoxin genes. The most frequent virulence genes detected belong to the HBL complex, being the most abundant hblA (98%), followed by hblD (64%), hblB (54%), and hblC (32%), respectively. Ten strains (20%), present at all sampling points, carried all the subunits of the HBL complex. The non-hemolytic enterotoxic complex (nheAB) was found in 48 strains (96%), while seven strains (14%) present at all sampling points showed the amplification product for sphingomyelinase (sph). One cereulide-producer was isolated at the cleaning step; this strain contained all the components for the hemolytic enterotoxin complex HBL, the NHE complex, and cytotoxin K related to the foodborne diarrhoeal syndrome. In total, 11 different virulence patterns were observed, and also a correlation between rep-fingerprint and virulence patterns. The results suggest that bee-pollen can be contaminated at any point in the production process with potential enterotoxic B. cereus strains, emphasizing the importance of hygienic processing.

Effectiveness of tilmicosin against Paenibacillus larvae, the causal agent of American Foulbrood disease of honeybees.

American Foulbrood (AFB) of honeybees (Apis mellifera L.), caused by the Gram-positive bacterium Paenibacillus larvae is one of the most serious diseases affecting the larval and pupal stages of honeybees (A. mellifera L.). The aim of the present work was to asses the response of 23 strains of P. larvae from diverse geographical origins to tilmicosin, a macrolide antibiotic developed for exclusive use in veterinary medicine, by means of the minimal inhibitory concentration (MIC) and the agar diffusion test (ADT). All the strains tested were highly susceptible to tilmicosin with MIC values ranging between 0.0625 and 0.5 microg ml(-1), and with MIC(50) and MIC(90) values of 0.250 microg ml(-1). The ADT tests results for 23 P. larvae strains tested showed that all were susceptible to tilmicosin with inhibition zones around 15 microg tilmicosin disks ranging between 21 and 50mm in diameter. Oral acute toxicity of tilmicosin was evaluated and the LD(50) values obtained demonstrated that it was virtually non-toxic for adult bees and also resulted non-toxic for larvae when compared with the normal brood mortality. Dosage of 1000 mg a.i. of tilmicosin applied in a 55 g candy resulted in a total suppression of AFB clinical signs in honeybee colonies 60 days after initial treatment. To our knowledge, this is the first report of the effectiveness of tilmicosin against P. larvae both in vitro and in vivo.