, 2.5 × 10 5 and 9.7 × 10 7 cfu/cm 2 , respectively, in the different units. Phenotypic identification of isolates revealed predominance of Gram-positive bacilli belonging to Bacillus and notably the Bacillus cereus group, at maximal levels of 72 and 21% respectively. The other Gram-positive microflora included Staphylococcus (30%) and Micrococcus (10%). In contrast, the incidence of the Gram-negative bacteria was relatively low. Two genera, identified as Pseudomonas (9%) and Enterobacter (6%), were found only in two dairies. Three dairies were Gram-negative bacteria-free. That should be the result of common contamination sources or highly environmental selective pressure. Further studies have to address these hypotheses. Treatment of experimental Bacillus cereus sensu lato strains biofilms with a 50, 100 and 150 ppm of quaternary ammonium disinfectant, showed a significant resistance of biofilms to this product even after long exposure time (15 min). This study emphasized the importance of aerobic spore-forming bacteria in dairy-processing equipment as they are able to built recalcitrant biofilms on the inside equipment surfaces with subsequent resistance to conventional CIP system and potential transfer to pasteurized milk. Therefore, in order to reduce the contamination levels of spore-forming bacteria and improve the quality and shelf life of the product, these dairies have, besides improvement in the hygienic status of the plant equipments, also to monitor either the pasteurization process or the contamination from raw material (that is, milk powder).
In the dairy industry, the biofilms formed by spore forming bacteria are not well characterized. Microscopic methods are crucial for the study of biofilm structural and architectural features. Here, a simple surface-associated non-submerged model combined to environmental scanning electron microscope (ESEM) imaging was used for the study of Bacillus cereus and Geobacillus spp. dairy biofilms. To evaluate the utility of this approach, non-submerged biofilms were compared to those developed in situ on stainless steel coupons introduced inside milk processing lines. Results reveal that both B. cereus and Geobacillus spp. are able to form specific biofilm characteristics on nonimmersed surfaces, notably an original dispersion style not previously described. Non-submerged biofilms in vitro are elaborate three-dimensional or extensive complex structures well resolved in ESEM and comparable to dairy biofilms in situ. The non-submerged surface-associated biofilm combined to ESEM imaging revealed a relevant model for the study of dairy biofilms.
International audienceBacillus cereus is a food pathogen of major concern to the dairy industry. Strains of B. cereus were isolated in 2006 and 2010 from a pasteurized milk processing line in a dairy plant in Algeria. Pasteurized milk is obtained from reconstituted and processed milk powder. This study was designed to discriminate between the isolated B. cereus strains and to predict their food poisoning potential. Strains were fingerprinted by M13-PCR, and identified at the phylogenetic group level by amplified panC gene sequence analysis. They were clustered into three distinct M13-PCR groups: one major group which included 17 strains and two minor groups which contained two and one strains, respectively. Strains originating from both the process equipment and milk powder were from the major group indicating that milk powder was the main source of initial contamination. Strains from the major and second group were affiliated to the mesophilic phylogenetic group III while the unique strain of the third group was classified into the mesophilic phylogenetic group IV. The data presented in this study showed a very low genetic diversity among B. cereus strains identified in milk powder and secondly by milk processing systems, as well as persistence in the dairy environment of specific B. cereus genotypes, across 4 years. M13-PCR typing and phylogenetic affiliation were useful for characterizing B. cereus dairy isolates, permitting their differentiation within the B. cereus group, and showing homogeneous contamination throughout the pasteurized milk processing line. The recurrent genotypes which belong to the potentially toxigenic group III B. cereus could threaten pasteurized milk safety
Bacillus cereus is a foodborne pathogen that often persists on food processing surfaces due the formation of spores and biofilms. Spores of 12 selected B. cereus strains from genotypes that recurred in a pasteurized milk processing line were investigated in this study, for their surface and biofilm characteristics. The main objective was to have an insight into their persistence strategies. Spore surface hydrophobicity and acid-base properties, were assessed using the microbial adhesion to solvents (MATS) method. To determine how hydrophobicity was affected by cleaning procedures, this property was measured when spores were submitted to alkali or acidic stresses mimicking those of cleaning-in-place (CIP) procedures. Biofilms formation on stainless steel coupons by pH-treated spores was investigated in three culture media and imaged by using environmental scanning electron microscopy (ESEM). Results showed that spores were either hydrophilic or moderately hydrophobic. Alkali-stress reduced spore surface hydrophobicity, whereas acidic shock increased it. More limited hydrophobicity changes following alkaline stress suggest alkali adaptation of spores. In addition, spores submitted to pH-stresses produced specific biofilm features on stainless steel as shown by ESEM imaging. Alkali tolerance and the biofilm lifestyle are strategies that permit B. cereus recurrent genotypes to persist in the milk processing line. Overall, this study gives an insight into hydrophobicity and specific biofilm features of B. cereus spores submitted to chemical cleaning.
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