Contamination flows of Bacillus cereus and spore-forming aerobic bacteria in a cooked, pasteurized and chilled zucchini purée processing line.

A food processing plant producing pasteurized purées and its zucchini purée processing line were examined for contamination with aerobic and facultative anaerobic bacterial spores during a day's operation. Multiplication of spores was also monitored in the product stored under different conditions. High concentrations of Bacillus cereus spores were found in the soil in which the zucchinis were grown (4.6+/-0.3 log CFU/g), with a background spore population of 6.1+/-0.2 log CFU/g. In the processing plant, no B. cereus or psychrotrophic bacterial spores were detected on equipment. B. cereus and psychrotrophic bacterial spores were detected after enrichment in all samples of raw zucchinis, washed zucchinis, of two ingredients (starch and milk proteins) and in processed purée at each processing step. Steam cooking of raw zucchinis and pasteurization of purée in the final package significantly reduced spore numbers to 0.5+/-0.3 log CFU/g in the processed food. During storage, numbers of spore-forming bacteria increased up to 7.8+/-0.1 log CFU/g in purée after 5 days at 20-25 degrees C, 7.5+/-0.3 log CFU/g after 21 days at 10 degrees C and 3.8+/-1.1 log CFU/g after 21 days at 4 degrees C. B. cereus counts reached 6.4+/-0.5 log CFU/g at 20-25 degrees C, 4.6+/-1.9 log CFU/g at 10 degrees C, and remained below the detection threshold (1.7 log CFU/g) at 4 degrees C. Our findings indicate that raw vegetables and texturing agents such as milk proteins and starch, in spite of their low levels of contamination with bacterial spores and the heat treatments they undergo, may significantly contribute to the final contamination of cooked chilled foods. This contamination resulted in growth of B. cereus and psychrotrophic bacterial spores during storage of vegetable purée. Ways to eliminate such contamination in the processing line are discussed.

Research on factors allowing a risk assessment of spore-forming pathogenic bacteria in cooked chilled foods containing vegetables: a FAIR collaborative project.

Vegetables are frequent ingredients of cooked chilled foods and are frequently contaminated with spore-forming bacteria (SFB). Therefore, risk assessment studies have been carried out, including the following: hazard identification and characterisation--from an extensive literature review and expertise of the participants, B. cereus and C. botulinum were identified as the main hazards; exposure assessment--consisting of determination of the prevalence of hazardous SFB in cooked chilled foods containing vegetables and in unprocessed vegetables, and identification of SFB representative of the bacterial community in cooked chilled foods containing vegetables, determination of heat-resistance parameters and factors affecting heat resistance of SFB, determination of the growth kinetics of SFB in vegetable substrate and of the influence of controlling factors, validation of previous work in complex food systems and by challenge testing and information about process and storage conditions of cooked chilled foods containing vegetables. The paper illustrates some original results obtained in the course of the project. The results and information collected from scientific literature or from the expertise of the participants are integrated into the microbial risk assessment, using both a Bayesian belief network approach and a process risk model approach, previously applied to other foodborne hazards.

Molecular characterization of the food-borne fungus Neosartorya fischeri (Malloch and Cain).

The food-borne fungus Neosartorya fischeri, which is phenotypically related to the human opportunistic pathogen Aspergillus fumigatus, causes spoilage of heat-processed fruit products. Genomic methods were used to type N. fischeri strains and identify the genomic relationship between A. fumigatus and N. fischeri and between the different varieties of N. fischeri. EcoRI restriction fragment length polymorphism (RFLP) patterns obtained after ethidium bromide staining could differentiate most of N. fischeri var. glabra and N. fischeri var. spinosa strains. On the contrary, all N. fischeri var. fischeri strains tested exhibit the same RFLP pattern, which was similar to the A. fumigatus pattern. Similarly, Southern hybridization with a ribosomal probe showed some polymorphism between N. fischeri var. glabra and N. fischeri var. spinosa strains but could not distinguish between N. fischeri var. fischeri and A. fumigatus strains. By using the endonucleases EcoRI, HindIII, and BglII to generate Southern blot patterns with a fragment of the A. fumigatus gene coding for a 33-kDa protease, it was possible to differentiate N. fischeri var. fischeri from A. fumigatus. The difference between N. fischeri and A. fumigatus was confirmed by the use of moderately repetitive nonribosomal A. fumigatus sequences. These results are in agreement with previous studies that showed important infraspecific polymorphism within N. fischeri var. glabra and N. fischeri var. spinosa and, in contrast, the homogeneity of N. fischeri var. fischeri strains. A unique Southern blot pattern was seen for each strain of N. fischeri fingerprinted with the A. fumigatus repetitive sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of DNA probes for fingerprinting Aspergillus fumigatus.

Several different DNA fragments containing nonribosomal repetitive sequences have been isolated from the genome of Aspergillus fumigatus and tested as potential DNA fingerprinting probes. Eight of these clones generate 19 or more bands when hybridized to EcoRI-digested DNA of a reference strain in Southern blots, and they fall into four families. Individual clones from two families were tested and were found to generate complex Southern blot hybridization patterns which are stable within a single strain over many generations, which vary among unrelated strains, and which are amenable to computer-assisted analyses involving large numbers of strains in epidemiological studies. Clones from three of the families clustered a majority of test strains in a similar fashion in individual dendrograms based on similarity coefficients computed from band positions in Southern blot hybridization patterns. These clones therefore fulfill the major requisites for effective DNA fingerprinting probes.

[Tools, progress and questions in the molecular study of Aspergillus fumigatus and invasive aspergillosis]. / Outils, progrès et questions dans l'étude moléculaire de Aspergillus fumigatus et de l'aspergillose invasive.

Development of A. fumigatus in the host tissues is due to the intrinsic biological characteristics of this fungus and to the impairment of the cellular defence reactions of the host. However, even today the understanding of the factors governing the infectivity of A. fumigatus remains very limited. For example, the cellular mechanisms involved in the killing of A. fumigatus are still not elucidated. The cellular site(s) of infection and the role of the different lung epithelia in the establishment of the fungus are unknown. No specific fungal virulence factors have been identified until now. Molecular biology techniques are powerful tools to investigate the pathogenesis of invasive aspergillosis. Recent developments in the study of this mycosis are presented in this review.

Use of DNA moderately repetitive sequence to type Aspergillus fumigatus isolates from aspergilloma patients.

Polymorphism of forty-seven sequential clinical isolates from 3 patients with an aspergilloma was analyzed. DNA from each isolate was digested with EcoRI and hybridized in Southern blots with a 32P-labeled nonribosomal DNA repetitive sequence. Most isolates from each patient displayed the same hybridization pattern. Southern blot patterns obtained with DNA repetitive sequences can be used to type clinical isolates of Aspergillus fumigatus and have shown that aspergilloma patients are most probably infected by a single strain.

Molecular epidemiology of nosocomial invasive aspergillosis.

Moderately repeated DNA sequences were used to fingerprint strains of Aspergillus fumigatus isolated from patients with invasive aspergillosis and their hospital environment. Most strains sampled from the environment displayed different Southern blot hybridization patterns. A temporal survey of air contaminants showed that some strains can persist in the same environment for at least 6 months. Patients with invasive aspergillosis were infected by a single strain. In two patients, a nosocomial origin of infection was suggested.

The role of the rodlet structure on the physicochemical properties of Aspergillus conidia.

Physico-chemical properties of Aspergillus conidia rely on their outer cell-wall rodlet layer. In A. fumigatus and A. nidulans, the rodlet structure is due to an hydrophobin encoded by homologous rodA genes. To evaluate the role of the rodlet structure on the physico-chemical properties of conidia, we compared hydrophobicity, Lewis acid-base (i.e. electron donor/acceptor) characteristics and electrostatic charge of hydrophobin-less (rodletless) mutant and wild-type conidia of A. fumigatus and A. nidulans. The results obtained by aqueous-solvent partitioning assays, microsphere adhesion assays and microelectrophoresis showed that the disruption of the rodA gene modifies surface properties of A. fumigatus and A. nidulans conidia, and confirmed that the rodlet layer plays a key role in their physico-chemical behaviour. The absence of this layer on A. fumigatus spores led to the appearance of weakly basic and acidic characteristics, and had a slight effect on the hydrophobicity of conidia. Whereas in A. nidulans, it induced a basic character, a marked decrease in hydrophobicity and in the polarization capacity (electronegativity) of conidia. These physico-chemical differences between A. fumigatus and A. nidulans rodletless conidia may be attributed to differences in the composition of the conidial outer cell-wall of the two species.

Ruminococcin A, a new lantibiotic produced by a Ruminococcus gnavus strain isolated from human feces.

When cultivated in the presence of trypsin, the Ruminococcus gnavus E1 strain, isolated from a human fecal sample, was able to produce an antibacterial substance that accumulated in the supernatant. This substance, called ruminococcin A, was purified to homogeneity by reverse-phase chromatography. It was shown to be a 2,675-Da bacteriocin harboring a lanthionine structure. The utilization of Edman degradation and tandem mass spectrometry techniques, followed by DNA sequencing of part of the structural gene, allowed the identification of 21 amino acid residues. Similarity to other bacteriocins present in sequence libraries strongly suggested that ruminococcin A belonged to class IIA of the lantibiotics. The purified ruminococcin A was active against various pathogenic clostridia and bacteria phylogenetically related to R. gnavus. This is the first report on the characterization of a bacteriocin produced by a strictly anaerobic bacterium from human fecal microbiota.