Modeling the recovery of heat-treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 spores at suboptimal temperature and pH using growth limits.

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.

Comparison of three Bacillus amyloliquefaciens strains growth behaviour and evaluation of the spoilage risk during bread shelf-life.

This study aims at the characterisation of growth behaviour of three strains of Bacillus amyloliquefaciens, isolated from ropy bread (ATCC8473), wheat grain (ISPA-S109.3) and semolina (ISPA-N9.1) to estimate rope spoilage risk in pan bread during shelf-life using the Sym'Previus tool. Cardinal values and growth/no growth boundaries were determined in broth, while artificial spore inoculations were performed in dough for various pan bread recipes to compare experimental counts with in silico growth simulations. Finally, two storage scenarios were tested to determine the probability to reach a spoilage threshold during bread shelf-life. Similarly to the safety criteria fixed for Listeria monocytogenes contamination in foodstuff complying with EC regulation, a potential rope spoilage threshold was arbitrary fixed at 5 log CFU/g for B. amyloliquefaciens. This study further underlines a higher rope spoilage potential of the ISPA strains as compared to the ATCC strain, thus emphasizing the interest to characterise both wild strains and reference strain to account for biological variability. In conclusion, this study showed that available decision making tools which are largely recognized to predict behaviour of pathogenic strains, shall also be used with spoilage strains to help maintain food quality and extend shelf-life.

Sensitivity of Bacillus weihenstephanensis to acidic changes of the medium is not dependant on physiological state.

This study aims to quantify the effect of salt and acid preliminary exposure on acid resistance of vegetative cells of Bacillus weihenstephanensis. The psychrotolerant strain KBAB4 was cultured until the mid-exponentially phase (i) in BHI, (ii) in BHI supplemented with 2.5% salt or (iii) in BHI acidified at pH 5.5 with HCl. The growing cells were subsequently inactivated in lethal acid conditions ranging from 4.45 to 4.70. Based on statistical criteria, a primary mixed-Weibull model was used to fit the acid inactivation kinetics. The acid resistance was enhanced for acid-adapted cells and decreased for salt-adapted cells. The secondary modelling of the bacterial resistance allowed the quantification of the change in pH leading to a ten folds variation of the bacterial resistance, i.e. cells sensitivity (zpH). This sensitivity was not significantly affected whatever the preliminary mild exposure and the presence of sub-populations with different acid resistances. These results highlighted that pre-incubation conditions influence bacterial acid resistance without affecting the sensitivity to acidic modifications, with a 10 fold reduction of Bacillus acid resistance observed for a reduction of 0.37 pH unit. Quantification of such adaptive stress response might be instrumental in quantitative risk assessment more particularly in food formulation, particularly for low-acid minimally processed foods.

Prediction of Bacillus weihenstephanensis acid resistance: the use of gene expression patterns to select potential biomarkers.

Exposure to mild stress conditions can activate stress adaptation mechanisms and provide cross-resistance towards otherwise lethal stresses. In this study, an approach was followed to select molecular biomarkers (quantitative gene expressions) to predict induced acid resistance after exposure to various mild stresses, i.e. exposure to sublethal concentrations of salt, acid and hydrogen peroxide during 5 min to 60 min. Gene expression patterns of unstressed and mildly stressed cells of Bacillus weihenstephanensis were correlated to their acid resistance (3D value) which was estimated after exposure to lethal acid conditions. Among the twenty-nine candidate biomarkers, 12 genes showed expression patterns that were correlated either linearly or non-linearly to acid resistance, while for the 17 other genes the correlation remains to be determined. The selected genes represented two types of biomarkers, (i) four direct biomarker genes (lexA, spxA, narL, bkdR) for which expression patterns upon mild stress treatment were linearly correlated to induced acid resistance; and (ii) nine long-acting biomarker genes (spxA, BcerKBAB4_0325, katA, trxB, codY, lacI, BcerKBAB4_1716, BcerKBAB4_2108, relA) which were transiently up-regulated during mild stress exposure and correlated to increased acid resistance over time. Our results highlight that mild stress induced transcripts can be linearly or non-linearly correlated to induced acid resistance and both approaches can be used to find relevant biomarkers. This quantitative and systematic approach opens avenues to select cellular biomarkers that could be incremented in mathematical models to predict microbial behaviour.

Study of the bacterial ecosystem in tropical cooked and peeled shrimps using a polyphasic approach.

The characterization of the microbial ecosystem of cooked tropical shrimps was carried out using a polyphasic approach. First, culture-dependent methods were used for bacterial enumeration and the phenotypic and molecular identification of bacterial isolates. Then, culture-independent methods, including PCR-TTGE (V3 region of the 16S rRNA gene), provided a fingerprinting of bacterial DNA directly extracted from shrimps. Two batches of cooked and peeled tropical shrimps were stored at 5 and 15 degrees C for 5 and 3 weeks, respectively. Trained panelists carried out a sensory evaluation and microbiological enumerations were performed. When spoilage of samples was perceived, several colonies were isolated from the total viable count media. Thus, 137 bacterial strains were identified by phenotypic and molecular tests. Lactic acid bacteria (LAB) constituted the major group with the most represented genera being Carnobacterium (C. divergens, C. maltaromaticum and indiscernible C. alterfunditum/pleistocenium), Vagococcus (indiscernible V. carniphilus/fluvialis) and Enterococcus (E. faecalis and E. faecium). The other groups corresponded to Brochothrix thermosphacta and Enterobacteriaceae (Serratia liquefaciens). In PCR-TTGE profiles some of DNA fragments were assigned to those of standard strains (S. liquefaciens, B. thermosphacta, E. faecalis, C. divergens and C. maltaromaticum) or identified isolates from culture-dependent analysis (E. faecium). Other additional informations were provided by fragment cloning (Psychrobacter sp, Citrobacter gillenii and Firmicute). In conclusion, TTGE is an excellent tool to monitor the evolution of the microbial ecosystem in seafood products.

Characterization of Bacillus and Pseudomonas strains with suppressive traits isolated from tomato hydroponic-slow filtration unit.

Bacillus and Pseudomonas spp. are known to be involved in plant pathogenic fungi elimination during the slow filtration process used in tomato soilless cultures. We isolated 6-8 strains of both Bacillus and Pseudomonas from the top, middle, and bottom sections of filters and identified them after 16S rDNA sequencing. Four Pseudomonas strains were identified as Pseudomonas fulva, 5 as Pseudomonas plecoglossicida, and 12 as Pseudomonas putida. The use of specific oligonucleotide polymerase chain reaction primer sets designed from gyrB gene sequences additionally permitted the identification of 17 Bacillus cereus and 3 Bacillus thuringiensis strains. Ribotyping with EcoRI pointed out an important polymorphism within Bacillus and Pseudomonas strains. Molecular characterization did not reveal a correlation between the location of isolates within the filter (top, middle, or bottom) and bacterial identification or riboclusters. Functional aspects assessed by community-level physiological profiling showed marked phenotypic differences between Pseudomonas communities isolated from the top and bottom filter layers; differences were lower between Bacillus communities of different layers and far less noticeable between mixed communities of Bacillus and Pseudomonas. These strains were tested for several suppressive activities. Conversely to most Bacillus, the majority of Pseudomonas strains were auxin producers and promoted the growth of tomato plantlet roots. On the other hand, only Bacillus strains displayed antagonistic activities by inhibiting the growth of pathogenic fungi frequently detected in soilless cultures. Siderophores were produced by nearly all bacteria, but at higher amounts by Pseudomonas than Bacillus strains. The biocontrol agent potentiality of certain strains to optimize the slow filtration process and to promote the suppressive potential of nutrient solution is discussed.

Identification of Geotrichum candidum at the species and strain level: proposal for a standardized protocol.

In this study, the M13 primer was used to distinguish Geotrichum candidum from the anamorphic and teleomorphic forms of other arthrospore-forming species (discriminatory power = 0.99). For intraspecific characterization, the GATA4 primer showed the highest level of discrimination for G. candidum among the 20 microsatellite primers tested. A molecular typing protocol (DNA concentration, hybridization temperature and type of PCR machine) was optimized through a series of intra- and interlaboratory trials. This protocol was validated using 75 strains of G. candidum, one strain of G. capitatum and one strain of G. fragrans, and exhibited a discrimination score of 0.87. This method could therefore be used in the agro-food industries to identify and to evaluate biodiversity and trace strains of G. candidum. The results show that the GATA4 primer might be used to differentiate strains according to their ecological niche.

Heat resistance of coliform species isolated from cooked ham, snail flesh, and 'bouchées à la reine'.

AIMS: In this study, the heat resistance of coliform species isolated from cooked ham and ready-made meals was determined. METHODS AND RESULTS: Thirteen coliform strains belonging to 12 different species were studied using laboratory medium in order to determine delta (first decimal reduction time) and z(T) values (temperature increase leading to a 10-fold reduction of delta) using the Weibull model. For seven strains, delta-values were determined at temperatures ranging from 55 to 60 degrees C, with, delta values between 0.52 and 2.98 min, at 59 degrees C. For the other six strains, lower temperature values were determined with delta-values ranging from 0.47 to 1.64 min at 54 degrees C. z(T) values calculated for the 13 strains were 3.1 to 7.5 degrees C. For eight strains, plotting of the log of survivors was not linear but rather showed shoulders or shoulders and tails. CONCLUSIONS: Coliform species were sensitive to heat treatment with a decimal reduction time under 2 min at 60 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The better knowledge of coliform heat resistance by determining thermal resistance parameters with confidence intervals will be useful for evaluating the efficiency of industrial thermal processes.

Molecular identification of Lactobacillus hilgardii and genetic relatedness with Lactobacillus brevis.

Conventional phenotypic methods lead to misidentification of the lactic acid bacteria Lactobacillus hilgardii and Lactobacillus brevis. Random amplified polymorphic DNA (RAPD) and repetitive element PCR (REP-PCR) techniques were developed for a molecular study of these two species. The taxonomic relationships were confirmed by analysis of the ribosomal operon. Amplified DNA fragments were chosen to isolate L. hilgardii-specific probes. In addition to rapid molecular methods for identification of L. hilgardii, these results convincingly proved that some strains first identified as L. brevis must be reclassified as L. hilgardii. The data clearly showed that these molecular methods are more efficient than phenotypic or biochemical studies for bacterial identification at the species level.