Collaborative Cohort of Cohorts for COVID-19 Research (C4R) Study: Study Design

The Collaborative Cohort of Cohorts for COVID-19 Research (C4R) is a national prospective study of adults at risk for coronavirus disease 2019 (COVID-19) comprising 14 established United States (US) prospective cohort studies. For decades, C4R cohorts have collected extensive data on clinical and subclinical diseases and their risk factors, including behavior, cognition, biomarkers, and social determinants of health. C4R will link this pre-COVID phenotyping to information on SARS-CoV-2 infection and acute and post-acute COVID-related illness. C4R is largely population-based, has an age range of 18-108 years, and broadly reflects the racial, ethnic, socioeconomic, and geographic diversity of the US. C4R is ascertaining severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and COVID-19 illness using standardized questionnaires, ascertainment of COVID-related hospitalizations and deaths, and a SARS-CoV-2 serosurvey via dried blood spots. Master protocols leverage existing robust retention rates for telephone and in-person examinations, and high-quality events surveillance. Extensive pre-pandemic data minimize referral, survival, and recall bias. Data are being harmonized with research-quality phenotyping unmatched by clinical and survey-based studies; these will be pooled and shared widely to expedite collaboration and scientific findings. This unique resource will allow evaluation of risk and resilience factors for COVID-19 severity and outcomes, including post-acute sequelae, and assessment of the social and behavioral impact of the pandemic on long-term trajectories of health and aging.

Water safety plans for water supply utilities in China, Cuba, France, Morocco and Spain: costs, benefits, and enabling environment elements.

Water Safety Plans (WSPs) are a management tool to identify and prioritize risks and implement appropriate control measures throughout the water supply chain, from catchment to consumer. WSPs have been implemented in over 90 countries; yet, costs, benefits and the enabling environment elements necessary for WSP implementation are under-studied. To better understand these factors, we conducted interviews with WSP implementation management teams from 20 private urban water utilities in China, Cuba, France, Morocco and Spain in 2014. Collectively, these utilities serve 10.6 million consumers and supply over 2.2 million m3/day of water to consumers. Time for WSP implementation to achieve certification averaged 13 months. The main startup cost was staff time, averaging 16.2 full-time equivalent person-months. Additional costs, averaging €16,777, were for training staff, hiring consultants, purchasing equipment, and certifying WSPs. Benefits commonly reported included improved hazard control, treatment practices, record keeping, and client and health agency confidence.

Risk ranking of pathogens in ready-to-eat unprocessed foods of non-animal origin (FoNAO) in the EU: initial evaluation using outbreak data (2007-2011).

Foods of non-animal origin (FoNAO) are consumed in a variety of forms, being a major component of almost all meals. These food types have the potential to be associated with large outbreaks as seen in 2011 associated with VTEC O104. In order to identify and rank specific food/pathogen combinations most often linked to human cases originating from FoNAO in the EU, a semi-quantitative model was developed using seven criteria: strength of associations between food and pathogen based on the foodborne outbreak data from EU Zoonoses Monitoring (2007-2011), incidence of illness, burden of disease, dose-response relationship, consumption, prevalence of contamination and pathogen growth potential during shelf life. The top ranking food/pathogen combination was Salmonella spp. and leafy greens eaten raw followed by (in equal rank) Salmonella spp. and bulb and stem vegetables, Salmonella spp. and tomatoes, Salmonella spp. and melons, and pathogenic Escherichia coli and fresh pods, legumes or grains. Despite the inherent assumptions and limitations, this risk model is considered a tool for risk managers, as it allows ranking of food/pathogen combinations most often linked to foodborne human cases originating from FoNAO in the EU. Efforts to collect additional data even in the absence of reported outbreaks as well as to enhance the quality of the EU-specific data, which was used as input for all the model criteria, will allow the improvement of the model outputs. Furthermore, it is recommended that harmonised terminology be applied to the categorisation of foods collected for different reasons, e.g. monitoring, surveillance, outbreak investigation and consumption. In addition, to assist future microbiological risk assessments, consideration should be given to the collection of additional information on how food has been processed, stored and prepared as part of the above data collection exercises.

Improving quantitative exposure assessment by considering genetic diversity of B. cereus in cooked, pasteurised and chilled foods.

The natural contamination of foods with a bacterial pathogen frequently consists of a mixture of strains with their own characteristics of survival, growth potential and virulence. Quantitative Microbial Risk Assessment (QMRA) must account for this genetic diversity to reflect the variability of the pathogen risk and to identify the genetic groups present at key stages of the food pathway. To describe the transmission dynamics of a heterogeneous population of B. cereus, we developed an exposure model that covers a food processing chain from "farm to table". The studied food was a cooked, pasteurised and chilled courgette purée used as an example of Refrigerated Processed Food of Extended Durability (REPFED). The B. cereus population consists of a continuum of genetic groups ranging from mesophilic and highly heat resistant, to psychrotrophic and moderately-heat resistant ones. At each step in a processing chain comprising cooking, blending, mixing with ingredients providing a secondary contamination, pasteurisation and chilling for several weeks, the prevalence of contaminated units (batches or packages) and the spore load within the units was determined for each genetic group, as well as their proportion to the total B. cereus population in the units. The model predicted that all packages contain mesophilic groups just after partitioning. The addition of mesophilic strains by the ingredients during the process of the courgette purée was an important contribution. At the end of the domestic storage, the model predicted a dominance of the mesophilic groups, while only some psychrotrophic groups were present.

Diversity of spore germination in response to inosine and L-alanine and its interaction with NaCl and pH in the Bacillus cereus group.

AIMS: Our aim was to assess the diversity of the nutrient germination response of Bacillus cereus spores. METHODS AND RESULTS: B. cereus spore germination was monitored by decrease in optical density using a Bioscreen C analyser in response to the major germinant substances inosine and l-alanine. Spores of a set of 12 strains taken to illustrate the diversity of the B. cereus group showed ranging germination capacities. Two strains never germinated in the presence of l-alanine, at any of the germinant concentrations tested. Both the extent and rate of spore germination were affected by low pH and high NaCl concentration, but differently according to the strain. CONCLUSIONS: A broad diversity was observed in nutrient-triggered spore germination among the members of the B. cereus group. Spore germination of some strains occurred at low concentrations of inosine or l-alanine, suggesting high receptor sensitivity to germinants. The activity of these receptors was also affected by pH or high NaCl concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: The greater ability of some strains to germinate in response to l-alanine and inosine is one criterion among others for B. cereus strain selection in food processing or storage studies, before confirmation in complex food or laboratory media. The diversity in response to germinants found among the B. cereus strains suggests a differential expression and (or) absence of some germination genes involved in the response, mainly to l-alanine.

Glycine betaine improves Listeria monocytogenes tolerance to desiccation on parsley leaves independent of the osmolyte transporters BetL, Gbu and OpuC.

AIMS: To investigate the effect of glycine betaine (GB) on the survival of Listeria monocytogenes on leaf surfaces under low relative humidity (RH). METHODS AND RESULTS: The addition of GB (> or = 25 mmol l(-1)) improved the survival of L. monocytogenes under low RH on parsley leaves, thus suggesting that GB can improve the tolerance of L. monocytogenes to desiccation. Ten times less GB was needed to improve L. monocytogenes survival under low RH on nonbiological surfaces compared with parsley leaves, suggesting that, on the leaf surface, L. monocytogenes may have to compete for the available GB with autochthonous bacteria and/or the plant itself. Wild type and mutants carrying deletions in the three GB uptake systems, BetL, Gbu and OpuC, behaved similarly with and without added GB on parsley leaves (P > 0.05). In addition, preaccumulation of GB, triggered by osmotic stress prior to inoculation, failed to improve survival under low RH compared with osmotic stress without GB accumulation. CONCLUSIONS: Exogenous GB had a protective effect on L. monocytogenes cells from desiccation during survival on parsley leaves. This effect was independent of intracellular GB accumulation by the known uptake systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Presence of GB could improve the survival of L. monocytogenes to desiccation on leaf surfaces and nonbiological surfaces.

Effects of porcine bile on survival of Bacillus cereus vegetative cells and Haemolysin BL enterotoxin production in reconstituted human small intestine media.

AIMS: To determine the effects of porcine bile (PB) on Bacillus cereus vegetative cells and Haemolysin BL (HBL) enterotoxin production in reconstituted small intestine media (IM). METHODS AND RESULTS: The effects of PB on the growth of B. cereus vegetative cells in reconstituted IM at PB concentrations ranging between 0 and 3.0 g l(-1) were examined. Four gastric media (GM) named GM-J broth (JB), GM-chicken, GM-milk and GM-pea were prepared by mixing equal volumes of a gastric electrolyte solution containing pepsin with JB, chicken, semi-skimmed milk and pea soup, respectively. Bacillus cereus was inoculated at approx. 2 x 10(4) CFU ml(-1) into each GM at pH 5.0 for 30 min at 37 degrees C, then mixed to the same volume of double-strength JB (IM) and PB to give concentrations of between 0 and 3.0 g of PB per litre at pH 6.5 and incubated at 37 degrees C. The diarrhoeal B. cereus strain F4430/73 grew in IM-JB, IM-chicken and IM-milk at PB concentrations of up to 0.6, 1.5 and 1.2 g l(-1), respectively. Growth was observed in IM-pea at all concentrations tested. The highest PB concentrations allowing a 3 log B. cereus increase in IM-JB, IM-chicken, IM-milk and IM-pea after a 7-10 h incubation period were 0.3, 0.9, 0.9 and 3.0 g l(-1), respectively. The effect of PB on B. cereus cells was strongest in IM-JB, followed by IM-chicken, IM-milk and IM-pea. Haemolysin BL enterotoxin was detectable in IM-chicken, IM-whole milk, IM-semi-skimmed milk and IM-pea up to PB concentrations of only 0.6, 0.6, 0.3 and 0.9 g l(-1), respectively. The diarrhoeal B. cereus strain F4433/73 behaved similarly to B. cereus strain F4430/73, whereas the food strain TZ415 was markedly more susceptible to bile. CONCLUSIONS: The tolerance of B. cereus cells to PB strongly depends on the type of food contained in the IM. Bile tolerance is also subject to strain variation. SIGNIFICANCE AND IMPACT OF THE STUDY: The probability that B. cereus cells will grow in the small intestine, produce toxins and cause diarrhoea is likely to depend on the food they are ingested with, on the bile tolerance of the B. cereus strain, and on bile concentration.

Fate of Listeria spp. on parsley leaves grown in laboratory and field cultures.

AIMS: To investigate the population dynamics of Listeria monocytogenes and Listeria innocua on the aerial surfaces of parsley. METHODS AND RESULTS: Under 100% relative humidity (RH) in laboratory and regardless of the inoculum tested (10(3)-10(8) CFU per leaf), counts of L. monocytogenes EGDe, LO28, LmP60 and L. innocua CIP 80-12 tended towards approx. 10(5) CFU per leaf. Under low RH, Listeria spp. populations declined regardless to the inoculum size (10(4)-10(8) CFU per leaf). L. innocua CIP 80-12 survived slightly better than L. monocytogenes in the laboratory and was used in field cultures. Under field cultures, counts of L. innocua decreased more rapidly than in the laboratory, representing a decrease of 9 log(10) in 2 days in field conditions compared to a decrease of 4.5 log(10) in 8 days in the laboratory. Counts of L. innocua on tunnel parsley cultures were always higher (at least by 100 times) than those on unprotected parsley culture. CONCLUSIONS: Even with a high inoculum and under protected conditions (i.e. plastic tunnels), population of L. monocytogenes on the surface of parsley on the field would decrease by several log(10) scales within 2 days. SIGNIFICANCE AND IMPACT OF THE STUDY: Direct contamination of aerial surfaces of parsley with L. monocytogenes (i.e. through contaminated irrigation water) will not lead to contaminated produce unless it occurs very shortly before harvest.

Viable but non-culturable Listeria monocytogenes on parsley leaves and absence of recovery to a culturable state.

AIMS: To investigate the presence of viable but non-culturable Listeria monocytogenes during survival on parsley leaves under low relative humidity (RH) and to evaluate the ability of L. monocytogenes to recover from VBNC to culturable state under satured humidity. METHODS AND RESULTS: Under low RH (47-69%) on parsley leaves, the initial number of L. monocytogenes populations counted on non selective media (10(9) L. monocytogenes per leaf on TSA) was reduced by 6 log10 scales in 15 days, whereas number of viable L. monocytogenes counted under the microscope was reduced by 3-4 log10 scales, indicating the presence of VBNC cells. This was demonstrated on three L. monocytogenes strains (EGDe, Bug 1995 and LmP60). Changing from low to 100% RH permitted an increase of the culturable counts of L. monocytogenes and this growth was observed only when residual culturable cells were present. Moreover, VBNC L. monocytogenes inoculated on parsley leaves did not become culturable after incubation under 100% RH. CONCLUSIONS: Dry conditions induced VBNC L. monocytogenes on parsley leaves but these VBNC were likely unable to recover culturability after transfer to satured humidity. SIGNIFICANCE AND IMPACT OF STUDY: Enumeration on culture media presumably under-estimates the number of viable L. monocytogenes on fresh produce after exposure to low RH.

Survival of Bacillus cereus spores and vegetative cells in acid media simulating human stomach.

AIMS: To determine the fate of Bacillus cereus spores or vegetative cells in simulated gastric medium. METHODS AND RESULTS: The effects of acidity on the survival of B. cereus in a medium simulating human stomach content was followed on spores at pH 1.0-5.2, and on vegetative cells at pH 2.5-5.7. Gastric media (GM) were prepared by mixing equal volumes of a gastric electrolyte solution with J broth (JB), half-skim milk, pea soup and chicken. At pH 1.0 and 1.4, the number of spores slightly decreased in GM-JB and GM-pea soup and remained stable in GM-milk and GM-chicken. A rapid marked decrease (always higher than 2.0 log CFU ml(-1) in 2 h) in vegetative cell counts was observed at pH below 4.2, 4.0, 3.6 and 3.5 in GM-chicken, GM-JB, GM-milk and GM-pea soup, respectively. Between pH 5.0 and 5.3, B. cereus growth was observed in GM-JB (1.2 log CFU ml(-1) increase after 4 h) and in GM-pea soup (1.8 log CFU ml(-1) increase after 4 h). CONCLUSIONS: Bacillus cereus spores are very much more resistant to gastric acidity than vegetative cells. This resistance strongly depends on the type of food present in the GM. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that the probability that viable B. cereus cells enter the small intestine, where they can cause diarrhoea, strongly depends on the form of the ingested cells (spores or vegetative cells), on what food they are ingested with, and on the level of stomach acidity.

Grouping of tropical mid-altitude maize inbred lines on the basis of yield data and molecular markers.

The classification of maize inbred lines into heterotic groups is an important undertaking in hybrid breeding. The objectives of our research were to: (1) separate selected tropical mid-altitude maize inbred lines into heterotic groups based on grain yield data; (2) assess the genetic relationships among these inbred lines using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers; (3) examine the consistency between yield-based and marker-based groupings of the inbred lines. Thirty-eight tropical mid-altitude maize inbred lines were crossed to two inbred line testers representing the flint and dent heterotic pattern, respectively. The resulting testcrosses were evaluated in a trial at three locations for 2 years. Significant general combining ability (GCA) and specific combining ability (SCA) effects for grain yield were detected among the inbred lines. The tester inbred lines classified 23 of the 38 tested inbred lines into two heterotic groups based on SCA effects and testcross mean grain yields. This grouping was not related to endosperm type of the inbred lines. The outstanding performance of testcrosses of the remaining 15 inbred lines indicates the presence of significant genetic diversity that may allow the assignment of the lines into more than two heterotic groups. Diversity analysis of the 40 maize inbred lines using AFLP and SSR markers found high levels of genetic diversity among these lines and subdivided them into two main groups with subdivision into sub-groups consistent with breeding history, origin and parentage of the lines. However, heterotic groups formed using yield-based combining ability were different from the groups established on the basis of molecular markers. Considering the diversity of the genetic backgrounds of the mid-altitude inbred lines, the marker-based grouping may serve as the basis to design and carry out combining ability studies in the field to establish clearly defined heterotic groups with a greater genetic similarity within groups.

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.

Identification of bacteria in pasteurized zucchini purées stored at different temperatures and comparison with those found in other pasteurized vegetable purées.

One hundred nineteen isolates from a commercial zucchini purée stored at 4, 10, and 20 to 25 degrees C were fingerprinted using repetitive sequence-based PCR (REP-PCR) and classified into 35 REP types. One representative isolate of each REP type was subsequently identified by API50CHB/20E profile and partial rrs gene sequence analysis. Nine REP types were misidentified by the API system. Strains were misidentified as being in the Bacillus circulans (group 2) API taxon or in taxa with a low number of positive API characters such as Brevibacillus brevis. A phylogenetic analysis pointed to one new species of Bacillus and three new species of Paenibacillus among the misidentified REP types. Bacterial components in zucchini purée were compared phenotypically with those obtained in previous work on broccoli, carrot, leek, potato, and split pea purées, based on simple matching coefficient and unweighted pair group method with averages cluster analysis. Out of 254 strains, 69 strains previously identified as B. circulans (group 2) or B. circulans/B. macerans/B. polymyxa were assigned to a new Paenibacillus taxon phylogenetically related to P. azotofixans. Storage conditions at 4 degrees C favored the development of "B. macroides/B. maroccanus" and Paenibacillus spp. in zucchini purées and Paenibacillus spp. in other purées. Storage conditions at 20 to 25 degrees C favored the development of B. subtilis group (B. licheniformis and B. subtilis) and B. cereus group strains. At 10 degrees C, Paenibacillus spp. were always present at high frequencies, whereas the occurrence of B. macroides/B. maroccanus (in zucchini purées), B. cereus, and B. pumilus varied with the experiment.

Effects of epiphytic Enterobacteriaceae and pseudomonads on the growth of Listeria monocytogenes in model media.

Four Enterobacteriaceae (Enterobacter agglomerans and Rhanella aquatilis) and six pseudomonads (Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas putida) isolated from minimally processed green endive were coinoculated at 10 degrees C with Listeria monocytogenes in a minimal medium. Pseudomonads did not modify the growth of L. monocytogenes, whereas Enterobacteriaceae reduced its maximal population by 2 to 3 log CFU/ml. The same effect was observed in a diluted yeast extract medium supplemented with amino acids and glucose, in which L. monocytogenes grown alone reached 10(9) to 10(10) CFU/ml. In the same diluted yeast extract medium, not supplemented with glucose and amino acids, the maximal population of L. monocytogenes in the presence of both Enterobacteriaceae and pseudomonads was only slightly reduced (less than 0.5 log CFU/ml). Culture filtrates of the Enterobacteriaceae had no inhibitory activity on L. monocytogenes. The effect of the Enterobacteriaceae on L. monocytogenes growth was presumably due to a competition for glucose and/or amino acids.

Screening for clostridium botulinum type A, B, and E in cooked chilled foods containing vegetables and raw material using polymerase chain reaction and molecular probes.

A molecular method was used for the detection of Clostridium botulinum spores of type A, B, and E in commercial cooked and pasteurized vegetable purées and in the raw materials (vegetables and other ingredients). The method allowed the detection of less than 8 spores/g of product for C. botulinum type A, less than 1 spore/g for proteolytic type B, less than 21 spores/g for nonproteolytic type B, and less than 0.1 spore/g for type E. Thirty-seven samples of raw vegetables and ingredients were tested for the presence of C. botulinum type A, B, and E; 88 and 90 samples of vegetable purées were tested, respectively, for the presence of C. botulinum type A and B and for the presence of C. botulinum type E. All samples were negative, suggesting that the prevalence of C. botulinum in these vegetable purées and the raw ingredients is probably low.

Antimicrobial effect of rosemary extracts.

A rosemary extract commercially exploited (Oxy'less) as an antioxidant of lipids in foods was dissolved in ethanol (100 mg/ml), and the solution was tested against foodborne microorganisms. For gram-positive bacteria, the MIC of the ethanolic solution was 1% for Leuconostoc mesenteroides, 0.5% for Listeria monocytogenes, 0.5% for Staphylococcus aureus, 0.13% for Streptococcus mutans, and 0.06% for Bacillus cereus. It slowed the growth of Penicillium roquefortii and Botrytis cinerea. Up to 1% of the ethanolic solution had no activity on the gram-negative bacteria Escherichia coli, Salmonella Enteritidis, and Erwinia carotovora and on the yeasts Rhodotorula glutinis and Cryptococcus laurentii. Antibacterial activity of the rosemary extract was strongly influenced by the composition of the media. The MIC was reduced by low pH, high NaCl contents, and low temperatures. Low pH and high NaCl concentration had a synergistic effect on the MIC of the rosemary extract for S. aureus. Lipids, surface-active agents, and some proteins decreased its antibacterial activity, whereas pectin had no effect. The inhibitory effect was little modified by heat treatment (100 degrees C). The natural microflora of pasteurized zucchini broth was inhibited by 0.5% of the rosemary extract. The antibacterial activity was linked to the compounds extracted with hexane, which are presumably phenolic diterpenoids.

Effect of temperature on growth characteristics of Bacillus cereus TZ415.

The effect of temperature on the maximal specific growth rate was studied in Bacillus cereus between 5 and 40 degrees C cultivated in courgette broth and rich medium (J broth). B. cereus grown from 5 to 38 degrees C in rich medium. No growth was observed in courgette broth below 10 degrees C. The Arrhenius plot was fitted from experimental data of B. cereus grown in rich medium and at regulated pH, oxygen and temperature. Two domains which are separated by a critical temperature around 13 degrees C can be distinguished with regard to temperature dependence of maximal specific growth rate. Over the cold domain from 5 to 13 degrees C, the temperature characteristic was 2.6 fold higher than over the sub-optimal domain from 13 to 38 degrees C suggesting that the growth temperature regulates several metabolic pathways.

Prevalence, characterization and growth of Bacillus cereus in commercial cooked chilled foods containing vegetables.

In cooked-chilled and pasteurized vegetable products, initial numbers of Bacillus cereus were below 10 cfu g-1. Before the appearance of spoilage, numbers reached 6-8 log cfu g-1 at 20 degrees C and 4-6 log cfu g-1 at 10 degrees C. Bacillus cereus was not detected in samples stored at 4 degrees C. Ten percent of strains isolated from the products were able to grow at 5 degrees C and 63% at 10 degrees C. Bacillus cereus strains unable to degrade starch, a feature linked to the production of emetic toxin, did not grow at 10 degrees C and had a higher heat resistance at 90 degrees C. Using immunochemical assays, enterotoxin was detected in the culture supernatant fluid of 97.5% of the strains. All culture supernatant fluids were cytotoxic but important variations in the level of activity were found. Psychrotrophic isolates of B. cereus were unable to grow in courgette broth at 7 degrees C whereas they grew in a rich laboratory medium. At 10 degrees C, these isolates grew in both media but lag time in courgette broth was 20-fold longer than in the rich laboratory medium.

Isolation and characterization of antagonists for the biocontrol of the postharvest wound pathogen Botrytis cinerea on strawberry fruits.

Antagonistic bacteria and yeasts were isolated from the epiphytic flora of stored strawberry fruits and evaluated for their ability to protect strawberry fruit wounds after harvest against Botrytis cinerea. Among selected potential antagonists, three strains of Candida reukaufii (5L3, 10CL4, 10L2) and one strain of Candida pulcherima (10L8) still protected fruit wounds when applied at 10(3) CFU/wound, reducing lesion or conidiophore development. In the same conditions, two Enterobacteriaceae (10B1, 5B4) highly reduced pathogen development. Strain 5B4 was still highly inhibitory when inoculated at 10(2) CFU/wound. The six strains applied on fruits did not produce any significant change in color, brightness, and firmness of fruits. The two yeasts, 5L3 and 10L8, and particularly the two bacteria, 5B4 and 10B1, were selected for further studies. The four antagonists effectively colonized fruit wounds and strongly inhibited spore germination of B. cinerea in vitro. The bacterial cells surrounded the germinating spores of B. cinerea and attachment of 5L3 cells on germinating spores were additionally observed. Bacterial antagonists, particularly the strain 5B4, multiplied and rapidly used carbohydrates in strawberry fruit juice despite the low pH (pH 3.5). The efficiency of the bacterial antagonists on fruit wounds was related to their growth and nutritional properties.