Toxigenic potential and antimicrobial susceptibility of Bacillus cereus group bacteria isolated from Tunisian foodstuffs

International audienceBackground: Despite the importance of the B. cereus group as major foodborne pathogens that may cause diarrheal and/or emetic syndrome(s), no study in Tunisia has been conducted in order to characterize the pathogenic potential of the B. cereus group. The aim of this study was to assess the sanitary potential risks of 174 B. cereus group strains isolated from different foodstuffs by detecting and profiling virulence genes (hblA, hblB, hblC, hblD, nheA, nheB, nheC, cytK, bceT and ces), testing the isolates cytotoxic activity on Caco-2 cells and antimicrobial susceptibility towards 11 antibiotics. Results: The entertoxin genes detected among B. cereus isolates were, in decreasing order, nheA (98.9%), nheC (97.7%) and nheB (86.8%) versus hblC (54.6%), hblD (54.6%), hblA (29.9%) and hblB (14.9%), respectively encoding for Non-hemolytic enterotoxin (NHE) and Hemolysin BL (HBL). The isolates are multi-toxigenic, harbouring at least one gene of each NHE and HBL complexes associated or not to bceT, cytK-2 and ces genes. Based on the incidence of virulence genes, the strains were separated into 12 toxigenic groups. Isolates positive for cytK (37,9%) harbored the cytK-2 variant. The detection rates of bceT and ces genes were 50.6 and 4%, respectively. When bacteria were incubated in BHI-YE at 30°C for 18 h and for 5 d, 70.7 and 35% of the strains were shown to be cytotoxic to Caco-2 cells, respectively. The cytotoxicity of B. cereus strains depended on the food source of isolation. The presence of virulence factors is not always consistent with cytotoxicity. However, different combinations of enterotoxin genetic determinants are significantly associated to the cytotoxic potential of the bacteria. All strains were fully sensitive to rifampicin, chloramphenicol, ciprofloxacin, and gentamycin. The majority of the isolates were susceptible to streptomycin, kanamycin, erythromycin, vancomycin and tetracycline but showed resistance to ampicillin and novobiocin. Conclusion: Our results contribute data that are primary to facilitate risk assessments in order to prevent food poisoning due to B. cereus group

Identification of the bacteria and their metabolic activities associated with the microbial spoilage of custard cream desserts

The famous French dessert “ile flottante” consists of a sweet egg white foam floating on a vanilla custard cream,which contains highly nutritive raw materials, including milk, sugar and egg. Spoilage issues are therefore a keyconcern for the manufacturers. This study explored the bacterial diversity of 64 spoiled custard cream dessertsmanufactured by 2 French companies. B. cereus group bacteria, coagulase negative Staphylococcus, Enterococcus and Leuconostoc spp. were isolated from spoiled products. Thirty-one bacterial isolates representative of the main spoilage species were tested for their spoilage abilities. Significant growth and pH decrease were observed regardless of species. While off-odours were detected with B. cereus group and staphylococci, yoghurt odours were detected with Enterococcus spp. and Leuconostoc spp. B. cereus group bacteria produced various esters and several compounds derived from amino acid and sugar metabolism. Most Staphylococci produced phenolic compounds. Enterococcus spp. and Leuconostoc spp. isolates produced high levels of compounds derived from sugar metabolism. Each type of spoilage bacteria was associated with a specific volatile profile and lactic acid was identified as a potential marker of spoilage of custard cream-based desserts. These findings provide valuable information for manufacturers to improve food spoilage detection and prevention of chilled desserts made with milk and egg

Spoilage of egg-based chilled desserts : bacteria and mechanisms involved

Parmi les desserts à base d’ovoproduits, l’île flottante est reconnue comme particulièrement sensible d’un point de vue microbiologique car sa commercialisation souffre de la survenue intempestive d’altérations que les industriels souhaitent maîtriser. Les travaux réalisés au cours de cette thèse avaient pour objectif de mieux appréhender ces phénomènes afin de mieux les contrôler. Nous avons montré que l’altération de l’île flottante concernait principalement la crème anglaise et qu’elle s’accompagnait d’un développement bactérien conséquent, d’une fréquente diminution du pH et de modifications sensorielles liées à l’aspect et à l’odeur. Les principales bactéries détectées ont été identifiées comme appartenant au groupe Bacillus cereus et aux genres Staphylococcus et Enterococcus. Le blanc d’œuf pasteurisé, utilisé pour la fabrication des œufs en neige, s’est avéré être une source de contamination possible.Cependant, l’implication de bactéries installées sous forme de biofilms dans l’environnement de production ou véhiculées par d’autres ingrédients a aussi été fortement envisagée. La ré-inoculation, en culture pure, d’une collection bactérienne représentative dans de la crème anglaise stérile a montré que différents types de modifications sensorielles et physico-chimiques s’exprimaient d’un genre bactérien à l’autre et qu’ils étaient notamment corrélés à la capacité des bactéries à consommer les sucres et les protéines de la crème anglaise et à produire des métabolites dont des composés volatils odorants. Ces résultats à l’appui, différents tests ont pu être proposés, pAmong the chilled egg products-based desserts, the French dessert “île flottante” is recognized as particularly sensitive from a microbiological point of view, because marketing is suffering from untimely spoilage occurrence that manufacturers wish to control. The work done in this thesis aimed to better understand these phenomena in order to better control them. We have shown that the dessert spoilage mainly concerned the custard cream and it was characterized by high bacterial count, frequent pH decreasing and sensory changes of appearance and smell. The main bacteria detected were identified as belonging to the Bacillus cereus group and Staphylococcus and Enterococcus genera. The possible involvement of bacteria from the pasteurized egg white, used for the egg white foaming, in the dessert spoilage issue was established.However, the involvement of bacteria from biofilms installed in the production environment or provided by other ingredients was also strongly suspected. The spoilage potential assessment of pure culture of a representative bacterial collection in sterile custard cream has shown that different types of sensory and physicochemical changes were expressed according to bacterial genus and that these changes were particularly correlated with the ability of bacteria to consume sugars and proteins of custard and to produce various volatile compounds with specific odorous. With these results, various tests have been proposed for a better control of the white egg batches orientation according to their microbiological quality and so to guarantee their safety wit

Microbial Spoilage of Eggs and Egg Products

This chapter focuses on microbial spoilage of shell eggs and egg products and reviews the spoilage characteristics, according to the type of product and the flora involved. The methods available for monitoring, reducing, and detecting the spoilage of egg and egg products also are reviewed.[br/][br/][br/]Under healthy breeding conditions, the egg content is generally sterile. It can be contaminated by a diversified microbiota-containing food spoilage microorganisms. Moreover, egg breaking systematically involves the contamination of egg white and egg yolk through contact with the spoiled shells, thus giving rise to highly perishable egg products. This article describes the spoilage characteristic and microbial flora of eggs and egg products. Additionally, several methodologies are highlighted, allowing for the control, reduction, or detection of the spoilage of egg and egg product

Microbial Milk Spoilage

This article focuses on liquid milk spoilage (raw, pasteurized, and UHT), the characteristics of the spoilage, the species and the enzymatic activity involved, and the methods used to detect or predict milk spoilage.[br/][br/][br/]Milk and dairy products are important components of the diet worldwide. The quality and shelf life of liquid milk as well as dairy products are often compromised by flavor, odors, and visual defects arising from the bacterial growth and activities of heat-stable enzymes produced by psychrotrophic bacteria before processing. This article focuses on the liquid milk spoilage characteristics, as well as the flora and enzymatic activities involved, and reviews some methods allowing for the detection or prediction of milk spoilage

Specific industrial bacteria adhering on stainless steel equipments are able to further contaminate and spoil liquid egg products

Introduction: Egg products resulting from the breaking of shell eggs, giving rise to egg yolk and egg white, or whole egg, are used in highly diversified sectors for their functionalities (coloring, thickening, emulsifying and gelling properties), such as in dairy, meat and seafood products. Liquid egg products are generally pasteurized at moderate temperatures because of the heat sensitivity of the proteins and stored at refrigeration temperatures. Contamination by Salmonella, which remains the major safety preoccupation of this sector, is now under the control of breeding surveillance and of effective pasteurization processes. However, the manufacturers now encounter new types of microorganisms selected by the preservation processes themselves. The spoilage flora resisting to pasteurization may adhere on the surfaces of the pipelines or inside the stainless steel equipment. This flora may lead to the contamination of the egg product passing through the pipelines, hence causing a massive and fast contamination of the final egg product. The spoilage flora can lead to technological and/or organoleptic problems (coagulation, changes in colour or in flavour) due to the production of hydrolytic enzymes, even at low temperature. The aim of this study was to investigate the whole egg spoilage potential of isolates collected from stainless steel equipment in the egg processing industry.[br/]Material and methods: Sterile stainless steel chips were suspended inside the pipelines of 7 egg processing companies, at two process levels, before and after the pasteurizer, and at two seasons (summer and winter). After two months, the adhering flora was harvested by sonication and scrapping in tryptone salt, followed by plating onto BHI-YE agar and incubation for 1 to 7 day(s) at 30°C, with or without oxygen. After incubation, the colonies were identified by 16S rDNA sequencing. Isolates were selected for further in vitro investigation on their ability to adhere on stainless steel surfaces. After bacterial adhesion, sterile liquid whole egg was added on the chips and incubated at various temperatures. Enumeration and visual observations were carried out in order to detect contamination and spoilage, i.e. transformation of whole egg into a white slurry cream.Results: A broad range of diversity was highlighted even if a majority of facultative aero-anaerobic Gram positive bacteria was found, such as Streptococcus, Micrococcus, Staphylococcus, Microbacterium, Lysinibacillus and Bacillus. The adhesion capacity of the harvested isolates did not depend on their location in the process (before or after pasteurization), neither on the season (cold or warm) nor on the company. The level of adhesion depended more on the species than on the genus. While the whole collection was able to grow in liquid whole egg, only the genera Lysinibacillus and Bacillus were able to provoke a detectable spoilage.Significance: The adhesion of microorganisms on industrial surfaces is frequently observed in the food industry. Poorly controlled, adhering bacteria can be the source of food product contamination and/or food spoilage, and therefore can lead to high economic losses for the industry

Microbial Quality of Industrial Liquid Egg White: Assumptions on Spoiling Issues in Egg-Based Chilled Desserts

As a 1st step, this study aimed at investigating the microbial quality of liquid egg white in a French eggprocessing company. Thirty raw and 33 pasteurized liquid egg white samples were analyzed. Pasteurization was globally found efficient on mesophilic contaminants (1.7 ± 1.6 and 0.8 ± 0.9 log CFU/mL in raw and pasteurized samples, respectively), including for the control of Salmonella. However,Gram-positive enterococci were still detected in the pasteurized samples. As a 2nd step, a representative bacterial collection was built for exploring the spoilage issue in egg-based chilled desserts. Custard cream was chosen as growth medium since this food is widely used for the production of French chilled desserts. All of the 166 isolates of the bacterial collection were shown to be able to grow and to induce spoilage of the custard cream at refrigeration temperature (10 °C). Several spoilage types were highlighted in the custard cream, on the basis of changes regarding pH, consistency, production of holes or gas. As a 3rd step, bacterial enzymatic activities were explored on custard cream-based agar media. The bacterial collection was reduced to 43 isolates, based on further selection regarding the genera and the spoilage types previously highlighted. Albeit to different degrees, all these isolates were able to produce proteases. A large part of these isolates also expressed lipolytic and amylolytic activities. This studyemphasizes the need to control egg white contamination and especially with Gram-positive heat-resistant Enterococi, in order to guarantee the shelf life of egg-based chilled desserts

Detection and quantification of staphylococcal enterotoxin A in foods with specific and sensitive polyclonal antibodies

International audienceStaphyloccocus aureus is reported to be one of the most frequent pathogens involved in food-borne diseases associated with dairy products, especially with raw milk cheese. Some strains produce enterotoxins as Staphylococcal enterotoxin A (SEA) which is involved in 75% of food poisoning outbreaks. Actually few methods are both sensitive and specific enough for confirming the diagnosis of staphylococcal food poisoning. In this work, an Enzyme-Linked ImmunoSorbent Assay (ELISA) was set up and optimised to detect SEA in milk and cheese. Various anti-SEA antibodies: polyclonal sera, anti-peptide polyclonal sera and monoclonal antibodies, used for the capture and or detection steps were compared and analysed. The mouse anti-whole SEA polyclonal serum, used as detection detected 32 pg/ml SEA in buffer and 64 pg/ml in milk. These concentrations were well under the limit set for food safety. This assay was also highly specific of SEA and no cross-reaction was observed with the other staphylococcal enterotoxins. In contaminated cheese samples the time of enterotoxin extraction was reduced by using ultrafiltration method instead of dialysis and the detection limits were 1.5-2.5 and 1.9-3 times more sensitive than a commercial kit and the official method, respectively. Preliminary study of SEA detection with the piezoelectric immunosensor allowed detecting and quantifying SEA within 10 min in unprocessed food but the sensitivity was not sufficient. The ELISA assay with mouse antibodies is likely suitable for SEA routine detection not only in dairy product but also in various foods as sauce and liver mousse. (C) 2012 Elsevier Ltd. All rights reserved

The volatilome allows assigning custard cream spoilage to a specific bacterial genus

National audienceCustard cream-based desserts, made up of highly nutritive raw materials, are easily spoiled by specific microbial contaminants. Little is known about these spoilage issues. The objective of this study was to fully characterize the type of bacteria and the respective metabolic activities involved in the spoilage of custard cream-based desserts. Materials &Methods (700) The level of bacterial contamination of 64 spoiled commercial custard cream-based desserts was measured (PCA, 24h, 30°C) and colonies were identified based on their 16SrDNA sequences. Over a bacterial collection of 565 isolates, 33 isolates of the 4 dominant genera were submitted to individual challenge tests (inoculum size of 3 log CFU/mL, incubation at 10°C for 21d in sterile custard cream). Microbial growth, sensorial evaluation, and biochemical changes (acidification; hydrolysis of sugar, protein and fats; production of volatile compounds and organic acids) were measured and the set of data was subjected to a principal component analysis (R software). Results (360) The spoilage was characterised by high bacterial counts (> 6 log CFU/mL) with predominance of Gram-positive bacteria, including Bacillus cereus group cells. The metabolic activities were highly dependent on the genus. The volatilome fingerprint appeared as a specific marker allowing assigning each type of spoilage to a specific bacterial genus. Significance (125) A better understanding of custard cream spoilage issues could help manufacturers improve detection and prevention