The three lipocalins of egg-white: only Ex-FABP inhibits siderophore-dependent iron sequestration by Salmonella Enteritidis

Salmonella Enteritidis is the most prevalent food-borne pathogen associated with egg-related outbreaks in the European Union. During egg colonization, S. Enteritidis must resist the powerful anti-bacterial activities of egg white (EW) and overcome ovotransferrin-imposed iron-restriction (the most important anti-bacterial mechanism of EW). Many pathogens respond to iron restriction by secreting iron-chelating chemicals called siderophores but EW contains a siderophore-sequestering “lipocalin” protein (Ex-FABP) that is predicted to limit the usefulness of siderophores in EW. S. Enteritidis produces two siderophores: enterobactin, which is strongly bound by Ex-FABP; and the di-glucosylated enterobactin-derivative, salmochelin (a so-called “stealth” siderophore), which is not recognized by Ex-FABP. Thus, production of salmochelin may allow S. Enteritidis to escape Ex-FABP-mediated growth inhibition under iron restriction although it is unclear whether its EW concentration is sufficient to inhibit pathogens. Further, two other lipocalins (Cal-γ and α-1-ovoglycoprotein) are found in EW but their siderophore sequestration potential remains unexplored. In addition, the effect of EW lipocalins on the major EW pathogen, S. Enteritidis, has yet to be reported. We overexpressed and purified the three lipocalins of EW and investigated their ability to interact with the siderophores of S. Enteritidis, as well as their EW concentrations. The results show that Ex-FABP is present in EW at concentrations (5.1 μM) sufficient to inhibit growth of a salmochelin-deficient S. Enteritidis mutant under iron restriction but has little impact on the salmochelin-producing wildtype. Neither Cal-γ nor α-1-ovoglycoprotein bind salmochelin or enterobactin, nor do they inhibit iron-restricted growth of S. Enteritidis. However, both are present in EW at significant concentrations (5.6 and 233 μM, respectively) indicating that α-1-ovoglycoprotein is the 4th most abundant protein in EW, with Cal-γ and Ex-FABP at 11th and 12th most abundant. Further, we confirm the preference (16-fold) of Ex-FABP for the ferrated form (Kd of 5.3 nM) of enterobactin over the iron-free form (Kd of 86.2 nM), and its lack of affinity for salmochelin. In conclusion, our findings show that salmochelin production by S. Enteritidis enables this key egg-associated pathogen to overcome the enterobactin-sequestration activity of Ex-FABP when this lipocalin is provided at levels found in EW

Bacterial diversity on stainless steel surfaces of egg processing companies and potential of selected isolates to spoil liquid whole egg products

AimsTo assess the bacterial diversity in the French egg processing industry and to explore the adhesion and spoilage potential of selected bacteria.Methods and ResultsSterile stainless steel chips were suspended for 2 months inside the pipelines of seven egg processing companies, before and after the pasteurizer, at warm and cold seasons. After exposure, the bacterial diversity was assessed by 16S rDNA sequencing. The 231 collected isolates were mainly facultative anaerobic Gram positive bacteria, such as Streptococcus, Staphylococcus, Bacillus and Kocuria. Sixty‐five representative isolates were further characterized in vitro regarding the potential for adhesion and egg product spoilage. A high diversity was observed from one genus to another. Kocuria and Rothia isolates showed significantly higher adhesion than the isolates of the other genera. Only the isolates belonging to the genera Bacillus and Lysinibacillus, associated with high enzymatic activities on a solid egg‐based medium, were able to induce spoilage of liquid whole egg.ConclusionsBacteria collected on stainless steel surfaces placed in egg processing industries could be associated to liquid egg product spoilage.Significance and Impact of the StudyThis study provides new insights on the bacterial contamination in egg processing companies and represents a first step for the effective control of undesirable bacteria in liquid egg products

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

Native lysozyme and dry-heated lysozyme interactions with membrane lipid monolayers: lateral reorganization of LPS monolayer, model of the E. coli outer membrane

International audienceLysozyme is mainly described active against Gram-positive bacteria, but is also efficient against some Gram-negative species. Especially, it was recently demonstrated that lysozyme disrupts Escherichia coli membranes. Moreover, dry-heating changes the physicochemical properties of the protein and increases the membrane activity of lysozyme. In order to elucidate the mode of insertion of lysozyme into the bacterial membrane, the interaction between lysozyme and a LPS monolayer mimicking the E. coli outer membrane has been investigated by tensiometry, ellipsometry, Brewster angle microscopy and atomic force microscopy. It was thus established that lysozyme has a high affinity for the LPS monolayer, and is able to insert into the latter as long as polysaccharide moieties are present, causing reorganization of the LPS monolayer. Dry-heating increases the lysozyme affinity for the LPS monolayer and its insertion capacity; the resulting reorganization of the LPS monolayer is different and more drastic than with the native protein

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

Global gene-expression analysis of the response of Salmonella Enteritidis to egg-white exposure reveals multiple egg-white-imposed stress responses

Chicken egg white protects the embryo from bacterial invaders by presenting an assortment of antagonistic activities that combine together to both kill and inhibit growth. The key features of the egg-white anti-bacterial system are iron restriction, high pH, antibacterial peptides and proteins, and viscosity. Salmonella enterica serovar Enteritidis is the major pathogen responsible for egg-borne infection in humans, which is partly explained by its exceptional capacity for survival under the harsh conditions encountered within egg white. However, at temperatures up to 42 ˚C, egg white exerts a much stronger bactericidal effect on S. Enteritidis than at lower tempertaures, although the mechanism of egg-white-induced killing is only partly understood. Here, for the first time, the impact of exposure of S. Enteritidis to egg white under bactericidal conditions (45 ˚C) is explored by global-expression analysis. A large-scale (18.7% of genome) shift in transcription is revealed suggesting major changes in specific aspects of S. Enteritidis physiology: induction of egg-white related stress-responses (envelope damage, exposure to heat and alkalinity, and translation shutdown); shift in energy metabolism from respiration to fermentation; and enhanced micronutrient provision (due to iron and biotin restriction). Little evidence of DNA damage or redox stress was obtained. Instead, data are consistent with envelope damage resulting in cell death by lysis. A surprise was the high degree of induction of hexonate/hexuronate utilisation genes, despite no evidence indicating the presence of these substrates in egg white

Longitudinal brain metabolic changes from amnestic mild cognitive impairment to Alzheimer's disease.

International audienceA sensitive marker for monitoring progression of early Alzheimer's disease would help to develop and test new therapeutic strategies. The present study is aimed at investigating brain metabolism changes over time, as a potential monitoring marker, in patients with amnestic mild cognitive impairment, according to their clinical outcome (converters or non-converters), and in relation to their cognitive decline. Seventeen amnestic mild cognitive impairment patients underwent magnetic resonance imaging and 18FDG-positron emission tomography scans both at inclusion and 18 months later. Baseline and follow-up positron emission tomography data were corrected for partial volume effects and spatially normalized using magnetic resonance imaging data, scaled to the vermis and compared using SPM2. 'PET-PAC' maps reflecting metabolic per cent annual changes were created for correlation analyses with cognitive decline. In the whole sample, the greatest metabolic decrease concerned the posterior cingulate-precuneus area. Converters had significantly greater metabolic decrease than non-converters in two ventro-medial prefrontal areas, the subgenual (BA25) and anterior cingulate (BA24/32). PET-PAC in BA25 and BA24/32 combined allowed complete between-group discrimination. BA25 PET-PAC significantly correlated with both cognitive decline and PET-PAC in the hippocampal region and temporal pole, while BA24/32 PET-PAC correlated with posterior cingulate PET-PAC. Finally, the metabolic change in BA8/9/10 was inversely related to that in BA25 and showed relative increase with cognitive decline, suggesting that compensatory processes may occur in this dorso-medial prefrontal region. The observed ventro-medial prefrontal disruption is likely to reflect disconnection from the hippocampus, both indirectly through the cingulum bundle and posterior cingulate cortex for BA24/32, and directly through the uncinate fasciculus for BA25. Altogether, our findings emphasize the potential of 18FDG-positron emission tomography for monitoring early Alzheimer's disease progression

Impact of comprehensive geriatric assessment on survival, function, and nutritional status in elderly patients with head and neck cancer: protocol for a multicentre randomised controlled trial (EGeSOR)

BACKGROUND: Survival is poorer in elderly patients with head and neck squamous cell carcinomas [HNSCCs] than in younger patients. Possible explanations include a contribution of co-morbidities to mortality, frequent refusal of standard therapy, and the use of suboptimal treatments due to concern about toxicities. The Comprehensive Geriatric Assessment [CGA] is a multidimensional assessment of general health that can help to customise treatment and follow-up plans. The CGA has been proven effective in several health settings but has not been evaluated in randomised studies of patients with cancer. Our aim here was to assess the impact of the CGA on overall survival, function, and nutritional status of elderly patients with HNSCC. METHODS/DESIGN: EGeSOR is an open-label, multicentre, randomised, controlled, parallel-group trial in patients aged 70 years or older and receiving standard care for HNSCC. The intervention includes four components: the CGA conducted by a geriatrician before cancer treatment, participation of the same geriatrician in cancer treatment selection, a standardised geriatric therapeutic intervention designed by the same geriatrician; and geriatric follow-up for 24 months. The primary endpoint, assessed after 6 months, is a composite criterion including death, functional impairment [Activities of Daily Living score decrease ≥2], and weight loss ≥10%. Secondary endpoints include progression-free survival, unscheduled admissions, quality of life, treatment toxicities, costs, and completion of the planned cancer treatment. A centralised online system is used to perform 1:1 randomisation with a minimisation algorithm for centre, age, T and N stages, and tumour site [oral, oropharyngeal, hypopharyngeal, or laryngeal]. The estimated sample size is 704 patients, who are being recruited by 14 centres in 9 French cities. DISCUSSION: EGeSOR is the first randomised trial of the CGA in elderly cancer patients. We expect the CGA to have direct clinical benefits on the management of elderly patients with HNSCC. If this expectation is fulfilled, the trial may lead to modifications of the management model for elderly patients with cancer. TRIAL REGISTRATION: Trial registration: NCT0202506

Egg white versus Salmonella Enteritidis! A harsh medium meets a resilient pathogen

Salmonella enterica serovar Enteritidis is the prevalent egg-product-related food-borne pathogen. The egg-contamination capacity of S. Enteritidis includes its exceptional survival capability within the harsh conditions provided by egg white. Egg white proteins, such as lysozyme and ovotransferrin, are well known to play important roles in defence against bacterial invaders. Indeed, several additional minor proteins and peptides have recently been found to play known or potential roles in protection against bacterial contamination. However, although such antibacterial proteins are well studied, little is known about their efficacy under the environmental conditions prevalent in egg white. Thus, the influence of factors such as temperature, alkalinity, nutrient restriction, viscosity and cooperative interactions on the activities of antibacterial proteins in egg white remains unclear. This review critically assesses the available evidence on the antimicrobial components of egg white. In addition, mechanisms employed by S. Enteritidis to resist egg white exposure are also considered along with various genetic studies that have shed light upon egg white resistance systems. We also consider how multiple, antibacterial proteins operate in association with specific environmental factors within egg white to generate a lethal protective cocktail that preserves sterility