Isolation, Identification, and Characterization of Bacillus cereus Group Bacteria Isolated from the Dairy Farm Environment and Raw Milk in Tunisia.

Members of the Bacillus cereus group are well-known opportunistic foodborne pathogens. In this study, the prevalence, hemolytic activity, antimicrobial resistance profile, virulence factor genes, genetic diversity by enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) genotyping, and adhesion potential were investigated in isolates from a Tunisian dairy farm environment and raw milk. A total of 200 samples, including bedding, feces, feed, liquid manure, and raw bovine milk, were examined. Based on PCR test targeting sspE gene, 59 isolates were detected. The prevalence of B. cereus group isolates in bedding, feces, liquid manure, feed, and raw milk was 48%, 37.8%, 20%, 17.1%, and 12.5%, respectively. Out of the tested strains, 81.4% showed ß-hemolytic on blood agar plates. An antimicrobial resistance test against 11 antibiotics showed that more than 50% of the isolates were resistant to ampicillin and novobiocin, while a high sensitivity to other antibiotics tested was observed in most isolates. The distribution of enterotoxigenic genes showed that 8.5% and 67.8% of isolates carried hblABCD and nheABC, respectively. In addition, the detection rate of cytotoxin K (cytk), enterotoxin T (bceT), and ces genes was 72.9%, 64.4%, and 5.1%, respectively. ERIC-PCR fingerprinting genotype analysis allowed discriminating 40 different profiles. The adhesion potential of B. cereus group on stainless steel showed that all isolates were able to adhere at various levels, from 1.5 ± 0.3 to 5.1 ± 0.1 log colony-forming unit (CFU)/cm2 for vegetative cells and from 2.6 ± 0.4 to 5.7 ± 0.3 log CFU/cm2 for spores. An important finding of the study is useful for updating the knowledge of the contamination status of B. cereus group in Tunisia, at the dairy farm level.

Into the Jungle of Biological Agents of Foodborne Diseases: Time to Put Some Order for the French Risk Manager.

Consumers can be exposed to many foodborne biological hazards that cause diseases with varying outcomes and incidence and, therefore, represent different levels of public health burden. To help the French risk managers to rank these hazards and to prioritize food safety actions, we have developed a three-step approach. The first step was to develop a list of foodborne hazards of health concern in mainland France. From an initial list of 335 human pathogenic biological agents, the final list of "retained hazards" consists of 24 hazards, including 12 bacteria (including bacterial toxins and metabolites), 3 viruses and 9 parasites. The second step was to collect data to estimate the disease burden (incidence, Disability Adjusted Life Years) associated with these hazards through food during two time periods: 2008-2013 and 2014-2019. The ranks of the different hazards changed slightly according to the considered period. The third step was the ranking of hazards according to a multicriteria decision support model using the ELECTRE III method. Three ranking criteria were used, where two reflect the severity of the effects (Years of life lost and Years lost due to disability) and one reflects the likelihood (incidence) of the disease. The multicriteria decision analysis approach takes into account the preferences of the risk managers through different sets of weights and the uncertainties associated with the data. The method and the data collected allowed to estimate the health burden of foodborne biological hazards in mainland France and to define a prioritization list for the health authorities.

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 key concern for the manufacturers. This study explored the bacterial diversity of 64 spoiled custard cream desserts manufactured 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.

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

BACKGROUND: 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.

Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages.

BACKGROUND: A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity. RESULTS: We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages. CONCLUSIONS: Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.

Antimicrobial activity of lysozyme isoforms: Key molecular features.

Increasing bacterial resistance towards antibiotics has stimulated research for novel antimicrobials. Proteins acting on bacterial membranes could be a solution. Lysozyme has been proven active against E. coli by disruption of both outer and cytoplasmic membranes, with dry-heating increasing lysozyme activity. Dry-heated lysozyme (DH-L) is a mixture of isoforms (isoaspartyl, native-like and succinimide lysozymes), giving rise to two questions: what effects does each form have, and which physicochemical properties are critical as regards the antibacterial activity? These issues were investigated by fractionating DH-L, analyzing structural properties of each fraction, and testing each fraction in vivo on bacteria and in vitro on membrane models. Positive net charge, hydrophobicity and molecular flexibility of the isoforms seem key parameters for their interaction with E. coli membranes. The succinimide lysozyme fraction, the most positive, flexible and hydrophobic, shows the highest antimicrobial activity, induces the strongest bacterial membrane disruption and is the most surface active on model lipid monolayers. Moreover, each fraction appears less efficient than DH-L against E. coli, indicating a synergetic cooperation between lysozyme isoforms. The bacterial membrane modifications induced by one isoform could facilitate the subsequent action of the other isoforms.

A mixed methods study of ruminant brucellosis in central-eastern Tunisia.

In this study, we conducted an investigation to determine the true prevalence of bovine and ovine brucellosis in central-eastern Tunisia. A total of 1134 veterinary samples taken from 130 ruminant herds were screened for brucellosis using IS711-based real-time PCR assay. Sera collected from the ruminants were tested using the Rose Bengal test and indirect enzyme-linked immunosorbent assay. Based on serological and molecular results, the true adjusted animal population level prevalence was 23.5 % in cattle, against 13.5 % in sheep. In addition, the true adjusted herd level prevalence of brucellosis was 55.6 % in cattle and 21.8 % in sheep. A statistically significant association was found between vaginal and milk shedding for ruminants. In addition, our results showed that Brucella abortus could be responsible for bovine and ovine brucellosis. Multivariable logistic regression analysis at the animal population level indicated that age and origin variables were important risk factors for cattle. However, age and abortion variables were found to be associated with ovine brucellosis. At the herd level, risk factors for Brucella positivity were as follows: abortion and herd composition for cattle against herd composition, mortality rates, and hygiene for sheep. Animal hygiene, food quality, and sanitary practices on the farm should be applied as strategies to control brucellosis in herds.

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

Lysozyme 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.

Native and dry-heated lysozyme interactions with membrane lipid monolayers: Lipid packing modifications of a phospholipid mixture, model of the Escherichia coli cytoplasmic membrane.

Antimicrobial resistance is currently an important public health issue. The need for innovative antimicrobials is therefore growing. The ideal antimicrobial compound should limit antimicrobial resistance. Antimicrobial peptides or proteins such as hen egg white lysozyme are promising molecules that act on bacterial membranes. Hen egg white lysozyme has recently been identified as active on Gram-negative bacteria due to disruption of the outer and cytoplasmic membrane integrity. Furthermore, dry-heating (7 days and 80 °C) improves the membrane activity of lysozyme, resulting in higher antimicrobial activity. These in vivo findings suggest interactions between lysozyme and membrane lipids. This is consistent with the findings of several other authors who have shown lysozyme interaction with bacterial phospholipids such as phosphatidylglycerol and cardiolipin. However, until now, the interaction between lysozyme and bacterial cytoplasmic phospholipids has been in need of clarification. This study proposes the use of monolayer models with a realistic bacterial phospholipid composition in physiological conditions. The lysozyme/phospholipid interactions have been studied by surface pressure measurements, ellipsometry and atomic force microscopy. Native lysozyme has proved able to absorb and insert into a bacterial phospholipid monolayer, resulting in lipid packing reorganization, which in turn has lead to lateral cohesion modifications between phospholipids. Dry-heating of lysozyme has increased insertion capacity and ability to induce lipid packing modifications. These in vitro findings are then consistent with the increased membrane disruption potential of dry heated lysozyme in vivo compared to native lysozyme. Moreover, an eggPC monolayer study suggested that lysozyme/phospholipid interactions are specific to bacterial cytoplasmic membranes.

Locked nucleic acid probe-based real-time PCR for the diagnosis of Listeria monocytogenes in ruminants.

Because of its high fatality rate, listeriosis ranks among the most important infectious diseases worldwide. Although ruminants are known as natural reservoirs for Listeria monocytogenes and a possible source of human listeriosis, studies of the prevalence and risk factors associated with ruminant listeriosis are limited to some developed countries. Therefore, this report describes the development of a real-time PCR targeting the hly gene for the absolute quantification of L. monocytogenes based on circular and linear DNA standards. Results show that the PCR that uses circular plasmid as a template gave a 2.6-7.89 greater threshold cycle number than did equimolar linear standards. No cross-amplification was observed when bacteria commonly found in bovine and ovine diseases were tested. The PCR achieved good intra and inter-run reproducibility and a detection limit of 6.1 copies of linear plasmid per reaction. This PCR was then applied to 1134 samples taken from 378 Tunisian ruminants. Based on the test sensitivity (90%) and specificity (100%), the true individual animal prevalence of listeriosis was 5.7% in cattle and 10.2% in sheep. In addition, the true herd-level prevalence was 50.1% in cattle and 26.7% in sheep. A multivariable logistic regression analysis at the animal-population level indicated that for cattle, the variables strata and mastitis were important risk factors, whereas for sheep, the variables strata, age and abortion were found to be associated with listeriosis. At the herd level, risk factors for Listeria test-positivity they were: abortion, herd composition and silage storage for cattle, whereas for sheep were: management system, cleaning frequency, silage storage and floor type. Animal hygiene, food quality and sanitary practices on the farm should be applied as strategies to control this pathogen in ruminant herds.

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.

Prevalence, PFGE typing, and antibiotic resistance of Bacillus cereus group isolated from food in Morocco.

This article reports the prevalence and antibiotic resistance of the Bacillus cereus group isolated from different foods (milk and dairy products, spices, and rice salad) in Morocco. In total, 402 different food samples collected from 2008 to 2010 were analyzed by microbiological methods to isolate B. cereus. The strains were subjected to a polymerase chain reaction test in order to verify whether they belonged to the B. cereus group. Sixty-four of all isolates (15.9%) were found to be positive. Among the sources, B. cereus strains from milk and dairy products constituted the largest proportion of isolates (33/64; 51.6%) followed by spices (22/64; 34.4%) and salad with rice (9/64; 14.1%). The genetic diversity of the strains of B. cereus group was examined by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA digested with SmaI. The enzyme restriction profiles showed a high degree of polymorphism among the strains. The results showed that PFGE analysis could reveal the genetic differences among B. cereus strains. Investigation of antibiotic-resistance profiles showed that isolates were resistant to ampicillin (98.4%), tetracycline (90.6%), oxacillin (100%), cefepime (100%), and penicillin (100%), and were susceptible to chloramphenicol (67.2%), erythromycin (84.4%), and gentamicin (100%). The results of this study indicated that B. cereus could be a significant etiological agent of food poisoning in Morocco because of its high prevalence. Also, we demonstrated that the majority of strains came from milk and dairy products. However, additional research involving cytotoxicity tests is needed to more evaluate this sanitary risk.

Isolation, Partial Characterization and Application of Bacteriophages in Eradicating Biofilm Formation by Bacillus cereus on Stainless Steel Surfaces in Food Processing Facilities.

The Bacillus cereus (B. cereus) group is a widespread foodborne pathogen with a persistent ability to form biofilm, and with inherent resistance to traditional treatment in the food industry. Bacteriophages are a promising biocontrol agent that could be applied to prevent or eliminate biofilms formation. We have described, in this study, the isolation from sewage samples and preliminary characterization of bacteriophages that are active against the B. cereus group. The effectiveness of phage treatment for reducing B. cereus attachment and biofilms on stainless steel surfaces has been also assessed using three incubation periods at different titrations of each phage. Out of 62 phages isolated, seven showed broad-spectrum lytic action against 174 B. cereus isolates. All selected phages appeared to be of the Siphoviridae family. SDS-PAGE proved that two phages have a similar profile, while the remainder are distinct. All isolated phages have the same restriction pattern, with an estimated genome size of around 37 kb. The isolated bacteriophages have been shown to be effective in preventing biofilm formation. Reductions of up to 1.5 log10 UFC/cm2 have been achieved, compared to the untreated biofilms. Curative treatment reduced the bacterial density by 0.5 log10 UFC/cm2. These results support the prospect of using these phages as a potential alternative strategy for controlling biofilms in food systems.

The Role of Ovotransferrin in Egg-White Antimicrobial Activity: A Review.

Eggs are a whole food which affordably support human nutritional requirements worldwide. Eggs strongly resist bacterial infection due to an arsenal of defensive systems, many of which reside in the egg white. However, despite improved control of egg production and distribution, eggs remain a vehicle for foodborne transmission of Salmonella enterica serovar Enteritidis, which continues to represent a major public health challenge. It is generally accepted that iron deficiency, mediated by the iron-chelating properties of the egg-white protein ovotransferrin, has a key role in inhibiting infection of eggs by Salmonella. Ovotransferrin has an additional antibacterial activity beyond iron-chelation, which appears to depend on direct interaction with the bacterial cell surface, resulting in membrane perturbation. Current understanding of the antibacterial role of ovotransferrin is limited by a failure to fully consider its activity within the natural context of the egg white, where a series relevant environmental factors (such as alkalinity, high viscosity, ionic composition, and egg white protein interactions) may exert significant influence on ovotransferrin activity. This review provides an overview of what is known and what remains to be determined regarding the antimicrobial activity of ovotransferrin in egg white, and thus enhances understanding of egg safety through improved insight of this key antimicrobial component of eggs.

Identification of New Antimicrobial Peptides that Contribute to the Bactericidal Activity of Egg White against Salmonella enterica Serovar Enteritidis at 45 °C.

A recent work revealed that egg white (EW) at 45 °C exhibits powerful bactericidal activity against S. enterica serovar Enteritidis, which is surprisingly little affected by removal of the >10 kDa EW proteins. Here, we sought to identify the major EW factors responsible for this bactericidal activity by fractionating EW using ultrafiltration and nanofiltration and by characterizing the physicochemical and antimicrobial properties of the resulting fractions. In particular, 22 peptides were identified by nano-LC/MS-MS and the bactericidal activities of representative peptides (with predicted antimicrobial activity) were further assessed. Two peptides (FVPPVQR and GDPSAWSWGAEAHS) were found to be bactericidal against S. enterica serovar Enteritidis at 45 °C when provided in an EW environment. Nevertheless, these peptides contribute only part of this bactericidal activity, suggesting other, yet to be determined, antimicrobial factors.

Isolation of Harveyi clade Vibrio spp. collected in aquaculture farms: How can the identification issue be addressed?

Aquaculture is a fast growing industry with its development hampered by bacterial diseases. Vibriosis caused by Harveyi clade strains is known for causing heavy loss especially in shrimp aquaculture farms. For farm treatment and pathogen spread management, veterinarians and researchers need reliable bacterial identification tools. A range of identification methods have been presented for Vibrio spp. in recent literature but little feedback on their performance have been made available to this day. This study aims at comparing Vibrio spp. identification methods and providing guidance on their use. Fifty farms were sampled and bacterial colonies were isolated using specific culture media before microscopic analysis and genomic profiling using ERIC-PCR. A preliminary identification step was carried out using MALDI-ToF mass spectrometry. Four methods were compared for strain identification on 14 newly isolated Harveyi clade Vibrio spp. strains: whole genome sequencing (digital DNA DNA Hybridization (dDDH)), 5 MLSA schemes, ferric uptake regulation (fur) and lecithin-dependent haemolysin (ldh) single gene based identification methods. Apart from dDDH which is a reference method, no technique could identify all the isolates to the species level. The other tested techniques allowed a faster, cheaper but sub genus clade identification which can be interesting when absolute precision is not required. In this regard, MALDI-ToF and fur based identification seemed especially promising.

Effect of temperature and time of storage on protein stability and anti-salmonella activity of egg white.

Hen egg white contains numerous molecules of interest for human health, including antimicrobial proteins. Little information is available concerning changes in the antimicrobial activity of egg white during storage; therefore, we analyzed the potential of egg white to inhibit growth of Salmonella enterica serovar Enteritidis following storage at 4, 20, or 37°C for 30 days prior to inoculation. Egg white displayed higher anti-Salmonella activity after a few days of storage at 20 and 37°C. The rate of increase in activity was more rapid and pronounced at the higher temperature. However, egg white stored at 20°C retained higher antimicrobial activity than that of egg white stored at 4 or 37°C, when the entire storage period is taken in consideration. In contrast, storage of egg at 37°C for more than 14 days reduced the bacteriostatic potential of egg white. Statistical analyses revealed a correlation between pH and the antimicrobial activity of egg white. Moreover, diminished antimicrobial activity was associated with degradation of ovalbumin and ovotransferrin, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry. However, the fluctuation in anti-Salmonella activity of egg white could not be related to any variation of trypsin-like, chymotrypsin-like, or gelatinolytic activities that potentially account for degradation of antimicrobial egg white proteins.

Egg-White Proteins Have a Minor Impact on the Bactericidal Action of Egg White Toward Salmonella Enteritidis at 45°C.

Salmonella enterica serovar Enteritidis is noted for its ability to survive the harsh antibacterial activity of egg white which is presumed to explain its occurrence as the major food-borne pathogen associated with the consumption of eggs and egg products. Liquid egg white is a major ingredient for the food industry but, because of its thermal fragility, pasteurization is performed at the modest temperature of 57°C (for 2-6 min). Unfortunately, such treatment does not lead to sufficient reduction in S. Enteritidis contamination, which is a clear health concern when the product is consumed without cooking. However, egg white is able to limit S. Enteritidis growth due to its alkaline pH, iron deficiency and multiple antimicrobial proteins. This anti-Salmonella activity of egg white is temperature dependent and becomes bactericidal once the incubation temperature exceeds 42°C. This property is exploited in the highly promising pasteurization treatment (42-45°C for 1-5 days) which achieves complete killing of S. Enteritidis. However, the precise mechanism and the role of the egg-white proteins are not fully understood. Here, the impact of exposure of S. Enteritidis to egg white-based media, with or without egg-white proteins (>10 kDa), under bactericidal conditions (45°C) was explored by measuring survival and global expression. Surprisingly, the bactericidal activity of egg white at 45°C was only slightly affected by egg-white proteins indicating that they play a minor role in the bactericidal activity observed. Moreover, egg-white proteins had minimal impact on the global-gene-expression response to egg white such that very similar, major regulatory responses (20% genes affected) were observed both with and without egg-white proteins following incubation for 45 min at 45°C. Egg-white proteins caused a significant change in expression for just 64 genes, including the psp and lysozyme-inhibitor responses genes which is suggestive of an early membrane perturbation effect. Such damage was supported by disruption of the proton motive force by egg-white proteins. In summary, the results suggest that low-mass components of egg white are largely responsible for the bactericidal activity of egg white at 45°C.

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 (K d of 5.3 nM) of enterobactin over the iron-free form (K d 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.

Staphylococcus aureus virulence expression is impaired by Lactococcus lactis in mixed cultures.

Staphylococcus aureus is responsible for numerous food poisonings due to the production of enterotoxins by strains contaminating foodstuffs, especially dairy products. Several parameters, including interaction with antagonistic flora such as Lactococcus lactis, a lactic acid bacterium widely used in the dairy industry, can modulate S. aureus proliferation and virulence expression. We developed a dedicated S. aureus microarray to investigate the effect of L. lactis on staphylococcal gene expression in mixed cultures. This microarray was used to establish the transcriptomic profile of S. aureus in mixed cultures with L. lactis in a chemically defined medium held at a constant pH (6.6). Under these conditions, L. lactis hardly affected S. aureus growth. The expression of most genes involved in the cellular machinery, carbohydrate and nitrogen metabolism, and stress responses was only slightly modulated: a short time lag in mixed compared to pure cultures was observed. Interestingly, the induction of several virulence factors and regulators, including the agr locus, sarA, and some enterotoxins, was strongly affected. This work clearly underlines the complexity of L. lactis antagonistic potential for S. aureus and yields promising leads for investigations into nonantibiotic biocontrol of this major pathogen.