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.

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.

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.

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.

Isolation, Identification, Prevalence, and Genetic Diversity of Bacillus cereus Group Bacteria From Different Foodstuffs in Tunisia.

Bacillus cereus group is widespread in nature and foods. Several members of this group are recognized as causing food spoilage and/or health issues. This study was designed to determine the prevalence and genetic diversity of the B. cereus group strains isolated in Tunisia from different foods (cereals, spices, cooked food, fresh-cut vegetables, raw and cooked poultry meats, seafood, canned, pastry, and dairy products). In total, 687 different samples were collected and searched for the presence of the B. cereus group after selective plating on MYP agar and enumeration of each sample. The typical pink-orange uniform colonies surrounded by a zone of precipitate were assumed to belong to the B. cereus group. One typical colony from each sample was subcultured and preserved as cryoculture. Overall, 191 (27.8%) food samples were found positive, giving rise to a collection of 191 B. cereus-like isolates. The concentration of B. cereus-like bacteria were below 103 cfu/g or ml in 77.5% of the tested samples. Higher counts (>104 cfu/g or ml) were found in 6.8% of samples including fresh-cut vegetables, cooked foods, cereals, and pastry products. To verify whether B. cereus-like isolates belonged to the B. cereus group, a PCR test targeting the sspE gene sequence specific of the group was carried out. Therefore, 174 isolates were found to be positive. Food samples were contaminated as follows: cereals (67.6%), pastry products (46.2%), cooked food (40.8%), cooked poultry meat (32.7%), seafood products (32.3%), spices (28.8%), canned products (16.7%), raw poultry meat (9.4%), fresh-cut vegetables (5.0%), and dairy products (4.8%). The 174 B. cereus isolates were characterized by partial sequencing of the panC gene, using a Sym'Previous software tool to assign them to different phylogenetic groups. Strains were distributed as follows: 61.3, 29.5, 7.5, and 1.7% in the group III, IV, II, and V, respectively. The genetic diversity was further assessed by ERIC-PCR and PFGE typing methods. PFGE and ERIC-PCR patterns analysis allowed discriminating 143 and 99 different profiles, respectivey. These findings, associated to a relatively higher prevalence of B. cereus group in different foods, could be a significant etiological agent of food in Tunisia.

Real time PCR gene profiling and detection of Salmonella using a novel target: The siiA gene.

The objective of this study was to develop and evaluate a SYBR Green real time PCR method for the specific detection of Salmonella spp using a novel target, the siiA gene. Primer specificity testing was done on a panel of 76 Salmonella strains and 32 non-Salmonella strains. The primers directed against the siiA gene amplified all Salmonella strains tested, while non-Salmonella strains were not amplified. The melting temperatures of the 107 bp amplicons were consistently specific as they gave melting peaks around 75.5°C. The precision of the assay, based on intra and inter-run variations, was shown to be widely acceptable. In the second part of this study, 45 Salmonella strains were screened for the presence of 6 virulence-associated genes (sopB, cat2, safC, sefB and SC1248) located in several Salmonella Pathogenicity Islands (SPIs) and the spvC gene from the Salmonella virulence plasmid. The prevalence of these genes ranged from 51% to 100%. Variable virulence gene profiles were obtained even within the same serotype.

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.

Genetic diversity of food-isolated Salmonella strains through Pulsed Field Gel Electrophoresis (PFGE) and Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR).

All over the world, the incidence of Salmonella spp contamination on different food sources like broilers, clams and cow milk has increased rapidly in recent years. The multifaceted properties of Salomnella serovars allow the microorganism to grow and multiply in various food matrices, even under adverse conditions. Therefore, methods are needed to detect and trace this pathogen along the entire food supply network. In the present work, PFGE and ERIC-PCR were used to subtype 45 Salmonella isolates belonging to different serovars and derived from different food origins. Among these isolates, S. Enteritidis and S. Kentucky were found to be the most predominant serovars. The Discrimination Index obtained by ERIC-PCR (0.85) was slightly below the acceptable confidence value. The best discriminatory ability was observed when PFGE typing method was used alone (DI = 0.94) or combined with ERIC-PCR (DI = 0.93). A wide variety of profiles was observed between the different serovars using PFGE or/and ERIC-PCR. This diversity is particularly important when the sample origins are varied and even within the same sampling origin.

Biochemical and micrographic evidence of Escherichia coli membrane damage during incubation in egg white under bactericidal conditions.

Bacterial membranes are often thought to be the main targets of the antimicrobial activity of egg white. In order to test this hypothesis, the state of the membranes of Escherichia coli K-12 cells during either bactericidal (45°C) or bacteriostatic (30°C) incubation in egg white at natural alkaline pH was studied by biochemical methods. Namely, the permeability of the outer membrane was evaluated through its ability to incorporate a hydrophobic fluorescent probe (1-N-phenylnaphthylamine), and the permeability of the cytoplasmic membrane was evaluated through the release of a specific intracellular enzyme (ß-galactosidase). The bacteria were observed by atomic force microscopy in order to support the biochemical results. At 45°C, the outer membrane of E. coli K-12 incorporated the hydrophobic probe, suggesting that it was disrupted. In addition, the cytoplasmic ß-galactosidase was released at this temperature. The atomic force microscopy analysis revealed the formation of spheroplasts, which provided further evidence of the cell wall disruption and a progressive release of cellular contents. At 30°C, biochemical and micrographic experiments confirmed that membrane integrity was preserved. These techniques provide a useful approach for studying the mechanisms of bacterial cell death in egg white.

Use of pulsed-field gel electrophoresis to characterize the heterogeneity and clonality of Salmonella serotype Enteritidis, Typhimurium and Infantis isolates obtained from whole liquid eggs.

Salmonella is a well-documented pathogen known to occur in a wide range of foods, especially poultry products. The most frequently reported food-sources of human infection are eggs and egg products. In this study, in order to describe Salmonella contamination of egg products, 144 liquid egg samples were collected from 3 different egg-breaking plants during the 3 sampling periods. Salmonella detection was performed on raw samples stored at 2 degrees C for 2 days (D+2) and on pasteurised samples stored at 2 degrees C at D+2 and at shelf-life date. Salmonella was detected in 130 of the 144 raw egg samples collected and in 11 of the 288 pasteurised egg samples analysed. 740 Salmonella isolates were collected and serotyped: 14 serovars were demonstrated. A great diversity, particularly during summer, was noted. The dominant serovars were S. Enteritidis, S. Typhimurium and S. Infantis, mainly found in whole raw egg products. Typing of 325 isolates of S. Enteritidis, 54 isolates of S. Typhimurium and 58 isolates of S. Infantis was carried out by macrorestriction of the genomic DNA with XbaI and SpeI enzymes followed by pulsed field gel electrophoresis (PFGE). The Salmonella Enteritidis isolates could be grouped into 3 clusters. Cluster 1 was predominant at all 3 egg-breaking companies during the different sampling periods. This cluster seemed to be adapted to the egg-breaking plants. Cluster 2 was linked to plant 1 and cluster 3 to plant 3. Two main clusters of Salmonella Typhimurium were demonstrated. Cluster A was mainly found at plant 2 during autumn. Plant 3 was contaminated by all the Salmonella Typhimurium genotypes but in a more sporadic manner during the three seasons studied. Plant 1 seemed to be less contaminated by Salmonella Typhimurium than the others. Three clusters and 2 genotypes of Salmonella Infantis were shown. The main cluster, cluster alpha, consisted of 75% of the S. Infantis isolates and was mainly found during summer at plants 1 and 3. Plant 2 seemed to be less contaminated by S. Infantis. In this study, molecular typing demonstrated that, although certain clusters were common to all three companies, specific clusters, notably of S. Enteritidis were present at each plant.

Isolation and characterization of a psychrotolerant toxin producer, Bacillus weihenstephanensis, in liquid egg products.

A psychrotolerant bacteria of the Bacillus cereus group was found responsible for the spoilage of whole liquid egg products. By sequencing a 16S rRNA region and performing a PCR amplification of specific 16S rRNA and cspA signatures, a Bacillus weihenstephanensis was identified. Characterization of this strain shows its ability to grow in defined medium as well as in whole liquid egg at refrigerated temperatures. The strain isolated possesses genes encoding for hemolysin BL, nonhemolytic enterotoxin, and B. cereus enterotoxins and produces enterotoxins with cytotoxic activity in whole liquid egg, even at refrigerated temperatures. The isolate exhibits a clear ability to stick and form biofilms on stainless steel, the most common material used in egg breaking factories, as well as on model hydrophilic (glass) and hydrophobic (polytetrafluoroethylene) materials. These findings show the necessity to monitor for Bacillus contamination in egg products that are often used in the composition of particularly susceptible finished products such as cream, dessert, dairy, meat, and seafood.