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

Detection of AmpC and ESBL-producing Enterobacterales isolated from urinary tract infections in Tunisia

Urinary tract infections (UTIs) are the most frequent human infections in community and hospitals. This study aimed to determine the distribution of bacterial uropathogens among urinary tract infections diagnosed within the regional hospital Houcine Bouzaiene (Gafsa, South West Tunisia) during a survey of 54 days from the 8th of November to the 31st of December 2017. Enterobacterales strains were tested for antimicrobial resistance by disk diffusion method and extended-spectrum beta-lactamase (ESBL) production was tested by double-disc synergy test. Strains were further subjected to a molecular assessment of ESBL and AmpC beta-lactamase production by PCR. Overall, 173 bacterial isolates were studied, out of which 91.3% were Enterobacterales. Escherichia coli was the dominant pathogen, followed by Klebsiella pneumoniae. High to moderate resistance rates were observed, ranging from 66% to 90.7% for penicillin, from 6.7% to 18.6% for cephalosporins and from 16.2% to 25.4% for fluoroquinolones. Enterobacterales with decreased susceptibility to third-generation cephalosporins (3rd GC) carried several resistance genes: bIaCTX-M group 1 and group 9, and ACC and FOX AmpC beta-lactamase genes. Overall, ESBLs and AmpC beta-lactamases were detected in 57% and 14% of the 3rd GC-resistant isolates, respectively. This study proved the high potential of K. pneumaniae species to develop resistance against commonly used antibiotics. Thus, rigorous monitoring of the antibiotic resistance of clinical pathogens have to be implemented in Tunisia. Our results are very relevant to evaluate efficiency of the Tunisian therapeutic strategies against UTIs and adapt them to the emerging problem of antimicrobial resistance

Maitrise des risques de contamination des produits alimentaires tunisiens par le groupe Bacillus cereus

This thesis focused on evaluating the level of risk represented by Bacillus cereus group bacteria in Tunisian food and testing the effectiveness of their control by treating industrial surfaces with bacteriophages. A collection of 191 isolates was created from 687 food matrices. Nearly 40% of the isolates were found to belong to the group, with high genetic diversity (143 PFGE profiles and 99 ERIC-PCR profiles) and an intermediate thermal profile (signatures 16S rDNA-1 m and-2 p). Nearly 60% of the group's isolates belong to the phylogenetic group III, which is potentially pathogenic. Spores have a higher rate of adhesion than vegetative cells. Twelve toxigenic groups have been identified.At least one of the genes of each of the NHE and HBL complexes are present, whether or not associated with bceT, cytK 2 and these. After 18 hours of incubation at 30°C, nearly 71% of the isolates are cytotoxic. Different combinations of virulence factors are associated with cytotoxic potential and a clear link appears between cytotoxicity and food type. The collection has been shown to be sensitive to many antibiotics, while it is resistant to ampicillin and novobiocin. Of the 7 bacteriophages selected, 5 have a unique protein profile while all have similar genome size and restriction profiles. They are used to prevent the formation of biofilms and to treat them. This work confirms the health risk associated with the presence of the B. cereus group in Tunisian foods and the promising role of bacteriophages as biocontrol tools.Cette thèse s’est intéressée à évaluer le niveau de risque représenté par les bactéries du groupe Bacillus cereus dans des aliments tunisiens et à tester l’efficacité de leur contrôle par le traitement des surfaces industrielles par des bactériophages. Une collection de 191 isolats a été créée à partir de 687 matrices alimentaires. Près de 40 % des isolats se sont avérés appartenir au groupe, avec une forte diversité génétique (143 profils PFGE et 99 profils ERIC-PCR) et un profil thermique intermédiaire (signatures 16S rDNA-1 m et-2 p). Près de 60 % des isolats du groupe appartiennent au groupe phylogénétique III, potentiellement pathogène. Les spores présentent majoritairement un taux d’adhésion plus fort que les cellules végétatives. Douze groupes toxinogènes ont été mis en évidence.Au moins un des gènes de chacun des complexes NHE et HBL sont présents, associés ou non à bceT, cytK 2 et ces. Après 18 h d’incubation à 30°C, près de 71% des isolats sont cytotoxiques. Différentes combinaisons de facteurs de virulence sont associées au potentiel cytotoxique et un lien apparait clairement entre cytotoxicité et type d’aliment. La collection s’est montrée sensible à de nombreux antibiotiques, alors qu’elle présente une résistance à l'ampicilline et à la novobiocine. Sur les 7 bactériophages sélectionnés, 5 possèdent un profil protéique unique alors qu’ils présentent tous une taille de génome et des profils de restriction similaires. Ils permettent de prévenir et de les traiter la formation de biofilms. Ce travail confirme le risque sanitaire lié à la présence du groupe B. ce

Contamination risks control of food products by B. cereus group.

Cette thèse s’est intéressée à évaluer le niveau de risque représenté par les bactéries du groupe Bacillus cereus dans des aliments tunisiens et à tester l’efficacité de leur contrôle par le traitement des surfaces industrielles par des bactériophages. Une collection de 191 isolats a été créée à partir de 687 matrices alimentaires. Près de 40 % des isolats se sont avérés appartenir au groupe, avec une forte diversité génétique (143 profils PFGE et 99 profils ERIC-PCR) et un profil thermique intermédiaire (signatures 16S rDNA-1 m et-2 p). Près de 60 % des isolats du groupe appartiennent au groupe phylogénétique III, potentiellement pathogène. Les spores présentent majoritairement un taux d’adhésion plus fort que les cellules végétatives. Douze groupes toxinogènes ont été mis en évidence.Au moins un des gènes de chacun des complexes NHE et HBL sont présents, associés ou non à bceT, cytK 2 et ces. Après 18 h d’incubation à 30°C, près de 71% des isolats sont cytotoxiques. Différentes combinaisons de facteurs de virulence sont associées au potentiel cytotoxique et un lien apparait clairement entre cytotoxicité et type d’aliment. La collection s’est montrée sensible à de nombreux antibiotiques, alors qu’elle présente une résistance à l'ampicilline et à la novobiocine. Sur les 7 bactériophages sélectionnés, 5 possèdent un profil protéique unique alors qu’ils présentent tous une taille de génome et des profils de restriction similaires. Ils permettent de prévenir et de les traiter la formation de biofilms. Ce travail confirme le risque sanitaire lié à la présence du groupe B. cerThis thesis focused on evaluating the level of risk represented by Bacillus cereus group bacteria in Tunisian food and testing the effectiveness of their control by treating industrial surfaces with bacteriophages. A collection of 191 isolates was created from 687 food matrices. Nearly 40% of the isolates were found to belong to the group, with high genetic diversity (143 PFGE profiles and 99 ERIC-PCR profiles) and an intermediate thermal profile (signatures 16S rDNA-1 m and-2 p). Nearly 60% of the group's isolates belong to the phylogenetic group III, which is potentially pathogenic. Spores have a higher rate of adhesion than vegetative cells. Twelve toxigenic groups have been identified.At least one of the genes of each of the NHE and HBL complexes are present, whether or not associated with bceT, cytK 2 and these. After 18 hours of incubation at 30°C, nearly 71% of the isolates are cytotoxic. Different combinations of virulence factors are associated with cytotoxic potential and a clear link appears between cytotoxicity and food type. The collection has been shown to be sensitive to many antibiotics, while it is resistant to ampicillin and novobiocin. Of the 7 bacteriophages selected, 5 have a unique protein profile while all have similar genome size and restriction profiles. They are used to prevent the formation of biofilms and to treat them. This work confirms the health risk associated with the presence of the B. cereus group in Tunisian foods and the promising role of bacteriophages as biocontrol tools

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