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.

Introduction Bacteria and/or their antigens have been implicated in the pathogenesis of reactive arthritis (ReA). Several studies have reported the presence of bacterial antigens and nucleic acids of bacteria other than those specified by diagnostic criteria for ReA in joint specimens from patients with ReA and various arthritides. The present study was conducted to detect any bacterial DNA and identify bacterial species that are present in the synovial tissue of Tunisian patients with reactive arthritis and undifferentiated arthritis (UA) using PCR, cloning and sequencing. Methods We examined synovial tissue samples from 28 patients: six patients with ReA and nine with UA, and a control group consisting of seven patients with rheumatoid arthritis and six with osteoarthritis (OA). Using broad-range bacterial PCR producing a 1,400-base-pair fragment from the 16S rRNA gene, at least 24 clones were sequenced for each synovial tissue sample. To identify the corresponding bacteria, DNA sequences were compared with sequences from the EMBL (European Molecular Biology Laboratory) database. Results Bacterial DNA was detected in 75% of the 28 synovial tissue samples. DNA from 68 various bacterial species were found in ReA and UA samples, whereas DNA from 12 bacteria were detected in control group samples. Most of the bacterial DNAs detected were from skin or intestinal bacteria. DNA from bacteria known to trigger ReA, such as Shigella flexneri and Shigella sonnei , were detected in ReA and UA samples of synovial tissue and not in control samples. DNA from various bacterial species detected in this study have not previously been found in synovial samples. Conclusion This study is the first to use broad-range PCR targeting the full 16S rRNA gene for detection of bacterial DNA in synovial tissue. We detected DNA from a wide spectrum of bacterial species, including those known to be involved in ReA and others not previously associated with ReA or related arthritis. The pathogenic significance of some of these intrasynovial bacterial DNAs remains unclear.

IntroductionTunisia has one of the highest burdens of extrapulmonary tuberculosis (EPTB) among tuberculosis (TB) cases but the contribution of MTBC-mediated human EPTB is unknown. EPTB diagnosis is challenging due to the paucibacillary nature of clinical samples. Therefore, a need of a simplified molecular method for sensitive and specific TB detection and differentiation of MTBC members caused EPTB remains a priority to an early diagnosis, optimize successful anti-TB treatment and minimize transmission. We evaluated the performance of a single tube tetraplex Taq Man real time PCR for EPTB detection and differentiation between MTBC members directly on extrapulmonary samples.Materials and methodsExtrapulmonary samples obtained from clinically suspected EPTB patients from 2013 to April 2015 were tested by Ziehl Neelsen Staining, mycobacterial culture and qPCR assay for RD1, RD9, RD12 and ext-RD9 targets (MTBC-RD qPCR). The performance of qPCR was compared to a reference standard based on MTBC culture and/or at least two criteria of a composite reference standard (CRS) including clinical, radiological, histopathological and therapeutic findings.ResultsEPTB was identified in 157/170 (92.4%) of included patients of whom 99 (63%) were confirmed by culture and 58 (36.9%) by CRS criteria. The sensitivity and specificity of qPCR, in comparison to the reference standard were 100% (157/157) and 92.3% (12/13), respectively. The sensitivity of qPCR was statistically significant as compared to culture and smear microscopy (P< 0.001). QPCR results showed M. bovis identification in 77.1% of extrapulmonary samples in occurrence to lymphadenitis infection. M. tuberculosis and M.bovis BCG were detected in 21.6% and 1.3% of cases, respectively.ConclusionsMTBC–RD qPCR proved to be a rapid and sensitive assay for simultaneously TB detection and MTBC members identification on extrapulmonary samples within 1.5 days after sample receipt. Its high sensitivity could make this method a useful tool in diagnosing TB in addition to routine conventional methods and TB clinical parameters.

BackgroundThe genetic diversity of M. bovis in Tunisia is still underestimated despite the implementation of an eradication program. The lack of data about spatial distribution of the M. bovis population hinders the control of bovine tuberculosis (bTB) progress. This study represents the largest molecular analysis of M. bovis isolates in Tunisia. It is aimed to upgrade the understanding of bTB epidemiology and the geographical distribution of the infection. Tuberculosis research was performed in cattle (n = 149) with TB-compatible lesions collected over 5 months from a slaughterhouse located in Sfax, Tunisia.ResultsNinety-four animals were found to be infected by M. bovis and two others by M. caprae. Spoligotyping revealed twenty-five patterns, SB0120, SB0134, and SB0121 being the most prevalent profiles (36.4%, 11.4%, and 7.2%, respectively). Three new spoligotypes were detected: SB2345, SB2344 and SB2343. MIRU-VNTR analysis classified the isolates in seventy-three profiles and showed a large genotypic variety observed within the main spoligotype which was split into several MIRU-VNTR types: 29 in SB0120 (h = 0.983), 10 in SB0134 (h = 0.981) and 7 in SB0121 (h = 1). Genotyping revealed a common pattern in different geographic regions. It also showed that Sfax, located in southern-Tunisia, represents a high-risk area with an elevated genetic diversity.ConclusionsSpatial analysis may provide insights into disease transmission, which affects the effectiveness of eradication campaigns in cattle.Electronic supplementary materialThe online version of this article (10.1186/s12917-017-1314-y) contains supplementary material, which is available to authorized users.

A combined enrichment/ newly developed invA TaqMan® real-time PCR (qPCR) method as a screening assay to detect Salmonella spp. in 500 naturally food matrices is evaluated. DNA template for qPCR was extracted from an overnight pre-enriched sample in buffered peptone water using lysis–guanidine isothiocyanate method. Heterologous internal amplification control (IAC) was incorporated during qPCR assays and co-amplified with the invA gene of the target pathogen. InvA qPCR exhibited 100% specificity when testing 94 Salmonella strains (inclusivity) and 32 non-Salmonella strains (exclusivity). The qPCR showed a consistent detection of two copies of the invA gene/PCR reaction, a good intra- and inter-run reproducibility with a good PCR efficiency (89.6%). QPCR was sensitive and showed Salmonella detection at 8.5 × 100 CFU mL-1 of artificially spiked poultry meat -BWP solution in less than 40 cycles. When analyzing 500 different food matrices and comparing the results with the ISO 6579:2002 conventional culture method, the sensitivity and specificity were 100 and 76.6%, respectively. QPCR showed Salmonella spp. DNA in raw poultry meat 27/45 (60%), milk 31/93 (33.3%), raw red meat 5/13 (38.5%), and fish 11/46 (23.9%) samples. The prevalence of Salmonella spp. in cakes, dairy, cooked meals, charcuterie products using qPCR was 11/14 (26.8%), 5/22 (22.7%), 32/150 (21.3%), and 5/20 (25%), respectively, compared to 0% as demonstrated by culture. S. Anatum was the most common serovar found associated with red meat compared to S. kentucky isolated from fish and poultry meat. In conclusion, our study is the first to use a combined enrichment/invA qPCR method as a screening assay to detect Salmonella DNA in different types of commercialized food in Southern Tunisia. QPCR results indicate that Salmonella contamination is common in milk and in other types of food samples.