A combined enrichment/ newly developed
TaqMan
real-time PCR (qPCR) method as a screening assay to detect
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
gene of the target pathogen.
qPCR exhibited 100% specificity when testing 94
strains (inclusivity) and 32 non-
strains (exclusivity). The qPCR showed a consistent detection of two copies of the
gene/PCR reaction, a good intra- and inter-run reproducibility with a good PCR efficiency (89.6%). QPCR was sensitive and showed
detection at 8.5 × 10
CFU mL
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
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
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.
Anatum was the most common serovar found associated with red meat compared to
kentucky isolated from fish and poultry meat. In conclusion, our study is the first to use a combined enrichment/
qPCR method as a screening assay to detect
DNA in different types of commercialized food in Southern Tunisia. QPCR results indicate that
contamination is common in milk and in other types of food samples.
stems were harvested in two seasons: winter and spring (February and May 2021). In this study, we investigated the antioxidant (DPPH, ABTS, FRAP and TAC) and antimicrobial activities, total phenolic ...contents and total flavonoid contents of the obtained extracts (hexane, ethyl acetate and methanol). The results showed that ethyl acetate extract from stems harvested in winter exhibited the highest antioxidant activity, while ethyl acetate extract from the stems harvested in spring showed the most potent antibacterial and antifungal activities. To explain these differences, we investigated the phytochemical composition of these two extracts using liquid chromatography coupled with mass spectrometry. Therefore, 45 compounds were detected, from which we identified 20 compounds (flavonoids, triterpenoids, chalcones and phenolic acids); some were specific to the harvest month while others were common for both periods. Some of the major compounds detected in ethyl acetate (spring) were dihydrochalcone (Kanzonol Y, 8.2%) and flavanone (sophoraflavanone G, 5.9%), previously recognized for their antimicrobial effects. We therefore concluded that the difference in activities observed for the two harvest periods depends on the chemical composition of the extracts and the relative abundance of each compound.
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
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
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
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
-like isolates. The concentration of
-like bacteria were below 10
cfu/g or ml in 77.5% of the tested samples. Higher counts (>10
cfu/g or ml) were found in 6.8% of samples including fresh-cut vegetables, cooked foods, cereals, and pastry products. To verify whether
-like isolates belonged to the
group, a PCR test targeting the
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
isolates were characterized by partial sequencing of the
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
group in different foods, could be a significant etiological agent of food in Tunisia.
In Tunisia, almost 77% of clinically and bacteriologically diagnosed cases of extrapulmonary tuberculosis (EPTB) are zoonotic TB, caused by M. bovis. Although several studies have analyzed bovine TB ...in cattle in Tunisia, no study has evaluated the risk of transmission to humans in such an endemic country. We aimed to study the genetic diversity of M. bovis human isolates, to ascertain the causes of human EPTB infection by M. bovis and to investigate the distribution and population structure of this species in Tunisia.
A total of 110 M. bovis isolates taken from patients with confirmed EPTB were characterized by spoligotyping and MIRU-VNTR typing methods.
Among the 15 spoligotypes detected in our study, 6 (SB0120, SB0121, SB2025, SB1200, SB1003 and SB0134) were the most prevalent (83.5%) of which SB0120, SB0121 and SB2025 were the most prevailing. MIRU-VNTR typing method showed a high genotypic and genetic diversity. The genetic differentiation based on MIRU-VNTR was significant between populations from South East (Tataouine, Medenine) and Central West (Gafsa, Sidi Bouzid, Kasserine) regions. Of note, 13/15 (86.7%) spoligotypes detected in our study were previously identified in cattle in Tunisia with different frequencies suggesting a peculiar ability of some genotypes to infect humans. Using combined spoligotyping and MIRU-VNTR method, a high clustering rate of 43.9% was obtained. Our results underlined that human EPTB due to M. bovis was more commonly found in female gender and in young patients. Most of our patients, 66.4% (73/110) were raw milk or derivatives consumers, whereas 30.9% (34/110) patients would have contracted EPTB through contact with livestock. The findings suggest that the transmission of Zoonotic TB caused by M. bovis to humans mainly occurred by oral route through raw milk or derivatives.
Our study showed the urgent need of a better veterinary control with the implementation of effective and comprehensive strategies in order to reach a good protection of animals as well as human health.
Tunisia 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.
Extrapulmonary 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.
EPTB 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.
MTBC-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.
The 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.
Ninety-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.
Spatial analysis may provide insights into disease transmission, which affects the effectiveness of eradication campaigns in cattle.
A rising isolation trend of drug-resistant M. bovis from human clinical cases is documented in the literature. Here we assessed Mycobacterium tuberculosis complex isolates from cattle for drug ...susceptibility by the gold standard agar proportion method and a simplified resazurin microtitre assay (d-REMA). A total of 38 M. tuberculosis complex strains, including M. bovis (n = 36) and M. caprae (n = 2) isolates, from cattle in Tunisia were tested against isoniazid, rifampin, streptomycin, ethambutol, kanamycin and pyrazinamide.
M. caprae isolates were found to be susceptible to all test drugs. All M. bovis strains were resistant to pyrazinamide, as expected. In addition, one M. bovis isolate showed high-level resistance to streptomycin (MIC > 500.0 μg/ml). Concordant results with the two methods were found. The most common target genes associated with streptomycin resistance, namely the rrs, rpsL and gidB genes, were DNA sequenced. A non-synonymous mutation at codon 43 (K43R) was found in the rpsL gene. To the best of our knowledge, this is the first report describing the isolation of a streptomycin-resistant M. bovis isolate from animal origin.
Antitubercular drug susceptibility testing of M. bovis isolates from animals should be performed in settings where bTB is endemic in order to estimate the magnitude of the risk of drug-resistant tuberculosis transmission to humans.
Whole genome sequencing (WGS) has made impressive progress in the field of molecular biology. Its most common application for public health is in the area of surveillance of food-borne diseases. WGS ...has the potential for providing a large amount of information, such as the identification of the strain type, the characterization of antibiotic resistance and virulence, and phylogeny. In our study, thirty-nine non-typhoidal Salmonella strains were isolated from diverse sources in Tunisia. Non-typhoidal Salmonella are among the most common pathogens contaminating food animals. The presence of virulence and antimicrobial resistance determinants in those strains were investigated using whole genome sequencing (WGS) and appropriate data analysis. The genomes were screened for several Salmonella virulence genes using the Virulence Factor Database VFDB. Twelve different virulence profiles, which correspond to the 12 identified serovars, were recognized. Several antimicrobial resistance genes were also detected: aac (6′)-Iaa, sul1, tetA, bla-TEM and qnrS genes. Phylogenetic relationships among the strains were further assessed by a cgMLST analysis. The resulting phylogenetic tree consisted of several clusters consistently with the in silico multilocus sequence typing (MLST) and serotyping. Our findings demonstrated that WGS and subsequent data analysis provided an accurate tool for genetic characterization of bacterial strains compared to usual molecular typing techniques. To the best of our knowledge, this is the first report of an application of WGS for genetic characterization of food-borne Tunisian strains.
•Whole genome sequencing of foodborne Salmonella isolated in Tunisia•Identification of Salmonella serovar, virulence, and antimicrobial resistance genes•Implementing WGS in low-income countries to control the food production chain
Multidrug-resistant
isolates have been recovered from food in Tunisia.
isolates from food are resistant to fluoroquinolones and cephalosporins. Surveillance of the antimicrobial susceptibility of ...foodborne bacteria is needed in Tunisia.
Anthyllis henoniana stems were harvested in two seasons: winter and spring (February and May 2021). In this study, we investigated the antioxidant (DPPH, ABTS, FRAP and TAC) and antimicrobial ...activities, total phenolic contents and total flavonoid contents of the obtained extracts (hexane, ethyl acetate and methanol). The results showed that ethyl acetate extract from stems harvested in winter exhibited the highest antioxidant activity, while ethyl acetate extract from the stems harvested in spring showed the most potent antibacterial and antifungal activities. To explain these differences, we investigated the phytochemical composition of these two extracts using liquid chromatography coupled with mass spectrometry. Therefore, 45 compounds were detected, from which we identified 20 compounds (flavonoids, triterpenoids, chalcones and phenolic acids); some were specific to the harvest month while others were common for both periods. Some of the major compounds detected in ethyl acetate (spring) were dihydrochalcone (Kanzonol Y, 8.2%) and flavanone (sophoraflavanone G, 5.9%), previously recognized for their antimicrobial effects. We therefore concluded that the difference in activities observed for the two harvest periods depends on the chemical composition of the extracts and the relative abundance of each compound.
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