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.

The Archaeome's Role in Colorectal Cancer: Unveiling the DPANN Group and Investigating Archaeal Functional Signatures.

BACKGROUND AND AIMS: Gut microbial imbalances are linked to colorectal cancer (CRC), but archaea's role remains underexplored. Here, using previously published metagenomic data from different populations including Austria, Germany, Italy, Japan, China, and India, we performed bioinformatic and statistical analysis to identify archaeal taxonomic and functional signatures related to CRC. METHODS: We analyzed published fecal metagenomic data from 390 subjects, comparing the archaeomes of CRC and healthy individuals. We conducted a biostatistical analysis to investigate the relationship between Candidatus Mancarchaeum acidiphilum (DPANN superphylum) and other archaeal species associated with CRC. Using the Prokka tool, we annotated the data focusing on archaeal genes, subsequently linking them to CRC and mapping them against UniprotKB and GO databases for specific archaeal gene functions. RESULTS: Our analysis identified enrichment of methanogenic archaea in healthy subjects, with an exception for Methanobrevibacter smithii, which correlated with CRC. Notably, CRC showed a strong association with archaeal species, particularly Natrinema sp. J7-2, Ferroglobus placidus, and Candidatus Mancarchaeum acidiphilum. Furthermore, the DPANN archaeon exhibited a significant correlation with other CRC-associated archaea (p < 0.001). Functionally, we found a marked association between MvhB-type polyferredoxin and colorectal cancer. We also highlighted the association of archaeal proteins involved in the biosynthesis of leucine and the galactose metabolism process with the healthy phenotype. CONCLUSIONS: The archaeomes of CRC patients show identifiable alterations, including a decline in methanogens and an increase in Halobacteria species. MvhB-type polyferredoxin, linked with CRC and species like Candidatus Mancarchaeum acidiphilum, Natrinema sp. J7-2, and Ferroglobus placidus emerge as potential archaeal biomarkers. Archaeal proteins may also offer gut protection, underscoring archaea's role in CRC dynamics.

Colorectal Cancer Archaeome: A Metagenomic Exploration, Tunisia.

Colorectal cancer (CRC) is a serious public health problem known to have a multifactorial etiology. The association between gut microbiota and CRC has been widely studied; however, the link between archaea and CRC has not been sufficiently studied. To investigate the involvement of archaea in colorectal carcinogenesis, we performed a metagenomic analysis of 68 formalin-embedded paraffin fixed tissues from tumoral (n = 33) and healthy mucosa (n = 35) collected from 35 CRC Tunisian patients. We used two DNA extraction methods: Generead DNA FFPE kit (Qiagen, Germantown, MD, USA) and Chelex. We then sequenced the samples using Illumina Miseq. Interestingly, DNA extraction exclusively using Chelex generated enough DNA for sequencing of all samples. After data filtering and processing, we reported the presence of archaeal sequences, which represented 0.33% of all the reads generated. In terms of abundance, we highlighted a depletion in methanogens and an enrichment in Halobacteria in the tumor tissues, while the correlation analysis revealed a significant association between the Halobacteria and the tumor mucosa (p < 0.05). We reported a strong correlation between Natrialba magadii, Sulfolobus acidocaldarius, and tumor tissues, and a weak correlation between Methanococcus voltae and healthy adjacent mucosa. Here, we demonstrated the feasibility of archaeome analysis from formol fixed paraffin-embedded (FFPE) tissues using simple protocols ranging from sampling to data analysis, and reported a significant association between Halobacteria and tumor tissues in Tunisian patients with CRC. The importance of our study is that it represents the first metagenomic analysis of Tunisian CRC patients' gut microbiome, which consists of sequencing DNA extracted from paired tumor-adjacent FFPE tissues collected from CRC patients. The detection of archaeal sequences in our samples confirms the feasibility of carrying out an archaeome analysis from FFPE tissues using a simple DNA extraction protocol. Our analysis revealed the enrichment of Halobacteria, especially Natrialba magadii, in tumor mucosa compared to the normal mucosa in CRC Tunisian patients. Other species were also associated with CRC, including Sulfolobus acidocaldarius and Methanococcus voltae, which is a methanogenic archaea; both species were found to be correlated with adjacent healthy tissues.

Epstein-Barr virus in breast carcinoma and in triple negative cases impact on clinical outcomes.

BACKGROUND: The contribution of viral infection in tumors pathogenesis has currently attracted attention. Epstein-Barr virus is an infectious agent involved in numerous human malignancies, including breast cancer. Although, their prognostic impact in breast tumor is rarely investigated. Therefore, we sought in our study to evaluate the prevalence of EBV in Tunisian breast carcinoma and to examine their potential association with clinicopathological features and overall survival. METHODS: Our retrospective study included 100 formalin fixed paraffin embedded samples from Tunisian breast carcinoma. EBV infection was evaluated by immunohistochemical analysis, using monoclonal antibody against latent membrane protein 1 (LMP-1) and polymerase chain reaction. A subset of PCR positive specimens was subjected to in situ hybridization for the detection of EBER expression. Biomarker's expression was evaluated by immunohistochemistry method. Statistical analysis was also explored. RESULTS: The expression status of ER, PR and HER2 was 81%, 71.4% and 33.7% respectively. The triple negative profile was present in 10.84% of cases. LMP-1 expression was negative in all breast cancer specimens. PCR assay showed that 44% of patients were positive for EBV genome. None of the 15 PCR positive cases showed positive results for EBV by ISH. According to the molecular phenotype, there was a statistically significant difference in EBV DNA prevalence between breast cancer subgroups including TN (67%), Lum B (64%), HER2 + (50%) and Lum A (30%). Bivariate analysis showed that EBV DNA was significantly associated with HER2 + (p = 0.035), tumor size (p = 0.018) and high SBR grade (p = 0.009). Multiple logistic regression analysis confirms the positive correlation of EBV with tumor size (p = 0.048) and SBR grade (p = 0.042). Kaplan-Meier analysis showed that patients with EBV+ had significantly shorter overall survival than those with EBV- (p = 0.032). CONCLUSIONS: Our study demonstrated the presence of EBV DNA in Tunisian breast carcinoma. EBV DNA was associated with aggressive features and poor overall survival. Further investigations will be required in large samples size to clarify the potential role of EBV in breast tumor progression.

Virulence Factors in Colorectal Cancer Metagenomes and Association of Microbial Siderophores with Advanced Stages.

Colorectal cancer (CRC) is a growing public health challenge, featuring a multifactorial etiology and complex host-environment interactions. Recently, increasing evidence has pointed to the role of the gut microbiota in CRC development and progression. To explore the role of gut microbes in CRC, we retrieved metagenomic data from 156 stools from the European Nucleotide Archive database and mapped them against the VFDB database for virulence factors (VFs). GO annotations of VFs and KEGG pathways were then performed to predict the microbial functions and define functional pathways enriched in the tumor-associated microbiota. Interestingly, 306 VFs were detected in the metagenomic data. We revealed the enrichment of adenomas with VFs involved in cell adhesion, whereas in the early stages of CRC they were enriched in both adhesins and isochorismatase. Advanced stages of CRC were enriched with microbial siderophores, especially enterobactin, which was significantly associated with isochorismate synthase. We highlighted higher abundances of porins and transporters involved in antibiotic resistance and the development of biofilm in advanced stages of CRC. Most VFs detected in CRC, particularly in advanced stages, were shown to be included in siderophore biosynthesis pathways. This enrichment of predicted VFs supports the key role of the gut microbiota in the disease.

Phylogeny and mycotoxin profiles of pathogenic Alternaria and Curvularia species isolated from date palm in southern Tunisia.

Date palm (Phoenix dactylifera L.), is a widely cultivated crop across North Africa, with about 300 thousand tons of fruits produced per year, in Tunisia. A wide range of fungal pathogens has been associated with leaf spots of date palm, Alternaria species being the most frequently reported. Symptomatic leaves of Deglet Nour variety were randomly collected in six localities in Tunisia. We used a polyphasic approach to identify 45 Alternaria and five Curvularia strains isolated from date palm, confirming their pathogenicity. Sequencing of allergen Alt-a1, glyceraldehyde-3-phosphate dehydrogenase (gpd) and calmodulin genes allowed us to group 35 strains in Alternaria Section, and 10 strains in Ulocladioides section. Based on sequencing analyses of Internal Transcribed Spacer, gpd and elongation factor genomic regions, all Curvularia strains were identified as Curvularia spicifera. All Alternaria and Curvularia species tested on date palm plantlets proved to be pathogenic, fulfilling Koch's postulates. Although no significant differences were observed among the species, the highest mean disease severity index was observed in A. arborescens, while the lowest corresponded to C. spicifera. The capability of these strains to produce mycotoxins in vitro was evaluated. None of the A. consortialis strains produced any known Alternaria mycotoxin, whereas more than 80% of the strains included in Alternaria section Alternaria produced variable amounts of multiple mycotoxins such as alternariol, alternariol monomethyl ether, altenuene, tenuazonic acid and tentoxin. Curvularia spicifera strains produced detectable traces of fumonisins B. This work reports a first comprehensive multidisciplinary study of mycotoxigenic Alternaria species and C. spicifera associated with leaf spot disease on date palm.

Detection of Clones B2-ST131-C2 and A-ST617 in Escherichia coli Producing Both CTX-M-15 and CTX-M-27 from Tunisian Community Patients.

During a two-month period (2017-2018), 336 urine samples positive for Escherichia coli were collected from Tunisian patients. Of the 336 samples, 266 were collected from community patients and 70 from hospital settings. In all, 15 ESBL producers were identified (8 and 7, respectively) and assigned to 13 pulsotypes, including four ESBL-producing E. coli (ESBL-E) with E1 and E2 profiles (2 isolates each) from community patients. The two strains E1 were identified as B2-ST131 subclade C2 and the two isolates E2, A-ST617. The four strains carrying both CTX-M-15 and CTX-M-27, exhibited the multireplicon IncFII/F1A/F1B with the same formula F31:A4:B1. Two isolates with patterns E3 and E4 (Dice coefficient, 78.7%) isolated from community and hospital settings of two geographic areas were assigned to the emerging ST131 C1-M27 subclade and contained the replicon F1:A-:B20. The remaining ESBL-E divided into different sequence types/associated CTX-M: 2 ST131-C2/CTX-M-15 and ST744/CTX-M-55, ST617/CTM-15, ST2973/CTX-M-55, ST6448/CTX-M-15, ST224/CTX-M-15, ST1431/CTX-M-15, and ST38/CTX-M-27, one isolate each. Our study reports for the first time the presence in the Tunisian community of two clones of E. coli, including the virulent clone ST131-C2 harboring both CTX-M-15 and CTX-M-27, and confirms the spread of the emergent clone ST131-C1-M-27, notably in community urinary tract infections.

An unusual familial Xp22.12 microduplication including EIF1AX: A novel candidate dosage-sensitive gene for premature ovarian insufficiency.

We report on the results of array-CGH and Whole exome sequencing (WES) studies carried out in a Tunisian family with 46,XX premature ovarian insufficiency (POI). This study has led to the identification of a familial Xp22.12 tandem duplication with a size of 559.4 kb, encompassing only three OMIM genes (RPS6KA3, SH3KBP1and EIF1AX), and a new heterozygous variant in SPIDR gene: NM_001080394.3:c.1845_1853delTATAATTGA (p.Ile616_Asp618del) segregating with POI. Increased mRNA expression levels were detected for SH3KBP1 and EIF1AX, while a normal transcript level for RPS6KA3 was detected in the three affected family members, explaining the absence of intellectual disability (ID). To the best of our knowledge, this is the first duplication involving the Xp22.12 region, reported in a family without ID, but rather with secondary amenorrhea (SA) and female infertility. As EIF1AX is a regulatory gene escaping X-inactivation, which has an extreme dosage sensitivity and highly expressed in the ovary, we suggest that this gene might be a candidate gene for ovarian function. Homozygous nonsense pathogenic variants of SPIDR gene have been reported in familial cases in POI. It has been suggested that chromosomal instability associated with SPIDR molecular defects supports the role of SPIDR protein in double-stranded DNA damage repair in vivo in humans and its causal role in POI. In this family, the variant (p.Ile616_Asp618del), present in a heterozygous state, is located in the domain that interacts with BLM and might disrupt the BLM binding ability of SPIDR protein. These findings strengthen the hypothesis that the additional effect of this variant could lead to POI in this family. Although the work represents the first evidence that EIF1AX duplication might be responsible for POI through its over-expression, further functional studies are needed to clarify and prove EIF1AX involvement in POI phenotype.

Study of Virulence Genes, Antimicrobial Resistance, and Genetic Relatedness of Foodborne Salmonella Isolates from Tunisia.

ABSTRACT: Nontyphoidal Salmonella strains are among the major foodborne pathogens with emerging multidrug-resistant phenotypes. In this study, antimicrobial susceptibility testing of a collection of Salmonella isolates (n = 54) recovered from poultry and bivalve molluscs was performed. The study also investigated profiling of virulence and resistance genes as well as phylogenetic relationships through pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting. Results revealed the presence of multiple virulence genes among Salmonella isolates. Salmonella intestinal infection A (siiA), Salmonella outer protein (sopB and sopE), putative 4-hydroxybutyrate coenzyme A transferase (cat2), Salmonella atypical fimbria C (safC), and Salmonella Enteritidis fimbria B (sefB) were present in most (83.32 to 100%) of the isolates, whereas the remaining tested genes (Salmonella plasmid virulence [spvC and spvB]), and the sopE gene, were exclusively detected within the serotype Enteritidis. The highest resistance rates were observed for oxacillin (94.4%), ampicillin (37%), and nalidixic acid (27.7%), followed by cefotaxime and amoxicillin-clavulanic acid (14.8%), trimethoprim-sulfamethoxazole (9.3%), and ciprofloxacin (5.5%). The results indicate that the Salmonella Enteritidis serotype possessed the widest range of virulence determinants and increasing levels of resistance. Such high-risk clones should be particularly controlled in Tunisia. Overall, increased resistance and virulence confer a selective advantage for the evolution of these bacteria and represent an alarming problem for global public health. The genetic study via PFGE and ERIC-PCR showed the high diversity of the clonal origins of these bacteria and the sources of contamination and revealed the great capacity of Salmonella to diversify within food-producing animals.

Rapid Detection of Beta-Lactamases Genes among Enterobacterales in Urine Samples by Using Real-Time PCR.

The objective of this study was to develop and evaluate newly improved, rapid, and reliable strategies based on real-time PCR to detect the most frequent beta-lactamase genes recorded in clinical Enterobacterales strains, particularly in Tunisia (blaSHV12 , blaTEM , blaCTX-M-15 , blaCTX-M-9 , blaCMY-2 , blaOXA-48 , blaNDM-1 , and blaIMP ) directly from the urine. Following the design of primers for a specific gene pool and their validation, a series of real-time PCR reactions were performed to detect these genes in 78 urine samples showing high antibiotic resistance after culture and susceptibility testing. Assays were applied to DNA extracted from cultured bacteria and collected urine. qPCR results were compared for phenotypic sensitivity. qPCR results were similar regardless of whether cultures or urine were collected, with 100% sensitivity and specificity. Out of 78 multiresistant uropathogenic, strains of Enterobacterales (44 E. coli and 34 K. pneumoniae strains) show the presence of the genes of the bla group. In all, 44% E. coli and 36 of K. pneumoniae clinical strains harbored the bla group genes with 36.4%, 52.3%, 70.5%, 68.2%, 18.2%, and 4.5% of E. coli having blaSHV-12 , blaTEM , blaCTX-M 15 , blaCTX-M-9 , blaCMY-2 , and blaOXA-48 group genes, respectively, whereas 52.9%, 67.6%, 76.5%, 35.5%, 61.8, 14.7, and 1.28% of K. pneumoniae had blaSHV-12 , blaTEM , blaCTX-M 15 , blaCTX-M-9 , blaCMY-2 , blaOXA-48 , and blaNDM-1 group genes, respectively. The time required to have a result was 3 hours by real-time PCR and 2 to 3 days by the conventional method. Resistance genes of Gram-negative bacteria in urine, as well as cultured bacteria, were rapidly detected using qPCR techniques. These techniques will be used as rapid and cost-effective methods in the laboratory. Therefore, this test could be a good candidate to create real-time PCR kits for the detection of resistance genes directly from urine in clinical or epidemiological settings.

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.

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 ß-lactamase (ESBL) production was tested by double-disc synergy test. Strains were further subjected to a molecular assessment of ESBL and AmpC ß-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 penicillins, 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: blaCTX-M group 1 and group 9, and ACC and FOX AmpC ß-lactamase genes. Overall, ESBLs and AmpC ß-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.

A New Lavender (Lavandula multifida L.) Ecotype from Arid Tunisia, with Differential Essential Oil Composition and Higher Antimicrobial Potential.

The lavender Lavandula multifida L., a medicinal plant grown in arid regions of Tunisia, was recently considered an endangered species; thus, its habitats regressed to some difficult zones in terms of access, such as the watershed of Oued Agareb in central-eastern Tunisia. This species was recorded only in deep and narrow shady Wadi of the watershed and benefited from protection against overgrazing, erosion and sunlight. L. multifida was rarely observed in an open area, such as a plateau or large-bed valley. The plant's metabolism is linked to its response to environmental conditions, which is of particular interest to understanding the components of the considered population of L. multifida. Consequently, biochemical and antimicrobial analyses have been evaluated. Using gas chromatography-mass spectrometry (GC-MS) analysis reveals that among the 58 compounds identified in L. multifida essential oil extracted from aboveground plant tissues, camphor was the major component (15.68%), followed by 1,8-cineole (14.14%) and alpha-pinene (13.82%). Moreover, it has been observed that Escherichia coli was more susceptible than Staphylococcus aureus to the antimicrobial properties of L. multifida essential oil, while in the case of camphor, S. aureus was more susceptible than E. coli. The protected population of L. multifida exhibits a distinctive vegetative development and growth cycle, resulting in specific secondary metabolites and distinguished antimicrobial activity.

Electrochemical Detection of Dopamine and Riboflavine on a Screen-Printed Carbon Electrode Modified by AuNPs Derived from Rhanterium suaveolens Plant Extract.

A AuNP-modified screen-printed carbon electrode (AuNP/SPCE) for monitoring important biomolecules, such as dopamine (DA) and riboflavin (RF), having a high potential for personalized medicine and for continuous monitoring of human health is here proposed. AuNPs were synthesized using the extract of Rhanterium suaveolens as a reducing medium and were characterized by UV-vis spectroscopy, dynamic light scattering (DLS), and scanning and transmission electron microscopy (SEM and TEM). The synthesized AuNPs appear spherical and present a bimodal size distribution with a maximum centered at around 30-50 nm. Cyclic voltammetry (CV) experiments demonstrated that the modified AuNP/SPCE sensor exhibits superior electrochemical performances to the bare SPCE. Low limits of detection (LODs) of 0.2 and 0.07 µM at S/N = 3 and sensitivities of 550.4  and 2399 µA mM-1 cm-2 were registered for DA and RF detection, respectively. Results demonstrate the promising electrochemical characteristics of the synthesized AuNPs and developed AuNP/SPCE electrochemical sensor for the determination of these important biomolecules.

Phylogeny and Mycotoxin Profile of Pathogenic Fusarium Species Isolated from Sudden Decline Syndrome and Leaf Wilt Symptoms on Date Palms (Phoenix dactylifera) in Tunisia.

In 2017-2018, extensive symptoms of sudden decline and fruit rot were observed on date palms in southern Tunisia. Samples of diseased plants were randomly collected in six localities. Based on morphological identification, Fusarium was the most frequent fungal genus detected. A sequencing of translation elongation factor, calmodulin, and second largest subunit of RNA polymerase II genes was used to identify 63 representative Fusarium strains at species level and investigate their phylogenetic relationships. The main species detected was Fusariumproliferatum, and at a much lesser extent, Fusariumbrachygibbosum, Fusariumcaatingaense, Fusariumclavum, Fusariumincarnatum, and Fusariumsolani. Pathogenicity on the DegletNour variety plantlets and the capability to produce mycotoxins were also assessed. All Fusarium species were pathogenic complying Koch's postulates. Fusariumproliferatum strains produced mainly fumonisins (FBs), beauvericin (BEA), and, to a lesser extent, enniatins (ENNs) and moniliformin (MON). All F.brachygibbosum strains produced low levels of BEA, diacetoxyscirpenol, and neosolaniol; two strains produced also T-2 toxin, and a single strain produced HT-2 toxin. Fusariumcaatingaense, F.clavum, F.incarnatum produced only BEA. Fusariumsolani strains produced MON, BEA, and ENNs. This work reports for the first time a comprehensive multidisciplinary study of Fusarium species on date palms, concerning both phytopathological and food safety issues.

Can leukocytospermia predict prostate cancer via its effects on mitochondrial DNA?

Leukocytospermia was previously reported to affect sperm quality by the production of reactive oxygen species (ROS) leading to oxidative stress (OS). In turn, OS decreases sperm functional integrity, increases sperm DNA damage and ultimately alters fertility status. To elucidate the impact of leukocytospermia on sperm nuclear DNA integrity and mitochondrial DNA (mtDNA) structure, we conducted a study including 67 samples from infertile patients with low level of leucocytes (Group 1: n = 20) and with leukocytospermia (Group 2: n = 47). In addition to standard sperm parameters' assessment, we measured the levels of inflammation biomarkers [interleukin-6 (IL-6) and interleukin-8 (IL-8)] and evaluated the oxidative status [malondialdehyde (MDA) and enzymatic and non-enzymatic antioxidants]. In addition, we evaluated the level of sperm nuclear DNA fragmentation and analysed mitochondrial DNA (mtDNA) of sperm cells by sequencing of 5 genes [cytochrome oxidase I (COXI), cytochrome oxidase II (COXII), cytochrome oxidase III (COXIII), adenosine triphosphate synthase 6 (ATPase 6) and adenosine triphosphate synthase 8 (ATPase 8)]. As expected, patients with leukocytospermia had significantly higher MDA levels (32.56 ± 24.30 nmole/ml) than patients without leukocytospermia (17.59 ± 9.60 nmole/ml) (p < .018). Also, sperm DNA fragmentation index (DFI) was significantly higher in Group 2 (33.05 ± 18.14%) as compared to Group 1 (14.19 ± 9.50%) (p < .001). The sequencing of mtDNA revealed a high number of substitutions in Group 2 (n = 102) compared to Group 1 (n = 5). These substitutions were observed mainly in COXI. Among COXI substitutions found in Group 2, twelve changes were previously described in patients with prostate cancer and six of them were shown associated with this pathology. These findings suggest that leukocytospermia may predispose to the manifestation of prostate cancer through modification of mitochondrial DNA and this may be promoted by OS.

Synthesis of Silver and Gold Nanoparticles from Rumex roseus Plant Extract and Their Application in Electrochemical Sensors.

The room-temperature synthesis of silver (AgNPs) and gold (AuNPs) nanoparticles from aqueous solution of AgNO3 and HAuCl4 respectively, using Rumex roseus (RR) plant extract as a reducing agent, is reported here for the first time. The nanoparticles obtained were characterized by UV-Vis spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). The formation of nanoparticles with spherical-shaped morphology was verified by TEM and confirmed by UV-Vis spectroscopy through the analysis of Ag and Au plasmon resonance peak and DLS measurements. New electrochemical sensors have been developed by employing the synthesized Ag and Au nanoparticles as modifiers of glassy carbon electrode (GCE) and screen-printed carbon electrode (SPCE), respectively. The AgNPs-modified GCE was investigated for the electrochemical determination of hydrogen peroxide (H2O2). Further enhancement of electrochemical performances was obtained using a nanocomposite made of AgNPs and reduced graphene oxide (rGO)-modified GCE. The AuNPs-SPCE sensor was instead tested in the electrochemical sensing of riboflavin (RF). To our knowledge, this is the first paper reporting Rumex roseus plant extract as a source for the synthesis of metal nanoparticles and their use for developing simple, sensitive and reliable electrochemical sensors for H2O2 and RF.

Relationship between nuclear DNA fragmentation, mitochondrial DNA damage and standard sperm parameters in spermatozoa of infertile patients with leukocytospermia.

The association of leukocytospermia with male fertility is still under debate. Our objective was to evaluate the association of leukocytospermia with sperm parameters, mitochondrial DNA (mtDNA) variations, and seminal concentration of several oxidative stress and inflammatory cytokines in Tunisian infertile men. The studied patients were divided into two groups: patients without leukocytospermia (Group 1) and patients with leukocytospermia (Group 2). DNA fragmentation significantly increased in group 2 (31.41 %) compared to group 1 (14.68 %) ; (p < 0.001). A total of 115 nucleotide substitutions in mitochondrial DNA were depicted, among which 113 were previously identified. The number of substitutions was more elevated in group 2. Leukocytospermic group had significantly higher MDA (nmole/mL) levels than patients without leukocytospermia (34±24.43 vs 18.94±15.96 ; p=0.001), GSH (µg/mL) levels were also higher compared to the control group (126.53±22.87 vs 79.4±19.38 ; p < 0.001), SOD (U/mg of protein) levels were higher but without reaching the statistical significance (89.74±74.85 vs 67.56±37.11 ; p = 0.25) ; whereas seminal CAT (µmole H2O2/min/mg of protein) levels were lower in this group (10.66±14.32 vs 27.35±25.28 ; p = 0.012). No statistically significant differences between the two groups of patients were found in the levels of inflammatory cytokines. However, IL-8 level was positively correlated with DNA fragmentation and negatively correlated with vitality. These findings confirm the association between leukocytospermia and sperm DNA damage.

Virulence and resistance genes profiles and clonal relationships of non-typhoidal food-borne Salmonella strains isolated in Tunisia by whole genome sequencing.

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.

First report of Leaf Wilt caused by Fusarium proliferatum and F. brachygibbosum on Date Palm (Phoenix dactylifera) in Tunisia.

Since 2017, a new leaf wilt syndrome was observed in plantations of date palm in Tunisia. Its incidence increases sharply from year to year, especially in 'Deglet Nour' trees, aged between 5 and 15 years. In severe cases, the large number of dried leaves per tree can lead to complete cessation of date production. Symptoms appear on one or more leaves in the center of the crown. Whitening and drying start at the top of the leaflets and proceed to their base, while the midrib remains green. Then the whole leaf dies. Small white-creamy leaflet fragments and roots were collected from five different regions in the Djerid Oases. They were disinfected with diluted bleach (0,8 % NaOCl) and ethanol (80%) (each 2 min), rinsed with sterile distilled water, dried and finally plated in Petri dishes containing Potato Dextrose Agar (PDA) amended with 50mg/l neomycin. After incubation for 7 days at 25ºC±2, emerging fungal colonies were single-spored by serial dilution. They were transferred to PDA, Carnation Leaf Agar (CLA) and Spezieller Nahrstoffarmer Agar (SNA) for morphological identification. Based on the colony color on PDA, conidial morphology and phialide structures on CLA and/or SNA, of the 85 Fusarium isolates, around 90% were identified as F. proliferatum and around 10% as F. brachygibbosum (Leslie and Summerell, 2006). Fusarium proliferatum colonies rapidly developed white aerial mycelium that became purple in old cultures. Microconidia were abundant in the aerial mycelium and formed chains of variable length, on monophialides and polyphialids, a characteristic that distinguishes F. proliferatum from F. verticilloides. Less often, they were observed in false heads. Chlamydospores were absent. On CLA, microconidia were mostly 2 × 15 µm in size, a large number of sickle shaped macroconidia (2 × 25 µm) had one septum, some were larger (2 × 50 µm) with 3 septa and tips at both ends. Molecular identification was carried out based on elongation factor (EF-1α) gene sequencing. The region between the EF1 and EF2 primers (O'Donnell et al., 1998) was amplified and the sequences were compared to Fusarium reference sequences (GenBank). The sequences of the isolates Fus 1953 (539 bp), Fus 1962 (618 bp), and Fus 1965 (605 bp) shared respectively 100%, 99.51% and 99.51% homology with that of F. proliferatum JF740713.1 and were deposited in GenBank with the following accession numbers: MT630418, MT630419, and MT630420, respectively. The sequences of isolates 7F, 28F, Fus 1955 and Fus 1956 shared 100 % homology with that of F. brachygibbosum (GQ505418.1) while those of Fus 1955 and Fus 1956 showed 99.02 and 98.91 % identity, respectively, with F. brachygibbosum JX118981.1. The sequences of 7F (535 bp), 28F (535 bp), Fus 1955 (608 bp), and Fus 1956 (647 bp) were deposited in GenBank with the following accession numbers: MT630409, MT630410, MT630411, and MT630412, respectively. Two ml suspension of 106 conidia / ml of each isolate was sprayed separately or in combinations on in vitro cloned 'Deglet Nour' plants, placed in a greenhouse at 28°±2 °C and 70% R.H.. Isolates of F. proliferatum led to dryness and wilting leaflets after 3 weeks. Fusarium brachygibbosum only induced mild leaf yellowing, while in combination they were more virulent. Fungal isolates of both species were re-isolated and their identity confirmed to be the same of those isolated from leaflets infected in the open field, confirming Koch's postulates. Control plants lacked symptoms. Fusarium proliferatum is known as date palm pathogen in many countries (Saleh et al. 2017), however, to our knowledge, this is the first report of F. proliferatum and also F. brachygibbosum causing Leaf Wilt symptoms on P. dactylifera in Tunisia.