Exploring Bacterial and Fungal Biodiversity in Eight Mediterranean Olive Orchards (Olea europaea L.) in Tunisia.

A wide array of bacteria and fungi are known for their association with pests that impact the health of the olive tree. The latter presents the most economically important cultivation in Tunisia. The microbial diversity associated with olive orchards in Tunisia remains unknown and undetermined. This study investigated microbial diversity to elucidate the microbial interactions that lead to olive disease, and the bio-prospects for potential microbial biocontrol agents associated with insect pests of economic relevance for olive cultivation in the Mediterranean area. Bacterial and fungal isolation was made from soil and olive tree pests. A total of 215 bacterial and fungal strains were randomly isolated from eight different biotopes situated in Sfax (Tunisia), with different management practices. 16S rRNA and ITS gene sequencing were used to identify the microbial community. The majority of the isolated bacteria, Staphylococcus, Bacillus, Alcaligenes, and Providencia, are typical of the olive ecosystem and the most common fungi are Penicillium, Aspergillus, and Cladosporium. The different olive orchards depicted distinct communities, and exhibited dissimilar amounts of bacteria and fungi with distinct ecological functions that could be considered as promising resources in biological control.

Optimization of gluten-free sponge cake fortified with whey protein concentrate using mixture design methodology.

This study aimed to optimize mixtures of whey protein concentrate (WPC) and two flours of rice and maize flours for the production of gluten-free sponge cakes. This was obtained by using mixture design methodology. WPC incorporation had positive effects on specific volume and baking loss of cakes, whilst, their incorporation increased their hardness. Considering all cakes properties, two formulas F1 (78.5% Maize, 15% Rice and 6.5% WPC) and F2 (82.4% Maize, 12% Rice and 5.6% WPC) were optimized using a mixture design. The microstructure F1 was more organized and very well structured with smaller aggregates. According to the organoleptic evaluation, F1 was also most appreciated by the tasting panel. The findings of the present study indicated that maize and rice flours, and WPC could be used as a substitute for wheat flour in producing sponge cakes of high quality.

Identification of fungi in Tunisian olive orchards: characterization and biological control potential.

BACKGROUND: Olive production is the main agricultural activity in Tunisia. The diversity of fungi was explored in two different olive groves located in two distant geographical zones in Sfax (Tunisia) with different management practices. RESULTS: Fungal isolation was made from soil and the major olive tree pests, namely the Olive fly, Bactrocera oleae Gmelin (Diptera: Tephritidae), and the Olive psyllid, Euphyllura olivina Costa (Homoptera: Psyllidae). A total of 34 fungal isolates were identified according to their phenotypic, genotypic, biochemical and biological activities. Twenty fungal species were identified belonging to six different genera (Alternaria, Aspergillus, Cladosporium, Fusarium, Lecanicillium and Penicillium) by the analysis of their ITS1-5.8S-ITS2 ribosomal DNA region. Different bioassays performed in this work revealed that 25/34 (73.5%) of the identified fungal isolates showed an entomopathogenic and/or antagonistic activity, 9/34 (26.5%) of them displayed phytopathogenic features. CONCLUSIONS: Fungal species that showed entomopathogenic and/or antagonistic potentialities and that are non-phytopathogenic, (17/34; 50%) of our fungal isolates, could be explored for olive protection against fungal diseases and pests, and might have a future application as biocontrol agents.

Antioxidant and antibacterial activities, interfacial and emulsifying properties of the apo and holo forms of purified camel and bovine α-lactalbumin.

The antioxidant and antibacterial activities of camel and bovine α-lactalbumin (α-La) in both calcium-loaded (holo) and calcium-depleted (apo) forms were investigated and compared. Antioxidant assay showed that camel and bovine α-La exhibited significant Ferric-reducing antioxidant power (FRAP), ferrous iron-chelating activity (FCA) and antiradical activities especially in their apo form. Camel apo α-La also exhibited attractive antibacterial activities against Gram-negative bacteria (Pseudomonas aeruginosa) and against fungal pathogens species (Penicillium bilaiae, Aspergillus tamari and Aspergillus sclerotiorum). Likewise, emulsifying properties (emulsification ability (EAI) and stability (ESI) indexes) and the surface characteristics (surface hydrophobicity, ζ-potential and interfacial tension) of the α-La were assessed. Maximum EAI were found at pH 7.0, with higher EAI values for the camel apo α-La (EAI ~19.5 m2/g). This behavior was explained by its relative high surface hydrophobicity and its greater efficiency to reduce the surface tension at the oil-water interface. Furthermore, emulsions were found to be more stable at pH 7.0 compared to pH 5.0 (ESI ~50%) due to the higher electrostatic repulsive forces between oil droplets at pH 7.0 in consistence with the ζ-potential results. This study concluded that the camel apo α-La has antibacterial, antioxidant, and emulsifying properties in agricultural and food industries.

Providencia entomophila sp. nov., a new bacterial species associated with major olive pests in Tunisia.

Bioprospection for potential microbial biocontrol agents associated with three major insect pests of economic relevance for olive cultivation in the Mediterranean area, namely the olive fly, Bactrocera oleae, the olive moth, Prays oleae, and the olive psyllid, Euphyllura olivina, led to the isolation of several strains of readily cultivable Gram-negative, rod-shaped bacteria from Tunisian olive orchards. Determination of 16S ribosomal RNA encoding sequences identified the bacteria as members of the taxonomic genus Providencia (Enterobacterales; Morganellaceae). A more detailed molecular taxonomic analysis based on a previously established set of protein-encoding marker genes together with DNA-DNA hybridization and metabolic profiling studies led to the conclusion that the new isolates should be organized in a new species within this genus. With reference to their original insect association, the designation "Providencia entomophila" is proposed here for this hypothetical new taxon.

Isolation and structural characterization of levan produced by probiotic Bacillus tequilensis-GM from Tunisian fermented goat milk.

The probiotic features of strain GM newly isolated from Tunisian spontaneously fermented goat milk and identified as Bacillus tequilensis-GM were assessed. Strain GM showed high resistance to saliva (90.64%), gastric juice (88.55%), intestinal juice (72.83%) and resistance to bile salts (65.22%), was able to act against Listeria monocytogenes ATCC 15313, Escherichia coli ATCC 25922 and Enterococcus feacalis ATCC 25912, showed high surface hydrophobicity (77.3%) and was sensitive to most of the studied antibiotics. Strain GM did not exhibit any hemolytic activity whereas it was able to produce protease, amylase and ß-galactosidase. Moreover, results showed that strain GM produced high molecular weight ß-(2 → 6)-levan with high ability to inhibit and to disrupt pathogenic biofilms and with high ability to reduce syneresis of sucrose-supplemented skimmed milk. B. tequilensis-GM can therefore be suitable to be used as starter culture in fermented dairy products, since it possesses desirable probiotic properties in addition to its ability to produce levan.

Lipopeptides from Bacillus amyloliquefaciens strain 32a as promising biocontrol compounds against the plant pathogen Agrobacterium tumefaciens.

Despite the potential biological importance of lipopeptides from Bacillus amyloliquefaciens as antimicrobial compounds, their effects on Agrobacterium tumefaciens biofilms have not been previously studied. These latter are important virulence factors for the development and re-occurrence of crown gall disease. As part of the development of a new biopesticide acting as anti-biofilm and biocontrol agent, we investigated for the first time the ability of a mixture of lipopeptides produced by B. amyloliquefaciens strain 32a to inhibit the tumor formation on plants and to reduce the formation of biofilms by the phytopathogenic A. tumefaciens strains C58 and B6. The mixture was found to display a strong biosurfactant activity as well as bactericidal activity against planktonic Agrobacterium cells. Moreover, the lipopeptide treatment inhibited biofilm formation in the range of 79.58 ± 0.60-100.00 ± 0.00% and dislodged 43.42 ± 0.91-93.89 ± 2.70% of preformed biofilm. For these assays, fluorescence microscopy did not show any adherent cell in the anti-adhesive assay and only few ones in the cell-dislodging assay. More importantly, lipopeptide-enriched extract inhibits tumor formation on tomato stem when treatments were applied after pathogen adhesion to wounded tissues. By virtue of its ability to inhibit biofilms formed on biotic and abiotic surfaces and to control efficiently tumor development, the 32a lipopeptide mixture may represent an excellent new tool for an efficient biocontrol of crown gall disease.

Three-dimensional mapping of the joint space for the diagnosis of knee osteoarthritis based on high resolution computed tomography: Comparison with radiographic, outerbridge, and meniscal classifications.

One of the most important characteristic of knee osteoarthritis (OA) is the joint space (JS) width narrowing. Measurements are usually performed on two dimensional (2D) X-rays. We propose and validate a new method to assess the 3D joint space at the medial knee compartment using high resolution peripheral computed tomography images. A semi-automated method was developed to obtain a distance 3D map between femur an tibia with the following parameters: volume, minimum, maximum, mean, standard deviation, median, asymmetry, and entropy. We analyzed 71 knee specimens (mean age: 85 years), radiographs were performed for the Kellgren Lawrence (KL) score grading. In a subgroup of 41 specimens, the histopathological Outerbridge and meniscal classifications were performed and then cores were harvested from the tibial plateau in three different positions (posterior, central, and peripheral) and imaged at 10 µm of resolution to measure the cartilage thickness. Minimum, maximum, mean, and median were statistically lower and entropy higher between knee specimens classified as KL = 0 and KL = 3-4. Gr1 and 2 were statistically different from Gr3-4 for minimum, asymmetry, entropy using the Outerbridge classification and Gr1 was statistically different from Gr3-4 using the meniscal classification. Asymmetry, minimum, mean, median and entropy were significantly correlated with cartilage thickness. Parameters extracted from a 3D map of the medial joint space indicate local variations of JS and are related to local measurements of tibial cartilage thickness, and could be consequently useful to identify early OA. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 36:2380-2391, 2018.

Comparison of reverse hybridization and ompA sequencing methods applied on Chlamydia trachomatis strains from Tunisia.

Two techniques based on ompA amplification of Chlamydia trachomatis were compared, being reverse hybridization (RHM) and ompA sequencing (OSA), to investigate the concordance between them and to study the epidemiological relevance of each method. In addition, phylogenetic analysis was performed on the ompA sequences. One hundred and seven C. trachomatis positive samples from Tunisian patients and female sex workers were analyzed using both the RHM and ompA sequencing. The overall genovar distribution obtained with both techniques was very similar. The RHM identified nine genovars, being B, D, E, F, G, H, I, J and K, where B, I, J, and K were only found in mixed infections versus 7 types for the OSA being D, E, F, G, H, I, and K. The agreement between both typing techniques was 87.8%. Both methods showed that genovar E was the most predominant type. In 24.3% of the analyzed samples, mixed infections were detected. In 96.1% of these, the genovar identified by OSA was also detected using the RHM. OmpA sequencing allowed determination of six genovar types that could not be typed using RHM. The analyses of ompA nucleotide variation in the 107 clinical specimens detected ompA genovar variants with distinct ompA mutational patterns for types D2, G1, G2, and H1. In conclusion, RHM and OSA showed a high agreement in C. trachomatis genotyping results with each having their specific benefits.

Production and structural characterization of exopolysaccharides from newly isolated probiotic lactic acid bacteria.

In this work, four exopolysaccharide-producing lactic acid bacteria (LAB) strains, newly isolated from Tunisian spontaneously fermented foods and beverages, namely bovine and turkey meat sausages (BMS and TMS), date palm sap (DPS) and cow milk (CM), were identified as Leuconostoc citreum-BMS, Leuconostoc mesenteroides-TMS, Pediococcus pentosaceus-DPS and Leuconostoc pseudomesenteroides-CM, respectively. The isolated strains showed the ability to withstand simulated human gastrointestinal (GI) tract conditions (low pH, lysozyme, bile salts, pepsin and pancreatin) and showed high surface hydrophobicity (79-90%), besides their ability to act against Escherichia coli and Listeria monocytogenes and to produce exopolysaccharides (EPS). Therefore, these isolates can be served as potential probiotics. The produced EPS were growth-associated suggesting that they are primary metabolites. The molecular weights were higher than 106Da using HPLC-SEC. 2D-NMR results indicated that all the samples were mixtures of dextran and levan, except for EPS-CM which was a levan-type EPS. Furthermore, the EPS samples showed an abitlity to inhibit and to disrupt pathogenic biofilms and showed high thermostability studied via differential scanning calorimetry (DSC) with melting points higher than 224°C making them promising to be used in thermal processed foods.

Identification of a dominant Chlamydia trachomatis strain in patients attending sexual transmitted infection clinic and female sex workers in Tunisia using a high resolution typing method.

BACKGROUND: The distribution of Chlamydia trachomatis genotypes in Tunisia was previously studied using the reverse hybridization method. In this study, we used multilocus sequence typing (MLST) to describe Chlamydia trachomatis genetic diversity among heterosexual populations in Tunisia. The obtained sequence types (STs) were compared with those from a heterosexual population from Amsterdam, the Netherlands. METHODS: Clinical Tunisian patients and female sex workers provided 107 Chlamydia trachomatis positive samples that were used for MLST. Samples from 256 heterosexuals visiting the Amsterdam STI clinic were included as a reference group. Six highly variable genetic regions including the ompA gene were amplified and sequenced. The ST numbers were derived from a Chlamydia typing database (http://mlstdb.uu.se) and used to draw minimum spanning trees. RESULTS: ompA sequencing detected 7 genotypes among the Tunisian populations of which genotype E was the most prevalent (66.3%). This genotype E resolved into 23 different STs and among these the ST3 was predominant (53.5%). MLST displayed 43 STs, of which 28 (65%) were new in the database. Minimum spanning tree analysis of all Tunisian samples identified 4 clusters of which one formed a clonal cluster with samples presenting the most prevalent ST3. When comparing samples from the Tunisian and Dutch populations in one minimum spanning tree, there was little overlap between the Chlamydia trachomatis samples. CONCLUSION: The CT-hrMLST scheme allowed us to identify that the Tunisian distribution was dominated by one genotype E (ST3) strain which is also highly prevalent in many other countries worldwide.

Molecular epidemiological characteristics of Salmonella enterica serovars Enteritidis, Typhimurium and Livingstone strains isolated in a Tunisian university hospital.

Enteritidis, Typhimurium and Livingstone are the main Salmonella enterica serovars recovered in Tunisia. Here, we aimed to assess the genetic diversity of fifty-seven Salmonella enterica strains from different sampling periods, origins and settings using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST) and multi-locus variable-number tandem repeat analysis (MLVA). Salmonella Enteritidis, isolated from human and food sources from two regions in Sfax in 2007, were grouped into one cluster using PFGE. However, using MLVA these strains were divided into two clusters. Salmonella Typhimurium strains, recovered in 2012 and represent sporadic cases of human clinical isolates, were included in one PFGE cluster. Nevertheless, the MLVA technique, divided Salmonella Typhimurium isolates into six clusters with diversity index reaching (DI = 0.757). For Salmonella Livingstone which was responsible of two nosocomial outbreaks during 2000-2003, the PFGE and MLVA methods showed that these strains were genetically closely related. Salmonella Enteritidis and Salmonella Livingstone populations showed a single ST lineage ST11 and ST543 respectively. For Salmonella Typhimurium, two MLST sequence types ST19 and ST328 were defined. Salmonella Enteritidis and Salmonella Typhimurium strains were clearly differentiated by MLVA which was not the case using PFGE.

Chlamydia trachomatis genovar distribution in clinical urogenital specimens from Tunisian patients: high prevalence of C. trachomatis genovar E and mixed infections.

BACKGROUND: This epidemiological study was carried out in Sfax (south of Tunisia) and focused on genital Chlamydia trachomatis (C. trachomatis) genovar distribution. METHODS: One hundred and thirty seven genital samples from 4067 patients (4.2%) attending the Habib Bourguiba University hospital of Sfax over 12 years (from 2000 to 2011) were found to be C. trachomatis PCR positive by the Cobas Amplicor system. These samples were genotyped by an in house reverse hybridization method. RESULTS: One hundred and eight (78.8%) samples contained only one genovar and 29 (21.2%) samples contained two or three genovars. Genovar E was the most prevalent (70.8%) single genovar and it was detected in 90.6% of all the cases. Genovars J, C and L1-L3 were not detected in our samples whereas ocular genovars A and B were in 5 cases. All the five cases were mixed infections. Men had more mixed infections than women (p=0.02) and were more frequently infected by genovars F and K (p<0.05). No associations between current infection, infertility and the genovar distribution were observed. Patients coinfected with Neisseria gonorrhoeae were also significantly more frequently infected with mixed genovars (p=0.04). CONCLUSIONS: In conclusion, we have reported a high prevalence of genovar E and of mixed infections in our study population. Such data could have implications for the control and vaccine development of C. trachomatis in Tunisia.

Antibacterial, anti-chlamydial, and cytotoxic activities of a marine snail (Hexaplex trunculus) phospholipase A2: an in vitro study.

In order to report pharmacological characterization of marine snail (Hexaplex trunculus) hepatopancreatic phospholipase A(2) (mSDPLA(2)), we have talked for the first time the antimicrobial activity against different pathogenic bacterial strains, anti-chlamydial activity as well as its cytotoxic activity against McCoy cell lines. mSDPLA(2), showed a high level of activity towards Gram-positive bacteria as Staphylococcus aureus and Staphylococcus epidermidis. Whereas Gram-negative bacteria, unfortunately, exhibited a higher resistance, mSDPLA(2) was also found to have a strong cytotoxic activity, causing significant morphological alterations of the McCoy cell lines surfaces and to be a hinder to the proliferation. Moreover, mSDPLA(2) proved to have a very potent anti-chlamydial activity. Over 95 % inhibition of chlamydial inclusions were obtained at a concentration of 10 µg/ml of mSDPLA(2) after 24 h postinfection. Interestingly, at a concentration of 10 µg/ml of mSDPLA(2), the proliferation of McCoy cells was not affected. Approximately 50 % inhibition of cell growth was obtained with a concentration of 37 µg/mL of mSDPLA(2). mSDPLA(2) could be considered as an excellent candidate for the development of a new anti-infective agent. This enzyme showed significant antimicrobial activities.