Maha Mastouri

Population Structure of Pseudomonas aeruginosa from Five Mediterranean Countries : Evidence for Frequent Recombination and Epidemic Occurrence of CC235

Several studies in recent years have provided evidence that Pseudomonas aeruginosa has a non-clonal population structure punctuated by highly successful epidemic clones or clonal complexes. The role of recombination in the diversification of P. aeruginosa clones has been suggested, but not yet demonstrated using multi-locus sequence typing (MLST). Isolates of P. aeruginosa from five Mediterranean countries (n = 141) were subjected to pulsed-field gel electrophoresis (PFGE), serotyping and PCR targeting the virulence genes exoS and exoU. The occurrence of multi-resistance (≥3 antipseudomonal drugs) was analyzed with disk diffusion according to EUCAST. MLST was performed on a subset of strains (n = 110) most of them had a distinct PFGE variant. MLST data were analyzed with Bionumerics 6.0, using minimal spanning tree (MST) as well as eBURST. Measurement of clonality was assessed by the standardized index of association (IA S). Evidence of recombination was estimated by ClonalFrame as well as SplitsTree4.0. The MST analysis connected 70 sequence types, among which ST235 was by far the most common. ST235 was very frequently associated with the O11 serotype, and frequently displayed multi-resistance and the virulence genotype exoS −/exoU +. ClonalFrame linked several groups previously identified by eBURST and MST, and provided insight to the evolutionary events occurring in the population; the recombination/mutation ratio was found to be 8.4. A Neighbor-Net analysis based on the concatenated sequences revealed a complex network, providing evidence of frequent recombination. The index of association when all the strains were considered indicated a freely recombining population. P. aeruginosa isolates from the Mediterranean countries display an epidemic population structure, particularly dominated by ST235-O11, which has earlier also been coupled to the spread of ß-lactamases in many countries.

Characterization of ST80 Panton-Valentine leukocidin-positive community-acquired methicillin-resistant Staphylococcus aureus clone in Tunisia

The spread of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has been reported in communities worldwide. In this study, we characterized 64 Tunisian CA-MRSA by agr typing, polymerase chain reaction assay for 20 virulence genes, staphylococcal chromosomal cassette mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and protein A gene (spa) typing. All our isolates were lukS-PV-lukF-PV positive, etd positive, and edin positive. They harbored SCCmec type IV and belonged to agr group 3. PFGE typing showed that our isolates were distributed in 11 different pulsotypes. spa typing and MLST, performed with isolates representative of each PFGE pattern, revealed that all isolates had a unique spa type (t044) and a common sequence type (ST80). The isolates showed susceptibility to the majority of antibiotics, and resistance to kanamycin, erythromycin, and tetracycline, but intermediate resistance to fusidic acid. Full analysis of our results revealed that our isolates were nonmultiresistant and belonged to a single clonal type ST80.

Chemical composition and antibacterial, antifungal and antioxidant activities of the flower oil of Retama raetam (Forssk.) Webb from Tunisia

The chemical composition of the essential oils obtained by hydrodistillation from the flowers of Retama raetam (Forssk.) Webb cultivated in Tunisia was determined by GC and GC/MS analysis. A total of 50 components representing 98.58% of the oil were identified: nonanal (35.75%), α-humulene (29.29%), acetaldehyde (7.84%), linalool (5,62%), myrcene (3.38%), tridecanal (2.21%), β-caryophyllene (1.79%), α-terpinyl acetate (1.46%), terpinolene (1.26%) and methyl anthranilate (1.06%) were found to be the major components. The oil was evaluated for antibacterial and antifungal activities using a microdilution assay against some bacteria and yeasts. The minimal inhibitory concentrations (MIC) of the essential oil varied between 0.625 and 5 mg mL−1 and the minimum bactericidal concentrations (MBC) were superior to 5 mg mL−1 of oil for most strains. The antioxidant potential of the essential oil was evaluated using the 2,2′-diphenyl-1-picrylhydrazyl free radical scavenging method. The essential oil possesses good antioxidant properties (IC50 = 0.800 mg mL−1). The results may suggest that the flower oil of R. raetam possesses compounds with antibacterial, antifungal and antioxidant capacities, and thus the oil can be explored as a natural preservative ingredient in food and/or pharmaceutical preparations.

Antibacterial, antifungal and cytotoxic activities of two flavonoids from Retama raetam flowers.

We have investigated the antibacterial, antifungal and cytotoxic activities of two flavonoids isolated from Retama raetam flowers using the disc diffusion and micro-dilution broth methods. The cytotoxic activity was tested against Hep-2 cells using the MTT assay. The compounds licoflavone C (1) and derrone (2) were active against Pseudomonas aeruginosa and Escherichia coli (7.81–15.62 µg/mL) and showed important antifungal activity. Strong antifungal activity against Candida species (7.81 µg/mL) was for example found with compound 2. The tested compounds also showed strong cytotoxicity against Hep-2 cells. These two compounds may be interesting antimicrobial agents to be used against infectious diseases caused by many pathogens.

Flower and Root Oils of the Tunisian Daucus carota L. ssp. maritimus (Apiaceae): Integrated Analyses by GC, GC/MS, and 13C‐NMR Spectroscopy, and in vitro Antibacterial Activity

The essential oils from flowers and roots of Daucus carota L. ssp. maritimus were obtained by hydrodistillation and analyzed by combination of GC, GC/MS, and (13)C-NMR. The chemical composition of the flower and root oils of this subspecies is reported here for the first time. Thirty-two and six compounds were identified in flower and root oils, respectively. A remarkable difference was found between the constituent percentages of the two organs. The chemical composition of the essential oil from flowers was characterized by a high proportion of monoterpene hydrocarbons (68.4%) and belonged to the sabinene (4; 51.6%) chemotype. The oxygenated monoterpenes represented the second major fraction of the same oil among which terpinen-4-ol (11.0%) was the predominant compound. Root oil exhibited a surprisingly different composition. Phenolic derivatives (76.3%), myristicin (31; 29.7%), and dillapiole (32; 46.6%) constituted the main fraction. The antibacterial effect resulted in the inhibition of a series of common human pathogenic bacteria, and of some clinically and environmentally isolated strains with significant MIC and MBC values.

Chemical composition and biological activities of volatile fractions from three Tunisian cultivars of olive leaves

The chemical composition, antibacterial, and antifungal activities of the volatile fractions from fresh and dried leaves of three Olea europaea L. cultivars from Tunisia (Neb jemel, Chemchali and Chemlali) have been studied. The volatile components were obtained via hydrodistillation and analyzed by GC–MS. The major constituents were (E)-3-hexenol, 3-ethenylpyridine, (E)-β-damascenone and phenylethyl alcohol, but their percentages varied according to the treatment of the leaves. Antioxidant activities were determined applying DPPH and ABTS+ radical-scavenging assays. In general, antioxidant activity of the volatile fraction obtained from fresh leaves was superior to that obtained from dried leaves. The antibacterial and antifungal activities of the volatile fractions from fresh and dried leaves were evaluated against four bacterial and four fungal strains. The volatile fractions showed significant antibacterial and antifungal effects. However, some differences were observed in the response for several microorganisms, because of the variability of the composition. This work gives further knowledge for extensive development of this medicinal plant.

Aichi Virus IgG Seroprevalence in Tunisia Parallels Genomic Detection and Clinical Presentation in Children with Gastroenteritis

ABSTRACT Aichi virus has been described as a novel causative agent of gastroenteritis in humans. In this study, we report the seroprevalence distribution of Aichi virus in Tunisia. A panel of 1,000 sera was screened by applying an enzyme-linked immunosorbent assay for immunoglobulin G specific for Aichi virus. A considerable prevalence (92%) of antibody to Aichi virus was found across all age groups. The specific anti-Aichi virus antibodies increased with age, from a high rate (68.8%) in children under 10 years old to about 100% in persons more than 60 years old. We found a statistically significant increase in levels of antibody to Aichi virus according to the age of patients. Immunoglobulin M antibodies were detected among five children. A high frequency of Aichi virus monoinfections in hospitalized children with severe gastroenteritis was previously observed in Tunisia. Aichi virus causes diarrhea with dehydration, fever, and vomiting. This work is the first to establish a correlation between the high seroprevalence of specific Aichi virus antibodies, clinical presentation, and a high frequency of isolation of Aichi virus by genomic characterization in stools of children suffering from gastroenteritis. Our data show the importance and emerging character of Aichi virus in the viral etiology of pediatric gastroenteritis.

Chemical composition and in vitro evaluation of antioxidant and antibacterial activities of the root oil of Ridolfia segetum (L.) Moris from Tunisia

The present work describes the chemical composition and evaluates the antibacterial and the antioxidant properties of root oil from Ridolfia segetum, a traditional medicinal plant widely distributed in Tunisia. The essential oil, analysed by combination of GC, GC/MS and 13C-NMR, was dominated by dillapiole (47.4%) and myristicin (19.2%). The antioxidant potential of the essential oil was evaluated using the 2,2′-diphenyl-1-picrylhydrazyl free radical scavenging method. The phenylpropanoid-rich root oil possesses good antioxidant properties (IC50 = 38 mg mL−1). The essential oil was evaluated for its antibacterial activity using the microdilution assay, resulting in the inhibition of a number of common human pathogenic bacteria as well as of some clinical and environmental isolated strains. The minimum inhibitory concentrations of the essential oil varied between 1.25 and 5 mg mL−1 and the minimum bactericidal concentrations were superior to 5 mg mL−1 of oil for most strains. These results may suggest that the root oil of R. segetum possesses compounds with antibacterial and antioxidant capacities, and therefore can be explored as a natural preservative ingredient in food and/or for pharmaceutical preparations.

Chemical Composition and Antimicrobial Activity of Essential Oils from Scabiosa arenariaForssk. Growing Wild in Tunisia

The essential oils isolated from three organs, i.e., fruits, stems and leaves, and flowers, of the endemic North African plant Scabiosa arenaria Forssk. were screened for their chemical composition, as well as their possible antibacterial, anticandidal, and antifungal properties. According to the GC‐FID and GC/MS analyses, 61 (99.26% of the total oil composition), 79 (98.43%), and 51 compounds (99.9%) were identified in the three oils, respectively. While α‐thujone (34.39%), camphor (17.48%), and β‐thujone (15.29%) constituted the major compounds of the fruit oil, chrysanthenone (23.43%), together with camphor (12.98%) and α‐thujone (10.7%), were the main constituents of the stem and leaf oil. In the case of the flower oil, also chrysanthenone (38.52%), camphor (11.75%), and α‐thujone (9.5%) were identified as the major compounds.

Adhesive properties and extracellular enzymatic activity of Staphylococcus aureus strains isolated from oral cavity

Staphylococcus aureus is one of prominent bacterial pathogen that occurs in oral region. In this study, 21 strains of S. aureus isolated from the oral cavity of Tunisian patients were investigated for slime production using Congo red agar method (CRA) and adherence assay. Biofilm formation of oral isolates on orthodontic biomaterials (Bis-GMA and PMMA) was also evaluated by MTT reduction assay. In addition, the production of hydrolytic enzymes by S. aureus strains was analyzed and the presence of protease, lipase and β-hemolysin genes (sspA, sspB, geh, hlb) was achieved by polymerase chain reaction (PCR). Qualitative biofilm production tested on CRA revealed that 91% of strains were slime producers. The result of OD570 showed that five strains isolated from the oral cavity were highly biofilm positive. The metabolic activity of S. aureus biofilm formed on Bis-GMA and PMMA did not differ between tested strains. The atomic force micrographs demonstrated that biofilm formed by S. aureus strains was organized in typical cocci cells attached to each other through production of exopolymeric substances. The production of hydrolytic enzymes showed that all S. aureus strains were protease positive. Lipase (77%) and beta hemolytic (59%) activities were also detected. Among the tested strains, 17 were positive for sspA, sspB and hlb genes. While only ten S. aureus strains harbor the geh gene (48%). These data highlight the importance of evaluation of biofilm formation and exoenzyme production in oral S. aureus isolates to investigate the role of this pathogen and its impact in oral pathology.