Tarek Zmantar

Antioxidant properties of the essential oil of Eugenia caryophyllata and its antifungal activity against a large number of clinical Candida species.

Many essential oils are known to possess an antioxidant activity and antifungal properties and therefore they potentially act as antimycotic agents. Essential oil of clove (Eugenia caryophyllata) was isolated by hydrodistillation. The chemical composition of the essential oil was analysed by gas chromatography and gas chromatography/mass spectroscopy. The antioxidant effect of the tested oil was evaluated by measuring its 2,2‐diphenyl‐l‐1‐picrylhydrazil radical scavenging ability and the antiradical dose required to cause a 50% inhibition (IC50) was recorded. The antifungal activity of essential oils was evaluated against 53 human pathogenic yeasts using a disc paper diffusion method. Our results show that the major components present in the clove bund oil were eugenol (88.6%), eugenyl acetate (5.6%), β‐caryophyllene (1.4%) and 2‐heptanone (0.9%). The tested essential oil exhibited a very strong radical scavenging activity (IC50u2003=u20030.2u2003μgu2003ml−1) when compared with the synthetic antioxidant (tert‐butylated hydroxytoluene, IC50u2003=u200311.5u2003μgu2003ml−1). On the other hand, this species displayed an important antifungal effect against the tested strains. It is clear that clove oil shows powerful antifungal activity; and it can be used as an easily accessible source of natural antioxidants and in pharmaceutical applications.

Cell surface hydrophobicity, biofilm formation, adhesives properties and molecular detection of adhesins genes in Staphylococcus aureus associated to dental caries.

Staphylococcus aureus is an important pathogen that forms biofilm. In this study, 22 S. aureus have been isolated from the oral cavity of Tunisian children and investigated for slime production using Congo red agar method (CRA) and semi quantitative adherence assay. The hydrophobicity of strains was evaluated by the microbial adhesion to solvent (MATS) test. The adherence of S. aureus to Hep2 cells was examined by light microscopy. The genes implicated in adhesion (icaA, icaD, fnbA, cna, clfA) were detected. Polymerase chain reaction was used. The affinity to hexadecane was low proving a hydrophilic character of all the studied strains. Qualitative biofilm production revealed that 50% of strains were slime producers. The result of OD(570) showed that four strains isolated from the caries-active children were highly biofilm positive. In addition, 50% of strains were icaA and icaD positive. The fnbA gene was present in 59.1% of isolated strains. Furthermore, 54.5% of strains harboured the cna gene, 9.1% were clfA positive and 50% were hla positive. Quantitative adherence varied considerably among the tested strains. All strains showed adherence to Hep2 cells. However, the level of adhesion varied between strains as follows. Seven strains were defined as moderately adherent, nine as strongly adherent and six as weakly adherent. The percentage of infected cells ranged from 15+/-0.0376 (B374) to 96+/-0.019 (B295) and the total number of bacteria per 100 cells ranged from 15+/-5.1 (B374) to 1824+/-30.1 (B295).

Detection of macrolide and disinfectant resistance genes in clinical Staphylococcus aureus and coagulase-negative staphylococci

BackgroundStaphylococcus aureus and Coagulase-negative staphylococci (CoNS) are a major source of infections associated with indwelling medical devices. Many antiseptic agents are used in hygienic handwash to prevent nosocomial infections by Staphylococci. Our aim was to determine the antibiotic susceptibility and resistance to quaternary ammonium compound of 46 S. aureus strains and 71 CoNS.MethodsS. aureus (n = 46) isolated from auricular infection and CoNS (n = 71), 22 of the strains isolated from dialysis fluids and 49 of the strains isolated from needles cultures were investigated. Erythromycin resistance genes (erm A, erm B, erm C, msr A and mef) were analysed by multiplex PCR and disinfectant-resistant genes (qac A, qac B, and qac C) were studied by PCR-RFLP.ResultsThe frequency of erythromycin resistance genes in S. aureus was: erm A+ 7.7%, erm B+ 13.7%, erm C+ 6% and msr A+ 10.2%. In addition, the number of positive isolates in CoNS was respectively erm A+ (9.4%), erm B+ (11.1%), erm C+ (27.4%), and msr A+ (41%). The MIC analyses revealed that 88 isolates (74%) were resistant to quaternary ammonium compound-based disinfectant benzalkonium chloride (BC). 56% of the BC-resistant staphylococcus isolates have at least one of the three resistant disinfectants genes (qac A, qac B and qac C). Nine strains (7.7%) among the CoNS species and two S. aureus strains (2%) harboured the three-qac genes. In addition, the qac C were detected in 41 strains.ConclusionsMulti-resistant strains towards macrolide and disinfectant were recorded. The investigation of antibiotics and antiseptic-resistant CoNS may provide crucial information on the control of nosocomial infections.

Anti-cariogenic and anti-biofilms activity of Tunisian propolis extract and its potential protective effect against cancer cells proliferation

Propolis is a multifunctional substance used by bees to maintain the safety of their hives. It is worldwide used for its potential therapeutic effects. In this study, Tunisian propolis ethanol extract (EEP) was tested for their anti-cariogenic, anti-biofilms and antiproliferative effects of many cell lines. The Tunisian EEP was evaluated in vitro against 33 oral pathogens including streptococci and enterococci using broth microdilution method. The anti-biofilms activity of EEP was assessed via Crystal Violet staining and MTT assays. The Tunisian EEP antiproliferative effect was evaluated on normal (MRC-5) and cancer cell lines (HT-29, A549, Hep-2, raw 264.7, Vero) by the ability of the cells to metabolically reduce MTT to a formazan dye. Our results revealed that Tunisian EEP possessed excellent protective effects against cariogenic and biofilms activity of oral streptococci. Furthermore, EEP showed a strong antiproliferative potencies against all studied cancer cell lines as judged by IC50 and its value ranges from 15.7 ± 3.4 to 200 ± 22.2 μg mL⁻¹. These results suggest that EEP is able to inhibit cancer cell proliferation, cariogenic bacteria and oral biofilms formation. It could have a promising role in the future medicine and nutrition when used as antibiotic or food additive.

Detection by PCR of adhesins genes and slime production in clinical Staphylococcus aureus

The presence of the ica loci and adhesins genes in clinical Staphylococcus aureus strains were considered important factors of virulence. In this study, 46 strains of Staphylococcus aureus were isolated from auricular infection, and were investigated for slime production using Congo Red Agar method (CRA). In order to detect the adhesins genes (ica A, ica D, fnb A, cna, Clf A) Polymerase Chain Reaction was used. Qualitative biofilm production of S. aureus using CRA plates revealed that 56.5% of strains were slime producers. In addition 78.26% of strains were ica A and ica D positive. While the fnbA gene was present in 76.1% of isolated strains. Furthermore, 56.5% of strains have the cna gene and 30.4% were clfA positives. Overall this study confirms the presence of fnb A and ica A/ica D genes in the majority of studies S. aureus strains isolated from Staphylococcal sepsis. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

In vitro effect of pH and ethanol on biofilm formation by clinicalica-positiveStaphylococcus epidermidis strains

Biofilm production is an important step in the pathogenesis ofStaphylococcus epidermidis associated biomaterial infections.Staphylococcus epidermidis strains isolated from dialysis fluid (n=9) and needle cultures (n=14) were phenotyped and genotyped for extracellular polysaccharide production and were examined for their ability to produce slime in a medium at various pH levels (3, 5, 7, 9 and 12) and with ethanol supplementation (0, 2, 5, 10 and 15%) using a semi-quantitative adherence assay. A total of 23 clinicalicaADBC positiveS. epidermidis, one reference strain (S. epidermidis CIP 106510) used as positive control, and oneicaADBC negative strain (E21) were investigated. Qualitative biofilm production analysis revealed that 15 of the 23icaADBC positive strains (65.21%) produced slime on Congo Red agar plates. Quantitative biofilm was determined by measuring the optical density at 570 nm (OD570). The results show that the slime production depended on the pH value of the medium and the ethanol concentration. At highly acidic (pH 3) and alkaline (pH 12) levels, the OD570 was lower, while at pH 7 the adhesion was moderate. In addition the cells adhered strongly with 2% ethanol than with the other concentrations. Our results suggest that pH and ethanol were stress factors that led toS. epidermidis biofilm formation and also play a possible role in the pathogenesis of biomaterial-related infections.

Antibacterial and resistance-modifying activities of thymoquinone against oral pathogens

BackgroundThe presence of resistant bacteria in the oral cavity can be the major cause of dental antibiotic prophylaxis failure. Multidrug efflux has been described for many organisms, including bacteria and fungi as part of their drugs resistance strategy. The discovery of a new efflux pump inhibitor could extend the useful lifetime of some antibiotics.MethodsIn this study, the MICs of thymoquinone (TQ), tetracycline and benzalkonium chloride (BC) were determined in absence and in presence of a sub-MIC doses of thymoquinone (1/2 MIC). In addition the 4,6-diamidino-2-phenylindole (DAPI) efflux assay was carried out to determine the effect of TQ on DAPI cells accumulation.ResultsTQ induced a selective antimicrobial activity. Its synergic effect resulted in at least a 4-fold potentiation of the tested antibiotics and antiseptic. In addition, TQ inhibited the DAPI efflux activity in a concentration-dependent manner. The rate of DAPI accumulation in clinical isolates was enhanced with TQ (0 to 200 μg/ml). There is also a decrease in loss of DAPI from bacteria in the presence of TQ. The concentration causing 50% of DAPI efflux inhibition after 15 minutes was approximately 59 μg/ml for Pseudomonas aeroginosa and 100 μg/ml and Staphylococcus aureus respectively.ConclusionsTQ possesses a selective antibacterial activity against oral bacteria. It is therefore suggested that TQ could be used as a source of natural products with resistance-modifying activity. Further investigation is needed to assess their clinical relevance.

Anticariogenic and cytotoxic activity of clove essential oil (Eugenia caryophyllata) against a large number of oral pathogens

The occurrence of dental caries is mainly associated with oral pathogens, especially cariogenic bacteria. Numerous studies have validated the traditional use of medicinal plants by investigating the biological activity of essential oils. The Eugenia caryophyllata (clove) essential oil was tested in vitro against a large number of oral pathogens (114 streptococci and 46 yeast strains) using a disc diffusion method. The cytotoxicity assay of Eugenia caryophyllata essential oil on cancer cells (HT29, A549, Hep2, raw 264.7) and normal cells (MRC-5) was determined by the ability of the cells to metabolically reduce MTT to a formazan dye. Our results revealed that Eugenia essential oil possessed an excellent antibacterial activity against oral streptococci including the cariogenic bacteria as well as an excellent antifungal activity. Furthermore, the Eugenia caryophyllata essential oil showed significant cytotoxic effects against all studied cancer cell lines as judged by IC50 and its value ranges from 15.75 to 200xa0μg/ml. In conclusion, it is clear that clove oil shows powerful antibacterial and antifungal activity. The cytotoxic activity of the essential oil was dependent on the tested cell lines.

Multiplex PCR detection of the antibiotic resistance genes in Staphylococcus aureus strains isolated from auricular infections

Thirty-five Staphylococcus aureus strains from auricular infections were isolated. The identification of strains was confirmed by Api ID 32 Staph strips, the antibiotic susceptibility test was performed using ATB Staph kit. PCR assay was used to detect the oxacillin resistance gene (mecA) and the erythromycin genes (ermA, ermB, ermC, msrA and mef). The susceptibility profile of all strains revealed a low resistance level to oxacillin and erythromycin. The PCR results show that 60 % of the strains are mecA positive. The frequency of erythromycin genes was: ermA+ 22.8 %, ermB+ 45.7, ermC+ 17.1, msrA+ 28.6. The mef gene was not detected in any strain. No correlations between genotypic and phenotypic methods for the determination of oxacillin and erythromycin resistance was found. However, multiplex PCR technique was shown to be a fast, practical and economic technique for the detection of methicillin-and erythromycin-resistant staphylococci.

Antibiotic resistance and adhesion properties of oral Enterococci associated to dental caries

BackgroundEnterococci are increasingly associated with opportunistic infections in Humans but the role of the oral cavity as a reservoir for this species is unclear. This study aimed to explore the carriage rate of Enterococci in the oral cavity of Tunisian children and their antimicrobial susceptibility to a broad range of antibiotics together with their adherence ability to abiotic and biotic surfaces.ResultsIn this study, 17 E. faecalis (27.5%) and 4 E. faecium (6.5%) were detected. The identified strains showed resistance to commonly used antibiotics. Among the 17 isolated E. faecalis, 12 strains (71%) were slime producers and 5 strains were non-producers. Among the 4 E. faecium, 2 strains were slime producers. All the tested strains were able to adhere to at least one of the two tested cell lines. Our result showed that 11 E. faecalis and 2 E. faecium strains adhered strongly to Hep-2 as well as to A549 cells.ConclusionsDrugs resistance and strong biofilm production abilities together with a high phenotypic adhesion to host cells are important equipment in E. faecalis and E. faecium which lead to their oral cavity colonization and focal infections.