Bochra Kouidhi

Cytotoxicity and genotoxicity induced by aflatoxin B1, ochratoxin A, and their combination in cultured Vero cells.

Aflatoxin B1 (AFB1) and ochratoxin A (OTA) are important food‐borne mycotoxins that have been implicated in human health. In this study, independent and combinative toxicities of AFB1 and OTA were tested in cultured monkey kidney Vero cells. The experiments reported here were conducted to evaluate the effect of these toxins on cell viability followed by the determination of cell death pathways, using the quantification of DNA fragmentation and the expression of p53 and bcl‐2 protein levels. Our results showed that AFB1 and OTA caused a marked decrease of cell viability in a dose‐dependent manner. Under the same conditions, these mycotoxins increased fragmented DNA levels. In addition, p53 was activated in response to DNA damage and the expression of the antiapoptotic factor bcl‐2 decreased significantly. According to these data, AFB1 and OTA seemed to be involved in an apoptotic process. Moreover, combined AFB1 and OTA induced all the toxicities observed with the mycotoxins separately. Therefore, this combination was classified as an additive response of the two mycotoxins.

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.

Drug resistance of bacterial dental biofilm and the potential use of natural compounds as alternative for prevention and treatment

Oral diseases, such as dental caries and periodontal disease are directly linked with the ability of bacteria to form biofilm. The development of dental caries involves acidogenic and aciduric Gram-positive bacteria colonizing the supragingival biofilm (Streptococcus, Lactobacillus and Actinomycetes). Periodontal diseases have been linked to anaerobic Gram-negative bacteria forming a subgingival plaque (Porphyromonas gingivalis, Actinobacillus, Prevotella and Fusobacterium). Cells embedded in biofilm are up to 1000-fold more resistant to antibiotics compared to their planctonic ones. Several mechanisms have been proposed to explain biofilms drug resistance. Given the increased bacterial resistance to antibiotics currently used in dentistry, a great importance is given to natural compounds for the prevention of oral bacterial growth, adhesion and colonization. Over the past decade, interest in drugs derived from medicinal plants has markedly increased. It has been well documented that medicinal plants and natural compounds confer considerable antibacterial activity against various microorganisms including cariogenic and periodontal pathogens. This paper provides a review of the literature focusing on the studies on (i) biofilm in the oral cavity, (ii) drug resistance of bacterial biofilm and (iii) the potential use of plant extracts, essential oils and natural compounds as biofilm preventive agents in dentistry, involving their origin and their mechanism of biofilm inhibition.

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.

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.

Molecular investigation of macrolide and Tetracycline resistances in oral bacteria isolated from Tunisian children

OBJECTIVE This study aims to investigate the antibiotic susceptibility of strains isolated from the oral cavity of Tunisian children. DESIGN Strains were isolated from the oral cavity of Tunisian children (60 caries-actives and 30 caries-free). Molecular characterization was assessed by PCR assay to detect erythromycin methylase gene (ermB), macrolide efflux (mefI) and tetracycline resistance genes (tetM and tetO). RESULTS A total of 21 species were isolated and identified. Antimicrobial susceptibility revealed that the resistance rate to antibiotics was as follow: erythromycin (22%), tetracycline (15.6%), cefotaxim, (7.3%), trimethoprim-sulfamethoxazol (37.6%), nitrofurantoine (2.8%), pristinamycin (17.4%), quinupristin-dalfopristin (15.6%), and rifampicin (3.7%). The majority of mefI positive strains (31.2%) were isolated from the carious children (n=34) in comparison with 8.25% from the control group (n=9). In addition, frequency of strains caring resistance genes were as follow: 12.84% for ermB, 9.17% for tetM and 27.52% for tetO from the carious children in comparison to 0.092%, 3.67% and 3.67% from the caries free group respectively. CONCLUSION Multi-resistance strains towards macrolides and tetracycline were recorded. The majority of strains carrying antibiotics resistance genes were isolated from the caries active children. The presence of multi-resistant bacteria in the oral cavity can be the major cause of antibiotic prophylaxis failure in dental practise.

Detection of disinfectant and antibiotic resistance genes in Staphylococcus aureus isolated from the oral cavity of Tunisian children

Staphylococcus aureus is an important pathogen associated to dental infection. Many antiseptic agents are used in hygienic handwash to prevent nosocomial infections by methicillin-resistant staphylococci. In this study, 22 Staphylococcus aureus strains isolated from the oral cavity of Tunisian children were investigated for their susceptibility to benzalkonium chloride (0.5–512 μg/ml) and antibiotics. The β-lactams resistance gene blaZ, the erythromycin resistance methylase genes (ermA, ermB and ermC), the macrolide efflux gene (msrA) and the disinfectant resistance genes (qacH, qacA, qacB and qacC) were also investigated. Determination of the minimal inhibitory concentration values revealed that 54.5, 54.5, 68, and 82% of isolates were resistant to benzalkonium chloride, oxacillin, tetracycline and erythromycin respectively. The frequency of strains positive for the antibiotic resistance genes tested was 100 (blaZ), 50 (ermA), 36.4 (ermB), 22.7 (ermC) and 13.6% (mrsA). The qacH and the qacA genes were found in 22.7% of isolates and qacB and qacC in 13.6%. Two strains harboured three qac (qacH, qacA and qacB or qacC) genes. These data highlight the importance of determining the susceptibility to antibiotics and disinfectants of strains isolated in dental medicine in order to monitor the epidemiology and spread of multi-drug resistant staphylococci.

Cytotoxic effects exerted by polyarylsulfone dialyser membranes depend on different sterilization processes

Polyarylsulfone group is one of the most important polymeric materials used in the biomedical field, due to its excellent properties, such as good thermal, chemical, and mechanical stability. There are three important polyarylsulfone polymers, all of which have excellent electrical properties: polysulfone (PSu), polyarylsulfone (PAS) and polyarylethersulfone (PAES). All these polymers have excellent creep, radiation and high temperature resistance. In this study, we aimed to determine the effect of three sterilization processes (steam, ethylene oxide and gamma rays) on cytotoxicity of polyarylsulfone dialysis membranes. Ten long-term dialysis patients and ten age-matched healthy controls were enrolled in our study. We analysed (1) serum effect on cultured endothelial cell viability using MTT assay and (2) lipid peroxidation assessed by serum malondialdehyde (MDA) formation at the beginning (T0), the middle (T2) and the end (T4) of haemodialysis (HD) session. Our results clearly showed that steam-sterilized membranes improve endothelial cell viability when compared to ethylene oxide or gamma rays-sterilized ones. Moreover, there is a increased generation of MDA in patients sera during HD session. The serum MDA concentration was about 3, 6 and 10 times higher, respectively, for steam, ethylene oxide and gamma rays sterilization procedures when compared to the MDA amount in healthy subject sera. We concluded that using steam instead of ethylene oxide or gamma rays for sterilization may improve the biocompatibility of polyarylsulfone membranes.

Adsorption of aflatoxin B1, zearalenone and ochratoxin A by microorganisms isolated from Kefir grains

A strategy to reduce the deleterious effects of mycotoxins is to use dietary supplements that contain microorganisms that bind mycotoxins and decrease their gastrointestinal absorption. Novel strains were isolated from a Kefir culture and assessed for their mycotoxin adsorption and biotransformation ability. The most active strains were identified using DNA sequencing, and the stability of microorganism/mycotoxin complexes was evaluated using buffer solutions to simulate the pH conditions in the gastrointestinal tract. Our results showed that the microorganism consortium of Kefir grains adsorbed 82 to 100% of aflatoxin B1 (AFB1), zearalenone (ZEA) and ochratoxin A (OTA) when cultivated in milk. The main strains that were capable of mycotoxin adsorption were identified as Lactobacillus kefiri, Kazachstania servazzii and Acetobacter syzygii. The strain L. kefiri KFLM3 was the most active, adsorbing 80 to 100% of the studied mycotoxins when cultivated in milk. Nonetheless, the strain K. servazzii KFGY7 retained more mycotoxin after the desorption experiments (65, 69 and 67% for AFB1, OTA and ZEA, respectively). These findings suggest that Kefir consumption may help to reduce gastrointestinal absorption of these mycotoxins and consequently reduce their toxic effects. The isolated strains may be of interest for the development of fermented dairy products for human consumption that have a new probiotic characteristic, the adsorption of mycotoxins.