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In the current study, a simple method for the synthesis of 3-methyl-2-propyl-2H-[1,2,4]triazolo[4,3b][1,2,4,6]thiatriazine-1,1-dioxide (2) was carried out. In the presence of pyridine, a reaction between amidine (1) and sulfuryl chloride occurs. FTIR spectroscopy, 1H and 13C NMR, mass spectra, and elemental analysis were utilized in order to verify the structure of a novel synthetic molecule. The antibacterial activities of compound (2) were tested against eight pathogenic bacteria and the minimum inhibitory concentration as well as minimum bactericidal concentration were determined. Moreover, the possible antibiofilm effect of compound (2) was evaluated. Molecular docking was investigated to determine the interaction between compound (2) and eight crystal structures of bacterial and yeast proteins associated with virulence activity and antimicrobial resistance. Our results showed that the new 3-methyl-2-propyl-2H-[1,2,4]triazolo[4,3b][1,2,4,6]thiatriazine-1,1-dioxide (2) compound has a moderate antibacterial activity toward the selected pathogenic bacteria. The obtained MICs varied from 32 to 512 µg/mL being the lowest values attributed to Staphylococcus epidermidis ATCC 14990 and Streptococcus mutans ATCC 25175 (MIC = 32 µg/mL).We noted also that heterocyclic compound (2) may inhibit bacterial biofilm formation at concentration depend manner with a lowest value obtained against S.mutans ATCC 25175 (BIC50= 490 µg/mL). Molecular docking showed a promising inhibitory activity of compound (2) on TetM-mediated tetracycline resistance (3J25) and Staphylococcus aureus gyrase (3G7B) with lower binding energy compared to the other target proteins. KEY WORDS: Synthesis, Thiatriazine-1,1-dioxide, Antibacterial, Antibiofilm, Molecular docking Bull. Chem. Soc. Ethiop. 2022, 36(1), 109-117. DOI: https://dx.doi.org/10.4314/bcse.v36i1.10 &nbsp

The Aims of the study was to evaluate the antibacterial susceptibility and the biofilm eradication of three natural compounds carvacrol (CAR), thymol (TH) and eugenol (EUG), alone or in combination with nalidixic acid (NA) against twelve Salmonella Typhimurium strains. The minimum inhibitory concentration (MIC) and the minimum biofilm eradication concentration (BEC50) of the tested compounds (CAR, TH and EUG) and their combinations with NA were evaluated. In order to assess whether these bacteria had active efflux pumps, ethidium bromide (EtBr) accumulation assays was achieved using spectrophotometric accumulation assays. Moreover, scanning electron microscopy was used to visualize the bacterial biofilm formation on stainless steel surfaces after exposed to NA, CAR, TH and EUG alone and in combination. TH was the most effective essential oil, with the lowest MICs values ranging from 32 to 128 μg/mL followed by EUG and CAR. In addition, the combination of NA with the different compounds enhances antibiotic susceptibility of the tested bacterial strains. These results were confirmed by EtBr accumulation assays. A pronounced effect in decreasing biofilm mass was also noticed. Moreover, SEM revealed that bacterial membrane was disrupted and a complete loss of membrane integrity was also evident. The combination of natural compounds with antibiotic enhances bacterial susceptibility to NA. This combination ameliorates eradication of biofilm formed by S. Typhimurium on polystyrene microtitre plates. Additionally, this synergy induces an alteration of the bacterial cell surface visualized by SEM.

Azo dyes are recalcitrant pollutants, which are toxic, carcinogenic, mutagenic and teratogenic, that constitute a significant burden to the environment. The decolorization and the mineralization efficiency of Remazol Brillant Orange 3R (RBO 3R) was studied using a probiotic consortium (Lactobacillus acidophilus and Lactobacillus plantarum). Biodegradation of RBO 3R (750 ppm) was investigated under shaking condition in Mineral Salt Medium (MSM) solution at pH 11.5 and temperature 25°C. The bio-decolorization process was further confirmed by FTIR and UV-Vis analysis. Under optimal conditions, the bacterial consortium was able to decolorize the dye completely (>99%) within 12 h. The color removal was 99.37% at 750 ppm. Muliplex PCR technique was used to detect the Lactobacillus genes. Using phytotoxicity, cytotoxicity, mutagenicity and biototoxicity endpoints, toxicological studies of RBO 3R before and after biodegradation were examined. A toxicity assay signaled that biodegradation led to detoxification of RBO 3R dye.

In this study thymol (THY) and carvacrol (CAR), two monoterpenic phenol produced by various aromatic plants, was tested for their antibacterial and efflux pump inhibitors potencies against a panel of clinical and foodborne pathogenes. Our results demonstrated a substantial susceptibility of the tested bacteria toward THY and CAR. Especially, THY displayed a strong inhibitory activity (MIC's values ranged from 32 to 64 μg/mL) against the majority of the tested strains compared to CAR. Moreover, a significant reduction in MIC's of TET and benzalkonium chloride (QAC) were noticed when tested in combinations with THY and CAR. Their synergic effect was more significant in the case of THY which resulted a reduction of MIC's values of TET (2–8 fold) and QAC (2–8 fold). We noted also that THY and CAR inhibited the ethidium bromide (EtBr) cell efflux in a concentration-dependent manner. The rate of EtBr accumulation in food-borne pathogen was enhanced with THY and CAR (0, 250 and 500 μg/mL). The lowest concentration causing 50% of EtBr efflux inhibition (IC 50) was noticed in Salmonella enteritidis (1129) at 150 μg/mL of THY and 190 μg/mL of CAR respectively. These findings indicate that THY and CAR may serve as potential sources of efflux pump inhibitor in food-borne pathogens.

Aim The aim of this study was to explore the caries features in hypoplastic Amelogenesis Imperfecta (AI) patients. Materials and methods A cross-sectional study was performed including 28 patients, 14 with hypoplastic AI and 14 controls for whom Decayed (D), Missed (M) and Filled (F) Teeth (T) were checked for a DMFT index evaluation. Twenty-eight saliva samples, 4 bacterial plaques and 19 teeth were used. Decayed teeth were observed under polarized light and scanning electron microscopy. Salivary pH was measured and saliva bacterial strains were biochemically identified and confirmed by PCR. Bacterial adhesions to tooth surfaces were observed by Scanning Electron Microscopy (SEM) and evaluated by colony enumeration after in vitro culture of Streptococcus mutans and Lactobacillus casei with dental fragments. Results DMFT indexes were significantly lower in AI patients (mean DMFT = 0.8) compared to controls (mean DMFT = 2.9). Decayed teeth revealed sclerotic, demineralized, invaded and disintegrated zones in dentine. Dental plaques were rich with filamentous bacteria in AI patients. Oral microbiotome of the saliva showed a low rate of Streptococci and a significant high level of Bacillus spp, Enterococcus faecalis and Enterococcus faecium in AI patients. In vitro study showed a significant high adhesion of Lactobacillus casei and a weak adhesion of Streptococcus mutans on AI dental hard tissues. Conclusion Our study showed that hypoplastic AI patients have (i) a low DMFT index, (ii) an alkaline pH of saliva enriched with Bacillus spp, Enterococcus faecalis and Enterococcus faecium and (iii) dental tissues more easily invaded by Lactobacilli than Streptococci. The combination of these bacteria seems to give AI patients protection against dental caries.

To explore the efficiency of xylitol chewing gum enriched or not with remineralizing agents to protect tooth against cariogenic biofilm formation and demineralization. Six groups of chewing gums were prepared; Group 1: isomalt (1.8%), Group 2: casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) (3%) + isomalt (1.8%), Group 3: hydroxyapatite (HAP) (2.5%) + isomalt (1.8%), Group 4: xylitol (1.8%), Group 5: CPP-ACP (3%) + xylitol (1.8%) and Group 6: HAP (2.5%) + xylitol (1.8%). The antibiofilm properties of different chewing gum extracts using seven oral bacterial species including Streptococcus mutans, Streptococcus constellatus, Streptococcus Salivarius and Streptococcus oralis were explored via the crystal violet staining assay. The remineralizing effects of those products were assessed on thirty human permanent teeth, half-protected with varnish before chemical erosion and thermocycling process with chewing gum. Remineralization was evaluated using scanning electron microscopy and microscopic measurements on polarized light microscopy. The ratio R comparing the thickness between unvarnished and varnished sides was evaluated. While the minimum biofilm inhibitory concentration (MBIC50) was low for xylitol alone compared to isomalt, it was inconsistent when enriched with remineralizing agents. The minimum biofilm eradication concentration (MBEC50) was low for xylitol groups compared to isomalt, for all the studied strains. R was significantly lower in Group 1 and Group 2, while Group 6 showed the highest ratio. Xylitol chewing gums confirmed good antibiofilm properties and showed remineralized potential on eroded teeth. When xylitol is associated to CPP-ACP or HAP, antibiofilm activity decreased while remineralization of eroded teeth increased.

Dental caries remains the most prevalent oral infectious disease worldwide. In this study, the antibacterial and the antibiofilm activities of five essential oils (EO's): eugenol (EUG), carvacrol (CAR), thymol (TYH), p-cymene (CYM) and γ-terpinene (TER) were tested (alone or in combinaison with tetracycline) against oral bacteria. In addition, their potential roles to enhance the accumulation of ethidium bromide (EtBr) in bacterial cells were tested. Our results indicated that EO's induced a selective antimicrobial activity. A synergistic effect of EO's and tetracycline (TET) was noticed with a reduction rate ranged from 2 to 8-fold. In addition, the efflux of EtBr was inhibited with a decrease in loss of EtBr from the bacteria. On the other hand a significant anti-biofilm activities of EO's (alone or combined with antibiotics) was noticed. In conclusion the tested EO's may be considered as a potential natural source with a resistance-modifying activity and may be applied to eradicate bacterial biofilm.

Tunisian Juglans regia bark ethanol extract (JREE) was examined for antibacterial, antibiofilm, and antioxidant activity. Its anti-proliferative effect was evaluated on normal (MRC-5) and cancer (HT29 and HEp-2) cell lines. The data revealed that JREE inhibited the growth of pathogenic bacteria (MICs value ranged from 32 to 128 μg.mL-1) and induced a strong antibiofilm effect (BIC50 was 23, 30, and 43 μg.mL-1 for Micrococcus luteus, Staphylococcus epidermidis, and Staphylococcus aureus, respectively). The JREE showed high scavenging property against 2,2-diphenyl-l-1-picrylhydrazil (DPPH) radical (IC50 = 3.5 μg.mL-1) and exhibited anti-proliferative effects with IC50 values ranging from 6.6 to 25.5 μg.mL-1.

Three Lactobacilli were isolated from traditional Tunisian fermented food and then characterized for its ability to inhibit Listeria monocytogenes growth. Antagonistic effect of Lactobacillus plantarum on four L. monocytogenes strains was tested in soft artisanal cheese. Anti-biofilm activity of Lactobacillus extracts was also tested. Our results demonstrate that the selected lactic acid bacteria (LAB) extract exhibited a good antibacterial effect against L. monocytogenes ATCC 19115 with low MICs values: 8.33 2.1, 20 1.2 and 23.33 1, respectively. L. plantarum also exhibited a stronger inhibitory effect against L. monocytogenes when grown in cheese. Moreover, a potential anti-biofilm effect of the three LAB extracts with BIC50 values ranging from 5% to 15% for L. monocytogenes ATCC 19115 was demonstrated. Although LAB extracts were able to eradicate significantly a preformed L. monocytogenes biofilm (P < 0.05). Growth inhibition of preformed biofilm was more difficult to achieve. LAB could be used as a bioprotective culture in cheese ripening to prevent Listeria growth.

In this study the minimal inhibitory concentration (MICs) of tetracycline (Tet), erythromycin (Ery) and benzalkonium chloride (BC) in absence and in presence of a sub-MIC of juglone (Jug) were determined. In addition, the Ethidium bromide (EtBr) efflux assay was performed to assess the effect of Jug on EtBr cells accumulation. Our results showed a selective antimicrobial activity of Jug against the tested strains. A synergistic effect of Jug, drugs (Tet and Ery) and disinfectant (BC) was noticed with a reduction rate varied from 2 to 16-fold. In addition, the efflux of EtBr was inhibited depending on the Jug concentration. In the presence of Jug, a decrease in loss of EtBr from bacteria was observed. The concentration inducing 50 % of EtBr efflux inhibition after 15 min was about 182 μg ml-1 for S. aureus ATCC 25923, 236 μg ml-1 for S. aureus B193 and 195 μg ml-1 for S. aureus B456. It appears from this study that Jug may be used as a natural source for resistance-modifying activity in same bacteria.

Candida is a major Human pathogen causing a variety of infections and can survive for extended period of time in aquatic environment including marine and fresh water. In this study we compared a colorimetric XTT assay to colony forming units (CFU) count to evaluate the survival potential of Candida albicans incubated in water microcosms. Our results showed that cells maintain cultivability within a long period followed by a decline in cultivability and a drop of plate counts to less than 20 cell ml(-1) after 150 days in tap water, 190 days in rain water and 200 days in seawater. In addition we noted that 10% of cells viability was reached after 150 days in seawater, 180 days in rain water and 210 days in tap water. Molecular method confirms the persistence of C. albicans cells in water during long time starvation period.

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.

International audience; Understanding the mechanism of the bacterial cell adhesion to solid surfaces is of great medical and industrial importance. Bacterial adhesion to inert surfaces, such as a catheter, and other indwelling devices can form biofilm, consequently cause severe morbidity and often fatal infections. Initial bacterial adhesion to the material surfaces is a complicated process that is affected by various physicochemical properties of both bacterial cells and substratum surfaces. The surface properties of the cells were characterized by the sessile drop technique. Moreover, the interfacial free energy of Staphylococcus aureus adhesion to the supporting materials was determined. The results showed that S. aureus examined at different pH levels could be considered hydrophilic. We noted hat the electron-donor character of S. aureus was important at intermediate pH (pH 5, pH 7, and pH 9) and it decreased at both limits acidic and basic conditions. In addition, the adhesion of Staphylococcus aureus ATCC 25923 to the hydrophilic glass and hydrophobic indium tin oxide (ITO)-coated glass surfaces at different pH values (2, 3, 5, 7, 9 and 11) was investigated using atomic force microscopy (AFM) and image analysis was assessed with the Mathlab(A (R)) program. The data analysis showed that cells (number of adhering cells to glass and ITO-coated glass surface) adhered strongly at acidic pH and weakly at alkaline pH. Also, S. aureus has the ability to attach to both hydrophobic and hydrophilic surfaces, but the adhesion was higher on hydrophobic surface.

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 200 μg/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.

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).

In this study we investigated the phenotypic slime production of Vibrio alginolyticus and Vibrio parahaemolyticus strains, food-borne pathogens, using a Congo red agar plate assay. Furthermore, we studied their ability to adhere to abiotic surfaces and Vero cells line. Our results showed that only V. alginolyticus ATCC 17749 was a slime-producer developing almost black colonies on Congo red agar plate. Adherence to glace tube showed that all V. alginolyticus strains were more adherent than V. parahaemolyticus. Only V. alginolyticus ATCC 17749 was found to be able to form biofilm on polystyrene microplate wells (OD570 = 0.532). Adherence to Vero cells showed that all tested strains were non adherent after 30 min, however after 60 min all the studied strains become adherent. The percentage of adherence ranged from1.23% to 4.66%. Neste estudo, investigou-se a produção de muco por cepas de Vibrio alginolyticus e Vibrio parahaemolyticus através do teste em placa de ágar com vermelho congo. Estudou-se também a capacidade de adesão à superfícies abióticas e células Vero. Os resultados indicaram que somente V. alginolyticus ATCC 17749produziu muco, formando colônias quase negras nas placas de ágar com vermelho congo. O teste de adesão a tubos de vidro indicou que as cepas de V. alginolyticus foram mais aderentes do que as de V. parahaemolyticus. Somente V. alginolyticus ATCC 17749 foi capaz de formar biofilme nos poços das microplacas de poliestireno (OD570=0,532). Testes de adesão a células Vero mostraram que nenhuma das cepas apresentou adesão em 30 min, mas todas aderiram após 60 min. A porcentagem de adesão variou de 1,23% a 4,66%.

In this study we investigated the phenotypic slime production of Vibrio alginolyticus and Vibrio parahaemolyticus strains, food-borne pathogens, using a Congo red agar plate assay. Furthermore, we studied their ability to adhere to abiotic surfaces and Vero cells line. Our results showed that only V. alginolyticus ATCC 17749 was a slime-producer developing almost black colonies on Congo red agar plate. Adherence to glace tube showed that all V. alginolyticus strains were more adherent than V. parahaemolyticus. Only V. alginolyticus ATCC 17749 was found to be able to form biofilm on polystyrene microplate wells (OD570 = 0.532). Adherence to Vero cells showed that all tested strains were non adherent after 30 min, however after 60 min all the studied strains become adherent. The percentage of adherence ranged from1.23% to 4.66%.Neste estudo, investigou-se a produção de muco por cepas de Vibrio alginolyticus e Vibrio parahaemolyticus através do teste em placa de ágar com vermelho congo. Estudou-se também a capacidade de adesão à superfícies abióticas e células Vero. Os resultados indicaram que somente V. alginolyticus ATCC 17749produziu muco, formando colônias quase negras nas placas de ágar com vermelho congo. O teste de adesão a tubos de vidro indicou que as cepas de V. alginolyticus foram mais aderentes do que as de V. parahaemolyticus. Somente V. alginolyticus ATCC 17749 foi capaz de formar biofilme nos poços das microplacas de poliestireno (OD570=0,532). Testes de adesão a células Vero mostraram que nenhuma das cepas apresentou adesão em 30 min, mas todas aderiram após 60 min. A porcentagem de adesão variou de 1,23% a 4,66%.

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.

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.

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.

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%), beta-caryophyllene (1.4%) and 2-heptanone (0.9%). The tested essential oil exhibited a very strong radical scavenging activity (IC50 = 0.2 microg ml-1) when compared with the synthetic antioxidant (tert-butylated hydroxytoluene, IC50 = 11.5 microg ml-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.

In clinicalStaphylococcus aureus strains, the presence of theica genes, biofilm formation and susceptibility to antibiotics are considered important factors of virulence. In this study, 35 strains ofS. aureus, isolated from auricular infection, were investigated for slime production using Congo red agar (CRA) method, antibiotic susceptibility, presence ofmecA gene, and presence oficaA andicaD gene. The results show that 60% of strains weremecA positive when tested by PCR although 25.7% of strains were oxacillin resistant when tested with ATB STAPH. Qualitative slime production ofS. aureus using CRA revealed that 74.3% ofS. aureus were slime producers. All the strains carried theica gene.

Vibrio alginolyticus was isolated from the internal organs of diseased gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) cultured in two fish farms located on the Tunisian Mediterranean coast, from 2003 to 2005. After phenotypic characterisation, a selection of 34 isolates from gilthead sea bream and sea bass were molecularly typed by repetitive intergenic consensus PCR (ERIC-PCR) showing a high polymorphism among the isolated strains (19 genotypes). Most of the isolates were resistant to atleast two antimicrobial agents. All the tested strains were resistant to ampicillin. However, 91.17% were resistant to nitrofurantoin and 35.29% to tetracycline. Several strains isolated from diseased gilthead sea bream and sea bass were tested for virulence in both fish species by intraperitoneal injection. The selected isolates (n=7) were pathogenic for gilthead sea bream and sea bass. LD(50) values ranged from 5.01 x 10(4) to 6.20 x 10(7)CFU/fish. This is the first report on characterisation and virulence of V. alginolyticus for sea bass and sea bream in Tunisia.

To analyze the degree of biofilm formation on three ica-positives Staphylococcus epidermidis as a function of biocides, the medium was supplemented with increasing concentrations of isopropanol, ethanol, and methanol at 0, 1, 4, 6, 8, 10, 12 and 14% (v/v), hydrogen peroxide (0, 0.125, 0.25, 0.5, 1, 2, 3, 4 and 5% v/v) and benzalkonium chloride (0, 0.125, 0.25, 0.5, 1, 2, 3, 4 and 6 μg ml⁻¹). In biocide-free biofilms, the results showed that two strains (S. epidermidis CIP106510 and E24) were strongly biofilm positive displaying a high oxidative activity (1.254 and 0.855, respectively) in comparison with the non-adherent one (S22). In addition biofilm formation was induced with 1% alcohol (isopropanol and ethanol) supplementation. The three studied strains cultured in TSB supplemented with 2% methanol displayed a strong oxidative activity (P=0.008). Moreover wells with 0.125% hydrogen peroxide enhanced increasing oxidative activity of S. epidermidis CIP106510 and S22. A significant induction of biofilm was noted after treatment with 1 μg ml⁻¹ of benzalkonium chloride. This study suggests that some biocides currently used in hospitals are ineffective against nosocomial pathogens growing in biofilms when used at weak concentration and fail to control this reservoir for hospital-acquired infection.

Biofilm formation is the leading cause of the pathogenesis of S. aureus associated with biomaterial infections. In S. aureus polysaccharide intercellular adhesin (PIA) was encoded by icaA and icaD genes. Production of PIA is currently responsible for staphylococcal biofilm development. In this study, S. aureus strains isolated from auricular infection (n = 46) and S. aureus ATCC 25923 were phenotyped and genotyped. Slime production was assessed using Congo red agar plate assay. In order to determine the biofilm formation capacity at various pH levels of the studied S. aureus strains, microtiter plate assay was performed. The strains were grown in medium adjusted at various pH levels (3, 5, 7, 9 and 12) and medium supplemented with hydrogen peroxide 3% (v/v). Qualitative biofilm production of S. aureus revealed that 56.5% of strains were slime producers. In addition 78.26% of strains were icaA and icaD positive. Quantitative biofilm showed that biofilm production depended on the pH value of the medium. At highly acidic (pH 3) and alkaline (pH 12) levels, biofilm formation was lower, while at pH 7 the adhesion was moderate. In addition, the cells adhered weakly after 3% hydrogen peroxide supplementation. Our results suggest that pH was a stress factor that led some S. aureus strains to produce the biofilm.

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.

The rate of the onset of methicillin-resistant Staphylococcus epidermidis infections is increasing in Tunisia. We have isolated 32 S. epidermidis strains from dialysis fluid and needle cultures in dialysis service. The strains were identified by classic methods (colonial morphology, Gram staining, catalase test, coagulase test, and DNase test) as well as by API ID32 Staph. Susceptibilities to 18 antibiotics were tested with the ATB Staph kit. Most of the tested strains were resistant to penicillin. In addition, the presence of multidrug resistant strains that showed resistance to different antibiotics was recorded. We have characterized these strains by multiplex PCR assay to identify intercellular adhesion genes icaA/icaD associated with the adhesiveness of staphylococci in biomaterials, and to identify representative resistant genes: oxacillin resistance, mecA; erythromycin methylase (ermA, ermB, and ermC), and macrolide efflux gene (msrA and mef). The frequency of the carriage of these genes was icaA/icaD (71.9%), mecA (78.1%), ermA (12.5%), ermB (31.3%), ermC (53.1%), msrA (68.8%), and mef (O%). Although the carriage of the genes and the results of susceptibility testing did not match exactly, it could be judged that the PCR identification of antibiotic resistance genes is rapid and supplementary methods for identifying staphylococci or epidemiological study used for the control of nosocomial infection.

The essential oil extracted from the dried flower buds of clove, Eugenia caryophyllata L. Merr. & Perry (Myrtaceae), is used as a topical application to relieve pain and to promote healing and also finds use in the fragrance and flavouring industries. The main constituents of the essential oil are phenylpropanoids such as carvacrol, thymol, eugenol and cinnamaldehyde. The biological activity of Eugenia caryophyllata has been investigated on several microorganisms and parasites, including pathogenic bacteria, Herpes simplex and hepatitis C viruses. In addition to its antimicrobial, antioxidant, antifungal and antiviral activity, clove essential oil possesses antiinflammatory, cytotoxic, insect repellent and anaesthetic properties. This short review addresses the chemical composition and biological effects of clove essential oil, and includes new results from GC/MS analysis and a study of its antimicrobial activity against a large number of multi-resistant Staphylococcus epidermidis isolated from dialysis biomaterials.

Background Enterococci 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. Results In 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. Conclusions Drugs 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.

Background Staphylococcus 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. Methods S. 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 (ermA, ermB, ermC, msrA and mef) were analysed by multiplex PCR and disinfectant-resistant genes (qacA, qacB, and qacC) were studied by PCR-RFLP. Results The frequency of erythromycin resistance genes in S. aureus was: ermA+ 7.7%, ermB+ 13.7%, ermC+ 6% and msrA+ 10.2%. In addition, the number of positive isolates in CoNS was respectively ermA+ (9.4%), ermB+ (11.1%), ermC+ (27.4%), and msrA+ (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 (qacA, qacB and qacC). Nine strains (7.7%) among the CoNS species and two S. aureus strains (2%) harboured the three-qac genes. In addition, the qacC were detected in 41 strains. Conclusions Multi-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.