Quinolone resistance among Salmonella Kentucky and Typhimurium isolates in Tunisia: first report of Salmonella Typhimurium ST34 in Africa and qnrB19 in Tunisia.

AIMS: Characterization of quinolone-resistant Salmonella Kentucky and Typhimurium isolates in Tunisia from various sources, detection of some plasmid-mediated quinolone resistance genes and the genetic relatedness. METHODS: A total of 1404 isolates of S. Kentucky (n = 1059)/S. Typhimurium (n = 345) from various sources from all over Tunisia were tested for quinolone resistance by disk diffusion method. Minimum inhibitory concentrations of nalidixic acid, ciprofloxacin and ofloxacin were determined. Quinolone-resistant isolates were screened for plasmid-mediated quinolone-resistance genes (qnrA,qnrB,qnrS, aac(6')-Ib-cr and qepA) by polymerase chain reaction (PCR). Mutations in the quinolone-resistance-determining regions of the gyrA and parC genes were detected by PCR and DNA sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were accomplished for isolates harbouring plasmid-mediated quinolone-resistance genes. RESULTS: According to our selection criteria (NAL = resistance phenotype; CIP = resistant with diameter 0, or intermediate), only 63 S. Kentucky/41 S. Typhimurium isolates were investigated: 49% (5/104) were multidrug resistant. Two S. Typhimurium isolates harboured qnrB19 with different PFGE profiles. A mutation was detected in the gyrA gene for each of these two isolates. MLST revealed the presence of ST313 and ST34, an endemic sequence type. CONCLUSION: Our study highlights the presence of quinolone multidrug-resistant Salmonella in humans and animals in Tunisia. This is the first report of S. Typhimurium ST34 in Africa and qnrB19 in Tunisia. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that describes not only the current epidemiological situation of the quinolone resistance in S. Kentucky and Typhimurium isolated from various sources and regions in Tunisia, but also, the genetic resistance determinants associated with phenotypic antibiotic resistance and the molecular mechanisms of their quinolone-resistance. Also, we provide the first report of S. Typhimurium ST34 in Africa, and the first report of qnrB19 in Salmonella in Tunisia.

Cytotoxic, genotoxic and antigenotoxic potencies of oligorutins.

Rutin has been enzymatically oligomerized by laccase from Trametes versicolor. Five fractions of oligomers were obtained from the monomers having high solubility in water, which can reach 351-times that of rutin. Cytotoxicity of rutin and oligorutin fractions was evaluated towards K562 cells. Oligorutin fractions showed a lower antiproliferative effect compared with its monomer. The genotoxic potential of rutin and oligorutin fractions was assessed, at the limit of the solubility of each molecule, using the comet test. None of the tested concentrations of either rutin or oligorutin fractions has showed a genotoxic effect. Similarly, the antigenotoxic effect of these flavonoids was tested using the same assay. The obtained results showed a higher ability of oligorutin fractions to reduce the genotoxicity induced by hydrogen peroxide compared with monomeric rutin.

Ethyl acetate extract and its major constituent, isorhamnetin 3-O-rutinoside, from Nitraria retusa leaves, promote apoptosis of human myelogenous erythroleukaemia cells.

OBJECTIVE: Fractionation of ethyl acetate extract (EA) obtained from Nitraria retusa leaves was assessed using different methods of chromatography, and isorhamnetin3-O-rutinoside (I3-O-R) was isolated from this extract. Its structure was determined using data obtained from (1) H and (13) C NMR spectra, as well as by various correlation experiments (COSY, HMQC and HMBC). Both EA extract and I3-O-R were investigated for their ability to induce apoptosis in human chronic myelogenous erythroleukaemia cells (K562). MATERIALS AND METHODS: Apoptosis of cells from the K562 line was detected by DNA fragmentation, PARP cleavage and by evaluating activities of caspases 3 and 8. RESULTS: Apoptosis, revealed by DNA fragmentation and PARP cleavage, was observed after 48-h incubation of these human myelogenous erythroleukaemia cells (K562), with the tested products. Likewise, caspase 3 and caspase 8 activities were induced in the presence of the EA extract and I3-O-R after 48 h of incubation. CONCLUSION: Our results strongly suggest the involvement of the extrinsic pathway of apoptosis in cells treated by both the original EA extract and its major component, I3-O-R.

Antimutagenic, antigenotoxic and antioxidant activities of phenolic-enriched extracts from Teucrium ramosissimum: combination with their phytochemical composition.

The evaluation of the mutagenic and antimutagenic actions of extracts obtained from aerial part of Teucrium ramosissimum was assayed using the Salmonella typhimurium assay system. The effect of the same extracts on genotoxicity and SOS response induced by aflatoxin B(1) as well as nitrofurantoin was investigated in a bacterial assay system, i.e., the SOS chromotest with Escherichia coli PQ37. The different extracts showed no mutagenicity when tested with Salmonella typhimurium strains TA100, TA98 and TA1535 either with or without S9 mix. In contrast, our results prove that T. ramosissimum extracts possess antimutagenic effects against sodium azide, aflatoxin B1, benzo[a]pyrene and 4-nitro-o-phenylenediamine. Moreover, the T. ramosissimum tested extracts exhibited no genotoxicity either with or without the external S9 activation mixtures. However, all the extracts significantly decreased the genotoxicity induced by aflatoxin B(1) and nitrofurantoin. The result obtained by the Ames test confirms those of SOS chromotest. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) and the non enzymatic (NBT/riboflavine, DPPH and ABTS assays) systems. All extracts exhibited high antioxidant activity except the chloroform and the methanol extracts in DPPH and NBT/riboflavine assays respectively. Our results underline the potential of T. ramosissimum to avoid mutations and also its antioxidant potential.

Induction of apoptosis in human lymphoblastoid cells by kaempferol 3-O-ß-isorhamninoside and rhamnocitrin 3-O-ß-isorhamninoside from Rhamnus alaternus L. (Rhamnaceae).

OBJECTIVE: Kaempferol 3-O-ß-isorhamninoside (K3O-ir) and rhamnocitrin 3-O-ß-isorhamninoside (R3O-ir) from Rhamnus alaternus L leaves are investigated for their ability to induce apoptosis in human lymphoblastoid cells. We have attempted to characterize apoptotic pathway activated by these two flavonoids. MATERIAL AND METHODS: Apoptosis of the human TK6 lymphoblastoid cell line was detected by DNA fragmentation, PARP cleavage and by evaluating caspase activity. RESULTS: Apoptosis was observed after 24- and 48-h incubation of the cells with the tested compounds. DNA fragmentation was observed after treatment with flavonoids; this was confirmed by demonstration of PARP cleavage. Caspase-3 and caspase-8 activities were induced by both K3O-ir and R3O-ir flavonoids showing highest activity with compound concentration of 400 µg/ml. CONCLUSION: We have demonstrated that K3O-ir and R3O-ir induce apoptosis in human lymphoblastoid cells by the extrinsic pathway of apoptosis.

Chemical investigation of different crude extracts from Teucrium ramosissimum leaves. Correlation with their antigenotoxic and antioxidant properties.

The effect of extracts obtained from Teucrium ramosissimum leaves on genotoxicity and SOS response induced by aflatoxin B(1) (0.5 µg/assay) as well as nitrofurantoin (5 µg/assay) was investigated in a bacterial assay system, i.e., the SOS chromotest with Escherichia coli PQ37. The T. ramosissimum tested extracts exhibited no genotoxicity either with or without the external S9 activation mixture. However, all the extracts, particularly the total oligomers flavonoids (TOF) extract significantly decreased the genotoxicity induced by aflatoxin B(1) and nitrofurantoin. Antioxidant capacity of the tested extracts was evaluated using the enzymatic (xanthine/xanthine oxidase assay) (X/XOD) and the non-enzymatic (NBT/Riboflavine assay) systems. TOF extract was the most effective one in inhibiting both xanthine oxidase activity and NBT reduction. Our findings emphasize the potential of T. ramosissimum to prevent mutations and also its antioxidant effect.

Chloroform extract from Moricandia arvensis inhibits growth of B16-F0 melanoma cells and promotes differentiation in vitro.

OBJECTIVES: Poor therapeutic results have been reported for treatment of malignant melanoma; therefore in this study we have investigated inhibitory capacity of ethyl acetate, chloroform (Chl) and methanol extracts from Moricandia arvensis on mouse melanoma (B16-F0) and human keratinocyte (HaCaT) cell proliferation. Influence of Chl extract on percentage distribution in cell cycle phases and melanogenesis was also studied. MATERIAL AND METHODS: Cell viability was determined at various periods using the MTT assay, and flow cytometry was used to analyse effects of Chl extract on progression through the cell cycle and apoptosis. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. RESULTS: Chl extract exhibited significant anti-proliferative activity after incubation with the two types of tumour skin cells. Morphological changes in B16-F0 cells, accompanied by increase of tyrosinase activity, and of melanin synthesis were observed, which are markers of differentiation of malignant melanoma cells. Furthermore, cell cycle analysis revealed that B16-F0 cells treated with Chl extract were arrested predominantly in G(1) phase. CONCLUSION: Chl extract had the ability to reverse malignant melanoma cells from proliferative to differentiated state, thus providing a new perspective in developing novel strategies for prevention and treatment of malignant melanoma, possibly through consumption of the extract in an appropriate cancer prevention diet. Moreover, there is scope for the extract being introduced into cosmetic products as a natural tanning agent.

Assessment of phenolic content, free-radical-scavenging capacity genotoxic and anti-genotoxic effect of aqueous extract prepared from Moricandia arvensis leaves.

The present study was undertaken to provide a set of data on the safety of an aqueous extract (AQE) from Moricandia arvensis. For this reason, Escherichia coli tested strains PQ35 and PQ37 were used to detect induction of DNA lesions by AQE. The SOS Chromotest showed that AQE induced a marginally genotoxic effect, as expressed by the induction factor (IF) value only with E. coli PQ37 tested strain (IF=1.77 at a dose of 250 microg/assay). The measurement of the anti-genotoxic activity of the AQE was also studied by inhibition of beta-galactosidase induction. A significant anti-genotoxic effect was observed with different tested doses of AQE, which suggests that M. arvensis extract has the potential to protect DNA from the action of nitrofurantoïn (NF) and free radicals generated by hydrogen peroxide (H2O2). In addition to anti-genotoxic activity, AQE showed a free-radical-scavenging capacity towards ABTS+* and DPPH*. Total phenolic content was also evaluated following Folin-Ciocalteu method and results indicated high correlation between total phenol content and anti-genotoxic and antioxidant activities for AQE, but the highest correlation was showed with its capacity to stabilize ABTS+* (R2=0.9944).

Antigenotoxic and antioxidant activities of Pituranthos chloranthus essential oils.

The SOS-chromotest in Escherichia coli is a widely used bacterial genotoxicity assay to test potential carcinogens. The aim of this work is to evaluate the genotoxic and antigenotoxic activities of essential oils obtained from aerial parts of Pituranthos chloranthus. The tested essential oils were not genotoxic towards both E. coli PQ37 and PQ35 strains. These essential oils reduced significantly Nifuroxazide and H(2)O(2)-induced genotoxicity. Essential oils showed a protective effect against damages induced by radicals, obtained from the photolysis of H(2)O(2), on DNA plasmid through free radical scavenging mechanisms. The scavenging capacity of these essential oils was also estimated by evaluating the inhibition of ABTS(+.) radical.

Antigenotoxic and free radical scavenging activities of extracts from Moricandia arvensis.

This study evaluates genotoxic and antigenotoxic effects of extracts from leaves of Moricandia arvensis, which are used in traditional cooking and medicines. Extracts showed no genotoxicity when tested with the SOS Chromotest using E. coli PQ37 and PQ35 strains, except for the total oligomers flavonoids enriched extract. Petroleum ether and methanol extracts are the most active in reducing nitrofurantoin genotoxicity, whereas methanol and total oligomers flavonoids enriched extracts showed the most important inhibitory effect of H2O2 genotoxicity. In addition, these two extracts showed important free radical scavenging activity toward the DPPH. radical, whereas the chloroform extract exhibited the highest value of TEAC against ABTS+. radical.

Antiinflammatory and analgesic activities of Zizyphus lotus root barks.

The root barks of Zizyphus lotus were extracted with water, chloroform, ethyl acetate and methanol to determine their antiinflammatory and analgesic activities. Aqueous extract (50, 100 and 200 mg/kg) given intraperitoneally (i.p.) showed a significant and dose-dependent antiinflammatory and analgesic activity.

Anti-genotoxic and free-radical scavenging activities of extracts from (Tunisian) Myrtus communis.

The effect of extracts from leaves of Myrtus communis on the SOS reponse induced by Aflatoxin B1 (AFB1) and Nifuroxazide was investigated in a bacterial assay system, i.e. the SOS chromotest with Escherichia coli PQ37. Aqueous extract, the total flavonoids oligomer fraction (TOF), hexane, chloroform, ethyl acetate and methanol extracts and essential oil obtained from M. communis significantly decreased the SOS response induced by AFB1 (10 microg/assay) and Nifuroxazide (20 microg/assay). Ethyl acetate and methanol extracts showed the strongest inhibition of the induction of the SOS response by the indirectly genotoxic AFB1. The methanol and aqueous extracts exhibited the highest level of protection towards the SOS-induced response by the directly genotoxic Nifuroxazide. In addition to anti-genotoxic activity, the aqueous extract, the TOF, and the ethyl acetate and methanol extracts showed an important free-radical scavenging activity towards the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical. These results suggest the future utilization of these extracts as additives in chemoprevention studies.

Dammarane saponins from Zizyphus lotus.

Four dammarane-type saponins were isolated by means of centrifugal partition chromatography from the root bark of Zizyphus lotus. Their structures were elucidated using a combination of 1D and 2D 1H and 13C NMR spectra and mass spectroscopy. One of these glycosides is the known jujuboside A. The others are three new dammarane saponins, identified as 3-O-beta-D-glucopyranosyl(1-->6)-beta-D-glucopyranosyl (1-->3)-[alpha-L-rhamnopyranosyl(1-->2)]-alpha-L-arabinopyranosyl jujubogenin = jujuboside C, 3-O-alpha-L-rhamnopyranosyl (1-->2)-[beta-D-glucopyranosyl(1-->3)]-beta-D-galactopyranosyl lotogenin = lotoside I, and 3-O-alpha-L-rhamnopyranosyl(1-->2)-[beta-D-glucopyranosyl (1-->3)]-beta-D-glucopyranosyl lotogenin = lotoside II. Lotogenin is a new dammarane derivative identified as (15R, 16R, 20R, 22R)-16 beta, 22-epoxydammar-24-ene-3 beta, 15 alpha, 16 alpha, 20 beta-tetraol.

Adenosine triphosphate in human semen: a quantitative estimate of fertilizing potential.

The concentration of adenosine triphosphate (ATP) per milliliter of ejaculate was found to be significantly correlated with the following parameters: sperm concentration, number of motile spermatozoa per milliliter, capacity of spermatozoa to migrate against gravity, and in vitro potential to penetrate zona-free hamster ova. The ATP concentration was significantly lower in the semen of infertile men with normal sperm concentration and motility, compared with matched fertile donors. The ATP concentration of deep-frozen donor semen was significantly correlated with its fertilizing potential as estimated from the pregnancy per cycle index in an artificial insemination by donor semen program. These qualities suggest that measurement of semen ATP may be a possible biochemical method for the quantitation of the actual fertilizing potential of the ejaculate.