The aim of the present research was to study the effects of olive leaf addition (0 and 3%) on the major antioxidants and the antioxidant activity of Neb Jmel and Oueslati olive oils. Olives and leaves of the two Tunisian varieties were harvested during the 2016/2017 crop season. Both leaves and oils were characterised for their concentrations in phenolics, tocopherols and antioxidant power. Other parameters such as free acidity, peroxide value, chlorophyll and carotenoid concentrations were also taken into consideration. Compared to Oueslati, the Neb Jmel oil showed a lower free acidity (50%) and peroxide value (5.6-fold), and higher chlorophyll (1.6-fold), total phenolics (1.3-fold), flavonoid (3-fold) and oleuropein derivative (1.5-fold) concentrations, in addition to an increased antioxidant activity (1.6-fold). Leaf addition promoted a significant increment in total chlorophyll, α-tocopherol and phenolics in both varieties, above all in Oueslati oil, due to a higher abundance of bioactive constituents in the corresponding leaves. In particular, chlorophyll and carotenoid concentrations reached values twice higher than in Neb Jmel leaves, and flavonoids and oleouperin derivatives were three-fold higher. This prevented the oxidation and the formation of peroxides, reducing the peroxide value of the fortified oil to the half. The results provide evidence on the performance of the Tunisian Neb Jmel and Oueslati varieties, showing that their oils present a chemical profile corresponding to the extra virgin olive oil category and that, after leaf addition, their nutritional value was improved.

c Antifungal resistance of Candida species is a clinical problem in the management of diseases caused by these pathogens. In this study we identified from a collection of 423 clinical samples taken from Tunisian hospitals two clinical Candida species (Candida albicans JEY355 and Candida tropicalis JEY162) with decreased susceptibility to azoles and polyenes. For JEY355, the fluconazole (FLC) MIC was 8 g/ml. Azole resistance in C. albicans JEY355 was mainly caused by overexpression of a multidrug efflux pump of the major facilitator superfamily, Mdr1. The regulator of Mdr1, MRR1, contained a yet-unknown gain-of-function mutation (V877F) causing MDR1 overexpression. The C. tropicalis JEY162 isolate demonstrated cross-resistance between FLC (MIC > 128 g/ml), voriconazole (MIC > 16 g/ml), and amphotericin B (MIC > 32 g/ml). Sterol analysis using gas chromatography-mass spectrometry revealed that ergosterol was undetectable in JEY162 and that it accumulated 14␣-methyl fecosterol, thus indicating a perturbation in the function of at least two main ergosterol biosynthesis proteins (Erg11 and Erg3). Sequence analyses of C. tropicalis ERG11 (CtERG11) and CtERG3 from JEY162 revealed a deletion of 132 nucleotides and a single amino acid substitution (S258F), respectively. These two alleles were demonstrated to be nonfunctional and thus are consistent with previous studies showing that ERG11 mutants can only survive in combination with other ERG3 mutations. CtERG3 and CtERG11 wild-type alleles were replaced by the defective genes in a wild-type C. tropicalis strain, resulting in a drug resistance phenotype identical to that of JEY162. This genetic evidence demonstrated that CtERG3 and CtERG11 mutations participated in drug resistance. During reconstitution of the drug resistance in C. tropicalis, a strain was obtained harboring only defective Cterg11 allele and containing as a major sterol the toxic metabolite 14␣-methyl-ergosta-8,24(28)-dien-3␣,6␤-diol, suggesting that ERG3 was still functional. This strain therefore challenged the current belief that ERG11 mutations cannot be viable unless accompanied by compensatory mutations. In conclusion, this study, in addition to identifying a novel MRR1 mutation in C. albicans, constitutes the first report on a clinical C. tropicalis with defective activity of sterol 14␣-demethylase and sterol ⌬ 5,6 -desaturase leading to azole-polyene cross-resistance.

In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a rapid method to identify yeasts isolated from patients in Tunisian hospitals. When identification could not be exstablished with this procedure, sequencing of the internal transcribed spacer with 5.8S ribosomal DNA (rDNA) (ITS1-5.8S-ITS2) and D1/D2 domain of large-subunit (LSU rDNA) were employed as a molecular approach for species differentiation. Candida albicans was the dominant species (43.37% of all cases), followed by C. glabrata (16.55%), C. parapsilosis (13.23%), C. tropicalis (11.34%), C. dubliniensis (4.96%), and other species more rarely encountered in human diseases such as C. krusei, C. metapsilosis, C. lusitaniae, C. kefyr, C. palmioleophila, C. guilliermondii, C. intermedia, C. orthopsilosis, and C. utilis. In addition, other yeast species were obtained including Saccharomyces cerevisiae, Debaryomyces hansenii (anamorph known as C. famata), Hanseniaspora opuntiae, Kodamaea ohmeri, Pichia caribbica (anamorph known as C. fermentati), Trichosporon spp. and finally a novel yeast species, C. tunisiensis. The in vitro antifungal activities of fluconazole and voriconazole were determined by the agar disk diffusion test and Etest, while the susceptibility to additional antifungal agents was determined with the Sensititre YeastOne system. Our results showed low incidence of azole resistance in C. albicans (0.54%), C. tropicalis (2.08%) and C. glabrata (4.28%). In addition, caspofungin was active against most isolates of the collection with the exception of two K. ohmeri isolates. This is the first report to describe caspofungin resistant isolates of this yeast.

No abstract

dFrom a collection of yeast isolates isolated from patients in Tunisian hospitals between September 2006 and July 2010, the yeast strain JEY63 (CBS 12513), isolated from a 50-year-old male that suffered from oral thrush, could not be identified to the species level using conventional methods used in clinical laboratories. These methods include matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), germ tube formation, and the use of CHROMagar Candida and metabolic galleries. Sequence analysis of the nuclear rRNA (18S rRNA, 5.8S rRNA, and 26S rRNA) and internal transcribed spacer regions (ITS1 and ITS2) indicated that the ribosomal DNA sequences of this species were not yet reported. Multiple gene phylogenic analyses suggested that this isolate clustered at the base of the Dipodascaceae (Saccharomycetales, Saccharomycetes, and Ascomycota). JEY63 was named Candida tunisiensis sp. nov. according to several phenotypic criteria and its geographical origin. C. tunisiensis was able to grow at 42°C and does not form chlamydospores and hyphae but could grow as yeast and pseudohyphal forms. C. tunisiensis exhibited most probably a haploid genome with an estimated size of 10 Mb on at least three chromosomes. Using European Committee for Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) Candida albicans susceptibility breakpoints as a reference, C. tunisiensis was resistant to fluconazole (MIC ‫؍‬ 8 g/ml), voriconazole (MIC ‫؍‬ 0.5 g/ml), itraconazole (MIC ‫؍‬ 16 g/ml), and amphotericin B (MIC ‫؍‬ 4 g/ml) but still susceptible to posaconazole (MIC ‫؍‬ 0.008 g/ml) and caspofungin (MIC ‫؍‬ 0.5 g/ml). In conclusion, MALDI-TOF MS permitted the early selection of an unusual isolate, which was still unreported in molecular databases but could not be unambiguously classified based on phylogenetic approaches.

Sero‐epidemiological studies of human papillomavirus (HPV) have been undertaken over the last two decades. In this study, the prevalences of nine serum antibodies (anti‐E6, E7 and L1 antibodies of HPV types 16, 18, and 58) were evaluated in normal (control) Korean women and women with cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III, and cervical cancer. The frequencies of all types of anti‐HPV antibodies were higher in the CIN stages and cervical cancer than in normal women, and those of anti‐HPV16 E6 and E7, anti‐HPV18 E6 and E7, and anti‐HPV58 E7 antibodies were higher in the cervical cancer group than in the CIN stages. The frequencies of antibodies against HPV16, 18, and 58 E7 tended to increase with increasing severity of cervical lesions. However, there were few differences in the frequencies of antibodies against the L1 antigens of HPV16, 18 and 58 in cervical cancer versus CIN stages. The anti‐HPV antibodies were detected in 26.5% of normal, 46.3% of CIN I, 62.5% of CIN II, 51.6% of CIN III, and 75% of cancers when any of the nine antigens was used as a criterion. Correlations between HPV DNA positivity and seropositivity for anti‐HPV E6, E7, or L1 antibodies were found only in HPV16 DNA‐positive cervical cancers for anti‐HPV16 E6 and L1 antibodies. In addition, strong positive correlations in seropositivity were found between anti‐HPV16 E7 and anti‐HPV58 E7 antibodies, and between anti‐HPV18 E6 and anti‐HPV58 E6 antibodies. These findings should advance global profiling of the seroprevalences of antibodies against HPV antigens.

No abstract

Control of opportunistic yeast is one of the factors reducing nosocomial infection in hospital. In this study, we investigated the presence of virulence factors including antifungal susceptibility, haemolytic potency, proteinase and phospholipase production. In addition biofilm formation was assessed using the XTT assay. A total of 24 Candida strains were isolated and identified. The most common species were Candida albicans and Candida glabrata (40.7%), followed by Candida dubliniensis (8.3%), Candida kefyr (4.2%) and Candida krusei (4.2%). In addition, 62.5% of strains were resistant to itraconazole and 16.7% of strains were resistant to fluconazole. All the strains were susceptible to amphotericin B. Phospholipase activity showed that 8.3% of the total strains were very strong producers and 41.7% were strong producers. Furthermore, three strains (12.5%) were found to have very strong proteinase activity. All the tested strains revealed beta haemolytic potency. All Candida strains were adhesive to polystyrene 96-well microtitre plates to differing degrees. The OD values of XTT reduction at 492 nm ranged from 0.025 (ca12) to 0.927 (ca2). The majority of C. albicans strains were highly metabolically active in comparison with the other species. Control of fungal infections in hospitalised patients is important to prevent the emergence of multi-resistant strains to commonly used antifungal agents.