Uso directo de identificación cromogénica y antibiograma directo (E-test) sobre muestra clínica para la identificación y estudio de sensibilidad de uropatógenos / Direct use of chromogenic identification and antibiogram (E-test) on clinical samples in order to perform identification and susceptibility tests on urinary pathogens

La obtención rápida y asequible de resultados es prioritaria. Para ello propusimos la realización de identificación y antibiograma sobre muestras de orina directa, mediante placa cromogénica CPS® y E-test (BioMérieux, Francia). Se evaluó la concordancia con los procedimientos standard, estableciendo un protocolo paralelo a la rutina haciendo lecturas de identificación y antibiograma a las 6 y 24 horas sobre 54 muestras clínicas de orina. La identificación concordó en un 88,88% a las 6 horas y en un 100% a las 24. El antibiograma solo detectó errores menores en amoxicilina-clavulánico y cefuroxima en la lectura a las 6 horas. Concluimos que en el caso de infecciones urinarias causadas por bacilos Gram negativos no Pseudomonas spp. la lectura a las 6 h del E-test sobre muestra directa es muy fiable como antibiograma presuntivo y para la detección de BLEE (AU)

Getting fast and affordable laboratory results is a priority. A method for the direct identification and susceptibility testing on direct urine samples by chromogenic plate CPS ® and E-test (BioMérieux, France) is presented. Tests were performed in parallel in order to compare the direct identification and susceptibility results at 6 h and 24 h on 54 samples of urine with the standard procedure. Identification agreement was 88.88% (6 h) and 100% (24 h). Antimicrobial susceptibility testing detected only minor errors in amoxicillin-clavulanate and cefuroxime (6 h). It is concluded that six hours testing is a very reliable susceptibility and extended-spectrum beta-lactamase (ESBL) detection method for urinary tract infection caused by non-Pseudomonas spp Gram negative bacteria (AU)

Antifolates as antimycotics? Connection between the folic acid cycle and the ergosterol biosynthesis pathway in Candida albicans.

The increased incidence of invasive mycoses and the emerging problem of antifungal drug resistance have encouraged the search for new antifungal agents or effective combinations of existing drugs. Infections due to Candida albicans are usually treated with azole antifungals such as fluconazole, ketoconazole or itraconazole. Whilst azoles may have little or no toxicity, they generally offer rather poor fungicidal activity. Even in the absence of resistance, treatment failures or recurrent infections are not uncommon, especially in immunocompromised individuals. Here we demonstrate that the non-classical antifolate pyrimethamine shows synergy with azole antifungal compounds and interferes with the ergosterol biosynthesis pathway in C. albicans. By disturbing folate metabolism in this fungus, pyrimethamine can inhibit ergosterol production. The molecular connection between the folic acid cycle and the ergosterol biosynthesis pathway is discussed and we show that the filamentous form of this fungus is more susceptible to methotrexate than the yeast form because the drug is more effectively transported through the membrane of the filamentous form. When used to treat the hyphal form, methotrexate showed synergy with other antifungals such as azoles and terbinafine. This finding could have important clinical applications, as a combination of azoles with antifolates and/or inhibitors of folic acid synthesis could represent an attractive alternative for the treatment of C. albicans infections.

Tea polyphenol epigallocatechin-3-gallate inhibits ergosterol synthesis by disturbing folic acid metabolism in Candida albicans.

OBJECTIVES: Elucidation of the mechanism of action of epigallocatechin-3-gallate (EGCG) against Candida albicans and demonstration of the connection between its antifolate activity and other metabolic pathways involved in C. albicans survival are the major objectives of this study. METHODS: C. albicans ATCC 10231 and 12 clinical isolates were used. MICs of EGCG against C. albicans were determined according to NCCLS. C. albicans dihydrofolate reductase (DHFR) was purified using methotrexate-affinity chromatography and its inhibition by EGCG studied by spectroscopic techniques. Synergy experiments were performed by chequerboard tests by combining eight doubling concentrations of EGCG with another eight dilutions of azole compounds or terbinafine. Reversion experiments with leucovorin or S-adenosylmethionine were carried out, and the content of ergosterol was determined by a spectrophotometric method. RESULTS: EGCG is an efficient inhibitor of C. albicans DHFR (K(i) = 0.7 microM). As with other antifolates, the effects of EGCG on C. albicans can be highly attenuated by growing the cells in the presence of leucovorin. EGCG showed synergy with inhibitors of the ergosterol biosynthesis pathway in C. albicans such as azole antifungals and terbinafine. We demonstrate that by disturbing the folate metabolism, EGCG can inhibit ergosterol production. The molecular connection between the pathways is discussed. CONCLUSIONS: EGCG acts as an antifolate compound on C. albicans, disturbing its folic acid metabolism. This effect could explain the molecular mechanism for the synergy between EGCG and azole antifungals, and could represent a starting point for therapies involving antifolates and azoles used as an alternative for the treatment of C. albicans infections.

Antifolate activity of epigallocatechin gallate against Stenotrophomonas maltophilia.

The catechin epigallocatechin gallate, one of the main constituents of green tea, showed strong antibiotic activity against 18 isolates of Stenotrophomonas maltophilia (MIC range, 4 to 256 microg/ml). In elucidating its mechanism of action, we have shown that epigallocatechin gallate is an efficient inhibitor of S. maltophilia dihydrofolate reductase, a strategic enzyme that is considered an attractive target for the development of antibacterial agents. The inhibition of S. maltophilia dihydrofolate reductase by this tea compound was studied and compared with the mechanism of a nonclassical antifolate compound, trimethoprim. Investigation of dihydrofolate reductase was undertaken with both a trimethoprim-susceptible S. maltophilia isolate and an isolate with a high level of resistance. The enzymes were purified using ammonium sulfate precipitation, gel filtration, and methotrexate affinity chromatography. The two isolates showed similar levels of dihydrofolate reductase expression and similar substrate kinetics. However, the dihydrofolate reductase from the trimethoprim-resistant isolate demonstrated decreased susceptibility to inhibition by trimethoprim and epigallocatechin gallate. As with other antifolates, the action of epigallocatechin gallate was synergistic with that of sulfamethoxazole, a drug that blocks folic acid metabolism in bacteria, and the inhibition of bacterial growth was attenuated by including leucovorin in the growth medium. We conclude that the mechanism of action of epigallocatechin gallate on S. maltophilia is related to its antifolate activity.

Molecular properties and prebiotic effect of inulin obtained from artichoke (Cynara scolymus L.).

A high molecular weight inulin has been prepared from artichoke (Cynara scolymus L.) agroindustrial wastes using environmentally benign aqueous extraction procedures. Physico-chemical analysis of the properties of artichoke inulin was carried out. Its average degree of polymerization was 46, which is higher than for Jerusalem artichoke, chicory, and dahlia inulins. GC-MS confirmed that the main constituent monosaccharide in artichoke inulin was fructose and its degradation by inulinase indicated that it contained the expected beta-2,1-fructan bonds. The FT-IR spectrum was identical to that of chicory inulin. These data indicate that artichoke inulin will be suitable for use in a wide range of food applications. The health-promoting prebiotic effects of artichoke inulin were demonstrated in an extensive microbiological study showing a long lasting bifidogenic effect on Bifidobacterium bifidum ATCC 29521 cultures and also in mixed cultures of colonic bacteria.