Reduction of cytotoxicity of ciprofloxacin and production/release of shiga toxin by fruit extracts.

PELLARIN G; ROJAS MJ; ALBRECHT C; BECERRA MC; AGUILAR JJ; ALBESA I; ERASO AJ

Cordoba

Congreso; 1° Reunión Internacional de Ciencias Farmacéuticas.; 2012

Universidades Nac.Cordoba y Rosario

Introduction: Plants useful for inhibiting the growth of Escherichia coli strains producing Stx toxins (STEC) that cause uremic hemolytic syndrome (UHS) have been reported, including TJ Kim et al. (1), which found antimicrobial activity in grape seed extracts rich in phenolic compounds. Voravuthikunchai et al (2) achieved antimicrobial effects and inhibition of toxin production with extracts of Pomegranate (Punica granatum) and Quercus infectoria (oak variety). These extracts inhibited the release of Shiga toxins Stx1 and Stx2, as well as reducing the toxic effect on Vero cells. The aim of this study was to investigate whether the therapeutic administration of Ziziphus mistol or Prosopis alba extracts with ciprofloxacin (CIP) could interfere the production or release of Stx caused by the antibiotic and reduce the cytotoxicity. Materials and methods: The Minimum Inhibitory Concentration (MIC) was determined for CIP, alcoholic and ketonic extracts of Z.mistol or P.alba, and the combination of CIP and extracts. Overnight cultures of E. coli O157: H7 EDL 933 in cysteine trypticase soy medium supplemented with 0.3% yeast extract, according to Clinical and Laboratory Standards Institute (CLSI) was employed. Cytotoxicity assays of supernatants from culture free of bacterial cells were carried out on subconfluent monolayers of Vero cells. Data were obtained at least in triplicate and statistically analyzed by ANOVA, considering p<0.05 as a significant difference. InfoStat Statistical software was used. Results: The MIC of the antibiotic was 0.039 µg/ml and none of the extracts showed antimicrobial activity at the concentrations tested. In addition, it was found that alcoholic and ketonic extracts of P.alba and the ketone extract of Z.mistol did not change the MIC of the antibiotic. However, a slight increase (1 dilution) of the MIC with the alcoholic extract of Z.mistol was detected. Moreover, CIP favored the production/release of toxin in the assays in which the bacterium was exposed to sublethal doses of the antibiotic. By cultivating the bacterium with a combination of Stx and extract, it was observed that the alcoholic extracts of both fruits were protectors for Vero cells, with significant decreases in toxicity found in cultures treated only with CIP. The percentages of reduction of the cytotoxicity of Stx were 70% and 60%, respectively for Z.mistol and P.alba. Notably, it was found that the plant extracts reduced the Stx damage on the Vero cells when they were previously incubated with the toxin for 1 h. Discussion: The protective effect of the extracts on the Vero cell line coupled with the significant decrease in the production/release of Stx are two beneficial effects that could prove useful for developing a complementary treatment of antibiotic therapy, especially if we consider that various antibiotics in addition to CIP, are capable of generating oxidative stress damage in different eucaryotic cells. Conclusion: This study shows that the extracts of both regional fruits may have a preventive role or as part of palliative strategy against UHS, either as potential pharmacological applications or as diet therapy to reduce long-term consequences of this infectious disease. References: (1)-Kim TJ, Silva JL, Jung YS. Antibacterial activity of fresh and processed red muscadine juice and the role of their polar compounds on Escherichia coli O157:H7. J Appl Microbiol. 2009, 107:533-9. (2)-Voravuthikunchai SP, Limsuwan S. Medicinal plant extracts as anti-Escherichia coli O157:H7 agents and their effects on bacterial cell aggregation. J Food Prot. 2006.69:2336-41. Alberto Eraso. Escuela de Nutrición. Facultad de Ciencias Médicas. UNC. aeraso@fcm.unc.edu.ar TE-FAX: 4333024 int 102.