Cavalcante, Fernanda S.

Coelho, Filipa Alexandra Baltar Lobo

Ferreira, Dennis de C.

Iorio, Natália L.

Lyra, Yuri C.

Matos, Pricilla D. M. de

Pereira, Maria Olívia

Santos, Kátia Regina N. dos

Sedaca, Stefanie

Sousa, Cláudia

Toledo, Vivian C. S.

Universidade do Minho: RepositoriUM

Identification of molecular targets associated with resistance or sensibility to rebeccamycin analogs using Saccharomyces cerevisiae as a model cell SOUSA, G. F.1  ; DREKENER, R. L.2; CORREIA, C. R. D.2; MONTEIRO, G.1* 1 Departamento de Tecnologia Bioquímico-Farmacêutica, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo - USP,  São Paulo-SP, Brazil 2 Departamento de Química Orgânica, Instituto de Química, Universidade Estadual de Campinas, Campinas-SP, Brazil * Correspondence: smgisele@usp.brSeveral antitumor drugs have been described to possess antimicrobial effect. In this project we investigated the antimicrobial effect of rebeccamycin (which is still in phase II clinical trials in respect to antitumor activity), which have the ability to inhibit the catalytic action of topoisomerase I. Therefore, our proposal aims to identify the genetic markers present in the yeast Saccharomyces cerevisiae associated with resistance and sensibility to rebeccamycin analogs and to characterize their antimicrobial activity. In this line, BY4741 strain of S. cerevisiaeand the yeast Knock-out (YKO) deletion collection (Invitrogen) were divided into pools and inoculated at 30 °C in liquid YPD for 36 hours. Then, cells were diluted to OD600nm 0.2 in 96-well plate and exposed to 1mM rebeccamycin analogs for 4 hours. Using a replicator, pools were transferred to solid media and placed in an incubator at 30 °C for 72 hours and cell viability was evaluated by the ability to form colonies. After growth analysis of the wild type strain as well as the YKO deletion collection, the Reb analog that presented pronounced antimicrobial effect observed was Reb C, on pools 32 and 39 in relation to control and wild type strain. We observed that the minimum lethal dose of analog Reb C was around 1 mM, killing mutant pools 32 and 39. With this result we want to analyze the individual mutant growth from plates 32 and 39 from YKO in the established dose of analog Reb C, in order to identify possible molecular targets associated with resistance or sensibility to this drug. Keywords: Saccharomyces cerevisiae; rebeccamycin. II International Conference on Antimicrobial Research - ICAR2012               Lisbon (Portugal) 21-23 November 2012407   In vitro activity of antibiotics against biofilms produced by MRSA SCCmec IV  isolates from hospitals in Rio de Janeiro 1Pricilla D. M. de Matos; 1Fernanda S. Cavalcante; 1Stefanie Sedaca; 1Dennis de C. Ferreira; 2Natália L Iorio; Vivian C. S. Toledo; 1Yuri C  Lyra; 3Filipa Lobo Coelho; 3Cláudia Sousa; 1Kátia Regina N. dos Santos, 3Maria Olívia Pereira;1Hospital Infection Laboratory, Institute of Microbiology Professor Paulo de Goes, Rio de Janeiro Federal University, Brazil; 2Basic Science Department, Fluminense Federal University, Nova Friburgo, Rio de Janeiro, Brazil; 3IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710–057 Braga, Portugal. Methicillin resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens. Biofilm formation is common in medical devices, such as catheters and prostheses, and allows the pathogen to resist to the host immune response and antimicrobials. To quantify the biofilm formation ability and to evaluate the invitro activity of different antimicrobial agents, alone or in combination, against prevalent MRSA SCCmec IV isolates from hospitals in Rio de Janeiro. The in vitro biofilm production ability was assessed for 43 clinical isolates, including 14 of sequence type (ST) 5, 9 ST1, 4 ST30 and 16 from other ST's, according to Stepanovic et al (2000). The biofilm susceptibility to gentamicin (Gen), linezolid (Lin), rifampicin (Rif) and vancomycin (Van) was determined for 6 isolates from three prevalent lineages (USA400/ST1, USA800/ST5, USA1100/ST30). After biofilm formation (24 h), the isolates were exposed to concentrations from 0.25 to 64g/mL of each antimicrobial agent, applied alone or in combination. Before and after antibiotic treatment, biofilms were characterized in terms of total biofilm mass (by crystal violet) and number of viable cells (CFU/cm2). The biofilm production was considered positive for 8 (57%) USA800/ST5 isolates (6 weak and 2 moderate), a single isolate USA400/ST1 was considered weak producer, and 2 USA1100/ST30 isolates were weak biofilm producers. After biofilm exposure to concentrations equal to or greater than 4 g/ml of Rif or Lin, a biomass reduction of 50 % was observed. For Gen or Van, it was verified a reduction of about 45 % but only after exposure to concentrations equal to or greater than 16g/mL. The synergism experiments showed improved biofilm mass reduction (55 %) using antimicrobials in concentrations lower than those mentioned above (Gen16g/mL + Rif2g/mL, Lin2g/mL + Rif2g/mL, Rif2g/mL + Van4g/mL, or Lin2g/mL + Van4g/mL). These latter results were equivalent to those obtained with high concentrations of the drugs used alone. Regarding biofilm cell viability, it was verified reductions of up to 1 log to Van64g/mL or Rif64g/mL, or 2 logs to Gen64g/mL or Lin64g/mL and between 2 and 3 logs in the synergisms of Lin2g/mL + Rif2g/mL, Rif2g/mL + Van4g/mL or Gen16g/mL + Rif2g/mL). Data demonstrated that antibiotic synergisms involving Lin2g/mL + Rif2g/mL and Rif2g/mL + Van4g/mL appear to be good therapy choices, since both produced greater reductions in biomass and number of cells in staphylococcal biofilms.  II International Conference on Antimicrobial Research - ICAR2012               Lisbon (Portugal) 21-23 November 2012408

In vitro activity of antibiotics against biofilms produced by MRSA SCCmec IV isolates from hospitals in Rio de Janeiro

In vitro activity of antibiotics against biofilms produced by MRSA SCCmec IV isolates from hospitals in Rio de Janeiro

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