ANTIBACTERIAL ACTIVITY OF THE COMBINATION OF CYSTEINE AND ANTIBIOTICS. RELATIONSHIP WITH THE GENERATION OF OXIDATIVE STRESS IN CLINICAL ESCHERICHIA COLI STRAINS.

MARTÍNEZ SR ; BONGIOVANNI ME ; PIERSIGILLI A; ALBESA I; BECERRA MC

Rosario

Congreso; RICiFa 2° Reunión Internacional de Ciencias Farmacéuticas.; 2012

Univ. Nac Cordoba y Univ Nac Rosario

ANTIBACTERIAL ACTIVITY OF THE COMBINATION OF CYSTEINE AND ANTIBIOTICS. RELATIONSHIP WITH THE GENERATION OF OXIDATIVE STRESS IN CLINICAL ESCHERICHIA COLI STRAINS.   Martínez SR  (1), Bongiovanni ME (2), Piersigilli A (3), Albesa I (1,4), Becerra MC (1,4). (1)Dpto. Farmacia, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Ciudad Universitaria. (2)Laboratorio de Microbiología del Hospital Privado de Córdoba, (3) Sanatorio Aconcagua de Córdoba, (4) IMBIV-CONICET. 5000 Córdoba. República Argentina.   Introduction. The emergence of Escherichia coli being resistant to antibiotics is a problem that affects different countries. Quinolone resistance and Extended-spectrum beta-lactamase (ESBL) production are often associated in Enterobacteriaceae, being cross-resistant to the quinolones and beta-lactams frequent in this family (1). Previously, we have demonstrated that antibiotics used in clinical treatments induced oxidative stress in different bacterial species (2). It was previously described that L-cysteine affects the growth of E.coli, leading to hydrogen peroxide production (3). On the basis of these antecedents, the aim of the present study was to evaluate whether L-cysteine increases the antibiotic susceptibility of E.coli ESBL strains when combined with ciprofloxacin or ceftazidime, and to identify the role of reactive oxygen species (ROS) in the antimicrobial activity. Materials and methods. E.coli ESBL strains (1 and 2) were provided by Sanatorio Aconcagua from Córdoba. ESBLs were detected according to CLSI (Clinical and Laboratory Standard Institute) (4). Suspensions of approximately 1x109 CFU/mL (colonies forming units per mL) were prepared from overnight cultures and each suspension was incubated with L-cysteine (0.05 to 3.2 mM). The minimum inhibitory concentrations (MICs) of ciprofloxacin and ceftazidime and the combination of cysteine with these antibiotics were determined in a minimal medium M-9 by the tube dilution method (3). The lowest concentration of the compound that prevented bacterial growth at 24 h of incubation was considered to be the MIC. To evaluate the generation of ROS, chemiluminescence with lucigenin as a probe was used. Suspensions of ESBL 1 and ESBL 2 were incubated with ciprofloxacin or ceftazidime at subMICs, and with the combination of L-cysteine and each antibiotic. The light emitted by ROS was expressed as relative light unities (RLU) (2). Results. The MICs of ciprofloxacin obtained for ESBL 1 were 2048 µg/mL and 1024 µg/mL for ESBL 2, while the MICs obtained for ceftazidime were 32 µg/mL for ESBL 1 and 8 µg/mL for ESBL 2. Cultures of strains ESBL 1 and ESBL 2 presented a growth inhibition of almost 3 log10 when they were incubated with 0.8 or 1.6 mML-cysteine compared to the control. Since L-cysteine inhibited ESBL strain growth, a subCIM concentration of ciprofloxacin for each strain was selected to combine with this amino acid. Thus, ciprofloxacin at 512 µg/mL or 256 µg/mL for strains ESBL 1 and ESBL 2 were used, respectively. In strain ESBL 1, when ciprofloxacin at subMIC was combined with L-cysteine, there was a clear growth inhibition respect to the control. When the combination of L-cysteine with ceftazidime at subMIC (4 µg/mL or 1 µg/mL for ESBL 1 and ESBL 2, respectively) was assayed, there was a growth inhibition of almost 3 log10, similarl to the results obtained with ciprofloxacin. Chemiluminescence assays suggested that ROS were generated during the incubation of bacterial suspensions with a combination of L-cysteine and antibiotics in both strains. Conclusions. In the search for compounds that enhance the susceptibility of resistant strains, the effect of L-cysteine could be an interest aspect to investigate. This work provides evidence of the ability of this amino acid to increase the antimicrobial activity of clinical antibiotics through an effect in the respiratory chain of E.coli, leading to the induction of ROS formation that may impact in the susceptibility to antibiotics.     Acknowledgments. CONICET, SeCyT.   References. 1-Frank T, Mbecko JR, Misatou P, Monchy D. Emergence of quinolone resistance among extended-spectrum beta-lactamase-producing Enterobacteriaceae in the Central African Republic: genetic characterization.                  Res. Notes. 2011; 25 (4):309. 2- Becerra MC, Albesa I. Oxidative stress induced by ciprofloxacin in Staphylococcus aureus. Biochem. Biophys. Res. Commun. 2002; 297:1003-1007. 3- Gomez RF, Montville T, Blais K. Toxic effect of cysteine against Salmonella typhimurium. Appl. Environ. Microbiol.1980; (39):1081-1083