The efficacy of immediate versus delayed antibiotic administration on bacterial growth and biofilm production of selected strains of uropathogenic Escherichia coli and Pseudomonas aeruginosa

Vol. 41 (1): 67-77, January - February, 2015 ORIGINAL ARTICLE doi: 10.1590/S1677-5538.IBJU.2015.01.10 The efficacy of immediate versus delayed antibiotic administration on bacterial growth and biofilm production of selected strains of uropathogenic Escherichia coli and Pseudomonas aeruginosa _______________________________________________ Leah Gandee1, Jer-Tsong Hsieh1, Vanessa Sperandio1, Cristiano G. Moreira1, Chih-Ho Lai1, Philippe E. Zimmern1 1 UT Southwestern Medical Center, Dallas, Texas, USA ABSTRACT ARTICLE INFO ______________________________________________________________ ______________________ Purpose: The treatment of urinary tract infections (UTI) with antibiotics is commonly used, but recurrence and antibiotic resistance have been growing and concerning clinicians. We studied whether the rapid onset of a protective biofilm may be responsible for the lack of effectiveness of antibiotics against selected bacteria. Materials and Methods: Two established uropathogenic Escherichia coli strains, UTI89 and CFT073, and two Pseudomonas aeruginosa strains, PA01 and Boston-41501, were studied to establish a reliable biofilm formation process. Bacterial growth (BG) was determined by optical density at 600 nm (OD 600) using a spectrophotometer, while biofilm formation (BF) using crystal violet staining was measured at OD 550. Next, these bacterial strains were treated with clinically relevant antibiotics, ciprofloxacin HCl (200 ng/mL and 2 μg/mL), nitrofurantoin (20 μg/mL and 40 μg/mL) and ampicillin (50 μg/mL) at time points of 0 (T0) or after 6 hours of culture (T6). All measurements, including controls (bacteria -1% DMSO), were done in triplicates and repeated three times for consistency. Results: The tested antibiotics effectively inhibited both BG and BF when administered at T0 for UPEC strains, but not when the antibiotic administration started 6 hours later. For Pseudomonas strains, only Ciprofloxacin was able to significantly inhibit bacterial growth at T0 but only at the higher concentration of 2 μg/mL for T6. Conclusion: When established UPEC and Pseudomonas bacteria were allowed to culture for 6 hours before initialization of treatment, the therapeutic effect of selected antibiotics was greatly suppressed when compared to immediate treatment, probably as a result of the protective nature of the biofilm. Key words: urinary tract infection; antibiotic therapy; biofilm formation; bacterial growth INTRODUCTION Int Braz J Urol. 2015; 41: 67-77 _____________________ Submitted for publication: October 29, 2013 _____________________ Accepted after revision: June 01, 2014 rank among the most common bacterial infectious diseases encountered in clinical practice, with the occurrence of UTI in the United States estimated at 12-50% for women and 3% for men (3, 5). In patients with bladder or catheter-associated infections, the primary etiologic agents associated Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infection (UTI) and, in the United States alone, health care costs for UTI surpass $1.5 billion per year (1-4). UTIs 67 IBJU | THE EFFICACY OF IMMEDIATE VERSUS DELAYED ANTIBIOTIC ADMINISTRATION ON BACTERIAL GROWTH AND BIOFILM PRODUCTION with UTIs are strains of E.coli and Pseudomonas aeruginosa (P.aeruginosa) respectively. Therefore, this study focused attention on well-established strains of these two bacteria. Currently, antibiotics represent the most effective treatment against UTI. However, some patients exhibit recurrent infections and/or appear to develop resistance to antibiotics (4, 6). Several studies have indicated that the production of bacterial biofilm, a large bacterial community that forms following bacterial adhesion and colonization to surfaces and in which bacteria are held together by exopolysaccharides (7) secreted by the bacteria, contributes to antibiotic resistance (8). A mature biofilm can contain a community of cells and provide a matrix for chemical signaling and message relay between individual cells (5, 9). Thus, we were interested in determining the role of biofilm production by E.coli and P.aeruginosa bacteria strains as one of the major contributors to antibiotic resistance. To study the biofilm formation by bacteria treated with antibiotics, two established strains of E.coli (UTI89 and CFT073) and two well-characterized strains of P.aeruginosa (Boston-41501 and PA01) were selected. We first determined the time course of the growth of each bacteria strain and biofilm under different culture conditions. We also examined whether different pH conditions, consistent with the range of urine pH in humans, had any impact on bacterial growth and biofilm formation. Based on these optimized culture conditions, we examined the effect of clinically relevant antibiotics on the bacterial growth and biofilm formation. Furthermore, we evaluated whether different treatment schedules of antibiotics have any impact on bacterial growth and biofilm formation. biofilm formation and bacterial growth was also studied to determine the effect of delayed therapy. Bacterial strains E.coli UTI89 and E.coli CFT073 were obtained from Harry Mobley (University of Michigan); both strains have been sequenced. E.coli UTI89 is a prototypic cystitis isolate (1) and E. coli CFT073 is a prototypical UPEC isolate cultured from blood and urine of a patient with pyelonephritis (10). P.aeruginosa-Boston 41501 was purchased from American Type Culture Collection (Manassas, VA), and P.aeruginosa-PA01 was obtained from Kevin McIver; only PA01 has been sequenced. Antibiotics (Ampicillin, Ciprofloxacin and Nitrofurantoin) were purchased from Sigma-Aldrich (St. Louis, MO) and used at the following concentration ranges: Ciprofloxacin HCl (Cipro: 200 ng/mL and 2 μg/mL), Nitrofurantoin (Nitro: 20 μg/mL and 40 μg/mL), and Ampicillin (Amp: 50 μg/mL). DMSO was used as a solvent to dilute all the antibiotics. The final concentration of DMSO was 0.1%. Biofilm formation was not affected at this low concentration of DMSO. MATERIALS AND METHODS Determination of Minimum Inhibitory Concentration (MIC) by the microdilution method MIC for E.coli and P.aeruginosa strains were assessed by varying concentrations of Ciprofloxacin, Nitrofurantoin, and Ampicillin assessing bacterial growth through CFU counts as previously described (11). An adjusted inoculum of the overnight growth organism was introduced into LB broth containing serial dilutions in 96 well plates, from 0.0015 μg/mL to 100,000 μg/mL of an initial antibiotic solution, with approximately 5x105 CFU/mL inoculum. Results were observed after 18 hours of incubation at 37ºC. The MIC was defined as the lowest concentration to inhibit visible growth (11). Study Design Following the evaluation of the impact of biofilm performance under different culture conditions and pH ranges, biofilm formation obtained from established strains of E.coli and P.aeruginosa was assessed in the presence of commonly used antibiotics in the treat (...truncated)