Conclusion The technique to assess cell wall integrity may be a rapid and simple procedure to discriminate resistant and susceptible strains to antibiotics that interfere with peptidoglycan biosynthesis. The methodology may be useful not only at the clinical level but also to perform basic studies about the mechanisms of action of antibiotics that act Oligomycin A in vivo at the cell wall. Methods Cultures, bacterial strains and experiments In an initial approach to evaluate the procedure to determine cell wall
integrity, ten clinical strains from Escherichia coli, isolated from urine samples in the selleckchem microbiology service, were tested blind for susceptibility or resistance to amoxicillin/clavulanic acid. According to the Clinical and Laboratory Standards Institute (CLSI) criteria (susceptible: minimum inhibitory concentration – MIC
≤ 8/4; 8 μg/ml amoxicillin/4 μg/ml clavulanic acid; resistant: MIC ≥ 32/16; 32 μg/ml amoxicillin/16 3-Methyladenine molecular weight μg/ml clavulanic acid), two strains were categorized as susceptible, five intermediate and three resistant. In this experiment, bacteria were growing in Mueller-Hinton agar at 37°C for 24 h. Then, they were diluted to an OD600 of 0.1 in Mueller-Hinton broth with 0, 8/4 and 32/16 μg/ml amoxicillin/clavulanic acid, incubated at 37°C for 60 min, and processed to determine cell wall integrity. In a second experiment, the effect of the incubation time with the antibiotic was analyzed, after treatment with 8/4 and 32/16 μg/ml amoxicillin/clavulanic acid, in three clinical
strains of E. coli isolated from urine samples, one susceptible (MIC: 8/4 μg/ml), one intermediate (MIC: 16/8 μg/ml) and one resistant (MIC: > 64/32 μg/ml). Moreover, it was tested both in cultures exponentially growing in Mueller-Hinton broth at 37°C, with aeration and shaking, and in cells cultured for 24 h in Mueller-Hinton agar dishes, as usual in the standard clinical microbiology laboratories. Cells were diluted to an OD600 of 0.1 in Mueller-Hinton broth, and incubated with the two doses of the antibiotic for 5, 10, 20, 30, 40, 60 and 75 min. Thirdly, a dose-response experiment at the cell wall level of one E. coli strain isolated from an urine sample, susceptible to ampicillin (MIC: 4 μg/ml), was performed. Bacteria exponentially growing in Mueller-Hinton broth were diluted to Cell press an OD600 of 0.1 in Mueller-Hinton broth and then incubated for 60 min with 0, 1, 2, 4, 8, 12, 16 μg/ml ampicillin. Afterwards, the cultures were processed to determine viability and cell wall integrity. The halo size of the nucleoid was measured in 250-400 bacteria per dose after image capture and digital image analysis, and included in one of four qualitative categories: undamaged, with low cell wall damage, with high cell wall damage where the residual body of the bacterium was retained, and with high cell wall damage where the residual core from the bacterium was not recognized.