If there were cells not lysed or insufficiently lysed, the condition of the DNA that remains inside is unknown.
Nevertheless, to assess the efficacy of antibiotics against the cell wall, the lysis must be adapted to only affect those bacteria whose cell wall has been damaged by GSK3 inhibitor the antibiotic. The liberation of the nucleoid must be the marker that indicates that the wall has been lysed, i.e., that has been affected by the antibiotic. In case of a resistant strain, bacteria would be practically unaffected by the lysis solution and so do not liberate the nucleoid, which retains its usual morphological appearance under the microscope. Results Identification ABT-199 chemical structure of susceptibility-resistance in E. coli strains The technique to evaluate cell wall integrity was initially assayed in E. coli strains from the clinical microbiology laboratory. Ten strains were processed blind after incubation with amoxicillin/clavulanic
acid at doses 0, 8/4 and 32/16 μg/ml, the CLSI breakpoints of susceptibility and resistance, respectively. Example images are presented in Figure 1. Control cultures without antibiotic (Figure 1 a, b, c) showed the bacteria practically unaffected by the lysis. After 8/4 μg/ml, only bacteria from susceptible strains appeared lysed, releasing the nucleoids (Figure 1a’). After 32/16 μg/ml, susceptible and intermediate bacteria appeared to be lysed (Figure 1a” and 1b”), whereas
the resistant strains did not spread their nucleoids (Figure 1c”). Nevertheless, resistance was not homogeneous and some occasional bacteria with damaged cell wall could be visible. Interestingly, a background of extracellular microgranular-fibrilar Oxymatrine material released by the bacteria was observed with a density dependent on the efficacy of the antibiotic, thus being especially intense in susceptible strains exposed to relative high doses. The coincidence of the results from the technique and the standard clinical laboratory was absolute, so the two susceptible, the five intermediate and the three resistant strains were correctly identified. Figure 1 Images of susceptible (above: a, a’, a”), intermediate (medium: b, b’, b”) and resistant (below: c, c’, c”) strains from E. coli incubated with 8/4 μg/ml and 32/16 μg/ml amoxicillin/clavulanic acid and processed by the technique to determine cell wall integrity. The strain is considered susceptible when its MIC is ≤ 8/4 and resistant when it is ≥ 32/16. a, b, c: control, without antibiotic. a’, b’, c’: 8/4 μg/ml; a”, b”, c”: 32/16 μg/ml. Controls without antibiotic (a, b, c) show the bacteria unaffected by the lysis. After 8/4, only bacteria from the first strain, sensitive, appear lysed, showing the spread nucleoids (a’).