Disks were observed for colour change up to 60 min. β-lactamase producer strain ATCC 29213 (#1) and β-lactamase negative strain ATCC 25923 (#2), were used as positive and negative controls respectively. Antibiotic susceptibility testing – disk diffusion and E-test The standard procedure recommended by CLSI was followed [41, 42]. Briefly, inoculum was prepared by the direct colony suspension method preferred for S.
aureus. Isolated colonies from non-selective overnight BHI agar plates were used to make a saline suspension, and turbidity was adjusted equivalent to a 0.5 McFarland turbidity standard. Thereafter, the standardized inoculum was spread uniformly on a Mueller Hinton II agar plate, allowed to dry, cefazolin disk applied to the centre of the plate, and plates incubated at 35°C for 20–24 h. The zones of inhibition Fludarabine clinical trial were GDC-0994 in vivo measured and compared
against CLSI Zone Diameter Interpretive Charts, to categorize Adriamycin in vivo isolates as susceptible, intermediate or resistant. (The CLSI 2012 charts were used, which were most current at the time of the experiments ). S. aureus ATCC 25923 (#2) was included in each experiment as the CLSI recommended quality control strain for disk diffusion . For the zone edge test comparison criteria, ATCC 29213 (#1) and ATCC 25923 (#2) were used as the CLSI recommended positive and negative controls, showing ‘sharp’ and ‘fuzzy’ inhibition zone edges respectively. For the E-test, cefoxitin or cefepime E-test strip was applied to the inoculated plate, and following incubation at 35°C for 24 h, the MIC value was read. The CLSI interpretive criteria, most current at the time of experiments, were used to categorize isolates as susceptible, intermediate or resistant . S. aureus ATCC 29213 (#1) was included in each experiment as the recommended quality control for MIC determination . Experiments were similarly performed
with ‘induced’ growth cultures, wherein bacteria grown in presence of penicillin disks overnight were used as ADAM7 the starting inoculum to prepare the saline suspension. The standard procedure described above was followed. Results β-LEAF assays determine β-lactamase production and assess cefazolin activity We used a panel of S. aureus comprising two ATCC strains and 25 clinical isolates (Table 1) as a model system. Isolate numbers (eg. #1, #4, etc.), rather than full names, are used to refer to isolates as per Table 1 throughout this study. ATCC strains with established β-lactamase status, β-lactamase producing strain 29213 (#1) and β-lactamase negative strain 25923 (#2) were used as positive and negative controls respectively. Cefazolin, a first generation cephalosporin, was used as the test antibiotic in these experiments. Each isolate was assayed under two conditions, with β-LEAF alone and with β-LEAF and saturating concentration of cefazolin (2500-fold higher concentration of cefazolin than β-LEAF) respectively.