Comparative in vitro study of the antimicrobial activities of different commercial antibiotic products of vancomycin

THE UNITED STATES PHARMACOPOEIA XXVII states that spores of Bacillus subtilis ATCC 6633 are the source of this microorganism used to develop a microbiological assay for evaluating the potencies of vancomycin products. For MIC and MBC studies, we used Acinetobacter baumanii strains 59, 139, 147 and 173, Enterococcus gallinarum, Streptococcus faecalis ATC 29212, a nosocomial strain 319623 and a vancomycin-sensitive strain, Escherichia coli strains 39, 50 and 69, Klebsiella pneumoniae strains 1, 43, 63, 65 and 207, Pseudomonas aeruginosa strains 42, 74, 151, 157, and HE1, Staphylococcus aureus strains 287, 291 and ATCC 25923, and Morganella morganii HE2. All of the microorganisms were grown in Mueller Hinton (MH) broth (incubated at 35°C for 24 h). Each strain was then plated on MH agar to obtain isolated colonies, which were then used to make larger cultures in MH medium. The cultures were harvested with cryopreservation broth. A portion of each was kept in a cryovial at -70°C, and the other portion was used to prepare a suspension with 25% transmittance at 600 nm (25%T) to develop in vitro assays. These suspensions were kept in cryovials at - 70°C.

An analytical bioassay was established and validated for vancomycin. First, the proper concentration range was determined, and then the linearity, precision, specificity and stability of the compound in question were assessed [2, 3]. All of the samples were evaluated with this analytical bioassay under the chosen conditions.

Assays to assess these parameters were developed in two parts. (1) Preparation of inocula: the number of colony forming units (CFUs) was determined for each suspension at 25%T to prepare inocula of 1-5 × 10⁶ CFUs/ml. (2) MIC and MBC determination by micro-dilution: samples were diluted to 2 mg/ml for evaluation. Using a multichannel pipette, 100 μl Mueller Hinton Broth was placed in each well of a 96-well ELISA plate, with 200 μl in column 12. Next, 100 μl of the antibiotic solution (2 mg/ml) was placed in the first column and thoroughly mixed by pipetting. From these wells, 100 μl was added to the second column and mixed, and this procedure was repeated up to column 10, after which the 100-μl portion was discarded. Columns 11 and 12 were positive and negative controls, respectively. Each row (A to H) represented a different sample to be analyzed. Each inoculum (100 μl) was then pipetted into each microplate, which was incubated at 37°C for 24 h. Growth in the wells was assessed. The lowest dilution showing no growth, the first dilution with growth, and the two controls were plated onto MH agar. The MIC was defined as the lowest dilution that showed no growth on the ELISA plate but showed growth on MH agar. The MBC was defined as the lowest dilution that did not show growth on either the ELISA plate or MH agar [10].

The CC was determined similarly to the analytical bioassay. The inocula for MIC and MBC determinations and two-fold serial dilutions of each sample from 993 to 31,03 μg/ml were used (The batch of Vancomycin USP standard has a potency of 99300 μg per vial). The halo of inhibition was measured, and the crown length (Χ) was calculated (the inhibition halo diameter minus the reservoir diameter divided by 2). The log concentration vs. Χ² was plotted, and a linear regression (y = mx + b) was applied. The y-intercept (b) is equivalent to the log of the CC [10].

Spontaneous mutation was analyzed similarly to the analytical bioassay. Again, the inocula for the MIC and MBC determinations were used. Specific microorganisms and dilutions were selected after determinations of critical concentrations. On each plate, a dilution of the USP standard and samples of the same concentration were used.

All the assays were performed three times, and the statistical tool of Microsoft Excel^® was applied to analyze the dates.

The United Stated Pharmacopoeia XXVII recommends Bacillus subtilis ATCC 6633 as the biological organism to use to develop the analytical bioassay for vancomycin products. Figure 1 shows the results of this bioassay.

The samples were analyzed with the previously validated assay. The results were quantified using the statistical method described by Hewitt (1977). Table 10 shows the content of vancomycin in the samples purchased, and in each case, the values fulfill the criteria laid out by USP XXV II for intravenous vancomycin: "...Contents no less than 90% and no more than 115% of Vancomycin, calculated on anhydrous base of the quantity registered of Vancomycin".

Using the previously described methods, the samples were analyzed in groups of seven per plate, and each plate was inoculated with a single bacterial strain. The first row of the plate contained the USP standard; the other seven rows contained the samples. Figure 3 shows the results for vancomycin products. The plates showed the same performance for the standard as for the samples.

Growth was inhibited at the same concentration of each sample. After transfer onto MH agar, there was no growth in concentrations C1 to C5 or C12, but there was growth in C6 to C11. This result means that the antibiotic has an MBC but no MIC. The MBC is C5 for the USP standard and for all the samples. For all of the samples, using all of the microorganisms evaluated, the results showed that the samples had the same performances at each repetition of the assay (Table 11 includes results for only some samples as an illustration).

The CC is the minimum concentration that inhibits microorganism growth. It occurs at the limit of the inhibition halo. It is a measure of a microorganism's sensitivity and can be different from the MIC, which is determined under different conditions. The CC can be defined mathematically as Ln(CC) = Ln(C_O) - X²/DT_O, where CC is the critical concentration, C_O is the antibiotic concentration in the reservoir, X is the length of the crown (see above), D is the diffusion coefficient, and T_O is the critical time. The intercept of a plot of Ln (C_O) vs. X² is the Ln of CC [7].

Figure 4 shows the different behaviors of the microorganisms tested with the vancomycin standard. In Figures 4A and 4B, the microorganisms exhibited growth of spontaneous mutants. Figure 4C shows a microorganism resistant to vancomycin, and, finally, Figures 4D, E and 4F correspond to microorganisms with well-defined haloes, allowing for a comparison of the performances of the products tested for development. A well-defined inhibition halo was the selection criterion for evaluating CCs. For the CC assays, E. faecalis, E. faecalis ATCC 29212, E. faecalis 319623, A. baumanii 59, E. gallinarum, P. aeruginosa 43 and 74, S. aureus 281, 291 and ATCC 25923 were selected. Figure 5 shows the correlation of X² with the log of antibiotic concentration. The regression equation is y= 0.0353x + 0.9297, and b is therefore 0.9287. The CC is equivalent to antilog (0.9297), i.e., 8.506 μg/ml.

The CC values for the different vancomycin products showed no significant differences, meaning that the products behaved in similar ways against the different microorganisms tested (Table 12). On this basis, the generic products meet all of the quality standards applied to the pharmaceutical products and perform as well as the newest versions of these products.

In addition, the ratio between the sample CCs and standard CCs are similar to the ratios of antibiotic contents. In other words, all samples perform the same with regard to their antimicrobial activities in vitro (Table 13).

It was noted in the previous assays that some strains produced spontaneous mutants (Figure 4A), as indicated by the appearance of colonies within the inhibition halo. Therefore, an assay to assess spontaneous mutation was developed with appropriate concentrations of antibiotics. Each experimental setup included an agar plate inoculated with a test strain. Of the six reservoirs, two contained standard solutions and the other four contained sample solutions. The numbers of mutants produced by the standard and sample solutions were counted after incubation.

For the spontaneous mutant assays, the strains selected were S. aureus 291 as a control strain (showing no production of spontaneous mutants) and A. baumanii 54 and E. gallinarum as mutant producing strains. After statistical analysis, the results (Table 14) showed no significant differences between the products in the production of spontaneous mutants for any of the strains tested (Figure 6).