Comparative in vitrostudy of the antimicrobial activities of different commercial antibiotic products for intravenous administration

To validate the microbiological assay for evaluating the potencies of Meropenem and Piperacillin, we used Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 29737 and ATCC 6538p, Pseudomonas aeruginosa ATCC 25619 and ATCC 9027, Micrococcus luteus ATCC 9341, Escherichia coli ATCC 10536, Klebsiella pneumoniae ATCC 10031 and Streptococcus faecalis. For MIC and MLC studies we used Acinetobacter baumanii strains 59, 139, 147 and 173, vancomycin-resistant Enterococcus gallinarum, Streptococcus faecalis ATCC 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 and 157, and Staphylococcus aureus strains 287, 291 and ATCC 25923. All 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, while 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.

This was established and validated for Meropenem and Piperacillin/Tazobactam. First, the most appropriate microorganisms were selected, the proper concentration range was determined, and the linearity, precision, specificity and stability of the compound in question were assessed [6]. All samples were then evaluated with the 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 (CFU) was determined for each suspension at 25%T in order to prepare inocula of 1-5 × 10⁶ CFU/ml. (2) MIC and MLC determination by micro-dilution: samples were diluted to 2 mg/ml for evaluation. Using a multichannel pipette, 100 μl of antibiotic sample 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 on to 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 MLC was defined as the lowest dilution that did not show growth on either the ELISA plate or MH agar [7].

The CC was determined similarly to the analytical bioassay. The inocula for MIC and MLC determination and serial two-fold dilutions of each sample from 500 to 0.244 μg/ml were used. The halo of inhibition was measured, and the crown length X was calculated (inhibition halo diameter minus reservoir diameter divided by 2). Log concentration vs. X² was plotted, and linear regression (y = mx + b) was applied. The y-intercept (b) is equivalent to the log of the CC [7].

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

Commercial products purchased from the pharmacies of different hospitals in Bogotá, D. C. Colombia, were analyzed. They included innovator (MERONEM^®, TAZOCIN^®), trademark products and generic products of Meropenem and Piperacillin/Tazobactam. All the samples declared the contents were 1 g. They were all diluted in sterile water in 100 ml volumetric flasks. The solutions were divided into 5 ml fractions for storage at -70°C; they were diluted to 1 mg/ml to develop the analytical bioassays and to 2 mg/ml for MIC and MLC assays.

The samples were analyzed in two groups. The first was used when the assay was being validated, and the second to evaluate antimicrobial activity by determining the MIC, MLC and CC values for each antibiotic. The results were quantified using the statistical method described by Hewitt (1977). Table 9 shows the content of each antibiotic in the samples purchased, and in each case the values fulfill the criteria laid out by USP 29 NF 24 for intravenous Piperacillin and Meropenem: "...Contents no less than 90% and no more than 120% of Piperacillin, calculated on anhydrous base of the quantity registered of Piperacillin" and "...Contents no less than 90% and no more than 120% of Meropenem, calculated on anhydrous base of the quantity registered of Meropenem."

Using previously described methods, the samples were analyzed in groups of seven per plate, each plate being inoculated with a single microorganism. The first row of the plate corresponded to the USP standard; the other seven rows contained the samples. Figure 5 shows the results for Piperacillin/Tazobactam. The plate shows the same performance for the standard as for the samples.

That is, growth was inhibited at the same concentration of each sample. After replication on to MH agar, there was no growth in concentrations C1 to C5 or C12, but there was growth in C6 to C11. This means that the antibiotic has an MLC but no MIC. The MLC is C5 for the USP standard and for all the samples. For all samples, using all microorganisms evaluated, the results showed the same performances for both molecules (Tables 10 and 11 include results for only some samples, as illustration).

The CC is the minimal concentration that inhibits microorganism growth. It is reached at the limit of the inhibition halo. It is a measure of the microorganism's sensitivity and can be two to four times greater than 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 6 shows the different behaviors of the microorganisms tested with Meropenem and Piperacillin/Tazobactam standards. In Figures 6A and 6B, the inhibition haloes are very diffuse, whereas Figures 6C and 6D correspond to microorganisms with well-defined haloes. Some microorganisms exhibited growth of spontaneous mutants (e.g., Figure 6D), allowing a new means of comparing the performances of the products tested to be developed. A well-defined inhibition halo was the selection criterion for evaluating CC. For the Meropenem assay, P. aeruginosa 151 and 157 and A. baumanii 148 and K. pneumoniae were selected, and for Piperacillin/Tazobactam, the selected microorganisms were E. faecalis, E. faecalis ATCC 29212, P. aeruginosa 42, 74 and 151 and S. aureus 287. Figure 7 shows the correlation of X² with the log of antibiotic concentration. The equation is y = 0.0203x + 1.4183, and b is therefore 1.4183. The CC is equivalent to antilog (1.4183), i.e., 26.2 μg/ml.

The CC values for the different Meropenem and Piperacillin/Tazobactam samples showed no significant differences, meaning that the products behaved in similar ways against the different microorganisms tested (Tables 12 and 13). On this basis, the generic products manufactured to meet all the quality standards applied to pharmaceutical products perform as well as the newest versions of those products.

In addition, the ratio between sample CC and standard CC is similar to the ratio of antibiotic contents. In others words, all samples perform the same with regard to their antimicrobial activities in vitro (Tables 14 and 15).

It was noted in the previous assays that some strains produced spontaneous mutants (Figure 6), 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 set-up included an agar plate inoculated with the test strain. Of the six reservoirs, two contained standard solutions and the other four contained sample solutions, as shown in Figure 8. The numbers of mutants produced by the standard and sample solutions were counted after incubation.

For Meropenem, the strains selected were A. baumanii 147 as a control strain (showing no production of spontaneous mutants), A. baumanii 189, E. coli 39 and 69, and K. pneumoniae 43 and 63. For Piperacillin/Tazobactam, the strains selected were P. aeruginosa 151 as a control strain (showing no production of spontaneous mutants), P. aeruginosa 74 and A. baumanii 189. After statistical analysis, the results (Tables 16 and 17) showed no significant differences in the production of spontaneous mutants for any of the strains tested.