INTRODUCTION

Top Abstract Introduction Materials & Methods Results & Discussion References

Magainins are a class of naturally occurring cationic peptides found in animals and have broad-spectrum antimicrobial activity through their interactions with anionic phospholipids of microbial cells, which result in disruption of the cell membranes (2, 6). MSI-78 is a 22-residue magainin analog that is being developed for use as a topical antimicrobial agent (1, 2, 6). In vivo studies have demonstrated a 5-log reduction in numbers of Pseudomonas aeruginosa organisms in swine skin wounds (5) and a 4-log reduction of perineal skin flora on human skin (3) following topical application of MSI-78. Two phase III clinical trials of MSI-78 have been completed for the treatment of infected diabetic foot ulcers. These trials were equivalency trials with orally administered ofloxacin as the comparator drug. MSI-78 applied topically was found to be statistically equivalent to orally administered ofloxacin with respect to clinical resolution of infection (3a).

The present study was designed to compare the in vitro antimicrobial activity of MSI-78 with those of ofloxacin and other antimicrobial agents against a wide variety of aerobic and anaerobic bacteria and yeasts.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results & Discussion References

Preliminary study. Because of the cationic nature of MSI-78, a variety of different media and incubation atmospheres were employed to test the susceptibility of 10 strains of Staphylococcus aureus to MSI-78 (see Table 1). Test procedures otherwise followed those outlined by the National Committee for Clinical Laboratory Standards (NCCLS) for broth microdilution and agar dilution (7).

Microorganisms. Fresh clinical isolates were obtained from local clinical microbiology laboratories and were supplemented by stock cultures of recent clinical isolates of some species to achieve target numbers. These included 411 aerobic and facultatively anaerobic bacteria representing 29 species, 61 anaerobic bacteria, and 10 yeast isolates, five of which were fluconazole resistant. (For individual species, see Tables 2 and 4.)

Antimicrobial agents. MSI-78 was provided by Magainin Pharmaceuticals, Inc., Plymouth Meeting, Pa., as a standardized powder with 100% potency (deionized water was used as the diluent). Standardized powders of ofloxacin, ceftazidime, cefuroxime, ciprofloxacin, and fluconazole were provided by their respective U.S. manufacturers, and ampicillin, cefazolin, gentamicin, oxacillin, penicillin G, piperacillin, cefoxitin, metronidazole, and amphotericin B were purchased from Sigma Chemicals, St. Louis, Mo.

Test methods. Bacteria that grew aerobically were tested by broth microdilution, following the procedure outlined by the NCCLS (7). Based on the results of the preliminary study and the recommendation of the supplier (3a), unsupplemented Mueller-Hinton broth was used for testing MSI-78, whereas cation-adjusted Mueller-Hinton broth was used for all other antibiotics tested against aerobes. Test concentrations of MSI-78 were serial twofold dilutions ranging from 256 to 0.125 µg/ml distributed in Dynatech microdilution trays. For the remaining antibiotics, only breakpoint concentrations were tested.

Quality control. On each day of testing, standard quality control organisms were also tested. These included S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and P. aeruginosa ATCC 27853 for aerobic bacteria; Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Eubacterium lentum ATCC 43055 for anaerobes; and Candida albicans ATCC 90028 for yeasts. Two inoculum colony counts were randomly performed from the growth control suspension of two organisms on each day of testing.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials & Methods Results & Discussion References

The results of the preliminary study evaluating the effects of different media and incubation atmospheres on the MICs of MSI-78 when 10 strains of S. aureus are tested are summarized in Table 1. Compared to the MICs observed with the standard method (cation-adjusted Mueller-Hinton broth incubated in ambient air), greatly increased MICs were observed with incubation in 5 to 7% CO₂ or in an anaerobic atmosphere and with the presence of agar or lysed horse blood in the medium. Using agarose to solidify the medium, however, significantly lowered the MICs, presumably due to its lower cation content. The use of unsupplemented Mueller-Hinton broth resulted in slightly lower MICs than did the use of the cation-adjusted medium. A larger difference was observed with other organisms (data not shown).

The modal MIC of MSI-78 for the 411 bacterial isolates that grew aerobically was 16 µg/ml, with a range of 0.125 to >256 µg/ml. The MIC distribution for MSI-78 is graphically displayed in Fig. 1. For purposes of data analysis in this report, we considered any organism for which the MIC was 64 µg/ml susceptible to MSI-78: that was based on the population statistics alone. Since the drug is being employed as a topical agent in a 1% formulation (10,000 µg/g), concentrations well above 64 µg/ml can be expected at the point of contact with surface bacteria.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Distribution of MSI-78 MICs for 411 bacterial isolates representing 29 species that grew aerobically.

The MICs of MSI-78 for each species are summarized in Table 2. MSI-78 was active against all but one species (E. faecalis). In this series, the MSI-78 MICs for 10 methicillin-resistant S. aureus strains were four- to eightfold higher than those for 15 methicillin-susceptible strains, but all were inhibited by 64 µg of MSI-78/ml. E. faecalis was the only enterococcus tested and it consistently required more than 64 µg of MSI-78/ml for inhibition. The percentages of isolates susceptible to 64 µg of MSI-78/ml compared to 10 other antimicrobial agents are also shown in Table 2. The percentage of all 411 isolates susceptible to concentrations of MSI-78 of 64 µg/ml (91%) was comparable to rates of susceptibility to ciprofloxacin (92%) and ofloxacin (91%), and these three drugs were the most active against the population studied.

Tests for bactericidal activity showed MSI-78 to be bactericidal and equivalent to ofloxacin (Table 3). The minimum bactericidal concentrations (MBCs) of MSI-78 were within one twofold dilution of the MICs for 93% of 161 organisms with on-scale endpoints, compared to 90% for ofloxacin. There was no cross-correlation for strains for which MBCs were 4 times the MIC for the two compounds.

The MICs of MSI-78 for anaerobes ranged from 0.25 to 128 µg/ml (Table 4). Of the 61 anaerobes tested, 97% were susceptible to MSI-78 at 64 µg/ml. Only two strains (both Prevotella bivia) required more than 64 µg/ml for inhibition. Of the six comparison drugs tested in parallel, only metronidazole had a greater percentage of susceptible isolates (100%); 57% were susceptible to ofloxacin.

For the 10 strains of C. albicans tested, the MSI-78 MICs ranged from 64 to >512 µg/ml. At the 24-h reading, three (30%) were inhibited by 64 µg of MSI-78/ml, but only two remained at this level at the 48-h reading. None were susceptible to ofloxacin (MICs of >16 µg/ml), half were susceptible to fluconazole (8.0 µg/ml), and all were susceptible to amphotericin B (1.0 µg/ml). Of the two MSI-78-susceptible strains (at 48 h), one was fluconazole susceptible and one was fluconazole resistant.

Since MSI-78 acts directly on the anionic phospholipid of the bacterial cell membrane and not on membrane receptors (2), the development of resistance is theoretically less likely to occur. At least one study supports this (4). In light of the current widespread increase in microbial resistance to most other antimicrobial agents, this characteristic plus its broad spectrum of antimicrobial activity makes MSI-78 an agent meriting further study. So far, however, such studies have been limited to topical applications.