In Vitro Antimicrobial Activity of MSI-78, a Magainin Analog

Abstract

MSI-78 is a cationic peptide with broad-spectrum antimicrobial activity and is being developed as a topical agent. We compared the in vitro activity of MSI-78 with those of ofloxacin and other antibiotics against fresh clinical isolates. Based on MIC distribution statistics, strains for which the MSI-78 MIC was ≤64 μg/ml were assumed to be susceptible for purposes of this report. Of 411 aerobic isolates tested, 91% were susceptible to MSI-78, compared to 91% for ofloxacin and 92% for ciprofloxacin. Only enterococci consistently required ≥64 μg of MSI-78/ml for inhibition. MSI-78 demonstrated bactericidal activity equivalent to that of ofloxacin. Of 61 anaerobes, 97% were susceptible to MSI-78. Of 10 isolates of Candida albicans, 3 were inhibited by MSI-78 at 24 h. Further studies of this compound appear to be warranted.

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

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).

TABLE 1.

MICs of MSI-78 for 10 S. aureus strains tested in different media and incubation conditions

Test medium	Incubation conditions	Geometric mean MIC (μg/ml)
MHa broth (unsupplemented)	Air	17.1
MH broth + 2–3% lysed horse blood	Air	34.3
MH broth + 2–3% lysed horse blood	5% CO2 in air	84.4
CAMHBb	Air	19.7
CAMHB + 2–3% lysed horse blood	Air	48.5
CAMHB + 2–3% lysed horse blood	5% CO2 in air	97.0
MH agar	Air	≥128
MH agar + 2–3% lysed horse blood	5% CO2 in air	≥128
CAMHB + 1% agarose	Air	0.32
CAMHB + 1% agarose	5% CO2 in air	1.52
CAMHB + 1% agarose + 2–3% lysed horse blood	5% CO2 in air	≥128
CAMHB + 1% agarose + HTMc supplements	5% CO2 in air	64
GC agar + XV supplementsd	5% CO2 in air	≥128
Wilkins-Chalgren agar	Anaerobic	≥128

^aMH, Mueller-Hinton. 

^bCAMHB, cation-adjusted Mueller-Hinton broth. 

^cHTM, haemophilus test medium. 

^dXV, defined supplement with hematin and NAD. 

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.)

TABLE 2.

MICs of MSI-78 for 411 aerobic bacteria and rates of susceptibility to MSI-78 and eight other antibiotics

Microorganism	No.	MSI-78 MIC (μg/ml)a			% of isolates for which MSI-78 MICs were ≤64 μg/ml	% of isolates susceptible tob:
		50%	90%c	Range		OFLX	AMP	CFU	CFZ	CTAZ	CIP	GEN	PIP
Acinetobacter sp.	10	8	8	4–8	100	100	40	0	0	100	100	100	60
Alcaligenes faecalis	5	32		4–64	100	80	100	0	0	100	80	100	100
Citrobacter diversus	10	8	16	8–16	100	100	0	90	100	100	100	100	100
Citrobacter freundii	25	16	16	8–>256	96	92	16	72	4	92	100	100	96
Corynebacterium jeikeium	10	2	4	≤0.125–4	100	100	50	30	40	0	100	40	30
Enterobacter aerogenes	25	16	16	8–16	100	100	0	64	4	76	100	96	88
Enterobacter cloacae	25	16	32	1–128	96	100	8	32	4	76	100	100	84
Enterococcus faecalis	30	>256	>256	256–>256	0	83	100	0	0	0	87	0	100
Escherichia coli	30	8	16	8–16	100	97	67	97	93	100	97	97	67
Klebsiella oxytoca	10	16	16	8–16	100	100	0	100	40	100	100	100	90
Klebsiella pneumoniae	25	16	16	8–32	100	100	12	92	96	100	100	92	88
Pseudomonas aeruginosa	25	8	16	8–32	100	100	0	0	0	88	100	100	88
Pseudomonas fluorescens	5	8		2–8	100	100	0	0	0	100	100	100	100
Pseudomonas stutzeri	5	4		4–8	100	100	100	0	0	100	100	100	100
Staphylococcus aureus (MRSA)d	10	32	64	16–64	100	20	0	0	0	0	20	60	0
Staphylococcus aureus (MSSA)e	15	4	16	≤0.125–16	100	93	7	100	100	67	93	100	87
Staphylococcus epidermidis	25	4	8	≤0.125–8	100	100	0	100	100	88	100	100	88
Staphylococcus haemolyticus	10	4	8	4–8	100	100	0	10	10	0	20	100	0
Staphylococcus hominis	5	4		2–8	100	100	60	100	100	20	100	80	80
Staphylococcus simulans	5	0.5		≤0.125–2	100	100	100	100	100	80	100	100	100
Staphylococcus warneri	5	1		≤0.125–8	100	100	60	100	100	40	100	80	60
Staphylococcus xylosus	1			≤0.125	100	100	100	100	100	100	100	100	100
Stenotrophomonas maltophilia	10	8	16	4–16	100	70	20	0	0	50	50	0	30
Streptococcus agalactiae	25	16	32	16–32	100	92	100	100	100	100	100	0	100
Streptococcus bovis	5	16		8–16	100	20	0	100	100	100	100	80	0
Streptococcus equinus	2	2		2–16	100	0	0	100	50	50	50	50	0
Streptococcus pyogenes	30	16	32	16–256	97	97	100	100	100	100	100	0	100
Streptococcus group C	5	32		16–64	100	100	100	100	100	100	100	0	100
Streptococcus group G	5	16		16–64	100	80	100	100	100	100	100	0	100
Viridans group streptococci	13	32	>256	4–>256	85	39	77	100	100	85	69	31	77

^a50% and 90%, MICs at which 50 and 90% of the isolates are inhibited, respectively. 

^bAbbreviations (susceptibility breakpoints are in parentheses): OFLX, ofloxacin (≤2.0 μg/ml); AMP, ampicillin (≤8.0 μg/ml); CFU, cefuroxime (≤8.0 μg/ml); CFZ, cefazolin (≤8.0 μg/ml); CTAZ, ceftazidime (≤8.0 μg/ml); CIP, ciprofloxacin (≤1.0 μg/ml); GEN, gentamicin (≤4.0 μg/ml); PIP, piperacillin (≤16 μg/ml). 

^cMIC₉₀s were not calculated for species of which <10 strains were tested. 

^dMRSA, methicillin-resistant S. aureus. 

^eMSSA, methicillin-susceptible S. aureus. 

TABLE 4.

MICs of MSI-78 for 61 anaerobic bacteria and rates of susceptibility to MSI-78 and six other antibiotics

Microorganism	No.	MSI-78 MIC (μg/ml)		% of isolates for which MSI-78 MICs were ≤64 μg/ml	% of isolates susceptible toa:
		Geometric mean	Range		OFLX	CFOX	CLIN	MET	PEN	PIP
Bacteroides fragilis	6	5.6	4–8	100	67	100	83	100	0	67
Bacteroides ovatus	7	5.9	4–8	100	0	29	43	100	0	29
Bacteroides ureolyticus	3	0.4	0.25–0.5	100	33	100	100	100	100	100
Bacteroides vulgatus	6	4.0	4–4	100	83	33	83	100	0	17
Clostridium difficile	4	1.7	0.5–4	100	25	0	100	100	100	100
Clostridium perfringens	5	18.4	8–32	100	100	100	100	100	100	100
Clostridium ramosum	2	16	16–16	100	0	100	0	100	100	100
Clostridium sporogenes	2	6	8–32	100	100	100	0	100	100	100
Clostridium sp.	1	32		100	100	100	0	100	100	100
Peptostreptococcus anaerobius	6	45	32–64	100	100	100	100	100	33	100
Peptostreptococcus asaccharolyticus	3	5	4–8	100	0	100	100	100	67	100
Peptostreptococcus magnus	4	0.7	0.5–1	100	0	100	75	100	100	100
Prevotella bivia	9	69	32–128	78	100	100	100	100	22	100
Prevotella melaninogenica	3	51	32–64	100	33	100	100	100	33	100

^aAbbreviations (susceptibility breakpoints are in parentheses): OFLX, ofloxacin (≤2.0 μg/ml); CFOX, cefoxitin (≤16 μg/ml); CLIN, clindamycin (≤2.0 μg/ml); MET, metronidazole (≤8.0 μg/ml); PEN, penicillin G (≤0.5 μg/ml); PIP, piperacillin (≤32 μg/ml). 

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.

For 161 of these aerobic isolates (approximately five per species), bactericidal endpoints were also determined, following the method recommended by the NCCLS (8).

Anaerobic bacteria were also tested by broth microdilution, which was performed in accordance with the procedure outlined by the NCCLS (9). This utilized a broth version of Wilkins-Chalgren medium with 3% horse serum added when needed to support growth. Test concentrations for MSI-78 were the same as for testing aerobic bacteria. Ofloxacin and cefoxitin concentrations tested were twofold dilutions ranging from 32 to 0.06 μg/ml. Clindamycin, metronidazole, penicillin G, and piperacillin were tested at breakpoint concentrations only.

Susceptibilities of yeasts were determined by the NCCLS broth macrodilution reference method (10), using RPMI 1640 broth. Test concentrations of MSI-78 were twofold dilutions ranging from 512 to 0.25 μg/ml. Test concentrations of ofloxacin, amphotericin B, and fluconazole were 16 to 0.03 μg/ml, 128 to 0.06 μg/ml, and 256 to 0.25 μg/ml, respectively.

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

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.

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.

TABLE 3.

MBCs of MSI-78 and ofloxacin compared to MICs for 161 strains of aerobic bacteria

Antimicrobial	No. on-scalea	No. (%) of isolates for which the MBC was:				No. off-scalea
		Equal to the MIC	Twice the MIC	Four times the MIC	Eight times the MIC
MSI-78	143	91 (63.6)	42 (29.4)	4 (2.8)	6 (4.2)	18
Ofloxacin	155	76 (49.0)	64 (41.3)	9 (5.8)	6 (3.9)	6

^aIf one or both values were off-scale, a valid comparison could not be made. 

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.