In Vitro Susceptibility to Pexiganan of Bacteria Isolated from Infected Diabetic Foot Ulcers

In Vitro Susceptibility to Pexiganan of Bacteria Isolated from Infected Diabetic Foot Ulcers Yigong Ge, Dorothy MacDonald, Marietta M. Henry, Howard I. Hait, Kimberly A. Nelson, Benjamin A. Lipsky, Michael A. Zasloff, and Kenneth J. Holroyd During two clinical trials involving the treatment of 835 outpatients with infected diabetic foot ulcers, 2515 bacterial isolates, including 2337 aerobes and 178 anaerobes, were grown from cultures of the ulcers. The in vitro susceptibility of these isolates was determined to pexiganan, a peptide anti-infective evaluated in these clinical trials, and to other classes of antibiotics. Pexiganan demonstrated broad spectrum antimicrobial activity against Gram-positive and Gram-negative aerobes and anaerobes. The MIC 90 values for the most common species among 1735 Gram-positive aerobes isolated, such as Staphylococcus aureus, coagulase-negative staphylococci, Group A streptococci, and Group B streptococci, were 16 g/ml or less. Of 602 Gram-negative aerobes tested, the MIC 90 values for pexiganan were 16 g/ml or less for Acinetobacter, Pseudomonas, Stenotrophomonas, Citrobacter, Enterobacter, Escherichia, Klebsiella, and Flavobacterium species. Pexiganan had a MIC 90 of4to16 g/ml against the anaerobic isolates of Bacteroides, Peptostreptococcus, Clostridium, and Prevotella species. Importantly, pexiganan did not exhibit cross-resistance with other commonly used antibiotics, including -lactams, quinolones, macrolides, and lincosamides. The broad spectrum in vitro antimicrobial activity of pexiganan against clinical isolates from infected diabetic foot ulcers supports its potential as a local therapy for infected diabetic foot ulcers. 1999 Elsevier Science Inc. INTRODUCTION From the Magainin Pharmaceuticals Inc. (YG, DM, HIH, KAN, MAZ, KJH), Plymouth Meeting, PA, USA, Covance Central Laboratory Services Inc. (MMH), Indianapolis, IN, USA, Medical Service, VA Puget Sound Health Care System, and Department of Medicine, University of Washington School of Medicine (BAL), Seattle, WA, USA. Address reprint requests to Department of Microbiology, Magainin Pharmaceuticals Inc., Plymouth Meeting, PA 19462. Received 5 January 1999; revised and accepted 21 April 1999. Lower extremity infections are among the most common and serious complications of diabetes mellitus. They are the most frequent cause of hospitalization for diabetics, and account for approximately 6% of all hospitalizations in the United States (Levin, 1997; Reiber et al., 1998). The management of infected diabetic foot ulcers imposes a substantial financial burden on society. According to recent estimates, medical costs run as high as $4600 to $9900 per ulcer episode (Holzer et al., 1998; Obrien et al., 1998). In addition to management of the blood glucose, treatment of diabetic foot ulcer infections requires vigorous wound care and antimicrobial chemotherapy. The antibiotic regimens most often recommended for the treatment of these potentially serious infections consist of orally or parenterally administered broad spectrum agents, such as amoxicillin/clavulanate, ampicillin/sulbactam, ofloxacin, ciprofloxacin, imipenem, piperacillin/tazobactam, and second or third generation cephalosporins (Grayson et al., 1994; Lipsky et al., 1990; Lipsky, 1999; West, 1995). At the present time, no local antibiotic therapy has been demonstrated to be effective in the treatment of diabetic foot ulcer infections. Magainins are a novel class of peptide antiinfectives originally isolated from the skin of the DIAGN MICROBIOL INFECT DIS 1999;35:45 53 1999 Elsevier Science Inc. All rights reserved. 0732-8893/99/$ see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0732-8893(99)00056-5

46 Y. Ge et al. African clawed frog, Xenopus laevis (Zasloff, 1988). Like other cationic peptide antimicrobials, magainins act by binding to anionic phospholipids on the outer leaflet of bacterial membranes, leading to perturbation of membrane permeability, and subsequently bacterial cell lysis and death (Boman, 1995; Hancock, 1997; Jacob and Zasloff, 1994; Nicolas and Mor, 1995). Based on this mechanism of action, the predicted likelihood of bacterial resistance against this class of antibiotic is low (Boman, 1995; Hancock, 1997; Jacob and Zasloff, 1994; Nicolas and Mor, 1995). Through extensive structure activity studies, pexiganan, an analog of Magainin 2 (a natural antimicrobial peptide compound isolated from the frog skin) was created with a potency and safety profile that supported its development as a therapeutic agent for the treatment of diabetic foot infections (Lipsky et al., 1997). Pexiganan (MSI-78) is a broad spectrum bactericidal agent active in vitro against both anaerobic and aerobic microorganisms (Fuchs et al., 1998; Ge et al., 1999). Recently, pexiganan has been evaluated in the treatment of outpatient diabetic foot ulcer infections in two placebo controlled double blind trials, comparing pexiganan cream 1% to orally administered ofloxacin (Lipsky et al., 1997). In this report, we present the in vitro pexiganan susceptibility of 2515 isolates cultured from infected diabetic foot ulcers. MATERIALS AND METHODS Patients A total of 835 adult diabetic outpatients clinically judged to have an infected foot ulcer were enrolled in two phase 3 studies comparing pexiganan cream 1% to oral ofloxacin. Patients were drawn from 81 medical centers widely distributed geographically across the United States. Institutional Review Board approval of the protocol was obtained at each medical center, and informal consent was obtained from each patient. Excluded were patients whose foot ulcer was complicated by osteomyelitis, advancing cellulitis, tendon or bone exposure, deep abscess requiring surgical drainage, or infections judged to require hospitalization. Specimen collection and Microorganism Isolation A standard and well validated (Lipsky et al., 1990) specimen collection protocol was established in all clinical centers. In brief, a culture sample was collected by tissue curettage during the debridement procedure; the tissue was obtained from the base of the ulcer, and placed in transport medium (Port-A- Cul Tube, Becton Dickinson Co., Cockeysville, MD, USA). Specimens were shipped by overnight mail to the testing center (Corning SciCor, Indianapolis, IN, USA) where the plating of cultures, identification of isolates, and susceptibility tests (see below) were performed. Cultures were obtained at the baseline visit, on Days 3, 10, 14, 21, 28, and at a 2 week post therapy follow-up visit. Where multiple isolates of the same species were obtained from a patient, only the first isolate collected is presented in this report. For example, if a Staphylococcus aureus was isolated at the baseline visit, on Day 3 and Day 28, only the result at the baseline visit is included in this report. Antimicrobial Agents The peptide pexiganan (Gly-Ile-Gly-Lys-Phe-Leu- Lys-Lys-Ala-Lys-Lys-Phe-Gly-Lys-Ala-Phe-Val-Lys- Ile-Leu-Lys-Lys-NH 2 ) was synthesized at Bachem Bioscience (Torrance, CA, USA). Pexiganan (powder) was dissolved in water prior to use. Other antimicrobial agents used were obtained from Sigma Chemicals (St. Louis, MO, USA) and their respective United States manufacturers. In Vitro Susceptibility Testing All susceptibility testing was performed at Corning SciCor (Indianapolis, IN, USA). The minimum inhibitory concentration (MIC) of pexiganan and other antimicrobials to both aerobic and anaerobic microorganisms was determined in broth and/or disk diffusion assays according to the NCCLS recommended procedures (NCCLS, 1987; NCCLS, 1993; NCCLS, 1997). Unsupplemented Mueller-Hinton broth (to avoid any interference of cations with in vitro pexiganan activity) and the anaerobic MIC broth (Wilkins-Chalgren) were used in testing most aerobic bacteria and anaerobic bacteria, respectively. Resistance breakpoints were determined by NCCLS criteria when available. When NCCLS criteria were not available, as was the case for anaerobic isolates tested with oxacillin, ofloxacin, cephalexin, and amikacin, the following breakpoints were used: ofloxacin ( 8.0 g/ml), oxacillin ( 8.0 g/ml), cephalexin ( 32 g/ml), and amikacin ( 32 g/ml). Eight quality control (QC) organisms, including Enterococcus faecalis ATCC 29212, S. aureus ATCC 29213, Escherichia coli ATCC 25922, E. coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Eubacterium lentum ATCC 43055, were tested along with clinical isolates to monitor testing quality.

In Vitro Susceptibility Pexiganan 47 RESULTS In Vitro Antibacterial Activity of Pexiganan To evaluate the activity of pexiganan against clinical isolates from diabetic foot ulcers, MICs were determined for a total of 2515 isolates. These isolates, that consisted of 2337 aerobes and 178 anaerobes, included pathogens commonly associated with complicated skin and soft tissue infections. Pexiganan demonstrated good activity against the majority of aerobic species tested (Table 1). Most aerobic bacteria, including staphylococci, some Streptococcus species, Corynebacterium spp., and Micrococcus spp., exhibited an MIC 90 of 16 g/ml or less. Importantly, the MIC 90 for most of the more prevalent Grampositive pathogens, such as S. aureus (375 isolates), Staphylococcus epidermidis (194 isolates), Staphylococcus hemolyticus (81 isolates), Streptococcus agalactiae (174 isolates), and Streptococcus pyogenes (11 isolates), was 16 g/ml or less. Limited numbers of other Gram-positive aerobic bacteria, including Aerococcus spp., Arcanobacterium spp., Bacillus spp., Brevibacterium spp., Gemella morbillorum, Oerskovia spp., and Rhodococcus equi, were also inhibited by pexiganan with an MIC of 16 g/ml or less. Pexiganan was less active against Streptococcus mitis (14 isolates), Streptococcus sanguis (38 isolates), and E. faecalis (325 isolates) with an MIC 90 of 128 to 256 g/ml. Enterococcus spp. other than E. faecalis exhibited an MIC 90 of 32 g/ml. These results suggest that pexiganan has in vitro activity against most Gram-positive aerobes. Pexiganan also exhibited good activity against Gram-negative bacteria (Table 2). Of the 602 isolates tested, pexiganan at a concentration of 16 g/ml or less inhibited the growth of 90% of isolates of Acinetobacter, Pseudomonas, Stenotrophomonas, Citrobacter, Enterobacter, Escherichia, Klebsiella, and Flavobacterium species. Several species not commonly implicated as pathogens in skin infections, including Capnocytophaga, Hemophilus, Neisseria, and Moraxella, were also isolated and inhibited by pexiganan (MICs 8 g/ ml). Pexiganan was less active against Alcaligenes faecalis with a MIC 90 of 256 g/ml, and a MIC 50 of 32 g/ml. Several closely related genera of the Enterobacteriaceae family, including Morganella, Proteus, Providencia, and Serratia, were found to be less susceptible in vitro to pexiganan, with both MIC 50 and MIC 90 greater than 256 g/ml. As shown in Table 3, pexiganan demonstrated excellent antimicrobial activity against anaerobic pathogens (177 isolates) recovered from diabetic foot ulcer infections. The MIC 90 for species of Bacteroides, Peptostreptococcus, and Prevotella, was 16 g/ml or less. All obligate anaerobes, including Clostridium, Propionibacterium, Mobiluncus, Fusobacterium, and Veillonella, were also inhibited by pexiganan at a concentration of 8 g/ml or less, although the number of isolates tested was limited. To monitor test quality and reproducibility, eight NCCLS quality control organisms were tested along with clinical isolates. A summary of all quality control testing results are shown in Table 4. Consistent and reproducible results were obtained with these quality control strains. The differences of the MICs in a specific QC strain were limited to one or two doubling dilutions (32 to 64 g/ml for E. faecalis ATCC 29212, 8 to 16 g/ml for S. aureus ATCC 29213, 4 to 8 g/ml for E. coli ATCC 25922 and E. coli ATCC 35218, 4 to 8 g/ml for Pseudomonas aeruginosa ATCC 27853, 2 to 8 g/ml for Bacteroides fragilis ATCC 25285, 2 to 8 g/ml for Bacteroides thetaiotaomicron ATCC 29741, and 4 to 16 g/ml for Eubacterium lentum ATCC 43055). Lack of Cross-Resistance between Pexiganan and other Classes of Antibiotics In addition to pexiganan, the in vitro susceptibility of the clinical isolates from diabetic foot ulcer infections was determined for other classes of antibiotics including -lactams, macrolides, quinolones, lincosamides, and aminoglycosides. The percentage and number of resistant isolates for each antibiotic tested are presented in Tables 1 to 3. To evaluate potential cross-resistance of pexiganan with other classes of antibiotics, the susceptibility data of isolates from the infected diabetic foot ulcers resistant to these other antibiotics were analyzed. These isolates included oxacillin (methicillin)-resistant staphylococci, quinolone-resistant staphylococci and Corynebacterium spp., erythromycin-resistant Corynebacterium species, clindamycin-resistant staphylococci, cephalosporin-resistant staphylococci, and imipenem-resistant S. epidermidis and S. hemolyticus. Table 5 lists the MIC distributions, MIC 50 and MIC 90 against pexiganan for these isolates in relationship to their susceptibility to other classes of antibiotics. There was no correlation between resistance to the other agents and higher MICs to pexiganan. The distribution of MICs against pexiganan was similar for organisms either resistant or susceptible to these other antibiotics. In each case, the differences in MIC 50 and MIC 90 between resistant and susceptible populations were no more than two-fold. Similarity of In Vitro Susceptibility to Pexiganan of Isolates Recovered from Infected Diabetic Foot Ulcers and Clinical Strains Isolated from Non-Diabetic Disorders The susceptibility patterns of clinical strains recovered from infections of non-diabetic foot ulcer origin (NDFU) were compared with identical species recov-

48 Y. Ge et al. TABLE 1 MICs of Infected Diabetic Foot Ulcer Gram-Positive Aerobic Bacteria to Pexiganan; % Resistant (by NCCLS Criteria) to Several Oral or Intravenous Antibiotics MIC ( g/ml) with Pexiganan Percentage of Resistant Isolates (number of resistant isolates) isolates MIC 50 MIC 90 Range Ofloxacin Oxacillin Amoxicillin/ Clavulanate Clindamycin Cephalexin Erythromycin Imipenem Staphylococcus S. aureus 375 8 16 0.5 256 16 (59) 13 (50) 9 (34) 14 (51) 12 (46) 27 (102) 4 (15) S. epidermidis 194 4 8 2 16 58 (112) 61 (118) 3 (5) 25 (48) 53 (72) 72 (139) 16 (31) S. haemolyticus 81 4 8 2 64 62 (50) 57 (46) 41 (33) 30 (24) 57 (46) 67 (54) 27 (22) S. hominis 20 4 8 2 16 0 (0) 0 (0) 0 (0) 0 (0) 5 (1) 15 (3) 0 (0) S. sciuri 36 4 16 2 256 17 (6) 33 (12) 3 (1) 14 (5) 19 (7) 28 (10) 0 (0) S. simulans 42 4 8 2 16 24 (10) 26 (11) 12 (5) 17 (7) 24 (10) 29 (12) 12 (5) Other coagulase negative staphylococci 72 4 8 1 128 18 (13) 14 (10) 0 (0) 11 (8) 10 (7) 28 (20) 1 (1) Streptococcus S. agalactiae 174 8 16 0.5 64 1 (2) 1 (2) 0 (0) 4 (7) 2 (4) 9 (16) 0 (0) S. canis 45 8 8 4 16 9 (4) 0 (0) 0 (0) 2 (1) 0 (0) 11 (5) 0 (0) S. mitis 14 32 128 8 128 7 (1) 29 (4) 0 (0) 7 (1) 29 (4) 29 (4) 0 (0) S. pyogenes 11 8 8 4 8 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) S. sanguis 38 64 256 4 256 13 (5) 13 (5) 0 (0) 3 (1) 11 (4) 5 (2) 0 (0) Other streptococci 66 8 16 0.25 128 17 (11) 12 (8) 0 (0) 8 (5) 9 (6) 8 (5) 2 (1) Enterococcus E. faecalis 325 128 256 0.25 256 10 (37) 99 (321) 1 (4) 98 (317) 99 (323) 34 (112) 1 (3) E. faecium 15 8 32 0.25 256 27 (4) 93 (14) 0 (0) 73 (11) 47 (7) 47 (7) 7 (1) Other enterococci 18 4 8 0.25 16 11 (2) 89 (16) 0 (0) 89 (16) 22 (4) 28 (5) 0 (0) Corynebacterium C. jeikeium 15 4 4 2 64 60 (9) 67 (10) 13 (2) 67 (10) 27 (4) 40 (6) 7 (1) C. minutissimum 31 4 8 1 8 77 (24) 55 (17) 0 (0) 74 (23) 3 (1) 48 (15) 3 (1) C. striatum 45 4 8 0.5 16 42 (19) 83 (38) 2 (1) 44 (20) 2 (1) 18 (8) 0 (0) C. xerosis 19 4 8 2 8 53 (10) 68 (13) 0 (0) 53 (10) 0 (0) 11 (2) 0 (0) Other Corynebacterium spp. 59 4 8 0.25 16 58 (34) 61 (36) 10 (6) 56 (34) 22 (13) 27 (16) 5 (3) Micrococcus spp. 14 4 8 0.5 8 36 (5) 43 (6) 14 (2) 14 (2) 21 (3) 14 (2) 7 (1) Other gram-positive bacteria Aerococcus spp. 10 4 4 2 4 10 (1) 80 (8) 0 (0) 10 (1) 0 (0) 40 (4) 0 (0) Arcanobacterium spp. 3 16 ND 8 16 ND (2) ND (0) ND (0) ND (0) ND (0) ND (0) ND (0) Bacillus spp. 3 8 ND 2 8 ND (0) ND (2) ND (0) ND (1) ND (2) ND (0) ND (0) Brevibacterium spp. 3 2 ND 0.25 2 ND (2) ND (0) ND (0) ND (1) ND (0) ND (0) ND (0) Gemella morbillorum 2 4 ND 4 8 ND (0) ND (1) ND (1) ND (0) ND (1) ND (0) ND (0) Oerskovia spp. 3 4 ND 2 8 ND (0) ND (3) ND (0) ND (2) ND (3) ND (1) ND (3) Rhodococcus equi 2 1 ND 1 ND (1) ND (1) ND (0) ND (1) ND (0) ND (0) ND (0) Total 1735 ND For species with less than 10 isolates, the MIC 90 and the percentage of resistance was not calculated, and for species with less than two isolates, the MIC 50 was not determined.

In Vitro Susceptibility Pexiganan 49 TABLE 2 MICs of Gram-Negative Aerobic Bacteria to Pexiganan; % Resistant (by NCCLS Criteria) to Several Oral or Intravenous Antibiotics MIC ( g/ml) with Pexiganan Percentage of Resistant Isolates (number of resistant isolates) Isolates MIC 50 MIC 90 Range Ofloxacin Ampicillin Amoxicillin/ Clavulanate Amikacin Ceftazidime Imipenem Acinetobacter A. anitratus 40 4 4 2 8 5 (2) 23 (9) 13 (5) 5 (2) 0 (0) 0 (0) A. baumanii 19 4 8 4 8 5 (1) 16 (3) 5 (1) 11 (2) 5 (1) 5 (1) Other Acinetobacter spp. 13 4 8 2 8 0 (0) 8 (1) 0 (0) 0 (0) 0 (0) 0 (0) Alcaligenes A. faecalis 21 32 256 8 256 14 (3) 10 (2) 0 (0) 0 (0) 0 (0) 0 (0) Other Alcaligenes spp. 9 16 ND 4 256 ND (2) ND (1) ND (0) ND (5) ND (1) ND (0) Pseudomonas P. aeruginosa 92 8 16 4 64 15 (14) 99 (91) 99 (91) 0 (0) 0 (0) 2 (2) Other Pseudomonas spp. 7 4 ND 1 8 ND (3) ND (3) ND (1) ND (0) ND (3) ND (0) Stenotrophomonas S. maltophilia 37 4 8 2 32 19 (7) 78 (29) 78 (29) 70 (26) 14 (5) 92 (34) Enterobacteriaceae Citrobacter C. diversus 9 4 ND 4 ND (0) ND (7) ND (0) ND (0) ND (0) ND (0) C. freundii 9 4 ND 2 8 ND (1) ND (2) ND (8) ND (0) ND (1) ND (0) Enterobacter E. cloacae 49 8 16 1 64 0 (0) 67 (33) 80 (39) 0 (0) 0 (0) 0 (0) Other Enterobacter spp. 12 8 16 4 16 0 (0) 33 (4) 25 (3) 0 (0) 0 (0) 0 (0) Escherichia E. coli 53 4 8 4 256 2 (1) 30 (16) 9 (5) 2 (1) 2 (1) 0 (0) Other Escherichia spp. 2 4 ND 4 8 ND (0) ND (0) ND (0) ND (0) ND (0) ND (0) Hafnia H. alvei 1 ND ND 4 ND (0) ND (0) ND (0) ND (0) ND (0) ND (0) Klebsiella K. oxytoca 31 4 8 2 16 3 (1) 65 (20) 0 (0) 0 (0) 0 (0) 0 (0) K. pneumoniae 24 8 8 4 16 0 (0) 54 (13) 0 (0) 0 (0) 4 (1) 3 (1) Morganella M. morganii 13 256 256 256 256 8 (1) 100 (13) 100 (13) 0 (0) 0 (0) 0 (0) Proteus P. mirabilis 60 256 256 8 256 0 (0) 7 (4) 2 (1) 0 (0) 3 (2) 2 (1) Other Proteus spp. 13 256 256 4 256 0 (0) 85 (11) 0 (0) 0 (0) 0 (0) 0 (0) Providencia P. rettgeri 12 256 256 256 256 8 (1) 17 (2) 42 (5) 0 (0) 0 (0) 0 (0) P. stuartii 3 256 ND 256 ND (0) ND (3) ND (3) ND (0) ND (0) ND (0) Serratia S. marcescens 35 256 256 8 256 0 (0) 57 (20) 89 (31) 0 (0) 0 (0) 0 (0) Other Serratia spp. 4 256 ND 4 256 ND (0) ND (2) ND (2) ND (0) 0 (0) ND (0) Yokenella regensburgi 1 ND ND 256 ND (0) ND (0) ND (1) ND (0) 0 (0) ND (0) Other gram-negative bacteria spp. Aeromonas spp. 3 64 ND 8 256 ND (0) ND (1) ND (1) ND (0) ND (0) ND (0) Capnocytophaga spp. 1 ND ND 4 ND (1) ND (1) ND (1) ND (0) ND (0) ND (0) Chryseomonas luteola 1 ND ND 4 ND (0) ND (1) ND (0) ND (0) ND (0) ND (0) Flavobacterium spp. 13 4 16 2 256 0 (0) 38 (5) 38 (5) 38 (5) 31 (4) 23 (3) Haemophilus spp. 8 4 ND 0.25 8 ND (0) ND (1) ND (0) ND (0) ND (0) ND (0) Neisseria meningitides 1 ND ND 4 ND (0) ND (0) ND (0) ND (0) ND (0) ND (0) Moraxella spp. 2 4 ND 4 ND (0) ND (0) ND (0) ND (0) ND (0) ND (0) Pasteuralla spp. 4 4 ND 1 8 ND (0) ND (0) ND (0) ND (0) ND (0) ND (0) Total 602 ND For species with less than 10 isolates, the MIC 90 and the percentage of resistance was not calculated, and for species with less than two isolates, the MIC 50 was not determined.

50 Y. Ge et al. TABLE 3 MICs of Anaerobic Bacteria to Pexiganan; % Resistant (by NCCLS Criteria) b to Several Oral or Intravenous Antibiotics MIC ( g/ml) with Pexiganan Percentage of Resistance (numbers of resistance isolates) Isolates Amoxicillin/ MIC 50 MIC 90 Range Ofloxacin a Oxacillin a Clavulanate Clindamycin Cephalexin a Amikacin a Gram-positive Clostridium C. clostridioforme 1 ND ND 4 ND (1) ND (0) ND (0) ND (0) ND (1) ND (1) C. paraputrificum 1 ND ND 1 ND (0) ND (0) ND (0) ND (0) ND (0) ND (0) Lactobacillus spp. 3 128 ND 64 128 ND (1) ND (0) ND (0) ND (0) ND (0) ND (2) Peptostreptococcus P. asaccharolyticus 17 2 4 0.25 8 76 (13) 12 (2) 0 (0) (1) 6 (1) 6 (1) P. magnus 36 2 8 0.25 64 53 (19) 3 (1) 0 (0) 11 (4) 0 (0) 8 (3) P. prevotii 11 2 8 0.25 32 55 (6) 9 (1) 0 (0) 20 (2) 0 (0) 9 (1) Other Peptostreptococcus spp. 11 2 8 1 8 36 (4) 0 (0) 0 (0) 27 (3) 9 (1) 9 (1) Propionibacterium P. acnes 1 ND ND 1 ND (0) ND (0) ND (0) ND (0) ND (0) ND (0) Mobiluncus spp. 1 ND ND 2 ND (0) ND (0) ND (0) ND (0) ND (0) ND (0) Gram-negative Bacteroides B. fragilis 27 8 16 2 64 48 (13) 96 (26) 0 (0) 7 (2) 93 (25) 100 (27) Other Bacteroides spp. 16 4 8 2 32 56 (9) 81 (13) 0 (0) 25 (4) 75 (12) 81 (13) Fusobacterium F. necrophorum 1 ND ND 4 ND (0) ND (0) ND (0) ND (0) ND (0) ND (0) F. nucleatum 1 ND ND 4 ND (1) ND (0) ND (0) ND (0) ND (0) ND (1) F. varium 1 ND ND 4 ND (0) ND (0) ND (0) ND (1) ND (0) ND (0) Prevotella P. bivia 34 4 4 0.25 8 24 (8) 56 (19) 6 (2) 12 (4) 24 (8) 85 (29) Other Prevotella spp. 11 4 8 0.25 32 27 (3) 64 (7) 9 (1) 18 (2) 45 (5) 91 (10) Veillonella spp. 5 2 ND 2 8 ND (3) ND (2) ND (0) ND (0) ND (0) ND (1) Total 178 a ND For species with less than 10 isolates, the MIC90 and the percentage of resistance was not calculated, and for species with less than two isolates, the MIC 50 was not determined. b Resistance breakpoints for anaerobes used the following breakpoints when no NCCLS criteria were available: ofloxacin ( 8.0 g/ml), oxacillin ( 8.0 g/ml), cephalexin ( 32 g/ml), and amikacin ( 32 g/ml); those for amoxicillin/clavulanate, and clindamycin were based on NCCLS criteria.

In Vitro Susceptibility Pexiganan 51 TABLE 4 MIC Distributions of Pexiganan With the QC Strains Tested MIC Distribution for the QC Strains ( g/ml) test 1 2 4 8 16 32 64 128 256 S. aureus ATCC 29213 238 96 138 4 E. faecalis ATCC 29212 238 4 124 109 1 E. coli ATCC 25922 235 1 6 179 45 4 E. coli ATCC 35218 237 2 106 121 7 1 P. aeruginosa ATCC 27853 238 67 159 11 1 B. fragilis ATCC 25285 111 49 54 7 1 B. thetaiotaomicron ATCC 29741 109 67 37 4 1 E. lentum ATCC 43055 109 39 59 10 1 TABLE 5 Comparison of MIC Distributions to Pexiganan for Infected Diabetic Foot Ulcer Isolates Resistant (Based on NCCLS Criteria) and Susceptible to Other Antibiotics isolates MIC ( g/ml) 0.25 0.5 1 2 4 8 16 32 64 128 256 256 MIC 50 MIC 90 Oxacillin resistant S. aureus 50 1 2 7 15 18 2 1 2 2 8 32 Oxacillin susceptible S. aureus 325 2 21 182 94 21 3 2 8 16 Oxacillin resistant S. epidermidis and S. haemolyticus 164 47 104 11 1 1 4 4 Oxacillin susceptible S. epidermidis and S. haemolyticus 111 33 59 14 3 1 1 4 8 Ofloxacin resistant S. aureus 59 1 2 6 22 21 4 1 2 8 32 Ofloxacin susceptible S. aureus 311 2 20 173 90 19 3 2 2 8 16 Ofloxacin resistant S. epidermidis and S. haemolyticus 162 49 101 10 2 4 4 Ofloxacin susceptible S. epidermidis and S. haemolyticus 91 38 35 13 4 1 4 8 Ofloxacin resistant Corynebacterium 93 2 2 2 20 54 11 1 1 4 8 Ofloxacin susceptible Corynebacterium 57 1 2 2 17 27 7 1 4 8 Ofloxacin resistant anaerobes 82 5 1 3 37 25 6 2 2 1 4 8 Ofloxacin susceptible anaerobes 73 5 1 9 16 23 11 2 4 2 4 16 Amoxicillin/clavulanate resistant S. haemolyticus 33 17 15 1 4 4 Amoxicillin/clavulanate susceptible S. haemolyticus 48 17 21 5 3 1 1 4 8 Erythromycin resistant Corynebacterium 56 1 2 2 19 25 4 2 1 4 8 Erythromycin susceptible Corynebacterium 60 1 3 1 11 31 12 1 4 8 Clindamycin resistant S. aureus, S. epidermidis and S. haemolyticus 123 29 47 22 17 2 2 2 2 4 16 Clindamycin susceptible S. aureus, S. epidermidis and S. haemolyticus 523 55 142 199 99 22 4 2 8 16 Imipenem resistance S. epidermidis and S. haemolyticus 53 27 22 3 1 2 4 Imipenem susceptible S. epidermidis and S. haemolyticus 216 51 137 22 4 1 1 4 4 Cephalexin resistant S. aureus 46 1 1 5 17 15 3 2 2 8 32 Cephalexin susceptible S. aureus 320 3 22 175 94 20 4 2 8 16 Cephalexin resistant S. epidermidis and S. haemolyticus 99 38 53 6 1 1 4 4 Cephalexin susceptible S. epidermidis and S. haemolyticus 98 28 53 12 3 1 1 4 8

52 Y. Ge et al. ered from diabetic foot ulcers (DFU). The pexiganan susceptibility data for the NDFU group used in Table 6 was adopted from our previous in vitro susceptibility study (Ge et al., 1999). Table 6 shows the distributions of MIC 50 and MIC 90 of the various isolates. For Gram-positive bacteria, there was no remarkable difference in the MIC distribution between the NFDU and DFU isolates, except for the lower MIC 50 and MIC 90 for E. faecalis in the DFU. Interestingly, MIC 50 and/or MIC 90 were lower in the DFU than those of the NDFU for the majority of Gram-negative aerobic bacteria, such as Enterobacteriaceae, Acinetobacter, Pseudomonas, and Stenotrophomonas. A similar pattern was also found in two species of anaerobic microorganisms; the MIC 90 of Peptostreptococcus and Prevotella in the DFU group were two-fold lower than those of the NDFU. In brief, the MIC 50 and MIC 90 of the DFU isolates were identical or lower than those of the NDFU isolates, with the exception of the MIC 90 for Alcaligenes faecalis. These findings suggest that clinical isolates from diabetic foot lesions exhibit a similar susceptibility to pexiganan as previously observed for clinical isolates recovered from other disease states. DISCUSSION Pexiganan demonstrated broad spectrum in vitro antimicrobial activity against most of the common pathogens isolated from infected diabetic foot ulcers. Several closely related genera of the Enterobacteriaceae family, including Morganella, Proteus, Providencia, and Serratia, were found to be the less susceptible in vitro to pexiganan, with both MIC 50 and MIC 90 greater than 256 g/ml. Interestingly, all four of these species have a close genetic relationship. The precise mechanism underlying the in vitro intrinsic relative resistance of these species is not yet understood. In its current formulation, pexiganan is administered as a 1% (10,000 g/ml) concentration in a cream base. Application at this concentration could provide anti-infective efficacy even against infected diabetic foot ulcer organisms less susceptible in vitro TABLE 6 Comparison of Susceptibility to Pexiganan for Clinical Isolates from Non-Diabetic Disorders and Infected Diabetic Foot Ulcers a MIC ( g/ml) MIC ( g/ml) 0.25 0.5 1 2 4 8 16 32 64 128 256 256 isolates MIC 50 MIC 90 Staphylococcus aureus DBF 1 4 28 197 112 23 4 4 2 375 8 16 NDFU 5 7 55 333 102 29 6 537 8 16 Coagulase-negative DBF 2 138 248 38 10 2 4 2 1 445 4 8 staphylococci NDFU 12 2 3 80 137 102 15 1 3 355 4 8 Streptococci b DBF 5 2 5 38 203 33 5 3 1 1 296 8 16 NDFU 1 160 97 96 36 4 4 1 1 519 8 16 Enterococcus faecalis DBF 1 2 2 2 3 14 54 151 66 30 325 128 256 NDFU 1 1 6 13 37 35 38 131 256 256 Corynebacterium spp. DBF 4 4 4 43 90 20 3 1 169 4 8 NDFU 4 2 8 35 6 3 1 59 4 8 Enterobacteriaceae c DBF 1 2 99 68 12 3 3 1 1 190 4 8 NDFU 2 10 77 368 368 56 18 7 2 2 910 8 16 Acinetobacter spp. DBF 9 45 18 72 4 8 NDFU 13 49 43 18 123 2 8 Alcaligenes spp. DBF 1 4 7 11 1 1 3 2 30 32 256 NDFU 2 2 5 6 4 3 4 1 2 29 32 128 Pseudomonas spp. DBF 2 21 58 15 1 1 1 99 8 16 NDFU 2 34 80 75 19 6 2 2 220 8 32 S. maltophilia DBF 2 30 4 1 37 4 8 NDFU 10 32 64 23 5 134 8 16 Bacteroides spp. DBF 1 10 14 10 5 2 1 43 4 16 NDFU 4 6 3 31 66 10 3 1 1 125 8 16 Peptostreptococcus spp. DBF 7 2 5 31 20 6 3 1 75 2 8 NDFU 5 3 13 20 19 13 5 5 4 1 2 90 4 32 Prevotella spp. DBF 3 3 8 26 4 1 45 4 8 NDFU 1 1 5 26 5 5 12 1 4 60 4 32 DBF diabetic foot ulcer infection isolates; NDFU non-diabetic foot ulcer isolates. a The data of the NDFU group were taken from the reference 16. b Excluded Streptococcus mitis and S. sanguis. c Excluded Morganella spp., Proteus spp., Providencia spp., Serratia spp., and Yokenella spp.

In Vitro Susceptibility Pexiganan 53 to pexiganan, such as Enterococcus faecalis, Proteus mirabilis, and Serratia marcescens. The determination of a susceptibility breakpoint for this topical antimicrobial agent is therefore difficult. To develop useful susceptibility breakpoints for pexiganan, close correlation with efficacy and microbial eradication in clinical infections will need to be performed. Pexiganan was found to be active against bacterial isolates from infected diabetic foot ulcers irrespective of their resistance or susceptibility to other classes of antibiotics. Pexiganan does not exhibit crossresistance with commonly used antibiotics, such as -lactams, quinolones, macrolides, and lincosamides. The lack of cross-resistance between pexiganan and other available antibiotics is likely a reflection of the membrane targeted mechanism of action of this antimicrobial peptide (Hancock, 1997; Jacob and Zasloff, 1994). These features, in combination with its rapidly bactericidal mechanism of action (Ge et al., 1999; Jacob and Zasloff, 1994), warrant further consideration of pexiganan as local therapy for infected diabetic foot ulcers. REFERENCES Boman HG (1995) Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 13:61 92. Fuchs PC, Barry AL, Brown SD (1998) In vitro antimicrobial activity of MSI-78, a magainin analog. Antimicrob Agents Chemother 42:1213 1216. Ge Y, MacDonald DL, Holroyd KJ, Thornsberry C, Wexler H, Zasloff M (1999) In vitro antibacterial properties of pexiganan, an analog of Magainin. Antimicrob Agents Chemother 43:782 788. Grayson ML, Gibbons GW, Habershaw GM, Freeman DV, Pomposelli FB, Rosenblum BI, Levin E, Karchmer AW (1994) Use of ampicillin/sulbactam versus imipenem/ cilastin in the treatment of life threatening foot infections in diabetic patients. Clin Infect Dis 18:683 693. Hancock REW (1997) Peptide antibiotics. Lancet 349:418 422. Holzer SE, Camerota A, Martens L, Cuerdon T, Crystal- Peters J, Zagari M (1998) Costs and duration of care for lower extremity ulcers in patients with diabetes. Clin Therpeutics 20:169 181. Jacob L, Zasloff MA (1994) Potential therapeutic application of magainins and other antimicrobial agents for animal origin. Ciba Found Symp 186:197 216. Levin M (1997) Diabetic foot wounds: pathogenesis and management. Adv Wound Care 10:24 30. Lipsky BA, Pecoraro RE, Larson SA, Hanley ME, Ahroni JH (1990) Outpatient management of uncomplicated lower extremity infections in diabetic patients. Arch Intern Med 150:790 797. Lipsky BA, Baker PD, Landon GC, Fernau R (1997) Microbial eradication and clinical resolution of infected diabetic foot ulcers treated with topical MSI-78 vs. oral ofloxacin. Abstract from the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy:74. Lipsky BA (1999) Antibiotic therapy of diabetic foot infections. Adv Wound Care, in press. National Committee for Clinical Laboratory Standards (1987) Proposed guideline (M26-P) Approved standard (M11-A): Methods for determining bactericidal activity of antimicrobial agents. Villanova, PA: NCCLS. National Committee for Clinical Laboratory Standards (1993) Approved standard (M11 A3): Methods for antimicrobial susceptibility testing of anaerobic bacteria, 3rd ed. Villanova, PA: NCCLS. National Committee for Clinical Laboratory Standards (1997) Approved standard (M7-A4): Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 4th ed. Villanova, PA: NCCLS. Nicolas P, Mor A (1995) Peptides as weapons against microorganisms in the chemical defense system of vertebrates. Annu Rev Microbiol 49:277 304. O Brien JA, Shomphe LA, Kavanagh PL, Raggio G, Caro JJ (1998) Direct medical costs of complications resulting from type 2 diabetes in the United States. Diabetes Care 7:1122. Reiber GE, Lipsky BA, Gibbons GW (1998) The burden of diabetic foot ulcers. Am J Surg 176 (2A Suppl.):5S 10S. West NJ (1995) Systemic antimicrobial treatment of foot infections in diabetic patients. Am J Health Syst Pharm 52:1199 1207. Zasloff MA (1988) Magainins, a class of antimicrobial peptides from Xenopus skin: Isolation, characterization of two active forms, and partial cdna sequence of a precursor. Proc Natl Acad Sci USA 84:5449 5453.