Comparative In Vitro Activities of Retapamulin (SB-275833) against 141 Clinical Isolates of Propionibacterium spp., Including 117 P. acnes Isolates

Abstract

Using the NCCLS agar dilution method, we studied the in vitro activity of retapamulin (SB-275833) against 141 clinical isolates of Propionibacterium species, including seven multiresistant strains, and found retapamulin to be the most active agent among those tested with MICs of ≤1 μg/ml against all isolates.

Propionibacterium species are associated with one of the most common inflammatory disorders in humankind, acne vulgaris, which affects ∼80% of adolescents and many adults as well (1). Propionibacterium acnes and Propionibacterium granulosum both produce fatty acids that, when liberated in hair follicles and sebaceous gland ducts, result in an intense inflammatory response which may result in an acne lesion (papular, pustular, or nodular) (6). P. acnes is isolated more frequently from these lesions than is P. granulosum (6). Therapy has been geared to either diminishing the sebaceous secretions, as with retinoids, or suppressing the propionibacteria with topical or oral antimicrobial agents (2, 5, 15 ).

Until the late 1970s, P. acnes was universally susceptible to oral and topical formulations of clindamycin, tetracyclines, and other antimicrobial agents (2, 10). However, since the 1980s, numerous reports of increasing Propionibacterium species resistance worldwide have been published (2, 5, 8, 10, 11, 12, 14). Resistance rates in 50% to 90% of isolates (in Spain and Hungary) have been associated with the widespread therapeutic use of various agents (10). This increased resistance has spurred trials of dual therapy using antimicrobials and retinoids (13, 15), as well as the search for new antimicrobial agents.

Retapamulin (SB-275833) is a novel semisynthetic pleuromutilin, representing a new class of antibacterial agents for use in humans. Retapamulin has been formulated as a topical antibiotic and is currently being developed for the treatment of uncomplicated bacterial skin infections. The drug selectively inhibits bacterial protein synthesis by way of a unique interaction with the ribosome that enables activity against target organisms, including those resistant to other antimicrobial agents. In particular, retapamulin demonstrates excellent in vitro activity against isolates resistant to other antimicrobial agents, such as methicillin-resistant Staphylococcus aureus and pathogens resistant to fusidic acid and mupirocin. There is no target-specific cross-resistance to other classes of antimicrobial agents. Retapamulin is active against bacterial pathogens commonly associated with skin infections (S. aureus and Streptococcus pyogenes) and respiratory tract infections (Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis), including bacterial isolates carrying resistance determinants to established agents, including β-lactams, macrolides, quinolones, fusidic acid, and mupirocin (N. Scangarella, GlaxoSmithKline, personal communication).

Strains were isolated from clinical skin specimens obtained between 1984 and 2003 from adult patients and identified by standard criteria (7) and stored in skim milk at −70°C. Frozen cultures were subcultured twice on brucella agar supplemented with hemin, vitamin K₁, and 5% sheep blood (Anaerobe Systems, Morgan Hill, CA) to ensure purity and good growth. Susceptibility testing was performed according to CLSI (formerly NCCLS) standards (NCCLS document M11-A6), with brucella agar supplemented with hemin, vitamin K₁, and 5% laked sheep blood and an inoculum of 10⁵ CFU per spot (9).

The susceptibility breakpoints established for anaerobic bacteria were used to characterize the susceptibility or resistance of Propionibacterium spp., although those breakpoints have not been specifically applied to these organisms. The breakpoints used to define susceptibility and resistance were MICs of ≤4 μg/ml and ≥16 μg/ml, respectively, for tetracycline and ≤2 μg/ml and ≥8 μg/ml, respectively, for clindamycin (9). Bacteria with intermediate values were considered nonsusceptible. There are no CLSI susceptibility guidelines for erythromycin or trimethoprim-sulfamethoxazole (TMP-SMX) for anaerobes. EUCAST breakpoints used to define resistance were ≥0.25 μg/ml for clindamycin, ≥0.5 μg/ml for erythromycin, ≥2 μg/ml for tetracycline, and ≥1 μg/ml for TMP-SMX (10, 12).

The antimicrobial agents were reconstituted according to the manufacturers' instructions. Retapamulin was dissolved in 0.2% glacial acetic acid. Serial twofold dilutions of antimicrobial agents were prepared on the day of the test and added to the agar medium. For retapamulin, the MIC ranges tested were 0.016 to 16 μg/ml. For TMP-SMX, lysed horse blood was used instead of sheep blood, as recommended by the CLSI for testing the susceptibility of aerobic organisms to sulfa drugs. The agar plates were inoculated with a Steers replicator (Craft Machine, Inc., Chester, PA) with an inoculum of 10⁵ CFU per spot and incubated in an anaerobic chamber (Anaerobe Systems, Morgan Hills, CA) at 37°C for 44 to 48 h and then examined. The MIC was defined as the lowest concentration of an agent that yielded no growth or a marked change in the appearance of growth compared to the growth on the control plate. Control strains, including Bacteroides fragilis ATCC 25285, Eubacterium lentum ATCC 43055, and Staphylococcus aureus ATCC 29213, were tested simultaneously.

The suppliers of standard laboratory powders were as follows: for retapamulin, GlaxoSmithKline, Philadelphia, PA; for erythromycin, Eli Lilly & Co., Indianapolis IN; for trimethoprim-sulfamethoxazole, Hoffman LaRoche, Nutley, NJ; for tetracycline, Bristol-Myers Squibb, Wallingford, CT; and for clindamycin, Voigt Global Distribution, Kansas City, MO.

The numbers and species of clinical isolates tested are given in Table 1. Overall, retapamulin was the most active compound tested, on a weight basis, against all Propionibacterium species tested. Against 117 P. acnes isolates, retapamulin had an MIC₉₀ of 0.25 μg/ml. It was active at ≤1 μg/ml against all strains resistant to the other agents tested, including five strains resistant to >128 μg/ml of erythromycin, three strains with tetracycline MICs of ≥8 μg/ml, and three strains with clindamycin MICs of ≥4 μg/ml. All 12 P. granulosum strains were susceptible to ≤0.015 μg/ml of SB-27583, and all had TMP-SMX MICs of ≥8 μg/ml. All 12 Propionibacterium avidum strains tested were susceptible to ≤0.015 μg/ml of retapamulin; all had TMP-SMX MICs of ≥4 μg/ml, and two had tetracycline MICs of 4 μg/ml.

TABLE 1.

Comparative in vitro activity of retapamulin (SB-275833) against P. acnes, P. avidum, and P. granulosum clinical isolates

Organism and drug	No. of strains	MIC (μg/ml)
		Range	50%	90%
P. acnes	117
Retapamulin		≤0.015-1	≤0.015	0.25
Clindamycin		≤0.015-32	0.03	0.25
Erythromycin		≤0.015->128	0.03	0.06
Tetracycline		≤0.015-32	0.5	1
Trimethoprim-sulfamethoxazole		≤0.25->8	≤0.25	1
P. avidum	12
Retapamulin		≤0.015	≤0.015	≤0.015
Clindamycin		≤0.015-0.06	0.03	0.06
Erythromycin		0.125	0.125	0.125
Tetracycline		0.5-4	1	4
Trimethoprim-sulfamethoxazole		4->8	>8	>8
P. granulosum	12
Retapamulin		≤0.015-0.03	≤0.015	≤0.015
Clindamycin		≤0.015-0.03	≤0.015	0.03
Erythromycin		0.06-0125	0.06	0.125
Tetracycline		1-4	1	2
Trimethoprim-sulfamethoxazole		8->8	>8	>8

The interpretive criteria for susceptibility and resistance breakpoints differ between the United States (9) and Europe (3), which makes it difficult to compare U.S. studies with many of the European studies in terms of their resistance rates. Breakpoints tend to be lower in Europe than in the United States, which results in higher resistance rates in Europe than in the United States.

The activity of retapamulin was evaluated against isolates that are characterized as antibiotic nonsusceptible based on interpretive criteria from the CLSI and EUCAST (11, 12). The results show that for tetracycline, five P. acnes isolates were nonsusceptible by European definitions and three by CLSI criteria; four of the five were susceptible to ≤0.25 μg/ml of retapamulin, and one isolate that had high-level resistance to tetracycline (MIC, 32 μg/ml), clindamycin, (MIC, 32 μg/ml), and erythromycin (MIC, >128 μg/ml) was susceptible to 1 μg/ml of retapamulin. Three of the 12 P. granulosum strains and 3 of the 12 P. avidum isolates were nonsusceptible to tetracycline by EUCAST standards. Fourteen P. acnes isolates were nonsusceptible to clindamycin by EUCAST standards (MIC, ≥0.25 μg/ml), but only three were nonsusceptible by CLSI standards (MIC, ≥4 μg/ml), and all were susceptible to ≤1 μg/ml of retapamulin. Based on EUCAST standards, seven P. acnes isolates were nonsusceptible to erythromycin (MIC, ≥0.5 μg/ml), and 12 P. acnes strains, all 12 P. granulosum strains, and all 12 P. avidum isolates were resistant to TMP-SMX (MIC, ≥1 μg/ml). No CLSI breakpoint standards exist for erythromycin or TMP-SMX for anaerobes. All Propionibacterium isolates tested were susceptible to ≤1 μg/ml of retapamulin.

Oprica et al. (10) studied the prevalence and antibiotic susceptibility patterns of P. acnes isolated from patients with moderate to severe acne in Stockholm, Sweden. They noted that resistant P. acnes strains were recovered in 37% of antimicrobial-treated patients, compared to 13% of nontreated controls. Using the European breakpoints, they found clindamycin and tetracycline resistance in 27% and 15% of 201 isolates, respectively, from treated patients. With CLSI breakpoints, the level would become ∼10% for both groups. Oprica et al. (10) also noted that “a person could become colonized with different strains with various degrees of antibiotic resistance.” Oprica et al. (12) recently noted that the susceptibilities of P. acnes varied between different countries in Europe, “ranging from 83% in Croatia and 60% in Italy to 0% in The Netherlands.” Of the 304 isolates obtained from 13 laboratories in 13 countries, 2.6% were resistant to tetracycline, 15.1% to clindamycin, and 17.1% to erythromycin. In an associated study, Oprica et al. (11) noted “that different resistance genotypes” were distributed throughout Europe. While resistance is clearly present in increasing numbers of patients, a comparison of prevalence rates and susceptibility studies performed in Europe, England, and the United States is difficult. Propionibacteria are generally considered resistant to metronidazole (6). Goldstein et al. (4) studied the comparative activities of four fluoroquinolones (gemifloxacin, levofloxacin, gatifloxacin, and moxifloxacin), two macrolides (azithromycin and clarithromycin), and two beta-lactams (amoxicillin and cefuroxime) against 28 isolates of Propionibacterium species isolated from antral sinus puncture specimens from patients with sinusitis and found that all were susceptible to ≤1 μg/ml of the agents tested.

Retapamulin showed good in vitro activity against the 141 clinical isolates of Propionibacterium species tested, including isolates resistant to erythromycin, TMP-SMX, clindamycin, and tetracycline. Based on this result, SB-275833 merits further consideration and exploration of its clinical utility. This is especially important with current prescribing practices contributing to rising rates of tetracycline and clindamycin resistance.