Abstract
The in vitro activity of retapamulin was determined and compared to that of topical and community antibiotics. The MIC90s of retapamulin against Staphylococcus aureus and Streptococcus pyogenes were 0.12 μg/ml and 0.016 μg/ml, respectively. Retapamulin has a low propensity to select resistance and produces an in vitro postantibiotic effect.
The emergence and spread of antibiotic resistance in hospital and community pathogens has significantly eroded the utility of established antibiotics, a problem that has been widely publicized and poses a serious threat to public health worldwide (6, 8). Novel mechanism antibiotics are needed to address rising resistance to established classes of both systemic and topical agents. Resistance has developed to two of the most commonly used topical antibiotics, fusidic acid and mupirocin, and thus dictates the need for novel mechanism topical agents for managing the treatment of bacterial skin infections.
Retapamulin {mutilin 14-(exo-8-methyl-8-azabicyclo[3.2.1]oct-3-yl-sulfanyl)-acetate} is a novel semisynthetic pleuromutilin that was discovered by GlaxoSmithKline as part of a program to identify novel compounds with the appropriate balance of drug developability and microbiological attributes to be formulated and developed as a topical antibiotic. In this work, we describe the initial microbiology evaluation that led to the selection of retapamulin for development.
The isolates evaluated were obtained from the GlaxoSmithKline culture collection (Collegeville, PA). The panel of methicillin-resistant Staphylococcus aureus isolates (based on oxacillin MICs) was supplemented with 32 isolates obtained from International Health Management Associates (Schaumburg, IL). Broth microdilution, agar dilution, and cidality experiments were performed using the CLSI recommended procedures (3, 4). In the absence of approved quality control limits for retapamulin and specific comparator antibiotics, correlation with previous internal testing of these compounds was used. Modifications were made to the standard broth microdilution method (pH, inoculum density, serum) to identify potential factors that might influence the in vitro activity of retapamulin. Antibiotics were obtained as follows: retapamulin, amoxicillin, and mupirocin were from GlaxoSmithKline Pharmaceuticals, Harlow, United Kingdom; bacitracin, cefaclor, and fusidic acid were from Sigma Chemical Co., St. Louis, MO; levofloxacin and azithromycin were deformulated by GlaxoSmithKline Pharmaceuticals, Harlow, United Kingdom. The postantibiotic effect (PAE) of retapamulin and mupirocin were determined using a filtration method (staphylococci) or a dilution method (Streptococcus pyogenes) as previously described (7). The spontaneous frequencies of resistance in S. aureus and S. pyogenes were determined by plating the test isolate on agar containing 4× and 10× MIC of retapamulin and mupirocin. Detection of resistant colonies was performed 48 h after inoculation. The development of resistance was determined by daily passage of the test isolates in sub-MIC drug concentrations for 10 days. Baseline MICs and subsequent passages were performed by broth macrodilution. The inoculum used for subsequent passages was obtained from the 0.5× MIC macrodilution tube of the previous day.
A summary of the MIC results is shown in Table 1. The MIC90 of retapamulin was 0.12 μg/ml against the S. aureus and Staphylococcus epidermidis isolates. This level of activity was retained against methicillin- and mupirocin-resistant isolates. Retapamulin was very active against the S. pyogenes isolates tested (MIC90 = 0.016 μg/ml), and based on MIC90s, was 32- and >1,024-fold more active than mupirocin and fusidic acid, respectively. The MIC90s (μg/ml) of retapamulin against the other organisms tested were as follows: S. epidermidis, 0.12; Staphylococcus saprophyticus, 0.12; Streptococcus agalactiae, 0.03; viridans group streptococci, 0.12; Streptococcus pneumoniae, 0.12; Haemophilus influenzae, 2; Moraxella catarrhalis, 0.03. Cross-resistance to retapamulin was not observed with any of the clinical isolates or reference strains tested.
TABLE 1.
In vitro antimicrobial activity of retapamulin and other comparator antibiotics against a panel of clinical isolates consisting of staphylococci, streptococci, H. influenzae, and M. catarrhalis
| Organism (no. of isolates tested) | Compound | MIC (μg/ml)
|
||
|---|---|---|---|---|
| 50% | 90% | Range | ||
| S. aureus | ||||
| Methicillin susceptible (100) | Retapamulin | 0.06 | 0.12 | 0.016-0.12 |
| Mupirocin | 0.25 | 0.5 | 0.12->16 | |
| Fusidic acid | 0.25 | 0.5 | 0.12-1 | |
| Bacitracin | >16 | >16 | 4->16 | |
| Cefaclor | 4 | 8 | 0.5-16 | |
| Amoxicillin | 2 | 8 | 0.12-64 | |
| Azithromycin | 1 | >64 | 0.25->64 | |
| Levofloxacin | 0.25 | 0.5 | 0.12-16 | |
| Methicillin resistant (86) | Retapamulin | 0.06 | 0.12 | 0.03-0.25 |
| Mupirocin | 0.25 | 32 | 0.12->64 | |
| Fusidic acid | 0.25 | 0.5 | 0.12-2 | |
| Bacitracin | >16 | >16 | 2->16 | |
| Cefaclor | >64 | >64 | 64->64 | |
| Amoxicillin | 64 | >64 | 8->64 | |
| Azithromycin | >64 | >64 | 1->64 | |
| Levofloxacin | 8 | 32 | 0.12-64 | |
| Methicillin resistant/mupirocin resistant (20) | Retapamulin | 0.06 | 0.12 | 0.03-0.25 |
| Mupirocin | 32 | 32 | 8->64 | |
| Fusidic acid | 0.25 | 1 | 0.12-2 | |
| Bacitracin | >16 | >16 | 2->16 | |
| Cefaclor | >64 | >64 | >64 | |
| Amoxicillin | 64 | >64 | 32->64 | |
| Azithromycin | >64 | >64 | 64->64 | |
| Levofloxacin | 16 | 32 | 4-64 | |
| S. epidermidis (40) | Retapamulin | 0.06 | 0.12 | 0.03-0.12 |
| Mupirocin | 0.25 | >16 | 0.12->16 | |
| Fusidic acid | 0.25 | 0.25 | 0.12->16 | |
| Bacitracin | >16 | >16 | 8->16 | |
| Cefaclor | 1 | 1 | 0.25-8 | |
| Amoxicillin | 0.25 | 0.5 | ≤0.06->64 | |
| Azithromycin | 0.5 | >64 | 0.25->64 | |
| Levofloxacin | 0.25 | 8 | 0.03->16 | |
| S. saprophyticus (28) | Retapamulin | 0.12 | 0.12 | 0.03-2 |
| Mupirocin | 0.25 | 8 | 0.06-16 | |
| Fusidic acid | 4 | 8 | 0.25-8 | |
| Bacitracin | >16 | >16 | 8->16 | |
| Cefaclor | 4 | 4 | 2->64 | |
| Amoxicillin | 0.25 | 0.5 | 0.25-16 | |
| Azithromycin | >64 | >64 | 0.25->64 | |
| Levofloxacin | 0.5 | 0.5 | 0.25-4 | |
| S. pyogenes (101) | Retapamulin | 0.008 | 0.016 | ≤0.004-0.12 |
| Mupirocin | 0.12 | 0.5 | 0.12->16 | |
| Fusidic acid | 16 | 16 | 8-16 | |
| Bacitracin | 1 | 2 | 0.5->16 | |
| Cefaclor | 0.12 | 0.25 | 0.12-4 | |
| Amoxicillin | ≤0.008 | ≤0.008 | ≤0.008-0.5 | |
| Azithromycin | 0.03 | 4 | ≤0.016->16 | |
| Levofloxacin | 0.5 | 2 | 0.25-2 | |
| S. agalactiae (27) | Retapamulin | 0.016 | 0.03 | 0.008-0.03 |
| Mupirocin | 1 | 1 | 0.25-1 | |
| Fusidic acid | 16 | 16 | 16-32 | |
| Bacitracin | >16 | >16 | 2->16 | |
| Cefaclor | 2 | 2 | 0.5-2 | |
| Amoxicillin | 0.06 | 0.06 | 0.03-0.06 | |
| Azithromycin | 0.016 | 2 | 0.016-4 | |
| Levofloxacin | 0.5 | 1 | 0.5-1 | |
| Viridans group streptococci (23) | Retapamulin | 0.03 | 0.12 | 0.008->4 |
| Mupirocin | 1 | 2 | 0.25->16 | |
| Fusidic acid | 32 | 32 | 16->32 | |
| Bacitracin | >16 | >16 | 2->16 | |
| Cefaclor | 4 | >8 | 0.03->8 | |
| Amoxicillin | 0.03 | 8 | ≤0.008->8 | |
| Azithromycin | 1 | >8 | ≤0.008->8 | |
| Levofloxacin | 1 | 2 | 0.25-4 | |
| S. pneumoniae (27) | Retapamulin | 0.06 | 0.12 | 0.03-0.12 |
| Mupirocin | 0.25 | 4 | 0.12-4 | |
| Fusidic acid | 16 | 32 | 4-32 | |
| Bacitracin | 16 | >16 | 0.06->16 | |
| Cefaclor | 1 | >8 | 0.25->8 | |
| Amoxicillin | 0.016 | 2 | ≤0.008-2 | |
| Azithromycin | 0.03 | >8 | 0.016->8 | |
| Levofloxacin | 1 | 1 | 0.5-2 | |
| H. influenzae (27) | Retapamulin | 0.5 | 2 | 0.25-2 |
| Mupirocin | 0.03 | 0.12 | ≤0.016-0.25 | |
| Fusidic acid | 8 | 16 | 2->16 | |
| Bacitracin | >16 | >16 | >16 | |
| Cefaclor | 8 | >16 | 2->16 | |
| Amoxicillin | 0.5 | >64 | 0.25->64 | |
| Azithromycin | 2 | 2 | 1-4 | |
| Levofloxacin | 0.016 | 0.016 | 0.008-0.016 | |
| M. catarrhalis (27) | Retapamulin | 0.3 | 0.3 | 0.016-0.06 |
| Mupirocin | 1 | 2 | 0.5-4 | |
| Fusidic acid | 0.06 | 0.06 | ≤0.03-0.25 | |
| Bacitracin | 2 | 4 | 1-4 | |
| Cefaclor | 1 | 2 | 0.12-4 | |
| Amoxicillin | 2 | 8 | ≤0.008->8 | |
| Azithromycin | 0.016 | 0.03 | ≤0.008-0.03 | |
| Levofloxacin | 0.03 | 0.03 | ≤0.016-0.06 | |
By agar dilution, the MICs for retapamulin were identical to or within a twofold dilution of the broth microdilution MICs (Table 2). At pH 6.0, the MICs of retapamulin generally increased two- to eightfold. In contrast, retapamulin MICs decreased 2- to >4-fold at an alkaline pH of 8.0. The activity of retapamulin was compromised using a high inoculum density (5 × 107 CFU/ml). In the presence of human serum, the in vitro activity of retapamulin increased two- to eightfold, which may have been influenced by pH changes in the broth caused by the addition of serum.
TABLE 2.
Comparison of retapamulin MICs determined under various test conditions
| Isolatea | MIC (μg/ml) determined by:
|
||||||
|---|---|---|---|---|---|---|---|
| Broth microdilution
|
Agar dilution
|
||||||
| CLSIb (pH 7.2) | pH 6.0 | pH 8.0 | 50% serumc | High inoculumd | Mueller-Hinton | 5% Lysed horse blood | |
| S. aureus | |||||||
| SA5 | 0.12 | 0.5 | 0.03 | 0.06 | >16 | 0.12 | 0.25 |
| SA29213 | 0.12 | 0.5 | 0.03 | 0.06 | >16 | 0.06 | 0.25 |
| Methr SA3 | 0.06 | 0.5 | 0.03 | 0.06 | >16 | 0.06 | 0.25 |
| Methr SA7 | 0.06 | 0.5 | 0.03 | 0.06 | 0.5 | 0.06 | 0.25 |
| Mupr SA206 | 0.06 | 0.5 | 0.03 | 0.06 | >16 | 0.06 | 0.12 |
| S. epidermidis | |||||||
| SE1 | 0.12 | 0.5 | 0.03 | 0.03 | 4 | 0.12 | 0.25 |
| SE2 | 0.06 | 0.5 | 0.03 | 0.03 | 8 | 0.06 | 0.12 |
| SE3 | 0.06 | 0.5 | 0.03 | 0.03 | 2 | 0.06 | 0.25 |
| Mupr SE25 | 0.06 | 0.25 | ≤0.016 | ≤0.016 | >16 | 0.06 | 0.12 |
| Mupr SE26 | 0.12 | 0.25 | 0.03 | ≤0.016 | 4 | 0.06 | 0.12 |
| S. pyogenes | |||||||
| SP1 | 0.03 | 0.06 | ≤0.016 | ≤0.016 | 0.12 | NTe | 0.06 |
| SP2 | ≤0.016 | 0.06 | ≤0.016 | ≤0.016 | 0.03 | NT | 0.03 |
| SP3 | 0.03 | 0.06 | ≤0.016 | ≤0.016 | 0.12 | NT | 0.06 |
| SP4 | 0.03 | 0.06 | ≤0.016 | ≤0.016 | 0.5 | NT | 0.06 |
| SP5 | 0.03 | 0.06 | ≤0.016 | ≤0.016 | 0.12 | NT | 0.03 |
Methr, methicillin resistant; Mupr, mupirocin resistant.
CLSI, Clinical and Laboratory Standards Institute.
Due to poor growth, S. pyogenes isolates were tested in the presence of 25% human serum.
The inoculum density was 5 × 107, and trailing endpoints were observed with most of the staphylococci.
NT, not tested.
As shown in Table 3, the minimum bactericidal concentrations (MBCs) of retapamulin and mupirocin were ≥16 μg/ml against the S. aureus isolates, suggesting a bacteriostatic effect. With the exception of a single S. epidermidis isolate (MBC > 16 μg/ml), the bactericidal concentration of retapamulin against S. epidermidis and S. pyogenes ranged from 0.5 to 8 μg/ml. In comparison, mupirocin was bacteriostatic against the S. epidermidis isolates (MBC ≥ 16 μg/ml) and showed cidal activity against S. pyogenes at concentrations ranging from 8 to >16 μg/ml.
TABLE 3.
Minimum inhibitory and bactericidal concentrations of retapamulin and mupirocin against clinical isolates commonly associated with skin and skin structure infections
| Isolatea | Retapamulin
|
Mupirocin
|
||
|---|---|---|---|---|
| MIC (μg/ml) | MBC (μg/ml) | MIC (μg/ml) | MBC (μg/ml) | |
| S. aureus | ||||
| SA5 | 0.12 | 16 | 0.06 | >16 |
| Methr SA3 | 0.06 | >16 | 0.12 | >16 |
| Methr SA7 | 0.06 | >16 | 0.12 | >16 |
| Mupr SA206 | 0.06 | >16 | 16 | >16 |
| SA29213 | 0.12 | >16 | 0.12 | 16 |
| S. epidermidis | ||||
| Sepi 1 | 0.12 | 2 | 0.12 | 16 |
| Sepi 2 | 0.06 | 8 | 0.12 | 16 |
| Sepi 3 | 0.06 | >16 | 0.06 | >16 |
| Mupr Sepi25 | 0.06 | 0.5 | >16 | >16 |
| Mupr Sepi26 | 0.12 | 1 | >16 | >16 |
| S. pyogenes | ||||
| Spy 1 | 0.03 | 4 | 0.25 | >16 |
| Spy 2 | ≤0.016 | 1 | 0.12 | 8 |
| Spy 3 | 0.03 | 0.5 | 0.25 | 8 |
| Spy 4 | 0.03 | 1 | 0.25 | 8 |
| Spy 5 | 0.03 | 4 | 0.12 | 16 |
Methr, methicillin resistant; Mupr, mupirocin resistant.
At 4× MIC, the retapamulin PAEs ranged from 3.1 to 3.4 h and the mupirocin PAEs ranged from 2.2 to 2.9 h against S. aureus (n = 3). Similar values were obtained with the S. pyogenes isolates (n = 4), ranging from 3.5 to 4.2 h for retapamulin and 2.2 to 3.2 h for mupirocin. The frequency of spontaneous resistance to retapamulin in S. aureus WCUH29 and S. pyogenes 257 is low. At 4× and 10× MIC of retapamulin, neither organism produced resistant colonies, indicating that frequencies of spontaneous resistance are <5.0 × 10−9 and <2.6 × 10−9 for these strains, respectively. In comparison, spontaneous resistance to mupirocin was detected at a frequency of 1.0 × 10−8 (S. aureus WCUH29 at 10× MIC) and 2.6 × 10−9 (S. pyogenes 257 at 4× MIC). Following 10 passages in the presence of subinhibitory concentrations, the baseline MICs of retapamulin against S. aureus (n = 3) increased to 0.12 μg/ml, representing only a two- to fourfold increase (Table 4). The mupirocin MICs increased to 4 to >16 μg/ml (8 to >512-fold) from the baseline value. Against S. pyogenes 257, the retapamulin MIC increased from 0.016 to 0.06 μg/ml (4-fold), and the mupirocin MIC increased from 0.03 to 16 μg/ml (512-fold).
TABLE 4.
Antibacterial activity of retapamulin and mupirocin following serial passage of S. aureus and S. pyogenes in subinhibitory concentrations (0.5× MIC) for 10 days
| Isolate | MIC (μg/ml) of drug at time point
|
|||
|---|---|---|---|---|
| Retapamulin
|
Mupirocin
|
|||
| Baseline | 10 passages | Baseline | 10 passages | |
| S. aureus MRSA3a | 0.03 | 0.12 | 0.5 | 4 |
| S. aureus ATCC 29213 | 0.03 | 0.12 | 0.03 | >16 |
| S. aureus WCUH29a | 0.06 | 0.12 | 0.06 | 8 |
| S. pyogenes 257 | 0.016 | 0.06 | 0.03 | 16 |
Methicillin-resistant isolate.
Retapamulin binds to a unique site on the bacterial ribosome, and by virtue of its novel mode of action (9), exhibits excellent in vitro activity against antibiotic-sensitive and -resistant S. aureus and S. pyogenes, two of the most common pathogens associated with skin infections (1, 2). As shown, retapamulin retains excellent in vitro activity against isolates resistant to currently used antibiotics, including mupirocin, β-lactams, macrolides, and quinolones. In addition, in vitro studies show that retapamulin has a low potential to select for resistance in S. aureus and S. pyogenes. Furthermore, retapamulin was found to produce a substantial PAE against S. aureus and S. pyogenes, which may contribute to the efficacy observed with twice-daily application of 1% retapamulin ointment in animal infection models (5).
The excellent in vitro activity observed in these studies led to the selection and development of retapamulin 1% (wt/wt) ointment as a novel topical agent for the treatment of impetigo and secondarily infected traumatic lesions and dermatoses. The significance of these in vitro effects is being evaluated in clinical trials.
Acknowledgments
This work was supported by GlaxoSmithKline Pharmaceutical Company.
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