JAC Advance Access originally published online on March 9, 2007
Journal of Antimicrobial Chemotherapy 2007 59(4):646-651; doi:10.1093/jac/dkm019
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In vitro activity against anaerobes of retapamulin, a new topical antibiotic for treatment of skin infections
1 Faculty of Pharmacy, 3 rue du Professeur Laguesse, BP83, 59006 Lille Cedex, France 2 Bichat Hospital, Paris, France
* Corresponding author. Tel: +33-3-20-96-40-43; Fax: +33-3-20-95-90-09; E-mail: marie-francoise.odou{at}univ-lille2.fr
Received 13 September 2006; returned 20 November 2006; revised 27 December 2006; accepted 11 January 2007
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Objectives: Retapamulin is the first agent of the pleuromutilin class formulated as a topical antibacterial for treating skin infections. The aim of this study was to determine the antimicrobial activity of retapamulin by determining the minimal inhibitory concentration (MIC) values of this new drug and comparators against a wide range of anaerobic bacteria of human origin.
Methods: The in vitro activity of retapamulin and six comparators (amoxicillin, amoxicillin/clavulanic acid, ceftriaxone, imipenem, clindamycin and metronidazole) was evaluated against 232 anaerobic clinical isolates. MICs were determined by the CLSI reference agar dilution method (M11-A6).
Results: Ceftriaxone, clindamycin and amoxicillin/clavulanic acid resistance rates were 54%, 42% and 9.6%, respectively, within the Bacteroides fragilis group. Despite high resistance rates to various antibiotics, retapamulin inhibited 37/52 (71%) strains of the B. fragilis group and 85/87 (98%) of the other Gram-negative bacilli at a concentration of 2 mg/L or less. All the investigated strains of Clostridium perfringens were inhibited by 1 mg/L retapamulin. Three strains of C. difficile and one strain of C. clostridioforme demonstrated decreased susceptibility to retapamulin. Based on inhibitory concentrations, retapamulin was more active than clindamycin, metronidazole and ceftriaxone against Propionibacterium acnes and anaerobic Gram-positive cocci, as all isolates were inhibited by 
2 mg/L.
Conclusions: At 2 mg/L, retapamulin inhibited 90% of all 232 anaerobes tested, whereas overall resistance rates for the comparators were as follows: co-amoxiclav, 2%; metronidazole, 12%; clindamycin, 15% and ceftriaxone, 20%. The broad anaerobic spectrum demonstrated by retapamulin in vitro is attractive. Pending further clinical investigation, retapamulin may offer an alternative treatment for anaerobic skin infections in this era of increasing resistance.
Keywords: pleuromutilins , skin infections , antianaerobic activity
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Introduction |
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Pleuromutilins are naturally occurring antibiotics extracted from a basidiomycete: Pleurotus mutilus. As results of structure–activity studies, 14-carbamate derivatives have been developed,1 such as tiamulin, an antibiotic for veterinary use, and, more recently, retapamulin (SB-275833), which has been formulated as a topical antibiotic for the treatment of skin infections.
Pleuromutilins exert their antibacterial activity through a unique mode of action. They inhibit bacterial protein synthesis through an interaction with the prokaryotic ribosome.2 They show no target-specific cross-resistance with other antibacterial classes. Therefore, this new class of antibacterial agents could provide a solution in response to the emergence and spread of resistance to existing antibiotics.
Retapamulin shows very good in vitro activity against most of the facultative aerobic organisms involved in skin and soft tissue infections: methicillin-susceptible and methicillin-resistant Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus pyogenes, S. agalactiae, S. pneumoniae, viridans group streptococci, Haemophilus influenzae, Moraxella catarrhalis, Corynebacterium spp. and Micrococcus species.3–5 Its activity against Propionibacterium spp., largely associated with skin infections and especially acne vulgaris, has also been demonstrated by Goldstein et al.6
Antibiotic resistance among anaerobes is increasing worldwide and especially in southern Europe.7–12 Bacteroides fragilis group and Clostridium spp. (endogenous bacteria from the gut) are important pathogens involved in intra-abdominal infections and severe sepsis. They may also be found in decubitus ulcers or skin infections around the sacrum and in diabetic foot ulcers. Propionibacterium, Prevotella, Porphyromonas, Fusobacterium and anaerobic Gram-positive cocci are the predominant anaerobes involved in skin and soft-tissue infections, human and animal bites, ear, nose and throat infections, dental infections and most respiratory infections.
The aim of this study was to determine the antimicrobial activity of retapamulin against a wide range of anaerobic bacteria from human origin by determining the MICs of this new drug compared with reference drugs with known activity against anaerobic clinical isolates.
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Bacterial strains
Strains of anaerobic bacteria were isolated from human clinical samples during the years 2003 and 2004. To be representative of the clinical situation, the panel of strains tested included 52 isolates of the B. fragilis group, 33 Prevotella spp., 25 Fusobacterium spp., 29 Porphyromonas spp., 29 Clostridium spp. (including 14 C. perfringens, 7 C. innocuum, 3 C. difficile, 2 C. paraputrificum, 1 C. sporogenes, 1 C. tertium and 1 C. clostridioforme), 25 P. acnes and 39 anaerobic Gram-positive cocci (including 10 Finegoldia magna, 13 Micromonas micros and 16 other anaerobic Gram-positive cocci). The source of the clinical strains was focused mostly on isolates from skin and soft-tissue infections of patients hospitalized in Bichat hospital (Paris, France) or Dron hospital (Tourcoing, France). A total of 232 clinical anaerobic isolates were assessed for susceptibility testing.
The strains were identified according to standard procedures,13 then subcultured in a Rosenow medium (Bio-Rad, France).
Appropriate reference and control strains were also assessed in the study: B. fragilis ATCC 25285, B. thetaiotaomicron ATCC 29741 and Eggerthella lenta ATCC 43055 as advocated by the CLSI (formerly the NCCLS) for MIC determinations; P. acnes ATCC 6919 and C. perfringens ATCC 13124 were also added in each series of tests as quality control strains.
ß-Lactamase testing was performed with nitrocefin discs on all B. fragilis and Prevotella species.
Retapamulin was obtained from GlaxoSmithKline Pharmaceuticals. The other compounds were obtained from the manufacturers: amoxicillin, amoxicillin/clavulanic acid (2/1), ceftriaxone, imipenem, clindamycin and metronidazole.
Stock solutions of 512 mg/L of each antibiotic were prepared in an appropriate dissolution mix. Retapamulin was dissolved in 1.5 mL of methanol and then distilled water was added to make up the desired stock concentration. Two-fold dilutions were done in distilled water according to Ericsson and Sherris recommendations.14 Each antibiotic was incorporated in Brucella blood agar (Difco, France) with the addition of 5% sterile defibrinated horse blood (Eurobio, France) to provide adequate support for the growth of anaerobes. Plates contained serial doubling dilutions of antimicrobial agents (64–0.06 mg/L for amoxicillin, 64/32–0.06/0.03 mg/L for amoxicillin/clavulanic acid, 128–0.03 mg/L for ceftriaxone, 64–0.03 mg/L for imipenem, 256–0.06 mg/L for clindamycin, 64–0.06 mg/L for metronidazole and 64–0.03 mg/L for retapamulin).
MICs were determined by a reference agar dilution method according to approved M11-A6 standards of the CLSI.15
An actively growing culture in Rosenow medium was diluted in Brucella broth (Difco) to a turbidity equivalent to that of a 0.5 McFarland standard. The inocula were 
7.5 x 107 to 108 cfu/mL. The inocula (2–3 µL) were delivered by a Steers replicator (Mast Systems, London, UK) with a final inoculum of 105 cfu per spot of inoculation onto the agar plates prepared as described above.
At the end of each series of tests, two culture plates of Brucella blood agar were inoculated without antimicrobial agent. One was incubated anaerobically to determine the viability of the organisms and to serve as a control for comparison of growth and the other one was incubated aerobically to indicate possible aerobic contamination. Incubation of the testing plates containing the antibiotics was done in an anaerobic chamber (Don Whitley, AES, France), at 35–36°C. The MIC values were read after 48 h of incubation. The MIC of an antibiotic for an organism is defined as the concentration where a marked reduction occurs in the appearance of growth on the test plate as compared with that of growth on the anaerobic control plate.
The current CLSI breakpoints used to interpret the MIC values are indicated in the legend of the tables. Amoxicillin CLSI breakpoints were used for Gram-negative anaerobes, but as recommended by the CLSI, all ß-lactamase-producing strains were reported as resistant.
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For all the control strains, the MIC values were within the range of the expected values.
Tables 1 and 2 present the range of MICs, MIC50s and MIC90s of retapamulin and the antibiotics used as comparators in addition to the percentage susceptibilities obtained for the comparators for the different groups of anaerobes: B. fragilis group and other Gram-negative bacilli (Table 1); Gram-positive bacilli and Gram-positive cocci (Table 2).
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B. fragilis group
At a concentration of 2 mg/L, retapamulin inhibited 37/52 strains of the B. fragilis group (71%). The resistance rates against B. fragilis of the other antibiotics were: ceftriaxone, 54%; clindamycin, 42% and co-amoxiclav, 10%. One strain of B. fragilis demonstrated intermediate susceptibility to imipenem (MIC = 8 mg/L) and one other demonstrated intermediate susceptibility to metronidazole (MIC = 16 mg/L). No resistance could be detected to either metronidazole or imipenem. All strains of the B. fragilis group were ß-lactamase-positive (tested by nitrocefin). As per CLSI guidelines, these strains were considered resistant to amoxicillin.
At a concentration of 0.25 mg/L, retapamulin inhibited all strains of Prevotella spp. and Porphyromonas spp. Retapamulin inhibited all strains of Fusobacterium nucleatum at a concentration of 2 mg/L. Two multiresistant strains of Fusobacterium varium were inhibited by 8 mg/L retapamulin. ß-Lactamase production was detected among 2/33 Prevotella spp. and 3/25 strains of Fusobacterium spp.; thus, these strains were reported as resistant to amoxicillin. No resistant strains to co-amoxiclav, metronidazole and imipenem among Prevotella spp., Porphyromonas spp. or Fusobacterium spp. could be found; only one strain of Prevotella spp. was resistant to clindamycin. Resistance to ceftriaxone was detected for one strain of Prevotella spp. and four strains of Fusobacterium spp.
Anaerobic Gram-positive bacilli
Among the sporulating anaerobic Gram-positive bacilli, C. perfringens was susceptible to all the antibiotics. At a concentration of 1 mg/L, retapamulin was able to inhibit all tested strains of C. perfringens. Three strains of C. difficile demonstrated decreased susceptibility to retapamulin (MIC from 16 to >64 mg/L). In this study, higher MIC values for retapamulin were observed with one strain of C. clostridioforme (MIC = 64 mg/L). Clindamycin resistance was frequent among Clostridium spp. other than C. perfringens. All strains were susceptible to metronidazole. ß-Lactamase production was not observed among the Clostridium spp. All strains of P. acnes were resistant to metronidazole (intrinsic resistance). No strains were resistant to amoxicillin, co-amoxiclav, clindamycin, ceftriaxone and imipenem. MIC values of retapamulin for P. acnes ranged from 0.015 to 2 mg/L. Retapamulin also had good activity against the reference strain of E. lenta (MIC = 0.25–1 mg/L).
All anaerobic Gram-positive cocci were susceptible to all the ß-lactams. Thirteen percent and 8% of the investigated strains were resistant to clindamycin and metronidazole, respectively. All strains of F. magna were inhibited by 0.125 mg/L or less of retapamulin. M. micros and all other species of anaerobic Gram-positive cocci were inhibited by 2 mg/L or less of retapamulin with the exception of one strain of Ruminococcus spp. (MIC value 16 mg/L).
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B. fragilis group
Retapamulin activity was good against B. fragilis but was less active against the other members of the B. fragilis group, notably B. thetaiotaomicron. This activity was comparable to those of imipenem, clindamycin and metronidazole. All strains of the B. fragilis group were ß-lactamase-positive, thus resistant to amoxicillin and explaining the poor activity of ceftriaxone. Resistance to imipenem, due to carbapenemase production, is associated with cross-resistance to all ß-lactams; it is still rare and was not found in this study. Five strains, susceptible to imipenem but resistant to co-amoxiclav were observed. The resistance mechanism was probably an increased production of ß-lactamase and/or a lack of porin, as we emphasized some years ago.16 Twenty-two of these strains in the B. fragilis group were also resistant to clindamycin. As previously demonstrated, resistance to clindamycin is still increasing in Europe.17,18
Prevotella spp., Porphyromonas spp. and Fusobacterium spp. are Gram-negative bacilli involved in dental, pulmonary, ear, nose and throat infections and soft-tissue infections.19 Antibiotic resistance is emerging in this group of anaerobic bacteria.20 Retapamulin was as potent as co-amoxiclav, imipenem, metronidazole and clindamycin against these bacteria.
Anaerobic Gram-positive bacilli
C. perfringens is generally susceptible to most antibiotics, and this is actually the case in our study. Meanwhile, resistance to antibacterial agents is more common with C. difficile but this organism is rarely isolated from skin infections.21–23 Among the other species, resistance has often been observed among the RIC group (C. ramosum, C. innocuum and C. clostridioforme).24,25 In our study, one strain of C. clostridioforme was susceptible to ß-lactams and metronidazole and resistant to clindamycin. Although retapamulin has a novel mechanism of action and its activity is not affected by resistance to other agents, this isolate also demonstrated an elevated MIC of retapamulin. The activity of retapamulin can be considered comparable to the other antibiotics tested. Goldstein et al.6 showed that retapamulin had a very good in vitro activity against Propionibacterium spp. (MIC90 0.25 mg/L), which is widely associated with acne vulgaris, one of the most common skin inflammatory disorders in humankind. This is in accordance with the results of our study. As Propionibacterium spp. resistance to antibiotics is increasing worldwide,26–28 the good in vitro activity of retapamulin is of great interest for clinical use.
Anaerobic Gram-positive cocci are generally known to be susceptible to all ß-lactams. Some strains are resistant to clindamycin and metronidazole. This was also the case in this study. Retapamulin was more active than clindamycin, metronidazole and ceftriaxone against anaerobic Gram-positive cocci.
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Against Gram-positive anaerobes, retapamulin demonstrated excellent activity. At a concentration of
2 mg/L, retapamulin was able to inhibit 96% of the Gram-positive anaerobes investigated in this study (89/93) and 211/232 of all anaerobes tested (91%) whereas resistance rates for the comparators were as follows: co-amoxiclav, 2%; metronidazole, 12%; clindamycin, 15% and ceftriaxone, 16%. The most resistant strains belonged to B. fragilis group and Clostridium genus. These bacteria are rarely involved in infections of skin and soft tissues in the head and neck regions. The anaerobic bacteria commonly isolated from skin infections are the following: Prevotella spp., Porphyromonas spp., Fusobacterium spp., P. acnes and all anaerobic Gram-positive cocci. Considering these groups of bacteria, a concentration of 2 mg/L retapamulin was able to inhibit 149/151 strains (99%). The in vitro activity of retapamulin seems to indicate that it has potential for the treatment for skin and soft-tissue infections when anaerobes are involved. Moreover, the use of retapamulin topically will induce high local concentrations that would be able to eradicate microorganisms even with increased MICs. These data, along with data from other studies of aerobic Gram-positive organisms and other aerobic pathogens associated most commonly with skin infections, suggest that retapamulin could potentially provide a valuable therapeutic option for the management of skin and skin structure infections. Further clinical studies are needed to assess its usefulness and efficacy in patients. |
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None to declare.
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Acknowledgements |
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This study was supported by a grant from GlaxoSmithKline Pharmaceuticals.
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