Antimicrobial Activity of the Investigational Pleuromutilin Compound BC-3781 Tested against Gram-Positive Organisms Commonly Associated with Acute Bacterial Skin and Skin Structure Infections

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The pleuromutilins were discovered as natural antimicrobial agents produced by Pleurotus mutilis in 1951 (7). Tiamulin was the first pleuromutilin compound to be approved for veterinary use, in 1979, followed by valnemulin in 1999. Tiamulin and valnemulin have been used in veterinary medicine mainly for the oral prevention and treatment of swine dysentery (Brachyspira hyodysenteriae) (12). Retapamulin (1% ointment) was the first pleuromutilin approved in 2007 to be used in humans for the topical, short-term treatment of impetigo due to Staphylococcus aureus (oxacillin susceptible [methicillin susceptible] only) and Streptococcus pyogenes and secondarily infected traumatic lesions (Europe only) (6, 17). The antibacterial spectrum of the pleuromutilin class of compounds comprises activity against Gram-positive pathogens, with the exception of Enterococcus faecalis. In addition, pleuromutilins demonstrate activity against Gram-negative organisms, including Haemophilus influenzae, Moraxella catarrhalis, and Legionella pneumophila, as well as pathogens responsible for “atypical” pneumonia, i.e., Chlamydophila pneumoniae and Mycoplasma pneumoniae. This spectrum makes these antimicrobials attractive candidates for systemic treatment of acute bacterial skin and skin structure infections (ABSSSI) and respiratory tract infections (RTI).

BC-3781 is an investigational semisynthetic pleuromutilin derivative developed by Nabriva Therapeutics (12). BC-3781, like other pleuromutilins, interferes with bacterial protein synthesis by binding to the peptidyl transferase center (PTC) of the ribosomal 50S subunit, subsequently preventing the correct positioning of the CCA ends of tRNAs for peptide transfer (8, 13, 15). This mechanism implies a low probability of cross-resistance with other antimicrobials currently used for systemic treatment. BC-3781 has successfully finished a phase II clinical trial in patients with ABSSSI (2, 14) (clinical trials.gov identifier NCT01119105). This study documents the in vitro activity of BC-3781 when tested against a diverse collection of clinical Gram-positive organisms commonly responsible for ABSSSI, including a subset of methicillin-resistant S. aureus (MRSA) strains characterized as belonging to the USA300 and USA400 clones and MRSA strains with defined SCCmec cassette types.

The organism collection evaluated included 1,748 nonduplicate clinical isolates of Gram-positive pathogens collected by the SENTRY Antimicrobial Surveillance Program from documented patient infections in medical centers located in the United States and Europe. The collection also included a subset of 145 molecularly characterized MRSA strains, including USA300 (45 strains) and USA400 (5 strains) and 95 MRSA strains with defined SCCmec cassette types (10, 10, and 75 strains with SCCmec types II, III, and IV, respectively).

Susceptibility testing (MIC) was performed by reference broth microdilution methods as described by Clinical and Laboratory Standards Institute (CLSI) document M07-A8 (3). Quality control (QC) ranges and interpretive criteria for comparator compounds were as published in CLSI document M100-S21 (4), and the EUCAST (2010) breakpoints were also applied (5). Tested QC strains included S. aureus ATCC 29213, Enterococcus faecalis 29212, and Streptococcus pneumoniae ATCC 49619.

SCCmec types were determined using a multiplex PCR approach according to the protocol described by Milheirico et al. (10). S. aureus strains carrying SCCmec types I to VI and S. aureus ATCC 29213 were concurrently tested for QC purposes. For characterization of USA300 and 400 clones, bacterial chromosomal DNA was digested with SmaI and subjected to pulsed-field gel electrophoresis (PFGE) as described by McDougal et al. (9). Gel pattern analysis was performed using the GelCompar II software (Applied Math, Kortrijk, Belgium), and the patterns obtained compared to those of the major USA and international MRSA clones.

BC-3781 was active against S. aureus (MIC_50/90, 0.12/0.25 μg/ml) (Tables 1 and 2) and showed similar activity against methicillin-susceptible S. aureus (MSSA) and MRSA isolates. The highest BC-3781 MIC among S. aureus was 0.5 μg/ml (Table 1). MICs of BC-3781 were lower than those of the comparator agents, especially against MRSA (Table 2). Only 47.7 and 73.2% of S. aureus strains were susceptible to azithromycin (MIC_50/90, 8/>16 μg/ml) and clindamycin (MIC_50/90, 0.12/>16 μg/ml). Linezolid (MIC₅₀ and MIC₉₀, 2 μg/ml) and vancomycin (MIC₅₀ and MIC₉₀, 1 μg/ml) showed complete activity (100.0% susceptible) against S. aureus, but they were 8- to 16-fold less potent than BC-3781 against this organism (Table 2).

Community-associated (CA)-MRSA strains of USA300/400 were also highly susceptible to BC-3781 (MIC₅₀ and MIC₉₀, 0.12 μg/ml) (Table 1). BC-3781 was active against the MRSA strains with SCCmec types II (MIC_50/90, 0.12/0.25 μg/ml), III (MIC₅₀ and MIC₉₀, 0.12 μg/ml), and IV (MIC₅₀ and MIC₉₀, 0.12 μg/ml) (Table 1).

BC-3781 was slightly more active against coagulase-negative staphylococci (CoNS) (MIC_50/90, 0.06/0.12 μg/ml) than S. aureus and showed very similar MIC distributions for oxacillin-susceptible and -resistant CoNS strains (Table 1). Furthermore, MICs of BC-3781 for CoNS were lower than those documented for the comparators azithromycin, clindamycin, and linezolid (Table 2).

β-Hemolytic streptococci were the organisms most susceptible to BC-3781, with MIC_50/90 of 0.03/0.06 μg/ml. Overall, 99.7% of strains were inhibited at ≤0.12 μg/ml (Table 1). Group A (S. pyogenes) and B (S. agalactiae) streptococci showed similar BC-3781 MIC distributions (88.0 to 91.6% inhibited at ≤0.03 μg/ml and highest MIC of 0.12 μg/ml) (Table 1). MICs of BC-3781 were modestly lower (2- to 4-fold) than those of clindamycin (MIC_50/90, 0.06/0.12 μg/ml) and azithromycin (MIC_50/90, 0.12/8 μg/ml) and

16- to 32-fold lower than those of linezolid (MIC₅₀ and MIC₉₀, 1 μg/ml) when tested against β-hemolytic streptococci (Table 2). BC-3781 showed potent activity against viridans group streptococci (MIC_50/90, 0.12/0.5 μg/ml). Against these organisms, BC-3781 was slightly less active than clindamycin (MIC_50/90, 0.03/0.12 μg/ml; 91.7% susceptible), as active as azithromycin against susceptible isolates (MIC_50/90, 0.12/8 μg/ml; 55.0% susceptible), and 8-fold more active than linezolid (MIC_50/90, 1/1 μg/ml; 100.0% susceptible) (Table 2).

BC-3781 was active against E. faecium with 77.6% of isolates being inhibited at ≤1 μg/ml (MIC_50/90, 0.12/16 μg/ml). BC-3781 MICs were lower among vancomycin-nonsusceptible (MIC₅₀, 0.12/2 μg/ml; defined as vancomycin MIC of ≥8 μg/ml) (4) strains than for vancomycin-susceptible strains (MIC_50/90, 0.12/16 μg/ml) (Table 1). Furthermore, MICs of BC-3781 were 8- and 16-fold lower than those of vancomycin (MIC₅₀, 1 μg/ml) and linezolid (MIC₅₀, 2 μg/ml), respectively, when tested against E. faecium. In contrast, BC-3781 exhibited limited activity against E. faecalis (MIC₅₀, >16 μg/ml) and other enterococcal species (MIC_50/90, 4/>16 μg/ml; Table 2).

ABSSSI are very common and include a varied group of anatomically and etiologically different infections. The majority of cases are mild and may be treated with orally administered antimicrobial agents or local care; however, moderate or severe cases of ABSSSI may require hospitalization and parenteral therapy (16). Regardless of site or severity, ABSSSI are predominantly caused by aerobic Gram-positive cocci, particularly S. aureus and β-hemolytic streptococci, and empirical choices of antimicrobials must include agents with activity against these pathogens (16). Among the organisms dominantly associated with ABSSSI, S. aureus is an evolving management challenge for various reasons, including the increasing prevalence of MRSA as the principal pathogen in surgical site infections and the growing importance of CA-MRSA and its contribution to health care-acquired infection, including necrotizing fasciitis and myositis (1, 11). Although CA-MRSA and health care-associated (HA)-MRSA initially had distinct susceptibility patterns, it is becoming more difficult to distinguish between CA-MRSA and HA-MRSA due to the evolution of resistant clones that can be isolated from both of these environmental settings. HA-MRSA is considered to be a serious pathogen among very ill patients and is generally multidrug resistant, while CA-MRSA is recognized as a major cause of ABSSSI, especially in the United States (11).

The results of the present study indicate that BC-3781 is very active against S. aureus, including HA-MRSA and CA-MRSA. The majority (>85%) of S. aureus isolates tested in this investigation were inhibited at ≤0.12 μg/ml, and the highest BC-3781 MIC was only 0.5 μg/ml. The overall potency of BC-3781 against S. aureus was 4- to 16-fold greater than that of linezolid and vancomycin, which represent major treatment options currently available for treatment of complicated ABSSSI. When tested against strains from the USA300/400 clones, the highest BC-3781 MIC was only 0.12 μg/ml. Furthermore, the activity of BC-3781 does not appear to be adversely affected by the SCCmec type. BC-3781 was similarly active against β-hemolytic streptococci. The highest BC-3781 MIC among group A β-hemolytic streptococci (S. pyogenes) was only 0.12 μg/ml, and 99.4% of strains were inhibited at ≤0.06 μg/ml of BC-3781 (MIC₉₀, 0.03 μg/ml).

In summary, BC-3781 demonstrated potent in vitro activity against a large worldwide collection of staphylococci, streptococci, and E. faecium strains, organisms commonly associated with ABSSSI. Toxicological and pharmacokinetic/pharmacodynamic studies and phase II clinical trial results indicate that BC-3781 is a promising adjunct for management of ABSSSI.