Abstract
The in vitro activities of LFF571, a novel analog of GE2270A that inhibits bacterial growth by binding with high affinity for protein synthesis elongation factor Tu, fidaxomicin, and 10 other antimicrobial agents were determined against 50 strains of Clostridium difficile and 630 other anaerobic and aerobic organisms of intestinal origin. LFF571 possesses potent activity against C. difficile and most other Gram-positive anaerobes (MIC90, ≤0.25 μg/ml), with the exception of bifidobacteria and lactobacilli. The MIC90s for aerobes, including enterococci, Staphylococcus aureus (as well as methicillin-resistant S. aureus [MRSA] isolates), Streptococcus pyogenes, and other streptococci were 0.06, 0.125, 2, and 8 μg/ml, respectively. Comparatively, fidaxomicin showed variable activity against Gram-positive organisms: MIC90s against C. difficile, Clostridium perfringens, and Bifidobacterium spp. were 0.5, ≤0.015, and 0.125 μg/ml, respectively, but >32 μg/ml against Clostridium ramosum and Clostridium innocuum. MIC90 for S. pyogenes and other streptococci was 16 and >32 μg/ml, respectively. LFF571 and fidaxomicin were generally less active against Gram-negative anaerobes.
INTRODUCTION
Toxigenic strains of Clostridium difficile are responsible for a spectrum of antibiotic-associated diarrheal diseases (C. difficile infection [CDI]) through elaboration of toxins A and B and other virulence factors (3, 9). In recent years, a hypervirulent strain (NAP-1, 027, BI) has emerged causing more severe disease and higher mortality, especially in more susceptible elderly patients. It is also seen increasingly in outpatients, including pregnant and postpartum women and people without previous antibiotic exposure (24, 25). Current antibiotic therapy for patients with CDI relies heavily on vancomycin or metronidazole, each of which has drawbacks, including treatment failure and frequent recurrence of disease. In addition, decreased susceptibility to metronidazole and vancomycin with emerging resistance to metronidazole (1, 2, 19) has potentiated the therapeutic dilemma. Only one new drug, fidaxomicin, has been developed during the past 30 years (17, 22). Therefore, there is an unmet need for other new drugs for this serious illness.
The current theory of CDI pathogenesis (15) is that the use of antimicrobials leads to unintended changes in the normal gastrointestinal microbiota that leave patients vulnerable to the effects of toxigenic C. difficile. Several strategies have emerged for the prevention and treatment of CDI, which include the use of probiotics (14), the restoration of the protective fecal microbiota (fecal biotherapy) (29), and the development of new agents that are less disruptive to the normal microbiota, especially the anaerobic component.
The thiopeptide LFF571 is a novel analog of the natural product GE2270 A, both of which inhibit bacterial growth by binding with high affinity for protein synthesis elongation factor Tu (10). GE2770 A has demonstrated excellent activity against a variety of Gram-positive organisms (16). In a study characterizing the mechanism of activity of LFF571, there was no evidence of inhibition of other biosynthetic pathways or disruption of bacterial membranes (20). LFF571 inhibits C. difficile in vitro and has proved more efficacious than vancomycin in an experimental hamster model of primary and relapsing C. difficile infection (26).
In order to fully assess LFF571's effect on fecal microbiota, we compared its in vitro activity with that of fidaxomicin, which has been shown to have a lesser effect on the levels of Bacteroides species and the gut microbiota than vancomycin and with 10 other antimicrobial agents against 50 strains of C. difficile and 630 other intestinal aerobic and anaerobic bacterial isolates representing 25 genera and 48 species.
MATERIALS AND METHODS
LFF571 was prepared by Novartis (Basel, Switzerland). Fidaxomicin (lipiarmycin A3) was prepared by fermentation of Catellatospora sp. Bp3323-81 at Novartis and supplied as a reference powder. Other laboratory reference powders were obtained from their manufacturer, USP or Sigma (St. Louis, MO), reconstituted according to the manufacturers' instructions, and stored at −70°C. On the day of testing, a tube of each stock solution was thawed and diluted according to the instructions in CLSI M7 and M11 documents (7, 8).
C. difficile strains were recovered from toxin-positive fecal specimens. The restriction endonuclease analysis (REA) groups included 16 BI, 6 Y, 4 J, 2 G, 2 CF, 1 BK, 1 Z, and 20 nonspecific strains. REA typing was conducted at Dale Gerding's laboratory using the method of Clabots (6). Other organisms representing 25 different genera and 48 species were cultured from clinical samples and identified by standard methods or by partial sequencing of the 16S rRNA gene and stored in 20% skim milk at −70°C (18, 23, 28). Strains were taken from the freezer and subcultured at least twice on supplemented brucella agar for anaerobes and on blood Trypticase soy agar for aerobes to ensure good growth. Anaerobes were incubated for 48 h and aerobes for 24 h prior to testing. Inocula were prepared by direct suspensions of cells into brucella broth for anaerobes or cation-adjusted Mueller-Hinton broth (CAMHB) for aerobes.
Quality control strains included Clostridium difficile ATCC 700057, Bacteroides fragilis ATCC 25285, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and Escherichia coli ATCC 25922 (for the comparator drugs).
For anaerobic organisms, supplemented brucella agar deeps were obtained from Anaerobe Systems (Morgan Hill, CA). Defibrinated sheep blood (Hardy Diagnostics, Santa Maria, CA) was frozen and thawed to produce laked blood. On the day of testing, laked blood and the antimicrobial agents were added to the tubes of molten agar before pouring the agar dilution plates. The strains were applied to the plates using a Steers multipronged inoculator for a final concentration of approximately 105 CFU/spot. After 44 h of incubation at 36°C in the anaerobic chamber incubator, the plates were examined for growth and the MICs interpreted (7).
MIC panels for testing aerobic organisms were prepared in-house using the Quick-Spense apparatus (Sandy Spring Instrument Co. Inc., Germantown, MD) at double antimicrobial strength, 50 μl/well, using CAMHB, and stored at −70°C until used. Tests for Streptococcus, Lactococcus, Leuconostoc, Pediococcus, and Aerococcus strains were supplemented with 2.5% lysed horse blood (LHB; Hardy Diagnostics) by adding 5% LHB to the inoculum tube and then adding 50 μl of inoculum to each well for a final concentration of ∼5× 105 CFU/ml. Panels were incubated for 20 h at 35°C before reading and interpreting the MICs (8).
RESULTS AND DISCUSSION
Table 1 summarizes and compares the activities of LFF571 and fidaxomicin against the major groups of organisms. Table 2 shows the ranges, MIC50/0.90, and percent resistance for all antimicrobial agents. Overall, LFF571 had excellent activity against the 50 C. difficile strains studied (MIC90, 0.25 μg/ml), which was one dilution lower than that of fidaxomicin (MIC90, 0.5 μg/ml) and three dilutions lower than both vancomycin and metronidazole (MIC90s, 2 μg/ml).
Table 1.
Summary of the in vitro activities (μg/ml) of LFF571 and fidaxomicin against diverse species of anaerobic and aerobic bacteriaa
| Organism/group | No. tested | LFF571 |
Fidaxomicin |
||
|---|---|---|---|---|---|
| Range | MIC50/90 | Range | MIC50/90 | ||
| Gram-positive anaerobes | |||||
| C. difficile | 50 | 0.125–0.5 | 0.25/0.25 | 0.03–0.5 | 0.25/0.5 |
| C. perfringens | 20 | ≤0.015–0.25 | 0.03/0.03 | ≤0.015–0.03 | <0.015/<0.015 |
| C. innocuum | 20 | 0.125–0.25 | 0.125–0.25 | >32 | >32–>32 |
| C. ramosum | 20 | ≤0.015–0.03 | ≤0.015/<0.015 | >32 | >32–>32 |
| Lactobacillus speciesb | 24 | 0.06–>32 | 2/>32 | ≤0.015–>32 | 8/32 |
| Bifidobacterium speciesc | 22 | >32 | >32/>32 | ≤0.015–0.125 | 0.06/0.125 |
| Eggerthella lenta | 20 | ≤0.015–0.06 | 0.03/0.06 | ≤0.015–0.25 | <0.015/0.125 |
| Eubacterium limosum | 20 | 0.06–0.25 | 0.125/0.25 | 16–>32 | 32/>32 |
| Eubacterium groupd | 28 | ≤0.015–1 | 0.125/0.5 | 0.125–>32 | 2/>32 |
| Finegoldia magna | 20 | ≤0.015–0.25 | 0.125–0.125 | 0.5–2 | 1/2 |
| Parvimonas micra | 20 | ≤0.015–0.25 | 0.125–0.125 | ≤0.015–2 | 0.06–0.06 |
| Peptostreptococcus anaerobius groupe | 20 | ≤0.03–0.06 | 0.06/0.06 | ≤0.015–0.03 | <0.015/<0.015 |
| Gram-negative anaerobes | |||||
| B. fragilis | 21 | 4–8 | 4/8 | >32 | >32/>32 |
| B. ovatus | 20 | 4–>32 | 16/>32 | >32 | >32/>32 |
| B. thetaiotaomicron | 20 | 4–>32 | 32/>32 | >32 | >32/>32 |
| B. vulgatus | 20 | 4–>32 | 16/32 | >32 | >32/>32 |
| P. distasonis | 20 | 4–>32 | 16/32 | >32 | >32/>32 |
| P. bivia | 20 | 0.5–>32 | 4/>32 | >32 | >32/>32 |
| P. melaninogenica groupf | 21 | 8–>32 | >32–>32 | >32 | >32/>32 |
| Porphyromonas uenonis | 20 | 0.06–0.25 | 0.125/0.25 | 8–>32 | >32/>32 |
| Porphyromonas asaccharolytica | 20 | 0.06–0.25 | 0.125/0.25 | 8–>32 | 32/>32 |
| Fusobacterium nucleatum | 22 | 2–>32 | 16/32 | 0.125–>32 | >32/>32 |
| F. mortiferum-varium groupg | 20 | >32 | >32/>32 | >32 | >32/>32 |
| Veillonella species | 20 | >32 | >32/>32 | 16–>32 | 32/>32 |
| Aerobic organisms | |||||
| Aerococcus speciesh | 10 | 0.06–1 | 0.5/1 | 0.5–16 | 2/2 |
| E. faecalis | 22 | ≤0.015–0.06 | 0.03/0.03 | 0.5–4 | 2/4 |
| E. faecium | 20 | ≤0.015–0.06 | 0.03/0.06 | 2–8 | 4/4 |
| S. pyogenes | 21 | 0.5–4 | 1/2 | 4–16 | 8/8 |
| Streptococcus anginosus | 21 | 1–16 | 2/8 | 4–>32 | 32/>32 |
| Streptococcus constellatus/intermediusi | 26 | 1–32 | 2/8 | 4–>32 | 32/>32 |
| Staphylococcus aureus | 20 | 0.125–0.25 | 0.125/0.125 | 4–8 | 8/8 |
| Unusual Gram-positive coccij | 12 | 8–>32 | 32/>32 | 2–>32 | >32/>32 |
Anaerobic organisms were tested by the agar dilution method; aerobic organisms were tested by broth microdilution (7, 8).
Lactobacillus antri (1), L. casei (6), L. catenaformis (4), L. crispatus (1), L. gasseri (4), L. reuteri (1), L. rhamnosus (6), and L. salivarius (1).
Bifidobacterium adolescentis (2), B. bifidum (4), B. breve (4), B. dentium (5), B. longum (5), and B. pseudocatenulatum (2).
Table 2.
In vitro activities (μg/ml) of LFF571 and comparator antimicrobial agents against C. difficile and other intestinal organismsa
| Organism (no. tested)/antimicrobial agents | MIC (μg/ml) |
Percent resistant | ||
|---|---|---|---|---|
| Range | 50% | 90% | ||
| Gram-positive anaerobes | ||||
| Clostridium difficile (50) | ||||
| LFF571 | 0.125–0.5 | 0.25 | 0.25 | NAk |
| Fidaxomicin | 0.03–0.5 | 0.25 | 0.5 | NA |
| Vancomycin | 1–4 | 1 | 2 | 0 |
| Imipenem | 4–16 | 8 | 16 | 18 |
| Piperacillin-tazobactam | 2–16 | 8 | 16 | 0 |
| Ampicillin | 1–2 | 2 | 2 | 0 |
| Ampicillin-sulbactam | 0.5–2 | 2 | 2 | 0 |
| Cefoxitin | >64 | >64 | >64 | 100 |
| Ceftriaxone | 32–>64 | 64 | 64 | 50 |
| Metronidazole | 0.25–4 | 1 | 2 | 0 |
| Clindamycin | 1–>128 | 8 | 8 | 66 |
| Moxifloxacin | 1–>32 | 2 | 16 | 26 |
| Clostridium perfringens (20) | ||||
| LFF571 | ≤0.015–0.25 | 0.03 | 0.03 | NA |
| Fidaxomicin | ≤0.015–0.03 | ≤0.015 | ≤0.015 | NA |
| Vancomycin | 0.5–1 | 0.5 | 1 | 0 |
| Imipenem | 0.06–0.5 | 0.125 | 0.5 | 0 |
| Piperacillin-tazobactam | ≤0.06–1 | ≤0.06 | 0.5 | 0 |
| Ampicillin | ≤0.06–1 | 0.125 | 0.5 | 0 |
| Ampicillin-sulbactam | ≤0.06–1 | 0.125 | 0.5 | 0 |
| Cefoxitin | 0.5–2 | 1 | 2 | 0 |
| Ceftriaxone | ≤0.06–4 | 0.25 | 1 | 0 |
| Metronidazole | 0.5–2 | 1 | 2 | 0 |
| Clindamycin | ≤0.06–2 | 1 | 2 | 0 |
| Moxifloxacin | 0.25–8 | 0.5 | 0.5 | 5 |
| Clostridium innocuum (20) | ||||
| LFF571 | 0.125–0.25 | 0.125 | 0.25 | NA |
| Fidaxomicin | >32 | >32 | >32 | NA |
| Vancomycin | 4–16 | 16 | 16 | 55 |
| Imipenem | 1–8 | 4 | 8 | 0 |
| Piperacillin-tazobactam | 0.5–4 | 1 | 2 | 0 |
| Ampicillin | ≤0.06–1 | 0.25 | 1 | 0 |
| Ampicillin-sulbactam | ≤0.06–1 | 0.25 | 1 | 0 |
| Cefoxitin | 64–>64 | >64 | >64 | 100 |
| Ceftriaxone | 8–32 | 16 | 32 | 0 |
| Metronidazole | 0.125–2 | 1 | 1 | 0 |
| Clindamycin | 0.125–>128 | 1 | >128 | 15 |
| Moxifloxacin | 1–>32 | 2 | 8 | 20 |
| Clostridium ramosum (20) | ||||
| LFF571 | ≤0.015–0.03 | ≤0.015 | ≤0.015 | NA |
| Fidaxomicin | >32 | >32 | >32 | NA |
| Vancomycin | 2–4 | 4 | 4 | 0 |
| Imipenem | 0.25–1 | 1 | 1 | 0 |
| Piperacillin-tazobactam | ≤0.06–2 | 0.125 | 0.25 | 0 |
| Ampicillin | 0.125–2 | 0.25 | 1 | 0 |
| Ampicillin-sulbactam | 0.125–2 | 0.5 | 1 | 0 |
| Cefoxitin | 1–>64 | 16 | 16 | 5 |
| Ceftriaxone | 0.25–0.5 | 0.25 | 0.5 | 0 |
| Metronidazole | 0.25–1 | 0.5 | 1 | 0 |
| Clindamycin | 2–>128 | 4 | 8 | 10 |
| Moxifloxacin | 1–16 | 4 | 4 | 5 |
| Lactobacillus speciesb (24) | ||||
| LFF571 | 0.06–>32 | 2 | >32 | NA |
| Fidaxomicin | ≤0.015 > 32 | 8 | 32 | NA |
| Vancomycin | ≤0.25–>64 | >64 | >64 | 63 |
| Imipenem | ≤0.25–4 | <0.25 | 2 | 0 |
| Piperacillin-tazobactam | ≤0.06–16 | 1 | 4 | 0 |
| Ampicillin | ≤0.06–4 | 0.5 | 2 | 0 |
| Ampicillin-sulbactam | ≤0.06–4 | 0.5 | 2 | 0 |
| Cefoxitin | 0.25–>32 | >32 | >32 | 67 |
| Ceftriaxone | ≤0.06–>32 | 8 | >32 | 50 |
| Metronidazole | 0.125–>64 | >64 | >64 | 83 |
| Clindamycin | ≤0.06–4 | 0.125 | 2 | 0 |
| Moxifloxacin | 0.125–8 | 0.25 | 1 | 4 |
| Bifidobacterium speciesc (22) | ||||
| LFF571 | >32 | >32 | >32 | NA |
| Fidaxomicin | ≤0.015–0.125 | 0.06 | 0.125 | NA |
| Vancomycin | 0.25–1 | 0.5 | 1 | 0 |
| Imipenem | ≤0.03–0.25 | ≤0.03 | 0.25 | 0 |
| Piperacillin-tazobactam | ≤0.06–1 | ≤0.06 | 0.125 | 0 |
| Ampicillin | ≤0.06–1 | ≤0.06 | 0.25 | 0 |
| Ampicillin-sulbactam | ≤0.06–1 | ≤0.06 | 0.125 | 0 |
| Cefoxitin | ≤0.06–32 | 1 | 4 | 0 |
| Ceftriaxone | ≤0.06–4 | 0.125 | 0.5 | 0 |
| Metronidazole | 1–>64 | 8 | >64 | 36 |
| Clindamycin | ≤0.06–0.125 | ≤0.06 | ≤0.06 | 0 |
| Moxifloxacin | 0.25–8 | 0.5 | 1 | 5 |
| Eggerthella lenta (20) | ||||
| LFF571 | ≤0.015–0.06 | 0.03 | 0.06 | NA |
| Fidaxomicin | ≤0.015–0.25 | ≤0.015 | 0.125 | NA |
| Vancomycin | 0.5–2 | 2 | 2 | 0 |
| Imipenem | ≤0.03–1 | 1 | 1 | 0 |
| Piperacillin-tazobactam | 0.5–32 | 16 | 32 | 0 |
| Ampicillin | 0.5–8 | 4 | 4 | 75 |
| Ampicillin-sulbactam | 0.5–8 | 4 | 4 | 80 |
| Cefoxitin | 8–32 | 16 | 16 | 0 |
| Ceftriaxone | 0.5–>64 | >64 | >64 | 95 |
| Metronidazole | 0.25–1 | 0.5 | 0.5 | 0 |
| Clindamycin | ≤0.06–0.5 | 0.25 | 0.5 | 0 |
| Moxifloxacin | 0.125–8 | 0.5 | 2 | 5 |
| Eubacterium limosum (20) | ||||
| LFF571 | 0.06–0.25 | 0.125 | 0.25 | NA |
| Fidaxomicin | 16–>32 | 32 | >32 | NA |
| Vancomycin | 2–4 | 2 | 2 | 0 |
| Imipenem | 0.06–0.125 | 0.06 | 0.06 | 0 |
| Piperacillin-tazobactam | ≤0.06–0.5 | 0.25 | 0.5 | 0 |
| Ampicillin | 0.125–0.25 | 0.25 | 0.25 | 0 |
| Ampicillin-sulbactam | ≤0.06–0.5 | 0.25 | 0.5 | 0 |
| Cefoxitin | 0.5–4 | 1 | 4 | 0 |
| Ceftriaxone | ≤0.06–1 | 0.5 | 1 | 0 |
| Metronidazole | 0.125–1 | 0.25 | 1 | 0 |
| Clindamycin | ≤0.06–64 | 1 | 2 | 7 |
| Moxifloxacin | 1–2 | 1 | 2 | 0 |
| Eubacterium groupd (28) | ||||
| LFF571 | ≤0.015–1 | 0.125 | 0.5 | NA |
| Fidaxomicin | 0.125–>32 | 2 | >32 | NA |
| Vancomycin | 0.25–4 | 0.5 | 1 | 0 |
| Imipenem | ≤0.03–0.5 | ≤0.03 | 0.06 | 0 |
| Piperacillin-tazobactam | ≤0.06–2 | 0.25 | 1 | 0 |
| Ampicillin | ≤0.06–1 | ≤0.06 | 0.5 | 0 |
| Ampicillin-sulbactam | ≤0.06-1 | 0.125 | 1 | 0 |
| Cefoxitin | ≤0.06–16 | 4 | 16 | 0 |
| Ceftriaxone | ≤0.06–8 | 0.5 | 4 | 0 |
| Metronidazole | ≤0.06–2 | 0.5 | 2 | 0 |
| Clindamycin | ≤0.06–>128 | ≤0.06 | 1 | 4 |
| Moxifloxacin | 0.125–1 | 0.25 | 1 | 0 |
| Finegoldia magna (20) | ||||
| LFF571 | ≤0.015–0.25 | 0.125 | 0.125 | NA |
| Fidaxomicin | 0.5–2 | 1 | 2 | NA |
| Vancomycin | 0.125–1 | 0.25 | 0.5 | 0 |
| Imipenem | ≤0.03–0.25 | 0.06 | 0.125 | 0 |
| Piperacillin-tazobactam | ≤0.06–1 | 0.125 | 0.25 | 0 |
| Ampicillin | ≤0.06–0.5 | 0.25 | 0.5 | 0 |
| Ampicillin-sulbactam | ≤0.06–0.5 | 0.25 | 0.25 | 0 |
| Cefoxitin | ≤0.06–1 | 0.5 | 1 | 0 |
| Ceftriaxone | 0.125–16 | 4 | 8 | 0 |
| Metronidazole | ≤0.06–2 | 0.5 | 1 | 0 |
| Clindamycin | ≤0.06–>128 | 0.5 | >128 | 25 |
| Moxifloxacin | 0.06–16 | 2 | 4 | 5 |
| Parvimonas micra (20) | ||||
| LFF571 | ≤0.015–0.25 | 0.125 | 0.125 | NA |
| Fidaxomicin | ≤0.015–2 | 0.06 | 0.06 | NA |
| Vancomycin | 0.5–1 | 1 | 1 | 0 |
| Imipenem | ≤0.03–0.25 | ≤0.03 | 0.06 | 0 |
| Piperacillin-tazobactam | ≤0.06–1 | ≤0.06 | 0.125 | 0 |
| Ampicillin | ≤0.06–2 | 0.125 | 1 | 0 |
| Ampicillin-sulbactam | ≤0.06–1 | 0.125 | 0.5 | 0 |
| Cefoxitin | 0.5–8 | 1 | 2 | 0 |
| Ceftriaxone | 0.125–2 | 0.25 | 1 | 0 |
| Metronidazole | 0.125–1 | 0.25 | 0.5 | 0 |
| Clindamycin | ≤0.06–32 | 0.25 | 16 | 15 |
| Moxifloxacin | 0.125–32 | 0.25 | 16 | 15 |
| P. anaerobius/stomatis groupe (20) | ||||
| LFF571 | ≤0.015–0.06 | 0.06 | 0.06 | NA |
| Fidaxomicin | ≤0.015–0.03 | ≤0.015 | ≤0.015 | NA |
| Vancomycin | 0.125–0.5 | 0.25 | 0.5 | 0 |
| Imipenem | ≤0.03–2 | 0.06 | 1 | 0 |
| Piperacillin-tazobactam | 0.25–16 | 0.25 | 16 | 0 |
| Ampicillin | ≤0.06–32 | 0.125 | 8 | 20 |
| Ampicillin-sulbactam | ≤0.06–32 | 0.125 | 16 | 20 |
| Cefoxitin | 0.25–16 | 0.5 | 16 | 0 |
| Ceftriaxone | 0.5–16 | 2 | 16 | 0 |
| Metronidazole | ≤0.06–1 | 0.25 | 1 | 0 |
| Clindamycin | ≤0.06–0.5 | ≤0.06 | 0.25 | 0 |
| Moxifloxacin | 0.125–8 | 0.125 | 0.25 | 0 |
| Gram-negative anaerobes | ||||
| Bacteroides fragilis (21) | ||||
| LFF571 | 4–8 | 4 | 8 | NA |
| Fidaxomicin | >32 | >32 | >32 | NA |
| Vancomycin | 32–>32 | 32 | 32 | 100 |
| Imipenem | ≤0.03–>32 | 0.06 | 4 | 5 |
| Piperacillin-tazobactam | 0.25–>64 | 1 | 64 | 10 |
| Ampicillin | 16–>64 | 32 | >64 | 100 |
| Ampicillin-sulbactam | 1–>64 | 2 | 32 | 19 |
| Cefoxitin | 8–>64 | 16 | 32 | 10 |
| Ceftriaxone | 4–>64 | 64 | >64 | 62 |
| Metronidazole | 0.25–>64 | 1 | 1 | 5 |
| Clindamycin | ≤0.06–>128 | 0.5 | >128 | 14 |
| Moxifloxacin | 0.5–8 | 1 | 8 | 38 |
| B. ovatus (20) | ||||
| LFF571 | 4–>32 | 16 | >32 | NA |
| Fidaxomicin | >32 | >32 | >32 | NA |
| Vancomycin | >32 | >32 | >32 | 100 |
| Imipenem | 0.06–2 | 0.125 | 0.25 | 0 |
| Piperacillin-tazobactam | 2–>64 | 4 | 16 | 5 |
| Ampicillin | 8–>64 | 32 | >64 | 100 |
| Ampicillin-sulbactam | 0.5–32 | 1 | 8 | 5 |
| Cefoxitin | 8–>64 | 32 | 32 | 10 |
| Ceftriaxone | 4–>64 | >64 | >64 | 95 |
| Metronidazole | 0.25–2 | 1 | 1 | 0 |
| Clindamycin | 1–>128 | 2 | >128 | 25 |
| Moxifloxacin | 1–>32 | 2 | 32 | 20 |
| B. thetaiotaomicron (20) | ||||
| LFF571 | 4–>32 | >32 | >32 | NA |
| Fidaxomicin | >32 | >32 | >32 | NA |
| Vancomycin | 32–>32 | >32 | >32 | 100 |
| Imipenem | 0.125–2 | 0.25 | 0.5 | 0 |
| Piperacillin-tazobactam | 8–>64 | 16 | 32 | 5 |
| Ampicillin | 16–>64 | >32 | >64 | 100 |
| Ampicillin-sulbactam | 1–16 | 2 | 8 | 0 |
| Cefoxitin | 16–>64 | 32 | 64 | 5 |
| Ceftriaxone | >64 | >64 | >64 | 100 |
| Metronidazole | 0.5–2 | 1 | 2 | 0 |
| Clindamycin | 0.5–>128 | 4 | >128 | 45 |
| Moxifloxacin | 1–>32 | 2 | 32 | 20 |
| B. vulgatus (20) | ||||
| LFF571 | 4–>32 | 16 | >32 | NA |
| Fidaxomicin | >32 | >32 | >32 | NA |
| Vancomycin | 16–>32 | 32 | >32 | 100 |
| Imipenem | 0.06–8 | 0.25 | 1 | 0 |
| Piperacillin-tazobactam | ≤0.06–32 | 4 | 16 | 0 |
| Ampicillin | 4–>64 | >64 | >64 | 100 |
| Ampicillin-sulbactam | 1–32 | 8 | 16 | 5 |
| Cefoxitin | 4–>64 | 8 | 32 | 5 |
| Ceftriaxone | 1–>64 | 64 | >64 | 50 |
| Metronidazole | 0.25–4 | 0.5 | 1 | 0 |
| Clindamycin | ≤0.06–>128 | 1 | >128 | 45 |
| Moxifloxacin | 0.5–>32 | 1 | 32 | 45 |
| Parabacteroides distasonis (20) | ||||
| LFF571 | 4–>32 | 16 | 32 | NA |
| Fidaxomicin | >32 | >32 | >32 | NA |
| Vancomycin | 32–>32 | >32 | >32 | 100 |
| Imipenem | 0.125–1 | 0.5 | 0.5 | 0 |
| Piperacillin-tazobactam | 2–32 | 4 | 32 | 0 |
| Ampicillin | 2–>64 | 4 | >64 | 100 |
| Ampicillin-sulbactam | 2–32 | 4 | 32 | 15 |
| Cefoxitin | 8–64 | 16 | 32 | 10 |
| Ceftriaxone | 0.25–>64 | 8 | >64 | 45 |
| Metronidazole | 0.5–8 | 2 | 4 | 0 |
| Clindamycin | ≤0.06–>128 | 4 | >128 | 45 |
| Moxifloxacin | 0.125–32 | 0.5 | 16 | 30 |
| Prevotella bivia (20) | ||||
| LFF571 | 0.5–>32 | 4 | >32 | NA |
| Fidaxomicin | >32 | >32 | >32 | NA |
| Vancomycin | >64 | >64 | >64 | 100 |
| Imipenem | ≤0.03–0.06 | ≤0.03 | 0.06 | 0 |
| Piperacillin-tazobactam | ≤0.06 | ≤0.06 | ≤0.06 | 0 |
| Ampicillin | ≤0.06–>64 | 8 | 32 | 60 |
| Ampicillin-sulbactam | ≤0.06–4 | 1 | 2 | 0 |
| Cefoxitin | 0.25–2 | 0.5 | 1 | 0 |
| Ceftriaxone | ≤0.06–64 | 8 | 32 | 10 |
| Metronidazole | 0.25–4 | 1 | 4 | 0 |
| Clindamycin | ≤0.06–>128 | ≤0.06 | >128 | 30 |
| Moxifloxacin | 1–>32 | 2 | 32 | 15 |
| P. melaninogenica groupf (21) | ||||
| LFF571 | 8–>32 | >32 | >32 | NA |
| Fidaxomicin | >32 | >32 | >32 | NA |
| Vancomycin | >64 | >64 | >64 | 100 |
| Imipenem | ≤0.03–0.06 | ≤0.03 | 0.06 | 0 |
| Piperacillin-tazobactam | ≤0.06–0.125 | ≤0.06 | ≤0.06 | 0 |
| Ampicillin | ≤0.06–64 | 1 | 32 | 38 |
| Ampicillin-sulbactam | ≤0.06–2 | 0.25 | 2 | 0 |
| Cefoxitin | ≤0.06–4 | 0.5 | 2 | 0 |
| Ceftriaxone | ≤0.06–64 | 2 | 64 | 14 |
| Metronidazole | ≤0.06–2 | 0.25 | 1 | 0 |
| Clindamycin | ≤0.06–>128 | ≤0.06 | ≤0.06 | 10 |
| Moxifloxacin | 0.5–32 | 0.5 | 32 | 19 |
| Porphyromonas asaccharolytica (20) | ||||
| LFF571 | 0.06–0.25 | 0.125 | 0.25 | NA |
| Fidaxomicin | 8–>32 | >32 | >32 | NA |
| Vancomycin | 0.5–2 | 1 | 2 | 0 |
| Imipenem | ≤0.03 | ≤0.03 | ≤0.03 | 0 |
| Piperacillin-tazobactam | ≤0.06–0.125 | ≤0.06 | ≤0.06 | 0 |
| Ampicillin | ≤0.06–0.25 | ≤0.06 | ≤0.06 | 0 |
| Ampicillin-sulbactam | ≤0.06 | ≤0.06 | ≤0.06 | 0 |
| Cefoxitin | ≤0.06–0.5 | 0.125 | 0.25 | 0 |
| Ceftriaxone | ≤0.06–0.25 | ≤0.06 | ≤0.06 | 0 |
| Metronidazole | ≤0.06–0.125 | ≤0.06 | ≤0.06 | 0 |
| Clindamycin | ≤0.06–64 | ≤0.06 | 64 | 25 |
| Moxifloxacin | 0.125–8 | 0.25 | 1 | 5 |
| P. uenonis (20) | ||||
| LFF571 | 0.06–0.25 | 0.125 | 0.25 | NA |
| Fidaxomicin | 8–>32 | >32 | >32 | NA |
| Vancomycin | 0.5–4 | 2 | 2 | 0 |
| Imipenem | ≤0.03 | ≤0.03 | ≤0.03 | 0 |
| Piperacillin-tazobactam | ≤0.06 | ≤0.06 | ≤0.06 | 0 |
| Ampicillin | ≤0.06–2 | ≤0.06 | ≤0.06 | 5 |
| Ampicillin-sulbactam | ≤0.06–0.25 | ≤0.06 | ≤0.06 | 0 |
| Cefoxitin | ≤0.06–2 | 0.125 | 0.25 | 0 |
| Ceftriaxone | ≤0.06–8 | ≤0.06 | 0.25 | 0 |
| Metronidazole | ≤0.06 | ≤0.06 | ≤0.06 | 0 |
| Clindamycin | ≤0.06 | ≤0.06 | ≤0.06 | 0 |
| Moxifloxacin | 0.125–0.5 | 0.5 | 0.5 | 0 |
| Fusobacterium nucleatum (22) | ||||
| LFF571 | 2–>32 | 16 | 32 | NA |
| Fidaxomicin | 0.125–>32 | >32 | >32 | NA |
| Vancomycin | 32–>32 | >32 | >32 | 100 |
| Imipenem | ≤0.03–≤0.03 | ≤0.03 | ≤0.03 | 0 |
| Piperacillin-tazobactam | ≤0.06 | ≤0.06 | ≤0.06 | 0 |
| Ampicillin | ≤0.06 | ≤0.06 | ≤0.06 | 0 |
| Ampicillin-sulbactam | ≤0.06 | ≤0.06 | ≤0.06 | 0 |
| Cefoxitin | ≤0.06–0.25 | 0.125 | 0.25 | 0 |
| Ceftriaxone | ≤0.06–0.125 | ≤0.06 | 0.125 | 0 |
| Metronidazole | ≤0.06–1 | ≤0.06 | 0.5 | 0 |
| Clindamycin | ≤0.06–0.25 | ≤0.06 | 0.125 | 0 |
| Moxifloxacin | ≤0.06–16 | 0.125 | 2 | 11 |
| F. mortiferum/varium groupg (20) | ||||
| LFF571 | >32 | >32 | >32 | NA |
| Fidaxomicin | >32 | >32 | >32 | NA |
| Vancomycin | >32 | >32 | >32 | 100 |
| Imipenem | 0.06–1 | 1 | 1 | 0 |
| Piperacillin-tazobactam | 0.125–4 | 0.25 | 4 | 0 |
| Ampicillin | 0.125–4 | 1 | 4 | 25 |
| Ampicillin-sulbactam | 0.125–4 | 1 | 4 | 0 |
| Cefoxitin | 0.25–8 | 4 | 8 | 0 |
| Ceftriaxone | ≤0.06–>64 | 16 | >64 | 50 |
| Metronidazole | ≤0.06–2 | 1 | 2 | 0 |
| Clindamycin | ≤0.06–32 | 0.125 | 4 | 5 |
| Moxifloxacin | 0.5–>32 | 0.5 | 16 | 20 |
| Veillonella species (20) | ||||
| LFF571 | >32 | >32 | >32 | NA |
| Fidaxomicin | 16–>32 | 32 | >32 | NA |
| Vancomycin | 64–>64 | >64 | >64 | 100 |
| Imipenem | 0.06–1 | 0.5 | 1 | 0 |
| Piperacillin-tazobactam | 4–>64 | 64 | >64 | 35 |
| Ampicillin | 0.25–2 | 0.5 | 2 | 25 |
| Ampicillin-sulbactam | 0.125–2 | 0.5 | 2 | 0 |
| Cefoxitin | 0.5–16 | 4 | 16 | 0 |
| Ceftriaxone | 0.25–8 | 4 | 8 | 0 |
| Metronidazole | 0.25–8 | 2 | 4 | 0 |
| Clindamycin | ≤0.06–128 | ≤0.06 | 0.25 | 5 |
| Moxifloxacin | 0.06–>32 | 0.5 | 4 | 10 |
| Gram-positive aerobes | ||||
| Aerococcus speciesh (10) | ||||
| LFF571 | 0.06–1 | 0.5 | 1 | NA |
| Fidaxomicin | 0.5–16 | 2 | 2 | NA |
| Vancomycin | ≤0.25–0.5 | ≤0.25 | ≤0.25 | 100 |
| Imipenem | ≤0.25–<0.25 | ≤0.25 | ≤0.25 | 100 |
| Piperacillin-tazobactam | ≤0.25–4 | 2 | 4 | 100 |
| Ampicillin | ≤0.125–<0.125 | ≤0.125 | ≤0.125 | 100 |
| Ampicillin-sulbactam | ≤0.125–<0.125 | ≤0.125 | ≤0.125 | 100 |
| Cefoxitin | ≤0.25–32 | 4 | 16 | 100 |
| Ceftriaxone | ≤0.25–8 | 2 | 8 | 100 |
| Clindamycin | ≤0.125–0.5 | ≤0.125 | 0.25 | 100 |
| Moxifloxacin | ≤0.06–>8 | 0.25 | 0.5 | 100 |
| Enterococcus faecalis (22) | ||||
| LFF571 | ≤0.015–0.06 | 0.03 | 0.03 | NA |
| Fidaxomicin | 0.5–4 | 2 | 4 | NA |
| Vancomycin | 0.5–>16 | >16 | >16 | 64 |
| Imipenem | 0.5–16 | 2 | 16 | 0 |
| Piperacillin-tazobactam | 2–16 | 8 | 16 | 0 |
| Ampicillin | 0.25–4 | 1 | 4 | 0 |
| Ampicillin-sulbactam | 0.25–4 | 1 | 4 | 0 |
| Cefoxitin | >32 | >32 | >32 | 100 |
| Ceftriaxone | >32 | >32 | >32 | 100 |
| Clindamycin | 2–>16 | >16 | >16 | NA |
| Moxifloxacin | 0.125–>8 | 4 | 8 | 27 |
| Enterococcus faecium (20) | ||||
| LFF571 | ≤0.015–0.06 | 0.03 | 0.06 | NA |
| Fidaxomicin | 2–8 | 4 | 4 | NA |
| Vancomycin | 0.5–>16 | >16 | >16 | 90 |
| Imipenem | >32 | >32 | >32 | 100 |
| Piperacillin-tazobactam | >32 | >32 | >32 | 100 |
| Ampicillin | 16–>16 | >16 | >16 | 100 |
| Ampicillin-sulbactam | 16–>16 | >16 | >16 | 100 |
| Cefoxitin | >32 | >32 | >32 | 100 |
| Ceftriaxone | >32 | >32 | >32 | 100 |
| Clindamycin | 0.25–>16 | >16 | >16 | NA |
| Moxifloxacin | 2–>8 | >8 | >8 | 75 |
| Streptococcus pyogenes (21) | ||||
| LFF571 | 0.5–4 | 1 | 2 | NA |
| Fidaxomicin | 4–16 | 8 | 8 | NA |
| Vancomycin | ≤0.25 | ≤0.25 | ≤0.25 | 0 |
| Imipenem | ≤0.25 | ≤0.25 | ≤0.25 | 0 |
| Piperacillin-tazobactam | ≤0.25 | ≤0.25 | ≤0.25 | 0 |
| Ampicillin | ≤0.125 | ≤0.125 | ≤0.125 | 0 |
| Ampicillin-sulbactam | ≤0.125 | ≤0.125 | ≤0.125 | 0 |
| Cefoxitin | ≤0.25–2 | 1 | 1 | 0 |
| Ceftriaxone | ≤0.25 | ≤0.25 | ≤0.25 | 0 |
| Clindamycin | ≤0.125–1 | ≤0.125 | ≤0.25 | 0 |
| Moxifloxacin | ≤0.06–0.25 | 0.125 | 0.25 | 0 |
| S. anginosus (22) | ||||
| LFF571 | 1–16 | 2 | 8 | NA |
| Fidaxomicin | 4–>32 | 32. | >32 | NA |
| Vancomycin | ≤0.25–1 | 0.5 | 0.5 | 0 |
| Imipenem | ≤0.25 | ≤0.25 | ≤0.25 | 0 |
| Piperacillin-tazobactam | ≤0.25 | ≤0.25 | ≤0.25 | 0 |
| Ampicillin | ≤0.125 | ≤0.125 | ≤0.125 | 0 |
| Ampicillin-sulbactam | ≤0.125 | ≤0.125 | ≤0.125 | 0 |
| Cefoxitin | 2–8 | 4 | 8 | 0 |
| Ceftriaxone | ≤0.25–0.5 | ≤0.25 | ≤0.25 | 0 |
| Clindamycin | ≤0.125–>16 | ≤0.125 | >16 | 15 |
| Moxifloxacin | ≤0.06–0.5 | 0.125 | 0.25 | 0 |
| S. constellatus/intermediusi (26) | ||||
| LFF571 | 1–32 | 2 | 8 | NA |
| Fidaxomicin | 4–>32 | 32 | >32 | NA |
| Vancomycin | ≤0.25–1 | 0.5 | 0.5 | 0 |
| Imipenem | ≤0.25 | ≤0.25 | ≤0.25 | 0 |
| Piperacillin-tazobactam | ≤0.25–0.5 | ≤0.25 | 0.5 | 0 |
| Ampicillin | ≤0.125–4 | ≤0.125 | 2 | 0 |
| Ampicillin-sulbactam | ≤0.125–0.25 | ≤0.125 | ≤0.25 | 0 |
| Cefoxitin | ≤0.25–16 | 4 | 8 | 0 |
| Ceftriaxone | ≤0.25–0.5 | ≤0.25 | 0.5 | 0 |
| Clindamycin | ≤0.125–>16 | ≤0.125 | 0.5 | 5 |
| Moxifloxacin | ≤0.06–0.5 | 0.125 | 0.25 | 0 |
| Staphylococcus aureus (20) | ||||
| LFF571 | 0.125–0.25 | 0.125 | 0.125 | NA |
| Fidaxomicin | 4–8 | 8 | 8 | NA |
| Vancomycin | 0.5–1 | 0.5 | 1 | 0 |
| Imipenem | ≤0.25–4 | ≤0.25 | 2 | 0 |
| Piperacillin-tazobactam | 1–>32 | 32 | >32 | 55 |
| Ampicillin | 0.25–>16 | 16 | >16 | 95 |
| Ampicillin-sulbactam | 0.25–16 | 2 | 16 | 0 |
| Cefoxitin | 4–>32 | 16 | >32 | 50 |
| Ceftriaxone | 2–>32 | 32 | >32 | 55 |
| Clindamycin | ≤0.125–>16 | ≤0.125 | 0.25 | 10 |
| Moxifloxacin | ≤0.06–>8 | 0.125 | 4 | 10 |
| Unusual Gram-positive coccij (12) | ||||
| LFF571 | 8–>32 | 32 | >32 | NA |
| Fidaxomicin | 2–>32 | >32 | >32 | NA |
| Vancomycin | 0.5–>16 | >16 | >16 | 75 |
| Imipenem | ≤0.25–4 | ≤0.25 | 4 | 0 |
| Piperacillin-tazobactam | 2–8 | 2 | 4 | 0 |
| Ampicillin | ≤0.125–2 | 0.25 | 1 | 0 |
| Ampicillin-sulbactam | ≤0.125–2 | 0.25 | 1 | 0 |
| Cefoxitin | >32 | >32 | >32 | 100 |
| Ceftriaxone | ≤0.25–>32 | 32 | >32 | 58 |
| Clindamycin | ≤0.125–0.25 | ≤0.125 | 0.25 | 0 |
| Moxifloxacin | 0.25–2 | 0.5 | 2 | 0 |
Anaerobic organisms were tested by the agar dilution method; aerobic organisms were tested by broth microdilution (7, 8).
Lactobacillus antri (1), L. casei (6), L. catenaformis (4), L. crispatus (1), L. gasseri (4), L. reuteri (1), L. rhamnosus (6), and L. salivarius (1).
Bifidobacterium adolescentis (2), B. bifidum (4), B. breve (4), B. dentium (5), B. longum (5), and B. pseudocatenulatum (2).
Collinsella aerofaciens (6), Pseudoramibacter alactolyticus (8), Eubacterium cylindroides (1), Slackia exigua (5), Solobacterium moorei (5), Olsenella uli (2), and Eubacterium species (1).
NA, not available.
LFF571 demonstrated consistently excellent activity against all anaerobic Gram-positive rods and cocci (MIC50/90, 0.125/0.25 μg/ml for 284 strains), with the exception of bifidobacteria and some species of lactobacilli. Activity against lactobacilli was species dependent with all strains of Lactobacillus catenaformis susceptible to ≤0.125 μg/ml, while MICs for the other species ranged from 2 to 16 μg/ml for the vancomycin-resistant Lactobacillus casei-rhamnosus group but >32 μg/ml for the vancomycin-susceptible Lactobacillus gasseri strains. Against the Gram-negative anaerobes, the 40 strains of Porphyromonas spp. were susceptible to ≤0.25 μg/ml of LFF571, similar to their relatively unusual susceptibility to vancomycin (MIC 0.5 to 4 μg/ml). MICs for Bacteroides fragilis were 4 and 8 μg/ml, although the other species in the B. fragilis group, including Bacteroides thetaiotaomicron, Bacteroides ovatus, and Parabacteroides (Bacteroides) distasonis, were less susceptible, with an overall MIC90 of >32 μg/ml. There was no apparent difference in the range of MICs for the individual Bacteroides species. Similarly, Prevotella bivia, Prevotella melaninogenica/denticola, and Veillonella spp. also displayed MIC90 of >32 μg/ml, although some of the P. bivia strains had MICs as low as 0.5 μg/ml. Similar to fidaxomicin, the relatively poor activity against Gram-negative anaerobes suggests that LFF571 might have a lesser impact on the normal gut microbiota that maintain colonization resistance (21, 27).
Fidaxomicin results for the Gram-positive organisms were more variable. While activity against C. difficile and Clostridium perfringens was excellent (MIC90, 0.5 and ≤0.015 μg/ml, respectively), MICs for Clostridium ramosum and Clostridium innocuum were all >32 μg/ml. Unlike LFF571, fidaxomicin inhibited all strains of Bifidobacterium species with MIC90 at 0.125 μg/ml, but similar to LFF571, activity against lactobacilli was species dependent. While Eggerthella lenta strains were inhibited by ≤0.25 μg/ml of fidaxomicin, Eubacterium limosum strains required 16 to >32 μg/ml for inhibition. All anaerobic Gram-positive coccus strains were very susceptible with fidaxomicin MICs ranging from ≤0.015 to 2 μg/ml. Against the anaerobic Gram-negative organisms, fidaxomicin showed poor activity with MIC50/90 of 32/>32 μg/ml for all strains, including Veillonella spp.
Among the aerobic strains, LFF571 was most active against vancomycin-resistant and -susceptible strains of Enterococcus faecalis and Enterococcus faecium with MIC90 at 0.03 and 0.06 μg/ml, respectively. It was equally active against methicillin-susceptible and -resistant strains of staphylococci with MIC90 of 0.125 μg/ml. Against the streptococci, LFF571 was slightly less active: the MIC90 for Streptococcus pyogenes was 2 μg/ml and for the S. milleri group, 8 μg/ml. Aerococcus strains were inhibited by 0.06 to 1 μg/ml, although other unusual cocci such as Lactococcus, Leuconostoc, Pediococcus, and Weissella were less susceptible with MICs ranging from 8 to >32 μg/ml. There was no relationship in resistance by other classes of antimicrobial agents and LFF571. Fidaxomicin was less active than LFF571 against the aerobic strains. The MIC90 against enterococci was 4 μg/ml, with no apparent difference between vancomycin-resistant and -susceptible strains. The MIC90 for Aerococcus species was 2 μg/ml, for S. pyogenes, 16 μg/ml, and for the S. milleri group, >32 μg/ml.
Susceptibilities for the comparator agents were typical for what has been reported in other surveys of anaerobic intestinal organisms (4, 5, 12, 13, 11). C. difficile resistance to cefoxitin, imipenem, clindamycin, and moxifloxacin was present in 100, 18, 66, and 26% of our isolates, respectively, while elevated MICs of 4 μg/ml were found in two strains for vancomycin and in one for metronidazole. Moxifloxacin resistance was present in 10 of 14 (71%) REA-BI (027, NAP1) strains, 1 of 4 type J, the single type Z strain, and 4 of 20 (20%) nonspecific type strains. All strains of C. innocuum were also resistant to cefoxitin and 55% to vancomycin. Eggerthella lenta displayed resistance to ampicillin (75%) and ceftriaxone (95%) while the other nonsporeforming Gram-positive rods were mostly susceptible to these drugs. Among the Gram-positive cocci, 25% of Finegoldia magna and 15% of Parvimonas micra strains were resistant to clindamycin while 5% and 15%, respectively, were also resistant to moxifloxacin. Ampicillin resistance was present in 4 of 12 (33%) Peptostreptococcus anaerobius strains, although all of the phenotypically similar Peptostreptococcus stomatis strains were susceptible.
Through a novel mechanism, LFF571 shows excellent activity against C. difficile and good activity against other Gram-positive anaerobes but little activity against the anaerobic Gram-negative organisms. All strains of enterococci, regardless of vancomycin susceptibility, were inhibited. With this relatively narrow spectrum of activity, LFF571 shows promise as a new drug for treating CDI. It is currently in phase II clinical trials.
ACKNOWLEDGMENTS
This study was sponsored by a grant from Novartis Pharmaceuticals.
We thank Eliza Leoncio for excellent technical assistance.
Footnotes
Published ahead of print 30 January 2012
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