Sub - inhibitory concentrations of LFF 571 reduce toxin production by 1 Clostridium difficile

17 LFF571 is a novel semi-synthetic thiopeptide antibacterial that is undergoing investigation for 18 safety and efficacy in patients with moderate C. difficile infections. LFF571 inhibits bacterial 19 protein synthesis by interacting with elongation factor Tu (EF-Tu) and interrupting complex 20 formation between EF-Tu and aminoacyl-tRNA. Given this mechanism of action, we 21 hypothesized that concentrations of LFF571 below those necessary to inhibit bacterial 22 growth would reduce steady state toxin levels in C. difficile cultures. We investigated C. 23 difficile growth and toxin A/B levels in the presence of LFF571, fidaxomicin, vancomycin and 24 metronidazole. LFF571 led to strain-dependent effects on toxin production, including 25 decreased toxin levels after treatment with sub-inhibitory concentrations, and more rapid 26 declines in toxin production compared to inhibition of colony formation. Fidaxomicin, which is 27 an RNA synthesis inhibitor, conferred a similar pattern to LFF571 with respect to toxin levels 28 versus viable cell counts. Incubation of two toxigenic C. difficile strains with sub-inhibitory 29 concentrations of vancomycin, a cell wall synthesis inhibitor, increased toxin levels in the 30 supernatant compared to untreated cultures. A similar phenomenon was observed with one 31 metronidazole-treated strain of C. difficile. These studies indicate that LFF571 and 32 fidaxomicin generally result in decreased C. difficile toxin levels in culture supernatants, 33 whereas treatment of some strains with vancomycin or metronidazole had the potential to 34 increase toxin levels. Although the relevance of these findings remains to be studied in 35 patients, reducing toxin levels with sub growth-inhibitory concentrations of antibiotic is 36 hypothesized to be beneficial in alleviating symptoms. 37 38 on O cber 2, 2017 by gest httpaac.asm .rg/ D ow nladed fom Introduction 39 Clostridium difficile infection (CDI) is a serious gastrointestinal disease. Approximately 40 500,000 cases occur in the United States each year, making CDI the most common hospital41 acquired infection (1), (2). The ability of C. difficile to form spores makes it difficult to remove 42 from surfaces and allows C. difficile to spread easily within a healthcare setting. Recently, the 43 incidence of the disease has been increasing and hyper-virulent strains, such as 44 B1/NAP1/027 have been recognized (reviewed in (3)). The epidemiology of CDI has also 45 been changing, and the disease is now more commonly seen outside the hospital 46 environment. The standard of care for CDI is treatment with the antibiotics metronidazole or 47 vancomycin. Fidaxomicin was approved in 2011 for treatment of C. difficile associated 48 diarrhea. 49 C. difficile are anaerobes that opportunistically colonize the gut, often after treatment 50 with broadly-acting antibacterials. CDI is an enterotoxin-mediated disease that can be sub51 clinical or have symptoms ranging from mild diarrhea to severe pseudomembranous colitis, 52 megacolon, bowel perforation, sepsis and death (1). C. difficile-encoded toxins are termed A, 53 B and CDT. There has been substantial debate about the roles of each toxin in 54 pathogenesis, and some consider both toxins A and B to be essential virulence factors (4, 5). 55 Toxins A and B are large, multi-domain proteins that catalyze the glucosylation of Rho56 GTPases. Inactivation of the cellular enzymes leads to deregulation of cytoskeleton 57 arrangement and cell death, followed by mucosal inflammation and diarrhea (6). Toxin CDT 58 is an ADP-ribosylating binary toxin with an unclear contribution to disease (6). 59 LFF571 (7) is a semi-synthetic thiopeptide antibiotic with potent in vitro activity 60 against C. difficile (8, 9) and is efficacious in the treatment of C. difficile infection in animal 61 models (10). LFF571 has recently been shown to be non-inferior to vancomycin in patients 62 with moderate C. difficile infections (Mullane et al, submitted). LFF571 inhibits bacterial 63 protein synthesis by binding to elongation factor Tu (EF-Tu) and preventing this translation 64 factor from delivering an aminoacyl-tRNA to the ribosome (11) (12). Because of this 65 mechanism of action, we hypothesized that LFF571 would prevent toxin production at doses 66 on O cber 2, 2017 by gest httpaac.asm .rg/ D ow nladed fom below the growth-inhibitory concentration of the compound. In theory, this could reduce toxin67 mediated diarrheal symptoms at lower concentrations and provide an advantage over other 68 antibiotics with unrelated modes of action. Here, we show that sub-inhibitory or inhibitory 69 concentrations of LFF571 and fidaxomicin decrease toxin levels in supernatants of C. difficile 70 cultures, while subto inhibitory levels of vancomycin and metronidazole increase toxin 71 levels from select strains. 72 Materials and Methods 73 Antibiotics. LFF571 and fidaxomicin (Lipiarmycin A3, prepared by fermentation of 74 Catellatospora sp. Bp3323-81) were obtained from Novartis. Vancomycin and metronidazole 75 were purchased from US Pharmacopeia (Rockville, MD). 76 Organisms. Bacterial strains used in this study were from the American Type Culture 77 Collection (ATCC) or kindly provided by D. Low at Mount Sinai Hospital, Toronto, Canada 78 (Table 1). All strains were routinely cultured on Brucella agar containing 5% sheep blood, 0.5 79 μg/ml vitamin K and 5 μg/ml hemin. Cultures were incubated at 37°C in an anaerobic 80 chamber (Coy Laboratory Products, Inc.) with a gas mix environment of 10% hydrogen, 10% 81 carbon dioxide and a balance of nitrogen. 82 In vitro susceptibility testing, cell titering, and toxin sampling. Minimal inhibitory 83 concentrations (MIC) of the test agents were determined using the agar dilution method 84 recommended by the Clinical and Laboratory Standards Institute (CLSI) (13). To select the 85 appropriate test agent concentration ranges for toxin level and cell titer determinations, 86 antibacterial activity was determined under the specific growth conditions used to generate 87 the toxin assay samples. Briefly, 5-6 colonies of C. difficile strains from an overnight Brucella 88 agar plate were suspended in pre-reduced tryptone yeast plus sodium thioglycolate (TY) 89 broth supplemented with 0.5 μg/ml vitamin K, 5.0 μg/ml hemin. Duplicate suspensions of C. 90 difficile (0.5 ml) were transferred to 96 deep-well plates and serial two-fold dilutions of the 91 test agents were added to the appropriate wells. Cultures were incubated for 24 hrs at 37oC, 92 anaerobically, unless otherwise indicated. 93 on O cber 2, 2017 by gest httpaac.asm .rg/ D ow nladed fom Once the optimal test agent concentration range was determined, experimental 94 samples were generated from 0.5 ml cultures inoculated and incubated as described above. 95 Viable cells were quantified by removing 110 μl and plating 10-fold serial dilutions onto 96 Brucella agar plates, followed by incubating anaerobically at 37oC for 48 hours unless stated 97 otherwise. The remaining culture was centrifuged for 10 min at 3500 xg and the supernatants 98 were removed and stored at -20oC for 48-96 hours prior to assaying for toxin A/B levels. 99 Toxin A/B analysis. Total toxin A/B levels in C. difficile culture supernatants were measured 100 by ELISA (Wampole, Techlab, Blacksburg, VA), according to the manufacturer’s 101 recommendations. The assay uses microtiter plates pre-coated with polyclonal goat 102 antibodies against toxins A and B. Briefly, 50 μl of horseradish peroxidase (HRP)-conjugated 103 secondary antibodies (mouse monoclonal anti-toxin A and goat polyclonal anti-toxin B) were 104 added to each well, immediately followed by 100 μl of undiluted culture supernatant. The 105 plates were incubated for 60 minutes at 37oC before washing five times with phosphate 106 buffered saline (PBS). Subsequently, 100 μl of substrate (tetramethyl benzidine) were added 107 and the samples were incubated for 10 minutes at room temperature. The reactions were 108 stopped by the addition of 50 μl H2SO4 and signals were detected at A450 using a 109 spectrophotometer (SPECTRAmax, Molecular Devices, Sunnyvale, CA). Purified toxin A and 110 toxin B (tgcBIOMICS) were used as controls. The lower limits of detection in this assay were 111 1.25 ng/ml of purified toxin A and 6 ng/ml of purified toxin B. 112 To monitor toxin A and B levels separately, culture supernatants were analyzed by 113 ELISA (tgcBIOMICS, Mainz, Germany), according the manufacturer’s instructions. Briefly, 114 100 μl of undiluted C. difficile culture supernatants were transferred to microtiter plates 115 coated with antibodies to both toxins A and B. Individual toxins were detected by immediately 116 adding 50 μl of HRP-conjugated specific anti-toxin A or anti-toxin B antibodies. Coated plates 117 containing specimens plus conjugate were incubated for 60 minutes at 37oC before washing 118 three times with wash buffer. Subsequently, 100 μl of substrate (tetramethylbenzidine) were 119 added and the samples were incubated for 20 minutes at room temperature. The reactions 120 were stopped by the addition of 50 μl of H2SO4 and signals were detected by 121 on O cber 2, 2017 by gest httpaac.asm .rg/ D ow nladed fom spectrophotometry (SPECTRAmax), followed by subtracting background (A620) from signal 122 (A450). Purified toxin A and toxin B (tcgBIOMICS) were used as controls. The lower limits of 123 detection in this assay were 1.25 ng/ml of purified toxin A and 0.6 ng/ml of purified toxin B. 124