In vitro susceptibility of the Borrelia burgdorferi sensu lato complex to ABT-773, a novel ketolide.

Abstract

Sir, Macrolides and azalides frequently fail in the treatment of Lyme borreliosis and the recent Infectious Diseases Society of America (IDSA) guidelines therefore discourage their routine application in the chemotherapy of Lyme disease. Nevertheless, pregnancy and -lactam allergy remain important indications for the administration of macrolides in acute cases, when -lactams and tetracyclines cannot be administered owing to the possibility of side effects. For these indications, new therapeutic alternatives with increased effectiveness against Borrelia burgdorferi are needed. Recently, the 3-keto, 6-methoxy, 11,12-carbamate clarithromycin derivatives, known as the ketolides, were shown to have potent antibacterial activity against a number of Gram-positive and Gram-negative microorganisms, including Chlamydia pneumoniae, Legionella spp. and, in part, macrolide-resistant bacteria. Accordingly, this new class of antimicrobials also merits consideration for the stagedependent therapy of Lyme disease. ABT-773 is a new ketolide agent; it is acid-stable and, as such, can be administered orally. The present pilot study was designed to determine the in vitro activity of ABT-773 against 17 isolates of the B. burgdorferi sensu lato (s.l.) complex. The origins of the borrelial isolates used in this study are summarized in the Table. For MIC and MBC determination in all cases except B. burgdorferi strain B31, low passage isolates (10–20 passages) were employed. Genospecies were identified by restriction fragment length polymorphism (RFLP) analysis after MluI digestion of DNA and by application of plasmid analysis. For control purposes, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were tested. The 96-well microtitre trays containing concentrations of ABT-773 (Abbott, Chicago, IL, USA) ranging from 0.002 to 2 mg/L were prepared in cooperation with MerlinDiagnostika GmbH (Bornheim-Hersel, Germany). In order that these data can be related to our recent publications on the in vitro susceptibility of Borreliae, MICs and MBCs were determined under the same standardized conditions as described previously: for MIC determination, a colorimetric assay and conventional subculture experiments in combination with dark-field microscopy were performed. To determine the drug concentrations that provided complete killing of the inoculum, after 72 h aliquots were taken in triplicate from all vials lacking detectable growth, diluted (1:1000) below the MIC, and subcultured for an additional 3 weeks in liquid medium. Moreover, the time–kill curve technique was representatively applied to isolate PSth at 2.5 10 cells/mL in duplicate using ABT-773 at a test range of 0.002 mg/L (MIC) to 0.125 mg/L (60 MIC) for 120 h. Significant antibiotic–medium interactions were excluded because for the ATCC reference strains the MIC ranges of ABT-773 read in triplicate after 24, 72 and 120 h of pre-incubation of the antibiotic–medium test preparation were within the tentative MIC ranges supplied by the manufacturer. The individual MIC and MBC values of ABT-773 for the 17 borrelial strains are summarized in the Table. After 72 h, the colorimetric MIC determination demonstrated 100% agreement with the results of conventional cell counts. The novel ketolide ABT-773 proved to be uniformly effective against Borreliae displaying an MIC90 of 0.002 mg/L. By using a 100% rather than a 99.9% killing criterion after 72 h, which constitutes a very stringent standard for any antibiotic against B. burgdorferi, the MBC50 and the MBC90 for ABT-773 were found to be 0.25 and 0.75 mg/L, respectively (MBC range 0.002–1 mg/L), thus indicating a slow killing of Borreliae. In a second approach in which motile organisms were considered viable, our time–kill experiments with isolate PSth revealed that after 96–120 h, drug concentrations of ABT-773 required for a 3 log10 unit (99.9%) reduction of the final inoculum decreased to 0.008 mg/L, which is 5 log2 unit dilutions below the MBC obtained after 72 h by subculture experiments. Proposed tentative interpretative criteria for susceptibility testing of ABT-773 against fastidious organisms have determined an MIC of 0.5 mg/L for susceptible and