ARTICLE IN PRESS. Ticks and Tick-borne Diseases

Ticks and Tick-borne Diseases 1 (2010) 30 34 Contents lists available at ScienceDirect Ticks and Tick-borne Diseases journal homepage: www.elsevier.de/ttbdis Comparison of in vitro activities of tigecycline, doxycycline, and tetracycline against the spirochete Borrelia burgdorferi Louis Ates a, c, Christa Hanssen-Hübner a, Douglas E. Norris b, Dania Richter d, Peter Kraiczy a, Klaus-Peter Hunfeld c, a Institute of Medical Microbiology and Infection Control, University Hospital of Frankfurt, Frankfurt/Main, Germany b The Harry W. Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland c Institute of Laboratory Medicine, Northwest Medical Centre, Academic Teaching Hospital, Medical Faculty, University of Frankfurt, Steinbacher Hohl 2 26, D-60488 Frankfurt/Main, Germany d Abt. Parasitologie, Institut für Pathologie, Charité Universitätsmedizin Berlin, Malteserstrasse 74 100, 12249 Berlin, Germany article info Article history: Received 23 August 2009 Received in revised form 22 November 2009 Accepted 23 November 2009 Available online 7 January 2010 Keywords: Borrelia burgdorferi Tetracyclines Tigecycline Susceptibility testing Lyme borreliosis abstract Tigecycline is a new glycylcycline that has recently been revealed to be very effective in vitro against a variety of Gram-negative and Gram-positive bacteria including multi-drug resistant microorganisms. Using a standardized microdilution susceptibility testing method, we determined the minimal inhibitory concentrations (MICs) and the minimal bactericidal concentrations (MBCs) of tigecycline, in parallel with doxycycline, tetracycline, and other antibiotic agents relevant for Lyme borreliosis treatment such as ceftriaxone and cefotaxime. The activity of all agents against 16 different Borrelia isolates belonging to all borrelial genospecies known to be pathogenic for humans was investigated and analyzed under standardized conditions. The overall rank order of MIC 90 s was tigecycline (r0.016 mg/l) 4 ceftriaxone (0.03 mg/l) 4 cefotaxime (r0.125 mg/l) 4 doxycycline (0.25 mg/l) 4 tetracycline (0.25 mg/l). The rank order of MBC 90 s was tigecycline (0.5 mg/l) 4 ceftriaxone (2 mg/l) 4 tetracycline (16 mg/l) 4 doxycycline (16 mg/l) 4 cefotaxime (416 mg/l). High in vitro activity of the new glycylcycline against Borrelia was further substantiated by time-kill experiments performed with B. afzelii isolate EB1. Parallel testing of tigecycline and ceftriaxone demonstrated a bacteriostatic effect for 0.016 mg/l of tigecycline and for 0.03 mg/l for ceftriaxone after 72 h of incubation. Moreover, tigecycline was bactericidal at a concentration of 0.25 mg/l showing a 43 log 10 unit reduction of the initial inoculum, whereas for ceftriaxone a concentration of 2 mg/l was needed. & 2009 Elsevier GmbH. All rights reserved. Introduction Lyme borreliosis is a vector-borne disease that is transmitted by ixodid ticks and is caused by the spirochete Borrelia burgdorferi sensu lato (s.l.) complex. The 5 genospecies that are currently considered to be human pathogens are B. burgdorferi sensu stricto (s.s.), B. afzelii, B. garinii, and B. spielmanii, and the proposed but not yet validated novel species B. bavariensis (Margos et al., 2009). In Europe, the incidence of Lyme borreliosis is estimated to range from 3.9 to 168/ 100,000 (Hunfeld et al., 2005; Stanek et al., 1996). The disease can manifest itself progressively as a multisystem disorder exhibiting a large variety of clinical symptoms (Steere, 2001). Early on in the course of the infection, a bull s-eye rash called erythema migrans is the most common disease manifestation appearing in 80 90% of the patients. If left untreated, however, the infection may cause Corresponding author. Tel.: +49 69 7601 3252; fax: +49 69 7601 3907. E-mail address: K.Hunfeld@em.uni-frankfurt.de (K.-P. Hunfeld). neurological disorders and late manifestations including acrodermatitis chronica atrophicans or chronic arthritis (Steere, 2001; Weinstein and Britchkov, 2002). Lyme borreliosis is frequently treated by orally administered doxycycline or intravenous ceftriaxone, which appear to be equally effectiveineuropeanpatients(ljostad et al., 2008; Loewen et al., 1999). Other often administered antibiotics are penicillin, amoxicillin, cefuroxime, cefotaxime, and tetracycline. Although these drugs have been reported to be clinically effective in the majority of cases under controlled study conditions, treatment failures have been repeatedly published for most of these compounds (Hodzic et al., 2008; Hunfeld et al., 2005; Miklossy et al., 2008; Preac-Mursic et al., 1989). Tigecycline, a glycylcycline and structural analog of minocycline is a relatively new antibiotic agent that has been found to be effective against many Gram-positive and Gram-negative bacteria including multi-drug resistant pathogens such as ESBL-producing enterobacteriaceae, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and multi-drug resistant Acinetobacter baumanii (Jamal et al., 2009; Zhanel et al., 1877-959X/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.ttbdis.2009.11.004

L. Ates et al. / Ticks and Tick-borne Diseases 1 (2010) 30 34 31 2008). Tigecycline is a primarily bacteriostatic agent that binds to thebacterial30ssubunitofthebacterialribosome(greer, 2006). Recently, tigecycline was also found to be effective in vitro against several strains of B. burgdorferi s.l. (Yang et al., 2009). However, the number of isolates tested in this study was small and so far tigecycline has not been tested in parallel with other tetracyclines against borreliae under well-controlled conditions. In this study, we used a well-evaluated and standardized methodology to investigate the in vitro activity and bactericidal properties of tigecycline against 16 isolates of the B. burgdorferi s.l. complex in comparison to a series of compounds relevant for the treatment of Lyme borreliosis in the clinical setting, including doxycycline, tetracycline, ceftriaxone, and cefotaxime. Materials and methods Isolates The susceptibility of a total of 16 isolates belonging to 5 human pathogenic species of the B. burgdorferi s.l. complex as well as 2 B. valaisiana and 2 B. lusitaniae isolates were tested. The clinical, geographic, and phenotypic characteristics of 12 of the tested isolates (Table 1) have been previously described (Hunfeld et al., 2000a, 2001, 2003; Morgenstern et al., 2009; Preac-Mursic et al., 1986). In the present study, we also tested B. lusitaniae isolate MT0407 M3 (tick isolate, Portugal), B. lusitaniae isolate IP10907 N1 (tick isolate, Germany), B. valaisiana isolate ZWU3 0507 N3 (tick isolate, Germany), and B. valaisiana isolate Bv9 (tick isolate, Germany). Genospecies were identified by restriction fragment length polymorphism patterns after digestion with endonuclease MluI and by application of plasmid analysis as described previously (Belfaiza et al., 1993) or by amplifying and sequencing a 600-nucleotide fragment of the gene encoding the 16SrRNA and comparing it to the same fragment of various genospecies type strains (Richter and Matuschka, 2006). Determination of MICs MIC values were obtained by applying our previously described standardized colorimetric microdilution method Table 1 Individual antibiotic susceptibilities of 16 B. burgdorferi s.l. isolates to tigecycline, doxycycline, tetracycline, ceftriaxone, and cefotaxime (MICs and MBCs in milligram per liter) a. Antibiotic substances (MIC and MBC in mg/l) b Isolates Tigecycline Doxycycline Tetracycline Ceftriaxone Cefotaxime MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC B. burgdorferi s.s. B31-e2 r0.016 1 0.125 16 0.25 16 0.03 2 r0.125 8 LW2 r0.016 0.5 0.25 32 0.25 >16 0.03 2 r0.125 8 297 r0.016 0.5 0.25 16 0.25 16 0.03 2 r0.125 8 B. bavariensis PBi r0.016 0.5 0.125 8 0.25 8 0.03 2 r0.125 16 B. garinii G1 r0.016 0.5 0.25 16 0.25 16 0.03 2 r0.125 16 PSth r0.016 0.5 1 16 1 16 0.06 2 r0.125 8 B. afzelii FEM1 r0.016 ND 0.25 16 0.25 16 0.06 0.5 0.5 8 EB1 r0.016 0.25 0.25 16 r0.125 16 0.06 2 r0.125 16 PKo r0.016 0.25 1 8 2 16 0.06 2 0.25 Z16 B. spielmanii A14S r0.016 0.25 0.125 8 r0.125 8 0.03 2 r0.125 16 PSigII r0.016 0.25 0.125 32 r0.125 8 0.03 2 r0.125 Z16 TIsar2 r0.016 ND 0.25 8 r0.125 4 0.06 2 r0.125 4 B. valaisiana Bv9 r0.016 ND 0.125 2 r0.125 4 0.06 4 r0.125 16 ZWU3 0507 N3 r0.016 0.25 0.25 8 0.25 8 0.03 2 r0.125 Z16 B. lusitaniae IP10907 N1 r0.016 0.25 0.5 8 0.5 8 0.06 0.25 r0.125 1 MT0407 M3 r0.016 0.5 0.125 8 0.25 8 0.03 2 r0.125 2 Medium controls c, S. aureus (ATCC 29213) Median MIC in BSK 0.06 0.5 0.25 8 2 MIC range in BSK 0.03 0.06 0.25 0.5 r0.125 0.25 4 8 2 CLSI range 0.03 0.25 0.12 0.5 0.12 1 1 8 1 4 E. coli (ATCC 25922) Median MIC in BSK medium 0.125 1 0.5 0.06 r0.125 MIC range in BSK 0.125 1 2 0.5 0.06 0.12 r0.125 CLSI range 0.03 0.25 0.5 2 0.5 2 0.03 0.12 0.03 0.12 ND, not determined. a Antimicrobial susceptibility was determined on 3 different days, and MIC and MBC values for each isolate were reported as the median of 3 experiments. b The test ranges (in mg/l) were as follows: tigecycline: 0.016 2; doxycycline: 0.016 32; tetracycline: 0.125 16; ceftriaxone: 0.004 8; cefotaxime: 0.125 16. c To investigate significant antibiotic medium interaction, MICs for S. aureus (ATCC 29213) and E. coli (ATCC 25922) were determined on 3 different days, referring to the CLSI method (CLSI, 2006) except for the use of BSK and preincubation of the antibiotic medium test preparation for 48 h before testing. Results were reported as the median of all 3 experiments.

32 L. Ates et al. / Ticks and Tick-borne Diseases 1 (2010) 30 34 (Hunfeld et al., 2000a, 2000b). The antibiotic substances used were provided in lyophilized form on 96-well microtiter plates in serially diluted concentrations. The plates were supplied and quality controlled by the manufacturer (Merlin GmbH, Bornheim- Hersel, Germany). An inoculum (200 ml) of 5 10 6 borreliae/well, in the log phase of growth, was incubated in Barbour-Stoenner- Kelly (BSK) medium (Preac-Mursic et al., 1986) supplemented with phenol red for 72 h at 33 1C. The absorbance of the test medium was measured at 0, 24, 48, and 72 h using a spectrophotometer (PowerWave 200, Bio-Tec Instruments, USA), and growth curves were calculated by utilizing a software-assisted method (MicroWin 3.0, Mikrotek Germany). Growth of samples and controls was determined for each well based on the decrease of absorbance after 72 h (E t72 ) in comparison to the initial absorbance values (E t0 ). A sample was judged to exhibit growth if the decrease from E t0 to E t72 accounted for more than 10% (Hunfeld et al., 2000b). The lowest concentration at which an isolate failed to grow in the presence of antibiotics was interpreted as the colorimetric MIC (Hunfeld et al., 2000b). Every substance was tested in triplicate on different days, but all substances were tested in parallel to each other, together with a control without antibiotic substance and a control without borreliae. The reported MIC is the median of all 3 independent experiments performed. As an additional quality control measure and to exclude significant antibiotic medium interactions, the method was also applied using Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 in triplicate experiments following the CLSI (formerly NCCLS) standard protocols except for preincubation of the used antibiotic BSK preparation for 48 h before testing (CLSI 2006). Determination of MBCs Conventional MBC values of doxycycline, tetracycline, ceftriaxone, and cefotaxime were determined for at least one isolate of each genospecies by obtaining aliquots of 18 ml from each well without growth after 72 h and transferring these samples into vials containing 1332 ml of fresh BSK medium (dilution factor 1:75) (Hunfeld et al., 2000b). Due to the very low MIC values measured for tigecycline, an aliquot of 1.8 ml was transferred into 1348 ml of BSK (dilution factor 1:750) for this agent. Subcultures were then incubated at 33 1C for another 3 weeks. After slight agitation of the medium, 10 microscopical fields were examined for the presence or absence of viable spirochetes using dark-field microscopy (Carl Zeiss MicroImaging, Göttingen, Germany). Time-kill experiments Time-kill experiments were performed by culturing B. afzelii isolate EB1 in the presence of 0.016, 0.03, 0.25, and 2 mg/l tigecycline or ceftriaxone as described in the MIC experiments. The time-kill curve was constructed by counting the number of viable spirochetes under a dark-field microscope using a KOVA counting chamber (Hycor Biomedical inc., Garden grove, CA) after 0, 24, 48, 72, and 96 h. Experiments were performed in duplicate and the data are depicted as the average of 2 experiments as described recently (Kraiczy et al., 2001). The concentration of the antibiotic agent that inhibited growth of the inoculum for 72 h was defined as the MIC. The concentration that achieved a 43 log 10 reduction of the inoculum after 72 h was defined as the MBC (Kraiczy et al., 2001). Statistics To detect possible variations in the antibiotic susceptibility pattern of the different borrelial genospecies, all MIC and MBC values were analyzed for intragenospecies-specific differences applying the Kruskal-Wallis test. Calculations were performed with the statistical program BIAS, version 8.3.8 (Epsilon Verlag, Hochheim, Germany). Results MIC determination When comparing antibiotic activities of the different substances against the tested isolates, the rank order of in vitro activity against all tested borrelial genospecies considering the median MICs of the individual substances for all tested isolates in milligram per liter was as follows: tigecycline (MIC 90 : r0.016 mg/l), ceftriaxone (MIC 90 : 0.03 mg/l), cefotaxime (MIC 90 : r0.125 mg/l), doxycycline (MIC 90 : 0.25 mg/l), tetracycline (MIC 90 : 0.25 mg/l) (Table 1). The most active antibiotic agent was tigecycline exhibiting an MIC of r0.016 mg/l in every tested isolate. Doxycycline and tetracycline had identical median MIC values, but since the average sensitivity for doxycycline in the tested isolates was lower, doxycycline was considered the more active substance. Antibiotic susceptibilities between the 7 tested genospecies did not differ significantly (Kruskal-Wallis test, po0.05). In addition, no significant MIC deviations attributable to interactions of the tested antimicrobial agents with components of the BSK medium were detected in our quality control experiments with the reference organisms S. aureus ATCC 29213 and E. coli ATCC 25922 for any of the antibiotics used (Table 1). MBC determination The MBC (defined as 100% killing) value of each isolate was obtained after a 72-h exposure to the antibiotic substances followed by an additional 3-week incubation of subcultures. When taking the median MBCs of all tested isolates in milligram per liter into account, the rank order of MBC 90 s in the tested isolates was: tigecycline (0.5 mg/l), ceftriaxone (2 mg/l), tetracycline (16 mg/l), doxycycline (16 mg/l), cefotaxime (Z16 mg/l) (Table 1). Time-kill experiments The acitivity of tigecycline and ceftriaxone against the B. afzelii isolate EB1 was further examined by time-kill experiments. Tigecycline inhibited growth of the initial inoculum for 72 h at a concentration of 0.016 mg/l, whereas ceftriaxone required concentrations of 0.03 mg/l (Fig. 1). Tigecycline achieved a 43 log 10 - unit reduction of the initial inoculum after 24 h at a concentration of 0.25 mg/l, whereas in ceftriaxone a concentration of 2 mg/l was needed. Discussion Doxycycline, and occasionally other tetracyclines, are frequently administered drugs for the treatment of early Lyme borreliosis (Ljostad et al., 2008) and have shown to be effective in the majority of clinical cases (Smith et al., 2002; Wormser et al., 2003). However, clinical treatment failures in early Lyme borreliosis occur in 5 10% of all patients including

L. Ates et al. / Ticks and Tick-borne Diseases 1 (2010) 30 34 33 Borrelia /well *10 6 Borrelia /well *10 6 100 10 1 0.1 0.01 0.001 100 10 1 0.1 0.01 0.001 0 24 48 72 96 Time (h) Control 0.016 mg/l 0.03 mg/l 0.25 mg/l 2 mg/l 0 24 48 72 96 Time (h) Control 0.016 mg/l 0.03 mg/l 0.25 mg/l 2 mg/l Fig. 1. Time-kill curves of B. afzelii isolate EB1 cultured with different concentrations of tigecycline (Fig. 1A), or ceftriaxone (Fig. 1B). Concentrations for ceftriaxone were chosen as half the MIC (0.016 mg/l), the MIC (0.03 mg/l), 8 times the MIC (0.25 mg/l), and the MBC (2 mg/l) as defined by the microdilution test method (Table 1). Concentrations for tigecycline were 0.016 mg/l corresponding to the MIC (growth inhibition of the initial inoculum), and 0.25 mg/l corresponding to the MBC (>3 log 10 reduction of the initial inoculum after 72 h). Two experiments were performed in parallel. Depicted is the mean of the 2 independent experiments. doxycycline-treated individuals (Ljostad et al., 2008; Preac- Mursic et al., 1989; Smith et al., 2002; Strle et al., 1995; Wormser et al., 2003). The clinical success rate in patients with early or late neuroborreliosis that were treated with doxycycline or ceftriaxone ranges between 33 and 66% (Kaiser, 2004; Ljostad et al., 2008). Doxycycline seems to be as effective as ceftriaxone in European patients affected by neuroborreliosis (Ljostad et al., 2008). However, treatment failures may be more likely in later stages of Lyme borreliosis than in the early phase characterized by erythema migrans or neuroborreliosis (Steere, 2001). In recent years, new substances for the treatment of bacterial infections have become available. In our present study, we performed in vitro susceptibility testing experiments with tigecycline, a new tetracycline and a structural analog of minocycline. Tigecycline was recently reported to be effective in vitro against B. burgdorferi (Yang et al., 2009). However, data on its in vitro effectiveness against a wider range of Borrelia isolates under standardized test conditions are missing. Here, for the first time, the in vitro activity of tigecycline was tested in well-controlled assays and compared to other clinically relevant antibiotics. In our present study, tigecycline clearly had a low MIC 90 (r0.016 mg/l) for all tested isolates. In fact, tigecycline was more active than doxycycline (MIC 90 : 0.25 mg/l) and appeared even more active in vitro than ceftriaxone (MIC 90 : 0.03 mg/l) for all isolates tested. Furthermore, tigecycline was the most borreliacidal compound of all tested substances resulting in an MBC 90 of 0.5 mg/l for the tested isolates. Taking all MBC 90 values into account, tigecycline was 4 times more effective in vitro than the second most active antibiotic, ceftriaxone (MBC 90 : 2 mg/l), and up to 32 times more effective than doxycycline (range of MBCs: 8 16 mg/l). Our observation of a high in vitro activity of tigecycline was further confirmed by time-kill experiments using the B. afzelii isolate EB1. Tigecycline inhibited the growth of the initial inoculum at 0.016 mg/l and reduced the inoculum by 43 log 10 at 0.25 mg/l after 72 h of incubation, thereby corresponding to the low MIC 90 and MBC 90 values as determined by our microdilution susceptibility experiments. As depicted in Table 1, the in vitro susceptibility values of the other tested tetracyclines and cephalosporins were found to be in good accordance with values published in the recent literature (Hunfeld and Brade, 2006). Moreover, our quality control experiments clearly confirmed that in vitro test results were not significantly hampered by possible interactions of the antibiotic substances with the BSK medium. Interestingly, tigecycline was shown to be more effective than related classical tetracyclines in many Gram-positive and Gramnegative bacteria including multi-drug resistant pathogens with RND-efflux pumps (Greer, 2006). Such RND-type efflux pumps are prevalent in a wide variety of other Gram-negative bacteriae, such as E. coli and Acinetobacter baumanii, where they appear to decrease the susceptibility of these organisms to a large number of antibiotic agents including tetracyclines (Chau et al., 2004). Interestingly, such a mechanism of antibiotic resistance has recently been described also for B. burgdorferi demonstrating the efflux of antibiotics from thespirochetebyuseofaresistancenodulation-celldivision(rnd) type efflux pump called BesABC (Bunikis et al., 2008). A knockout of BesC in a genetically modified B. burgdorferi s.s. strain was demonstrated to result in an increased susceptibility to multiple classes of antibiotics used in the therapy of Lyme borreliosis. For tetracycline, an 8-fold decrease of the MIC 90 (0.31 mg/l in the wildtype strain vs. 0.04 mg/l in the BesC knockout strain) and a 15-fold decrease of the MBC (2.5 mg/l in the wild-type strain vs. 0.16 mg/l in the BesC knockout strain) was observed using our standardized susceptibility-testing assay (Bunikis et al., 2008). One of the proposed reasons for the increased activity of tigecycline against borrelia compared to classical tetracyclines as observed in our study may be the fact that tigecycline is obviously more resistant to efflux as mediated by RND-type efflux systems than are the classical tetracyclines (Greer, 2006; Zhanel et al., 2004). Taking into consideration the results of our study, the susceptibility testing of borrelial mutants with altered tetracycline resistance against tigecycline would be a very interesting future approach of antibiotic research in borrelia. Furthermore, tigecycline has a much larger volume of distribution V d compared to the classical tetracyclines (Zhanel et al., 2004) (>10 L/kg vs. 0.14 1.6 L/kg) which is, however, similar to the V d of ceftriaxone (Patel and Kaplan, 1984). These considerations and its high in vitro activity against Borrelia clearly warrant further clinical studies on tigecyline in the treatment of disseminated late manifestations of Lyme borreliosis, i.e. arthritis or acrodermatitis chronica atrophicans. In summary, we have further confirmed that tigecycline is very effective in vitro against a wide variety of B. burgdorferi s.l. isolates. In fact, it actually appears more effective in vitro than other agents that are frequently used for the treatment of Lyme

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