In vitro susceptibilities of Leptospira spp. and Borrelia burgdorferi isolates to amoxicillin, tilmicosin, and enrofloxacin.

Antimicrobial susceptibility testing was conducted with 6 different spirochetal strains (4 strains of Leptospira spp. and 2 strains of Borrelia burgdorferi) against 3 antimicrobial agents, commonly used in equine and bovine practice. The ranges of MIC and MBC of amoxicillin against Leptospira spp. were 0.05 - 6.25 microgram/ml and 6.25 - 25.0 microgram/ml, respectively. And the ranges of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of amoxicillin against B. burgdorferi were 0.05 - 0.39 microgram/ml and 0.20 - 0.78 microgram/ml, respectively. The ranges of MIC and MBC of enrofloxacin against Leptospira spp. were 0.05 - 0.39 microgram/ml and 0.05 - 0.39 microgram/ml, respectively. Two strains of B. burgdorferi were resistant to enrofloxacin at the highest concentration tested for MBC (>or=100 microgram/ml). Therefore, the potential role of tilmicosin in the treatment of leptospirosis and borreliosis should be further evaluated in animal models to understand whether the in vivo studies will confirm in vitro results. All spirochetal isolates were inhibited (MIC) and were killed (MBC) by tilmicosin at concentrations below the limit of testing (<or=0.01 microgram/ml).

Introduction

Leptospirosis is a zoonotic infection with a worldwide distribution that is associated with both endemic disease and epidemics, with the incidence of disease being highest in tropical climates [17]. The number of cases of leptospirosis diagnosed has increased since 1983 in North America [36]. Lyme disease is a multisystemic disorder caused by infection with Borrelia burgdorferi and is the most common tick-associated illness in North America [4,7,8].

Leptospirosis and Lyme disease have some overlapping geographic distributions and both can produce acute febrile disease. Although they may be self-limiting diseases in some cases, they can also have significant morbidity and mortality if not prevented or treated. The lack of rapid and accurate diagnostic testing of leptospirosis and Lyme disease often dictate empirical therapy for acute febrile illness. As the differential diagnosis of an acute febrile illness in any particular setting may be extensive, therapeutics that may cover a broad range of infectious diseases are desirable. Doxycycline and penicillin are currently the drugs of choice in the treatment of human leptospirosis, based chiefly on the fact that they are the only agents that have been studied in randomized controlled clinical trials [12,21,29]. Previous studies have shown that the newer cephalosporins and other β-lactams, as well as fluoroquinolone antibiotics, all have good in vitro activity against strains of Leptospira [26,34]. Doxycyline and tetracycline have also been used in the treatment of equine and canine borreliosis [10,35].

Although many in vitro and in vivo studies have been performed to find an optimal treatment for spirochetal disease, preferred forms of treatment are still not quite clear. Moreover, the majority of studies concerning in vitro susceptibility testing of leptospires and borreliae against antimicrobial agents were conducted with organisms recovered from human infections. The antimicrobial susceptibility of leptospires and borreliae isolated from animals is not well known. In this investigation, in vitro antimicrobial susceptibility tests were conducted with 6 spirochetal strains (4 leptospires and 2 borreliae) isolated from animal clinical cases against 3 antimicrobial agents (amoxicillin, tilmicosin, and enrofloxacin), which are used in the treatment of animal diseases.

Materials and Methods

Bacterial strains

The four strains of Leptospira spp. (L. kirchneri serovar Grippotyphosa, L. interogans serovar Pomona, L. interogans serovar Canicola, and L. interogans serovar Icterohaemorrhagiae) were obtained from National Veterinary Service Laboratory (USA) and two strains of Borrelia burdgorferi (B. burdgorferei #15770 isolated from a dog and B. burdgorferi #2769 isolated from a horse in our laboratory) were used for this study. All leptospiral and borrelial organisms were not more than 5 passages. Leptospires had been passed through hamsters three times before used. Leptospires were maintained in Ellinghausen McCullough Johnson Harris (EMJH) medium and borreliae were maintained in Barbour-Stoenner-Kelly (BSK)-II medium, as previously reported [5,13].

Antimicrobial agents

Stock antimicrobial solutions of 1 mg/ml of amoxicillin and tilmicosin, and 0.2 mg/ml of enrofloxacin were prepared with EMJH or BSK-II medium as suggested in National Committee for Clinical Laboratory Standards (NCCLS) document M31-A2 or by their manufacturers [25]. Amoxicillin was purchased from Sigma-Aldrich (USA), enrofloxacin was obtained from its manufacturer (Bayer, USA) and tilmicosin obtained from IDEXX Laboratories (Greensboro, USA). All stock antimicrobial solutions were stored in onetime-use aliquots at -70℃.

Quality control and internal validity

L. interrogans serovar Icterohaemorrhagiae was evaluated by the broth microdilution and macrodilution methods. These methods run 5 times over numerous days with several different EMJH medium lots to determine the reproducibility of our methods. Each replicate run used an individually prepared medium and inoculum suspension. Quality control and internal validity test for B. burgdorferi was also conducted with B. burgdorferi #2769 strain and BSK-II medium.

Susceptibility testing for leptospirae

Broth microdilution and macrodilution susceptibility tests were performed with a previously described technique [24]. Testing of each combination of strain and drug was performed in parallel runs by each method, microdilution and macrodilution, to compare the variability of results within and between the methods. Two parallel runs were performed at different times to determine the reproducibility of results. For each isolate and substance, independent experiments were performed on different days, with minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) reported as the median of all three experiments.

Susceptibility testing for Borreliae

Broth macrodilution MICs and MBCs were obtained with a previously described technique [31].

Statistics

To investigate possible differences in the susceptibility patterns of the spirochetal species tested, the Kruskal-Wallis test was performed for all MICs and MBCs of the antibiotics as determined by our experiments with different spirochetal isolates. The level of significance was p < 0.05.

Results

Quality control

Quality control (internal-validity) assessment of L. interrogans serovar Icterohaemorrhagiae showed consistent results (data not shown). MICs determined by microdilution fell within 2 dilutions of each other for all (100%) of 15 triplicate runs. Fourteen (93.3%) of 15 MICs determined by the macrodilution technique were within 2 dilutions of each other. A single outlier for macrodilution was produced by enrofloxacin. In macrodilution MBCs, all 15 runs (100%) fell within 2 dilutions of each other, although the majority of results for amoxicillin fell outside the upper limit of the drug concentration tested. Quality control assessment of B. burgdorferi showed consistent results (data not shown). MICs and MBCs determined by macrodilution fell within 2 dilutions of each other for all (100%) of 15 runs. An assessment of the reproducibility of our technique with a single serovar over numerous days with various lots of media appears to have produced consistent results with the techniques.

MIC and MBC

MICs and MBCs of each antimicrobial agent for the same isolate spanned a maximum range of +/-1 dilution around the median except L. interogans serovar Pomona (Table 1). While the MICs of Leptospira spp. determined by the microdilution method tended to be higher than those obtained by the macrodilution method, the mean MICs of all three drugs were within one dilution of each other by the two methods. Tilmicosin was the most potent antimicrobial against all Leptospira and Borrelia isolates on a microgramper-milliliter basis. All spirochetal isolates were inhibited (MIC) and were killed (MBC) by tilmicosin at below the limit of testing (0.01 µg/ml). The ranges of MIC and MBC of amoxicillin against Leptospira spp. were 0.05-6.25 µg/ml and 6.25-25.0 µg/ml, respectively. And the ranges of MIC and MBC of amoxicillin against B. burgdorferi were 0.05-0.39 µg/ml and 0.20-0.78 µg/ml, respectively. The ranges of MIC and MBC of enrofloxacin against Leptospira spp. were 0.05-0.39 µg/ml and 0.05-0.39 µg/ml, respectively. Two strains of B. burgdorferi were resistant to enrofloxacin at the highest concentration tested for MBC (100 µg/ml) (Table 1 and 2).

Table 1

In vitro susceptibility of 4 strains of Leptospira spp. to 3 antimicrobial agents (µg/ml)

Table 2

In vitro susceptibility of 2 strains of Borrelia burgdorferi to 3 antimicrobial agents (µg/ml)

Statistical analysis

Statistical analysis, including all measured MICs and MBCs (n = 144), did not show significant differences in the tested genospecies. The overall reproducibility seen in our study was excellent, although amoxicillin had greater variability than other antimicrobial agents tested.

Discussion

A uniform and standardized method to test antimicrobial susceptibility against pathogenic spirochetes has not been established. For borreliae and leptospires, the MIC is generally considered the drug concentration at which no motile organisms are observed by dark-field microscopy after 48-72 h of incubation [2,18]. The MBCs for leptospires and borreliae are determined by the absence of spirochetes in subcultures on microscopic examination after various incubation periods [18]. The MBC determinations made by these methods require virtually 100% killing of the final inoculum, which is strict requirement for any antimicrobial agent [9]. In this study, the MBC of antibiotics for leptospires and borreliae was defined as the lowest concentration of the antibiotics at which no spirochetes were subcultured. The macrolide antibiotic tilmicosin produced excellent in vitro activity against all 6 strains of spirochetes tested. The MIC and MBC of amoxicillin against Leptospira spp. were higher than those of tilmicosin and enrofloxacin, and the MBC of amoxicillin against Leptospira spp. was higher than noted MICs. However, enrofloxacin was not effective against B. burgdorferi tested, as shown by its high MIC and MBC.

Tilmicosin is a novel long-acting macrolide antibiotic developed exclusively for veterinary use [28]. As Mycotil, it is used to treat bovine respiratory disease associated with pasteurellae [16,23]. Tilmicosin (10 mg/kg, once) and amoxicillin (15 mg/ml, once or twice) were effective in cattle for resolving experimental leptospirosis caused by L. borgpetersenii serovar Hardjo [3,32]. Enrofloxacin was used concurrently with ampicillin in canine leptospirosis and 83% of infected dogs survived [1]. Amoxicillin is effective in treatment of canine Lyme disease [15]. However, there are no data on the clinical use of enrofloxacin or tilmicosin in borreliosis of animals.

Based upon our findings, tilmicosin was superior in vitro on a microgram-per-milliliter basis when tested alongside amoxicillin and enrofloxacin under identical test conditions in EMJH or BSK-II media. Moreover, maximum concentrations of tilmicosin in plasma after a subcutaneous dose of 10 mg/kg to cattle are over 100 times higher than the MIC against borreliae and tilmicosin has high volume of distribution that selectively accumulates in solid tissues [22,38]. The accumulation and concentration of tilmicosin in tissues could be due to its lipophilic properties, which favor passage through the lipid membrane of the cells. One mechanism by which the diffusion of tilmicosin may have led to enhance intracellular accumulation is the tendency of basic compounds to be lysosomotropic and become trapped in lysosomes as a result of the acidic pH [30]. The Lyme borrelia reside in the acidic endosome and the use of a lysosomotropic agent augments the clinical activity of macrolide antibiotics in treatment of human patients with chronic borreliosis [11]. Therefore, the potential role of tilmicosin in the treatment of leptospirosis and Lyme disease in some animal species merits further evaluation. Although no significant adverse effects have been reported in cattle, injections of Mycotil to horses, goats, swine, or nonhuman primates can be fatal. Extensive studies on its safety in dogs have not been performed. The heart is the target of toxicity in animals, perhaps mediated via depletion of cardiac intracellular calcium, resulting in a negative inotropic effect [20]. Other formulations of the tilmicosin would be necessary before it could be used in these species.

Classical macrolides and azalides frequently fail in the therapy of early Lyme disease in human and clinical relapse has been observed following conclusion of treatment [14,19, 27,37]. Moreover, it has been speculated that resistance may develop in borreliae preexposed to erythromycin owing to resistant subpopulations. We have evaluated antibiotic treatment using tetracycline, doxycline, and ceftiofur in experimental borreliosis of dogs and horses, however, most of them can not completely eliminate the persistent infection [6,33]. The lack of effective agents in the treatment of animal borreliosis made it urgent to identify other potential antibiotics. Therefore, further evaluation of the potential use of a non-toxic formulation of tilmicosin in the treatment of equine or canine leptopsirosis and/or borreliosis should be confirmed.