Mice.

Five-week-old female C3H/HeN (C3H) mice were purchased from the Frederick Cancer Research Center, Frederick, MD, and C3H.C-Prkdc^scid/IcrSmnHsd (SCID) mice were purchased from Harlan Laboratories, Inc., Indianapolis, IN. Mice were specific pathogen free, maintained in an isolation room under static filter-top cages, and provided food and waterad libitum. Mice were necropsied following an American Veterinary Medical Association-approved euthanasia procedure of carbon dioxide narcosis and exsanguination by cardiocentesis. The presence of infectious spirochetes in tissues of various treated mice was assessed by subcutaneous transplantation of small pieces of tibiotarsal joint and heart base tissue into recipient SCID mice under ketamine/xylazine anesthesia, as described previously (6,8). Animal use was approved by the University of California at Davis (UCD) Animal Care and Use Committee. UCD has a Public Health Service Animal Welfare Assurance on file and is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.

Borrelia burgdorferi.

Mice were infected by subdermal inoculation of the dorsal thoracic midline with 10⁴ mid-log-phase clonalB. burgdorferi N40 (cN40) cells in 0.1 ml of modified Barbour-Stoenner-Kelly (BSK II) medium (5). All mice were inoculated with the same dose of spirochetes and at the same time. Infection status of mice was determined by culture and PCR. Tissues (urinary bladder and inoculation site) collected from mice at necropsy were cultured in BSK II medium, as described previously (7). Based upon serial dilutions of quantified cultured spirochetes, culture in BSK II medium is sensitive for detection down to the level of single organisms (27). Tissues were also processed forospA PCR (inoculation site, heart base, ventricular muscle, left tibiotarsus, and quadriceps muscle).

Antibiotic.

Mice were treated with 16 mg/kg of body weight ceftriaxone (Hoffman LaRoche, Nutley, NJ) in a 500-μl total volume of 0.9% normal saline solution administered intraperitoneally twice daily for 5 days and then once daily for 25 days. Mice were treated with low (12.5 mg/kg) or high (50 mg/kg) doses of tigecycline (Wyeth Pharmaceutical, Pearl River, NY), administered subcutaneously once daily for 10 days. The ceftriaxone treatment regimen served as a control group for comparison with previously published studies that demonstrated persistence ofB. burgdorferi after treatment (13,27). Tigecycline doses were based on recently published tigecycline pharmacokinetic data in mice (35), which showed the 12.5-mg/kg dose to be similar to the serum drug concentrations achieved in humans.

MICs of antibiotics were determined againstB. burgdorferi N40 andB. burgdorferi B31 using a 96-well microdilution method with minor modifications, as described previously (80). Duplicate wells containing BSK-H medium (Sigma-Aldrich, St. Louis, MO) with and without the appropriately diluted antimicrobial agents were inoculated to final density of 5 × 10⁶ cells/ml of the test organism. The ranges of antibiotic concentrations tested were 0.006 to 25.0 mg/liter. After incubation at 34°C for 72 h, 10-μl aliquots were extracted from each well and live (motile)Borrelia cells were examined under dark-field microscopy. The MIC was the lowest concentration of antibiotic at which the number of cells after incubation did not exceed the initial concentration. The minimum bactericidal concentration (MBC) was the lowest antibiotic concentration from which spirochetes could not be cultured after 72 h of coincubation with antibiotics. Following 72 h of incubation with antibiotic, an aliquot (20 μl) from each test well was transferred to 5 ml of fresh BSK-H medium and subcultures were assessed for the presence of motile spirochetes at 21 days.

Ticks.

Ticks were derived from a single population of laboratory-rearedIxodes scapularis larvae kindly provided by Durland Fish of Yale University. For xenodiagnosis, 40 larval ticks were placed on mice 1 week prior to necropsy and allowed to feed to repletion. Engorged larvae were allowed to molt into nymphs and harden, and samples of each tick cohort from each mouse were individually tested byospA PCR to determine the prevalence of infection within each cohort. Ten nymphal ticks from positive cohorts were allowed to attach and feed upon naïve SCID mice to determine the presence of infective spirochetes. Some of the nymphs were allowed to molt into adult ticks, which were then dipped in 70% ethanol and bisected sagittally using a sterilized razor blade. One-half of the tick was placed in BSK II medium containing 50 mg/ml rifampin, 20 mg/ml phosphomycin, and 2.5 mg/ml amphotericin B; incubated at 33°C for 11 to 14 days; and then examined for spirochete growth under dark-field microscopy. The other half of the tick was frozen in liquid nitrogen and then ground with a mortar and pestle, and DNA was extracted for PCR analysis.

PCR analysis.

Quantitative real-time PCR was utilized to detectospA DNA in mouse tissues and ticks, as previously described (25,26). TheospA target was chosen because of the known “target imbalance” ofospA over other gene targets in spirochetal DNA obtained from tissues (51), which has been confirmed in multiple studies in this laboratory. All samples were assayed with positive and negative controls. In addition, since copy numbers of target DNA are typically very low in antibiotic-treated mice, the specificity of the real-time PCR was verified on skin, heart base, ventricular muscle, quadriceps muscle, and tibiotarsal tissues from 4 uninfected mice (20 sites total) and subjected toflaB,ospA, anddbpA real-time PCR amplification. All samples were negative for all 3 gene targets, and 16 positive control tissues from previously archived antibiotic-treated mice were positive. DNA was extracted from tissues or ticks with DNeasy kits (Qiagen, Valencia, CA) according to manufacturer's instructions for tissue or insects, respectively. Prior to DNA extraction, tissue samples were weighed. Data are expressed as the number of DNA copies per mg of tissue or per tick.

A multiplex nested quantitative real-time reverse transcriptase PCR (RT-PCR) for cDNA of theflaB,ospA,ospC,arp,dbpA,fbp (bbk-32),oppA-2, and host β-actin genes was developed, using previously publishedB. burgdorferi N40-specific primer sequences (25). Although nested RT-PCR is not precisely quantitative, this assay was used to detect RNA transcription in tissues of mice subjected to the various treatments. Total RNA from each sample was purified using RNeasy minikits, according to the manufacturer's instruction for tissues (Qiagen, Valencia, CA). Samples were frozen in liquid nitrogen and homogenized with a mortar and pestle (pretreated with 0.1% diethylpyrocarbonate in water overnight, and then autoclaved). RNA was purified using the RNeasy protocol for animal tissues. After elution, RNA was treated with RNase-free DNase I and then repurified using the RNeasy cleanup protocol. Extracted total RNA was stored at −80°C until use. The extracted total RNA was subjected to two separate reactions: one to synthesize cDNA and the other to test for DNA contamination. The reverse transcription step was performed utilizing TaqMan reverse transcription reagents (PE Biosystems, Foster City, CA) with random hexamers. cDNA synthesis and the control reaction were amplified in a standard heat block instrument after incubation for 10 min at 25°C, following transcription at 48°C for 30 min and inactivation at 95°C for 5 min. For the preamplification step, a mixture of outer primers for each target gene (designed with Primers Express software), the Advantage cDNA polymerase system (Clontech, France), deoxynucleoside triphosphates (dNTPs), and cDNA was amplified in a thermocycler: 1 cycle at 94°C for 1 min; 25 cycles at 94°C for 15 s, 55°C for 15 s, and 70°C for 40 s; and 1 cycle at 70°C for 5 min. Following preamplification, individual RT-PCRs were set up using individual sets of inner primers and labeled probes. Amplification, data acquisition, and data analysis were performed in an ABI Prism 7700 sequence detector (Perkin-Elmer, Applied Biosystems, Foster City, CA).

Serology.

Antibody titers were determined againstB. burgdorferi N40 lysates by enzyme-linked immunosorbent assay (ELISA), using duplicate 3-fold serial dilutions of test serum and alkaline phosphatase-conjugated rat anti-mouse immunoglobulin (heavy and light chain), as described previously (18,27). Each assay included positive and negative controls. Cutoff points for each serum dilution were established by determining absorbance means of normal mouse sera and adding 3 standard deviations (SD) above the means.

Histology.

Tissues were fixed in neutral buffered formalin, and rear legs were demineralized in acid. Legs and hearts were embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin by standard methods. Legs and hearts were blindly examined and scored for inflammation, as described previously (2,7,8,10,27).

Statistical analysis.

Statistical analysis of quantitative PCR data between treatment groups and time intervals was performed by one-way analysis of variance, followed by multiple pairwise comparisons by Tukey's honestly significant difference (HSD) test (SPSS 16.0 for Mac; SPSS, Inc., Chicago, IL). Antibody titers were compared among treatment groups with Student's unpairedt test. CalculatedP values lower than 0.05 were considered significant.

Determination of MICs and MBCs.

The MICs and MBCs for tigecycline againstB. burgdorferi N40 and B31 were compared to doxycycline, ceftriaxone, and penicillin in order to evaluate the relative effectiveness of tigecycline and the relative sensitivity ofB. burgdorferi N40. Tigecycline and ceftriaxone had the lowest MICs against bothB. burgdorferi N40 and B31, compared to doxycycline and penicillin (Table1). When the MBCs ofB. burgdorferi were compared to peak serum levels obtained in previous mouse studies, the serum maximum concentrations of tigecycline and ceftriaxone were at least 100 times greater than theB. burgdorferi N40 tigecycline MBC and 70 times greater than theB. burgdorferi N40 ceftriaxone MBC (35,61,75). The MIC and MBC forB. burgdorferi N40 were essentially similar to those ofB. burgdorferi B31 for any of the antibiotics tested. Therefore,B. burgdorferi N40 was considered suitable for examining efficacy of tigecycline in the mouse model.

Experimental design and rationale.

Groups ofB. burgdorferi-infected C3H mice were treated with high-dose tigecycline, low-dose tigecycline, ceftriaxone, or saline. Saline-treated mice served as negative-effect controls, and ceftriaxone-treated mice served as positive-effect controls, using a previously published regimen that resulted in persistence of noncultivable spirochetes (13,27). Treatment was commenced at 1 week, 3 weeks, or 4 months of infection. Based upon previous studies in C3H mice, these stages of infection represented (i) an early preimmune phase of infection (1 week) in which spirochetes are in the process of disseminating from the site of inoculation, (ii) an early immune phase of infection (3 weeks) in which the host immune response is under way and beginning to resolve joint and heart inflammation and globally reducing spirochete burdens in tissues, and (iii) a late phase of infection (4 months) in which inflammation is generally absent and infection is persistent (7,9,25). Early and late stages of infection were evaluated because treatment regimens for later-stage Lyme disease in humans require longer and sometimes repeated courses of antibiotics (68,78). Mice were necropsied at 3 months after completion of treatment, which provided an interval that would test for survival and outgrowth of spirochetes well after antibiotic levels had dissipated.

Preimmune phase (1 week) treatment.

All of the antibiotic-treated mice were culture negative, but approximately half of the mice in each of the antibiotic treatment groups had one or more tissues with detectableospA DNA (Table2). Copy numbers per mg of PCR-positive tissue (negative samples not included in calculations) did not differ significantly (P ≥ 0.05) among antibiotic treatment groups, but were significantly lower than mean copy numbers per mg of PCR-positive tissues from saline-treated mice (P = 0.25). Cumulative percent positive PCR results from all tissues tested from mice treated with antibiotics suggested that antibiotic treatment reduced dissemination or the number of tissues involved compared to that in saline-treated control mice (P < 0.001).

Early immune phase (3 week) treatment.

All of the antibiotic-treated mice were culture negative, but all mice, regardless of antibiotic treatment, had one or moreospA PCR-positive tissue sites (Table3). There were no significant differences inospA copy numbers per mg of PCR-positive tissue among antibiotic treatment groups (P > 0.05). Cumulative percent positive PCR results from all tissues tested from mice treated with antibiotics suggested that antibiotic treatment reduced dissemination or the number of tissues involved compared to that in saline-treated control mice, but differences were only marginally insignificantly different (P = 0.069). There was no significant difference (P = 0.261) betweenospA copies in saline-treated mice versus antibiotic-treated mice due to the wide range of values in the saline group, reflecting the nonuniform distribution of spirochetes in tissues.

Late persistent-phase (4 months) treatment.

As with the other intervals, all of the antibiotic-treated mice were culture negative. Regardless of antibiotic treatment, all mice had one or moreospA PCR-positive tissues (Table4). There were no significant differences inospA copy numbers per mg of PCR-positive tissue among antibiotic treatment groups (P > 0.05), and differences were marginally insignificant compared to saline-treated control mice (P = 0.063). Cumulative percent positive PCR results from all tissues tested from mice treated with antibiotics suggested that antibiotic treatment reduced dissemination or the number of tissues involved compared to that in saline-treated control mice, but differences did not reach statistical significance (P = 0.086).

Differences among treatment intervals.

Analysis of mean copy numbers ofospA DNA in PCR-positive tissues across all treatment intervals and between treatment groups revealed significant (P = 0.023) differences among antibiotic-treated mice and saline-treated control mice. Likewise, there was a significant difference (P < 0.001) in the percent PCR-positive tissues across intervals between antibiotic-treated mice and saline-treated control mice.

Antibody response.

ELISA of antibody toB. burgdorferi lysates (Fig.1) indicated different titers among saline-treated mice, depending upon phase of infection. These differences reflected the different intervals after infection (approximately 4, 5, and 8 months). With the exception of mice treated with low-dose tigecycline during the late persistent phase of infection, antibiotic treatment resulted in significant reduction (P ≤ 0.05) in titers at 3 months after antibiotic treatment compared to those in saline-treated mice. Antibody titers were less reduced compared to saline-treated controls when mice were treated during the late persistent phase of infection.

RNA transcription.

Because of the high prevalence of positive DNA results among heart base tissues during the early immune phase interval, heart base tissues from DNA-positive samples were analyzed for RNA transcription by nested RT-PCR (Table5). Samples were tested for theflaB,ospA,ospC,arp,dbpA,bbk-32,oppA-2, and mouse β-actin (mouse positive control) genes. These targets were selected because they are considered to be constitutively expressed (flaB), downregulated (ospA), or upregulated (ospC,arp,dbpA,bbk-32, andoppA-2) during infection (25) or were involved in environmental adaptation as one of only a few transporters encoded by theB. burgdorferi genome (oppA-2) (42,76). Most of the samples from saline-treated mice were positive for all targets (exceptospA). RNA was detected variably among tissues from antibiotic-treated mice, depending upon the RNA target. Notably, none of 16 samples from antibiotic-treated mice had detectable levels offlaB, and the most common target detected in tissues from antibiotic-treated mice wasoppA-2. These results indicate viability and metabolic activity among the persisting spirochetes.

Xenodiagnosis and transmission of spirochetes.

Xenodiagnosis was performed on mice treated with high-dose tigecycline or saline during the late persistent phase of infection. Larval ticks were fed to repletion on each of the mice (one week prior to necropsy) and allowed to molt, and 15 to 23 nymphal ticks from mice in each of the treatment groups were allowed to feed upon individual SCID mice. Two SCID mice were infested with ticks from high-dose tigecycline-treated mice, and 3 SCID mice were infested with ticks from saline-treated mice. None of the tissues from SCID mice fed upon by ticks from antibiotic-treated mice were culture positive, whereas all 3 of the SCID mice fed upon by ticks from saline-treated mice were culture positive. Ventricular muscle and quadriceps muscle from each of the 2 SCID mice fed upon by ticks from tigecycline-treated mice wereospA positive (15.8 to 19.5 copies/mg tissue). All 3 SCID mice that were fed upon by ticks from saline-treated mice had multiple tissues that wereospA positive (range, 2,870 to 162,000 copies per mg tissue; mean ± SD, 40,673 ± 60,284). Inflammatory lesions were present in the hearts and joints of only the SCID mice fed upon by ticks from the saline-treated mice. Thus, mice treated with tigecycline during the late phase of infection remained infected with spirochetes that could be acquired by ticks and could be transmitted by ticks to SCID mice, but the spirochetes were present in very low numbers and did not induce disease.

Xenodiagnosis was also performed on 8 ceftriaxone-treated mice treated during the early immune phase of infection. This treatment group was selected to examine the phenomenon of tick acquisition and transmission through molting into adult stage, in order to confirm and extend previous studies with ceftriaxone (13,27). Forty larval ticks were fed to repletion on each mouse (one week prior to necropsy), and allowed to molt into nymphs. Cohorts of nymphs from each mouse were sampled to determine acquisition of infection by the ticks, and 3 of the cohorts were found to contain PCR-positive ticks. Six of 7, 6 of 8, and 3 of 4 nymphs from the 3 cohorts wereospA PCR positive. Copy numbers ofospA DNA ranged from 31.1 to 1,840 copies per tick. The remaining nymphal ticks were pooled, and 15 nymphs were allowed to feed on each of 7 SCID mice. The SCID mice were necropsied at 30 days after tick feeding, and tissues were cultured (skin near tick feeding sites, quadriceps muscle, heart, joint, and urinary bladder) and sampled forospA PCR reactivity (tick feeding site, ventricular muscle, quadriceps muscle, joint, and ear). Ticks on all 7 SCID mice fed to repletion and were collected, but 4 of the SCID mice died prior to necropsy (water bottle accident). None of the tissues from the remaining 3 SCID mice was culture positive, but all tissues from the SCID mice were positive forospA DNA, ranging from 1.8 to 98ospA copies per mg tissue. These results indicated that ticks feeding upon ceftriaxone-treated mice acquired infection and transmitted infection to SCID mice, as previously shown (27). None of the mice had inflammatory lesions in their joints or hearts.

In an effort to determine if spirochetes from ceftriaxone-treated mice survived molting from the nymphal stage into adults, the remaining nymphs that fed upon the SCID mice were allowed to molt into adults. The adult ticks were bisected, one-half was cultured, and the other half was assayed forospA DNA. All 78 adult ticks were culture negative, but 4 of the 78 ticks (2 of which fed upon the same mouse) wereospA PCR positive, with copy numbers ranging from 154 to 234 (mean ± SD, 150.2 ± 101.7)ospA copies/tick. Thus, spirochetes survived molting in at least some of the ticks, but continued to be resistant to culture, suggesting a continued state of nonreplication or low replication.

Tissue transplant transmission of spirochetes.

At the time of necropsy of mice treated during the late persistent phase of infection, samples of tibiotarsal tissue and heart base tissue from each mouse that was treated with high-dose tigecycline, ceftriaxone, or saline were transplanted subcutaneously into a single SCID mouse for each treated donor mouse (Table6). Previously published studies with ceftriaxone revealed a very low level of transmission by tissue transplant, but those studies used ear tissue, which was less apt to be persistently infected after antibiotic treatment (27). Therefore, tibiotarsal and heart base tissue from treated mice were utilized in the current study, since these sites were more consistently positive after treatment. The recipient SCID mice were necropsied at 4 weeks after transplantation, and blood, urinary bladder, and transplantation sites were cultured. None of 9 SCID mice transplanted with tissue from high-dose tigecycline-treated mice and none of 10 SCID mice transplanted with tissue from ceftriaxone-treated mice were culture positive. Four of nine SCID mice that received transplants from high-dose tigecycline-treated mice had one or more tissue sites that wereospA PCR positive. Four of 10 SCID mice that received transplants from ceftriaxone-treated mice had single tissue sites that wereospA PCR positive. Copy numbers ofospA DNA ranged from 1.2 to 18.6 copies per mg tissue. In contrast, all 10 SCID mice transplanted with tissues from the saline-treated mice were culture positive, including 9 of 10 with bacteremia. Furthermore, all tissue sites containedospA DNA, with copy numbers ranging from 144 to 112,000 copies per mg tissue. Histology of joint and heart tissue of the recipient SCID mice revealed inflammation in all knees and tibiotarsal joints (arthritis) and inflammation in all hearts (carditis) of mice receiving transplants from saline-treated donors, whereas there were no detectable lesions in joints or hearts of SCID mice receiving transplants from mice treated with ceftriaxone or high doses of tigecycline. These results demonstrated that tissues from antibiotic-treated mice contained spirochetes that were capable of infecting SCID mice following transplant of tissues and were capable of dissemination to multiple sites, but they did not induce arthritis or carditis.

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