Resurgence of persisting non-cultivable Borrelia burgdorferi following antibiotic treatment in mice

Emir Hodzic, Denise Imai, Sunlian Feng, Stephen W Barthold, Emir Hodzic, Denise Imai, Sunlian Feng, Stephen W Barthold

Abstract

The agent of Lyme borreliosis, Borrelia burgdorferi, evades host immunity and establishes persistent infections in its varied mammalian hosts. This persistent biology may pose challenges to effective antibiotic treatment. Experimental studies in dogs, mice, and non-human primates have found persistence of B. burgdorferi DNA following treatment with a variety of antibiotics, but persisting spirochetes are non-cultivable. Persistence of B. burgdorferi DNA has been documented in humans following treatment, but the significance remains unknown. The present study utilized a ceftriaxone treatment regimen in the C3H mouse model that resulted in persistence of non-cultivable B. burgdorferi in order to determine their long-term fate, and to examine their effects on the host. Results confirmed previous studies, in which B. burgdorferi could not be cultured from tissues, but low copy numbers of B. burgdorferi flaB DNA were detectable in tissues at 2, 4 and 8 months after completion of treatment, and the rate of PCR-positive tissues appeared to progressively decline over time. However, there was resurgence of spirochete flaB DNA in multiple tissues at 12 months, with flaB DNA copy levels nearly equivalent to those found in saline-treated mice. Despite the continued non-cultivable state, RNA transcription of multiple B. burgdorferi genes was detected in host tissues, flaB DNA was acquired by xenodiagnostic ticks, and spirochetal forms could be visualized within ticks and mouse tissues by immunofluorescence and immunohistochemistry, respectively. A number of host cytokines were up- or down-regulated in tissues of both saline- and antibiotic-treated mice in the absence of histopathology, indicating host response to the presence of non-cultivable, despite the lack of inflammation in tissues.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Borrelia burgdorferi levels resurge in…
Figure 1. Borrelia burgdorferi levels resurge in tissues at 12 months after antibiotic treatment.
Copy numbers of B. burgdorferi flaB DNA, determined by quantitative PCR, in ear, inoculation site (Inoc), heart base (HB), ventricular muscle (VM), quadriceps muscle (QM), and tibiotarsus (Tt) tissues of saline- and antibiotic-treated mice at 12 months after treatment.
Figure 2. Antibody titers to Borrelia burgdorferi…
Figure 2. Antibody titers to Borrelia burgdorferi are significantly lower following antibiotic treatment, and do not rise in response to resurgence at 12 months.
Reciprocal end-point dilutions of sera reacted against B. burgdorferi lysate antigen.
Figure 3. Spirochetes are present within xenodiagnostic…
Figure 3. Spirochetes are present within xenodiagnostic ticks that fed upon saline- or antibiotic-treated mice at 12 months after treatment.
Indirect immunofluorescent staining of B. burgdorferi in the midguts of ticks that fed upon saline-treated (A) or ceftriaxone-treated mice (B) at 12 months after treatment.
Figure 4. Persisting B. burgdorferi elicit host…
Figure 4. Persisting B. burgdorferi elicit host cytokine responses at 12 months following saline or antibiotic treatment.
Transcription of 19 host cytokine cDNAs, relative to uninfected, age-matched control mice in heart base (HB), ventricular muscle (VM), quadriceps muscle (QM) and tibiotarsus (Tt) at 12 months after treatment with saline or antibiotic.
Figure 5. Spirochetes can be visualized within…
Figure 5. Spirochetes can be visualized within tissue of a mouse at 12 months following antibiotic treatment.
Indirect immunohistochemical staining of B. burgdorferi spirochetes (arrows) in the heart base connective tissue.

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