Detection of Borrelia burgdorferi nucleic acids after antibiotic treatment does not confirm viability

Abstract

The persistence of dormant, noncultivable Borrelia burgdorferi after ceftriaxone treatment was examined. B. burgdorferi isolates were cultivated in Barbour-Stoenner-Kelly medium in the presence or absence of ceftriaxone, and cultures were monitored for up to 56 days. Viability of B. burgdorferi was assessed by subculture, growth, morphology, and pH (as a surrogate for metabolic activity). In addition, the presence of B. burgdorferi DNA and mRNA was assayed by PCR and by real-time reverse transcription (RT)-PCR, respectively. Spirochetes could not be successfully subcultured by day 3 after exposure to ceftriaxone. In cultures treated with ceftriaxone, the pH of the culture medium did not change through day 56, whereas it declined by at least 1 pH unit by 14 days in untreated cultures. These results suggest that B. burgdorferi viability is rapidly eliminated after antibiotic treatment. Nevertheless, DNA was detected by B. burgdorferi-specific PCR for up to 56 days in aliquots from both ceftriaxone-treated and untreated cultures. In addition, although ceftriaxone treatment resulted in a reduction in the quantities of transcript for ospC, ospA, flaB, and pfk, certain mRNAs could be detected through day 14. Transcript for all 4 genes was essentially undetectable after 28 days of treatment. Taken together, the results suggest that B. burgdorferi DNA and mRNA can be detected in samples long after spirochetes are no longer viable as assessed by classic microbiological parameters. PCR positivity in the absence of culture positivity following antibiotic treatment in animal and human studies should be interpreted with caution.

Fig 1

Survival of B. burgdorferi after ceftriaxone treatment. (A and B) Growth of B. burgdorferi B31A3 (A) or BL206 (B) in BSK medium. Values are means ± standard errors of the means (SEM) for measurements from duplicate cultures. (C) Metabolic activity of B. burgdorferi in BSK medium (based on acidification of medium). (D) Viable B. burgdorferi cells as assessed by fluorescence microscopy. ●, B31A3; ■, B31A3 plus ceftriaxone; ○, BL206; □, BL206 plus ceftriaxone.