Grapefruit seed extract is a powerful in vitro agent against motile and cystic forms of Borrelia burgdorferi sensu lato.

Lyme borreliosis [1], caused by Borrelia burgdorferi sensu lato, may lead to long-term tissue infection, which may be difficult to cure. The outcome of Lyme borreliosis is highly dependent on the antibiotic treatment [2]. The observation of the ability of B. burgdorferi sensu lato to convert (and reconvert) to cystic forms [3–5] may explain why the infection sometimes is persistent and reactivating. Therefore, it might be important to eradicate all germative forms (not only the motile form) of the bacterium to obtain a proper treatment for Lyme borreliosis. Grapefruit-seed extract (GSE) contains bioactive flavenoids (e.g., hesperitin, resveratrol, and naringenin) and has been shown to possess anti-microbiological effect against bacteria and fungus [6, 7]. Many studies indicate that GSE is a substance whose therapeutic effect ranks equal to or better than other known anti-bacterial agents. Positive effects of GSE are decreased levels of TNF-α, Nuclear factor Kb, NO, protection of the gastrointestinal tract against mechanical stress, and has anti-allergic and other antioxidative properties [8, 9]. Naringenin, hesperidin and other citrus flavones have been found in plasma and tissue after ingestion [10]. Lactobacillus and bifidobacteria in the gut seems to be insignificantly affected by GSE [6], and no severe side effects have been observed. B. burgdorferi sensu lato has a gene for efflux mechanism which may be responsible for antibiotic resistance [11]. GSE is an efflux inhibitor, which can be used to enhance the activity of antibacterial agents [12]. For the reasons mentioned above it is reasonable to test the hypothesis that motile and cystic forms of B. burgdorferi sensu lato will be susceptible to GSE, and this is the aim of our study. The bacterial strain used in our experiments was B. afzelii ACA-1. Production of mobile spirochetes and cystic forms was performed according to our previous procedure [13]. Grape fruit seed extract 33% (Citrosept; Cintamani Europe AS, 2071 Råholt, Norway) was diluted in distilled water, sterile filtered by a 0.2 μm filter, and diluted geometrically in 5 ml Nalgene tubes from 0.33%–0.00064% in 2 ml of diluted BSK-H medium (dilution 1:100 in distilled water). The control was diluted BSK-H. Two ml suspension of cystic forms at an age of 1 h was added to each of the tubes giving a final GSE concentration of 0.165%–0.00032%. Susceptibility testing of mobile spirochetes to GSE was performed in a final dilution of GSE from 0.165% to 0.00032% in BSK-H medium. Forty microliter of 107/ml bacteria in logarithmic growth was added, making the final volume 4 ml in each tube. One control with only BSK-H was used. To examine if GSE could prevent the conversion of mobile spirochetes to cystic forms, testing was also performed in distilled water for 1 h at 34 °C. One control with only distilled water was used. Motile bacteria in distilled water and BSK-H medium were incubated aerobically. The tubes with the mobile borrelia in BSK-H medium and the cysts in diluted BSK-H were examined by Dark Field Microscopy (DFM) (400×) after 1 h and 7 days to detect presence of eventual mobile spirochetes and intact cysts. Bacteria exposed to GSE in water were examined by DFM at 400× to examine the ratio of cyst/bacteria. Vital staining was performed on bacteria exposed to GSE for 1 week by mixing 10 μl of Live/dead BacLightTM bacterial viability kit (Molecular Probes L-13152 Eugene, OR, USA) with 10 μl of the culture. This mixture was placed on a glass slide protected with a coverslip. The BacLight-stained bacteria were examined by UV-microscopy (800×).