Study
Conducted at the University of Jyväskylä, Finland

Aim

To clarify the impact of the Lyme Plus Protocol (APP Plus, DTC Plus and TBB Plus) on Borrelia burgdorferi that has been cultured in vitro at +37°C

 

Hypothesis

An efficient therapy, antimicrobial agents that can eliminate all the forms of B. burgdorferi have to be found. Efflux pumps give Borrelia resistance to several antibiotics by antimicrobial efflux, and combining antimicrobial therapy with non-toxic compounds that inhibit efflux could increase antimicrobial efficiency against all the pleomorphic forms of B. burgdorferi.

 

Study objectives

Studying the effects of antibiotics and herbal compounds of the Lyme Plus Protocol on the different pleomorphic forms of B. burgdorferi. The aim of these experiments is to use sensitivity and specificity studies to find out what kind of effects the compounds individually have on different pleomorphic forms of B. burgdorferi. MIC and MBC of the individual compounds will be assessed.

Initial Experimental Setup


1. Minimum Inhibitory Concentration (MIC)

  • MIC will be determined according to Ates et al. (2010) with modifications to evaluate the antimicrobial properties of the compounds. The lowest antibiotic concentration at which Borrelia fails to grow will be interpreted as the MIC. 1 x 10e6 cells in volume of 200 ul. Cultured in vitro at +37°C for 24, 48, 72, 96 hrs post-exposure to antimicrobial agent. Fluorescent reading from the bacteria is recorded.
  • Pleomorphic forms are observed with the determine MIC.
  • Concentrations of antibiotics and Lyme Plus Protocol compounds were 5, 50, 100, 200, 400, 800 and 100 µg/ml or otherwise stated.

2. Minimum Bactericidal Concentration (MBC)

  • MBC will be determined according to Ates et al. (2010) with modifications. The cultures with antimicrobials that do not have bacterial growth after 72 h will be subcultured for 3 weeks in vitro at +37°C in fresh media.
  • The lowest antimicrobial concentration that will not contain live bacteria after this subculturing step will be considered as the MBC.

Results:
MIC of antibiotics

MIC 100 μg/ml confirmed by MBD
No Pleomorphic forms but Damaged Cells

 

Relative fluorescence units (RFU) of fluorescent B. burgdorferi strain with azithromycin, clindamycin and doxycycline over time when background media subtracted. B. burgdorferi spirochetes (1 x 106/ml) were various concentrations of the antibiotics. H2O2 was used as a control for death. Representative image of the similar results obtained from all three antibiotics. Data is presented from three different experiments.


Results:
MICs of Lyme Plus Protocol Compounds

 

Relative fluorescence units (RFU) of fluorescent B. burgdorferi strain with azithromycin, clindamycin and doxycycline over time when background media subtracted. B. burgdorferi spirochetes (1 x 106/ml) were various concentrations of the antibiotics. H2O2 was used as a control for death. Representative image of the similar results obtained from all three antibiotics. Data is presented from three different experiments.

MIC 5 μg/ml confirmed by MBD
No Pleomorphic forms but Damaged Cells

 

Relative fluorescence units (RFU) of fluorescent B. burgdorferi strain APP Plus, DTC Plus and TBB Plus over time when background media subtracted. B. burgdorferi spirochetes (1 x 106/ml) were various concentrations of the antibiotics. H2O2 was used as a control for death. Representative image of the similar results obtained from all three antibiotics. Data is presented from three different experiments.

Summary for Initial Experiments


  • Minimum Inhibitory Concentration (MIC): all Lyme Plus Protocol compounds had a 5 μg/mL which was confirmed by Minimum Bactericidal Concentration (MBC). MIC of antibiotics was 100 μg/ml and confirmed by MBD.
  • The Lyme Plus Protocol compounds did not induce pleomorphic forms. Antibiotics showed damaged cells.
  • Over growth of microbes was observed after the first 24 hrs in the MICs and also in the MBC.
  • Due to observed microbial growth, efflux studies were not conducted on the unfiltered Lyme Plus Protocol compounds.

Second Experimental Setup


  • Lyme Plus Protocol compounds were dissolved in sterile H2O for stock of 200 mg/ml
  • Antibiotics were used directly from solutions
  • Fluorescent B. burgdorferi GFP strain with initial concentration of 5 x 106 bacteria/ml in a total volume of 3.5 ml were grown at 37°C
  • Concentrations of 1000 and 2000 μg/ml herbal compounds were used unless otherwise stated. 100 μg/ml doxycycline was used as the negative control
  • Spirochete and Lyme Plus Protocol compounds without spirochetes were used as controls
  • The fluorescent readings were performed in 96-well plates with Victor X Multilabel Plate reader (PerkinElmer) at time points 0h, 24, 48h, 72h and 96h. Samples were analyzed as duplicates, with 200 μl of each sample/well Excitation/emission wavelengths of 485/535 nm were used
  • The data was normalized/scaled in each experiment with the smallest value of 0 h time point
  • The experiments were performed three times

Results:



TBB Plus Studie with background subtracted

Summary for Second Experiments


  • Phase Contrast Images indicated that the Lyme Plus Protocol compounds did NOT induce pleomorphic forms of Borrelia.
  • Phase Contrast Images indicated that the Lyme Plus Protocol compounds caused bacteria cell death. There was considerable cell death at 24 h post-treatment with APP, DTC and TBB Plus compounds.
  • Phase Contrast Images also indicated that there were unidentified microbes surviving in the APP, DTC and TBB Plus experiments.
  • Minimum Inhibitory Concentration (MIC) experiments of the Lyme Plus Protocol was 5 μg/mL – 50 μg/mL and was confirmed by minimal bacterial death (MBD) experiments.