This is shown by 7 day subculture of B. burgdorferi persister cells treated with 3 drug combinations daptomycin+ doxycycline+cefoperazone or Smx or Cfz, which all produced no detectable levels of any residual growth. However, extended click more info incubation to 15 days of subculture showed that only daptomycin, doxycycline and cefoperazone combination was able to completely eradicate biofilm- like microcolonies with no detectable spirochetes. These findings suggest that longer incubation to 15 days or more in post-antibiotic exposure may be needed to thoroughly assess the drug combinations that stably eradicate the persister forms without relapse. The subculture results do validate the SYBR Green I/PI viability assay and is a useful and more sensitive technique to assess the viability of B. burgdorferi persisters or microcolonies after drug treatment in identifying optimal drug combinations for killing more resistant persisters. B. burgdorferi spirochetes could develop morphological variants as in vitro cultures age or are subjected to adverse conditions. The proportions of these variants have not been well characterized over time in culture conditions. With careful measurement, the percentages of morphological variants were determined as they transitioned from spirochetes to progressively round body form to then microcolony form as log phase culture grew to stationary phase. Although previous studies reported the round body form or biofilm-like microcolony form is more resistant to antibiotics, their relative resistance was not fully studied. Here, we found a hierarchy or varying levels of stationary phase B. burgdorferi persisters in terms of their levels of persistence as the morphology of the variants changes from spirochetes, to round body, and to microcolony forms, with increasing antibiotic tolerance. Future studies are needed to address the molecular basis of morphological transitions and their relationship to persistence in vivo. The finding that persister frequencies are higher in stationary phase B. burgdorferi cultures than in log phase cultures is consistent with studies in other bacteria. However, the persister frequencies in B. burgdorferi log phase culture and stationary phase cultures determined by SYBR Green I/PI assay seem to be higher than those reported for E. coli. CFU is the gold standard of current persister assays for bacteria that form colonies readily on agar plates. However, because B. burgdorferi does not form colonies easily on plates, especailly after antibiotic treatment, we have to rely on viability staining using microscope counting of green cells stained by viability dye SYBR Green even though CFU and viability by viability staining are two different measures of bacterial viability. To more accurately assess the percentage of B. burgdorferi persisters after antibiotic treatment, we used E. coli as a control to determine the correlation between the CFU assay and the SYBR Green assay. We found that the SYBR Green I/PI assay tended to overestimate the persister frequency by about 5 fold based on the E. coli data. The overestimation of the persister frequency by SYBR Green I/PI assay may be due to some live persisters and/or injured cells that are unable to recover and form colonies on agar plates. Assuming the same correlation holds true for B. burgdorferi, the converted persister frequencies of 1-2% and 3-5% for B. burgdorferi log phase and stationary phase cultures would still suggest higher persister frequencies with B. burgdorferi than E. coli, which has 0.001% persisters in log phase and 1% in stationary phase. In this study, the log phase E. coli had a considerably higher persister frequency of 0.9% by the CFU assay, and this could reflect differences in the higher inoculum and use of non-shaking condition, which could greatly increase the persister frequency. On the other hand, the higher persister frequencies for B. burgdorferi than E. coli could indicate that B. burgdorferi may form persisters more readily or reflect differences in the speed of growth of the organisms, the age of culture when antibiotic is added, and the dilution factor which affects the number of persisters carried over during the subculture. In addition, we found that the persister frequencies vary according to antibiotic exposure, with the more effective antibiotic ceftriaxone having a lower persister frequency than amoxicillin, a finding that is consistent with previous studies. It remains to be determined if there are differences in persistence of B. burgdorferi strains and if the high persister frequencies in B. burgdorferi strains are associated with recalcitrance to antibiotic therapies. In conclusion, we found there is a hierarchy of B. burgdorferi persisters with increasing antibiotic tolerance as the culture ages from log phase to stationary phase with morphological changes from spirochetal form to round body and microcolony forms. Importantly, we identified drug combinations that have high activity against B. burgdorferi persisters with daptomycin- containing combinations achieving the best activity.