Prior studies have suggested that Akt1 is essential for proper lactation during being pregnant and nursing

Previous U0126 studies showed that tinidazole, metronidazole, and tigecycline were more active against B. burgdorferi round body and microcolonies than doxycycline and amoxicillin, but they could not completely kill the microcolonies even at high concentrations of antibiotics, indicating the limited activity of these individual antibiotics against B. burgdorferi persisters. Although tigecycline was the most active antibiotic against the round body form compared with tinidazole and metronidazole in that study, we found that by itself tigecycline was not very effective at killing the biofilm-like microcolonies. Remarkably, we found that daptomycin in combination with doxycycline and cefoperazone was able to completely eradicate the most resistant microcolonies, and this was further confirmed by subculture studies which showed lack of any growth. While various drug combinations showed improved activity against stationary phase B. burgdorferi persisters, daptomycin combinations had the best activity among drug combinations against persisters. The only non-daptomycin regimens that were close to daptomycin combinations contained cefoperazone. Unexpectedly, other antibiotics such as sulfamethoxazole, clofazimine and miconazole also had more activity against stationary phase B. burgdorferi persisters in combination with doxycycline and cefoperazone. These drugs are not currently used as antibiotics for treatment of Lyme disease clinically. Although sulfa drugs are bacteriostatic when used alone for growing bacteria, they could kill non-growing round body or aggregated microcolony form of B. burgdorferi during long-term treatment. Clofazimine with high anti-persister activity improved the combination with daptomycin or daptomycin plus doxycycline which may be due to its multiple mechanisms of action including membrane destabilization, reactive oxygen species production, and inhibition of membrane energy metabolism in M. tuberculosis. We also found that miconazole, an imidazole antifungal drug, had high activity against B. burgdorferi persisters when combined with doxycycline and cefoperazone. Miconazole has been shown to alter the integrity of lipid membrane and therefore may facilitate the penetration of other drugs such as doxycycline and cefoperazone for improved activity against B. burgdorferi persisters. The complete eradication of the B. burgdorferi biofilm-like microcolonies by the three drug combination of daptomycin+doxycycline+cefoperazone has not been achieved with any single, dual or even some three drug combinations in this study or any other previous studies. The mechanism by which this three drug combination was able to achieve this remarkable activity is worth commenting. Doxycycline and cefoperazone inhibits protein synthesis and cell wall peptidoglycan synthesis respectively. Either may be needed to kill the growing forms present in the B. burgdorferi microcolonies or those occasionally revert to growing forms from microcolonies, but these drugs are less effective against the round body or microcolony persisters themselves. This inability could be because of the reduced drug penetration into the microcolony structure, efflux mechanism, or decreased protein or cell wall synthesis in persisters. The high efficacy of daptomycin against B. burgdorferi persisters could be due to its effect on membrane disruption or depolarization, resulting in a loss of membrane potential and inhibition of energy metabolism, which is required for persister survival. Prior studies have suggested that the combination of beta-lactams plus daptomycin increase effectiveness even with daptomycin resistant Gram-positive infections. While drugs traditionally active against Gram-positive organisms are not thought to have activity against B. burgdorferi, in vitro studies have previously documented activity with drugs such as vancomycin but not teicoplanin or daptomycin, though this study was performed examining not persisters but log phase cultures. Though daptomycin is not used for Gram-negative pathogens, a drug such as colistin has been suggested to improve polyanionic lipopeptide activity due to outer membrane permeabilization. Regardless, our studies suggest that combined use of these agents that kill or inhibit both growing organisms and non-replicating organisms may be important for good activity against the highly resistant microcolonies, which is consistent with the proposition to use drugs targeting both growing and non-growing microbial populations for improved treatment of persistent infections. It is worth noting that short term incubation in subculture studies of antibiotic treated B. burgdorferi is not sufficient to assess the stable eradication of persisters.

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