In our previous study, we compared the activity of antibiotics against non-growing persisters with their activity against growing 18 ]. Here we also tested the minimum inhibitory concentration (MIC) of some active hits. We found these drugs showed good activity against the growingwith low MICs ( Table 2 ). The maximum blood drug concentrations (Cmax) of several active hits, including 3-formyl rifamycin, oltipraz, and fluconazole, were higher than the MICs of these drug candidates, indicating these drugs are likely to achieve clinically relevant drug concentrations. However, some drugs such as verteporfin, pidolic acid, and dextrorphan tartrate had lower Cmax values than their corresponding MICs. Some other active hits such as thonzonium bromide, benzododecinium chloride, and quinaldine blue did not have Cmax values as they are used only topically or not available.

We used a relatively high drug concentration of 50 μM in our drug screens with seven-day-old stationary phase cultures; as in our preliminary pilot screens, lower drug concentrations, such as 10 μM, commonly used for actively growing organisms or protein target screens, would not allow one to see the effect of any compounds—even the highly active compounds such as daptomycin—under this condition. The findings with 50 μM are still valid and relevant, because in the setting of drug combinations, the persister-active hits can be used at lower clinically relevant concentrations (10 μg/mL for daptomycin) and still allow one to see the effect and importance of the persister-active hits in eradicating more resistant microcolony forms ofpersisters [ 19 ]. In addition, taking into consideration some drugs such as pyrazinamide, with high activity against persistersbut poor activity 17 ], we used this higher concentration to increase the sensitivity of the screen. We plan to validate these results using clinically relevant dosages in future drug combination studiesand

In our previous study, we identified 165 hits with higher activity againstpersisters than the currently used Lyme antibiotics. In this study, we further analyzed and characterized these active hits. By removing redundant hits from the library and also those that could not be repeated, we obtained 113 that gave consistent results ( Supplementary Table S1 ). Of the 113 hits, the top 52 candidates that can be used in humans and killed 65% or more of the stationary phase bacteria according to either the SYBR Green I/Propidium Iodide (PI) assay or microscopic quantitation are presented in Table 1 . The remainder of active hits that may not be used in humans and also less active ones are presented in Supplementary Table S1 . However, these agents in general were not as active as the top hits such as daptomycin, clofazimine, cefoperazone, and anthracyclines from the previous screens [ 18 21 ]. These active hits are grouped into antimicrobial agents (antibiotics, antivirals, antifungals, anthelmintics or antiparasitics), agents used for treating other disease conditions, as well as agents that may only be used as topical agents or not used internally and are presented below ( Table 1 ).

Zanamivir is a clinically used antiviral agent that inhibits the neurominidase inhibitor that is inhaled as an aerosol to shorten the duration of influenza infections by preventing neuraminidase from releasing virions from the infected cells [ 22 ]. Recently, multiple bacterial species have been shown to express bacterial neuraminidases capable of cleaving α2,3-sialic acids [ 23 ]. These neuraminidases have been implicated in biofilm formation, with aneuraminidase mutant showing decreased ability to colonize the mouse respiratory tract and decreased biofilm production [ 23 ]. It remains to be seen if zanamivir acts in a similar manner in

As previously published, the SYBR Green I/PI assay is a high-throughput technique that uses the ratio of green:red fluorescence in each sample to quantitate the amount of residual viable cells remaining [ 18 ]. While this technique has the benefits of high-throughput analysis, discoloration of the culture medium by test drugs can result in altered readings [ 18 ]. Quinaldine blue and methylene blue are two drugs whose staining properties resulted in medium discoloration and required verification through microscopy. Careful microscopy analysis revealed that quinaldine blue and methylene blue had high activity againstpersisters ( Table 1 Figure 1 ). Quinaldine (2-methylquinoline) is a heterocyclic quinoline compound that is used as an antimalarial and in dye manufacturing, food colorants, pH indicators, and pharmaceuticals. Methylene blue was originally used as an antimalarial and is used to treat methemoglobinemia and urinary tract infections.

Readily available drugs with low toxicity are important objectives in this screen as they are the most likely to be used for the clinical treatment of Lyme disease. Here we focused on antimicrobial agents used in humans that had higher activity against the stationary phasethan the commonly used Lyme antibiotics. The antibacterial agents include rifamycins (3-formal-rifamycin, rifaximin, rifamycin SV) ( Figure 1 ), thiostrepton, quinolone drugs (sarafloxacin, clinafloxacin, tosufloxacin), carbenicillin, tazobactam, aztreonam, and puromycin ( Table 1 Supplementary Table S1 ). Some antifungal agents such as fluconazole ( Figure 1 ), mepartricin, bifonazole, climbazole, oxiconazole, and nystatin had reasonable activity against stationary phase Supplementary Table S1 ). Antiviral agents zanamivir, nevirapine, and tilorone (orally active interferon inducer) had good activity against stationary phaseAntimalarial agents artemisinin, methylene blue, and quidaldine blue were found to have good activity against stationary phase Table 1 ). Antihelmintic and antiparasitic agents that had activity againstincluded toltrazuril, tartar emetic, potassium antimonyl tartrate trihydrate, oxantel, closantel, hycanthone, pyrimethamine, and tetramisole ( Table 1 ). These drugs with high activity against stationary phaseare good potential candidates for drug combination studies and for further evaluation in animal models.

The significant number of highly active drugs that act upon this γ-glutamyl pathway suggests that this pathway is important for persisters and that ROS and peroxide-induced damage are important for killing persisters. We anticipate that inhibition of this pathway could be a good therapeutic target for B. burgdorferi persisters for the improved treatment of Lyme disease. Future studies are needed to further evaluate if these agents are active in drug combination studies and in animal models.

Verteporfin (Visudyne), a benzophorphyrin derivative, is a photosensitizing agent currently used to treat the macular degeneration that affects the γ-glutamyl pathway [ 24 28 ]. This intravenous drug is transported in oxygenated blood by lipoproteins, and is activated by laser light treatment allowing for precise chemotherapeutic application [ 24 ]. Verteporfin is a possible effector of cell membrane permeability through ROS lipid peroxidation [ 24 29 ]. Activated verteporfin has been shown to target the mitochondria, producing reactive oxygen radicals and nitric oxide that damage local endothelium and seal leaky vessels [ 24 27 ]. Verteporfin depletes GSH levels in HepG2 cells after activation, possibly through increased nitric oxide production [ 24 ]. Oltipraz is an organosulfur compound that belongs to the dithiolethione class. It has been shown to inhibit schistosome and prevent the formation of cancer. It activates phase II detoxification enzymes in mammalian cells, which results in the binding of glutathione to electrophilic compounds and subsequent protection against reactive oxygen species (ROS) damage [ 30 31 ]. Pyroglutamic acid (PCA) or pidolic acid (pidolate) or 5-oxoproline is an amino acid derivative that is involved in the γ-glutamyl cycle. PCA is a metabolite of the glutathione cycle which is broken down to glutamate and cysteine, which are then converted back into glutathione [ 26 ], and is used in humans as dietary supplement and skin moisturizer retainer.

It is interesting to note that several highly active drugs identified in our screen, including verteporfin, oltipraz, pyroglutamic acid, pidolic acid ( Figure 1 ), and dextrorphan tartrate, act on the glutathione/γ-glutamyl pathway used in mammalian cells which involved in protection against intracellular damage from free radicals and peroxides. Glutathione (GSH) is a reducing agent produced in the cytoplasm and transferred to the mitochondria by glutathione-S-transferase (GST), where it protects the mitochondria from reactive oxygen species (ROS) damage and functions in amino acid transport [ 24 25 ]. Reduced levels of GSH have been linked to increased sensitivity to ROS damage, resulting in mitochondrial swelling and subsequent damage [ 24 26 ].

2.4. Active Hits that Are Topical Agents or Toxic for Internal Use

B. burgdorferi (Supplementary Table S1, B. burgdorferi . However, thonzonium bromide is a cationic detergent and surfactant that is used as a topical agent in combination with other compounds to assist in the penetration of cellular membranes [ B. burgdorferi [33,34, C. albicans, thonzonium bromide was also shown to inhibit ATPases in isolated vacuoles and cause general cellular toxicity [ C. albicans biofilms [ S. aureus to have higher activity against the biofilm form of the bacteria than the free planktonic form in vitro [ B. burgdorferi persisters suggests that both the cell membrane and biofilms are potential targets for future persister drug design. Thonzonium bromide, benzododecinium chloride, and butyl chloride were found to have very high activities against stationary phase Figure 1 ). Thonzonium bromide even had comparable activity to daptomycin against stationary phase. However, thonzonium bromide is a cationic detergent and surfactant that is used as a topical agent in combination with other compounds to assist in the penetration of cellular membranes [ 32 ]. Thonzonium bromide has been shown to inhibit vacuolar ATPases in yeast, which is an enzyme that is closely related to the ATPase found in 35 ]. Inthonzonium bromide was also shown to inhibit ATPases in isolated vacuoles and cause general cellular toxicity [ 33 34 ]. Thonzonium bromide was also shown to be active against preformedbiofilms [ 32 ]. Benzododecinium chloride is a C12-substituted alkyl chain derivate of the quarternary ammonium detergent benzalkonium chloride that alters cell membrane permeability and can cause cell lysis through lipid dispersion [ 36 37 ]. Benzododecinium chloride was shown into have higher activity against the biofilm form of the bacteria than the free planktonic form 38 ]. Since thonzonium bromide and benzododecinium chloride have strong detergent properties causing generalized cellular damage in humans, they may not be used directly for Lyme treatment. However, the high activity of these drugs againstpersisters suggests that both the cell membrane and biofilms are potential targets for future persister drug design.

B. burgdorferi persisters [ M. tuberculosis and E. coli [39,40, B. burgdorferi persisters interfere with energy production (thonzonium bromide, oxantel) and ROS production (verteporfin, oltipraz, pyroglutamic acid, pidolic acid). Our data showed that these three types of agents, cell membrane disruptors, energy inhibitors, and ROS producers, are generally more active against the B. burgdorferi persisters than the more conventional antibiotics that inhibit cell wall, protein, RNA, and DNA syntheses ( B. burgdorferi persisters in vitro [ It is worth noting that the most active hits from the compound library screen are those that affect cell membranes (benzododecinium chloride, thonzonium bromide, zanamivir). This is consistent with our previous finding that daptomycin and clofazimine may act on the cell membrane to show their high activity againstpersisters [ 18 ]. Indeed, agents that target bacterial cell membranes have been found to be active against persisters in different bacterial pathogens such asand 41 ]. Other active hits that show good activity againstpersisters interfere with energy production (thonzonium bromide, oxantel) and ROS production (verteporfin, oltipraz, pyroglutamic acid, pidolic acid). Our data showed that these three types of agents, cell membrane disruptors, energy inhibitors, and ROS producers, are generally more active against thepersisters than the more conventional antibiotics that inhibit cell wall, protein, RNA, and DNA syntheses ( Table 1 Supplementary Table S1 ). Future studies are needed to assess the activity of these agents in combination with Lyme antibiotics for more effective eradication ofpersisters 19 ].

B burgdorferi persisters have not received any attention other than in the structural study of the adaptor protein of the tripartite efflux pump of the organism. Given our results in this study and also our recent observation that efflux and transporters are upregulated in B. burgdorferi persisters [ Efflux pumps ofpersisters have not received any attention other than in the structural study of the adaptor protein of the tripartite efflux pump of the organism. Given our results in this study and also our recent observation that efflux and transporters are upregulated inpersisters [ 42 ], it is very likely that over-expressed efflux pumps are one of the causes for the persisters and that compounds known to inhibit bacterial efflux pumps such as RND proton motive force-dependent pumps are possible lead compounds for the development of effective drugs for the treatment of persisting Lyme disease.

et al. used colony-forming unit (CFU) assay to assess “persisters” in B. burgdorferi [ B. burgdorferi and is not commonly used for viability assessment for B. burgdorferi. Instead, BacLight LIVE/DEAD viability assay is the most commonly used method for assessing drug activity in this field. In addition, it is well known that true B. burgdorferi persisters after antibiotic treatment in vivo cannot be cultured in Barbour-Stoenner-Kelly (BSK) media, and therefore the CFU assay may not be an appropriate method to evaluate the survival of persisters from in vivo . Furthermore, the CFU assay may be used only on relatively young cultures (3–5 days) that consist primarily of planktonic spirochetal forms, but cannot be used for older true stationary phase cultures (7–10 days) that consist mainly of aggregated microcolonies [ A recent study by Sharmaused colony-forming unit (CFU) assay to assess “persisters” in 43 ]. However, the CFU assay, while working well for bacteria that easily form colonies on agar plates, does not work well forand is not commonly used for viability assessment forInstead, BacLight LIVE/DEAD viability assay is the most commonly used method for assessing drug activity in this field. In addition, it is well known that truepersisters after antibiotic treatmentcannot be cultured in Barbour-Stoenner-Kelly (BSK) media, and therefore the CFU assay may not be an appropriate method to evaluate the survival of persisters from. Furthermore, the CFU assay may be used only on relatively young cultures (3–5 days) that consist primarily of planktonic spirochetal forms, but cannot be used for older true stationary phase cultures (7–10 days) that consist mainly of aggregated microcolonies [ 19 ]. Therefore, we did not use CFU assay in our studies involving seven-day-old cultures, but instead used SYBYR Green I/PI viability assay, which is similar in principle but more sensitive than BacLight LIVE/DEAD viability assay [ 44 ]; we also used subculture in liquid medium to assess the anti-persister activity of the drugs. In contrast to the CFU assay, the SYBYR Green I/PI viability assay and subculture in liquid medium can be readily used and are more suited to this setting. In addition, the CFU assay cannot be used for drug screens as it is not only infeasible due to the washing to remove drugs for the CFU assay in a high-throughput format, but it also requires a significant amount of drugs, which precluded us from testing the active hits with the CFU assay, as the FDA drug library did not have a sufficient amount for it. Nevertheless, the CFU assay will be performed in future studies on selected active hits using younger cultures with primarily planktonic spirochetes when a sufficient quantity of the drugs is secured for testing.

Drug screens are subject to false positive non-specific hits referred to as pan-assay interference compounds (PAINS) in protein target-based screens [ 45 ]. However, because this study used whole organism (stationary phase culture)-based screens, most of the factors that can lead to PAINS are not present in our assay system. Nevertheless, we looked into the possible presence of PAINS in our active hits derived from the FDA drug library using the software from the Scripps Institute program for chemical promiscuity evaluation [ 46 ]. We found the majority of our hits to be highly specific, and the only identified PAINS in our highly active hits included FDA-approved drugs that are also coloring chemicals including methylene blue, quinaldine blue, and ethacridine lactate. The possibility of the PAINS was excluded using manual verification of the anti-persister activity of these drugs through epifluorescence microscopy.