Persister Drug Research Articles

Eradication of Staphylococcus aureus Biofilm Infection by Persister Drug Combination.

Staphylococcus aureus can cause a variety of infections, including persistent biofilm infections, which are difficult to eradicate with current antibiotic treatments. Here, we demonstrate that combining drugs that have robust anti-persister activity, such as clinafloxacin or oritavancin, in combination with drugs that have high activity against growing bacteria, such as vancomycin or meropenem, could completely eradicate S. aureus biofilm bacteria in vitro. In contrast, single or two drugs, including the current treatment doxycycline plus rifampin for persistent S. aureus infection, failed to kill all biofilm bacteria in vitro. In a chronic persistent skin infection mouse model, we showed that the drug combination clinafloxacin + meropenem + daptomycin which killed all biofilm bacteria in vitro completely eradicated S. aureus biofilm infection in mice while the current treatments failed to do so. The complete eradication of biofilm bacteria is attributed to the unique high anti-persister activity of clinafloxacin, which could not be replaced by other fluoroquinolones including moxifloxacin, levofloxacin, or ciprofloxacin. We also compared our persister drug combination with the current approaches for treating persistent infections, including gentamicin + fructose and ADEP4 + rifampin in the S. aureus biofilm infection mouse model, and found neither treatment could eradicate the biofilm infection. Our study demonstrates an important treatment principle, the Yin–Yang model, for persistent infections by targeting both growing and non-growing heterogeneous bacterial populations, utilizing persister drugs for the more effective eradication of persistent and biofilm infections. Our findings have implications for the improved treatment of other persistent and biofilm infections in general.

Central carbon metabolism remodeling as a mechanism to develop drug tolerance and drug resistance in Mycobacterium tuberculosis.

Suboptimal efficacy of the current antibiotic regimens and frequent emergence of antibiotic-resistant Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis (TB), render TB the world’s deadliest infectious disease before the COVID-19 outbreak. Our outdated TB treatment method is designed to eradicate actively replicating populations of Mtb. Unfortunately, accumulating evidence suggests that a small population of Mtb can survive antimycobacterial pressure of antibiotics by entering a “persister” state (slowly replicating or non-replicating and lacking a stably heritable antibiotic resistance, termed drug tolerance). The formation of drug-tolerant Mtb persisters is associated with TB treatment failure and is thought to be an adaptive strategy for eventual development of permanent genetic mutation-mediated drug resistance. Thus, the molecular mechanisms behind persister formation and drug tolerance acquisition are a source of new antibiotic targets to eradicate both Mtb persisters and drug-resistant Mtb. As Mtb persisters are genetically identical to antibiotic susceptible populations, metabolomics has emerged as a vital biochemical tool to differentiate these populations by determining phenotypic shifts and metabolic reprogramming. Metabolomics, which provides detailed insights into the molecular basis of drug tolerance and resistance in Mtb, has unique advantages over other techniques by its ability to identify specific metabolic differences between the two genetically identical populations. This review summarizes the recent advances in our understanding of the metabolic adaptations used by Mtb persisters to achieve intrinsic drug tolerance and facilitate the emergence of drug resistance. These findings present metabolomics as a powerful tool to identify previously unexplored antibiotic targets and improved combinations of drug regimens against drug-resistant TB infection.

Nitroxoline Drug Combinations Are More Active Than Lyme Antibiotic Combination and Can Eradicate Stationary-Phase Borrelia burgdorferi

Abstract Lyme disease (LD), caused by Borrelia burgdorferi, is the most common vector-borne disease in the United States and Europe. Despite the standard 2–4 weeks' antibiotic treatment, approximately 10%–20% of patients will develop posttreatment LD syndrome, a condition that is poorly understood. One of the probable causes is thought to be the presence of B. burgdorferi persister forms that are not effectively killed by the current LD antibiotics. In this study, we evaluated nitroxoline, an antibiotic used to treat urinary tract infections, for its activity against a stationary-phase culture enriched with persister forms of B. burgdorferi. Nitroxoline was found to be more active than doxycycline and equally active as cefuroxime (standard LD antibiotics) against B. burgdorferi. Importantly, the nitroxoline two-drug combinations nitroxoline + cefuroxime and nitroxoline + clarithromycin, as well as the nitroxoline three-drug combination nitroxoline + cefuroxime + clarithromycin, were as effective as the persister drug daptomycin-based positive control three-drug combination cefuroxime + doxycycline + daptomycin, completely eradicating stationary-phase B. burgdorferi in the drug-exposure experiments and preventing regrowth in the subculture study. Future studies should evaluate these promising drug combinations in a persistent LD mouse model.

A model-based analysis identifies differences in phenotypic resistance between in vitro and in vivo: implications for translational medicine within tuberculosis

Proper characterization of drug effects on Mycobacterium tuberculosis relies on the characterization of phenotypically resistant bacteria to correctly establish exposure–response relationships. The aim of this work was to evaluate the potential difference in phenotypic resistance in in vitro compared to murine in vivo models using CFU data alone or CFU together with most probable number (MPN) data following resuscitation with culture supernatant. Predictions of in vitro and in vivo phenotypic resistance i.e. persisters, using the Multistate Tuberculosis Pharmacometric (MTP) model framework was evaluated based on bacterial cultures grown with and without drug exposure using CFU alone or CFU plus MPN data. Phenotypic resistance and total bacterial number in in vitro natural growth observations, i.e. without drug, was well predicted by the MTP model using only CFU data. Capturing the murine in vivo total bacterial number and persisters during natural growth did however require re-estimation of model parameter using both the CFU and MPN observations implying that the ratio of persisters to total bacterial burden is different in vitro compared to murine in vivo. The evaluation of the in vitro rifampicin drug effect revealed that higher resolution in the persister drug effect was seen using CFU and MPN compared to CFU alone although drug effects on the other bacterial populations were well predicted using only CFU data. The ratio of persistent bacteria to total bacteria was predicted to be different between in vitro and murine in vivo. This difference could have implications for subsequent translational efforts in tuberculosis drug development.

Identification of essential oils with activity against stationary phase Staphylococcus aureus

BackgroundStaphylococcus aureus is the most dominant human pathogen, responsible for a variety of chronic and severe infections. There is mounting evidence that persisters are associated with treatment failure and relapse of persistent infections. While some essential oils were reported to have antimicrobial activity against growing S. aureus, activity of essential oils against the stationary phase S. aureus enriched in persisters has not been investigated.MethodsIn this study, we evaluated the activity of 143 essential oils against both growing and stationary phase S. aureus by minimum inhibitory concentration (MIC) testing and by colony forming unit assay.ResultsWe identified 39 essential oils (Oregano, Cinnamon bark, Thyme white, Bandit “Thieves”, Lemongrass (Cymbopogon flexuosus), Sandalwood oil, Health shield, Allspice, Amyris, Palmarosa, Cinnamon leaf, Clove bud, Citronella, Geranium bourbon, Marjoram, Peppermint, Lemongrass, Cornmint, Elemi, Ho wood, Head ease, Lemon eucalyptus, Litsea cubeba, Myrrh, Parsley seed, Coriander oil, Dillweed, Hyssop, Neroli, Rosewood oil, Tea tree, Cajeput, Clove bud, Lavender, Sleep tight, Vetiver, Palo santo, Sage oil, Yarrow) at 0.5% (v/v) concentration, 10 essential oils (Cinnamon bark, Oregano, Thyme white, Bandit “Thieves”, Lemongrass, Sandalwood oil, Health shield, Allspice, Amyris, Palmarosa at 0.25% (v/v) concentration, and 7 essential oils (Oregano, Cinnamon bark, Thyme white, Lemongrass, Allspice, Amyris, Palmarosa at 0.125% (v/v) concentration to have high activity against stationary phase S. aureus with no visible growth on agar plates after five-day exposure. Among the 10 essential oils which showed high activity at 0.25% (v/v) concentration, 9 (Oregano, Cinnamon bark, Thyme white, Bandit “Thieves”, Lemongrass, Health shield, Allspice, Palmarosa, Amyris showed higher activity than the known persister drug tosufloxacin, while Sandalwood oil had activity at a higher concentration. In Oregano essential oil combination studies with antibiotics, Oregano plus tosufloxacin (or levofloxacin, ciprofloxacin) and rifampin completely eradicated stationary phase S. aureus cells, but had no apparent enhancement for linezolid, vancomycin, sulfamethoxazole, trimethoprim, azithromycin or gentamicin.ConclusionsOur findings indicate that some essential oils have excellent activity against both growing and stationary phase S. aureus. Further studies are needed to identify the active components, evaluate safety, pharmacokinetics, and their activity to eradicate S. aureus infections in vivo.

Selective Essential Oils from Spice or Culinary Herbs Have High Activity against Stationary Phase and Biofilm Borrelia burgdorferi.

Although the majority of patients with acute Lyme disease can be cured with the standard 2–4 week antibiotic treatment, about 10–20% of patients continue suffering from chronic symptoms described as posttreatment Lyme disease syndrome. While the cause for this is debated, one possibility is that persister bacteria are not killed by the current Lyme antibiotics and remain active in the system. It has been reported that essential oils have antimicrobial activities and some have been used by patients with persisting Lyme disease symptoms. However, the activity of essential oils against the causative agent Borrelia burgdorferi (B. burgdorferi) has not been well studied. Here, we evaluated the activity of 34 essential oils against B. burgdorferi stationary phase culture as a model for persister bacteria. We found that not all essential oils had activity against the B. burgdorferi stationary phase culture, with top five essential oils (oregano, cinnamon bark, clove bud, citronella, and wintergreen) at a low concentration of 0.25% showing high anti-persister activity that is more active than the known persister drug daptomycin. Interestingly, some highly active essential oils were found to have excellent anti-biofilm ability as shown by their ability to dissolve the aggregated biofilm-like structures. The top three hits, oregano, cinnamon bark, and clove bud completely eradicated all viable cells without any regrowth in subculture in fresh medium, whereas but not citronella and wintergreen did not have this effect. Carvacrol was found to be the most active ingredient of oregano oil showing excellent activity against B. burgdorferi stationary phase cells, while other ingredients of oregano oil p-cymene and α-terpinene had no apparent activity. Future studies are needed to characterize and optimize the active essential oils in drug combination studies in vitro and in vivo and to address their safety and pharmacokinetic properties before they can be considered as a novel treatment of persistent Lyme disease.

Enhanced respiration prevents drug tolerance and drug resistance in Mycobacterium tuberculosis

Persistence, manifested as drug tolerance, represents a significant obstacle to global tuberculosis control. The bactericidal drugs isoniazid and rifampicin kill greater than 99% of exponentially growing Mycobacterium tuberculosis (Mtb) cells, but the remaining cells are persisters, cells with decreased metabolic rate, refractory to killing by these drugs, and able to generate drug-resistant mutants. We discovered that the combination of cysteine or other small thiols with either isoniazid or rifampicin prevents the formation of drug-tolerant and drug-resistant cells in Mtb cultures. This effect was concentration- and time-dependent, relying on increased oxygen consumption that triggered enhanced production of reactive oxygen species. In infected murine macrophages, the addition of N-acetylcysteine to isoniazid treatment potentiated the killing of Mtb Furthermore, we demonstrate that the addition of small thiols to Mtb drug treatment shifted the menaquinol/menaquinone balance toward a reduced state that stimulates Mtb respiration and converts persister cells to metabolically active cells. This prevention of both persister cell formation and drug resistance leads ultimately to mycobacterial cell death. Strategies to enhance respiration and initiate oxidative damage should improve tuberculosis chemotherapies.

Ceftriaxone Pulse Dosing Fails to Eradicate Biofilm-Like Microcolony B. burgdorferi Persisters Which Are Sterilized by Daptomycin/ Doxycycline/Cefuroxime without Pulse Dosing.

Although the majority of Lyme disease patients can be cured, at least 10–20% of the patients continue to suffer from persisting symptoms such as fatigue, muscular and joint pain, and neurologic impairment after standard 2–4 week antibiotic treatment. While the causes for this post-treatment Lyme disease symptoms are unclear, one possibility is due to Borrelia burgdorferi persisters that are not effectively killed by current antibiotics such as doxycycline or amoxicillin used to treat Lyme disease. A previous study showed that four rounds of ceftriaxone pulse dosing treatment eradicated B. burgdorferi persisters in vitro using a relatively young late log phase culture (5 day old). In this study, we investigated if ceftriaxone pulse dosing could also eradicate B. burgdorferi persisters in older stationary phase cultures (10 day old) enriched with more resistant microcolony form of persisters. We found that ceftriaxone pulse dosing could only eradicate planktonic log phase B. burgdorferi spirochetal forms and round body forms but not more resistant aggregated biofilm-like microcolony persisters enriched in stationary phase cultures. Moreover, we found that not all drugs are suitable for pulse dosing, with bactericidal drugs ceftriaxone and cefuroxime being more appropriate for pulse dosing than bacteriostatic drug doxycycline and persister drug daptomycin. We also showed that drug combination pulse dosing treatment was more effective than single drug pulse dosing. Importantly, we demonstrate that pulse dosing treatment impaired the activity of the persister drug daptomycin and its drug combination against B. burgdorferi persisters and that the most effective way to kill the more resistant biofilm-like microcolonies is the daptomycin/doxycycline/ceftriaxone triple drug combination without pulse dosing. Our findings indicate pulse dosing may not always work as a general principle but rather depends on the specific drugs used, with cidal drugs being more appropriate for pulse dosing than static or persister drugs, and that drug combination approach with persister drugs is more effective at killing the more resistant microcolony form of persisters than pulse dosing. These observations may have implications for more effective treatment of Lyme disease. Future studies are required to validate these findings in animal models of B. burgdorferi persistence.

Mechanisms of Pyrazinamide Action and Resistance.

PZA is a unique anti-tuberculosis drug that plays a key role in shortening the TB therapy. PZA kills non-replicating persisters that other TB drugs fail to kill, and thus making it an essential drug for inclusion in any drug combinations for treating drug susceptible and drug-resistant TB such as MDR-TB. PZA acts differently from common antibiotics by inhibiting multiple targets such as energy production, trans-translation and perhaps pantothenate /coenzyme A required for persister survival. Resistance to PZA is mostly caused by mutations in the pncA gene encoding pyrazinamidase involved in conversion of the prodrug PZA to the active form POA. Mutations in the drug target RpsA are also found in some PZA-resistant strains. The recent finding that panD mutations are found in some PZA-resistant strains without pncA or rpsA mutations may suggest a third PZA resistance gene and a potential new target of PZA. Current phenotype based PZA susceptibility testing is not reliable due to false resistance, and sequencing of the pncA gene represents a more rapid, cost-effective and more reliable molecular test for PZA susceptibility testing and should be used for guiding improved treatment of MDR/XDR-TB. Finally, the story of PZA has important implications for not only TB therapy but also chemotherapy in general. PZA serves as a model prototype persister drug and hopefully a 'tipping point' that inspires new efforts at developing a new type of antibiotics or drugs that target non-replicating persisters for improved treatment of not only TB but also other persistent bacterial infections.

Aspartate decarboxylase (PanD) as a new target of pyrazinamide in Mycobacterium tuberculosis

Pyrazinamide (PZA) is a frontline anti-tuberculosis drug that plays a crucial role in the treatment of both drug-susceptible and multidrug-resistant tuberculosis (MDR-TB). PZA is a prodrug that is converted to its active form, pyrazinoic acid (POA), by a nicotinamidase/pyrazinamidase encoded by the pncA gene, the mutation of which is the major cause of PZA resistance. Although RpsA (ribosomal protein S1, involved in trans-translation) has recently been shown to be a target of POA/PZA, whole-genome sequencing has identified mutations in the panD gene encoding aspartate decarboxylase in PZA-resistant strains lacking pncA and rpsA mutations. To gain more insight into a possible new target of PZA, we isolated 30 POA-resistant mutants lacking mutations in pncA and rpsA from M. tuberculosis in vitro, and whole-genome sequencing of 3 mutants identified various mutations in the panD gene. Additionally, sequencing analysis revealed that the remaining 27 POA-resistant mutants all harbored panD mutations affecting the C-terminus of the PanD protein, with PanD M117I being the most frequent mutation (24/30, 80%). Conditional overexpression of panD from M. tuberculosis, M. smegmatis or E. coli, or of M. tuberculosis mutant PanD M117I, all conferred resistance to POA and PZA in M. tuberculosis. β-alanine and pantothenate, which are downstream products of PanD, were found to antagonize the antituberculosis activity of POA. In addition, the activity of the M. tuberculosis PanD enzyme was inhibited by POA at therapeutically relevant concentrations in a concentration-dependent manner but was not inhibited by the prodrug PZA or the control compound nicotinamide. These findings suggest that PanD represents a new target of PZA/POA. These results have implications for a better understanding of this peculiar persister drug and for the design of new drugs targeting M. tuberculosis persisters for improved treatment.

Trans-translation mediates tolerance to multiple antibiotics and stresses in Escherichia coli

Trans-translation mediated by SsrA (tmRNA) and its associated protein SmpB plays an important role in rescuing stalled ribosomes and detoxifying toxic protein products under stress conditions. However, the role of SsrA and SmpB in bacterial persister survival has not been studied. The recent finding that pyrazinamide as a unique persister drug inhibits trans-translation in Mycobacterium tuberculosis prompted us to examine the role of trans-translation in persister survival. Using Escherichia coli as a model, we constructed SsrA and SmpB mutants and assessed the susceptibility of the mutants to various antibiotics and stress conditions in MIC/MBC and persister assays. We found that mutations in SsrA and SmpB caused a defect in persister survival as shown by their increased susceptibility to a variety of antibiotics, including gentamicin, streptomycin, amikacin, norfloxacin, trimethoprim and tetracycline, and also stresses, such as acid, weak acid salicylate, heat and peroxide. Additionally, the SsrA and SmpB mutants were 2-8-fold more susceptible than the parent strain to various antibiotics in MIC and MBC tests. The SmpB mutant was more susceptible to antibiotics and stresses than the SsrA mutant. A particularly interesting finding is the hypersusceptibility of the SmpB mutant and the SsrA mutant to trimethoprim. The defect of various SsrA and SmpB mutant phenotypes could be complemented by functional ssrA and smpB, respectively. We conclude that SsrA and SmpB are important for persister survival and may serve as a good target for developing new antibiotics that kill persister bacteria for improved treatment of persistent bacterial infections.