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A drug combination screen identifies drugs active against amoxicillin-induced round bodies of in vitro Borrelia burgdorferi persisters from an FDA drug library

Front Microbiol. May 23;7:743. doi: 10.3389/fmicb.2016.00743.

YEAR: 2016 TOPICS: Chronic Lyme, Treatment.

In lab culture, Borrelia burgdorferi, the bacterium that causes Lyme disease, can survive amoxicillin treatment by transforming into a round-body form. Such round bodies persist in a slowly-growing state and are tolerant of antibiotics. In this study, the authors tested 1581 drugs that are already approved by the FDA, for antimicrobial activity against amoxicillin-induced round-bodied B. burgdorferi. They found 23 drugs with activity against the round bodies when used singly. In addition, they identified artemisinin, a malaria drug, in combination with cefoperazone and doxycycline, as well as sulfachlorpyridazine together with daptomycin and doxycycline as potent combinations that reduced round bodies. Importantly, these combinations also had some durable inhibitory effect, because re-growth after subculture was low. These findings provide evidence that the continued search for effective drug combinations against persister bacteria is warranted.

An optimized SYBR Green I/PI assay for rapid viability assessment and antibiotic susceptibility testing for Borrelia burgdorferi

PLoS One. 9(11)e111809

YEAR: 2014 TOPICS: Chronic Lyme, Treatment.

The authors analyzed various methods to efficiently and accurately count live and dead Borrelia burgdorferi in lab culture. This is important for calculating the efficacy of antibiotics in the killing of bacteria. They adapted a method that stains live bacteria green, and dead ones red, allowing simple, reproducible, and high-throughput means to count bacteria.

Antimicrobial action of calprotectin that does not involve metal withholding.

Metallomics doi: 10.1039c8mt00133b

YEAR: 2018 TOPICS: Basic Science, Treatment.

Heavy metals like iron, copper, manganese and zinc can be toxic in large amounts. But in low concentrations, they are essential micronutrients for humans as well as bacteria. As part of the defense against invasive microbes, our immune system exploits this property by starving bacteria of the metals they need to survive. One way of doing this is with calprotectin, an immune defense protein produced by neutrophils at the site of infections. Calprotectin depletes the local environment of metal nutrients, and can block Borrelia burgdorferi growth. The mechanism of how this works has now been explained in a newly published study, funded by GLA and led by Dr. Valeria Culotta.

Calprotectin was found in the dermal and epidermal layers of erythema migrans rashes of early Lyme disease patients. It is believed that epithelial keratinocytes in the skin produce calprotectin, which is more commonly associated with neutrophils, immune cells that are absent in this particular site. Surprisingly, calprotectin blocked B. burgdorferi growth not by depleting metals, as would be expected. Instead, it directly bound to the bacteria and changed their shape from spirochetes into a cyst-like form, when immersed in water. This differs from calprotectin’s mechanism of blocking another bacteria, E. coli, in which metal sequestration is essential to blocking bacterial replication. These findings suggest a unique new pathway that could be explored for a potential therapeutic that is specific to B. burgdorferi.

Biofilm formation by Borrelia burgdorferi sensu lato

FEMS Microbiol Lett 362:fnv120

Sapi E

YEAR: 2015 TOPICS: Chronic Lyme, Treatment.

The authors studied whether B. afzelii and B. garinii, two bacteria associated with Lyme disease in Europe, are capable of growing as biofilms in lab culture. Using chemical stains and microscopy, they showed that these two bacteria form biofilms, similar to B. burgdorferi, the North American species, when grown under specific lab culture conditions.

Borrelia burgdorferi, the causative agent of Lyme disease, forms drug-tolerant persister cells

Antimicrob Agents Chemother 59(8):4616-24. Epub 2015 May 26.

Lewis K, Sharma B

YEAR: 2015 TOPICS: Chronic Lyme, Treatment.

Persisting bacteria survive antibiotic treatment in lab culture. Here, the authors showed that antibiotics killed most of lab-grown B. burgdorferi, with a small surviving population that tolerated drug treatment. They found that mitomycin C, a cancer drug, efficiently killed persisters. They also showed how pulse-dosing, wherein four repetitive cycles that included treating bacterial cultures with ceftriaxone, followed by washing out the drug, resulted in eradication of persistent bacteria.

Distinct cerebrospinal fluid proteomes differentiate post-treatment Lyme disease from chronic fatigue syndrome

PLoS One. 6(2):e17287

YEAR: 2011 TOPICS: Chronic Lyme, Diagnostics, Treatment.

Chronic fatigue syndrome patients and post-treatment Lyme disease patients share some similar neurological symptoms. This study showed that the protein profiles in cerebrospinal fluid from the two groups were distinctive, suggesting that they are two different diseases.

Drug combinations against Borrelia burgdorferi persisters in vitro: eradication achieved by using daptomycin, cefoperazone and doxycycline

PLoS One. 10(3): e0117207

YEAR: 2015 TOPICS: Chronic Lyme, Treatment.

The persistence of Borrelia burgdorferi in the form of drug-tolerant bacteria may be one reason why some Lyme disease patients may continue to suffer symptoms despite antibiotic therapy. The authors determined which drug combinations were effective in eradicating persistent microcolonies of B. burgdorferi, and identified daptomycin, doxycycline and cefoperazone together as an effective way of eliminating bacteria. Even subculturing bacteria treated with this combination to fresh media did not result in bacterial growth, suggesting durable killing.

Epitope-Specific Evolution of Human B Cell Responses to Borrelia burgdorferi VlsE Protein from Early to Late Stages of Lyme Disease

J Immunol 196:1036-43

Alaedini A

YEAR: 2016 TOPICS: Chronic Lyme, Diagnostics, Treatment.

Lyme disease patients make antibodies that recognize Borrelia burgdorferi, the bacteria that cause illness. When the disease progresses from early to late stages, these antibodies may change. A protein on the outside of B. burgdorferi is called VlsE, and antibodies that bind to a portion of it called the membrane-proximal domain illustrate this. These specific antibodies were low in patients with early disease, and were increased in patients with symptoms typical of later illness, such as neurological symptoms.

Eradication of Biofilm-Like Microcolony Structures of Borrelia burgdorferi by Daunomycin and Daptomycin but not Mitomycin C in Combination with Doxycycline and Cefuroxime

Front Microbiol. Feb 10;7:62. doi: 10.3389/fmicb.2016.00062

YEAR: 2016 TOPICS: Chronic Lyme, Treatment.

The persistence of symptoms in Lyme patients treated with antibiotics may be due to the bacteria becoming tolerant to drugs. In lab culture, persistent Lyme bacteria grown into biofilm form were exposed to various antibiotic combinations. The authors found that double antibiotic combinations such as doxycycline and cefoperazone were not enough for biofilm eradication. The addition of either daptomycin or daunomycin was required for bacterial elimination.

Evaluation of Disulfiram Drug Combinations and Identification of Other More Effective Combinations against Stationary Phase Borrelia burgdorferi

Yumin-Zhang, Hector S. Alvarez-Manzo, Wanliang Shi, Ying Zhang

YEAR: 2020 TOPICS: Basic Science, Treatment.

Published in: Antibiotics Aug 26;9(9):542.

doi: 10.3390/antibiotics9090542

Identification of novel anti-persister activity against Borrelia burgdorferi from an FDA-approved drug library

Emerg Microbes Infect Jul 3 (e49). Epub Jul 2.

YEAR: 2014 TOPICS: Chronic Lyme, Treatment.

A library of drug compounds that has already been approved by the FDA was tested for efficacy against persister forms of Borrelia burgdorferi that were grown in lab culture. They identified daptomycin, clofazimine, sulfa drugs, and cefoperazone as potential drugs worth pursuing in further studies, which may include animal models of Lyme disease.

Identifying vancomycin as an effective antibiotic for killing Borrelia burgdorferi

Antimicrob Agents Chemother. Aug 20. Doi: 10.1128/AAC.01201-18

Lewis K, Sharma B

YEAR: 2018 TOPICS: Chronic Lyme, Treatment.

The persistence of symptoms in some Lyme disease patients treated with antibiotics has never been conclusively explained. This is known as post-treatment Lyme disease syndrome (PTLDS). It is possible that in common with animal models like mice and monkeys, human Borrelia burgdorferi infection results in residual, persistent infection even after treatment. Continued bacterial presence and/or an ongoing immune response could cause PTLDS. In an effort to identify antibiotics that might completely eradicate bacterial replication, the GLA-funded research team of Dr. Kim Lewis followed up earlier observations that beta-lactam antibiotics might be effective. The team found that vancomycin was effective against in vitro cultures of growing B. burgdorferi. However, this class of drugs is not expected to work on stationary cells, in which growth is very slow, and cell wall synthesis is expected to be minimal. Unexpectedly, when tested on stationary B. burgdorferi, they found that cell wall synthesis still occurred, and could be blocked by vancomycin. To extend their studies to an in vivo model, the group treated B. burgdorferi-infected, immunodeficient mice for five days. They found that vancomycin and ceftriaxone each completely blocked bacterial growth, compared with partial eradication by doxycycline. These studies suggest that more effective antimicrobial drugs, used early in infection, may prevent or reduce the occurrence of persisting infection. However, ceftriaxone and vancomycin are intravenously administered, and are not justifiable when most Lyme patients recover with oral doxycycline. Better identification of the subset of patients for whom doxycycline is inadequate would pave the way to studying whether vancomycin might reduce the incidence of PTLDS.

Increased IFN-α activity and differential antibody response in patients with a history of Lyme disease and persistent cognitive deficits

J Neuroimmunol. 255:85–91

Alaedini A

YEAR: 2013 TOPICS: Chronic Lyme, Treatment.

Some patients treated for Lyme disease continue to suffer neurological symptoms. In these patients compared to healthy controls, there was increased interferon-α (IFNα) activity observed in the blood. IFNα is a cytokine, or chemical messenger, and together with the observation of elevated antibodies against B. burgdorferi and neural proteins, suggests an ongoing inflammatory response in these patients.

Longitudinal Transcriptome Analysis Reveals a Sustained Differential Gene Expression Signature in Patients Treated for Acute Lyme Disease

mBio. Feb 12;7(1). pii: e00100-16. doi: 10.1128/mBio.00100-16.

Aucott JN, Chiu CY, Soloski MJ

YEAR: 2016 TOPICS: Chronic Lyme, Diagnostics, Treatment.

The genes that are expressed in sick people differ from those who are healthy. Using advanced sequencing techniques, these researchers found that immune cells in the blood of people with Lyme disease express different genes from those of healthy controls. Genes that are linked to a strong immune response were reduced in Lyme patients compared to those with other bacterial or viral infections. People with persisting symptoms attributed to Lyme disease shared some gene expression patterns with patients suffering chronic illnesses caused by immune system dysfunction.

Persister mechanisms in Borrelia burgdorferi: implications for improved intervention

Emerg Microbes Infect Aug 4 (e51). Epub 2015 Aug 19.

YEAR: 2015 TOPICS: Chronic Lyme, Treatment.

Lyme disease is caused by the bacteria called Borrelia burgdorferi. In lab cultures of B. burgdorferi that are treated with antibiotics, most are killed. However, some become persister bacteria that survive. The authors used RNA sequencing to study how the genes that are expressed may differ in persister compared to drug-free bacteria. They found that expression changes in 675 genes in persister bacteria.

Persisters, persistent infections and the Yin–Yang model

Emerg Microbes Infect 3(e3)

YEAR: 2014 TOPICS: Chronic Lyme, Treatment.

In this review, Dr. Zhang discusses the differences between B. burgdorferi persister and growing cells, and proposes a model for bacterial transition between the two states, with the balance tipped depending on the environment and stresses such as antibiotics.

Posttreatment Symptoms in Lyme Borreliosis

Klemen Strle

YEAR: 2020 TOPICS: Chronic Lyme, Treatment.

Lyme borreliosis (LB) is the most common vector-borne disease in the Northern Hemisphere, and its incidence is on the rise. With 5%–20% of Lyme patients continuing to suffer from symptoms of Lyme post antibiotic treatment, it’s important to understand what mechanisms are involved, how to predict which patients will develop what symptoms, and how best to treat them?

Published in Oxford Academic: Clinical Infectious Diseases: 

Serum inflammatory mediator as markers of human Lyme disease activity

PLoS One. 9(4):e93243.

Aucott JN, Soloski MJ

YEAR: 2014 TOPICS: Chronic Lyme, Diagnostics, Treatment.

The authors compared the blood of patients suffering more severe symptoms of Lyme disease against those with less. They found that the pattern of chemokines, which are chemical messengers produced by immune cells, varies between the two groups. This work may lead to better diagnostic tools and clinical treatments, and shows a biological difference between these two groups of patients.

Stationary Phase Persister/Biofilm Microcolony of Borrelia burgdorferi Causes More Severe Disease in a Mouse Model of Lyme Arthritis: Implications for Understanding Persistence, Post-Treatment Lyme Disease Syndrome (PTLDS), and Treatment Failure

Monica Embers, Ying Zhang

YEAR: 2019 TOPICS: Basic Science, Chronic Lyme, Treatment.

Although most patients with Lyme disease can be cured with a 2-4 week antibiotic therapy, about 10-20% of patients continue to suffer prolonged persistent symptoms, a condition called post-treatment Lyme disease syndrome (PTLDS). The cause for PTLDS is unclear and hotly debated. B. burgdorferi develops morphological variants under stress conditions but their significance is not clear. Here we isolated the biofilm-like microcolony (MC) and planktonic (spirochetal form and round body) (SP) variant forms from the stationary phase culture and showed that the MC and SP were not only more tolerant to the current Lyme antibiotics but also caused more severe arthritis in mice than the log phase spirochete form (LOG). We propose to divide the persistent Lyme disease into two categories: (1) early development of persistent disease from inoculation with persister/biofilm at the beginning of infection introduced by tick bites, or Type I persistent disease (i.e., PTLDS); and (2) late development of persistent disease due to initial infection not being diagnosed or treated in time such that the infection develops into late persistent disease, or Type II persistent disease. Importantly, we show that the murine infection caused by LOG could be eradicated by ceftriaxone whereas the persistent infection established with MC could not be eradicated by doxycycline (Doxy), ceftriaxone (CefT), or vancomycin (Van), or Doxy+CefT or Van+CefT, but could only be eradicated by the persister drug combination daptomycin+doxycycline+ceftriaxone. Our studies demonstrate that varying levels of persistence and pathologies of Borrelia infection can be established with heterogeneous inocula with different morphologies and have different treatment responses. These observations may have broad implications for understanding pathogenesis and treatment of not only persistent Lyme disease but also other persistent infections in general and call for treatment of persistent infections with persister drug combination regimens that are more effective than the current often single-antibiotic monotherapy treatment.

Published in Discovery Medicine: 

The Functional and Molecular Effects of Doxycycline Treatment on Borrelia burgdorferi Phenotype

Monica Embers

YEAR: 2019 TOPICS: Basic Science, Chronic Lyme, Treatment.

Recent studies have shown that Borrelia burgdorferi can form antibiotic-tolerant persisters in the presence of microbiostatic drugs such as doxycycline. Precisely how this occurs is yet unknown. The goal of this study was to examine gene transcription by B. burgdorferi following doxycycline treatment in an effort to identify both persister-associated genes and possible targets for antimicrobial intervention.

Published in Frontiers in Microbiology:

The Long-Term Persistence of Borrelia burgdorferi Antigens and DNA in the Tissues of a Patient with Lyme Disease

Sapi E

YEAR: 2019 TOPICS: Chronic Lyme, Treatment.

Whether Borrelia burgdorferi, the causative agent of Lyme disease, can persist for long periods in the human body has been a controversial question. The objective of this study was to see if we could find B. burgdorferi in a Lyme disease patient after a long clinical course and after long-term antibiotic treatment.

Published in Antibiotics:

TLR-mediated B cell activation results in ectopic CLIP expression that promotes B cell-dependent inflammation.

J Leuk Biol 88:779-789

YEAR: 2010 TOPICS: Chronic Lyme, Treatment.

In this article, the authors used a mouse model to study B cells, which when activated, produce antibodies and release inflammatory cytokines, or chemical messengers. They found that a protein called CLIP is important in controlling B cells, and speculated that it shows a potential pathway to control chronic immune activation diseases such as Crohn’s disease and post-treatment Lyme disease.

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