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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Abstract: 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: http://www.discoverymedicine.com/Jie-Feng/2019/03/persister-biofilm-microcolony-borrelia-burgdorferi-causes-severe-lyme-arthritis-in-mouse-model/ 

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.