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Professor Ying Zhang’s research is focused on resistance and persistent of bacteria against antibiotics. He has been working on Tuberculosis for years. Since 2009 he has been working on research on Lyme Disease as well. The results of his research is shedding a new light on chronic Lyme disease.

Can you please begin by introducing yourself?

My name is Ying Zhang and I am a professor at the Johns Hopkins University. My research is focused on bacterial persistence and antibiotic resistance. We have been doing research on Tuberculosis (TB), which is a persistent infection, for many years. Since 2009 we have been working on understanding the persistence problem of Lyme disease.

We started in vitro work by growing the Borrelia bacteria in a culture system and found persisters can form like any other bacteria. When we started there were no accurate tests to identify drugs that work against Borrelia persisters. So we developed a testing method called SYBR Green/PI viability assay, which allowed us to screen the FDA drugs-library for activity against Borrelia persisters.

We found that the current antibiotics for treating Lyme disease are unable to kill the more resistant persister forms of Borrelia. We identified that antibiotics like daptomycin, clofazimine, sulfa-drugs and other drug-candidates are more effective in killing these persister-forms than the current Lyme antibiotics.

We found that Borrelia is a very intriguing and complex organism because it changes forms: the spirochetal form, variant round-body forms, aggregated micro-colony forms and even larger biofilm-like structures. These persisters are very heterogeneous. There are various kinds of persisters and some are very resistant while others are less resistant. In these different forms they change antigen expression. This has implications for diagnosis as well as treatment. The current tests are based on antigens expressed by the growing organisms and have limitations. We are working on improving anti-borrelial treatments by finding antibiotics that target these persister-forms, as well as different drug-combinations and developing more sensitive diagnostics incorporating persister antigens.

What is your perspective on persistent Lyme disease?

I do think that this infection can develop into a persistent and chronic form that resists the current Lyme antibiotic treatment. There is a lot of debate about this chronic form whether or not it exists. The problem comes in with the unique features of the organism that causes Lyme disease. That is because Borrelia cannot be cultured after treatment in patients or in animal models in general. In some rare cases you do find culturable spirochetes after treatment in CSF or tissues but these are isolated cases.

While patients relapse in symptoms it is very difficult to isolate the organism. This is in stark contrast with other persistent infections like TB. When those patients relapse you can culture the organism from sputum. With Lyme this is quite different. You can’t see it. On the other hand I recognize that patients suffer and that patients can improve on re-treatment. I am sure you are aware of these clinical studies. These studies use the current Lyme antibiotics which are mainly active against the growing form of the organism but have very poor activity against the persister form. I do think that based on our experience with TB treatment, especially with persister drug pyrazinamide, studies looking at drug targeting Borrelia persisters will be important for improving the treatment of persistent Lyme disease.

Is there a correlation between the time needed for diagnosis and the persistence of the infection?

The more prolonged the infection or delayed diagnosis the more likely a persistent infection that is more difficult to treat would develop. This is also true for other persistent infections like Tuberculosis. In the mouse-model of TB infection or Borrelia infection, it is well known that if you allow the infection to go on longer before treatment it becomes harder to treat or eradicate. But it is not always as simple. Even though patients are diagnosed early, they sometimes still develop persistent symptoms.

It is not understood why 10-20% of patients don’t respond to current Lyme antibiotic treatments. There is a lot of debate. There are different theories:

auto-immune and immune-response to antigenic debris

residual tissue damage during infection and inflammation

co-infections with other organisms such as Bartonella or Babesia, etc

persistent infection

All these different possibilities may overlap and one is not exclusive of the other, that is, some of the conditions may co-exist. But I do think that persistent infection with the borrelia organism itself is important because that has been demonstrated in various animal-models in mice, monkeys, dogs as well as in humans. There are many reports and studies that demonstrate this persistence problem. Sometimes there is even isolation of the organism after antibiotic therapy.

There is this well-known case of Vicki Logan, a patient of Ken Liegner who unfortunately died. They were able to culture the Borrelia organism from her CSF. More recent studies from Adriana Marques at NIH show by xenodiagnosis that persistence is a problem. They use sterile ticks that bite patients who have been treated with antibiotics and in some cases the ticks can pick up the organism. The organism cannot be detected by culture but mainly by molecular tests like PCR. So it is a very complex organism.

The current culture technique is not very good. Despite the organism being viable and replicating in some ways, you can’t culture it in vitro so far. Even the mouse study by Emir Hodzic and Stephen Barthold in 2014 showed that, despite antibiotic treatment with ceftriaxone for 30 days, they could find increasing DNA content after treatment over a period of 12 months but were unable to culture the organism. They detected it in a form of increased DNA content. This indicates that the organism must have replicated in some way but then again you can’t culture it. So it is a tricky organism to work with.

In the Netherlands the PLEASE-study was done. Newspapers report that there is no pill that helps with complaints after Lyme. Doctors in the Netherlands say this study shows that chronic Lyme infection does not exist at all or should not be treated with antibiotics. What do you think of these interpretations and what can you conclude from your research?

First, the PLEASE study has various issues with the study design such as controls and enrollment criteria. It is peculiar that all patients were treated with ceftriaxone for 2 weeks and then divided into different treatment subgroups (placebo and different treatment regimens with current Lyme antibiotics) for 3 months. There is no real untreated control group, which makes the data hard to interpret. Also, some physicians would think the length of treatment is not long enough.

Another point is that the particular drugs used in the study: ceftriaxone, doxycycline, azithromycine/clarithromycine are not very good at killing persister forms. Compared with the short length of treatment it is not surprising you don’t see much improvement.

This is where our study may have impact because we found drugs that are more effective against the persister forms than the current Lyme antibiotics, and future studies are needed to evaluate the persister drugs. It is too early to say that antibiotics do not work. When you look at the comments from the media reporting the PLEASE study it sounded like all antibiotic-therapy does not work. They say that these patients with ongoing symptoms cannot be treated with antibiotics and that is not as simple as that. They don’t understand the importance of persister-drugs.

From Tuberculosis treatment we know that a persister-drug like pyrazinamide is important to shorten the therapy, and without it the therapy is longer, at least 9-12 months. With this persister-drug PZA we can shorten the therapy to 6 months. It is used in the context of a drug combination. We need a drug like PZA that targets the persister-form, a drug that targets the growing form and a drug that works on the growing form and the non-growing form.

In Tuberculosis we use:

Isoniazid that works on the growing form

Rifampin that works on the growing and non-growing form

Pyrazinamide that works on the non-growing persister-form

We need all these drugs in combination for 6 months to more effectively cure Tuberculosis. In the case of Lyme the current guideline is too simple. The current Lyme treatment is only good for the active form of the disease, but not so good for treating the persistent form of the disease probably due to persister forms of the organism. This is a very unusual organism that even in non-susceptible persister form it may produce disease.

Indeed many patients are treated successfully but there is also a significant group that doesn’t respond well to current Lyme antibiotics. This is where we think that our persister-drugs are likely to play a role. Indeed, we found an analogous 3 drug combination (doxycycline acting on growing form+cefuroxime acting on both growing and some non-growing persister form+daptomycin acting on non-growing persister form) as in TB treatment to be most active in eradicating aggregated biofilm-like structures of Borrelia. We are testing different persister drug combinations in vitro and doing animal studies. If the regimen is active in the animal model,then clinical trials will be conducted to prove their effectiveness in patients. From our experience with Tuberculosis I see the same treatment principle being applied to treating persistent Lyme disease.

Why is there so much controversy about Lyme disease?

It is very important to bring Lyme disease to people’s attention, especially the chronic Lyme disease patients who suffer tremendously for whom we don’t have an effective solution as yet. There is no FDA approved treatment for persistent Lyme disease. It is a huge unmet medical need.

Tuberculosis is a disease that is tangible. People can see it. If it infects a lung you can see cavities, you can do imaging. You can see the organism in a patient’s sputum. You can culture it. That is all visible. In Lyme this is different. The complexity of the spirochete makes it very difficult to isolate and culture the organism and patients continue to suffer, often silently, that is, they can appear well. I think this is part of the reason why the ‘orthodox camp’ continues to deny the problem.

Current science is not advanced enough to find a solution to isolate the organism or to provide a more effective treatment yet, but some promising studies are ongoing. Some physicians choose to only believe it when they see it but what one sees is actually very limited. A lot of things cannot be seen but they are there. With Lyme disease we don’t have a way to culture this organism like in Tuberculosis. Physicians mainly have patient treatment response to guide them. Maybe we could find bio-markers that reflect disease activity.

At the end of the day it all comes down to finding an effective regimen that more predictably cures these chronic patients. This is a hard problem as this is such a complex and heterogeneous disease, with spectrum ranging from active (acute) disease to persistent disease, some of which could merge with autoimmune disease. This is a huge challenge for modern medicine, and more research is needed to resolve the issue.

Do you think there is hope for patients who remain ill after treatment?

There is currently a lot of activity and interest in Lyme disease research. However, government funding for Lyme disease research is very limited. Patients and researchers are getting some support from major research-oriented foundations like the Cohen Foundation and the Global Lyme Alliance which support our work. For that, we are very grateful. They put increasing amounts of support for Lyme research, but on the other hand, to do clinical studies and develop better diagnostic tests, more funding from government agencies and commercial entities is needed.

Based on the recent studies coming out from different labs and improved funding environment, I do think there is hope. We are very encouraged by the recent results on Borrelia persister drugs and some highly active drug combinations we have found. Even though the data is in vitro we are hopeful that such persister drug-regimens may help to more effectively treat or cure persistent Lyme disease. In the meantime, we are working with our collaborators on validating the effective drug combinations in the animal model which is quite tedious, time consuming and expensive.

I get e-mails and phone-calls from patients all the time because they suffer and the current guidelines have failed them. I would tell them: “do not give up; there is hope”.

Animal models Hodzic E, Feng S, Holden K, Freet KJ en Barthold SW. Persistence of Borrelia burgdorferi following antibiotic treatment in mice. Antimicrob Agents Chemother 2008;52:1728–1736.

Yrjanainen H, Hyotenen J, Song SR, Oksi J, Hartiala K, Viljanen MK. Anti-tumor necrosis factor-alpha treatment activates Borrelia burgdorferi spirochetes in 4 weeks after ceftriaxone treatment in C3H/He mice. J Infect Dis 2007;195:1489–1496.

Yrjanainen H, Hytonen J, Hartiala P, Oksi J, Vijanen MK. Persistence of borrelial DNA in the joints of Borrelia burgdorferi-infected mice after ceftriaxone treatment. APMIS 2010; 118(9): 665–673.

Bockenstedt LK, Mao J, Hodzic E, Barthold SW, Fish D (2002) Detection of attenuated, non-infectious spirochetes after antibiotic treatment of Borrelia burgdorferi-infected mice. J Infect Dis 2002;186:1430–1437

Embers ME, Barthold SW, Borda JT, Bowers L, Doyle L, Hodzic E, Jacobs MB, Hasenkampf NR, Martin DS, Narasimhan S et al. Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection. PLoS One 2012;7(1):e29914.

Malawista SM, Barthold SW en Persing DH. Fate of Borrelia burgdorferi DNA in tissues of infected mice after antibiotic treatment. J Infect Dis 1994;170:1312–1316.

Straubinger RK. PCR-based quantification of Borrelia burgdorferi organisms in canine tissue over a 500-day postinfection period. J Clin Microbiol 2000; 38:2191–2199.

Straubinger RK, Straubinger AF, Summers BA en Jacobson RH. Status of Borrelia burgdorferi infection after antibiotic treatment and the effects of corticosteroids: an experimental study. J Infect Dis 2000; 181:1069–1081.

Straubinger RK, Summers BA, Chang YF, Appel MJG. Persistence of Borrelia burgdorferi in experimentally infected dogs after antibiotic treatment. J Clin Microbiol 1997; 35:111–116.

Hodzic e , Imai D, Feng S en Barthold SW. Resurgence of Persisting Non-Cultivable Borrelia burgdorferi following Antibiotic Treatment in Mice. PLoS One. 2014;9(1):e86907. Xenodiagnosis Marques A, Hu LT et al. Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-human study. Clin Infect Dis. 2014;58(7):937-45. Persisters Feng J, Zhang S, Shi W en Zhang Y. Activity of Sulfa Drugs and Their Combinations against Stationary Phase B. burgdorferi In Vitro. Antibiotics. 2017;6(1). pii: E10.

Feng J, Zhang S, Shi W en Zhang Y. Ceftriaxone Pulse Dosing Fails to Eradicate Biofilm-Like Microcolony B. burgdorferi Persisters Which Are Sterilized by Daptomycin/ Doxycycline/Cefuroxime without Pulse Dosing. Front Microbiol. 2016;7:1744.

Feng J, Shi W, Zhang S, Sullivan D, Auwaerter PG en Zhang Y. 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. 2016;7:743.

Feng J, Weitner M, Shi W, Zhang S en Zhang Y. 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. 2016;7:62.

Feng J, Zhang S, Shi W en Zhang Y. Persister mechanisms in Borrelia burgdorferi: implications for improved intervention. Emerg Microbes Infect. 2015;4(8):e51.

Feng J, Auwaerter PG en Zhang Y. Drug combinations against Borrelia burgdorferi persisters in vitro: eradication achieved by using daptomycin, cefoperazone and doxycycline. PLoS One. 2015;10(3):e0117207.

Feng J, Wang T, Zhang S, Shi W en Zhang Y. An optimized SYBR Green I/PI assay for rapid viability assessment and antibiotic susceptibility testing for Borrelia burgdorferi. PLoS One. 2014;9(11):e111809.

Feng J, Wang T, Shi W, Zhang S, Sullivan D, Auwaerter PG en Zhang Y.Identification of novel activity against Borrelia burgdorferi persisters using an FDA approved drug library. Emerg Microbes Infect. 2014;3(7):e49.

Sharma B, Brown AV, Matluck NE, Hu LT en Lewis K. Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells. Antimicrob Agents Chemother. 2015;59(8):4616-24.

Lewis K. Persister cells, dormancy and infectious disease. Nat Rev Microbiol 2007; 5(1):48–56.

Lewis K. Persister cells. Annu Rev Microbiol 2010; 64(1):357–372.

Barthold SW, Hodzic E, Imai D, Feng S, Yang X, Luft BJ. Ineffectiveness of tigecycline against persistent Borrelia burgdorferi. Antimicrob Agents Chemother 2010; 54:643–651.