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1. Challenges of Post-Treatment Lyme Disease Syndrome
(PTLDS): Will Persister Drugs
Help to Cure PTLDS?
Ying Zhang, MD, PhD
Department of Molecular Microbiology & Immunology
Bloomberg School of Public Health
Johns Hopkins University
Email: yzhang@jhsph.edu

2. Faculty/Presenter Disclosure
• Faculty:
Ying Zhang
• Relationships with commercial interests:
– Grants/Research Support:
Global Lyme Alliance
JHU Fisher Center
Lyme Disease Association
Cohen Foundation
CFPC CoI Templates: Slide 1

3. Disclosure of Commercial Support
• Potential for conflict(s) of interest:
– None to declare
CFPC CoI Templates: Slide 2

4. Mitigating Potential Bias
• The foundations that provide the support have no role in
research design or execution of the project, or decision to
publish. The research ideas are solely from the investigators
and not from the foundations.
CFPC CoI Templates: Slide 3

5. Lyme disease in United States
300,000 each year
Northeast, mid-Atlantic, upper
midwest, and northern California in US
Ticks that transmit Lyme disease found
in nearly half of all US counties
Central and eastern Europe, most of
northern Asia
Lyme Disease: Most common vector-borne disease in US
Reported Cases of Lyme Disease - United States, 2014

6. PTLDS
Morph changes
Treatment problem
Diverse disease expression
Immune dysfunction Diagnosis problem
VBNC
Peculiar Features of Borrelia and Lyme Disease

7. Lyme Disease:
Signs/Symptoms and Treatment
Current treatment: IDSA 2-4 weeks (Early Lyme) vs ILADS (Late Lyme):
Doxycycline, or Amoxicillin, or Cefuroxime (Ceftriaxone, iv)
Early localized Early disseminated Late disseminated PTLDS

8. • 10-20% (higher in Europe) Lyme patients after 2-4 week treatment have
Persistent and Recurrent Symptoms (fatigue, pain, or joint and muscle aches,
“brain fog”) “Post-Treatment Lyme Disease Syndrome” (PTLDS, CDC, 2014)
• Current Lyme antibiotic treatment has poor activity for PTLDS
Persistence Problem in Lyme Disease
(“Chronic Lyme”, PTLDS)
PTLDS: Biggest Challenge Facing Modern Medicine
Author Year Treatment Outcome
Klempner 2001 IV Ceftr 4 w+ Doxy 2
month vs placebo
No improvement in
fatigue or quality of life
Krupp 2003 IV Ceftr 4 w
vs placebo
Improvement in fatigue
but not cognition
Fallon 2005 IV Ceftr 10 w
vs placebo
Improvement in cognition
at 12 w but not 24 w
Berende
(PLEASE)
2016 IV Ceftr 2 w +
Doxy or Clari+HCQ
12 w
No improvement in
fatigue or quality of life

9. PTLDS: Elephant in the Room

10. PTLDS (Heterogeneous): Causes unclear:
(A) Host response to antigenic debris
(B) Autoimmune
(C) Co-infections / Secondary opportunistic infections
(D) Residual damage to tissues during infection
(E) Persisters not killed by current antibiotics
No FDA-approved treatment for PTLDS!
New York Times, 7/8/2013

11. Huge healthcare cost (1.3 B): but do not work well, getting worse

12. Antibiotic treatment is unable to clear persisting Borrelia
burgdorferi in mice, dogs and monkeys, but organism
nonculturable (Barthold S et al., 2010; AAC, 54:643-51;
Hodzic et al. 2014, PLoS One 9: e86907; Embers M et al.
PLoS One 2012;7:e29914):
Viable but non-culturable (VBNC)
Human study with tick xenodiagnosis showed patient after
treatment still had Borrelia bacteria (A Marques et al.
Clinical Infectious Diseases, 58:937-945, 2014)
CDC webinar on Lyme persistence (5/22/2014):
http://www.cdc.gov/lyme/resources/May2014_HHS_Lyme
_Disease_webinar_mazarin_508.pdf
Evidence for persisters not killed by current antibiotics

13. Resurgence Phenomenon: Analysis of flaB DNA in tissues from mice treated
with ceftriaxone 30 days after inoculation
Hodzic E, Imai D, Feng S, Barthold SW (2014). PLoS ONE 9(1): e86907. doi:10.1371/journal.pone.0086907
http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0086907

14. Log phase (3 Day) Stationary phase
Current Lyme antibiotics have poor activity against Borrelia persisters
in vitro!
Stationary phase (7 Day)
50 uM drugs 5 day treatment
Feng J, et al. (2014). Emerging Microbes and Infections
(www.nature.com/emi), July 2, 2014. 3, e49

15. Bacterial Persisters
• Bacterial persisters first described by Hobby in 1942;
“persister” given by Bigger in 1944
• Penicillin kills 99% bacteria, 1%, not growing, not killed
by antibiotic, called “persister”, still susceptible to
antibiotic upon subculture
• Resistance (tolerance) in persisters is phenotypic and
distinct from genetic resistance
• Persisters underlie persistent infections (TB, UTI, Lyme)

16. Why Persisters and Persister Drugs Matter?
Dandelion Phenomenon

17. Drug Combination (targeting both growing
bacteria and non-growing persisters)
Principle of TB Treatment:
A Model for Lyme Treatment?
INH
(Doxy,
Amox)
PZA
(Dapto)
RIF
(Cefuroxime)
Reverters
Persisters
(Y. Zhang, Emerging Microbes & Infections (2014) 3, e3; doi:10.1038/emi.2014.3)

18. • No antibiotics effective against B. burgdorferi persisters
• Developed a new high throughput SYBR Green/PI assay
Feng J, Wang T, Zhang S, Shi W, Zhang Y (2014) PLoS ONE 9(11): e111809
• Screened FDA drug library, NCI compound libraries against
B. burgdorferi persisters (150 FDA-approved drugs):
Problems and Solutions
1. Top 27 FDA-approved drugs active against Borrelia persisters:
daptomycin, clofazimine, sulfa drugs, etc.
Feng J, et al. (2014). Emerging Microbes and Infections (www.nature.com/emi),
July 2, 2014. 3, e49
2. Additional 113 active hits from FDA drug library that have higher activity
against Bb persisters than current Lyme antibiotics
Feng J et al. (2015). Antibiotics (www.mdpi.com) 4(3), 397-410
3. New NCI compound screens identify top 30 hits anthracycline antibiotics
having high activity against Borrelia persisters
Feng et al. (2015). Emerging Microbes and Infections, June 3, 2015
(www.nature.com/emi)

19. Drug combinations are more effective against Bb persisters
Feng et al. (2015). PLoS One, Mar 25;10(3):e0117207.
 Different persister forms: Aggregated microcolony forms
(biofilm-like) are more resistant than planktonic form
 Single drugs, or two or even most 3 drug combos failed to
eradicate microcolony persister forms
 Best drug combos: Dapto+Doxy+CefU eradicates all
persisters and even the most resistant microcolony form
(biofilm-like structure), failed to grow in subculture
a. Control b. CefP d. DAP
e. DOX+CefP f. DAP+CefP g. DAP+DOX h. DAP+DOX+CefP
c. DOX

20. Subculture to assess viability of drug-treated
stationary phase B. burgdorferi
Subculture (15 days) of 10 day old B. burgdorferi stationary phase culture
treated with different antibiotics alone or in combinations (10 μg/ml).
Feng et al. (2015). PLoS One, Mar 25;10(3):e0117207

21. Subculture to assess the viability of drug treated Bb
• 15 day old stationary phase Bb culture
• Low drug concentration 10 ug/ml
• Treated for 7 day,
• Subculture for 7 or 21 days
Live % (MC) Subculture 7 days
Subculture 21
days
Control 79% 6×106 2×107
Dox+Cefu 67% 1×106 1×107
Dox+Cefp 67% 9×105 1×107
Dox+Cefu+Dap 30% 0 0
Dox+CefP+Dap 29% 0 0
Dox+Cefu+MitC 45% 0 1×106
Dox+Cefu+Dau 12% 0 0
Cefu: Cefuroxime; CefP: Cefoperazone
Feng J et al., Front. Microbiol., 10 February 2016

22. Subculture to assess the viability of drug treated Bb
Feng J et al., Front. Microbiol., 10 February 2016

23. A Unified Yin-Yang Model for Lyme Disease?
Where Growing Bacteria/Active infection and Persisters/Latent infection
meet and interconvert: as a continuum
Current Lyme Antibiotics:
Doxycycline,
Amoxicillin, Macrolides
Reverters
Persisters
Persister
Antibiotics:
Daptomycin,
Clofazimine
Zhang Y. Emerging Microbes & Infections (2014) 3, e3; doi:10.1038/emi.2014.3
Cefoperazone (Ceftin), Ceftriaxone
Sulfa drugs, Daunomycin
Acute Lyme (IDSA)?PTLDS (ILADS)?
⊙

24. Key Questions/Future Directions
1. Can drug combinations that eradicated biofilm-like
microcolonies in vitro cure persistent Lyme disease in animal
models and patients?
2. What causes PTLDS ? Need to test other possibilities
3. What are the biomarkers of treatment response and cure?
4. Why can we not culture VBNC organisms after treatment?

25. “The greater the ignorance,
the greater the dogmatism.”
--- William Osler
“It’s there but you cannot see it”
--- Willy Burgdorfer

26. Acknowledgements
Johns Hopkins University
Jie Feng, Megan Weitner,
Wanliang Shi, Shuo Zhang
Global Lyme Alliance
Fisher Center
Cohen Foundation

27. Thank You!