1. Lyme Disease: Borrelia burgdorferi
Gretta Johnson Ashley Dahly

2. Organism Identification
Neither Gram positive or Gram negative*
Group: Spirochaetes
Gram stain
Borrelia burgdorferi is not classified as either Gram-positive or Gram-negative. When B. burgdorferi is Gram-stained, the cells stain a weak Gram-negative by default, as safrin is the last dye used.

3. Motility and Physiology
Morphology: spiral, wave-like body
Flagella enclosed between inner and outer membrane
Allows bacteria to move in high and low viscosity media (thickness)
Contributes to its high virulence factor

4. Organism Characteristics
Slow generation time (about 12 to 24 hrs)
Unusual genome: a linear chromosome approximately one megabase in size and numerous linear and circular plasmids
It can be cultivated on a modified Kelly medium called BSK (Barbour-Stoenner- Kelly).

5. Interesting History
Lyme disease was diagnosed as it’s own separate condition in 1975 in Old Lyme, Connecticut. It was originally mistaken for juvenile rheumatoid arthritis.

6. Early Signs & Symptoms (3-30 days after bite)
Fever, headache, chills, fatigue, muscle and joint aches, and swollen lymph nodes
Erythema migrans (EM):
Occurs in 70-80% of infected patients
Begins at the site of a tick bite after an average 7 day delay
Expands gradually over a period of time reaching up to 12in across
May feel warm to the touch but rarely painful or itchy

7. EM rash

8. Later Signs & Symptoms (days to months after bite)
Severe headaches and neck stiffness
Additional EM rashes on other areas of the body
Severe joint pain in knees and other large joints
Facial or Bell’s palsy
Pain in tendons, muscle joints, and bones
Heart palpitations or irregular heart rate
Dizziness or shortness of breath
Nerve pain

9. Late Signs & Symptoms

10. Transmission
Borrelia burgdorferi is transmitted through a bite from an infected blacklegged or deer tick
The blacklegged and deer tick spread the disease in the northeastern, mid-Atlantic, and North- Central regions of the US
The Western Blacklegged tick spreads the disease to the western part of the country
The ticks can attach to any part of the body but most often found to hide in hard to find places such as armpit, groin, and scalp.
In most cases the tick must be attached for 36 to 48 hours or more to transmit the bacterium
Most people are infected by the bite from an infected nymph

11. Transmission

12. Evading the Immune System
The outer membrane contains many different outer surface proteins (Osp)
These proteins work as an invisible cloak so the immune system of the host doesn’t make antibodies for weeks
Because of this factor, many people who have been bitten by a tick and go in to get tested for the disease and get a negative test result because the body hasn’t been alerted that there is an infection in the body.
Borrelia burgdorferi contains a flagella which allows the bacteria to get through body tissues and thick mucus which would normally stop other bacteria
It is also able to change its form by altering its proteins on the outer cell wall
Usually the immune system is easily able to recognize the invader and create the specific antibodies to fight it, but when Borrelia burgdorferi continually changes its outer proteins, the immune system is unable to keep up with making the correct antibodies to fight it.

13. Immune System Response
When the innate immune system recognizes Borrelia burgdorferi it releases inflammatory mediators such as interleukin (IL)-1β, IL-6, IL-10, IL-12, tumor necrosis factor (TNF) from monocytes, macrophages, and neutrophils
These components help recruit other parts of the innate immune system but also for signaling to the adaptive immune system
The adaptive immune system consists of T and B lymphocytes and acts as the second line of defense against Borrelia burgdorferi
(Skogman et al., 2012)

14. Treatment
Lyme disease can usually be treated completely with antibiotics when started in the early stages of the disease
Ex: doxycycline, amoxicillin, or cefuroxime axetil
There has been recent studies that suggest Borrelia burgdorferi is resistant to the antibiotic erythromycin
However, erythromycin is not a typical form of antibiotic treatment

15. Treatment

16. Prevention Avoid direct contact with ticks
Avoid wooded and brushy areas with high grass and leaf litter
Walk in the center of trails
Repel ticks with DEET or permethrin
Use repellents that contain at least 20-30% DEET on exposed skin or clothing
Use products that contain permethrin on clothing or pre-treat clothing
Find and remove ticks from the body
Bathe or shower as soon as possible after coming indoors
Conduct a full-body tick check
Examine gear and pets
Tumble dry clothes in the dryer on high for 10 min to kill ticks on clothing

17. Paper Review
Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells Bijaya Sharma, Autumn Brown, Nicole Matluck, Linden Hu, Kim Lewis

18. Background: What is a Persister Cell?
In 1944, Dr. Joseph Bigger identified persisters in a study on Staphylococcus pyogenes
Bacterial cells that can escape the effects of antibiotics without going through genetic change
Arise due to a state of dormancy (a state at which a cell is metabolically inactive)
Phenotypic variants that are tolerant to antibiotics

19. Persistence vs Resistance
Persistent
Resistant
Genetics
Genetic
Phenotypic
Reproduction
Same sensitivity of original colony
Purely resistant cells
Growth in presence of Antibiotics
None (loss of Ab causes growth)
Exponential
Overall Outcome
Recalcitrance of chronic infections
Antibiotic resistance

20. Background cont.
Biofilms: When bacteria adhere to surfaces and excrete glue-like, slimy substance that anchors attaches them to materials
Antibiotics kill regular cells but the dormant persisters survive, when antibiotics drop, the dormant cells resuscitate and repopulate in the biofilm
Many patients that receive the 1st round of antibiotics for Lyme arthritis do not fully get rid of the bacteria
This study reports that drug-tolerant persisters in B. burgdorferi are present and analyzes the possible solutions to this problem

21. Results: Characterizing Persisters
Biphasic pattern of persisters was shown in Amoxicillin and Ceftriaxone (Fig 1a.)
Previous persister studies show that bacteria levels stay the same, even as antibiotic levels rise (Fig. 1b and c)
Fig 1d. Decreased slightly, but not significant

22. Results: Characterizing Persisters cont.
Amoxicillin
Testing persisters vs resistant mutant
Colonies of surviving cells (persisters) were regrown and tested for MIC
Persister levels of the colonies recovered were not significantly different than the colonies prior to antibiotics
Shows no genetic variation
Ceftriaxone

23. Results: Eradication of Persisters
Only 2 system were successful in killing persisters
Pulse dosing: high dosing antibiotics interval followed by non antibiotic interval
Prodrug Mitomycin C: anticancer drug that destroys DNA

24. Conclusions
Persisters could be the reason that antibiotics do not effectively get rid of lyme disease
Similar to other pathogens, B. burgdorferi killing by antibiotics is biphasic and the surviving clones are not resistant mutants
Mitomycin C (cancer drug) was able to eradicate persisters after 24 hours, but this drug is very toxic and can result in serious negative side effects
Pulse dosing was successful in eradicating bacterial populations, so this is a place where more research can be done
The mechanisms by which they are able to survive still remain unknown

25. References
Wood, T. K., Knabel, S. J., & Kwan, B. W. (2013, September 13). Bacterial Persister Cell Formation and Dormancy. Applied and Environmental Microbiology, 79(23), Retrieved October 20, 2016.
Barbro H. Skogman, Sandra Hellberg, Christina Ekerfelt, et al., “Adaptive and Innate Immune Responsiveness to Borrelia burgdorferi sensu lato in Exposed Asymptomatic Children and Children with Previous Clinical Lyme Borreliosis,” Clinical and Developmental Immunology, vol. 2012, Article ID , 10 pages, doi: /2012/294587
Sharma, B., Brown, A. V., Matluck, N. E., Hu, L. T., & K. L. (2015, May 26). Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells. Antimicrobial Agents and Chemotherapy, 59(8), Retrieved October 20, 2016.