Lyme Disease: Borrelia burgdorferi Gretta Johnson Ashley Dahly

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.

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

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).

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. http://www.bayarealyme.org/about-lyme/history-lyme-disease/

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

EM rash

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

Late Signs & Symptoms

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

Transmission

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.

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)

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

Treatment

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

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

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

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

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

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

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

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

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

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), 7116-7121. 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 294587, 10 pages, 2012. doi:10.1155/2012/294587 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), 4616-4624. Retrieved October 20, 2016. https://www.holtorfmed.com/lyme-disease-evade-immune-system/ https://www.cdc.gov/lyme/index.html http://textbookofbacteriology.net/Lyme.html http://borreliaburgdorferi.org https://microbewiki.kenyon.edu/index.php/Borrelia_burgdorferi http://web.uconn.edu/mcbstaff/graf/Student%20presentations/Bburgdorferi/bburgdorferi.html