Biofilms of the Lyme disease spirochete

Thanks to a recent study published in PLoS One, we now know that free-swimming Borrelia burgdorferi are able to organize themselves into a sedentary community called a biofilm.  This is not too surprising since most other bacteria are capable of the same feat when provided the opportunity.  In fact, outside of the laboratory many bacteria, including those that live on and within us, spend much of their time within biofilms.

Prior to the 1990s biofilms were thought to be blobs of goo containing bacteria randomly distributed throughout their sticky matrix.  In reality, the bacteria and matrix are carefully organized into a complex three-dimensional structure. B. burgdorferi biofilms are no exception.  The organization of B. burgdorferi is apparent even at the earliest stages of biofilm development.  The images below show B. burgdorferi developing into a biofilm on a solid surface.  Instead of randomly associating with each other, the spirochetes organize themselves into "nets" of the type you see hanging from basketball hoops.  The spirochetes come together lengthwise to form the "strands" of the net.  With time, the biofilm thickens as the bacteria form additional layers.  Most of the spaces in the net close up with the rest probably ending up forming a network of channels.  The remaining holes can be seen as pits along the surface of the mature biofilm.  The pits appear to be entry points for the channels, which are thought to circulate nutrients to the members of the community and remove waste products.

Figure 2 from Sapi et al.  Atomic force microscopy of a developing B. burgdorferi biofilm.

Stalks can also rise up from the surface of microbial biofilms.  One stalk can be seen in this "flyover" along the surface of a mature B. burgdorferi biofilm.

Video S2 from Sapi et al.  Composte image from atomic force microscopy.

The matrix of B. burgdorferi biofilms includes DNA and an alginate-like substance bound to calcium.  The images below capture what appears to be matrix being laid down at an early stage of biofilm formation.

Figure 4 from Sapi et al.  Atomic force microscopy of an aggregate of B. burgdorferi in an early stage of biofilm development.  The matrix is colored blue in panel B.

What is the biological significance of B. burgdorferi biofilms?  To answer this question, the authors will need to determine whether B. burgdorferi assembles into biofilms at some point during its life cycle, which involves stages in the tick and vertebrate host.

Reference

Sapi, E., Bastian, S.L., Mpoy, C.M., Scott, S., Rattelle, A., Pabbati, N., Poruri, A., Burugu, D., Theophilus, P.A.S., Pham, T.V., Datar, A., Dhaliwal, N.K., MacDonald, A., Rossi, M.J., Sinha, S.K., & Luecke, D.F. (2012). Characterization of biofilm formation by Borrelia burgdorferi in vitro. PLoS ONE, 7 (10) DOI: 10.1371/journal.pone.0048277

A post-Thanksgiving story of leptospirosis

I'm about half way through 1491, a book that gives readers a view of the Americas before Columbus showed up.  It also describes the devastating impact that foreign infectious diseases had on the native population as Europeans explored the New World.

One chapter tells the story of Tisquantum (Squanto), who lived in the village of Patuxet, one of the many Indian communities thriving along the coast of New England at the time.  In 1614 Thomas Hunt, a British slave trader, kidnapped Tisquantum and other Indians and shipped them to Spain.  Fortunately, Tisquantum was rescued by Spanish priests before he could be sold.  After convincing the priests to let him return home, he left for London, where he learned English while staying at a shipbuilder's home, and eventually made his way back to North America.  As he sailed down the New England shoreline in 1619 on a British ship, he realized that the world familiar to him had vanished.  A mysterious disease had wiped out 90% of the population of coastal New England.  When he arrived at his home village of Patuxet, he found it deserted.  Tisquantum was soon captured and sent to Massasoit, the leader of the Wampanoag confederacy, which encompassed Patuxet.  Massasoit did not trust Tisquantum because of his recent association with the British, yet he would later use him as a translator in a negotiation that turned out to be a pivotal event in American history.

The epidemic had been blamed at one time or another on smallpox, the plague, yellow fever, typhus, and hepatitis.  As I've mentioned before, a recent analysis has added leptospirosis to the list of suspects.  The symptoms and signs of leptospirosis match those reported from first-hand accounts of the mystery ailment.  Here's a post on the Slate website about the epidemic.  I'm glad to see that the story is getting attention from popular news sites.

Leptospirosis can be deadly, but could it account for the devastating 90% motality rate of the 1616-1619 epidemic?  A hypervirulent strain of Leptospira or genetic susceptibility of the Indians could be an explanation.  However, the authors of the study thought that the most critical factor was the Indian lifestyle, which brought them into repeated contact with Leptospira in the environment.  The Europeans who fished nearby were spared because they did not engage in activities that exposed them to Leptospira.  Therefore, only the Indians contracted the illness, according to the hypothesis.

Figure 3 from Marr and Cathey, 2010.

Whatever its cause, it's hard to overstate the significance of the epidemic.  Prior to 1616, the New England native communities traded with the Europeans and even welcomed them for brief stays.  However, all attempts by the foreigners to establish permanent settlements were fiercely resisted.  Coastal New England was well defended by the large native population.  The Wampanoag confederacy became especially hostile towards the Europeans after having their citizens abducted.

By the time the Mayflower landed in Patuxet (Plymouth) in December of 1620, the thinking of the Wampanoag had changed.  Their depleted population was vulnerable to attack by their longtime enemies to the west, the Narragansett, who remained untouched by the epidemic.  To forestall an attack, Massasoit felt that the best course of action was to form an alliance with the Pilgrims rather than expel them.  In the spring of 1621, with Tisquantum serving as the translator, Massasoit arranged a peace treaty with the Pilgrims.

Reference

Marr J.S. & Cathey J.T. (2010). New hypothesis for cause of epidemic among Native Americans, New England, 1616–1619, Emerging Infectious Diseases, 16 (2) 281-286. DOI: 10.3201/eid1602.090276

Related post

• Did spirochetes kill off the Indians in Massachusetts before the Mayflower landed?

Inflammatory spirochete debris left behind following antibiotic treatment for Lyme disease

According to the CDC, 10-20% of Lyme disease patients who have completed antibiotic therapy continue to suffer from symptoms such as joint, muscle, and neurological pain.  The following hypotheses are often presented as possible reasons for the lingering symptoms:  autoimmunity triggered by the infection, tissue damage inflicted by the spirochetes, and (depending on whom you ask) failure of antibiotics to kill all the spirochetes.  A new paper from Linda Bockenstedt's group at Yale proposes that antibiotic treatment of disseminated Borrelia burgdorferi infection leaves behind inflammatory pieces of dead spirochetes that are responsible for the persisting symptoms.

Bockenstedt's group used the mouse model of Lyme disease for the study.  To ensure that the tissues harbored enough B. burgdorferi spirochetes to be visible by intravital microscopy, the mice were genetically deficient in the intracellular signaling protein MyD88.  MyD88 links the recognition of microbial parts by most Toll-like receptors to activation of certain nuclear genes whose products are involved in the inflammatory process.  Mice lacking MyD88 are unable to control the proliferation of a number of bacterial pathogens, including B. burgdorferi.  The load of B. burgdorferi in tissues is about 100-fold higher in MyD88-deficient mice than in mice with a complete immune system.

The spirochetes were genetically altered to express green fluorescent protein (GFP).  The GFP⁺ B. burgdorferi was introduced into the MyD88-deficient mice by tick inoculation.  21 days later some of the mice were treated for one month with doxycycline, one of the antibiotics used to treat Lyme disease in humans.

The researchers next peered into the thin layer of skin covering the ear by intravital microscopy.  In the mice that were left untreated, they saw lots of spirochetes scurrying about in the dermis.

At the deepest depths of the dermis, they noticed immobile specks and patches of green material deposited near the cartilage.  They also saw the deposits in the doxycycline-treated mice.  The material was detected by immunofluorescence of ear sections with antibody against B. burgdorferi up to 10 weeks after antibiotic treatment was completed, indicating that the immune system was unable to clear the deposits.

There was no evidence that any spirochetes survived antibiotic treatment.  The researchers did not see any motile spirochetes in the skin by intravital microscopy.  In addition, tissues were culture negative, ticks that fed on the treated mice were culture negative (xenodiagnosis), and transplantation of skin from the treated mice failed to transmit the infection to recipient mice.  Based on these results, the authors concluded that the deposits were remnants of dead spirochetes.  As expected, untreated mice tested positive by these assays.

Since chronic infection can lead to Lyme arthritis, the investigators also examined the joints.  In another set of mice, the infection was allowed to proceed for four months.  The mice were then treated with the antibiotic ceftriaxone for 18 days.  When the researchers looked in the joints by intravital microscopy, they again saw the green material (see figure below).

Fig. 5 from Bockenstedt et al. showing the surface of the patella where it meets the tendon (enthesis).   Panel A, from mouse infected for 4 months, untreated.  Panel B, from mouse infected for 4 months and then treated with ceftriaxone for 18 days. Scale bar, 30 µm.

A critical issue to address is whether the amorphous material left behind following antibiotic treatment inflames the joints.   The authors could not answer this question directly because of the limitations of the mouse model. Histopathology is unlikely reveal joint inflammation, even in the untreated animals, because laboratory mice do not reliably exhibit joint inflammation so late (4-5 months) during B. burgdorferi infection.  Instead, the authors conducted a test tube experiment to see whether the deposits had inflammatory potential.  They ground up joint tissue from antibiotic-treated mice in buffer and applied the homogenate to cultured mouse macrophages.  The macrophages responded by producing TNF, a key cytokine that promotes inflammation.  The more tissue that was added, the more TNF that was produced by the macrophages.  In contrast, joint tissue from uninfected mice did not promote TNF production by the macrophages.  Therefore, the deposits had the potential to spark inflammation, even after motile spirochetes were eliminated by antibiotics.  The debris would continue to inflame the tissues even after antibiotics killed all live spirochetes, explaining why symptoms persist in ~10% of Lyme arthritis cases even after antibiotic treament.

The relevance of the deposits to Lyme disease in humans could be questioned because the MyD88-deficient mice did not have a complete immune system.  The authors addressed this concern in the Discussion by mentioning a recent study that described a TLR1 variant linked to severe inflammation and treatment failure in Lyme arthritis patients.  Although the gene encoding MyD88 has never been examined in Lyme disease patients, it is conceivable that the TLR1 variant or different forms of other immune genes lead to deposits of Borrelia antigen in the joint and other host tissues.

The authors also addressed the possibility that the deposits are really biofilms, which generally resist killing by antibiotics.  Biofilms are believed to be populated by persister cells, which are in a nondividing state that allows bacteria to tolerate antibiotics.  According to the authors, if the deposits had harbored persister cells, those cells should have resumed growing when conditions became favorable for growth again.  Because the skin and joints from the treated mice were culture negative and because the skin also tested negative by xenodiagnosis and transplantation assays, the authors quickly dismissed the biofilm hypothesis.

Stricly speaking, the authors are correct.  Persister cells should start multiplying again in fresh culture medium.  However, it's hard to dismiss the biofilm hypothesis completely given the known examples of culture-negative chronic infections associated with biofilms (see this review for one example).  Electron microscopy of the joint tissue could reveal whether these deposits are intact spirochetes or debris.

Regardless of their exact nature, deposits of antigen have never been detected within the joints of Lyme arthritis patients.  Allen Steere's group failed to find such deposits in pieces of synovial membrane removed from 26 patients with antibiotic-refractory Lyme arthritis.   The findings of Bockenstedt and colleagues, who detected the deposits in a location outside of the synovial membrane, suggest that Steere's group was looking in the wrong place.


Featured paper

Bockenstedt, L., Gonzalez, D., Haberman, A., & Belperron, A. (2012). Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy Journal of Clinical Investigation, 122 (7), 2652-2660 DOI: 10.1172/JCI58813
 
Helpful references

Bolz DD, Sundsbak RS, Ma Y, Akira S, Kirschning CJ, Zachary JF, Weis JH, and Weis JJ (August 1, 2004).  MyD88 plays a unique role in host defense but not arthritis development in Lyme disease.  The Journal of Immunology 173(3):2003-2010.  Link

Strle K, Shin JJ, Glickstein LJ, and Steere AC (May 2012).  Association of a Toll-like Receptor 1 polymorphism with heightened Th1 inflammatory responses and antibiotic-refractory Lyme arthritis.  Arthritis and Rheumatism 64(5):1497-1507.  DOI: 10.1002/art.34383

Bakaletz LO (October 2007).  Bacterial biofilms in otitis media, evidence and relevance.  The Pediatric Infectious Disease Journal 26(10):S17-S19.  Link

Carlson D, Hernandez J, Bloom BJ, Coburn J, Aversa JM, Steere AC (December 1999).  Lack of Borrelia burgdorferi DNA in synovial samples from patients with antibiotic treatment-resistant Lyme arthritis.  Arthritis and Rheumatism 42(12):2705-2709.  DOI: 10.1002/1529-0131(199912)42:12<2705::aid-anr29>3.0.CO;2-H


Related posts

• Chronic Lyme disease in mice?
• Tigecycline fails to eradicate persisting Borrelia burgdorferi
• The magic of antibiotic tolerance

100 (micro)meter dash

A fun read in the current issue of Nature Reviews Microbiology is an essay entitled "The Microbial Olympics, " just in time for the Summer Olympics.  You will find stories about microbes competing in boxing, javelin, pathogen relay, diving, and other Olympic events.  Flagellated bacteria compete in the 100 micrometer dash, which you can watch below.  (Note that some of the contestants were genetically modified.)

 Source

Lane assignments:

1. E. coli chimera (has sodium-driven flagellar motors instead of its normal proton-driven motors)
2. E. coli (proton-driven flagellar motors)
3. Vibrio alginolyticus, puller (clockwise-locked flagellum "pulls" cell body from front)
4. Vibrio alginolyticus, pusher (counterclockwise-locked flagellum "pushes" cell body from back)
5. Pseudomonas aeruginosa
6. Rhodobacter sphaeroides
7. Rhodospirillum rubrum
8. Yersinia enterocolitica

Reference

Youle M, Rohwer F, Stacy A, Whiteley M, Steel BC, Delalez NJ, Nord AL, Berry RM, Armitage JP, Kamoun S, Hogenhout S, Diggle SP, Gurney J, Pollitt EJG, Boetius A, and Cary SC (August 2012).  Nature Reviews Microbiology 10(8):583-588.  DOI: 10.1038/nrmicro2837

Not so golden? Microscopic agglutination test for diagnosis of leptospirosis

The microscopic agglutination test (MAT) is designated the "gold standard" for the laboratory diagnosis of leptospirosis, a spirochete disease that can cause severe illness if not promptly treated.  Although imperfect, MAT is used as the benchmark when the performance of another diagnostic test for leptospirosis is being assessed.  It is also used to determine the prevalence of leptospirosis in a population.  How imperfect is MAT?  A recent study by Limmathurotsakul and colleagues, published in the journal Clinical Infectious Diseases, claims that its performance is much worse than scientists previously thought.

MAT involves mixing serial dilutions of patient sera with live suspensions of Leptospira.  If agglutinating antibodies against Leptospira are present, the spirochetes will clump.  The clumps can be seen by darkfield microscopy.  Although the idea behind MAT is simple to understand, the technique itself is cumbersome.  Since agglutinating antibodies react best with the specific Leptospira serovar infecting the patient, cultures of at least one serovar from each of the ~20 major Leptospira serogroups must be maintained.  To perform the assay, each serum dilution is mixed individually with a suspension from each culture and examined by microscopy one at a time.  The assay is time consuming, laborious, and potentially hazardous to laboratory personnel.  For these reasons MAT is not routinely employed for diagnostic testing outside of the research setting.

The performance of a diagnostic test is judged by its sensitivity and specificity.  The problem with leptospirosis is figuring out how many actually have the disease so that the sensitivity can be calculated accurately.  Since the sensitivity of culture is poor, researchers rely on antibody tests such as MAT to identify leptospirosis cases.  This approach assumes that the sensitivity and specificity of MAT are 100%.

In general there are two problems with using antibody tests for diagnostics.  The first is that it takes time for the immune system to generate enough antibody that can be detected.  The second problem is that those with previous exposure to the pathogen will test positive even if they are not currently infected.  To minimize these problems, patients with the signs and symptoms of leptospirosis are deemed to have a positive MAT if they fulfill one of the following criteria.

• At least a four-fold increase in MAT titer between paired sera. 
• At least a 1:400 MAT titer when only a single specimen is available.  This cutoff is sometimes adjusted based on the prevalence of leptospirosis in the population being examined.

Since some false negative MAT cases can be identified by culture, one way to calculate the sensitivity of MAT is to add the number of MAT-positive and culture-positive (but MAT-negative) cases together to estimate the number of patients with leptospirosis and then calculate the percentage of MAT-positive cases among these patients.  Limmathurotsakul and colleagues performed these calculations with data from their four earlier studies conducted in Thailand.  A total of 413 patients tested positive by MAT or culture (or both).  They found that the sensitivity of MAT was 86%-96% across the four sets of data.  The remaining 4-14% were false negatives, having tested positive by culture but not by MAT.

The authors next calculated the true sensitivity and specificity of MAT with a statistical tool called latent class analysis, which does not assume any perfect gold standard.  Since there is no perfect test, the true disease status of each patient is the unknown or "latent" variable.  Results from multiple diagnostic tests are related to the latent variable using statistical models.  The calculations go beyond the scope of this blog post, but the bottom line is that the true sensitivity and specificity of each diagnostic test can be estimated with these models.  In addition to MAT and culture, the authors tested some of their patients with an immunofluorescence assay (IFA), lateral flow test (LF), and/or PCR. Latent class analysis is more powerful when the diagnostic tests being evaluated detect different features of the infection.  MAT, IFA, and LF are antibody tests, and culture and PCR detect the pathogen itself.

The sensitivity of MAT calculated by this method turned out to be only 49.8%, much lower than the 86%-96% calculated using the standard method that assumes a perfect gold standard.  The sensitivity of culture alone was 10.5%.  Combining culture with MAT did not help much; the sensitivity of the combined approach was only 55.5%.  The low sensitivity of "MAT plus culture" suggests that the specificities calculated for the alternative tests may be underestimated by the standard method.  This is because some of the many false-negative cases may be correctly identified as having leptospirosis by the alternative tests.  This turned out to be the case for two of the tests.   Specificities for all tests were over 95% by latent class analysis.  However, the specificities for PCR (82.5%) and the lateral flow test (70.5%) were lower when "MAT plus culture" was assumed to be the perfect gold standard.

You can see that the accuracy of alternative leptospirosis tests is underestimated when MAT (or MAT plus culture) is assumed to be the perfect reference test.  Another implication of the study is that the prevalence of leptospirosis has been underestimated, at least in Thailand.  The only other study to evaluate the performance of MAT by latent class analysis was conducted by the CDC here in the U.S almost a decade ago.  In contrast to the Limmathurostsakul study, the CDC study determined that the sensitivity of MAT was high, at 98.2%.  There were many differences between the two studies, including the patient population, the alternative tests evaluated, the time interval between collection of paired sera, and the number of serovars included for MAT.  The poor performance of MAT in the Thailand study may therefore not be a universal finding.

References

Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NPJ, Cooper B, Peacock SJ (August 2012).  Fool's gold: why imperfect reference tests are undermining the evaluation of novel diagnostics: a reevaluation of 5 diagnostic tests for leptospirosis.  Clinical Infectious Diseases 55(3):322-331.  DOI: 10.1093/cid/cis403

Bajani MD, Ashford DA, Bragg SL, Woods CW, Aye T, Spiegel RA, Plikaytis BD, Perkins BA, Phelan M, Levett PN, and Weyant RS (February 2003).  Evaluation of four commercially available rapid serologic tests for diagnosis of leptospirosis.  Journal of Clinical Microbiology 41(2):803-809.  DOI:  10.1128/JCM.41.2.803-809.2003