Antibiotic cream is NOT 100% effective in preventing Lyme disease

A topical antibiotic cream applied to tick bites did not perform any better than placebo in preventing Lyme disease, according to results of a randomized clinical trial conducted in Europe.  The study was published in Lancet Infectious Diseases.

I wasn't planning to blog about the study, but I changed my mind after a reader emailed me a link to a news article reporting that the antibiotic cream was 100% effective.  The lead investigator even claimed, "None of the test subjects went on to develop Lyme borreliosis."  As described by the news sources, seven subjects in the control group developed Lyme disease.  But the abstract of the paper states clearly that the antibiotic cream (azithromycin being the antibiotic) was not any better than the control cream; the investigators were even told to stop recruiting additional patients because the results was so clear with the patients who had already completed the study:

The trial was stopped early because an improvement in the primary endpoint in the group receiving azithromycin was not reached.  At 8 weeks, 11 (2%) of 505 in the azithromycin group and 11 (2%) of 490 in the placebo group had treatment failure.

So how is it possible for the lead author to claim that "none" of the subjects treated with azithromycin came down with Lyme disease?  The answer lies with the very last sentence of the abstract:

A subgroup analysis in this study suggested that topical azithromycin reduces erythema migrans after bites of infected ticks.

The subgroup analysis was done post-hoc (after looking at the data).  I won't dwell on why we shouldn't make definitive conclusions from any post-hoc analysis since the investigators themselves emphasized its exploratory nature in the Discussion of their paper.  However, even if you set that aside, you'll find another problem with the post-hoc analysis if you dig into the numbers.

Before I tell you what the problem is, let me first describe the study in greater detail so that you understand the issues that led to the post-hoc analysis.

The subjects were adults who had been bitten by a tick within the previous 72 hours and were able to save the tick.  The subjects were randomized to receive a topical azithromycin cream or placebo cream.  The cream was applied over the tick bite twice a day for three straight days.  The patients were followed for 8 weeks.  They were monitored for erythema migrans (EM), the characteristic rash of Lyme disease.  Blood was drawn for serological testing at the beginning and at the end of the 8 week study period.  "Treatment failure" was defined as the appearance of EM, seroconversion, or both by the end of 8 weeks.  The ticks were tested for the bacteria that cause Lyme disease (Borrelia garinii, B. afzelii, and B. burgdorferi) by PCR.

As I alluded to earlier, the independent committee monitoring the trial recommended that the investigators stop recruiting new subjects.  Among the patients who already completed the study, the group receiving azithromycin did not fare any better than the placebo group, and recruiting more patients to the study was unlikely to change the conclusion.  I provided the numbers above, but you can also find them in the table below ("ITT population," first row of data).

The researchers also did a pre-planned subanalysis with the per-protocol group, an idealized situation to directly test the question, "Does topical azithromycin prevent Lyme disease in those who are bitten by an infected tick?".  Patients bitten by a PCR-negative tick were excluded from the subanalysis.  The small number of patients who failed to follow or complete the study protocol were also excluded.

Again, azithromycin was not any better than placebo in preventing EM or seroconversion (see table, "Per-protocol population").  Treatment failure was observed in 5% (3/62) of the azithromycin group and 7% (5/72) of the placebo group (P = 0.34).

The researchers could have stopped the analysis there and write up the study, but the monitoring committee pointed out that none of the patients in the azithromycin group had erythema migrans by day 30 whereas five in the placebo group did.  The committee suggested that the investigators do a post-hoc subgroup analysis using a modified definition of treatment failure as EM by 30 days.  Seroconversion was removed from the modified definition.

Looking at the numbers in the table ("Reanalyzed ITT population"), we now see where the news media got their numbers.  No one in the azithromycin group (0/87, 0%) had EM by day 30, but seven in the placebo group (7/87, 8%) did.  The difference was statiscially significant (absolute risk reduction in those receiving azithromycin: 8.05%, 95% CI 1.18-14.91).  So, it's true that azithromycin prevented Lyme disease in all who were bitten by an infected tick - but only if you ignored the two patients who came down with EM after day 30 and a third patient who seroconverted.

This is why I'm so baffled by the lead author's quote, which I will repeat:  "None of the test subjects went on to develop Lyme borreiosis."  I'm guessing that the two patients with delayed EM would disagree.


Reference

Schwameis M, Kündig T, Huber G, von Bidder L, Meinel L, Weisser R, Aberer E, Härter G, Weinke T, Jelinek T, Fätkenheuer G, Wollina U, Burchard GD, Aschoff R, Nischik R, Sattler G, Popp G, Lotte W, Wiechert D, Eder G, Maus O, Staubach-Renz P, Gräfe A, Geigenberger V, Naudts I, Sebastian M, Reider N, Weber R, Heckmann M, Reisinger EC, Klein G, Wantzen J, & Jilma B (2016). Topical azithromycin for the prevention of Lyme borreliosis: a randomised, placebo-controlled, phase 3 efficacy trial. The Lancet. Infectious Diseases PMID: 28007428


Related posts

• A flawed study claiming prevention of Lyme spirochete infection with topical antibiotics
• A tale of two more studies: topical antibiotics applied to tick bites to prevent Lyme disease

Xenodiagnosis to detect Borrelia burgdorferi in humans

We've seen that live Borrelia burgdorferi persists (in unculturable form) when infected mice are treated with antibiotics.  What we don't know is whether they persist in humans with post-treatment Lyme disease syndrome (PTLDS), which refers to the lingering long-term symptoms experienced by a minority of Lyme disease patients who have been treated with the standard course of antibiotics.

In theory, one could simply determine whether B. burgdorferi can be detected in bits of tissue or blood extracted from volunteers with post-treatment symptoms.  This is what was done in the mouse studies that I described in my previous post.  It's easy to culture B. burgdorferi from untreated mice that have been infected for a long time.  However, humans are not mice.  Except in those with Lyme arthritis, the spirochete is hard to detect by culture or PCR in patients at later stages of Lyme disease, even in those who haven't taken antibiotics.

In fact, three of the four randomized controlled retreatment trials that I keep on bringing up on this blog included attempts to detect B. burgdorferi in cerebral spinal fluid or blood of PTLDS patients by culture and PCR.  No specimen was culture positive except for one, and none were PCR positive.  The single positive culture turned out to be a contaminant.

The rest of the scientific literature is littered with claims that Lyme Borrelia can be detected by culture or PCR in blood, urine, or CSF of treated patients.  However, critics have raised several concerns about these studies.  For instance, alternative explanations for the findings such as contamination or reinfection weren't ruled out.

With all of this as background, Marques and colleagues decided to test a different approach – xenodiagnosis.  For this procedure, uninfected ticks are deliberately placed on the skin and left for several days to give them time to take a blood meal.  If there are any spirochetes in the skin nearby, they will move towards the feeding site because they are attracted to the tick's saliva.  The spirochetes then get drawn into the tick's feeding tube along with the blood meal.  The fed ticks are then removed and tested for the presence of B. burgdorferi.  The sensitivity of xenodiagnosis can be enhanced by placing multiple ticks to increase the chance that at least one tick will drink blood containing B. burgdorferi.  Xenodiagnosis is done routinely with mice in the research setting, and I mentioned in my previous post that B. burgdorferi can be detected in antibiotic-treated mice by xenodiagnosis.

The first thing to do was a pilot study to make sure that the procedure was safe for volunteers.  25 subjects who had been treated for Lyme disease took part in the study.  10 of the 25 had PTLDS.  Ten healthy volunteers and one subject with untreated erythema migrans (EM), the skin rash of early-stage Lyme disease, were included in the study.

As for the ticks, the investigators bred and maintained Ixodes scapularis in the laboratory.  The ticks were carefully screened to make sure they were free of known infectious agents.

25-30 ticks were placed on each volunteer and covered with a special dressing to keep them in place (see images below).  The ticks were left alone for a week so that they could consume a blood meal.  Some of the fed ticks were tested for the presence of B. burgdorferi DNA by standard PCR or by a more sensitive technique:  isothermal amplification followed by PCR and mass spectrometry (IA/PCR/ESI-MS).  The remaining ticks were cultured or were placed on immune-deficient mice to determine whether B. burgdorferi, if present, could be transmitted.

Figure 1 from Marques et al., 2014.  Left panel: ticks covered with a special dressing on forearm.  Right panel: feeding ticks attached to forearm, dressing removed.

So did anyone test positive by xenodiagnosis? Yes. B. burgdorferi DNA was detected in two subjects.  One was the subject with untreated EM.  This subject served as sort of a positive control.  I say "sort of" because antibiotic therapy was started at the same time that the ticks were placed on the EM lesion – it would not have been ethical to delay treatment while the ticks were feeding.  B. burgdorferi DNA was detected in two of the ten ticks tested.  The subject was tested by xenodiagnosis again seven months later, and all ten ticks that were tested were negative for B. burgdorferi DNA.

The other positive test came from one of the PTLDS subjects.  One of the five ticks that were tested was positive for B. burgdorferi DNA.  The same subject tested positive by xenodiagnosis again 8 months later:  one of three ticks tested positive for B. burgdorferi DNA by IA/PCR/ESI-MS.

Of course DNA doesn't equal viability.  The study didn't provide much evidence that the DNA detected in the single case of PTLDS came from spirochetes that were alive at the time that the ticks were placed.  A skin biopsy taken from where the xenodiagnostic ticks were feeding was culture negative, as were the fed ticks themselves.  The ticks also failed to transmit B. burgdorferi to immune-deficient mice, a process that probably requires live, motile spirochetes.  To be fair, this was just a pilot study with the primary goal to assess the safety of xenodiagnosis.  Nothing terrible happened to the volunteers, although half experienced mild itching at the feeding site.  The investigators are recruiting additional subjects for a larger study to determine whether positive test results by xenodiagnosis are associated with post-treatment symptoms.


References

Marques A, Telford SR 3rd, Turk SP, Chung E, Williams C, Dardick K, Krause PJ, Brandeburg C, Crowder CD, Carolan HE, Eshoo MW, Shaw PA, & Hu LT (2014). Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-human study. Clinical Infectious Diseases, 58 (7), 937-45 PMID: 24523212

Bockenstedt LK, & Radolf JD (2014). Xenodiagnosis for posttreatment Lyme disease syndrome: resolving the conundrum or adding to it? Clinical Infectious Diseases, 58 (7), 946-8 PMID: 24523213

Telford SR 3rd, Hu LT, & Marques A (2014). Is there a place for xenodiagnosis in the clinic? Expert Review of Anti-infective Therapy, 12 (11), 1307-10 PMID: 25301228


Related posts

• Video microscopy of ticks acquiring the Lyme disease spirochete from mice
• Resurgence of Borrelia burgdorferi in mice a year after antibiotic treatment

Resurgence of Borrelia burgdorferi in mice a year after antibiotic treatment

As a follow up to my previous post, I would like to say something about several mouse studies from Stephen Barthold's group.  These papers are often cited by those who believe that retreatment is needed in patients who continue to experience symptoms following treatment of Lyme disease with conventional antibiotic regimens.  The assumption is that post-treatment symptoms stem from spirochetes surviving the initial antibiotic therapy.

In the 2008 and 2010 studies (described in detail here and here), Barthold's group gave doxycycline, ceftriaxone, or tigecycline to mice with disseminated Borrelia burgdorferi infection.  As expected, all tissues were culture negative up to three months following antibiotic therapy.  Tissues from untreated mice were culture positive.  However, B. burgdorferi DNA and mRNA were detected by PCR in up to half the treated mice, and microscopy revealed a few intact spirochetes in collagen-rich tissues from these mice.  Ticks allowed to feed on the treated mice even transmitted the spirochetes to other mice (albeit immune deficient ones), where B. burgdorferi DNA was detected by PCR.  Clearly, the spirochetes that survived antibiotic treatment were alive despite being unculturable.

Although live spirochetes remained following antibiotic therapy, there was no evidence that they were capable of causing disease.  Lyme disease is driven by inflammation, but no inflammatory response in the form of infiltrating immune cells were seen in tissues harboring the spirochetes.  A critic of the work also pointed out that the number of spirochetes declined during the 3 months following treatment, implying that any lingering spirochetes would eventually disappear.  It seemed unlikely that a similar phenomenon was responsible for persisting symptoms following treatment of Lyme disease in human patients, who may suffer with disabling symptoms for years.

In 2014 Barthold's group came out with another paper, which I'm discussing here for the first time.  Again, mice with disseminated B. burgdorferi infections were treated with antibiotics, ceftriaxone in this case.  But this time, the mice were left for up to a year before their tissues were examined for the presence of B. burgdorferi.  Control mice were mock treated with saline and examined along with the treated mice.

There weren't any surprises when tissues were tested by culture.  Most of the control mice were culture positive at all time points (2, 4, 8, and 12 months) with both tissues tested, the urinary bladder and the skin where B. burgdorferi was inoculated to initiate infection.  None of the treated mice were culture positive at either site at any time point.

PCR testing for B. burgdorferi DNA was done with tissue obtained from six sites in the mice.  Ticks allowed to feed on the mice were also tested for the presence B. burgdorferi DNA by PCR in a method called xenodiagnosis.  All saline-treated mice were PCR positive in most tissues tested, and most tested positive by xenodiagnosis.

The results with the mice treated with ceftriaxone are shown in the table below.  Each row represents a single mouse.  Note that each tissue homogenate was tested three times.

Table 2 from Hodzic et al., 2014.  "Interval" = time after completion of treatment; "Inoc" = skin from inoculation site; "HB" = heart base; "VM" = ventricular muscle; "QM" = quadriceps muscle; "Tt" = tibiotarsus; "XenoDx" = xenodiagnostic ticks (# ticks testing positive/# ticks placed on mouse).

They saw something remarkable with the mice left for 12 months.  Although few tissues were positive at earlier time points, most tissues extracted from mice a year after treatment tested positive.  6 of the 8 mice also tested positive by xenodiagnosis.  So, instead of eventually disappearing, the spirochetes proliferated starting at some point after 8 months elapsed following treatment.  This resurgence occurred even though the spirochetes remained unculturable.

Barthold's group also looked for evidence of inflammation.  Despite the resurgence of spirochetes, they did not see much evidence of inflammation by microscopy of the tissues 12 months  following antibiotic treatment.  However, the researchers pointed out that no conclusions can be drawn about the ability of the persisting spirochetes to cause disease since inflammation was minimal even in saline-treated mice, which harbored culturable spirochetes.

The researchers next looked for molecular evidence of inflammation.  They measured transcript levels of 18 cytokines in the base of the heart, heart muscle, quadriceps muscle, and leg joint 12 months after treatment with ceftriaxone or saline.  The levels of cytokine transcripts in the two groups were compared to those in age-matched uninfected mice.  Not surprisingly, saline-treated mice had what the authors deemed a "proinflammatory" cytokine profile, most likely due to their ongoing infection.  Antibiotic-treated mice also had a proinflammatory cytokine profile, although it differed from that of the saline-treated mice.  This observation is the first to suggest that the mice were responding to persisting spirochetes that survived antibiotic treatment.

In conclusion, the evidence is convincing that B. burgdorferi persists in mice for a long time after antibiotic treatment.  They don't eventually disappear and may even proliferate.  Whether these unculturable spirochetes are capable of generating an inflammatory condition necessary for disease is less clear, though mice do appear to generate a unique cytokine profile in response to the persisting spirochetes.

Barthold's group caution readers from applying the findings too broadly:

Because of the controversial nature of these findings, they should not be over-interpreted and certainly not translated directly into clinical management of human Lyme borreliosis.

So is there any relevance of these findings to post-treatment symptoms in humans?  I will touch upon this issue in a future post.

Reference

Hodzic E, Imai D, Feng S, & Barthold SW (2014). Resurgence of persisting non-cultivable Borrelia burgdorferi following antibiotic treatment in mice. PLOS One, 9 (1) PMID: 24466286

Related posts

• Chronic Lyme disease in mice?
• Tigecycline fails to eradicate persisting Borrelia burgdorferi
• Long-term antibiotics for those with chronic symptoms that may or may not be related to Lyme disease

Long-term antibiotics for those with chronic symptoms that may or may not be related to Lyme disease

A Lyme disease study published a few weeks ago in the New England Journal of Medicine has received a lot of coverage in the press.  According to the abstract of the study, Berende and colleagues conducted a randomized placebo-controlled clinical trial to test the effectiveness of long-term "longer-term" antibiotics in treating "longer-term" chronic symptoms "attributed" to Lyme disease.

As many readers of this blog know, treatment of Lyme disease is a controversial topic.  Antibiotics are effective in treating Lyme disease, but 10-20% experience symptoms such as fatigue, muscular aches, and joint pain for at least 6 months following conventional treatment.  The cause of the persisting symptoms is not known.  They could be due to tissue damage caused by the infection, ongoing inflammation, or bacteria that survived antibiotic treatment.  Mainstream medical societies such as the IDSA do not believe that lingering infection is responsible for the persisting symptoms, and they do not recommend retreatment with antibiotics.  Four randomized controlled studies conducted in the U.S. showed little benefit of retreating these patients with antibiotics for up to 3 months.  On the other hand, not-so-mainstream groups such as ILADS dispute the interpretation of the data.  They insist that the treatment groups did show some improvement and that longer treatment regimens lasting longer than 3 months are needed for complete recovery of these patients.

There is another group of patients that also suffer from enduring fatigue, muscle aches, and joint pain.  They may or may not have had Lyme disease in the past, but their ongoing symptoms stem from some other condition.  Unfortunately, they may be misdiagnosed with "chronic Lyme disease" and end up being treated for a long time with antibiotics in an attempt to eradicate an infection that they don't have.

The new NEJM paper describes a randomized placebo-controlled trial that was conducted in the Netherlands.  281 subjects who had been experiencing chronic symptoms blamed on Lyme disease (fatigue, muscle aches, joint pain) were randomized into three groups.  All three groups were treated with ceftriaxone intravenously for two weeks.  The subjects were next given oral antibiotics or placebo for 12 weeks.  One group was treated with doxycycline, another group with both clarithromycin and hydroxychloroquine, and the third group was given placebo pills.

The graph below shows that the physical quality of life, the primary outcome measure, improved a little for all groups.  Because there was no difference in outcome among the three groups, the authors concluded that longer-term antibiotics were no better than short-term antibiotics in alleviating symptoms.  We don't know whether the initial two-week treatment with ceftriaxone had anything to do with the slight improvement since there was no true placebo (antibiotic-free) group.

Change in mean SF-36 physical component summary scores before and after treatment period.  Figure 2 from Berende et al., 2016.

Why wasn't a true placebo group?  The authors worried about withholding antibiotics from subjects who might have an infection that should be treated.  11% of the subjects hadn't been treated with antibiotics for their symptoms prior to their acceptance into the study.  This wasn't an issue with the earlier U.S. trials since previous treatment of Lyme disease with antibiotics was a requirement for acceptance into those studies, which included true placebo groups.

One baffling aspect of the study was the inclusion of subjects who might not have had Lyme disease prior to the appearance of their chronic symptoms. Only a third of the subjects had objective clinical features of Lyme disease (erythema migrans, meningoradiculitis, or acrodermatitis chronica atrophicans) immediately preceding their chronic symptoms, and a little more than a half recalled a tick bite.  Contrast this with the earlier U.S. studies, which only accepted patients with chronic symptoms that followed antibiotic treatment of a well-documented case of Lyme disease.

The remaining subjects did not have any objective features of Lyme disease before their chronic symptoms appeared.  The only evidence of a previous episode of Lyme disease was positive antibody testing by Western blot.  However, the antibodies may have been elicited by a Borrelia infection in the distant past.  Their past episode of Lyme disease may not be related to their chronic symptoms, which aren't specific for Lyme disease.  Another problem with relying solely on Western blots to diagnose Lyme disease is that false positives occur.  Without additional evidence, it is hard to be sure that their chronic symptoms were related to Lyme disease.

Nevertheless, the editorial accompanying the paper expressed support for the relaxed inclusion criteria used to select the subjects for the study:

Critics may rightly say that this trial does not truly capture with certainty the consequences of bona fide Lyme disease. However, studies with more stringent inclusion criteria have already been conducted, and the approach used by Berende and colleagues probably reflects the common practice in ambulatory care settings, in which patient presentations of fatigue or nonspecific pain prompt serologic checks for Lyme disease, despite evidence suggesting that these tests will not identify a probable cause or result in a treatment benefit.

The study population may reflect what's encountered by clinicians in the real world, but for a clinical trial it doesn't seem right to lump those whose chronic problems followed a real episode of Lyme disease with those whose issues had nothing to do with Lyme.  Any benefit of the antibiotics experienced by those who had genuine Lyme disease (assuming that there was any benefit) may have been obscured by the lack of benefit in those whose chronic symptoms aren't related to Lyme disease.

Berende and colleagues also defended the length of treatment, which is considered to be on the short end by those who support lengthy courses of antibiotic therapy:

...it may be argued that 14 weeks of treatment is insufficient to show a beneficial treatment effect. However, whereas prolonged antimicrobial treatment is not uncommon for various infectious diseases, the purpose of prolonged therapy for such diseases is for the prevention of microbiologic relapse rather than for a delayed onset of clinical alleviation of signs or symptoms. We are not aware of any infectious disease in which the initial effect on signs, symptoms, and laboratory findings is delayed beyond the first 3 months of effective therapy.

But the graph above clearly shows that the subjects felt better following the treatment period.  Unfortunately, as I mentioned earlier, the improvement in the quality of life can't be attributed to the antibiotics because there was no true placebo group.

With these issues, I'm not sure how this study got published in NEJM.  Regardless of my opinion, it will undoubtedly be cited as further proof that long-term antibiotics don't alleviate long-term symptoms that stem from Lyme disease.

Edit: Corrected quotes in first paragraph.

References

Berende A, ter Hofstede HJ, Vos FJ, van Middendorp H, Vogelaar ML, Tromp M, van den Hoogen FH, Donders AR, Evers AW, & Kullberg BJ (2016). Randomized Trial of Longer-Term Therapy for Symptoms Attributed to Lyme Disease. The New England Journal of Medicine, 374 (13), 1209-20 PMID: 27028911

Melia MT, & Auwaerter PG (2016). Time for a Different Approach to Lyme Disease and Long-Term Symptoms. The New England Journal of Medicine, 374 (13), 1277-8 PMID: 27028918

Lighting up Leptospira interrogans to test antibiotic treatment of chronically-infected mice

A group at the Pasteur Institute succeeded in generating a bioluminescent strain of Leptospira interrogans.  Their study was published last year in PLOS Neglected Tropical Diseases.

The benefit of having a bioluminescent strain is that infections of small laboratory rodents can be monitored without sacrificing the animals.  Instead, the animals are placed in a special whole-body imager that detects light emitted from the bodies.  The location of the infection in the animals can be determined from the images, and the light intensity measured by the imager gives an idea of the bacterial load in infected tissues.  After imaging, the animal can be returned to its cage, and additional images of the same animal can be taken later as the infection progresses.

Another advantage of using bioluminescent bacteria is that the luciferase reaction requires ATP, meaning that the bacteria must be metabolically active to light up.  Dead bacteria containing luciferase will not generate light, unlike green fluorescent protein (GFP), another reporter used to image bacteria (see this post that describes an experiment with Borrelia burgdorferi expressing GFP).

To generate bioluminescent L. interrogans, the researchers hooked the firefly luciferase gene up to a strong L. interrogans promoter and inserted the construct into a transposon carried on a suicide plasmid.  The plasmid was then introduced into L. interrogans by conjugation (see this blog post for details of the process).  Cultures of the engineered spirochetes lit up when luciferin, the luciferase substrate, was added.  The amount of light emitted depended on the culture density: more light was detected at higher culture densities.

Mice are ideal models to study persistent infection of the kidney since many rodents are chronic carriers of Leptospira out in nature.  These rodents don't get sick from the infection, but they contaminate the environment with the spirochete every time they urinate.

To see how chronic infection is established, the investigators injected a sublethal dose of 10⁷ bioluminescent L. interrogans cells into the abdominal cavity of C57BL6/J mice and took sequential images of the animals over the following months.  Albino mice were used because dark fur blocks the signal emitted by the bioluminescent bacteria.  (They later showed that standard C57BL6/J mice with black fur could be used as long as they shaved the fur off before placing them in the imager.)  The mice were injected with luciferin 10 minutes prior to imaging.

There turned out to be two phases of infection (see the "MFlum1" plot in the graph below).  In the acute phase, the bioluminescent signal rose to a peak by day 4 and quickly declined to background levels by day 7.  The signal then started increasing again slowly and plateaued after a month.

Figure 2A from Ratet et al., 2014.  Images of a single mouse taken sequentially are shown below the graph.  Click for larger image.  Source.

Images of a single mouse taken at different times after inoculation are shown below the graph.  Thirty minutes after inoculation, signal was detected in the abdominal cavity.  By day 3, the signal consumed the entire mouse.  At this point, the bacteria were probably circulating in the bloodstream.  By day 6, the signal was almost completely gone.  After day 6, the signal appeared again, but it was confined to the kidneys.  The intensity of the signal in the kidneys increased with time.  They did not detect signal anywhere else in the animals during the second phase.  They even sacrificed some of the infected mice 2 months into the infection to check the organs directly, but they failed to detect Leptospira by bioluminescence and qPCR in the brain, lungs, spleen, liver, or blood.  Not surprisingly, bioluminescence was detected in urine, confirming that the mice were shedding live L. interrogans.

Next, the investigators tested the effectiveness of antibiotics in treating mice infected with the bioluminescent L. interrogans.  Several antibiotics are used to treat acute leptospirosis in humans, including penicillin and azithromycin.  It is generally believed that antibiotics are more effective if provided early in acute illness.  Therefore, the investigators tested whether the timing of antibiotic treatment was important for effectiveness.

As expected, penicillin treatment was most effective when treatment was started at the beginning of the acute phase.  In mice treated with daily injections of penicillin for 5 days starting a day after infection, no bioluminescence was detected in the kidneys, and urine was free of L. interrogans as measured by qPCR.  However, if treatment was delayed until three days after inoculation, during the peak of acute infection, a low level of L. interrogans was detected in urine by qPCR even though no bioluminescence was detected in the kidneys.  It is likely L. interrogans was present in the kidneys but at levels too low to be detected by the imager. The bioluminescence approach clearly does not have the sensitivity of qPCR.  Additional experiments revealed that the limit of detection was 100 bioluminescent L. interrogans cells in 100 μl of buffer.

Penicillin was even less effective when administered after the spirochetes settled in the kidneys. When penicillin treatment was initiated at peak bacterial load in the kidneys, day 25 of infection, the signal diminished by over 90% but then bounced back to the level observed before treatment began (see figure below).  Ciprofloxacin also failed to eradicate the bacteria.

Figure 5A from Ratet et al., 2014.  Antibiotics were administered for 5 days started on day 25 of infection.  Cipr, ciprofloxacin; Pen, penicillin.  Source.

On the other hand, azithromycin managed to knock the signal in the kidneys down to background levels (see graph below).  However, the signal came back within a week, although not to the high levels seen in untreated mice.  A second course of antibiotics starting on day 112 knocked the signal back down to near background levels, but again, spirochete numbers rebounded, although not to the levels seen before retreatment.

Figure 5B from Ratet et al., 2014.  Azithromycin was administered for 5 days starting on day 25 and day 112 of infection.  Source.

Why are antibiotics ineffective in eradicating L. interrogans during the chronic phase?  Like other bacteria, L. interrogans can form biofilms in vitro.  Scientists who work with Leptospira believe that they also assemble into biofilms within the kidney tubules during chronic infection.  Biofilms are hard to eliminate in part because they harbor persister cells that tolerate antibiotics.  (See this post for some background on persister cells.)

I should caution readers from concluding that tolerance accounts for the poor effectiveness of antibiotics in treating human cases of acute leptospirosis.  As the authors point out, leptospirosis patients die because the infection severely injure vital organs.  By the time lethal damage occurs, it does not matter whether antibiotics kill all of the spirochetes.

So does the mouse model have any relevance to human leptospirosis?  The authors argue that asymptomatic carriage of Leptospira has been overlooked.  A 2013 study from the Netherlands revealed that 21% of patients who contracted  leptospirosis continued to suffer from headaches, muscle aches, and extreme fatigue two years later.  This may reflect unrepaired tissue damage inflicted during acute infection, but no one checked for the presence of Leptospira in these patients.  Another study from Peru (see this post) describes asymptomatic individuals who may have persistent Leptospira infection. Kidney function was not checked in the Peruvians, but there is reason to believe that chronic infection affects the kidneys despite the lack of symptoms.  Mice chronically infected with L. interrogans are not visibly sick, but they end up with scarred kidneys (fibrosis), as explained in this study.

If persistent asymptomatic infections really do occur in humans, it may be sensible to treat with antibiotics.  The chronically-infected mouse will serve as a nice model for testing antibacterial regimens that target Leptospira living in the kidneys.

References

Ratet G, Veyrier FJ, Fanton d'Andon M, Kammerscheit X, Nicola MA, Picardeau M, Boneca IG, & Werts C (2014). Live imaging of bioluminescent Leptospira interrogans in mice reveals renal colonization as a stealth escape from the blood defenses and antibiotics. PLoS Neglected Tropical Diseases, 8 (12) PMID: 25474719

Goris MG, Kikken V, Straetemans M, Alba S, Goeijenbier M, van Gorp EC, Boer KR, Wagenaar JF, & Hartskeerl RA (2013). Towards the burden of human leptospirosis: duration of acute illness and occurrence of post-leptospirosis symptoms of patients in the Netherlands. PloS One, 8 (10) PMID: 24098528

Fanton d'Andon M, Quellard N, Fernandez B, Ratet G, Lacroix-Lamandé S, Vandewalle A, Boneca IG, Goujon JM, & Werts C (2014). Leptospira Interrogans induces fibrosis in the mouse kidney through Inos-dependent, TLR- and NLR-independent signaling pathways. PLoS Neglected Tropical Diseases, 8 (1) PMID: 24498450


Related posts

• Life after leptospirosis, a pilot study
• Healthy human carriers of the spirochete Leptospira in the Peruvian Amazon
• The magic of antibiotic tolerance

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?
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• The magic of antibiotic tolerance

The magic of antibiotic tolerance

Bactericidal antibiotics are effective at killing proliferating bacteria as long as they don't carry mutated or acquired genes that encode resistance to the antibiotics. Unfortunately even antibiotic-sensitive bacteria can tolerate antibiotics under some circumstances. Bacteria that are in a nondividing "dormant" state often survive antibiotic exposure.  When the antibiotic is removed and growth resumes, the bacteria regain susceptibility to antibiotics.

At first glance antibiotic tolerance appears to be a passive process in which nondividing cells survive simply because the target of the antibiotic is inactive.  However, this is not correct.  Antibiotic tolerance requires an active response by the bacteria.  The nondividing bacteria that survive antibiotic treatment are called persisters.  Persisters may account for infections that are difficult to eradicate with antibiotics.

Persisters were first identified in 1944 by Joesph Bigger, who was testing the effectiveness of the new miracle drug penicillin on cultures of Staphylococcus.   However, interest in antibiotic tolerance waned as antibiotic resistance came to be a problem.  In the 1980s Harris Moyed revisited the issue of antibiotic tolerance, and his group harnessed the power of bacterial genetics to isolate mutants that exhibited abnormally high frequencies of persister formation and to map the mutations that caused the novel trait.

As Joseph Bigger discovered, very rare persisters can even be identified in growing cultures, around 1 in 100,000 bacteria.  How can these rare bacteria behave so differently from those surrounding them if antibiotic tolerance doesn't involve alterations to the bacterium's DNA?  The current thinking is that the persister state is triggered randomly in a small fraction of proliferating bacteria due to random fluctuations in the expression of a small number of persister genes.  In the rare bacterium (about 1 in 100,000), the persister genes will be expressed at a high enough level to induce the persister state and slow growth of the bacterium.

Certain environmental cues can also enhance the development of persisters.  For example, the fraction of persisters in a culture increases substantially as nutrients are depleted.  At stationary phase, when the bacteria stop increasing in number, at least 1% of the bacterial cells become antibiotic tolerant.

One of the active responses that stimulates persister cell formation is called the stringent response, which rapidly generates the unusual nucleotides ppGpp and pppGpp when bacteria are starving for nutrients.  Historically these derivatives of GDP and GTP were known as "magic spots" because they appeared as novel radioactive spots on thin-layer chromatograms when E. coli starved for amino acids were labeled with ³²P-phosphate. (p)ppGpp has wide-ranging effects on bacterial physiology.  The best-known activity of (p)ppGpp is its attachment, along with the protein DksA, to RNA polymerase during amino acid starvation, causing transcription of rRNA and tRNA genes to cease and transcription of amino acid biosynthetic operons to increase.  This make sense since there's no point in making more ribosomes until more amino acids are available to support protein synthesis and growth of the bacteria.

Magic spot!  Source

(p)ppGpp production is also necessary to generate the rare persisters in cultures of proliferating bacteria.  When the two genes coding for the enzymes that make (p)ppGpp, relA and spoT, are deleted, persister cells become even more rare in growing E. coli cultures.

A recent study published in the journal Science looked at the role of the stringent response in inducing antibiotic tolerance in biofilms, in which nondividing bacteria are embedded in a matrix secreted by the bacteria.  These studies were conducted with E. coli and Pseudomonas aeruginosa strains with deletion mutations in relA and spoT.  When the biofilms formed by the mutant and wild-type strains were treated with different antibiotics, the mutants turned out to be much more sensitive to the antibiotics even though the bacteria were not dividing (data for P. aeruginosa shown in bar graphs below).  Similar results were obtained when standard cultures of the P. aeruginosa mutant and wild-type strains were grown to stationary phase and then treated with antibiotics.

Figure 1E from Nguyen et al.  P. aeruginosa was allowed to form biofilms on polycarbonate membrane filters resting on agar culture medium.  The biofilms were incubated with different antibiotics or with no antibiotic (control).  To determine the number of bacteria surviving antibiotic treatment, the bacteria in the biofilms were dispersed by vigorous mixing and plated onto agar culture medium to determine cfu counts.  +SR, complementation of the ΔrelA ΔspoT mutant with intact copies of relA and spoT; *P ≤ 0.05, ***P ≤ 0.0005 versus wildtype.

Bactericidal antibiotics, regardless of their target, are now known to enhance production of reactive oxygen species (ROS) within bacteria.  If not detoxified by enzymes such as catalase and superoxide dismutase, ROS can fatally damage the bacteria by reacting with their DNA, protein, and lipids.  ROS is generated within bacteria during the course of their normal metabolic activities, even when antibiotics are not present.  When the authors measured the amounts of hydroxyl radical generated within untreated E. coli and P. aeruginosa biofilms, the ΔrelA ΔspoT mutants were burdened with higher levels of hydroxyl radicals than the wild-type strains. Why do the mutant strains have higher levels of hydroxyl radicals?  Further analysis of the bacteria in the biofilms showed that the ΔrelA ΔspoT mutants also had lower levels of catalase and superoxide dismutase activity.  It's possible that bactericidal antibiotics triggered production of lethal amounts of ROS that the mutants could not handle due to their insufficient production of catalase and superoxide dismutase, but this needs to be confirmed by further experimentation using bacterial strains with the superoxide dismutase and catalase genes knocked out.

The Science study also looked at infected laboratory mice treated with antibiotics.  Wild-type and ΔrelA ΔspoT P. aeruginosa strains were grown to stationary phase, and a lethal dose of the bacteria were injected into the abdominal cavity of mice.  Four hours later the animals were treated with the antibiotic ofloxacin.  The antibiotic was more effective at preventing lethal infection by the ΔrelA ΔspoT mutant than those caused by the wild-type strain.  Similarly, ofloxacin was more effective at reducing the number of bacteria in a biofilm chamber implanted underneath the mouse skin when the biofilm was composed of the ΔrelA ΔspoT mutant.

One must keep in mind that multiple pathways to persister cell formation have been identified (see figure below).  Note that for the animal experiments persisters were allowed to develop in the test tube prior to animal inoculation.  It is possible that the stringent response would not be involved at all if persisters were allowed to develop during infection instead.

Figure 4 from Lewis, 2010.  The redundant pathways to persister formation are shown.  FMN, flavin mononucleotide pool; pmf, proton motive force; TAs, toxin/antitoxin modules

Featured paper

Nguyen, D., Joshi-Datar, A., Lepine, F., Bauerle, E., Olakanmi, O., Beer, K., McKay, G., Siehnel, R., Schafhauser, J., Wang, Y., Britigan, B.E., & Singh, P.K. (2011). Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria Science, 334 (6058), 982-986 DOI: 10.1126/science.1211037

Key references

Bigger, J.W. (1944). Treatment of staphylococcal infections with penicillin by intermittent sterilisation The Lancet, 244 (6320), 497-500 DOI: 10.1016/S0140-6736(00)74210-3

Moyed H.S., & Bertrand K.P. (1983). hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. Journal of bacteriology, 155 (2), 768-775 PMID: 6348026

Cashel, M., & Gallant, J. (1969). Two compounds implicated in the function of the RC gene of Escherichia coli Nature, 221 (5183), 838-841 DOI: 10.1038/221838a0

Paul, B., Barker, M.M., Ross, W., Schneider, D.A., Webb, C., Foster, J.W., & Gourse, R.L. (2004). DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP. Cell, 118 (3), 311-322 DOI: 10.1016/j.cell.2004.07.009

Korch, S.B., Henderson, T.A., & Hill, T.M. (2003). Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis Molecular Microbiology, 50 (4), 1199-1213 DOI: 10.1046/j.1365-2958.2003.03779.x

Kohanski, M.A., Dwyer, D.J., Hayete, B., Lawrence, C.A., & Collins, J.J. (2007). A common mechanism of cellular death induced by bactericidal antibiotics Cell, 130 (5), 797-810 DOI: 10.1016/j.cell.2007.06.049

Lewis, K. (2010). Persister cells Annual Review of Microbiology, 64 (1), 357-372 DOI: 10.1146/annurev.micro.112408.134306

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• Chronic Lyme disease in mice?
• Tigecycline fails to eradicate persisting Borrelia burgdorferi

A tale of two more studies: topical antibiotics applied to tick bites to prevent Lyme disease

Feeding Ixodes ticks harboring Borrelia burgdorferi deposit the Lyme disease spirochete in the skin of the victim.  The spirochetes remain in the skin for a few days before entering the bloodstream to spread throughout the host.  The delay in dissemination provides a window of opportunity to stop the infection by simply applying antibiotics to the skin where the tick was feeding.  Topical application of antibiotics would allow patients to avoid experiencing side effects associated with ingesting antibiotics.

I recently posted a critique of a study by Knauer and colleagues, who tested the ability of a topical antibiotic to prevent B. burgdorferi infection in lab mice bitten by infected ticks.  As I explained in the post, the antibiotic appeared to prevent infection, but the investigators had used a weakened B. burgdorferi strain to inoculate the mice.  Consequently it wasn't possible to draw any conclusions about the effectiveness of their antibiotic formulation in preventing infection.

Now let's look at two more studies that tested the ability of topical antibiotics to prevent infection in the mouse model of Lyme disease.  These studies were conducted properly with highly infectious B. burgdorferi strains.  One study was published almost 20 years ago.  The other appeared online just last month.  Both studies were published in The Journal of Infectious Diseases.

In their 1993 study Shih and Spielman were able to prevent B. burgdorferi infection by applying at least 1 milligram of tetracycline starting up to two days following the tick bite.  The antibiotic had to be applied twice a day for at least three consecutive days (see table below).  The presence of infection four weeks after tick feeding was assessed by xenodiagnosis, which tests whether the spirochetes could be recovered by ticks placed on the skin at a location distant from the original tick bite.  The investigators also found that penicillin, amoxicillin, ceftriaxone, doxycycline, and erythromycin applied for three days beginning one day after tick feeding prevented infection.

Although this study demonstrated the promise of topical antibiotics in preventing Lyme disease in those who discover a tick feeding on them, a limitation of the study was that most of the antibiotics were dissolved in DMSO, which is not approved for use on humans.  The only antibiotic dissolved in a solvent suitable for humans was erythromycin, which was added into 70% ethanol.

For their 2011 study, Wormser and colleagues decided to dissolve the antibiotics in something else that would be acceptable to apply to human skin.  They rubbed a 2% erythromycin ointment or 3% tetracycline gel over the tick bite 1-2 days after the infected ticks finished feeding on the mice.  The antibiotics were applied twice daily for three days.  Four weeks later urinary bladder and ear tissue were cultured to see whether the mice had a disseminated infection.  The authors found that their antibiotic formulations failed to prevent systemic infection, although erythromycin was able to prevent a persistent infection at the tick bite site in some of the mice (see table below).

Erythromycin and tetracycline were tested in both studies.  Why the stark difference in the effectiveness of the same antibiotics in the two studies?   In the Discussion of their paper, Wormser and colleagues highlighted the major methodological differences between the studies:

• Different antibiotic concentrations. A much higher concentration of erythromycin was applied to the tick bites in the 1993 study.
• Different solvents.  DMSO, the solvent used for the 1993 study, may have promoted better penetration of tetracycline into the skin than the ointment and gel formulations selected for the Wormser study.
• Different placement of infected ticks.  In the 1993 study the infected ticks were placed on the ear for feeding.  In the Wormser study the ticks were placed on the back, where the skin may be thicker and hence more resistant to antibiotic penetration.
• Different B. burgdorferi strains.  Wormser and colleagues used ticks infected with the highly invasive BL206 strain to inoculate the mice, whereas Shih and Spielman used ticks infected with the less invasive JD1 strain.
• Different mouse strains.  The C3H mouse strain used in the Wormser study is highly susceptible to dissemination by B. burgdorferi.

For this simple treatment approach to effective, higher concentrations of the antibiotic in a penetrating solvent such as ethanol may be necessary.  Different B. burgdorferi and mouse strains should also be tested in future studies.


References

Shih, C.-M., & Spielman, A. (1993). Topical prophylaxis for Lyme disease after tick bite in a rodent model Journal of Infectious Diseases, 168 (4), 1042-1045 DOI: 10.1093/infdis/168.4.1042

Wormser, G.P., Daniels, T.J., Bittker, S., Cooper, D., Wang, G., & Pavia, C.S. (2011). Failure of topical antibiotics to prevent disseminated Borrelia burgdorferi infection following a tick bite in C3H/HeJ mice Journal of Infectious Diseases DOI: 10.1093/infdis/jir382
Knauer, J., Krupka, I., Fueldner, C., Lehmann, J., & Straubinger, R.K. (2011). Evaluation of the preventive capacities of a topically applied azithromycin formulation against Lyme borreliosis in a murine model Journal of Antimicrobial Chemotherapy DOI: 10.1093/jac/dkr371


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