9 Abstract: Evaluation of in-vitro antibiotic susceptibility of Bb

I was recently asked about this study in comments, since there has been much discussion about this research in the Lyme disease patient community online:

Evaluation of in-vitro antibiotic susceptibility of different morphological forms of Borrelia burgdorferi

My opinion of it thus far is one of waiting and seeing - I'm not sure what to think yet and I'm waiting to hear more information... I'm guessing this is an initial publication of findings and a more detailed paper similar to Dr. Sapi's cancer research papers will be published in the near future.

Tinidazole

I have a number of questions and thoughts on it, and prior to this publication, I had the impression that tinidazole has helped a number of Lyme disease patients - but there has been limited research on it (See: Brorsons).

The Brorsons stated in their 2003 paper, An in vitro study of the susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole:

"Acridine orange staining, dark-field microscopy and transmission electron microscopy revealed that, when the concentration of TZ (Tinidazole) was ≥ MBC, the contents of the cysts were partly degraded, core structures did not develop inside the young cysts, and the amount of RNA in these cysts decreased significantly. When cysts were exposed to TZ, both the spirochetal structures and core structures inside the cysts dissolved, and the production of blebs was significantly reduced."

But I digress...

To take each question and thought about Dr. Sapi's latest paper by point:

"Three morphological forms of B. burgdorferi (spirochetes, round bodies, and biofilm-like colonies) were generated using novel culture methods."

Is there a description of these novel culture methods? Have they been through a process of verification and validation? How are they superior to other methods?

If different media is used other than BSK/BSK-H, it would be good to know if that affects the results in some fashion.

"Minimum inhibitory concentration and minimum bactericidal concentration of five antimicrobial agents (doxycycline, amoxicillin, tigecycline, metronidazole, and tinidazole) against spirochetal forms of B. burgdorferi were evaluated using the standard published microdilution technique."

What exactly were the concentrations?

It's important to know, and compare against other studies which have been done in vitro. Of all the literature I've read on this, standardization of these documented concentrations is lacking.

And to note: This was an in vitro test, not in vivo. Given everything that is known about Bb's behavior in vivo, in vitro tests will only give us part of the picture. More on that in a minute, below...

"The susceptibility of spirochetal and round body forms to the antibiotics was then tested using fluorescent microscopy (BacLight™ viability staining) and dark field microscopy (direct cell counting), and these results were compared with the microdilution technique. Qualitative and quantitative effects of the antibiotics against biofilm-like colonies were assessed using fluorescent microscopy and dark field microscopy, respectively."

How do these tests compare to one another relative to results found for each? How do the novel culture methods affect outcome relative to standard culture methods?

How is something biofilm-like, versus a biofilm?

"Doxycycline reduced spirochetal structures ~90% but increased the number of round body forms about twofold. Amoxicillin reduced spirochetal forms by ~85%–90% and round body forms by ~68%, while treatment with metronidazole led to reduction of spirochetal structures by ~90% and round body forms by ~80%. Tigecycline and tinidazole treatment reduced both spirochetal and round body forms by ~80%–90%."

This is interesting... Some of these results match earlier research findings on specific antibiotics used to treat Bb.

In 2008, Xiaohua Yang, Andrew Nguyen, Dan Qiu, and Ben Luft did some research on the effectiveness of tigecycline and doxycycline on Borrelia burgdorferi in vitro in In vitro activity of tigecycline against multiple strains of Borrelia burgdorferi: http://jac.oxfordjournals.org/content/63/4/709.short

In this abstract it was stated:

"Tigecycline inhibited the growth of and killed the organism more rapidly than doxycycline. Tigecycline was able to kill B. burgdorferi within 24 h at clinically achievable concentrations (> 1 mg/L). In contrast, doxycycline was bacteriostatic and required 48–72 h to achieve its maximal inhibitory effect. The anti-Borrelia activity of the antibiotics was tested against 20 different isolates from three species. Tigecycline was 16- to 1000-fold more active than doxycycline at immobilizing Borrelia for the 20 isolates tested."

The MIC versus the MBC is important to know, and it affects outcomes on a timeline.

Comparing Tigecycline to doxycycline sounds like comparing an axe to a thousand papercuts - the former is just going to be more immediately effective than the latter... And if Bb happens to disseminate rather quickly in an individual patient, there's a chance the doxycycline prescribed may not be enough to adequately treat the patient if you're going by this MIC - especially if one of Bb's strategies is to evade the immune system. (Let's not even get into how ineffective doxycycline is for prophylaxis for now...)

More recently, in 2010, Louis Ates, Christa Hanssen-Hübner, Douglas E. Norris, Dania Richter, Peter Kraiczy and Klaus-Peter Hunfeld conducted the study, Comparison of in vitro activities of tigecycline, doxycycline, and tetracycline against the spirochete Borrelia burgdorferi.

In their abstract they stated:

"The overall rank order of MIC90s was tigecycline (≤0.016 mg/L) > ceftriaxone (0.03 mg/L) > cefotaxime (≤0.125 mg/L) > doxycycline (0.25 mg/L) > tetracycline (0.25 mg/L). The rank order of MBC90s was tigecycline (0.5 mg/L) > ceftriaxone (2 mg/L) > tetracycline (16 mg/L) > doxycycline (16 mg/L) > cefotaxime (>16 mg/L).

High in vitro activity of the new glycylcycline against Borrelia was further substantiated by time-kill experiments performed with B. afzelii isolate EB1. Parallel testing of tigecycline and ceftriaxone demonstrated a bacteriostatic effect for 0.016 mg/L of tigecycline and for 0.03 mg/L for ceftriaxone after 72 h of incubation. Moreover, tigecycline was bactericidal at a concentration of 0.25 mg/L showing a > 3 log₁₀ unit reduction of the initial inoculum, whereas for ceftriaxone a concentration of 2 mg/L was needed."

So as you can see in this earlier research, tigecycline is highly effective in vitro, and doxycycline is ranked much further down the list of effectiveness.

Stepping into our time machine again, and going back to 2004 to Klaus-Peter Hunfeld, Thomas A. Wichelhaus, Rebecca Rödel, Georg Acker, Volker Brade, and Peter Kraiczy's study, Comparison of In Vitro Activities of Ketolides, Macrolides, and an Azalide against the Spirochete Borrelia burgdorferi: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC310164/

Now this was measuring an entirely different group of antibiotics, with the following results:

"The ketolides were the most potent against borrelial isolates on a micrograms-per-milliliter basis. For all agents except cethromycin and telithromycin, the MIC at which 90% of isolates were inhibited (MIC90) and the MBC at which 90% of the isolates were killed were ≥0.01 μg/ml and > 0.25 μg/ml, respectively."

and

"In our study, the rank order of activity by classical macrolides and azalides against borreliae clearly corresponds to the effectiveness of these agents as revealed by in vitro susceptibility studies and clinical treatment trials to date (2, 4, 5, 7, 8, 9, 11, 23, 24), demonstrating higher in vitro effectiveness for azithromycin (MIC90, 0.0156 μg/ml) than for erythromycin (MIC90, 0.0625 μg/ml), roxitromycin (MIC90, 0.0625 μg/ml), and clarithromycin (MIC90, 0.0312 μg/ml). Median MICs of the different substances, however, tended to vary over a 10-fold range between individual strains, with the B. garinii isolate PSth and the B. afzelii isolate EB1 showing the highest MICs for both the classical macrolides and the ketolides."

So in this study, of the antibiotics looked at, ketolides were more effective than macrolides, and azithromycin was more effective than other macrolides.

We also see that MIC's are different for different European Bb isolates. More on that below, in another cited study.

And then they said this about treatment failure:

"Classical macrolides and azalides frequently fail in the therapy of early LD (7, 14, 17, 26), and clinical relapse has been observed following conclusion of treatment (14, 17, 26). Moreover, it has been speculated that resistance may develop in borreliae preexposed to erythromycin owing to resistant subpopulations (25). Based upon our findings, however, the ketolides were superior in vitro on a micrograms-per-milliliter basis when tested alongside classical macrolides under identical test conditions in BSK."

There are a number of studies out there showing that Borrelia burgdorferi can be antibiotic resistant, with some being erythromycin resistant. Because of this, it is critical for doctors to weigh the use of erythromycin in patients - especially pregnant women with Lyme disease - against the risks of another antibiotic which is more effective.

The surprising thing about Dr. Sapi's study, to me, is the part about amoxicillin being that effective at reducing both spirochetal and round body forms. I say this, because I thought earlier research points towards amoxicillin being a less effective treatment than doxycycline.

Did I miss something, though? I thought earlier research did not look at antibiotic impact on round body forms in general, though - other than the Brorsons' metronidazole and tinidazole research.

Image taken from Brosons' research, An in vitro study of the
susceptibility of mobile and cystic forms of Borrelia burgdorferi to tinidazole

But earlier research is out there which confirms the effectiveness of amoxicillin on Borrelia burgdorferi, and found in a 2003 publication, In Vitro Susceptibility Testing of Four Antibiotics against Borrelia burgdorferi: a Comparison of Results for the Three Genospecies Borrelia afzelii, Borrelia garinii, and Borrelia burgdorferi Sensu Stricto.

What's fascinating to note here, again, is that different isolates of Bb respond very differently to different antibiotics!

"In 7 out of 12 comparative evaluations (P  > 0.05), MBCs were significantly different among the three genospecies. B. garinii seemed to be especially susceptible to azithromycin, while amoxicillin had a significantly greater effect on B. burgdorferi sensu stricto compared to the other genospecies. Ceftriaxone had the lowest MBC with B. afzelii and increasingly higher MBCs with B. garinii and B. burgdorferi sensu stricto. Doxycycline did not show any remarkable differences in its effects on the three genospecies."

So amoxicillin apparently doesn't suck when it comes to treating Borrelia burgdorferi, but it's not as effective on the other genospecies. (C'mon, Dr. Luft, please get that test working so we know which Bb we have to treat it the most effectively right off the bat.)

If it is found in vivo that doxycycline creates round bodies that contribute to the spirochete's survival, then doxycycline - what is typically given to patients diagnosed early with Lyme disease - would be contraindicated.

Whether the round bodies are as relevant as a potential "stasis" of metabolism in Borrelia burgdorferi remains to be seen. Either way, in vivo findings are different from in vitro findings.

In vitro, tigecycline was said to be many times more powerful than other antibiotics in killing Borrelia burgdorferi...Yet as we can see from Barthold's experiments on mice, viable spirochetes are found after tigecycline treatment in vivo, and viable enough that they can be picked up by ticks and transmitted to a new host.

Is there any treatment which can be used that would ensure the destruction of these remaining spirochetes, and would their demise lead to the end of persisting symptoms in patients who have them - or would there be an ongoing immune dysregulation which was triggered by their existence which continues after they are all dead?

This is something I'd really like to see studied.

Getting back to the last bit of Dr. Sapi's paper...

"When quantitative effects on biofilm-like colonies were evaluated, the five antibiotics reduced formation of these colonies by only 30%–55%. In terms of qualitative effects, only tinidazole reduced viable organisms by ~90%. Following treatment with the other antibiotics, viable organisms were detected in 70%–85% of the biofilm-like colonies."

I'd like to see analysis of how each of the five antibiotics fared relative to one another within biofilm-like colonies, rather than a range. It'd be good to do a direct comparison of each antibiotic against each form in vitro including biofilm-like colonies.

So... On the whole, I'd say take note of the study, with the message that independent confirmation and reproducibility of the methods chosen and these findings are important - and it's good to make note of these findings in relationship to other research already completed.

If anyone heads to the University of New Haven on the 21st to see the presentation on this, I'd love to get a report from you in comments about what was said.

In the end, I'll leave you with this closing thought from a paper from 2005, In Vitro Susceptibility Testing of Borrelia burgdorferi Sensu Lato Isolates Cultured from Patients with Erythema Migrans before and after Antimicrobial Chemotherapy :

"... similar to failures of chemotherapy for Treponema pallidum in syphilis (24), clinical treatment failures have been reported to occur in early LB cases for almost every suitable antimicrobial agent (10, 12, 28, 38, 42). Furthermore, the currently available diagnostic techniques do not reliably discriminate among possible reinfection, true endogenous relapse, and coinfection with other tick-borne pathogens (12). These drawbacks together with the phenomenon of resistance to therapy in individual patients undoubtedly contribute to the inconsistencies surrounding the optimal treatment regimens for LB and are often misinterpreted and misused to support prolonged antibiotic treatment regimens. However, relatively few cases of culture-proven treatment failure have been published (19, 22, 28, 29, 37, 38, 39), and the underlying mechanisms of antimicrobial resistance in B. burgdorferi sensu lato remain unresolved."

And there you have it. This pretty much characterizes the scientific reasons contributing to the ongoing controversy, five years later: Yes, there are treatment failures; yes, they are hard to diagnose and distinguish from coinfection and reinfection; yes, there is antimicrobial resistance; yes, scientists state these issues contribute to what is viewed as a misuse of prolonged antibiotic treatment.

But if treatment is necessary - whether it is a relapse or a new infection - then treatment is necessary.

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