Sunday, May 8, 2011

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.

9 comments:

  1. This is a comment by a prominent doctor in the Lyme 'field' in 2006. I see nothing in his comments that aren't still true 5 years later. I received permission to reprint it which I did on Lymenet.org but has since been 'removed'.(grin)

    (Bolding is mine)

    Begin quote:
    Many, if not all, members of ILADS in my experience find chronic Lyme disease to be a very complex disease with multiple pathogenic mechanisms.

    The multiple mechanisms of dysfunction are well documented- from
    neurotransmitters, to immune modulators, to endocrine dysfunctions, to auto-immune markers and homologies of antigenic outer surface molecular
    structures and host tissue, mold and chemical sensitivities/ toxins and variant and persistent complex coinfection microbes only partially discovered yet.

    Paralleling the complexity of pathology is the complexity of patient response to treatment options.

    Compounding the progress in knowledge is the fact that parasites utilize existing host physiological pathways to their own benefit ie evasion of
    detection and conservation of energy/genomic content for vital functions: reproduction and modulation of antigenic presentations.

    Consequently, and I think I speak a chant from ILADs, no one researcher or one explanation for the illness, has been accepted as THE option in disease
    diagnosis and treatment to the exclusion of other options for the patients.

    For example, steering a course toward auto-immunity totally devoid of even small amounts of microbial presence as part of the driving machine is as fraught with danger of running aground -as is a course set on microbial persistence without
    awareness of complex mechanisms, which once set in place, show evidence of running on their own - even though microbial load in some forms has been
    diminished.

    ReplyDelete
  2. Hi cave76,

    Yes, you bring up a very good point - this is a complex disease, and Dr. Luft pretty much hit it on the head: It's more of a Lyme Borrelia Complex, because patients can be coinfected with more than one pathogen.

    Depending on the patient's history (where they lived, travel, exposure to animals with ticks from who-knows-where) there can be a number of possibilities as to what the doctor is looking at and needs to be treated - infections don't have to be contracted in one's own backyard.

    Most of us in the US are probably going to be infected with Bb ss and not garinii or afzelii, but a doctor shouldn't rule those strains out if the patient has visited Europe. From a baseline, though - even if one discounts exposure to European strains, it seems logical to not turn to doxycycline as one's first antibiotic of choice.

    As seen from the above studies, doxycycline takes a long time to reach even the MIC and it's not as effective in treating Lyme disease early on regardless of the existence of round bodies or their role. If there is a logic for the IDSA to recommend doxycycline, about the only reason I can see at the moment is that it might simultaneously take care of a coinfection.

    I haven't yet looked at all the research done in this area. I do know that some LLMDs have methods of combination antibiotics they use early on to address Lyme disease and coinfections at the same time if not forms and mechanisms.

    Anyway, the research above provides evidence that tigecycline is at least more effective than doxycycline in vitro, and that tinidazole is shown to degrade more round bodies in vitro.

    Now what would be useful is a study with three groups of newly diagnosed, clinically defined Lyme disease patients - one taking doxycycline, and one taking amoxicillin, and one taking tinidazole. Have them do so for a month using MBC (if tolerated by individual patients) levels. Then follow up at regular post-treatment intervals and see how patients fare. I'd be interested to see the results - I'd also like to see the experiment run using late stage and persisting symptom groups.

    ReplyDelete
  3. Actually, I wish to append to my above comment:

    How often are patients receiving a dose of antibiotics which is only reaching the MIC and not the MBC? From what I've read, different doctors offer somewhat different dosages and frequencies for antibiotic treatment. This will always vary by nature of accommodating certain patient morbidity such as kidney or liver issues; pregnancy - but outside of this, is it firmly established which dosage and frequency is optimal?

    ReplyDelete
  4. Buried back in the past many people who were tested for Lyme were also tested for HME and HGE (now called HGA) at the same time. And still are.

    Most people (and I don't have anything to go on except my memory) did test positive for those also. I know I did but I certainly wasn't alone!

    Doxy may have become the abx of choice because it addressed two bacterial infections at the same time. And perhaps that choice is still extant.

    Because of the potential for serious or even fatal infection (of Ehrlichia and Anaplasma), it is therefore recommended that all patients who have suspected or documented HME or HGA should be treated with oral or intravenous doxycycline hyclate in the absence of specific contraindications to tetracycline
    http://www.medscape.com/viewarticle/490468_5

    ReplyDelete
  5. From what I've seen of the research, Tigecycline is more effective for treating Bb and it will also handle Ehrlichiosis and Rickettsial diseases in general quite well. At least that's what the research has stated so far.

    ReplyDelete
  6. Oh, and by the way, I just posted this on LNE, where they've been speculating about Sapi's paper:

    http://www.newhaven.edu/unhtoday/archive/unh_12162008/pages/faculty_profile.html

    The reason why the paper is short on detail may be because her team is in the middle of the patent process for a new Lyme disease test.

    ReplyDelete
  7. Yeah, nothing talked about before a patent is conferred. That's a given.

    About tigecycline----- New drug. Needs more 'testing' in the general population. That's my concern.

    Plus this:

    WARNINGS AND PRECAUTIONS

    All-Cause Mortality

    An increase in all-cause mortality has been observed across Phase 3 and 4 clinical trials in TYGACIL-treated patients versus comparator-treated patients.

    In all 13 Phase 3 and 4 trials that included a comparator, death occurred in 4.0% (150/3788) of patients receiving TYGACIL and 3.0% (110/3646) of patients receiving comparator drugs.

    In a pooled analysis of these trials, based on a random effects model by trial weight, an adjusted risk difference of all-cause mortality was 0.6% (95% CI 0.1, 1.2) between TYGACIL and comparator-treated patients.

    The cause of this increase has not been established.

    This increase in all-cause mortality should be considered when selecting among treatment options


    http://www.pfizerpro.com/hcp/tygacil/safety/information

    When I read 'not been established' I go 'hmmmm'.

    That's just ONE reason I won't 'consider' tygecycline. (grin)


    Was it 'fast tracked' as so many drugs are now?

    ReplyDelete
  8. cave76,

    It might be more likely to see more antibiotics fast-tracked in general is my thought. If the "10 by 2020" initiative is getting off the ground, we'll be seeing 10 new antimicrobials hit the market within the next 9 years.

    The issue is more to get antibiotics out there to treat acute antibiotic resistant infections like MRSA and others - not so much Lyme disease.

    It may be a case of "damn the torpedoes, full steam ahead" if the choice is to treat, say, MRSA with a 4% chance of death versus, well, 80% or higher, depending on where the infection is and how resistant it is (See: VRSA. Oh, and by the way, apparently some Bb is Vancomycin resistant... so much for that idea.)

    We're inbetween (as it's been said in other contexts) a rock and a hard place. There isn't much choice, is there?

    ReplyDelete
  9. NB: Tigecycline was approved in 2005 to treat MRSA. It's used today for multi-drug resistant infection.

    ReplyDelete

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