Chronic Lyme Study Findings
1. There is no antibiotic resistance development with any antibiotics used to treat Lyme disease.
2. Combination therapy does not work. In fact, combinations of amoxicillin, ceftriaxone, and doxycycline were found to be no more effective than the drugs used individually for killing B. burgdorferi.
3. Pulse dosing with ceftrixone (after the 4th treatment) was effective in killing persister cells. The pulse-dosing experiment showed that a population of the pathogen can be eradicated with conventional antibiotics commonly used to treat the disease.
4. Increasing the strength of amoxicillin and ceftriaxone did not kill more of the surviving bacteria cells.
5. Increasing the strength of Doxycycline appeared to kill Borrelia burgdorferi.
6. B. burgdorferi forms persister cells capable of surviving very high concentrations of antibiotics, which exceed what is clinically achievable.
7. Surviving cells do not acquire a genetic mechanism for antibiotic resistance.
8. B. burgdorferi forms typical persister cells. The population grown from the surviving cells produces the same level of persisters as the original population.
9. Reintroducing antibiotics will kill the regrowing bacteria.
10. Doxycycline inhibits the action of amoxicillin.
11. Borrelia burgdorferi is poorly suceptible to fluoroquinolones and aminoglycosides.
12. Daptomycin kills the majority of cells in a stationary culture, but the level of surviving persisters was comparable to that of a stationary culture treated with ceftriaxone.
13. *Mitomycin C eradicated B. burgdorferi within 24 h, with no detectable persisters remaining, however- QUOTE- "Treatment with mitomycin C can result in serious negative side effects, and it should not be used for treatment of Lyme disease."
14. Vancomycin effectively killed growing cells of B. burgdorferi, but not persisters, and was comparable to ceftriaxone.
15. Persister cells were substantially diminished after four rounds of killing with amoxicillin, and were eradicated below the limit of detection after four rounds of killing with ceftriaxone.
16. Combinations of standard antibiotics are used in treatment of Lyme disease as well as a combination of a fluoroquinolone and an aminoglycoside, compounds that often synergize and are capable of killing nongrowing cells. However, there was no synergy in killing B. burgdorferi with any of the tested combinations.
17. Patients referred to as having “antibiotic-resistant Lyme arthritis” will continue to have arthritis with synovial fluid that is PCR negative for B. burgdorferi DNA.
18. B. burgdorferi establishes long-term infections lasting years to lifelong in its natural (i.e., mice) and incidental (i.e., humans) hosts in the absence of antibiotic therapy. Source
Selected Quotes Supporting Above Statements
Treatment of the late-stage disease may require multiple courses of antibiotic therapy. Given that antibiotic resistance has not been observed for B. burgdorferi, the reason for the recalcitrance of late-stage disease to antibiotics is unclear. In other chronic infections, the presence of drug-tolerant persisters has been linked to recalcitrance of the disease.
Combinations of antibiotics did not improve killing. Daptomycin, a membrane-active bactericidal antibiotic, killed stationary-phase cells but not persisters.
Mitomycin C, an anticancer agent that forms adducts with DNA, killed persisters and eradicated growing and stationary cultures of B. burgdorferi.
Finally, we examined the ability of pulse dosing an antibiotic to eliminate persisters. After addition of ceftriaxone, the antibiotic was washed away, surviving persisters were allowed to resuscitate, and the antibiotic was added again. Four pulse doses of ceftriaxone killed persisters, eradicating all live bacteria in the culture.
All pathogens studied to date form persisters, dormant variants of regular cells which are tolerant to killing by antibiotics.
The ability to produce persisters explains the puzzling recalcitrance of chronic infections to antibiotics that are effective against the same pathogen in vitro.
Indeed, many chronic infections are caused by drug-susceptible pathogens (1, 2).
The immune system can effectively remove sessile cells from the blood and many of the tissues, and this accounts for the efficacy of antibiotics, including bacteriostatic compounds, in treating uncomplicated infections. When the immune response is limited, the result is often a chronic infection (2).
Biofilms are a well-studied example of immune evasion and serve as a paradigm for understanding chronic infections. In biofilms, cells are protected from the large components of the immune system by a surface exopolymer (3,–5).
Antibiotics kill the regular cells, but dormant persisters survive, and when the concentration of antibiotic drops, they resuscitate and repopulate the biofilm (2). This scenario is supported by our finding of high-persister (hip) Pseudomonas aeruginosa isolates selected in the course of prolonged antibiotic treatment (6).
Isolated from patients with late-stage cystic fibrosis, hip mutants of P. aeruginosa can produce 1,000 times more persisters than the parent strain; this indicates that selection for increased tolerance (rather than resistance) provided the pathogen with a survival advantage.
In this regard, Lyme disease resembles other chronic infections where the pathogen is protected from the immune system, and persister cells may enable it to survive treatment with antibiotics.
In this study, we report formation of drug-tolerant persisters in B. burgdorferiand describe possible approaches to their elimination.
Previous studies have shown that the persister fraction in other bacteria remains relatively unchanged even as the antibiotic level increases. We sought to determine if B. burgdorferipersisters behaved similarly in a dose-dependent killing experiment.
As the concentration of amoxicillin and ceftriaxone increased, the fraction of surviving cells remained largely unchanged (Fig. 1b and andc).c). Doxycycline is a bacteriostatic antibiotic but at higher concentrations appeared to effectively kill B. burgdorferi (Fig. 1d).
Again, the fraction of surviving cells did not change significantly with increasing levels of the compound. Thus, B. burgdorferi forms persisters capable of surviving very high concentrations of antibiotics, which exceed what is clinically achievable.
In B. burgdorferi, we observe a very different picture; amoxicillin and ceftriaxone kill stationary cells fairly well, yet the fraction of persisters continues to increase. One possibility is that this “stationary” culture actually represents a steady state where some cells die and others grow.
Next, we tested whether the B. burgdorferi cells surviving antibiotic treatment are drug-tolerant persisters or resistant mutants. For this, colonies produced by the surviving cells were regrown and tested for MIC. The amoxicillin and ceftriaxone MIC remained unchanged, showing that surviving cells had not acquired or developed a genetic mechanism for antibiotic resistance. The population grown from the surviving cells produced the same level of persisters as the original population (Fig. 3). These experiments show that B. burgdorferi forms typical persister cells.
All possible two-drug combinations of amoxicillin, ceftriaxone, and doxycycline were tested with a late-exponential-phase culture in a time-dependent killing experiment and found to be no more effective than the drugs used individually for killing B. burgdorferi (Fig. 4a). Doxycycline actually inhibited the action of amoxicillin.
We have shown previously that fluoroquinolones and aminoglycosides can kill nongrowing cells (36, 37), and we next tested these compounds against B. burgdorferi. The pathogen is generally poorly susceptible to compounds from these classes.
However, the B. burgdorferi MICs for gemifloxacin (fluoroquinolone) and spectinomycin (aminoglycoside) are within achievable human dosing levels, so we chose to test them (38,–41) (Table 1). Gemifloxacin and spectinomycin were ineffective in killing B. burgdorferi at tested concentrations (Fig. 4b). Combining these compounds also did not improve killing (Fig. 4b).
Daptomycin was highly bactericidal against B. burgdorferi, but a remaining subpopulation of persisters survived (Fig. 5), suggesting that B. burgdorferi persisters can tolerate a drop in the energy level.
Next, we tested vancomycin. This large glycopeptide antibiotic binds to lipid II, a precursor of peptidoglycan, on the outside of the cytoplasmic membrane. Vancomycin is highly effective against Gram-positive bacteria, but does not penetrate across the outer membrane of Gram-negative species.
Surprisingly, the vancomycin MIC with B. burgdorferiis low, 0.25 μg/ml, which is similar to Gram-positive species. B. burgdorferi cells have an outer membrane; the basis for this anomaly is unclear.
Vancomycin effectively killed growing cells of B. burgdorferi, but not persisters, and was comparable to ceftriaxone (not shown).
We also tested teixobactin, a compound we recently discovered, which also binds lipid II (44). At 1.2 kDa, teixobactin is considerably smaller than vancomycin (1.8 kDa), but it did not exhibit good activity in killing B. burgdorferi (data not shown).
Mitomycin C eradicated a late exponential culture of B. burgdorferi within 24 h, with no detectable persisters remaining (Fig. 6a). This was observed with a low, clinically achievable dose of the compound, 1.6 μg/ml or 8× MIC. In a dose-dependent experiment, eradication of a late exponential culture was achieved within 5 days with a 0.8 μg/ml (4× MIC) dose of the compounds (Fig. 6b). Finally, mitomycin C was tested against a stationary culture of B. burgdorferi. Surprisingly, eradication was achieved with a low dose of 4× MIC within 24 h (Fig. 6c). It appears that a stationary population is more susceptible to this compound than an exponentially growing one.
Apart from identifying compounds capable of killing persisters, it may also be possible to eliminate them with conventional bactericidal antibiotics using pulse dosing. Based on our results, the level of persisters is lowest during early exponential growth (Fig. 2).
We reasoned that allowing growth to resume and then retreating them as they enter the exponential phase may kill persisters surviving an antibiotic challenge. Eradication of the culture can then be achieved after several rounds of killing and regrowth.
To test this, a culture of B. burgdorferi was exposed to amoxicillin or ceftriaxone. The surviving persisters were allowed to resuscitate for a short period of time in fresh medium, and then exposed to the antibiotic again for a second round of killing. Persisters were substantially diminished after four rounds of killing with amoxicillin, and were eradicated below the limit of detection after four rounds of killing with ceftriaxone (Fig. 7).
Additionally, we found that a ceftriaxone solution stored under experimental conditions (in BSK-II medium at 34°C, 3% O2, and 5% CO2) does not lose activity, as measured by MICs against B. burgdorferi, for up to 20 days.
The activity of amoxicillin measured similarly, however, dropped 20-fold over 20 days, which suggests degradation over time. The resulting MIC was still lower than the concentration used in killing experiments. This pulse-dosing experiment shows that a population of the pathogen can be eradicated with conventional antibiotics commonly used to treat the disease.
B. burgdorferi is a pathogen that can affect immunocompetent hosts. It establishes long-term infections lasting years to lifelong in its natural (i.e., mice) and incidental (i.e., humans) hosts in the absence of antibiotic therapy (14, 48).
Delays in diagnosis and treatment lead to sequelae that may require additional treatment. For example, patients who develop arthritis, which typically begins after 1 month of untreated infection, often do not respond fully to a first course of 28 days of antibiotics (49).
The majority of these patients have evidence of B. burgdorferi DNA in their synovial fluid and will respond to additional 1- or 2-month courses of antibiotics (13, 16). A smaller minority of patients referred to as “antibiotic-resistant Lyme arthritis” patients will continue to have arthritis with synovial fluid that is PCR negative for B. burgdorferi DNA.
These patients [PTLDS] typically respond to anti-inflammatory agents such as methotrexate or tumor necrosis factor (TNF) inhibitors. These two groups of patients should be distinguished from patients with “chronic Lyme disease” that exhibit fatigue, myalgias, and arthralgias without clear evidence for the presence of the pathogen.
For the first group of Lyme arthritis patients responsive to antibiotics, given that there is no reported resistance to clinically used tetracyclines, β-lactams, and cephalosporins in the pathogen, the need for lengthy courses of therapy is unclear.
The presence of persister cells is one possible explanation, and this is a pattern that is seen in other infections where persister cells are thought to be relevant for disease in vivo.
We tested combinations of standard antibiotics used in treatment of Lyme disease as well as a combination of a fluoroquinolone and an aminoglycoside, compounds that often synergize and are capable of killing nongrowing cells. However, there was no synergy in killing B. burgdorferi with any of the tested combinations.
We also tested daptomycin, a lipopeptide that acts by increasing the K+ permeability of the membrane. Being in a low-energy (microaerophilic) environment, the pathogen may be vulnerable to membrane-acting compounds. Daptomycin killed the majority of cells in a stationary culture, but the level of surviving persisters was comparable to that of a stationary culture treated with ceftriaxone.
In a recent publication, daptomycin was reported to kill B. burgdorferi persisters more effectively than regular cells (43). This conclusion was based on equating stationary cells with persisters. As follows from our experiments, a stationary culture harbors a small subpopulation of persisters. The actual level of stationary cells apparently surviving treatment by daptomycin in that study was very high, 28%, as determined by LIVE/DEAD staining. Under similar conditions, we detect about 103 (0.002%) surviving persisters by CFU count. It appears that LIVE/DEAD staining may be over-reporting the level of live B. burgdorferi cells.
Treatment with mitomycin C can result in serious negative side effects, and it should not be used for treatment of Lyme disease. This agent will be useful to examine the possible contribution of persisters to the disease in an animal model of infection.
Another peculiar feature of B. burgdorferi and a weakness of the pathogen is the lack of development of resistance to any antibiotic used to treat Lyme disease. Even attempts to raise mutants resistant to amoxicillin and ceftriaxone in vitro have been unsuccessful.
Joseph Bigger proposed an interesting strategy for elimination of persisters in 1944 in the first publication describing these cells (53). The rationale is to add an antibiotic to kill off regular cells, wash it away, and allow the culture to start regrowing, at which point persisters will resuscitate.
Reintroducing antibiotics will kill the regrowing bacteria. The argument against pulse dosing is that this protocol invites resistance development. Given that this is not a concern for B. burgdorferi, pulse dosing may be an effective strategy, and we performed pulse dosing with amoxicillin and ceftriaxone.
Persisters were eradicated with ceftriaxone in four pulses. These experiments form the basis for testing pulse dosing in an animal model and, if successful, in humans.
Source
Antimicrob Agents Chemother. 2015 Aug; 59(8): 4616–4624.
Published online 2015 Jul 16. Prepublished online 2015 May 26. doi: 10.1128/AAC.00864-15
PMCID: PMC4505243
PMID: 26014929
Borrelia burgdorferi, the Causative Agent of Lyme Disease, Forms Drug-Tolerant Persister Cells
Bijaya Sharma,a Autumn V. Brown,a Nicole E. Matluck,a Linden T. Hu,b and Kim Lewisa
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505243/