Lyme disease, the most prevalent tick-borne illness in North America and Europe, is caused by the bacterium Borrelia burgdorferi. While standard antibiotic therapies, such as doxycycline or amoxicillin, are generally effective in early stages, some patients continue to experience persistent symptoms even after treatment. This condition, often termed post-treatment Lyme disease syndrome (PTLDS), may be linked to antibiotic-tolerant bacterial forms that evade eradication. Our comprehensive review of in vitro studies reveals that certain antibiotic combinations, including daptomycin, cefoperazone, and doxycycline, demonstrate significant efficacy against the diverse morphological forms of B. burgdorferi, offering a promising approach to improve treatment outcomes and reduce relapse risk.
Best Antibiotic Combinations for Lyme Disease: Targeting Borrelia Persisters
Lyme disease, caused by Borrelia burgdorferi, is typically treated with antibiotics such as doxycycline or amoxicillin, which are effective in the early stages. Despite this, some patients experience persistent symptoms even after treatment completion, often referred to as post-treatment Lyme disease syndrome (PTLDS). This has raised concerns about the potential for antibiotic-tolerant forms of B. burgdorferi to survive standard therapies. The bacterium’s ability to exist in multiple morphological forms, including spirochetes, round bodies, and biofilm-like colonies, poses a challenge to eradication due to varying levels of antibiotic resistance. This review synthesizes findings from key in vitro studies evaluating the effectiveness of different antibiotics and combinations against B. burgdorferi, with a particular focus on persister cells. Notably, the combination of daptomycin, cefoperazone, and doxycycline emerged as highly effective in eliminating all morphological variants, suggesting a potential new therapeutic approach for Lyme disease. The results emphasize the importance of targeting multiple forms of the bacterium to improve treatment outcomes and reduce the risk of relapse.
Introduction
Lyme disease is the leading tick-borne infection in North America and Europe, with Borrelia burgdorferi being the principal causative pathogen. While antibiotic therapy typically involves the use of doxycycline or amoxicillin for early-stage disease, a subset of patients continues to report symptoms such as fatigue, joint pain, and neurocognitive dysfunction, even months or years after completing treatment. The underlying cause of these persistent symptoms, commonly known as PTLDS, is still debated, with proposed explanations including immune-mediated damage, residual bacterial debris, or the presence of surviving antibiotic-tolerant bacterial forms.
The pleomorphic nature of B. burgdorferi is a key factor in its ability to evade immune responses and antibiotic treatments. The bacterium can transform into different morphological forms, including the actively dividing spirochete form, dormant round bodies, and biofilm-like colonies that enhance its survival under hostile conditions. Each form exhibits distinct levels of antibiotic susceptibility, necessitating an approach that targets multiple bacterial states. This review consolidates data from various in vitro studies to assess the effectiveness of different antibiotic regimens against the different morphological forms of B. burgdorferi, with an emphasis on optimizing treatment for cases of persistent infection.
Morphological Variability of Borrelia burgdorferi and Antibiotic Resistance Mechanisms
Morphological Forms and Their Significance in Lyme Disease Treatment
B. burgdorferi is a highly adaptable pathogen capable of transitioning between different forms depending on environmental stimuli. These forms include:
Spirochetal Form
The helical, motile state associated with active infection and rapid proliferation. Spirochetes are the primary form encountered in early Lyme disease and are typically targeted by first-line antibiotics like doxycycline and amoxicillin. Their high metabolic activity renders them more susceptible to antibiotics that interfere with cell wall synthesis and protein production.
Round Bodies (Cystic Forms)
These arise in response to unfavorable conditions such as nutrient deprivation, immune stress, or exposure to antibiotics. Round bodies are characterized by a compact, spherical shape, with reduced metabolic activity, which makes them less susceptible to agents that target actively growing bacteria. Studies have shown that certain antibiotics, like metronidazole and tinidazole, can be more effective against these dormant forms.
Biofilm-Like Colonies
Biofilm formation is a defense mechanism used by many bacteria, including B. burgdorferi. Biofilms consist of aggregated bacterial cells encased in an extracellular matrix composed of polysaccharides, proteins, and nucleic acids. This structure confers significant resistance to antibiotics and immune responses by limiting drug penetration and protecting dormant cells within the biofilm. Biofilm-like forms of B. burgdorferi are believed to contribute to chronic and relapsing infections.
The ability to form biofilms and round bodies may explain why B. burgdorferi can persist in patients even after antibiotic therapy. Eradicating these forms requires a multi-pronged approach that can penetrate the biofilm, disrupt dormant cells, and kill actively replicating spirochetes.
Mechanisms of Resistance Across Morphological Forms
Resistance in B. burgdorferi varies based on its morphological state:
Active spirochetes are susceptible to cell wall-targeting antibiotics, such as beta-lactams, and protein synthesis inhibitors, such as doxycycline.
Round bodies exhibit increased tolerance to these antibiotics due to their reduced metabolic activity and thickened outer membrane.
Biofilm-associated cells are particularly resistant due to the protective nature of the extracellular matrix, which restricts the penetration of many antimicrobial agents and facilitates the survival of persister cells.
Persister cells, a subset of the bacterial population that enters a dormant state, are not resistant due to genetic mutations but can survive antibiotic exposure due to their metabolic inactivity. These cells can later "wake up" and resume growth once the antibiotic pressure is removed, potentially leading to relapse of infection. The challenge is to find combinations of antibiotics that can target both active and dormant cells.
Detailed Analysis of In Vitro Studies on Antibiotic Susceptibility
Study by Feng et al.: The Search for Effective Drug Combinations Against Borrelia burgdorferi Persisters
The study conducted by Feng et al. evaluated a wide range of antibiotics and their combinations to determine which regimens were most effective at eradicating stationary-phase B. burgdorferi. The focus was on identifying treatments that could eliminate persisters, including round bodies and biofilm-like forms, using a combination of fluorescence-based viability assays and microscopy.
Methods
The researchers used a 10-day-old stationary-phase culture of B. burgdorferi, enriched with persisters. A series of 81 different antibiotic combinations were tested at a concentration of 10 μg/ml for each drug. The primary antibiotics included in the testing were:
Doxycycline (10 μg/ml): A tetracycline-class antibiotic with known effectiveness against actively replicating bacteria.
Cefoperazone (10 μg/ml): A third-generation cephalosporin with broad-spectrum activity.
Daptomycin (10 μg/ml): A lipopeptide antibiotic effective against Gram-positive persisters.
Other agents such as clofazimine, carbenicillin, sulfamethoxazole, and metronidazole were included in different combinations.
The viability of treated cultures was measured using the SYBR Green I/PI assay, where live cells stained green and dead cells stained red. The cultures were also subjected to subculturing in fresh medium for 15 days to confirm whether any viable bacteria remained.
Results
The study found that:
Daptomycin alone significantly reduced the viability of stationary-phase B. burgdorferi but could not completely eradicate microcolony forms.
Doxycycline and cefoperazone, when used together, showed improved activity over either drug alone, but some biofilm-associated cells survived.
The combination of daptomycin, doxycycline, and cefoperazone achieved complete eradication of all bacterial forms, including biofilm-like microcolonies, with no regrowth observed during a 15-day subculture period. This combination exhibited a synergistic effect, with significantly reduced green/red fluorescence ratios and an absence of detectable viable cells.
Other three-drug combinations, such as daptomycin with doxycycline and clofazimine or sulfamethoxazole, showed considerable bactericidal activity but still allowed some cells to survive in the microcolony form.
The findings underscore the superior efficacy of the daptomycin, doxycycline, and cefoperazone combination, which effectively targeted different morphological states, including the highly resistant biofilm forms. The study suggests that daptomycin’s mechanism, which disrupts cell membrane integrity, might complement the action of doxycycline and cefoperazone in overcoming biofilm-associated resistance.
Statistical Analysis
The statistical significance of the results was confirmed through t-tests, with p-values of less than 0.01 for combinations that achieved complete eradication compared to single-drug regimens. The reduction in viable cell percentages was particularly pronounced for the daptomycin, doxycycline, and cefoperazone combination, which had a residual viable cell count of less than 10% immediately after treatment.
Study by Sapi et al.: Susceptibility of Different Borrelia Morphological Forms to Antimicrobial Agents
Sapi and colleagues conducted a study evaluating the in vitro susceptibility of different B. burgdorferi morphological forms—spirochetes, round bodies, and biofilm-like colonies—to five antibiotics: doxycycline, amoxicillin, tigecycline, metronidazole, and tinidazole. The study aimed to determine MIC and MBC values and to assess the qualitative and quantitative effects of antibiotics on the various forms.
Methods
A range of techniques was used, including the microdilution method to determine MIC (the lowest concentration inhibiting visible bacterial growth) and MBC (the minimum concentration at which no bacteria could be cultured after 21 days). Antibiotic effects were observed at different time points (24, 48, 72 hours, and three weeks) using dark field and fluorescence microscopy.
Biofilm-like colonies were generated using collagen-coated plates and treated with antibiotics for 72 hours. The biofilms were stained with crystal violet, and viability was quantified by optical density measurements at 570 nm.
Results
Doxycycline (250 μg/ml): Reduced the number of spirochetal structures by approximately 90%, but paradoxically led to a twofold increase in round body forms, indicating a survival strategy by morphologic transformation.
Amoxicillin (250 μg/ml): Demonstrated an 85–90% reduction in spirochete numbers but had limited activity against round bodies, achieving only 68% reduction.
Metronidazole and tigecycline (250 μg/ml and 20 μg/ml, respectively): Showed better efficacy against round bodies, with 80–90% reductions, but struggled to eliminate biofilm colonies.
Tinidazole (500 μg/ml): Reduced biofilm viability by approximately 55%, the highest among the tested antibiotics.
The study found significant variations in MIC/MBC values when using different methods. For instance, doxycycline’s MIC was reported to be 0.4 μg/ml using traditional microdilution, but direct cell counting methods showed an MIC above 25 μg/ml, indicating greater resistance under more realistic conditions.
These results support the notion that combination therapies may be necessary to effectively eliminate all forms of B. burgdorferi, especially biofilm-associated bacteria. The increase in round body formation following doxycycline treatment highlights the need for drugs that can target dormant forms, such as metronidazole or tinidazole, in combination regimens.
Study by Alvarez-Manzo et al.: Evaluation of Nitroxoline Combinations Against Stationary-Phase Borrelia burgdorferi
The study conducted by Alvarez-Manzo and colleagues explored the potential of nitroxoline (NTX), an FDA-approved antibiotic traditionally used for urinary tract infections, as a treatment for persistent forms of Borrelia burgdorferi. Given the limitations of current Lyme disease antibiotics in eradicating stationary-phase B. burgdorferi, the researchers investigated whether NTX and its combinations could outperform standard Lyme antibiotics in vitro. They compared the efficacy of NTX combinations to the established persister drug combination of cefuroxime (CefU), doxycycline (Doxy), and daptomycin (Dapto).
Rationale for Testing Nitroxoline
Nitroxoline (8-hydroxy-5-nitroquinoline) is a broad-spectrum antibiotic with known activity against Gram-positive and Gram-negative bacteria, as well as certain fungal pathogens. It has been used extensively in Europe for treating urinary tract infections. More recently, there has been interest in repurposing nitroxoline for other medical applications, including its use as an anticancer agent and in the treatment of persistent bacterial infections. Given its ability to inhibit biofilm formation and bacterial growth, nitroxoline was considered a promising candidate for testing against B. burgdorferi persister forms.
Methodology
The study evaluated the minimum inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC50) of nitroxoline and other antibiotics against stationary-phase cultures of B. burgdorferi. The antibiotics tested included:
- Nitroxoline (NTX)
- Cefuroxime (CefU)
- Doxycycline (Doxy)
- Artemisinin (ART) (A source of natural remedies)
- Azithromycin (AZM)
- Clarithromycin (Clari)
- Clofazimine (CFZ)
- Daptomycin (Dapto)
- Erythromycin (Ery)
- Linezolid (LNZ)
- Nitazoxanide (NTZ)
- Rifabutin (RFB)
The study involved treating 7-day-old stationary-phase cultures of B. burgdorferi for seven days with various single antibiotics, two-drug combinations, and three-drug combinations at a concentration of 5 μg/mL. The primary outcome was the percentage of viable bacteria after treatment, determined by SYBR Green I/PI viability staining and fluorescence microscopy.
Results
Minimum Inhibitory Concentrations (MICs) and IC50 Values
The MIC for NTX was found to be 1.25 μg/mL, which was higher than that of the standard Lyme antibiotics CefU (0.08 μg/mL) and Doxy (0.15 μg/mL). When evaluating the IC50 values in a 7-day-old stationary-phase culture, NTX had an IC50 of 5.3 μg/mL, which was slightly lower than CefU's IC50 of 7.9 μg/mL. This finding suggested that NTX exhibited comparable or slightly superior activity to CefU against stationary-phase B. burgdorferi.
Activity Against Stationary-Phase B. burgdorferi at 5 μg/mL
When tested individually at 5 μg/mL, NTX resulted in a 36.3% survival rate for stationary-phase B. burgdorferi, slightly lower than the 42.3% survival observed with CefU. Doxy and other antibiotics such as AZM, ART, and CFZ demonstrated higher survival rates (ranging from 47.2% to 56.9%), indicating that NTX and CefU had better activity against persisters compared to these standard antibiotics. However, neither NTX nor CefU as monotherapies achieved sufficient eradication of stationary-phase bacteria.
Two-Drug and Three-Drug Combinations
The study evaluated 19 two-drug combinations and 31 three-drug combinations at 5 μg/mL. Key findings included:
Two-Drug Combinations
NTX + CefU had a survival rate of 23.5%, demonstrating better activity compared to combinations without NTX, such as CefU + Doxy (42.5%). NTX + Clari also showed improved efficacy with a survival rate of 13.3%.
Overall, two-drug combinations with NTX were more effective than equivalent combinations with CefU alone, with survival rates ranging from 7.3% to 34.8%.
Three-Drug Combinations
The majority (87%) of NTX-based three-drug combinations resulted in bacterial viability below the 10% threshold, indicating strong activity against stationary-phase cultures.
The most effective three-drug combination was NTX + CefU + Clari, which achieved a survival rate of 2.7%. Other successful three-drug combinations included NTX + Clari + LNZ and NTX + Clari + NTZ, both with survival rates under 10%.
Comparison at Maximum Serum Concentration (Cmax)
Further testing was performed at Cmax for selected three-drug combinations to simulate therapeutic levels achieved in vivo. NTX-based combinations were compared to the positive control of CefU + Doxy + Dapto, known for its effectiveness against persister forms.
CefU + Clari + NTX emerged as the most effective three-drug combination, with a survival rate of 1.7%, comparable to the control combination CefU + Doxy + Dapto. This result suggests that NTX-based combinations can achieve levels of activity equivalent to the best available persister treatments.
Discussion
The study's findings indicate that NTX possesses significant activity against stationary-phase B. burgdorferi when used alone or in combination. The observed MIC and IC50 values for NTX, which were comparable to or better than those for CefU, support its potential as a treatment for persister forms. The superior performance of NTX in combination therapies, particularly NTX + CefU + Clari, suggests that it may be a viable alternative to standard Lyme disease antibiotics for patients with persistent symptoms.
The ability of NTX to perform as well as the established persister combination of CefU + Doxy + Dapto highlights its potential role in future treatment strategies for Lyme disease. Additionally, the study suggests that combinations involving NTX and macrolides (e.g., clarithromycin) could be especially effective, potentially providing a new therapeutic approach for eradicating persister forms.
Implications for Clinical Treatment and Future Research
The promising in vitro results warrant further investigation of NTX combinations in animal models and clinical trials. The study suggests that NTX, especially in three-drug combinations, could offer an effective alternative for patients with PTLDS. Future research should explore the pharmacokinetics and pharmacodynamics of NTX in vivo, as well as the optimal dosing strategies for combination therapy.
The findings also open avenues for repurposing NTX, which could potentially improve treatment outcomes for patients suffering from persistent Lyme disease by addressing the limitations of current antibiotic regimens.
Recommendations from the German Borreliosis Society for Treating Lyme Borreliosis
The German Borreliosis Society’s guidelines emphasize that treatment regimens should be tailored to target different morphological forms of B. burgdorferi, with special consideration for encysted and intracellular forms.
Encysted Forms
Hydroxychloroquine (200 mg daily) is recommended to enhance the activity of macrolides, while metronidazole (400–1200 mg daily) is suggested for its activity against round bodies.
Treatment Cycles for Persistent Symptoms
Long-term treatment, extending three months or more, is recommended for late-stage or relapsing cases. Recurrence should be treated with shorter cycles, varying from three days to three weeks depending on symptom severity.
Mechanisms of Biofilm Resistance and the Role of Combination Therapy
Biofilm formation serves as a protective strategy for B. burgdorferi, with its extracellular matrix limiting antibiotic access and promoting persister cell survival. Combination therapies that include drugs disrupting cell membranes (e.g., daptomycin) or interfering with DNA replication (e.g., tinidazole) show promise in overcoming biofilm-associated resistance.
Implications for Clinical Practice and Future Research
The combination of daptomycin, doxycycline, and cefoperazone warrants further investigation through clinical trials to evaluate its potential to improve outcomes for patients with PTLDS. Additionally, exploring novel adjunctive therapies that target biofilm disruption or persister cell activation could lead to more effective treatment regimens.
Practical Overview of Antibiotic Combinations and Real-World Dosages for Treating Borrelia burgdorferi Persisters
Several studies have evaluated different antibiotic combinations for their effectiveness against Borrelia burgdorferi in various morphological forms, including actively growing spirochetes, dormant round bodies, and biofilm-like colonies. This section summarizes these antibiotic regimens, providing practical dosage recommendations based on their in vitro efficacy and known clinical usage.
Doxycycline-Based Combinations
Doxycycline (Doxy) is a commonly used antibiotic for Lyme disease, with known efficacy against actively growing spirochetes but limited effectiveness against persister forms when used alone. Combining doxycycline with other antibiotics can improve its efficacy.
Doxycycline + Cefuroxime (CefU) + Daptomycin (Dapto)
Dosage:
- Doxycycline: 100 mg orally, twice daily. For more severe cases, 200 mg twice daily may be used.
- Cefuroxime: 500 mg orally, twice daily, or 1.5 g intravenously every 8 hours if severe.
- Daptomycin: 4–6 mg/kg intravenously once daily. The exact dose depends on patient weight and kidney function, with 6 mg/kg used for severe or persistent cases.
- Rationale: Doxycycline targets replicating bacteria, cefuroxime disrupts cell wall synthesis, and daptomycin acts on the cell membrane. This combination has shown to be more effective against persister cells.
Doxycycline + Cefoperazone + Daptomycin
Dosage:
- Doxycycline: 100 mg orally, twice daily (200 mg for severe cases).
- Cefoperazone: 2 g intravenously every 12 hours. This dose is chosen for its ability to reach effective levels in the central nervous system, which may be useful for neurological symptoms.
- Daptomycin: 4–6 mg/kg intravenously once daily, adjusted according to patient response and tolerability.
- Rationale: Cefoperazone, a third-generation cephalosporin, offers enhanced CNS penetration, and this combination eradicated B. burgdorferi persisters in in vitro studies.
Nitroxoline-Based Combinations
Nitroxoline (NTX) has shown promising activity against B. burgdorferi persisters, including biofilm forms, in laboratory settings.
Nitroxoline + Cefuroxime + Clarithromycin (Clari)
Dosage:
- Nitroxoline: 250 mg orally, three times daily. This dosage aligns with standard treatment for urinary tract infections and is well-tolerated in adults.
- Cefuroxime: 500 mg orally, twice daily. Alternatively, 1.5 g intravenously every 8 hours for severe cases.
- Clarithromycin: 500 mg orally, twice daily. This dosage targets intracellular bacteria and covers atypical pathogens.
- Rationale: The combination uses nitroxoline to target biofilm-associated B. burgdorferi, cefuroxime for cell wall inhibition, and clarithromycin to enhance intracellular coverage.
Metronidazole or Tinidazole-Based Combinations
Metronidazole and tinidazole target anaerobic bacteria and certain protozoans. Their activity against B. burgdorferi round body forms makes them useful in combination therapies.
Doxycycline + Metronidazole + Cefuroxime
Dosage:
- Doxycycline: 100 mg orally, twice daily.
- Metronidazole: 500 mg orally, three times daily, matching standard doses used in anaerobic infections.
- Cefuroxime: 500 mg orally, twice daily or intravenously for severe cases (1.5 g every 8 hours).
- Rationale: Metronidazole adds coverage against round body forms, while doxycycline and cefuroxime cover actively replicating bacteria. The combination aims to cover different morphological forms.
Tinidazole + Azithromycin (AZM) + Doxycycline
Dosage:
- Tinidazole: 500 mg orally, twice daily (commonly used for 5–10 days in other infections).
- Azithromycin: 500 mg orally once daily for the first 3–5 days, then may be reduced to 250 mg daily.
- Doxycycline: 100 mg orally, twice daily.
- Rationale: Tinidazole and azithromycin provide coverage against round bodies and intracellular forms, while doxycycline addresses active spirochetes.
Other Promising Combinations
Combining other antibiotics like linezolid (LNZ), clofazimine (CFZ), or nitazoxanide (NTZ) has shown potential:
Cefuroxime + Linezolid + Nitroxoline
Dosage:
- Cefuroxime: 500 mg orally, twice daily or intravenously at 1.5 g every 8 hours.
- Linezolid: 600 mg orally, twice daily, which is a standard dosing used in resistant Gram-positive infections.
- Nitroxoline: 250 mg orally, three times daily.
- Rationale: Linezolid adds Gram-positive activity, while nitroxoline enhances biofilm disruption. This combination may be useful for recalcitrant cases.
Clofazimine + Doxycycline + Cefuroxime
Dosage:
- Clofazimine: 100 mg orally, once daily, which is often used for mycobacterial infections.
- Doxycycline: 100 mg orally, twice daily.
- Cefuroxime: 500 mg orally, twice daily or 1.5 g intravenously every 8 hours.
- Rationale: Clofazimine's anti-biofilm and anti-inflammatory properties complement the action of doxycycline and cefuroxime, providing a comprehensive approach to target persisters.
Summary of Dosage Recommendations
The above regimens are based on commonly used dosages in clinical practice and consider each antibiotic's pharmacokinetic properties, safety profiles, and potential for combination therapy. It is important to adjust these doses based on patient weight, kidney function, and specific clinical scenarios, especially in cases of severe or disseminated Lyme disease.
Considerations for Clinical Application
Duration of Therapy: Depending on disease severity, treatment may last 6–12 weeks or longer, with intermittent dosing cycles for better tolerance.
Monitoring: Some antibiotics (e.g., daptomycin, linezolid) require monitoring for side effects like muscle toxicity or blood count changes.
Co-Infections: Presence of co-infections (e.g., Babesia) may necessitate adding other therapies, affecting antibiotic selection.
Conclusion
The persistence of Borrelia burgdorferi in various morphological forms, including spirochetes, round bodies, and biofilm-like colonies, poses a significant challenge to the successful treatment of Lyme disease, particularly in patients experiencing post-treatment Lyme disease syndrome (PTLDS). In vitro studies consistently demonstrate that single-agent therapies, while effective against actively growing spirochetes, fall short in eradicating persister forms, which can survive and contribute to symptom relapse. This review has highlighted several promising antibiotic combinations that target multiple bacterial states, offering a more comprehensive approach to overcoming B. burgdorferi's resilience.
Among the combinations tested, regimens including daptomycin, doxycycline, and cefoperazone have shown the greatest potential for completely eradicating persister cells, including biofilm-associated forms. Nitroxoline-based combinations, particularly when paired with cefuroxime and clarithromycin, have also demonstrated significant in vitro activity, suggesting a possible role in treating persistent or relapsing cases of Lyme disease. Other combinations, such as those incorporating metronidazole, tinidazole, or linezolid, offer additional options for addressing various morphological forms, especially when targeting round bodies or intracellular bacteria.
The translation of these findings into clinical practice requires careful consideration of dosage, duration, and patient-specific factors, including co-infections and tolerance to antibiotics. The promising results of multi-drug regimens in vitro underscore the need for clinical trials to validate these approaches in humans. Future research should focus on optimizing treatment protocols, exploring novel anti-biofilm agents, and developing strategies to prevent relapse by thoroughly addressing all bacterial forms.
Ultimately, a combination therapy approach that integrates antibiotics with complementary mechanisms of action may offer the most effective strategy for eradicating B. burgdorferi persisters and improving treatment outcomes for patients with Lyme disease, especially those with persistent symptoms after standard antibiotic therapy.