Biofilm/Persister/Stationary Phase Bacteria Cause More Severe Disease Than Log Phase Bacteria – I BiofilmBorrelia burgdorferiNot Only Display More Tolerance to Lyme Antibiotics But Also Cause More Severe Pathology In a Mouse Arthritis Model: Implications for Understanding Persistence, PTLDS and Treatment Failure

Abstract

24Lyme disease, caused by Borrelia burgdorferi, is the most common tick-borne illness in US and Europe. While 25 most patients can be cured with a 2-4 week antibiotic therapy, about 10%-20% patients continue to suffer 26 persistent symptoms of fatigue, pain or joint and muscle aches, and neurocognitive despite the treatment, a 27 condition called post-treatment Lyme disease syndrome (PTLDS). The cause for PTLDS is unclear but one 28 possibility is persistent infection with B. burgdorferi. B. burgdorferi is known to develop morphological variant 29 forms such as round bodies and aggregated biofilm-like microcolonies as a log phase culture consisting of 30 spirochete form grows into stationary phase. Here we isolated biofilm-like microcolony and planktonic form 31(spirochetal forms and round body) from stationary phase culture and found that the stationary phase planktonic 32 form (SP) and microcolony form (MC) were much more tolerant to the current antibiotics for Lyme disease, 33 doxycycline, ceftriaxone and cefuroxime than log phase spirochete form (LOG). In addition, we also compared 34 the ability of the variant forms to cause disease in a mouse arthritis model. Surprisingly, the MC in particular 35 and the SP caused a more severe arthritis with an earlier onset of inflammation and joint swelling than LOG. 36MC-infected mice showed significant joint swelling as early as 9 days post-infection, while the LOG and SP did 37 not cause significant swelling. At 21 days, the joint swelling of the MC group dramatically increased and 38 peaked, while the SP showed significant swelling at this time but less severe than the MC group. The LOG 39 infected mice were just beginning to develop joint swelling at 21-day post-infection, with only slight swelling. 40At 30-day post infection, the SP group mice also developed similar severity of joint swelling as the MC group, 41 but the LOG group still did not show significant swelling. However, at 35-day post infection, all three infected 42 groups showed similar degree of significant joint swelling. Thereafter, the joint swelling of the three infected 43 groups waxed and waned during the 90-day observation. Thus, we established a new biofilm-inocula mediated 44 visual arthritis model that could facilitate more efficient evaluation of treatment regimens for persistent B. 45 burgdorferi infections. Our findings provide new insight about disease pathogenesis and may have implications 46 for understanding PTLDS and PTLDS treatment failure, due to possible biofilm inoculation during tick-bite. 47 This biofilm/persister seeding model may be valid for different microbial infections and facilitate developing 48 more effective treatments of persistent infections in general. 49 50

Generality of Post-Antimicrobial Treatment Persistence of Borrelia burgdorferi Strains N40 and B31 in Genetically Susceptible and Resistant Mouse Strains

“…Our results indicate that disseminated spirochetes of two different B. burgdorferi strains can persist in mice at 12 and 18 months following antimicrobial treatment. Noncultivable spirochetes persisted in mice (105), dogs (29), and nonhuman primates (54) inoculated with alternate strains of B. burgdorferi, so we have confirmed that persistence is not unique to N40.…”

Effect of dapsone alone and in combination with intracellular antibiotics against the biofilm form of B. burgdorferi

“…Unfortunately, these researchers did not evaluate DDS combinations against attached B. burgdorferi biofilm, the most antibiotic resistant form in vitro [24,30] and a dominant form in a human autopsy study from a Lyme disease patient [31]. Furthermore, a recent mouse model study [42] showed that the biofilm like microcolony and stationary phase planktonic forms (free cells) caused more severe Lyme arthritis with an earlier onset of inflammation and joint swelling than the log phase spirochetes. Therefore, addressing biofilm forms is vital, and for that reason in this study, we added several assays to evaluate dapsone alone and in combination on B. burgdorferi biofilms.…”

The Long-Term Persistence of Borrelia burgdorferi Antigens and DNA in the Tissues of a Patient with Lyme Disease

“…In vivo animal studies with immunocompetent mice, dog, and non-human primate models have identified a surviving, but not cultivatable form of B. burgdorferi that can withstand antibiotic treatment [16][17][18][19][20]. Recent studies on rhesus macaques and mice confirmed these observations by demonstrating a metabolically active, persistent B. burgdorferi post-treatment in antibiotic-treated animals [20][21][22][23][24]. There are also human studies providing clinical evidence that a chronic form of Lyme disease could be caused by a persistent spirochetal infection, which could explain the lingering symptoms [25][26][27][28].…”