The author’s write this sample:
“After performing a literature review, we identified 56 studies showing that B. burgdorferi was detectable in long term culture (Table A1 and Table A2).
Viable spirochetes were identified by culture, histology and/or xenodiagnosis following antibiotic treatment in 10 animal studies and 25 human studies, demonstrating that live organisms can persist following this treatment. [My Bold and in instances below]
These findings contradict the earlier contention that the Lyme spirochete cannot survive antibiotics.
Further evidence for evasion of the immune response and antibiotic therapy is described below.
A monkey study by Embers et al. published in 2012 provides the best animal evidence for persistent infection as a mechanism for CLD [Chronic Lyme Disease [9]].
The study was conceived as an animal counterpart to the human trial by Klempner et al. that was published in 2001 [10], and the monkeys were treated with a regimen of intravenous ceftriaxone followed by oral doxycycline that was identical to the protocol used in the human trial.
The results of this [Embers] study showed that three-quarters of the monkeys failed treatment, and these animals had evidence of persistent infection in various tissues at necropsy using culture, immunofluorescence and PCR techniques [9]
Equally important, the [Embers] study showed that 25% of treated monkeys cleared their infection, thereby demonstrating antibiotic efficacy in some animals.
This finding contradicts the negative treatment results reported by Klempner et al. in humans to support the conclusion that antibiotics are not effective in treating patients with persistent Lyme disease symptoms [11]. In short, Embers was able to demonstrate persistence using an invasive approach (necropsy) that could not be used in human clinical trials [9] [12].
In another study, Bockenstedt et al. presented a mouse model of B. burgdorferi infection that on the surface appears to contradict the monkey study [13]. Follow DOI: 10.4236/aid.2026.161005 75 Advances in Infectious Diseases R. B. Stricker et al. ing infection, the mice were treated with subcutaneous ceftriaxone or doxycycline administered in drinking water. The authors arrive at the conclusion that non-infectious spirochetal “debris” gets deposited around the joints of these mice, and instead of being cleared by the reticuloendothelial system this “debris” is responsible for persistent inflammation in mouse tissues [13]. The “debris”, which contained both DNA and protein particles, could not be cultured, transmitted to other mice via ear transplants or to ticks that were allowed to feed on the mice (xenodiagnosis).
This novel hypothesis of non-infectious persistence of B. burgdorferi “debris” including the presence of DNA contradicts previous experimental results. For example, Malawista et al. showed that B. burgdorferi DNA is rapidly cleared from culture-negative ear and bladder tissues of mice following prompt antibiotic treatment [14], and Lazarus et al. demonstrated that DNA from dead spirochetes is routinely cleared from mouse skin within several hours [15]. The “debris” hypothesis fails to explain persistence of viable spirochetes in culture, histology and xenodiagnosis experiments following antibiotic therapy.
Furthermore, the study methods of Bockenstedt et al. may have been insufficient to rule out persistent spirochetal forms of B. burgdorferi, since ear transplants are often negative following antibiotic treatment, and using an insufficient number of animals for xenodiagnosis may fail to demonstrate transmissible infection [16] [17].
Of greater importance, there appear to be two alternative mechanisms of B. burgdorferi persistence that merit consideration in these mice: the persistence of cysts (L-forms) and the inability to detect spirochetes in biofilms.
In a commentary on the mouse study, Alan Barbour proposed the alternative hypothesis that cell-wall deficient cysts (L-forms) may be responsible for B. burgdorferi persistence in these animals [18]. He noted that these cystic structures, which Bockenstedt et al. observed in their infected animals, have been described as a persister mechanism employed by many bacteria, including B. burgdorferi [19]-[27].
Bockenstedt et al. claim that these are not true cysts because they form too fast, appearing in minutes rather than hours or days. However, Brorson and Brorson have demonstrated that cysts of B. burgdorferi may develop in minutes under appropriate culture conditions [28]. Thus the observation of Bockenstedt et al. supports B. burgdorferi cyst formation in their mouse model, and this cyst formation appears to be a better explanation for spirochetal persistence compared to the “debris” that the authors postulate. As noted above, the methods employed by Bockenstedt et al. may not have been sufficient to exclude other persistent spirochetal forms such as cysts (L-forms) in their animals.
Persistent viable organisms also may have been hidden in biofilms, the adherent polysaccharide-based matrices that protect bacteria against the host immune system and antibiotic therapy [1]. Biofilms of B. burgdorferi have been demonstrated in vitro by Sapi et al. [29]. These biofilms may take the form of “debris” on intravital microscopy, and they may contain organisms that are non- cultivable but still viable and prone to reactivation [29]-[31]. Biofilms of B. burgdorferi would also be consistent with the “amber hypothesis” proposed as Advances in Infectious Diseases DOI: 10.4236/aid.2026.161005 76 R. B. Stricker et al. mechanism of persistent Lyme disease symptoms due to “introduction into the joint space of non-viable spirochetes or spirochetal debris enmeshed in a host derived fibrinous or collagenous matrix” [32].
Like the “debris” hypothesis, the “amber” hypothesis fails to explain live Borrelia persistence after antibiotic therapy. Persister spirochetes in biofilms could explain the experimental results of Bockenstedt et al. and would offer a more plausible explanation than the “debris” and “amber” hypotheses for the reasons outlined above.
Recently McClune et al. presented evidence that B. burgdorferi peptidoglycan (PG) can persist in synovial fluid after murine infection and may cause symptoms compatible with CLD [33]. McCausland et al. demonstrated unusual properties of the spirochete-derived PG that allow it to persist in mouse liver for weeks [34]. Although these observations may support the “debris” hypothesis of CLD, they do not rule out persistent B. burgdorferi infection in cysts (L-forms) and biofilms.
Further work is needed to examine the relationship between PG-induced inflammation and persistent spirochete infection in CLD.
Like most aspects of Lyme disease, the role of cysts (L-forms) and biofilms in persistent B. burgdorferi infection has been controversial [1] [30] [35]. These spirochetal forms are resistant to common antibiotics due to reduced metabolic activity or protective matrices. Whether CLD arises from persisting spirochetal forms hidden in biofilms (as suggested by the monkey studies of Embers et al. and the work of Sapi et al.) or from cell wall-deficient cysts (L-forms) of B. burgdorferi (as suggested by the mouse study observations of Bockenstedt et al. and the interpretation of Barbour),…”
Persisting “forms of bacteria require treatment.”
“To date the treatment options for these bacterial persisters are extremely limited, but their recognition dictates a more aggressive approach to eradication of Lyme disease using combination antibiotic therapy modeled on treatment regimens for tuberculosis and HIV disease [2].
The fact that B. burgdorferi shares cyst (L-form) properties, biofilm configurations and resistance genes with pathogenic mycobacteria supports the need for this therapeutic approach [36] [37]. It remains to be seen which forms of B. burgdorferi are the true culprits in CLD and which treatments are most efficacious in clearing infection from patients [38]-[48]
Source: Stricker, R.B., Fesler, M.C. and Johnson, L. (2026) Persistent Borrelia Infection in Chronic Lyme Disease: A Review of the Medical Literature. Advances in Infectious Diseases, 16, 73-87.