Identifying vancomycin as an effective antibiotic for killing Borrelia burgdorferi.
Wu X1, Sharma B2, Niles S1, O'Connor K1, Schilling R1, Matluck N1, D'Onofrio A1, Hu LT2, Lewis K3.
Antimicrobial Discovery Center, Northeastern University, Boston, MA.
Tufts Medical Center, Boston, MA.
Antimicrobial Discovery Center, Northeastern University, Boston, MA. firstname.lastname@example.org.
Borrelia burgdorferi is the causative agent of Lyme borreliosis. Antibiotic therapy of early acute infection is effective for most patients, but 10-20% go on to develop Post-Treatment Lyme Disease Syndrome. The nature of PTLDS remains unknown, but currently approved antibiotics for treatment of Lyme disease do not appear to impact these symptoms after they have developed. We reason that minimizing the time the pathogen interacts with the host will diminish the probability of developing PTLDS, irrespective of its nature. This calls for an efficient eradication of the pathogen during acute infection. In search of a superior killing antibiotic, we examined approved antibiotics for their ability to kill B. burgdorferi Vancomycin proved more effective in killing the pathogen in vitro than ceftriaxone, the standard of care for disseminated B. burgdorferi infection. Both compounds were also the most effective in killing stationary phase cells. This is surprising, given that inhibitors of cell wall biosynthesis are known to only kill growing bacteria. We found that peptidoglycan synthesis continues in stationary cells of B. burgdorferi, explaining this paradox. A combination of vancomycin and gemifloxacin stehttps://www.ncbi.nlm.nih.gov/pubmed/30126963rilized a stationary phase culture of B. burgdorferi Examination of the action of antibiotics in immune-deficient SCID mice showed that doxycycline, a standard of care for uncomplicated acute infection, did not clear the pathogen. By contrast, both ceftriaxone and vancomycin cleared the infection. A trial examining early use of more potent antibiotics on development of PTLDS may be warranted.
Vancomycin efficiently clears B. burgdorferi in a mouse model of infection. Given
the potent killing activity of vancomycin against B. burgdorferi, we examined its efficacy
in vivo, and compared it to the two standard of care compounds – doxycycline and
ceftriaxone. Apart from its ability to kill the pathogen, vancomycin reduces cell wall
stiffness and decreases the rate of motility of B. burgdorferi, which improves its capture
by phagocytes (35The N40 strain used in the mouse infection model is also sensitive to doxycycline,
ceftriaxone and vancomycin. The MICs of doxycycline, ceftriaxone and vancomycin
against N40 strain are 0.25, 0.12 and 0.25 μg/ml, respectively. Vancomycin was
introduced into infected C3H mice at 110 mg/kg every 12 h by subcutaneous injection.
Vancomycin eliminated the infection after 5 days of treatment, similarly to doxycycline
and ceftriaxone (Table 3). An issue with this standard model is that inhibition of growth
alone may be sufficient for substantial clearing of the infection, since the remaining cells
will be eliminated by the immune system. In order to better differentiate between
antibiotics with varying capabilities to kill and taking into account that not all human
patients will mount a robust immune response, we decided to use SCID (severe
combined immunodeficiency) mice as a model. SCID mice are deficient for both B and
T lymphocytes, which results in a higher B. burgdorferi burden (36).
In this new model, doxycycline failed to clear the infection in the majority of mice after
5 days of administration (Table 3). Both ceftriaxone and vancomycin cleared the
infection after two doses in the first day of administration based on analysis of single ear
punch cultivation (Table 2). To better compare the efficacy between vancomycin and
ceftriaxone, mice were sacrificed after only one or two doses and larger amounts of
tissues were collected. A whole ear of every mouse was used for cultivation analysis.
The spleen, bladder, and heart were collected for whole DNA extraction and qPCR
analysis. After 1 dose, in the ceftriaxone group, 5 out of 7 mice remained culture-
positive while in the vancomycin group 3 out of 7 mice were positive. After 2 doses, both
groups only had 1 culture-positive mouse (Table 4). B. burgdorferi DNA could still be
detected from some tissues based on qPCR of the flaB gene of culture-negative mice,ranging from few to hundreds of copies (Table 4). For example, after 2 doses of
ceftriaxone, B. burgdorferi could be detected in ear punch culture of only one of seven
mice. Of the culture negative mice, 5 were positive by PCR from all tissues and the sixth
mouse was positive for PCR only from the heart tissue. Similarly, after 2 doses of
vancomycin, only 1 out of 7 mice was culture-positive. However, of the culture negative
mice, 4 were positive by PCR from at least one tissue while 2 were PCR negative for all
tested tissues. These signatures may either represent live pathogens or components
that had not been cleared from tissues. Both two-way Anova (ceftriaxone groups vs
vancomycin groups) and t-tests (ceftriaxone vs vancomycin, 1 dose and 2 doses separately) were performed to compare the efficacies of ceftriaxone and vancomycin.
However, there was no statistically significant difference between treatments by the two
antibiotics (P > 0.05).
In the current study, we sought to identify antibiotics with good bactericidal properties,
and ideally, with an ability to kill B. burgdorferi persisters. All bacteria studied to date
form persister cells in vitro in response to antibiotic exposure. Our current
understanding of persisters is still limited, but considerable progress has been made in
recent years, providing both a link to chronic infections, and molecular mechanisms for
formation of these drug tolerant cells. High level persister mutants (hip) have been
identified in Pseudomonas aeruginosa from patients with cystic fibrosis (37), in clinical
isolates of M. tuberculosis (38) and in E. coli from patients with urinary tract infection
(39). We recently found that a stochastic decrease in ATP levels is the main mechanism
of persister formation in S. aureus (33) and E. coli (40). These species are unrelated,
cells of the pathogen. While mitomycin C is an FDA-approved drug, it is a toxic anti-
cancer agent, unlikely to be introduced as an antibiotic. We also serendipitously found
that β-lactams, including ceftriaxone, were able to kill the majority of cells in a stationary
phase culture. This was unexpected, since cell wall acting antibiotics have been known
to only act against rapidly growing bacteria (42). In the current study, we tested
additional cell wall acting compounds and found that vancomycin was better than
ceftriaxone in killing B. burgdorferi in vitro when killing was observed over a longer time
period than previously reported (20). Of note, B. burgdorferi does not develop
resistance to antibiotics, at least in part due to the lack of human-to-human transmission
of the pathogen. Probability of resistance development to vancomycin is particularly low
due to its action against a non-protein target (lipid II, precursor of peptidoglycan), and
would not be a concern for an infection caused by B. burgdorferi.
Vancomycin in combination with gemifloxacin completely eradicated a stationary
phase culture. We sought to determine the nature of this vulnerability to cell wall acting
antibiotics. Tracking incorporation of a fluorescently labeled d-alanine, we found that
peptidoglycan continues to be synthesized in non-growing stationary phase cells of B.
burgdorferi, accounting for this susceptibility paradox.
We attempted to test the relative efficacy of doxycycline, ceftriaxone, and
vancomycin in vivo, but found that the standard mouse model of Lyme disease is poorly
suited for this purpose. Doxycycline cleared the infection of WT mice after one day of
treatment, similarly to ceftriaxone and vancomycin. We then turned to immune-deficient
SCID mice and found that doxycycline failed to clear the infection even after 5 days of
treatment. In contrast, vancomycin and ceftriaxone both cleared the infection rapidly
even in the immunocompromised mice.IIt would be difficult justifying the use of intravenous antibiotics such as vancomycin and ceftriaxone for the early treatment of B. burgdorferi infection given the added risks
and difficulty in administration and monitoring compared with oral doxycycline. However,
it is tempting to speculate that persistence of bacteria or their products that have been
seen in animals after treatment may trigger ongoing inflammatory responses in some
patients resulting in PTLDS. It is unknown what factors predispose patients to the
development of PTLDS and why only some patients are susceptible. One hypothesis is
that patients with immunodeficiencies may fare poorly with drugs such as doxycycline,
similar to what was seen with our immunocompromised mice. Indeed, Strle et al have
shown that an inactivating mutation in TLR1 is associated with persistent arthritis in
patients with Lyme arthritis after antibiotic treatment (43). These patients may benefit
from early use of more bactericidal antibiotics that kill both growing and stationary
bacteria as studies of IV ceftriaxone administered after establishment of PTLDS have
not shown a benefit compared with placebo (12, 44, 45). Unfortunately, there is
currently no good test, genetic or otherwise, to identify patients at higher risk for
developing PTLDS a priori who may benefit from more aggressive therapy with
intravenous antibiotics. There are several markers reported that may be related to
PTLDS, such as increased immune response to VlsE membrane-proximal epitopes in
PTLDS patients (46), innate acute C-reactive protein (47), T-cell chemokine CCL19 (48).....-