For decades, the routine treatment for Lyme disease has been standard antibiotics, which usually kill off the infection.
But for up to 20% of people with the tick-borne illness, the antibiotics don’t work, and lingering symptoms of muscle pain, fatigue and cognitive impairment can continue for years-sometimes indefinitely.
A new Stanford Medicine study in lab dishes and mice provides evidence that the drug azlocillin completely kills off the disease-causing bacteria Borrelia burgdorferi at the onset of the illness.
The study suggests it could also be effective for treating patients infected with drug-tolerant bacteria that may cause lingering symptoms.
“This compound is just amazing,” said Jayakumar Rajadas, Ph.D., assistant professor of medicine and director of the Biomaterials and Advanced Drug Delivery Laboratory at the Stanford School of Medicine.
“It clears the infection without a lot of side effects. We are hoping to repurpose it as an oral treatment for Lyme disease.”
Rajadas is the senior author of the study, which was published online March 2 in Scientific Reports. The lead author is research associate Venkata Raveendra Pothineni, Ph.D.
“We have been screening potential drugs for six years,” Pothineni said. “We’ve screened almost 8,000 chemical compounds. We have tested 50 molecules in the dish. The most effective and safest molecules were tested in animal models.
Along the way, I’ve met many people suffering with this horrible, lingering disease. Our main goal is to find the best compound for treating patients and stop this disease.”
Hunting for alternative drug
Frustrated by the lack of treatment options for Lyme disease patients with lingering symptoms, Rajadas and his team began hunting for a better alternative in 2011. In 2016, they published a study in Drug Design, Development and Therapy that listed 20 chemical compounds, from about 4,000, that were most effective at killing the infection in mice. All 20 had been approved by the Food and Drug Administration for various uses. One, for instance, is used to treat alcohol abuse disorder.
In this most recent study, azlocillin, one of the top-20 contenders, was shown to eclipse a total of 7,450 compounds because it is more effective in killing B. burgdorferi and causes fewer side effects.
Lyme disease affects more than 300,000 people annually, according to the Centers for Disease Control and Prevention. It can affect various organs, including the brain, skin, heart, joints and nervous system, and cause heart problems and arthritis if untreated.
Symptoms include fever, headaches, chills, and muscle and joint pain.
Traditional antibiotics, such as doxycycline, are effective as an early course of treatment for the infection in the majority of patients, but it remains unclear why these drugs fail to treat 10% to 20% of patients, Rajadas said.
“Some researchers think this may be due to drug-tolerant bacteria living in the body and continuing to cause disease,” said Rajadas, who is also a member of the Lyme Disease Working Group at Stanford.
“Others believe it’s an immune disorder caused by bacteria during the first exposure, which causes a perpetual inflammation condition. Whatever the cause, the pain for patients is still very real.”
Azlocillin comes out on top
The drug, which is not on the market, was tested in mouse models of Lyme disease at seven-day, 14-day and 21-day intervals and found to eliminate the infection.
For the first time, azlocillin was also shown to be effective in killing drug-tolerant forms of B. burgdorferi in lab dishes, indicating that it may work as a therapy for lingering symptoms of Lyme disease.
Pothineni and Rajadas have patented the compound for the treatment of Lyme disease and are working with a company to develop an oral form of the drug. Researchers plan to conduct a clinical trial.
Rajadas is also a professor of bioengineering and therapeutic sciences at the University of California-San Francisco.
Bacterial strains and culture
B. burgdorferi s.s. strains CA4 and CA8 originated biologically from Ixodes pacificus ticks, USA. The bacterial strains were generously provided by Dr Robert Lane, University of California, Berkeley, CA, USA. These strains are infectious low passage numbers that were cultured in Barbour-Stoenner-Kelly II (BSK-II) complete medium, with 6% rabbit serum (Sigma-Aldrich, St Louis, MO, USA). We have chosen these strains because these are infectious and prevalent in California, USA. The cultures were grown in sterile 50 mL Falcon tubes (Corning Incorporated, Corning, NY, USA) and incubated at 33°C. All culture media were sterilized with 0.2 µM filter units (EMD Millipore, Billerica, MA, USA). The B. burgdorferi cultures were grown for 7–10 days to reach the stationary phase with cell density >108/mL for performing all the assays. For HTS drug screening, 7–10-day-old stationary-phase B. burgdorferi cultures were transferred to 384-well culture microplates.
Drugs and drug libraries
All the information regarding purchase, solubility, and stock solutions of drugs used in this study have been provided in Table 1. All the drug stocks were filter sterilized with 0.2 µM filter units. The FDA-approved drug libraries, such as the LOPAC1280, the NIHCC, the Microsource Spectrum, and the Biomol FDA (now Enzo Life Sciences) were acquired from High-Throughput Bioscience Center, Stanford University. All the library stocks were maintained in dimethyl sulfoxide solutions at 10 mM compound concentrations. Plate-to-plate dilutions were performed in 384-well plates using an Evolution P3 system equipped with a 384-well head.
Details of drugs used
|Serial number||Name||Supplier||Solubility||Stock solution (mM)|
|1||Tetraethylthiuram disulfide||Cayman Chemical||DMSO||10|
|2||Doxorubicin hydrochloride||Cayman Chemical||Water||10|
|4||Cefotaxime acid||Cayman Chemical||DMSO||20|
|5||Cefazolin sodium||Cayman Chemical||Water||10|
|6||Epirubicin hydrochloride||Cayman Chemical||Water||10|
|7||Erythromycin ethylsuccinate||Santa Cruz Biotech||DMSO||20|
|8||A-23187 calcimycin||Cayman Chemical||DMSO||10|
|11||Gambogic acid||Cayman Chemical||DMSO||20|
|12||Cephalothin sodium||Santa Cruz Biotech||Water||10|
|14||Ticarcillin disodium||Santa Cruz Biotech||Water||20|
|16||Moxifloxacin hydrochloride||Santa Cruz Biotech||Water||20|
|17||Linezolide||Santa Cruz Biotech||Water||15|
|19||Tosufloxacin tosylate||Santa Cruz Biotech||DMSO||10|
|20||Azlocillin sodium||Santa Cruz Biotech||Water||10|
Abbreviation: DMSO, dimethyl sulfoxide.
HTS of chemical libraries with B. burgdorferi persisters
To identify the effect of chemical compounds on B. burgdorferi stationary-phase cultures qualitatively, HTS was performed using the following procedure. A total of 50 µL of BSK-II medium was added to white 384-well Corning plates (Corning Incorporated) using the Matrix Wellmate, and ~100 nL of each compound from the stock solution was added using the pin tool in the Staccato System (CaliperLS, Caliper Life Sciences Inc, Alameda, CA) to 1–22 columns. The last two columns of the 384 wells were left for culturing the controls. We screened one compound per well in a 384-well microplate format. These are screened in a 7-point titration ranging from 25 µM, 12.5 µM, 7.25 µM, 3.625 µM, 1.81 µM, 0.9 µM, and 0.45 µM. To these plates, 25 µL of 106/mL B. burgdorferi stationary-phase cultures were added using the Multidrop dispenser. Then the plates were incubated at 33°C for 96 hours in a humidified CO2 incubator (Forma Scientific, Waltham, MA, USA) having 5% CO2 and 95% air. After 96 hours, 25 µL of BacTiter-Glo reagent (Promega Corporation, Fitchburg, WI, USA) was added to the plates using the Multidrop dispenser. The plates were shaken for 2 minutes and incubated at room temperature for 5 minutes. Finally, the luminescence was measured using a Flexstation 3 (Molecular Devices LLC, Sunnyvale, CA, USA) (500 ms/well). The data were analyzed using the Assay Explorer software. Hits were identified as compounds that resulted in a decrease in the luciferase signal compared to controls with no compound.
Determination of MIC and MBC
The MIC of the small molecules identified through screening was determined by culturing 106/mL Borrelia in BSK-II medium with different concentrations (0.3–160 µM) of drugs. For MIC, 1 mL of cultures with respective drugs are grown in 48-well plates in triplicates, wrapped with paraffin film and placed for 72 hours at 33°C in a humidified CO2 incubator (Forma Scientific) having 5% CO2 and 95% air.23
Cell proliferation was assessed using the bacterial counting chamber (Petroff-Hausser Counter, Horsham, PA, USA) by phase-contrast microscopy. At the same time, cell proliferation was also assessed using the BacTiter-Glo™ assay.
The counting was performed in all the 25 squares of the central grid. The BacTiter-Glo™ assay is performed by mixing 100 µL of culture in each well with 100 µL of BacTiter-Glo™ reagent (Promega Corporation).
Then, the assay is performed according to the manufacturer’s instructions. Luminescence was measured using a Flexstation 3 microplate reader at luminescence 500 milliseconds.33
For determining MBC, 20 µL of the 106/mL Borrelia cultures grown in BSK-II medium for 72 hours at different drug concentrations were added to the fresh BSK-II medium and subcultured for 3 weeks.23,34–36 After a 3 week incubation period, the samples were observed microscopically for motile spirochetes in the culture. The MIC and MBC determinations were done thrice independently.
Time kill studies
Time kill studies were performed with borrelial isolate CA8 (B. burgdorferi s.s.) with azlocillin sodium and cefotaxime acid. To determine the rate of antimicrobial activity, 106/mL Borrelia cultures were grown in BSK-II medium with drugs at different concentrations. BSK-II medium with no drugs was used as a control.
The antibacterial activity was determined by counting bacteria at 24 hours, 48 hours, and 72 hours. The experiment was done once with triplicates.34
Development of BacTiter-Glo™ assay compatible to HTS
B. burgdorferi grows very slowly, typically taking from 12 hours to 18 hours to replicate. Due to this slow growth, it is difficult to measure B. burgdorferi culture quantitatively using direct optical density. To measure the bacterial viability and antibiotic susceptibility of B. burgdorferi, several rapid colorimetric assays, such as fluorogenic dye SYTO 9 (LIVE/DEAD) assay, Sytox Green/Hoechst 33342 assay, and SYBR Green I/PI assay, have been developed.7,28,37
It was reported that SYBR Green I/PI assay can only detect accurately the live/dead ratio of 105 cells/mL.37 In parallel to these assays, we have developed a highly sensitive BacTiter-Glo™ assay, which has been optimized for 384-well plate.33
It is a one-step, straightforward method to assess bacterial viability by measuring ATP from the given sample. As it is a single-step assay, BacTiter-Glo™ assay can screen drugs quickly and efficiently in a large scale. We have recently reported that the BacTiter-Glo™ assay is a very sensitive assay that can reliably detect signal in the range of minimum ten Borrelia cells in phosphate-buffered saline and 7×103 in BSK-II medium.33
The BacTiter-Glo™ assay can only detect cells that can generate ATP. It has not been tested whether BacTiter-Glo™ assay can detect nongrowing Borrelia, which may produce low levels of ATP. Due to the advantage of detecting low number of bacteria (7×103) by this method, false-positive candidates are eliminated in the HTS.
The BacTiter-Glo assay was validated by calculating Z′ values with the Assay Explorer software for CA4 and CA8 strains, which were >0.6, considered to be a good value to perform an HTS assay. The data indicate that the BacTiter-Glo™ assay provides a one-step, straightforward method to quantify B. burgdorferi with good sensitivity and dynamic range.
High-throughput primary screening of chemical libraries with B. burgdorferi persisters to identify potent drugs
To identify safe and effective molecules for Lyme disease treatment, repurposing FDA-approved drug molecules might be a fast and viable alternative in developing novel borrelicidal compounds. To achieve this, we utilized the BacTiter-Glo™ assay system. All the screening of drugs was done by stationary-phase CA8 borrelial cultures with cell density >108/mL, which are grown in BSK-II medium for 7–10 days.
During screening, the last two rows in the 384-well plate were taken as control by not adding any drugs. By using the HTS platform, we have screened 4,366 chemical compounds representing different libraries, including the Sigma LOPAC (1,280 compounds), the NIHCC (446 compounds), the Microsource Spectrum (2,000 compounds), and the Biomol FDA (640 compounds). The screening was repeated for seven times with a titration range from 0.45 µM to 25 µM to confirm the reproducibility.
That means each drug is tested at different concentrations in seven different plates. By successfully screening these libraries, we have identified ~150 hit molecules that showed the inhibition of bacterial growth >90% compared to the control. Out of these 150 hit molecules, 101 (67.3%) small molecules are FDA-approved compounds.
Based on the results from the primary screening, we chose the top 20 candidates and reconfirmed these by secondary screening with BacTiter-Glo™ assay. These candidates were chosen based on the >95% inhibition of bacteria in primary screening, FDA approval, and safety of the compounds (Table 2).
The doxycycline, which is one of the currently prescribed drugs inhibited 94.14% of borrelial growth compared to the control (no drug), which is less than the percentage of inhibition of the top 20 candidates (Table 2). Percentage inhibition was also one of the criteria for selecting top 20 candidates for testing.
The results of the remaining 130 compounds are shown in Table S1. Of the Borrelia cultured with some of the drugs, the viability was evaluated with BacTiter-Glo™ assay as shown in Figure 1. In Figure 1A, tetraethylthiuram disulfide shows the complete borrelial cell inhibition at 1.25 µM, while epirubicin hydrochloride and doxorubicin hydrochloride are uniformly effective against B. burgdorferi at 0.625 µM.
Vehicle control (dimethyl sulfoxide) did not show any significant effect on the cell survival. Drugs azlocillin and cephalothin showed ~99% efficacy at 0.31 µM (Figure 1B). After confirming from secondary screening, the MIC and MBC values were determined by microdilution method for all the potential candidates.
Structure and activity of Top 20 hits against B. burgdorferi
|Serial number||Drugs||Structure||% inhibition||MIC|
|Control (no drug)||–||0|
|9||Gramicidin||Formyl-L-X-Gly-L-Ala-D-Leu-L-Ala-D-Val-L-Val-D-Val-L-Trp-D-Leu-L-Y-D-Leu-L-Trp-D-Leu-L-Trp-ethanolamine X = Val/Ile, Y = Trp/Phe/Tyr||98.67||1.25||2.5|
Abbreviations: MBC, minimum bactericidal concentration; MIC, minimum inhibitory concentration.
Determinations of MIC and MBC values
To confirm the efficacy of the screening, MIC and MBC values were evaluated. The MIC is determined as the lowest concentration at which no motile spirochete is observed by microscopy. Of the top 20 candidates evaluated, the MIC values of epirubicin hydrochloride and doxorubicin hydrochloride are <1 µM. For the drugs cefazolin sodium, gramicidin, azlocillin sodium, cefotaxime, and cephalothin sodium, tetraethylthiuram disulfide, and linezolid, the MIC values are ≤3 µM. For the remaining drugs, the MIC values are ≥3 µM. The MBC was determined by subculturing 20 µL of the Borrelia cultures grown at different drug concentrations in fresh BSK-II medium for 21 days.23,34 The MBC was determined when no spirochete was observed microscopically in the culture. Of the top 20 compounds, the lowest MBC values (<1.5 µM) were observed for epirubicin hydrochloride and doxorubicin hydrochloride. The drugs gramicidin, azlocillin sodium, leucomycin, cefotaxime, idarubicin, and tetraethylthiuram disulfide show the MBC values of ≤10 µM. All the remaining drugs show the MBC values of >10 µM. For the cephalothin sodium, the MIC value is very low, but the MBC value is >80 µM, and for tosufloxacin tosylate, both the MIC and MBC values are very high. The compounds doxorubicin, cephalothin, ticarcillin, and cefdinir were also reported in the HTS performed by Feng et al.29,38
Time kill studies
To determine the rate of antimicrobial activity of azlocillin sodium and cefotaxime acid, CA8 strain of Borrelia was exposed to different concentrations (0.625 µM, 1.25 µM, 2.5 µM, and 5 µM) of each of the drugs. The initial Borrelia inoculum did not even decrease 1-log10-unit at concentrations 0.625 µM and 1.25 µM for azlocillin sodium (Figure 2A). For cefotaxime acid at concentration 1.25 µM, the microbial growth is decreased 1-log10-unit but not at 0.625 µM (Figure 2B). Both azlocillin sodium and cefotaxime acid reduced morphologically intact motile cells to 3-log10-unit (99.9%) between 48 hours and 72 hours at concentrations 2.5 µM and 5 µM. These specified concentrations of 2.5 µM and 5 µM are MIC values and twice the MIC values. In control, the Borrelia growth increased to 7-log10-unit.
More information: Venkata Raveendra Pothineni et al. Azlocillin can be the potential drug candidate against drug-tolerant Borrelia burgdorferi sensu stricto JLB31, Scientific Reports (2020). DOI: 10.1038/s41598-020-59600-4