Whole animal automated platform for drug discovery against multi-drug resistant Staphylococcus aureus

Abstract

Staphylococcus aureus, the leading cause of hospital-acquired infections in the United States, is also pathogenic to the model nematode Caenorhabditis elegans. The C. elegans-S. aureus infection model was previously carried out on solid agar plates where the bacteriovorous C. elegans feeds on a lawn of S. aureus. However, agar-based assays are not amenable to large scale screens for antibacterial compounds. We have developed a high throughput liquid screening assay that uses robotic instrumentation to dispense a precise amount of methicillin resistant S. aureus (MRSA) and worms in 384-well assay plates, followed by automated microscopy and image analysis. In validation of the liquid assay, an MRSA cell wall defective mutant, MW2ΔtarO, which is attenuated for killing in the agar-based assay, was found to be less virulent in the liquid assay. This robust assay with a Z'-factor consistently greater than 0.5 was utilized to screen the Biomol 4 compound library consisting of 640 small molecules with well characterized bioactivities. As proof of principle, 27 of the 30 clinically used antibiotics present in the library conferred increased C. elegans survival and were identified as hits in the screen. Surprisingly, the antihelminthic drug closantel was also identified as a hit in the screen. In further studies, we confirmed the anti-staphylococcal activity of closantel against vancomycin-resistant S. aureus isolates and other Gram-positive bacteria. The liquid C. elegans-S. aureus assay described here allows screening for anti-staphylococcal compounds that are not toxic to the host.

Figure 1. Flowchart representing the time line and work flow of the C. elegans-MRSA high throughput screening assay.

Figure 2. Worm survival quantification using CellProfiler.

Worms in 384-well plates were incubated with Sytox Orange, which specifically stains dead worms. The results of several processing steps using CellProfiler are shown. The total area of fluorescent and bright field worms are measured and worm survival in each well is calculated as a percentage. Top row: Raw fluorescent Sytox Orange and bright field images of an untreated and an antibiotic-treated well. Middle row: Correction for uneven illumination of the bright field images. Cropping of Sytox images so that total fluorescence measurements are only made within worm areas determined by the bright field images. Bottom row: Thresholding, identifying worms and filtering for object size.

Figure 3. Optimization of starting bacterial concentration used in the infection assay.

Worms display a dose dependent susceptibility to the S. aureus MW2 starting inoculum.

Figure 4. C. elegans-MRSA liquid infection assay in 384-well plates.

A) Assay plates were co-inoculated with nematodes, bacteria and either DMSO (negative control) or vancomycin (10 µg/ml, positive control). The plates were incubated at 25°C for 5 days, washed to remove residual bacteria and imaged. The tiled image was constructed from TRITC fluorescent images of each well from a 384-well plate. B) Worm survival was significantly enhanced in wells treated with vancomycin.

Figure 5. A cell wall defective MRSA strain displays attenuated killing of C. elegans in the liquid infection assay.

Survival of worms infected with either MW2 or MW2ΔtarO was assayed under the same assay conditions. MW2ΔtarO was significantly attenuated in killing compared to the wild type MW2 strain in the DMSO wells. Error bars represent standard error.

Figure 6. Sytox staining of assay plate.

Tiled image of an example assay plate constructed from Sytox fluorescence images. White boxes indicate compounds that enhanced survival of infected worms with Z score greater than 2, the red box indicates DMSO control wells, and the green box indicates vancomycin positive control wells.

Figure 7. Closantel has a low in vitro MIC against VRSA.

A) Structure of closantel. B) In vitro antimicrobial activity of closantel was compared with vancomycin against the vancomycin resistant strain VRS1.