A microfluidic platform for rapid, stress-induced antibiotic susceptibility testing of Staphylococcus aureus

Abstract

The emergence and spread of bacterial resistance to ever increasing classes of antibiotics intensifies the need for fast phenotype-based clinical tests for determining antibiotic susceptibility. Standard susceptibility testing relies on the passive observation of bacterial growth inhibition in the presence of antibiotics. In this paper, we present a novel microfluidic platform for antibiotic susceptibility testing based on stress-activation of biosynthetic pathways that are the primary targets of antibiotics. We chose Staphylococcus aureus (S. aureus) as a model system due to its clinical importance, and we selected bacterial cell wall biosynthesis as the primary target of both stress and antibiotic. Enzymatic and mechanical stresses were used to damage the bacterial cell wall, and a β-lactam antibiotic interfered with the repair process, resulting in rapid cell death of strains that harbor no resistance mechanism. In contrast, resistant bacteria remained viable under the assay conditions. Bacteria, covalently-bound to the bottom of the microfluidic channel, were subjected to mechanical shear stress created by flowing culture media through the microfluidic channel and to enzymatic stress with sub-inhibitory concentrations of the bactericidal agent lysostaphin. Bacterial cell death was monitored via fluorescence using the Sytox Green dead cell stain, and rates of killing were measured for the bacterial samples in the presence and absence of oxacillin. Using model susceptible (Sanger 476) and resistant (MW2) S. aureus strains, a metric was established to separate susceptible and resistant staphylococci based on normalized fluorescence values after 60 min of exposure to stress and antibiotic. Because this ground-breaking approach is not based on standard methodology, it circumvents the need for minimum inhibitory concentration (MIC) measurements and long wait times. We demonstrate the successful development of a rapid microfluidic-based and stress-activated antibiotic susceptibility test by correctly designating the phenotypes of 16 additional clinically relevant S. aureus strains in a blinded study. In addition to future clinical utility, this method has great potential for studying the effects of various stresses on bacteria and their antibiotic susceptibility.

Fig. 1

Cartoon showing the cross-sectional view of a single microfluidic channel. Bacterial cells (orange) are covalently bound to the bottom of the microfluidic channel. Under shear stress (τ) with or without enzymatic stress (green circles), susceptible cells die over time (lighter shade) in the presence of the antibiotic (red pentagons). The fluorescent dye (yellow diamonds) can only permeate the cell membrane when the bacteria die, producing fluorescence when it binds to DNA.

Fig. 2

Schematic drawing of the microfluidic channel geometry (not to scale).

Fig. 3

Microfluidic flow cell assembly (exploded view). The top metal plate provides connectivity with the outside fluidics. The top glass window provides an even sealing pressure on top of the PDMS-layer, while the bottom sealing plate provides sealing pressure to the bottom of the coated glass slide.

Fig. 4

Analyzed phase contrast and fluorescent fields at 60× magnification at T=59 min for channels with or without (control) antibiotic. Automated bacterial counts in phase contrast (red dots) and fluorescence (white speckles) images are shown in the top left corner of each panel.

Fig. 5

Normalized cell death percentage in the presence of 50 μg/mL oxacillin as a function of time for Sanger 476 (oxacillin MIC: 0.5 μg/mL, MSSA, blue) and MW2 (oxacillin MIC: 16 μg/mL, MRSA, red). Data are shown for mechanical stress only (Mech, circles) and for combined mechanical and enzymatic stress (Mech/Enz, triangles). The DP_{norm_max} for each experiment is highlighted with a black square.

Fig. 6

Distribution of DP_{norm_max} values for Sanger 476 (open, blue) and MW2 (solid, red) in the presence of 0.7 ng/mL lysostaphin and either 10 μg/mL (circles) or 50 μg/mL (triangles) oxacillin. Each data point is the result of a single experiment (i.e. 34 replicates of the Sanger 476 at 10 μg/mL) and is slightly offset on the x-axis for clarity. Note: data are plotted on a log-scale. Arrows and letters indicate susceptibility zones with 0.5% and 1% line borders; S=susceptible; R=resistant. Zone of indeterminate susceptibility is highlighted in gray.

Fig. 7

Average DP_{norm_max} values measured during the blinded study for various strains in the presence of mechanical and enzymatic stress at 50 μg/mL oxacillin. Susceptible strains (blue triangles) and resistant strains (red circles) are separated into groups. Each strain was measured three times, with the exception of Newman (N=6). The error bars show a single standard deviation. Note: data are plotted on a log-scale.