Simultaneous and quantitative monitoring of co-cultured Pseudomonas aeruginosa and Staphylococcus aureus with antibiotics on a diffusometric platform

Successful treatments against bacterial infections depend on antimicrobial susceptibility testing (AST). However, conventional AST requires more than 24 h to obtain an outcome, thereby contributing to high patient mortality. An antibiotic therapy based on experiences is therefore necessary for saving lives and escalating the emergence of multidrug-resistant pathogens. Accordingly, a fast and effective drug screen is necessary for the appropriate administration of antibiotics. The mixed pathogenic nature of infectious diseases emphasizes the need to develop an assay system for polymicrobial infections. On this basis, we present a novel technique for simultaneous and quantitative monitoring of co-cultured microorganisms by coupling optical diffusometry with bead-based immunoassays. This simple integration simultaneously achieves a rapid AST analysis for two pathogens. Triple color particles were simultaneously recorded and subsequently analyzed by functionalizing different fluorescent color particles with dissimilar pathogen-specific antibodies. Results suggested that the effect of the antibiotic, gentamicin, on co-cultured Pseudomonas aeruginosa and Staphylococcus aureus was effectively distinguished by the proposed technique. This study revealed a multiplexed and time-saving (within 2 h) platform with a small sample volume (~0.5 μL) and a low initial bacterial count (50 CFU per droplet, ~105 CFU/mL) for continuously monitoring the growth of co-cultured microorganisms. This technique provides insights into timely therapies against polymicrobial diseases in the near future.


Simultaneous detection with dual color particles
Green fluorescent particles were coated with anti-TNF-α Ab and served as a reference probe and orange fluorescent particles coated with anti-P. aeruginosa Ab to capture P.
aeruginosa. The bright field image provided visual evidence showing the successful binding between the mixed bacteria and their corresponding particles (green: environmental reference; orange: P. aeruginosa) (Fig. S3). This result proved that simultaneous detection of multipathogens could be achieved by immunoassays.

Bacterial quantification with mono-colored particles
The UV-sterilized P. aeruginosa was incubated with the Ab-functionalized particles. The diffusivity of particles decreased monotonically according to the number of dead bacteria they were attached to (Fig. S4A). A similar phenomenon was observed in particles bound with nonmotile bacteria, S. aureus, in that a high number of bacteria always resulted in low diffusivity (Fig. S4B). Overall, the experimental data showed favorable agreements with the predicted curves of 2-μm particles (Figs S4A and B, the blue and red solid lines).

Bacterial quantification with dual color particles
With dual color particles that were composed of equal amounts of anti-S. aureus and anti-TNF-α Ab-functionalized particles, the diffusivity values of the two functionalized particles at different S. aureus concentrations were recorded simultaneously. The diffusivity values of anti-S. aureus Ab-functionalized particles divided by those of anti-TNF-α Ab-functionalized particles were compared using the equivalent diameter theoretical model. The results showed favorable agreement with the predicted curves of 2-μm particles (Fig. S5).

Preventing sedimentation by flipping for continuous monitoring
Although the density of particles was close to water ( = 1.05 g/cm 3 ), sedimentation could still disturb its diffusivity when the particles were near the chip walls. For continuously monitoring the growth of polymicrobial infection pathogens over a long timescale (at least 2 h), sample droplets loaded in chips coated with or without BSA were flipped at rate of 0, 1, and 2 min -1 over 2 h. Numbers of particles at middle and near-wall plates were calculated. Results showed that flipping a chip with BSA coating at rate of 1 and 2 min -1 could effectively avoid particle sedimentation on the near-wall plate (Fig. S6).

Rapid antimicrobial susceptibility testing by diffusometry
An AST process was assessed by measuring the growth of P. aeruginosa and S. aureus in TSB with antibiotics. The initial density of P. aeruginosa and S. aureus was 10 5 CFU/mL according to the guideline of Clinical and Laboratory Standards Institute. The density ratio of the bacteria and the Ab-functionalized particles was maintained at 1:1. After 1 h of incubation, the bacteria were respectively mixed with antibiotics at different concentrations in the TSB medium at 37 °C and 800 rpm for 2 h. Particles images were recorded every 20 min with a 20× objective to monitor the bacterial activity.
In the P. aeruginosa mono-cultured condition, the diffusivity changes of the particles in the control group and in the group of 0.02 μg/mL gentamicin all exhibit increases in the first 20 min, followed by constant decreases. By contrast, the diffusivity changes of the particles in the presence of 0.5 and 2 μg/mL gentamicin exhibit slight decreases in the first 60 min and then show no change afterward (Fig. S7A).
In the S. aureus mono-cultured condition, the diffusivity changes in the functionalized particles in the control group and in the group of 0.1 μg/mL cefapime only displayed a constant decline, whereas the diffusivity of the particles in the presence of 1 and 4 μg/mL cefapime did not change (Fig. S7B). is the regression line slope of particles measured at 0 min. * p < 0.05, and ** p < 0.01.

Antimicrobial susceptibility testing by broth microdilution
Conventional AST was conducted to measure the growth of co-cultured P. aeruginosa and S. aureus (10 5 CFU/mL each) in TSB with 0, 0.2, 0.4, and 2 μg/mL gentamicin, according to the guideline of Clinical and Laboratory Standards Institute. Briefly, 100 μL mixed bacteria suspension was distributed to the 96-well plate. The mixed bacteria was incubated with 0, 0.2, 0.4, and 2 μg/mL gentamicin at 37°C for 48 h. The turbidity of each groups was determined by using a plate reader (530 nm) at 24 h and 48 h. Fig. S8. Broth microdilution. Co-cultured P. aeruginosa and S. aureus (10 5 CFU/mL each) in TSB with 0, 0.2, 0.4, and 2 μg/mL gentamicin were incubated at 37°C for 48 h. A plate reader (530 nm) was applied to determine the turbidity of each groups at 24 h and 48 h.