Differentiating methicillin resistant and susceptible Staphylococcus aureus from ocular infections using photoacoustic labeling

Abstract

Introduction: Antibiotic resistance in bacterial species constitutes a growing problem in the clinical management of infections. Not only does it limit therapeutic options, but application of ineffective antibiotics allows resistant species to progress prior to prescribing more effective treatment to patients. Methicillin resistance in Staphylococcus aureus is a major problem in clinical infections as it is the most common hospital acquired infection.

Methods: We developed a photoacoustic flow cytometer using engineered bacteriophage as probes for rapid determination of methicillin resistance in Staphylococcus aureus with thirteen clinical samples obtained from keratitis patients. This method irradiates cells under flow with 532 nm laser light and selectively generates acoustic waves in labeled bacterial cells, thus enabling detection and enumeration of them. Staphylococcus aureus isolates were classified from culture isolation as either methicillin resistant or susceptible using cefoxitin disk diffusion testing. The photoacoustic method enumerates bacterial cells before and after treatment with antibiotics. Decreasing counts of bacteria after treatment indicate susceptible strains. We quantified the bacterial cells in the treated and untreated samples.

Results: Using k-means clustering on the data, we achieved 100% concordance with the classification of Staphylococcus aureus resistance using culture.

Discussion: Photoacoustics can be used to differentiate methicillin resistant and susceptible strains of bacteria from ocular infections. This method may be generalized to other bacterial species using appropriate bacteriophages and testing for resistance using other antibiotics.

Keywords: flow cytometry; k-means; microbial; optoacoustics; ultrasonic.

Figure 1

DET7 bacteriophage showing polyhedral capsid, tail sheath, and tail fibers with recognition proteins that attach to specific bacteria.

Figure 2

(Left) Schematic showing photoacoustic setup used for testing S. aureus samples. (Right) The actual flow chamber is shown.

Figure 3

The flow chamber has alternating droplets of cell suspension and mineral oil so that detected cells are sequestered for downstream capture.

Figure 4

Posterior distributions for photoacoustic test using (A) uniform conjugate prior and (B) high accuracy conjugate prior.