Rapid antibiotic susceptibility phenotypic characterization of Staphylococcus aureus using automated microscopy of small numbers of cells

Abstract

Staphylococcus aureus remains a leading, virulent pathogen capable of expressing complex drug resistance that requires up to 2-4 days for laboratory analysis. In this study, we evaluate the ability of automated microscopy of immobilized live bacterial cells to differentiate susceptible from non-susceptible responses of S. aureus isolates (MRSA/MSSA, clindamycin resistance/susceptibility and VSSA/hVISA/VISA) to an antibiotic based on the characterization of as few as 10 growing clones after 4 h of growth, compared to overnight growth required for traditional culture based methods. Isolates included 131 characterized CDC isolates, 3 clinical isolates and reference strains. MRSA phenotype testing used 1 h of 1 μg/mL cefoxitin induction followed by 3 h of 6 μg/mL cefoxitin. Clindamycin susceptibility testing used 1h of induction by 0.1 μg/mL erythromycin followed by 3h of 0.5 μg/mL clindamycin. An automated microscopy system acquired time-lapse dark-field images, and then computed growth data for individual immobilized progenitor cells and their progeny clones while exposed to different test conditions. Results were compared to concurrent cefoxitin disk diffusion and D-test references. For CDC organisms, microscopy detected 77/77 MRSA phenotypes and 54/54 MSSA phenotypes, plus 53/56 clindamycin-resistant and 75/75 clindamycin susceptible strains. Automated microscopy was used to characterize heterogeneous and inducible resistance, and perform population analysis profiles. Microscopy-based hVISA population analysis profiles (PAPs) were included as an extended proof of concept, and successfully differentiated VSSA from hVISA and VISA phenotypes compared to plate-based PAP.

New and developing diagnostic technologies for urinary tract infections

“…The assay has been demonstrated to be successful at both pathogen identification and antimicrobial susceptibility assessment from blood culture 101 . Further preliminary tests indicate that this system is capable of direct detection of pathogens from patient blood, urine, and sputum samples 100,102,103 . The phenotypic measures used for pathogen identification are not precise, but the system is also amenable to process samples for species identification methods such as FISH.…”

“…Pathogen characterization can be completed in 1.5 hours, and introduction of antibiotics to the culture medium can be used to assess susceptibility, with results available in approximately 5 hours (REF. 100). The assay has been demonstrated to be successful at both pathogen identification and antimicrobial susceptibility assessment from blood culture 101 .…”

Current and emerging techniques for antibiotic susceptibility tests

“…With the increasing clinical demand for rapid AST, various new AST techniques based on optical imaging 18-20, micro-channel resonators 21-23 and other biosensors 24, 25 have been pursued. For example, optical detection of bacterial growth via the cell lengths and numbers 18, 20, 26, forward light scattering 25, and measuring vibrational amplitude changes of magnetic beads 24, 27, have been proposed. Micro-channel resonators have also been used to detect nanoscale fluctuations associated with bacteria growth 21.…”

A rapid antimicrobial susceptibility test based on single-cell morphological analysis

“…To overcome the limitations of conventional AST systems, researchers have developed various methods to observe bacterial division at early stages, in so-called rapid ASTs (11)(12)(13)(14)(15)(16)(17). Rapid ASTs have been designed to measure, for example, the number of bacteria in a microfluidic channel (11,(15)(16)(17); the rotation rate of magnetic beads, which is inversely proportional to bacterial mass (12,13); or the fluorescent signal from the metabolic activity of bacteria cells in droplets (14).…”