Traditional antimicrobial susceptibility tests include the d

Traditional antimicrobial susceptibility tests include the disk diffusion test, E-test gradient diffusion test and broth dilution susceptibility tests. Although the tests are robust, they each rely on the growth of the bacteria to a dense culture while being exposed to the antibiotic panel. Consequently, these methods require 24–48h to isolate the bacterium from the patient in pure culture and another 24–72h to complete susceptibility testing (Goff et al., 2012). Due to the long time required to deliver test results compared to the tempo of infection, patients are often treated “empirically” with broader-spectrum regimens under the assumption that the infection could be drug-resistant. To improve susceptibility testing times there are currently a few rapid platforms gaining acceptance in the clinical laboratory: polymerase chain reaction (PCR), poly-nucleic order NVP-TNKS656 fluorescence in situ hybridization (PNA-FISH), and nanosphere hybridization. PCR method detects coding sequences of known resistance genes while PNA-FISH identifies species and/or resistance by hybridizing synthetic oligo-nucleotide fluorescence-labeled probes to species-specific ribosomal RNA and mRNA. Similarly, nanosphere hybridization uses nanoparticle probes to detect DNA, RNA or protein targets. Rapid susceptibility methods can improve patient outcomes and decrease cost of care. In a study comparing PCR to traditional methods, time required for identification of methicillin-susceptible S. aureus (MSSA) versus methicillin-resistant S. aureus (MRSA) was reduced by 1.7days, cost per patient decreased from $69,737 to $48,350, and outcomes improved with 6.2 fewer days in the hospital and 20% lower mortality (Bauer et al., 2010). Unfortunately, both these current rapid methods depend on a priori knowledge of resistance genes. S. aureus is a prevalent Gram-positive human pathogen that is a leading cause of bacteremia, pneumonia, skin/soft tissue infections and endocarditis worldwide (Tong et al., 2015). Asymptomatic colonization of up to 30% of healthy individuals allows for the continual transmission and proliferation of this pathogen (Wertheim et al., 2005). S. aureus is responsible for community-acquired (CA-) and hospital-acquired (HA-) infections in both healthy and immune-compromised individuals, and numerous lineages of MRSA including the USA300 clone have spread throughout the US and internationally (Mediavilla et al., 2012). Currently, rPCR is used by many clinical laboratories to detect the mecA gene, which confers beta-lactam resistance in S. aureus by encoding the low-affinity penicillin-binding protein, PBP2a (Pinho et al., 2001). Since MSSA lack mecA, detection of this gene is considered a gold standard for molecular identification of MRSA. However, occurrence of oxacillin-susceptible mecA+ MRSA strains (Hososaka et al., 2007) and emergence of a mecA variant, mecC, which encodes a protein with <63% AA identity to PBP2a (Laurent et al., 2012; García-Álvarez et al., 2011), highlight limitations in tests that detect only the presence of this gene. In recent years, rapid phenotypic susceptibility assays for S. aureus have been proposed, but many of these assays are based solely on cell lysis and/or growth (Price et al., 2014; Kalashnikov et al., 2012; Choi et al., 2013; Kinnunen et al., 2012; Sinn et al., 2012). Recently, an imaging-based single-cell morphological analysis (SCMA) focused on early morphological changes in response to antibiotic exposure sought to determine susceptibility of five different bacterial pathogens to a variety of antibiotics within 3–4h (Choi et al., 2014). Despite the impressive scale and scope of this study, S. aureus antibiotic susceptibility determination in this assay depended solely on cell division, similar to standard broth microdilution assays, and had an error rate of 5.4% compared to conventional testing. Recently, we demonstrated the utility of bacterial cytological profiling (BCP) to determine specific antibacterial mechanisms of action (Lamsa et al., 2012; Nonejuie et al., 2013). BCP is based on the observation that treatment of a diverse range of bacterial species with different antibiotics yield unique and quantifiable changes in cytological profiles. BCP profiles are comprised of a multitude of parameters including DNA and cell size and shape and dye intensity on a single-cell level for hundreds of cells in a single run. Here, we applied BCP as a rapid method for determining the antibiotic susceptibility of S. aureus clinical isolates obtained from patient samples. With BCP, 100% (n=30) of blind test isolates were correctly categorize as MRSA and MSSA within 1h, and the MRSA strains were further subdivided into two groups that correlated with increased susceptibility to combinatorial drug therapy. Likewise, daptomycin susceptible (DS) and daptomycin non-susceptible (DNS) S. aureus strains (n=20) were correctly classified after 30min of antibiotic treatment. BCP provides a flexible alternative to current rapid susceptibility testing methods as it doesn\'t require prior knowledge of resistance genes and can be applied broadly to different antibiotics and species.