Rapid Differentiation of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus by Flow Cytometry after Brief Antibiotic Exposure

Abstract

We noticed that methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates yielded side-scatter (SSC) and fluorescence intensity (FI) differences on flow cytometry (FCM) following incubation in oxacillin broth. The purpose of this study was to determine whether MRSA and MSSA could be reliably differentiated by FCM. S. aureus isolates were incubated in oxacillin-containing MuellerHinton broth, stained using the FASTEST total viable organisms kit, and analyzed by FCM in the MicroPRO instrument. SSC versus FI were examined, and gates 1 and 2 were defined to encompass the majority of MSSA and MRSA signal events, respectively. A count ratio (CR) was defined as the ratio of counts in gate 2 to those in gate 1. Initially, 33 isolates were tested after 4 h of incubation for proof-of-concept. Twenty others were then tested after incubation intervals ranging from 30 min to 4 h to determine the earliest possible time for differentiation. Next, 100 separate isolates were tested to determine the best CR cutoff value. Finally, the CR was validated by using an independent cohort of 121 isolates. We noted that MRSA isolates had higher SSC and FI readings than did MSSA isolates after 2 h of incubation. The receiver-operator characteristics curve showed that a CR cutoff of 0.0445 reliably differentiated MRSA from MSSA. In the validation cohort, this cutoff had a sensitivity of 100% and a specificity of 98.7% for identifying MRSA from among S. aureus isolates, following 2 h of incubation. This study demonstrates that MRSA and MSSA can be accurately differentiated by FCM after 2 h of incubation in an oxacillin-containing liquid culture medium.Several methods have been developed in recent years to detect Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) rapidly. Peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) allows for rapid detection of S. aureus (12) but cannot identify methicillin resistance in S. aureus. Real-time PCR can detect S. aureus and MRSA in as short a period of time as 2 h (16), but the fact that that most of the currently available formats requires batch testing ensures that in reality detection of S. aureus and MRSA by PCR is usually not as rapid as is commonly believed. The BD-GeneOhm assay for the detection of S. aureus and MRSA has been designed to target the insertion site of the staphylococcal cassette chromosome mec (SCCmec) for detection. The advantage of this approach is that it allows for MRSA detection by targeting a single site, thus avoiding the loss of sensitivity that might occur with multiplex PCR. A disadvantage is that mutations at the target site lead to false-negative results, and mutations in the mecA gene itself that result in nonexpression of the mecA gene or deletion of the mecA gene despite the presence of the SCCmec yield false-positive results for MRSA (4, 7). Indeed, surveillance studies for nasal carriage of MRSA in communities have yielded sensitivities and specificities of only ca. 90% (5). The advantage of ...

Rapid Identification of Staphylococcus aureus and Methicillin Resistance by Flow Cytometry Using a Peptide Nucleic Acid Probe

“…Our previous study showed that given a suspension of S. aureus, one can differentiate MRSA from MSSA by flow cytometry using a dye that stains nucleic acid in a nonspecific manner (5). The present study shows that the same differentiation can be achieved using an S. aureus-specific dye instead of the nonspecific nucleic acid dye.…”

“…We have previously demonstrated that methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolates can be accurately and rapidly differentiated by flow cytometry based on fluorescence intensity and side scatter differences after brief exposure to oxacillin, using a nonspecific fluorescent nucleic acid dye (5). Flow cytometry to differentiate MRSA from MSSA would be even more useful if the method could incorporate S. aureus-specific probe labeling instead of using a nonspecific dye.…”

“…After allowing 5 to 10 min for cooling, 400 l was removed and added to 2.6 ml of phosphate buffer, to yield a final 3-ml sample. This sample was analyzed in a MicroPRO flow cytometer (Advanced Analytical Technologies, Inc., Ames, IA) using a sampling volume of 0.25 ml, with cytometer settings as previously described (5).…”

Rapid Clinical Bacteriology and Its Future Impact

“…During susceptibility testing, the ratio between dead and/or non-dividing cells changes over time and the use of fluorescent labeling and FACS can then help to determine the bactericidal or bacteriostatic effect of the antibiotic studied. It has been shown that methicillin susceptible S. aureus (MSSA) and methicillin resistant S. aureus (MRSA) can be distinguished by FACS after two-hour incubation with oxacillin [95]. When using micro-fluidic methods in combination with FACS, one can also distinguish cellular shapes and sizes.…”

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