Abstract
Antibiotic resistance continues to contribute significantly to morbidity and mortality across the world. Developing new tests for antibiotic-resistant bacteria is a core action to combat resistant infections. We describe a method that uses phage amplification detection (PAD) combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to rapidly identify Staphylococcus aureus and determine phenotypic susceptibility to cefoxitin. Samples tested for S. aureus are incubated together with bacteriophage in the presence and absence of cefoxitin and subjected to rapid trypsin digestion followed by MALDI-MS analysis. Tryptic peptides derived from amplified phage proteins can be detected by MALDI-MS, as validated by time-of-flight (TOF)/TOF analysis of each peptide combined with database searching. Methicillin-resistant S. aureus show significant phage amplification in the presence of cefoxitin, while methicillin-sensitive S. aureus show no phage amplification relative to a no-antibiotic control. We also show that PAD methodology can be implemented on an FDA-approved commercial MALDI-MS bacterial identification system to identify S. aureus and determine antibiotic susceptibility. The novelty of this assay includes the use of phage-derived tryptic peptides as detected by MALDI-MS to monitor the results of PAD on an instrument common to many modern microbiology laboratories.
Highlights
In 2013, the Centers for Disease Control and Prevention issued a detailed report on the specter of antibiotic resistance threats in the USA, describing the substantial burden of current and emerging antibiotic-resistant strains of bacteria imposed on the US healthcare system [1]
This study demonstrates phage amplification detection (PAD) as a tool to determine the presence of Staphylococcus aureus in a sample and determine its antibiotic susceptibility using MALDI-MS as a detector
PAD followed by trypsin digestion and MALDI-MS analysis can identify S. aureus in samples and determine antibiotic susceptibility
Summary
In 2013, the Centers for Disease Control and Prevention issued a detailed report on the specter of antibiotic resistance threats in the USA, describing the substantial burden of current and emerging antibiotic-resistant strains of bacteria imposed on the US healthcare system [1]. In the USA, at least two million people contract infections from antibioticresistant bacteria, resulting in approximately 23,000 deaths. Direct healthcare costs in the USA related to antibioticresistant bacteria are as high as $20 billion, with estimates for lost productivity ranging up to $35 billion [1]. While the report focuses on antibiotic threats within the USA, it recognizes the worldwide nature of the antibiotic resistance dilemma. The CDC outlines four core actions to combat antibiotic-resistant infections, which includes Bdeveloping new diagnostic tests for resistant bacteria^ [1]. Bacteriophages are virus particles that can infect a bacterial host, hijack the cell machinery of the host to produce clonal progeny virus particles, and lyse the bacterial host, spilling the
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