Test requires a few hours compared to a day or two for more traditional culturing techniques
Jan. 26, 2012 — Researchers from the Georgia Institute of Technology and the Centers for Disease Control and Prevention (CDC) have developed a new laboratory test that can rapidly identify the bacterium responsible for staph infections. This new test takes advantage of unique isotopic labeling combined with specific bacteriophage amplification to rapidly identify Staphylococcus aureus.
Viable Staphylococcus aureus Quantitation using 15N Metabolically Labeled Bacteriophage Amplification Coupled with a Multiple Reaction Monitoring Proteomic Workflow
A multiple reaction monitoring liquid chromatography method with tandem mass spectrometric detection for quantitation of Staphylococcus aureus via phage amplification detection is described. This phage amplification detection method enables rapid and accurate quantitation of viable S. aureus by detecting an amplified capsid protein from a specific phage. A known amount of metabolically labeled 15N reference bacteriophage, utilized as the input phage and as the internal standard for quantitation, was spiked into S. aureus samples. Following a 2-h incubation, the sample was subjected to a 3-min rapid trypsin digest and analyzed by high-throughput liquid chromatography tandem mass spectrometric detection targeting peptides unique to both the 15N (input phage) and 14N (progeny phage) capsid proteins. Quantitation was achieved by comparing peak areas of target peptides from the metabolically labeled 15N bacteriophage peptide internal standard with that of the wild-type 14N peptides that were produced by phage amplification and subsequent digestion when the host bacteria was present. This approach is based on the fact that a labeled species differs from the unlabeled one in terms of its mass but exhibits almost identical chemical properties such as ion yields and retention times. A 6-point calibration curve forS. aureus concentration was constructed with standards ranging from 5.0 × 104colony forming units (CFU) ml−1 to 2.0 × 106 CFU ml−1, with the 15N reference phage spiked at a concentration of 1.0 × 109 plaque forming units (PFU) ml−1. Amplification with 15N bacteriophage coupled with LC-MS/MS detection offers speed (3 h total analysis time), sensitivity (LOD: < 5.0 × 104 CFU ml−1), accuracy, and precision for quantitation of S. aureus.
Quickly and accurately detecting infections caused by S. aureus is critical because the pathogenic bacterium causes a broad spectrum of infections, ranging from acute to chronic disease, which need to be treated in a prompt manner with the correct antibiotic.
The test uses mass spectrometry to quantify the number of S. aureus organisms in a large number of samples in just a few hours, compared to a day or two for culturing techniques typically used to detect this bacterium.
“Our method for detecting staph infections using mass spectrometry will be valuable in a variety of situations, but will be crucial when a large number of people need to be tested very quickly, which will ultimately improve treatment,” said Facundo Fernández, an associate professor in the Georgia Tech School of Chemistry and Biochemistry.
Details of the new staph infection detection method were published in the January issue of the journal Molecular & Cellular Proteomics. Partial funding for this research was provided by 3M and the CDC/Georgia Tech seed award program.
Fernández, together with Carrie Pierce, Jon Rees, and John Barr from the CDC’s Division of Laboratory Sciences created this test.
“The simplicity of sample preparation, the low cost of required reagents and the increased availability of mass spectrometers in clinical laboratories make this new method a cost-effective way to rapidly and effectively detect staph infections, which must be treated quickly to prevent spread of the disease,” explained Pierce, a research chemist at the CDC who also worked on the project as a graduate student at Georgia Tech.
To run their test, the researchers first inject a known amount of bacteriophage labeled with nitrogen-15 into a sample. The phages -- which are viruses that infect bacteria -- infect only live S. aureus cells, which then multiply and amplify the phage signal. Following a two-hour incubation, the researchers break up proteins from the phage shell into component peptides using a trypsin digest technique.
Then they analyze the sample using liquid chromatography with tandem mass spectrometric detection. By detecting peptides from the protein shell of the phage, the researchers can measure the concentration of S. aureus in the sample.
“The strength of this technique is coupling a well-characterized method for identifying bacteria with a modern detection device, such as a mass spectrometer,” said Barr, biological mass spectrometry lead in the CDC’s Division of Laboratory Sciences. “By labeling input phage with heavy nitrogen isotopes, we were able to use mass spectrometry to effectively distinguish between the parent and progeny phage, thus enhancing the selectivity of the method.”
This prototype mass spectrometry-based technique has been optimized to detect low concentrations of bacteria that should allow clinicians to diagnose staph infections without the need for a significant culture period. Coupled with standard laboratory robotics, the test will reduce manual labor and subjective interpretation of results inherent in traditional techniques.
“An exciting aspect of this phage method is that with small modifications to the procedures, resistance and susceptibility to a number of different antibiotics can be determined in addition to bacterial identification,” said Rees. “This additional piece of information may be the key to wide acceptance of the method.”
Press release and photo courtesy of EurekaAlert! You can find the original press release here.