Rapid Intrinsic Method Identifies Blood Cultures Pathogen

A simple method has been described that identifies microorganisms from positive blood culture broth within the time it takes to perform a Gram stain.

The method is based on intrinsic fluorescence spectroscopy (IFS) of whole cells and required development of a selective lysis buffer, aqueous density cushion, optical microcentrifuge tube, and reference database.

Scientists from the Microbiology Department at bioMérieux (Durham, NC, USA) analyzed a total of 1,121 monomicrobial-positive broth samples from 751 strains to build a database representing 37 of the most commonly encountered species in bloodstream infections or present as contaminants. The method combines a selective lysis step in which blood cells in the sample are destroyed, a centrifugation step to collect any bacteria or fungi in the sample, and a fluorescence step that analyzes the particular fingerprint of any pathogens present in the sample.

In the technique developed, a sample of positive blood culture is treated with lysis buffer to lyse the blood cells, and then transferred to a specialized optical tube. The tube is centrifuged, which drives bacteria or fungi, which are denser than the solution, down through a liquid density cushion to form a pellet at the bottom of the tube. To use the intrinsic fluorescence spectroscopy (IFS), the microbial pellet is irradiated with light ranging from the deep ultraviolet to infrared, which excites certain organic molecules in the microorganisms. This includes tryptophan, reduced nicotinamide adenine dinucleotide (NADH), flavin adenine dinucleotide (FAD), porphyrins, and others, and causes them to fluoresce in a characteristic way depending on the identity of the microbe. The exact pattern of fluorescence is compared with a database of 37 of the most common known pathogens to identify the organism present.

In a controlled laboratory, the method can correctly identify the species in 96.5% of all test samples. In the 2.7% of samples for which no species identity was provided, the system was able to correctly identify 67% to the family level, which is often enough information to select an effective therapy. Among over a thousand samples tested, the method never gave an incorrect result as to the family level or the Gram type.

John D. Walsh, PhD, the lead author of the study, said, “We're using intrinsic fluorescence to identify the microorganisms. It's an innate property of most living organisms. Because it's intrinsic, no reagents are needed for the identification step, which removes many of the opportunities for mistakes and lowers test costs. Our vision is to have a system that will automatically identify the blood culture isolate within 15 minutes of the culture being called positive.” The study was published on November 19, 2013, in the journal mBio.

Related Links:
bioMérieux