Abstract
Rapid diagnostics that enable identification of infectious agents improve patient outcomes, antimicrobial stewardship, and length of hospital stay. Current methods for pathogen detection in the clinical laboratory include biological culture, nucleic acid amplification, ribosomal protein characterization, and genome sequencing. Pathogen identification from single colonies by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of high abundance proteins is gaining popularity in clinical laboratories. Here, we present a novel and complementary approach that utilizes essential microbial glycolipids as chemical fingerprints for identification of individual bacterial species. Gram-positive and negative bacterial glycolipids were extracted using a single optimized protocol. Extracts of the clinically significant ESKAPE pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. were analyzed by MALDI-TOF-MS in negative ion mode to obtain glycolipid mass spectra. A library of glycolipid mass spectra from 50 microbial entries was developed that allowed bacterial speciation of the ESKAPE pathogens, as well as identification of pathogens directly from blood bottles without culture on solid medium and determination of antimicrobial peptide resistance. These results demonstrate that bacterial glycolipid mass spectra represent chemical barcodes that identify pathogens, potentially providing a useful alternative to existing diagnostics.
Highlights
Infectious diseases pose an ongoing threat to public health
This process resulted in a mass spectral ESKAPE pathogen library of six unique species and an additional 44 non-ESKAPE pathogens used as decoys for further analyses (Supplementary Table S2)
We investigated a novel approach to bacterial identification, namely, analysis of microbial membrane glycolipids by MALDI-TOF-mass spectrometry (MS)
Summary
Infectious diseases pose an ongoing threat to public health. Rapid and accurate pathogen detection is needed to guide physicians in the treatment of infection to improve patient and economic outcomes[1]. Nucleic acid amplification and sequencing of essential bacterial genes, such as the 16S rDNA, for bacterial identification offers increased accuracy; high sensitivity can result in false positives and it may not provide valuable sub-species information[3] These methods are time intensive, require at least 24 hours of incubation of clinically obtained material, and often significantly increase the cost and burden of diagnostic laboratory support. Microbial membranes possess more complex, glycosylated lipids that are exclusive to bacterial cell membranes and are not produced by mammals They are present in high abundance, approximately 106 molecules per bacterium, and are readily extracted from bacteria grown under laboratory conditions or directly from biological fluids. We hypothesized that these glycolipids represent novel chemical fingerprints that would enable identification of bacteria by MS in a manner similar to bacterial proteins
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