Identification of the ESKAPE pathogens by mass spectrometric analysis of microbial membrane glycolipids

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: E nterococcus faecium, S taphylococcus aureus, K lebsiella pneumoniae, A cinetobacter baumannii, P seudomonas aeruginosa, and E nterobacter 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.

Figure 1

Strategy for glycolipid-based mass spectrometry platform for pathogen identification. (a) Microbes are isolated from pure culture or biological specimen and whole cell lipids are extracted by hot ammonium-isobutyrate (b) Lipid extracts are purified and analyzed by MALDI-TOF-MS (c) A mass spectrum of membrane glycolipids is acquired and compared against an extensive reference database of mass spectral profiles from known organisms via pattern-matching to (d) Generate a digital identification output and an assigned confidence score.

Figure 2

Representative mass spectra from ESKAPE pathogens: (a) Enterococcus faecium; (b) Klebsiella pneumoniae; (c) Pseudomonas aeruginosa; (d) Staphylococcus aureus; (e) Acinetobacter baumannii; and, (f) Enterobacter cloacae. m/z values of select ions are given.

Figure 3

Dot product analysis of mass spectra for differentiation of ESKAPE pathogens. Mass spectra were acquired from lipid extracts of each ESKAPE pathogen. Species were compared by calculating a pairwise dot product between mass lists of ions from each mass spectrum, a measure of spectrum similarity. A similarity score of 1.0 is an identical match (black squares). White squares represent a score of 0.0 where there is no match. (*) indicate colistin-resistant strains.

Figure 4

Consensus mass spectra were created by summation of detected ions from all replicates of colistin-resistant (top panels) and colistin-susceptible (bottom panels) A. baumannii (a) and K. pneumoniae (b). The mirrored consensus mass spectra highlight ions of core lipid A structures for A. baumannii (m/z 1376, 1404, 1728, 1910) and K. pneumoniae (m/z 1376, 1404, 1824, 1840, 2063, 2079). Importantly, these consensus spectra reveal consistent diagnostic ions for detecting colistin resistance in A. baumannii (m/z 1953, 2033) and K. pneumoniae (m/z 1891, 1955, 1971).

Figure 5

Detection of S. aureus and K. pneumoniae from blood culture. (a) Blood culture control containing sterile blood (b) Blood culture containing MRSA M2 after overnight growth (24 hours), and (c) Blood culture containing K. pneumoniae B6 after six hours growth. A 10⁴ CFU inoculate was seeded into 10 mL blood, transferred to standard aerobic culture bottles and sampled at 1, 2, 4, 6, and 24 hours. Differential centrifugation allowed separation of human cells. Extraction and mass analysis was performed.