Honghui Wang1, Steven K Drake1, Chen Yong2, Marjan Gucek2, Matthew A Lyes1, Avi Z Rosenberg3, Erik Soderblom4, M Arthur Moseley4, John P Dekker5, Anthony F Suffredini6. 1. Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD. 2. Proteomic Core Facility, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD. 3. Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD. 4. Proteomics and Metabolomics Shared Resource, Duke University School of Medicine, Durham, NC. 5. Microbiology Service, Department of Laboratory Medicine, Clinical Center, National Institutes of Health, Bethesda, MD. 6. Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, MD; asuffredini@cc.nih.gov.
Abstract
BACKGROUND: Rapid identification of respiratory pathogens may facilitate targeted antimicrobial therapy. Direct identification of bacteria in bronchoalveolar lavage (BAL) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is confounded by interfering substances. We describe a method to identify unique peptide markers of 5 gram-negative bacteria by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for direct pathogen identification in BAL. METHODS: In silico translation and digestion were performed on 14-25 whole genomes representing strains of Acinetobacter baumannii, Moraxella catarrhalis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Klebsiella pneumoniae. Peptides constituting theoretical core peptidomes in each were identified. Rapid tryptic digestion was performed; peptides were analyzed by LC-MS/MS and compared with the theoretical core peptidomes. High-confidence core peptides (false discovery rate <1%) were identified and analyzed with the lowest common ancestor search to yield potential species-specific peptide markers. The species specificity of each peptide was verified with protein BLAST. Further, 1 or 2 pathogens were serially diluted into pooled inflamed BAL, and a targeted LC-MS/MS assay was used to detect 25 peptides simultaneously. RESULTS: Five unique peptides with the highest abundance for each pathogen distinguished these pathogens with varied detection sensitivities. Peptide markers for A. baumannii and P. aeruginosa, when spiked simultaneously into inflamed BAL, were detected with as few as 3.6 (0.2) × 103 and 2.2 (0.6) × 103 colony-forming units, respectively, by targeted LC-MS/MS. CONCLUSIONS: This proof-of-concept study shows the feasibility of identifying unique peptides in BAL for 5 gram-negative bacterial pathogens, and it may provide a novel approach for rapid direct identification of bacterial pathogens in BAL.
BACKGROUND: Rapid identification of respiratory pathogens may facilitate targeted antimicrobial therapy. Direct identification of bacteria in bronchoalveolar lavage (BAL) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is confounded by interfering substances. We describe a method to identify unique peptide markers of 5 gram-negative bacteria by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for direct pathogen identification in BAL. METHODS: In silico translation and digestion were performed on 14-25 whole genomes representing strains of Acinetobacter baumannii, Moraxella catarrhalis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Klebsiella pneumoniae. Peptides constituting theoretical core peptidomes in each were identified. Rapid tryptic digestion was performed; peptides were analyzed by LC-MS/MS and compared with the theoretical core peptidomes. High-confidence core peptides (false discovery rate <1%) were identified and analyzed with the lowest common ancestor search to yield potential species-specific peptide markers. The species specificity of each peptide was verified with protein BLAST. Further, 1 or 2 pathogens were serially diluted into pooled inflamed BAL, and a targeted LC-MS/MS assay was used to detect 25 peptides simultaneously. RESULTS: Five unique peptides with the highest abundance for each pathogen distinguished these pathogens with varied detection sensitivities. Peptide markers for A. baumannii and P. aeruginosa, when spiked simultaneously into inflamed BAL, were detected with as few as 3.6 (0.2) × 103 and 2.2 (0.6) × 103 colony-forming units, respectively, by targeted LC-MS/MS. CONCLUSIONS: This proof-of-concept study shows the feasibility of identifying unique peptides in BAL for 5 gram-negative bacterial pathogens, and it may provide a novel approach for rapid direct identification of bacterial pathogens in BAL.
Authors: Jeffrey R Strich; Honghui Wang; Ousmane H Cissé; Jung-Ho Youn; Steven K Drake; Yong Chen; Avi Z Rosenberg; Marjan Gucek; Patrick T McGann; John P Dekker; Anthony F Suffredini Journal: J Clin Microbiol Date: 2019-04-26 Impact factor: 5.948
Authors: Honghui Wang; Ousmane H Cissé; Anthony F Suffredini; John P Dekker; Thomas Bolig; Steven K Drake; Yong Chen; Jeffrey R Strich; Jung-Ho Youn; Uchenna Okoro; Avi Z Rosenberg; Junfeng Sun; John J LiPuma Journal: J Clin Microbiol Date: 2020-10-21 Impact factor: 5.948
Authors: Roger Karlsson; Lucia Gonzales-Siles; Margarita Gomila; Antonio Busquets; Francisco Salvà-Serra; Daniel Jaén-Luchoro; Hedvig E Jakobsson; Anders Karlsson; Fredrik Boulund; Erik Kristiansson; Edward R B Moore Journal: PLoS One Date: 2018-12-10 Impact factor: 3.240
Authors: Roger Karlsson; Annika Thorsell; Margarita Gomila; Francisco Salvà-Serra; Hedvig E Jakobsson; Lucia Gonzales-Siles; Daniel Jaén-Luchoro; Susann Skovbjerg; Johannes Fuchs; Anders Karlsson; Fredrik Boulund; Anna Johnning; Erik Kristiansson; Edward R B Moore Journal: Mol Cell Proteomics Date: 2020-01-15 Impact factor: 5.911