Mass Spectrometry–Based Escherichia coli H Antigen/Flagella Typing: Validation and Comparison with Traditional Serotyping

Abstract

BACKGROUND:Escherichia coli H antigen typing with antisera, a useful method for flagella clinical identification and classification, is a time-consuming process because of the need to induce flagella growth and the occurrence of undetermined strains. We developed an alternative rapid and analytically sensitive mass spectrometry (MS) method, termed MS-based H antigen typing (MS-H), and applied it at the protein sequence level for H antigen typing. We also performed a comparison with traditional serotyping on reference strains and clinical isolates.

Phenotypic H-Antigen Typing by Mass Spectrometry Combined with Genetic Typing of H Antigens, O Antigens, and Toxins by Whole-Genome Sequencing Enhances Identification of Escherichia coli Isolates

“…Each culture was mixed in water, and the flagella were sheared from the cell body by vortexing for 60 s ( 8 ). The cell bodies were centrifuged at 16,000 × g for 20 min and the supernatant (containing detached flagella) was applied to a syringe filter ( 8 , 12 – 14 ). The flagella were then washed and digested on the filter for 2 h at 37°C.…”

“…(iii) Repeated jigsaw cleanups and LC-MS/MS analyses of samples were performed after blank runs showing emPAI values of >1. (iv) If the adjacent previous blank run had no significant flagellin identified (fewer than two specific peptides) and the emPAI value of the subsequent sample run was 0.10 to 0.99 because of low flagellum production from “sluggish” isolates, repeat testing should be performed with a larger sample amount to obtain an emPAI value of ≥1 ( 13 , 14 ).…”

“…For a proof-of-principle demonstration, MS-H typing and WGS-HOT were initially performed with well-known E. coli reference strains such as EDL933 (ATCC 43895; an Stx 1 - and Stx 2 -producing strain) and K-12 (MG1655, ATCC 47076; a nonpathogenic rough strain), as well as those examined in our earlier studies ( 13 , 15 ). Seventeen randomly chosen clinical isolates whose serotypes and MS-H types were in agreement were also tested to verify the method, but because of the high cost of performing WGS, WGS-HOT was focused mainly on problematic isolates (i.e., Hund, NM, and O rough isolates and those whose MS-H and serotyping results did not agree) ( 12 – 14 ). All databases were compiled in accordance with the strategy used for MS-H typing ( 8 , 12 – 14 ).…”

“…Over the past few years, we have focused our efforts on developing a new method for more accurate and rapid typing of E. coli H antigens by using mass spectrometry ( 8 , 12 ). This H typing method was named MS-H ( 8 ), and validation using clinical isolates proved that it displayed higher speed, better sensitivity, and better specificity than conventional serotyping ( 12 – 14 ). In this study, we formally applied whole-genome sequencing (WGS) to the MS-H typing process, with a focus on resolving clinical isolates that could not be assigned an H type by conventional serotyping and on those isolates whose H serotypes were in disagreement with MS-H-assigned types.…”

Ready, Set, Type! Proteomics vs Agglutination for Escherichia coli H Antigen Confirmation

“…This is where novel approaches, such as the mass spectrometric H antigen typing protocol (MS-H) 2 presented in this issue of Clinical Chemistry by Cheng and colleagues (2 ) can complement and update existing workflows for outbreak investigations. MS-H is a qualitative proteomics approach for typing H antigens.…”