Comparative Study of Traditional Flagellum Serotyping and Liquid Chromatography–Tandem Mass Spectrometry-Based Flagellum Typing with Clinical Escherichia coli Isolates

Abstract

T raditional methods of phenotyping Escherichia coli bacteria include the serotyping of surface O antigens (lipopolysaccharides), capsule K antigens, and H antigens found on the bacteria's flagellar filaments (1). Despite their usefulness, these conventional antibody-based assays can be costly and laborious to perform due to the wide-ranging quality of antibodies (serum) and the number of antibody agglutination reactions needed to assign a final classification (2, 3). H serotyping is further lengthened by the motility induction required before typing among many distinctive flagellar antigens (H1 to H56; designations H13, H22, and H50 are no longer in use [2,3]).Molecular typing methods using PCR-based amplification and genetic sequencing are gaining popularity for serotype classifications of E. coli due to their potential for high throughput and accuracy (4, 5). Problems with this approach, however, arise because genetics do not necessarily indicate phenotypes and because multiple primers need to be used for amplifying the sequences of unknown antigens. Matrix-associated laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) usage for whole-bacterial-protein profiling to classify and type bacteria has also shown some promising results due to its ease of use and highthroughput potential (6, 7). This platform can currently produce data for some successful bacterial subtyping at the species level but hardly the strain level (i.e., the H and O antigen levels [8]).Recently, we reported a new approach called MS-H for H typing E. coli by purifying and digesting E. coli flagella of reference strains of all 53 known H types on a 0.22-m-pore-size filter membrane, followed by online liquid chromatography-tandem mass spectrometry (LC-MS/MS) of the resulting flagellin peptides. The H-antigen serogroups from flagellin peptide data were identified using a minimum of two sequence-specific peptides (9), and MS-H types were assigned as the top-scoring hit in the identified protein list possessing the highest confidence score (10). An example of data output is shown in Fig. S1 in the supplemental material. This novel approach is thoroughly evaluated here by using 127 clinical isolates, including both motile and nonmotile strains collected from three provinces in Canada over the 1-year period of 2012, and by employing a standardized high-throughput method. The evaluation process comprises two steps: preliminary tests and confirmation assays. Preliminary tests were conducted using both the previously described MS-H typing method and the ISO-certified serotyping method in parallel (10). Rather than testing samples of known H types one by one, however, MS-H was conducted in a blind, high-throughput, batch-by-batch mode in order to replicate the clinical-sample scenario. In the absence of motility induction, flagella of 8 isolates of unknown motilities per batch were extracted and digested, together with 2 reference strains of known H types as control samples. Flagellar peptide sequence data were searched against...

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

“…Ultimately, data quality remained high because QC strain H11 demonstrated 100.0% accurate identification. MS-H was tested on 1 MALDI-TOF platform and 3 LC-MS/MS platforms, the LC-MS/MS selected for method validation because of its superior data quality and ease of use (sequence-based LC-MS/MS is routinely performed once per isolate whereas PMF-based MALDI-TOF should be tested daily on 3 separate sample preparations) (11)(12). The 2 LC-MS/MS systems, Orbitrap Velos and Orbitrap Fusion, showed higher detectability than the Orbitrap XL.…”

“…Clinical isolates from January 1, 2011, to April 1, 2015, were obtained from 5 provincial public health laboratories in Canada. All cells were cultured overnight on tryptic soy agar with 5% sheep blood for flagella extraction (10,11 ). Detailed sources and applications of the cells for different analyses are summarized in part 1 of the Supplemental Data File that accompanies the online version of this article at http://www.clinchem.org/content/vol62/issue6.…”

“…We then performed LC-MS/MS-based sequence analysis (10,11 ) or MALDI-TOF-based peptide mass fingerprinting (PMF) (12 ) through a curated E. coli flagella protein sequence database search (13 ). MS-H was able to identify and differentiate any E. coli H type within 5 min after sample loading by MALDI-TOF (12 ), or within 4 hours by LC-MS/MS after flagella extraction (10,11 ). Currently, overnight bacteria culture is typically needed to extract flagella, but motility induction is not required for the majority of isolates, especially when using LC-MS/MS (10 -12 ).…”

Rapid, Sensitive, and Specific Escherichia coli H Antigen Typing by Matrix-Assisted Laser Desorption Ionization–Time of Flight-Based Peptide Mass Fingerprinting

“…The method applied a unique flagellar extraction and on-filter trypsin digestion method together with a curated E. coli flagellar protein sequence database for targeted and specific identification (19). The approach was termed MS-H and was able to identify and differentiate an E. coli H type in only a few hours after cell culture, with higher sensitivity and specificity than traditional serotyping (18). Since the MALDI-TOF platform can be used with higher throughput and less cost than with LC-MS/MS and is gaining popularity in clinical microbiology laboratories due to its ease of operation, in this study we have challenged the method to obtain subspecies-level identification and typing of E. coli flagella for the purpose of applying this technique for rapid H typing during an E. coli outbreak.…”

“…Recently, we reported an MS-based E. coli H antigen identification and classification method at the flagellar protein sequence level using a liquid chromatography-tandem MS (LC-MS/MS) platform (17,18). The method applied a unique flagellar extraction and on-filter trypsin digestion method together with a curated E. coli flagellar protein sequence database for targeted and specific identification (19).…”