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

Abstract

i Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has gained popularity in recent years for rapid bacterial identification, mostly at the genus or species level. In this study, a rapid method to identify the Escherichia coli flagellar antigen (H antigen) at the subspecies level was developed using a MALDI-TOF MS platform with high specificity and sensitivity. Flagella were trapped on a filter membrane, and on-filter trypsin digestion was performed. The tryptic digests of each flagellin then were collected and analyzed by MALDI-TOF MS through peptide mass fingerprinting. Sixty-one reference strains containing all 53 H types and 85 clinical strains were tested and compared to serotyping designations. Wholegenome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (␣-cyano-4-hydroxycinnamic acid) as the matrix for MALDI-TOF MS, with higher confidence during protein identification. After method optimization, reference strains representing all 53 E. coli H types were identified correctly by MALDI-TOF MS. A custom E. coli flagellar/H antigen database was crucial for clearly identifying the E. coli H antigens. Of 85 clinical isolates tested by MALDI-TOF MS-H, 75 identified MS-H types (88.2%) matched results obtained from traditional serotyping. Among 10 isolates where the results of MALDI-TOF MS-H and serotyping did not agree, 60% of H types characterized by whole-genome sequencing agreed with those identified by MALDI-TOF MS-H, compared to only 20% byserotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coli H antigen identification, especially during E. coli outbreaks. Escherichia coli food contamination can have serious consequences, such as hemolytic-uremic syndrome (HUS) (1, 2), with the 2011 Germany E. coli outbreak presenting a clear example (3). Therefore, the fast identification and typing of E. coli is important for tracking sources of contamination and reducing health risks. Traditional typing of E. coli is based mainly on two surface antigens of the bacteria for antiserum-based agglutination reactions: lipopolysaccharide (LPS) or O antigens for O typing and flagellar proteins or H antigens for H typing (4). The procedure for performing serotyping is time-consuming (2 to 12 days) and barely meets the need for fast diagnosis in outbreak situations. This is mainly due to the need to induce flagellar growth for optimizing agglutination reactions for H antigens. In addition, since there are many serotypes of H antigens, multiple agglutination steps have to be performed. There are 53 H types of E. coli flagella in total (designated H1 to H56; H13, H22, and H50 no longer exist) (4, 5). They are composed of polymerized flagellin proteins, each with an average molecular mass of approximately 50 kDa (36 to 60 kDa).Since H antigens take longer to be typed with antisera, flagellar gene-based molecular methods, such as PCR-based flagellar gene sequen...

Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry as a reliable proteomic method for characterization of Escherichia coli and Salmonella isolates

“…On the other hand, Huixia et al . [ 63 ] was developed a rapid method to identify E. coli at subspecies level (identifying flagellar (H) antigen) using a MALDI-TOFMS platform with high sensitivity and specificity which could identify 100% of reference strains containing H types (53 strains) and could detect 75 out of 85 clinical isolates representing matched results obtained from traditional serotyping.…”

A molecular investigation of free-living and lichenized Nostoc sp. and symbiotic lifestyle determination

“…The nature of bacterial species in general is an unsolved problem (Fraser et al 2007). However, there have been decades of productive ecological and evolutionary research on bacteria that relate informal naming schemes such as pathovar (Cai et al 2011), serovar (Chui et al 2015), RAD type (Lee et al 2017), etc., to well resolved lineages in phylogenies inferred from the core genome. In the case of Nostoc, previous research has studied host association and geographic structuring of clusters defined by the rbcLX locus and supported by multi-locus sequence analysis (O'Brien et al 2013).…”

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.…”

“…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 ).…”