Identification and typing of bacteria occupy a large fraction of time and work in clinical microbiology laboratories. With the certification of some MS platforms in recent years, more applications and tests of MS‐based diagnosis methods for bacteria identification and typing have been created, not only on well‐accepted MALDI‐TOF‐MS‐based fingerprint matches, but also on solving the insufficiencies of MALDI‐TOF‐MS‐based platforms and advancing the technology to areas such as targeted MS identification and typing of bacteria, bacterial toxin identification, antibiotics susceptibility/resistance tests, and MS‐based diagnostic method development on unique bacteria such as Clostridium and Mycobacteria. This review summarizes the recent development in MS platforms and applications in bacteria identification and typing of common pathogenic bacteria.

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...

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.

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt. The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing.