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ABSTRACTMatrix-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 coliflagellar 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. Whole-genome sequencing was used to resolve conflicting results between the two methods. It was found that DHB (2,5-dihydroxybenzoic acid) worked better than CHCA (a-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. coliH types were identified correctly by MALDI-TOF MS. A custom E. coliflagellar/H antigen database was crucial for clearly identifying the E. coliH 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% by serotyping. This MALDI-TOF MS-H platform can be used for rapid and cost-effective E. coliH antigen identification, especially during E. colioutbreaks.

ABSTRACTForty-three reference strains involving the 24 most common serovars of Salmonella entericawere examined by using a mass spectrometry-based H antigen typing platform (MS-H). The results indicate that MS-H can be used as a sensitive, rapid, and straightforward approach for the typing of Salmonellaflagella at the molecular level without antiserum and phase inversion.