Serotyping is the long-standing gold standard method to determine E. coli H antigens; however, this method requires a panel of H-antigen specific antibodies and often culture-based induction of the H-antigen flagellar motility. In this study, a rapid and accurate method to isolate and identify the Escherichia coli (E. coli) H flagellar antigen was developed using membrane filtration and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Flagella were isolated from pure culture, digested with trypsin, and then subjected to LC-MS/MS using one of two systems (Agilent-nano-LC-QSTAR XL or Proxeon-nano-LC-LTQ-Orbitrap XL). The resulting peptide sequence data were searched against a custom E. coli flagella/H antigen database. This approach was evaluated using flagella isolated from reference E. coli strains representing all 53 known H antigen types and 41 clinical E. coli strains. The resulting LC-MS/MS classifications of H antigen types (MS-H) were concordant with the known H serogroup for all 53 reference types, and of 41 clinical isolates tested, 38 (92.7%) were concordant with the known H serogroup. MS-H clearly also identified two clinical isolates (4.9%) that were untypeable by serotyping. Notably, successful detection and classification of flagellar antigens with MS-H did not generally require induction of motility, establishing this proteomic approach as more rapid and cost-effective than traditional methods, while providing equitable specificity for typing E. coli H antigens.

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...

The development of rapid and sensitive molecular techniques for the detection of Vibrio species would be useful for the surveillance of sporadic infections and management of major outbreaks. Comparative sequence analysis of the ftsZ gene in the predominant Vibrio species that cause human disease revealed distinct alleles for each examined species, including Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Light Upon eXtension (LUX) real-time PCR assays were developed to target these species-specific polymorphisms, and were successful in rapidly differentiating the major pathogenic Vibrio species. Luminex liquid microsphere array technology was used to develop a comprehensive assay capable of simultaneously detecting V. cholerae, V. parahaemolyticus and V. vulnificus. These assays permitted the identification of a presumptive V. parahaemolyticus isolate as Vibrio alginolyticus, which was verified using additional molecular characterization. INTRODUCTIONPathogenic species of the genus Vibrio pose a considerable public health threat as the causative agents of both sporadic and epidemic human infections. Cholera, caused by Vibrio cholerae, continues to spread globally in a seventh pandemic (O1 El Tor biotype), and the emergence of a non-O1 serogroup (O139 Bengal) has led to a new pandemic (Sack et al., 2004;Faruque & Mekalanos, 2003). Unlike the watery diarrhoeal disease caused by V. cholerae, infection with Vibrio parahaemolyticus usually results in a self-limiting gastroenteritis with inflammatory diarrhoea and, in rare cases, septicaemia (Janda et al., 1988). Opportunistic infections with Vibrio vulnificus can cause severe wound infections (Oliver, 2005) and fulminant septicaemia, with highly virulent strains causing extensive host-tissue damage and producing mortality rates of up to 60 % (Linkous & Oliver, 1999). The threat posed by pathogenic Vibrio species has been highlighted by recent natural disasters, in which a number of survivors were infected in the aftermath of Hurricanes Katrina and Rita on the gulf coast of the USA (Centers for Disease Control, 2005Control, , 2006, and the Indian Ocean tsunami (Lim, 2005). Fortunately, major outbreaks did not occur, but these disasters have served as an impetus to develop rapid and sensitive molecular techniques that can be easily deployed to identify pathogenic Vibrio species during a public health emergency.Traditionally, identification of Vibrio spp. has consisted of isolation on selective agar medium followed by biochemical and serological testing (Harwood et al., 2004). The availability of genomic sequence data allows for Vibrio spp. comparative genomic studies, revealing not only a wealth of information on their evolution and pathogenesis, but potential targets for molecular typing and detection. Molecular techniques for Vibrio identification and subtyping have been developed, including oligonucleotide probes (Wright et al., 1993) and DNA microarray technologies which target species-specific virulence determinants . PCR-based methods have been d...

Several real-time polymerase chain reaction (PCR) methods are currently available to rapidly detect the presence of a specific DNA sequence. When used for detection of pathogenic organisms, the turnaround time for PCR-based methods is much lower than for traditional culture techniques. This study compared the sensitivity of three real-time PCR methods when detecting the Escherichia coli pathogenic gene eae to determine which method is most effective in identifying very low levels of the organism. The three methods were used to detect the eae gene over a range of DNA concentrations. The differences in sensitivity were statistically significant (p<0.05), and SYBR Green I PCR was found to have the lowest detection limit of the three; LUX primers had the highest detection limit. Therefore, using a defined DNA concentration for detecting the eae gene, SYBR Green I is the best alternative.