|NARM, KOH-EUN - University Of Illinois|
|Cooley, Michael - Mike|
Submitted to: Frontiers in Cellular and Infection Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/18/2012
Publication Date: 5/11/2012
Citation: Quinones, B., Swimley, M.S., Narm, K., Patel, R.N., Cooley, M.B., Mandrell, R.E. 2012. O-antigen and virulence profiling of Shiga toxin-producing Escherichia coli by a rapid and cost-effective DNA microarray colorimetric method. Frontiers in Cellular and Infection Microbiology. 10.3389/fcimb.2012.00061/full.
Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) is a food- and water-borne pathogen enteric pathogen known to cause human gastrointestinal illnesses with diverse clinical manifestations, ranging from bloody diarrhea and hemorrhagic colitis to the life-threatening hemolytic-uremic syndrome. The varying disease severity in humans has been associated with more than 100 serotypes of STEC. In particular, E. coli O157:H7 is the STEC most commonly reported in the United States. The reporting of non-O157 infections by public health laboratories has increased considerably in recent years. Epidemiological studies have indicated that several non-O157 serotypes, O26, O45, O103, O111, O121, and O145, were prominently associated with outbreaks and severe hemorrhagic symptoms in the United States. Additionally, strains with serotypes O55, O91, O113, and O128 have been considered as relevant causes of human infections. The prevalence of disease caused by non-O157 serotypes has led to the proposal that certain non-O157 STEC strains are potentially as virulent as O157:H7 strains. The onset of life-threatening disease symptoms caused by STEC has been associated with the production of Shiga toxins by at least one of the stx genes. However, epidemiological studies revealed that not all STEC strains that produce Shiga toxin are clinically significant. Sequence analyses have identified the presence of a group of chromosomal and plasmid-encoded virulence genes that are thought to also contribute significantly to STEC pathogenesis. Screening for these virulence genes in STEC strains would allow the identification of risk factors that could potentially lead to sporadic- and outbreak-related human illness. The use of DNA microarrays as genotyping method enables the examination of multiple gene targets in a single assay. However, DNA microarray platforms have been challenged with the development of sensitive and rapid procedures, indicating the positive signals on the array, in conjunction with cost-effective instrumentation for pathogen detection applications. The commonly-used fluorescent microarray methods follow procedures that can be inconsistent and highly variable and utilize expensive and non-portable scanners for data acquisition and analysis. Thus, improved procedures are required with sufficient cost-effectiveness and consistency for routine pathogen surveillance. To simultaneously detect and genotype STECs by using a simpler, rapid and inexpensive method, the present study developed the use of the ampliPHOX colorimetric technology, based on light-initiated signal amplification through polymerization, with low-density microarrays to genotype STEC strains, recovered from various sources and geographical locations. Results from the validation experiments indicated that this microarray method serotyped accurately the O-antigen of the reference STEC strain. Similar results were obtained when assessing the virulence profile of the STEC strains. Polymer formation was observed exclusively where the probe sequences were spotted on the microarray, and the patterns of photopolymerization correlated with the genotype of the tested STEC strains. Quantification analysis indicated that high signal-to-noise ratio (SNR) values were obtained for spots where polymer formation was detected. The positive signals detected for all O-antigen cluster and virulence gene spots resulted in average SNR values ranging from 24.68 ± 0.14 to 48.89 ± 0.31. This contrasted with average SNR values for spots where no specific hybridization was observed ranging from 0.08 ± 0.17 to 1.28 ± 0.22. Thus, the use of ampliPHOX colorimetric detection technology, in conjunction with low-density DNA microarrays, allowed a rapid and high-throughput O-antigen typing as well as an accurate assessment of the virulence potential of reference STEC strains by using reagents and instrumentat
Technical Abstract: Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne illness worldwide. To evaluate better methods to rapidly detect and genotype Shiga toxin-producing Escherichia coli strains, the present study evaluated the use of the ampliPHOX colorimetric detection technology, based on light-initiated signal amplification through polymerization, for pathogen identification with DNA microarrays. A low-density 30-mer oligonucleotide DNA microarray was designed to target O-antigen gene clusters of eleven serogroups, associated with the majority of STEC infections in the United States. In addition, the DNA microarray targeted twelve virulence factors implicated in conferring STEC strains an increased ability to cause disease. Results from the validation experiments demonstrated that the use of ampliPHOX with DNA microarray allowed the accurate genotyping of reference STEC strains, and positive hybridization signals were observed for only probes targeting virulence an O-antigen genes present in the tested strains. Quantification analysis indicated that the average signal-to-noise ratio values ranged from 36.27±1.41 to 80.29±1.44 for probes targeting O157 and non-O157 O-antigen cluster and virulence genes. Low signal-to-noise ratio values below 6.88±0.27 were determined for probes where no polymer was formed due to lack of specific hybridization. Thus, the use of ampliPHOX colorimetric detection technology in conjunction with low-density DNA microarrays, allowed a rapid and precise assessment of the virulence potential of reference STEC strains by using reagents and instrumentation that is low cost and suitable for routine pathogen surveillance.