|DEBROY, CHITRITA - Pennsylvania State University|
|WANG, WEI - J Craig Venter Institute|
|LOSADA, LILIANA - J Craig Venter Institute|
|BRINKAC, LAUREN - J Craig Venter Institute|
|RADUNE, DIANA - J Craig Venter Institute|
|TORO, MAGALY - University Of Maryland|
|MENG, JIANGHONG - University Of Maryland|
Submitted to: Biosensors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/29/2015
Publication Date: 2/2/2015
Citation: Liu, Y., Fratamico, P.M., Debroy, C., Yan, X., Needleman, D.S., Li, R.W., Wang, W., Losada, L., Brinkac, L., Radune, D., Toro, M., Meng, J. 2015. Escherichia coli O-antigen gene clusters of serogroups O62, O68, O131, O140, O142, and O163: DNA sequences and similarity between O62 and O68, and PCR-based serogrouping. Biosensors. 5:51-68.
Interpretive Summary: The bacterium, Escherichia coli, causes a variety of diseases in humans and animals, and non-harmful E. coli types, referred to as serogroups, also exist. Traditionally, a procedure called serotyping is used to distinguish among the ca. 180 different E. coli serogroups. This procedure, which relies on the use of antibodies raised in rabbits against different surface polysaccharides of the bacteria, can only be performed in specialized laboratories, is labor intensive and may require several days to complete, and one antiserum can react with multiple E. coli serogroups, rendering identification of the specific strain of E. coli difficult. Thus, due to the lack of simple, rapid, and reliable methods for detection and identification of harmful and non-harmful E. coli types, the incidence of disease caused by harmful strains of E. coli may be underestimated, and epidemiological studies are difficult to perform. To develop a more rapid and simple method for detection and typing of E. coli belonging to serogroups O62, O131, O140, O142 and O63, the DNA sequence of a cluster of genes involved in the production of specific surface polysaccharides of these bacteria was determined. Genes required for biosynthesis of the surface polysaccharides (O antigen) in each of the serogroups were identified, and polymerase chain reaction (PCR) assays were developed targeting unique DNA sequences of two genes present in each of these serogroups. The PCR assays were serogroup specific when tested against a variety of different bacterial strains. Thus, the use of PCR assays specific for E. coli O62, O131, O140, O142 and O163 facilitate the ability to identify, detect, and type these bacteria, and potentially can eliminate the use of the labor-intensive serotyping procedure.
Technical Abstract: The DNA sequences of the O-antigen gene clusters of the Escherichia coli serogroups O62, O131, O140, O142 and O163 were determined. There were 9-14 open reading frames (ORFs) identified, encoding genes required for O-antigen sugar biosynthesis, transfer, and processing. Primers based on the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes within the O-antigen gene clusters of each of the serogroups were used in PCR assays to identify each serogroup. Specificity testing using strains belonging to each of the serogroups isolated from various sources, representative strains of 167 other E. coli O serogroups, and 16 non-E. coli bacteria revealed that the PCR assays were specific for each serogroup. The PCR assays developed in this study can be used for the detection and identification of E. coli O 62, O131, O140, O142 and O163 strains isolated from different sources.