Multiplex polymerase chain reaction assay for detection of nonserotypable Shiga toxin-producing Escherichia coli strains of serogroup O147.
Nonserotypable Shiga toxin-producing Escherichia coli (STEC) strains (n = 72) from the collection of the E. coli Reference Center were O typed by a polymerase chain reaction (PCR)-restriction fragment length polymorphism method, and those that exhibited similar profiles (n = 17) were chosen for the study. These isolates, derived from pigs, carried genes for Shiga toxin variant 2e (100%), heat stable enterotoxins STa and STb (70% and 76%, respectively), and F107 (F18) fimbriae (82%). DNA sequencing and analysis of the O-antigen gene cluster of one of the nonserotypable strains exhibited 100% homology with O-antigen cluster of E. coli O147 although the lipopolysaccharide profiles differed significantly between the nonserotypable strains and O147 reference control strain normally used for antibody production. Scanning electron micrographs of the nonserotypable strains showed altered morphology as compared to E. coli O147. Therefore, nonserotypable strains may share 100% homology with O-antigen gene cluster of a certain serogroup but may not express that specific O-antigen. Highly specific multiplex PCR for detecting the nonserotypable STEC of serogroup O147 was developed targeting virulence genes stx2, stb, and fedA encoding for F107 fimbriae, and wzx and wzy of the O147 O-antigen cluster genes. The multiplex PCR method will allow identifying potentially pathogenic subgroup of STEC important in porcine and human health.
DebRoy
Chitrita
C
Department of Veterinary and Biomedical Sciences, E. coli Reference Center, The Pennsylvania State University, University Park, Pennsylvania 16802, USA. rcd3@psu.edu
Roberts
Elisabeth
E
Davis
Michael
M
Bumbaugh
Alyssa
A
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry; D004768:Enterotoxins / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D008855:Microscopy, Electron, Scanning; D019081:O Antigens / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D012703:Serotyping; D022641:Shiga Toxin 2 / Q000235:genetics; D054323:Shiga-Toxigenic Escherichia coli / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D013552:Swine / Q000382:microbiology
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D019081:O Antigens; D022641:Shiga Toxin 2; C034151:heat stable toxin (E coli); C027538:heat-labile enterotoxin, E coli