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. 2005 Oct;43(10):5143-9.
doi: 10.1128/JCM.43.10.5143-5149.2005.

Development of PCR assays targeting the genes involved in synthesis and assembly of the new Escherichia coli O 174 and O 177 O antigens

Lothar Beutin  1 , Qingke Kong, Lu Feng, Quan Wang, Gladys Krause, Luciana Leomil, Qi Jin, Lei Wang
Affiliations

Development of PCR assays targeting the genes involved in synthesis and assembly of the new Escherichia coli O 174 and O 177 O antigens

Lothar Beutin et al. J Clin Microbiol. 2005 Oct.

Abstract

Escherichia coli O 174 and O 177 are newly described O serogroups which were reported as human pathogens. Identification of these strains by serotyping has been restricted, as the required sera are not commercially available. In this study, a collection of 13 E. coli O 174 strains and 12 E. coli O 177 strains was studied on the O:H serotypes and virulence markers. The O-antigen gene clusters of E. coli O 174 and O 177 were sequenced, and associated genes were assigned functions on the basis of homology. Two genes, each specific for E. coli O 174 and O 177, were identified. PCR assays based on the O-antigen-specific genes were developed and tested on 25 clinical and environmental isolates of those two serogroups as well as 26 isolates of other O serogroups. As little as 1 pg per mul of chromosomal DNA and as few as 0.1 CFU per g of pork and water samples were detected for either strain. The PCR assays established in this study were shown to be highly sensitive and reliable and could be the method of choice for detection of these two human pathogens from clinical, food, and other environmental samples.

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Figures

FIG. 1.
FIG. 1.
O-antigen gene clusters of E. coli O174 and E. coli O177. All genes are transcribed in a direction from the JUMPStart sequence to gnd.
FIG. 2.
FIG. 2.
Amplification products obtained by PCR of 13 pools of DNA including 186 E. coli and Shigella O-antigen type strains and E. coli O174 strain G1609 or E. coli O174 strain G1606 using primer pairs wl-4397/wl-4398 (a), wl-4421/wl-4422 (b), wl-4399/wl-4400 (c), wl-4423/wl-4424 (d), wl-4393/wl-4394 (e), wl-4417/wl-4418 (f), wl-4395/wl-4396 (g), and wl-4419/wl-4420 (h). Lanes 1 and 15, DNA marker, bands with lengths of 100 bp, 250 bp, 500 bp, 750 bp, 1 kb, and 2 kb; lanes 2 to 14, pool 1 to pool 13, respectively.
FIG. 3.
FIG. 3.
Amplification products obtained by PCR of 13 clinical isolated E. coli O174 strains using primer pairs wl-4397/wl-4398 (a), wl-4421/wl-4422 (b), wl-4399/wl-4400 (c), and wl-4423/wl-4424 (d). Lanes 1 and 15, DNA marker, bands with lengths of 100 bp, 250 bp, 500 bp, 750 bp, 1 kb, and 2 kb; lane 2, G1609; lane 3, G1610; lane 4, G1611; lane 5, G2032; lane 6, G2033; lane 7, G2034; lane 8, G2035; lane 9, G2036; lane 10, G2037; lane 11, G2038; lane 12, G2039; lane 13, G2040; lane 14, G2041. Also shown are amplification products obtained by PCR of 12 clinically isolated E. coli O177 strains using primer pairs wl-4393/wl-4394 (e), wl-4417/wl-4418 (f), 4395/wl-4396 (g), and wl-4419/wl-4420 (h). Lanes 1 and 14, DNA marker, bands with lengths of 100 bp, 250 bp, 500 bp, 750 bp, 1 kb, and 2 kb; lane 2, G1606; lane 3, G1607; lane 4, G1608; lane 5, G2042; lane 6, G2043; lane 7, G2044; lane 8, G2045; lane 9, G2046; lane 10, G2047; lane 11, G2048; lane 12, G2049; lane 13, G2068.
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