Rapid and specific detection of Escherichia coli clonal group A by gene-specific PCR

Abstract

PCR primers specific for the recently described antimicrobial resistance-associated Escherichia coli clonal group A (CGA), a widespread cause of drug-resistant urinary tract infections in the United States, were devised on the basis of a novel single-nucleotide polymorphism identified within the housekeeping gene fumC, i.e., C288T. In comparison with two reference PCR-based fingerprinting methods, ERIC2 PCR and random amplified polymorphic DNA (RAPD) analysis, a PCR assay incorporating the new primers provided 100% sensitivity and 100% specificity for the detection of CGA among 138 diverse clinical and reference E. coli isolates. E. coli reference (ECOR) strain 47 was shown to be a member or a close relative of CGA (by ERIC2 PCR and RAPD analysis, respectively) and yielded a positive assay result. The new CGA-specific PCR assay, which exhibited interlaboratory reproducibility and stability under various experimental conditions, should allow the rapid and specific detection of CGA by any laboratory equipped for diagnostic PCR.

FIG. 1.

Phylogenetic relationships among diverse E. coli isolates as inferred from the fumC sequence. The tree was inferred from the partial coding sequence for fumC by the neighbor-joining method. Major phylogenetic groups (groups A, B1, B2, and D and nonaligned strains [Non]) are labeled in bold at the far right and bracketed; O:K:H serotypes and CGA are bracketed and labeled immediately adjacent to the tree. Salmonella was used as an outgroup to root the tree. EC, ECOR strain.

FIG. 2.

ERIC2 PCR profiles of diverse E. coli strains, including E. coli reference strain (ECOR) 47. SEQ102 (lane 3) is a CGA reference isolate. Urosepsis isolates V10 and V11 (lanes 5 and 6, respectively) and ECOR strain 47 (versions a and b; lanes 7 and 8, respectively) exhibit profiles indistinguishable from that of SEQ102, except for one irreproducible band (white arrowhead). In contrast, strain Py2 exhibits two reproducible bands (black arrowheads) not present in SEQ102. Non-CGA strain CFT073 (lane 2) is shown for reference.

FIG. 3.

RAPD profiles of diverse E. coli strains, including E. coli reference strain (ECOR) 47. The profiles were generated with arbitrary decamer primers 1247, 1281, 1287, 1254, and 1290, as listed above the gel lanes. ECOR strain 47 (lanes 1, 6, 11, 16, and 21) closely resembles, but (except with primer 1281) is distinct from, CGA reference isolates SEQ102 and UCB1002 (lanes 2, 7, 12, 17, and 22 and lanes 3, 8, 13, 18, and 28, respectively). Strain 21-P (O15:K52:H1; lanes 5, 10, 15, 20, and 25) is similar to the CGA reference isolates, especially with primers 1281, 1287, and 1254. Non-CGA strain CFT073 (lanes 4, 9, 14, 19, and 24) is shown for reference.

FIG. 4.

Gene-specific PCR for representative of E. coli CGA isolates, E. coli reference (ECOR) strain 47, and selected non-CGA reference isolates. The confirmed CGA members (lanes 2 to 5) and ECOR strain 47 (lane 7) all yielded the predicted 175-bp band, whereas non-CGA reference strains (lanes 8 to 12) did not. Lane M, marker (100-bp ladder). Sizes (in base pairs) are shown on the left.