Highly discriminatory single-nucleotide polymorphism interrogation of Escherichia coli by use of allele-specific real-time PCR and eBURST analysis

Abstract

In total, 782 Escherichia coli strains originating from various host sources have been analyzed in this study by using a highly discriminatory single-nucleotide polymorphism (SNP) approach. A set of eight SNPs, with a discrimination value (Simpson's index of diversity [D]) of 0.96, was determined using the Minimum SNPs software, based on sequences of housekeeping genes from the E. coli multilocus sequence typing (MLST) database. Allele-specific real-time PCR was used to screen 114 E. coli isolates from various fecal sources in Southeast Queensland (SEQ). The combined analysis of both the MLST database and SEQ E. coli isolates using eight high-D SNPs resolved the isolates into 74 SNP profiles. The data obtained suggest that SNP typing is a promising approach for the discrimination of host-specific groups and allows for the identification of human-specific E. coli in environmental samples. However, a more diverse E. coli collection is required to determine animal- and environment-specific E. coli SNP profiles due to the abundance of human E. coli strains (56%) in the MLST database.

FIG. 1.

Combined output of an eBURST analysis of E. coli STs from an MLST database and SNP profiles from SEQ isolates. Single-locus variants (STs that differ at 1/7 MLST alleles) are depicted by straight lines. Each circle represents a separate SNP profile. Each SNP profile is an 8-character “barcode” which indicates a particular SNP genotype (listed in an order of increasing discriminatory power): fadD(234) (D = 0.63), clpX(267) (D = 0.8), uidA(138) (D = 0.88), clpX(177) (D = 0.92), clpX(234) (D = 0.93), lysP(198) (D = 0.94), icdA(177) (D = 0.95), and mdh(450) (D = 0.96). SNP profile numbers were assigned to each individual barcode. All of the 114 SEQ isolates genotyped in this study are shown in italics and originated from cows (c), dogs (d), ducks (du), kangaroos (k), horses (h), human feces (hf), and human urine (hu). Host-specific SNP profiles are shaded in green for cattle, yellow for dogs, brown for horses, magenta for kangaroos, and pink for human E. coli. Animal-specific SNP profiles are depicted as hashed circles. SNP profiles with isolates originating from both animal and human sources were considered mixed and remain uncolored.