Identification of minimal predictors of avian pathogenic Escherichia coli virulence for use as a rapid diagnostic tool

Abstract

To identify traits that predict avian pathogenic Escherichia coli (APEC) virulence, 124 avian E. coli isolates of known pathogenicity and serogroup were subjected to virulence genotyping and phylogenetic typing. The results were analyzed by multiple-correspondence analysis. From this analysis, five genes carried by plasmids were identified as being the most significantly associated with highly pathogenic APEC strains: iutA, hlyF, iss, iroN, and ompT. A multiplex PCR panel targeting these five genes was used to screen a collection of 994 avian E. coli isolates. APEC isolates were clearly distinguished from the avian fecal E. coli isolates by their possession of these genes, suggesting that this pentaplex panel has diagnostic applications and underscoring the close association between avian E. coli virulence and the possession of ColV plasmids. Also, the sharp demarcation between APEC isolates and avian fecal E. coli isolates in their plasmid-associated virulence gene content suggests that APEC isolates are well equipped for a pathogenic lifestyle, which is contrary to the widely held belief that most APEC isolates are opportunistic pathogens. Regardless, APEC isolates remain an important problem for poultry producers and a potential concern for public health professionals, as growing evidence suggests a possible role for APEC in human disease. Thus, the pentaplex panel described here may be useful in detecting APEC-like strains occurring in poultry production, along the food chain, and in human disease. This panel may be helpful toward clarifying potential roles of APEC in human disease, ascertaining the source of APEC in animal outbreaks, and identifying effective targets of avian colibacillosis control.

FIG. 1.

MCA of 124 avian E. coli isolates of known pathogenicity, taking into account virulence genotypes, phylogenetic groups, and serogroups O1, O2, O11, O35, O36, O78, OT (other typeable serogroups are lumped together), and NT (nontypeable).

FIG. 2.

Left-most portion of this figure is the dendrogram resulting from the cluster analysis. There appears to be three main clusters of isolates. Cluster 1 (highlighted in red) contains most of the AFEC isolates. The majority of isolates in cluster 2 (highlighted in green) are APEC isolates, and cluster 3 (highlighted in blue) consists entirely of APEC isolates. Just to the right of the dendrogram is column 1, which identifies an isolate as APEC (green) or AFEC (pink). Columns 2 to 47 show the virulence genotype of each isolate tested. Each column in this group shows the results for a single gene or trait. The identity of each gene tested is shown in the horizontal bar at the top of the diagram, with those in blue being plasmid mediated and those in yellow being chromosomally located. Black indicates that a gene is present, and pale green indicates that a gene is absent. Column 48 shows the phylogenetic group of each isolate: blue, group A; orange, B1; brown, B2; and green, D. ompTp, episomal ompT; ompTc, chromosomal ompT; iss, episomal iss.

FIG. 3.

Results using the pentaplex panel for the 994 avian E. coli isolates. The left-most portion of this figure is the dendrogram resulting from the cluster analysis. Just to the right of the dendrogram is column 1, which identifies an isolate as APEC (green) or AFEC (pink). Columns 2 to 6 show the virulence genotype of each isolate tested for iroN, iutA, iss, ompTp, and hlyF. Each column in this group shows the results for a single gene. Black indicates that the gene is present, and light green indicates that the gene is absent.

FIG. 4.

Histogram comparing the prevalence of the genes targeted in the pentaplex procedure among APEC isolates of known pathogenicity (APEC high, high pathogenicity [n = 73]; APEC int, intermediate pathogenicity [n = 26]; APEC low, low pathogenicity [n = 25]). Also shown is the gene prevalence among 200 AFEC isolates. Letters above bars indicate levels of statistical significance according to Fisher's exact test (P < 0.05 is considered statistically significant).