Genotypes and pathogenicity of cellulitis isolates reveal traits that modulate APEC virulence

Abstract

We characterized 144 Escherichia coli isolates from severe cellulitis lesions in broiler chickens from South Brazil. Analysis of susceptibility to 15 antimicrobials revealed frequencies of resistance of less than 30% for most antimicrobials except tetracycline (70%) and sulphonamides (60%). The genotyping of 34 virulence-associated genes revealed that all the isolates harbored virulence factors related to adhesion, iron acquisition and serum resistance, which are characteristic of the avian pathogenic E. coli (APEC) pathotype. ColV plasmid-associated genes (cvi/cva, iroN, iss, iucD, sitD, traT, tsh) were especially frequent among the isolates (from 66.6% to 89.6%). According to the Clermont method of ECOR phylogenetic typing, isolates belonged to group D (47.2%), to group A (27.8%), to group B2 (17.4%) and to group B1 (7.6%); the group B2 isolates contained the highest number of virulence-associated genes. Clonal relationship analysis using the ARDRA method revealed a similarity level of 57% or higher among isolates, but no endemic clone. The virulence of the isolates was confirmed in vivo in one-day-old chicks. Most isolates (72.9%) killed all infected chicks within 7 days, and 65 isolates (38.1%) killed most of them within 24 hours. In order to analyze differences in virulence among the APEC isolates, we created a pathogenicity score by combining the times of death with the clinical symptoms noted. By looking for significant associations between the presence of virulence-associated genes and the pathogenicity score, we found that the presence of genes for invasins ibeA and gimB and for group II capsule KpsMTII increased virulence, while the presence of pic decreased virulence. The fact that ibeA, gimB and KpsMTII are characteristic of neonatal meningitis E. coli (NMEC) suggests that genes of NMEC in APEC increase virulence of strains.

Figure 1. Antimicrobial susceptibility of cellulitis isolates.

The susceptibility of 144 APEC isolates to 15 antimicrobials was tested individually using disc diffusion tests.

Figure 2. Association between resistance to 15 antimicrobials, presence of 31 VAGs¹ and ECOR group among 144 APEC isolates.

Numbers indicate the percentage of isolates that have both traits, while numbers in shadowed boxes indicate the percentage of isolates that have the corresponding trait; * p≤0.001 using χ². ND, not determined.¹ afa and cnf are not included, since they were not present in any strain, and csgA is not included, since it was present in all strains.

Figure 3. Pathogenicity scores for MG1655 and five cellulitis isolates.

Ten one-day old chicks were infected with each isolate and observed for 7 days as described in the Materials and Methods section. Data points represent the PS for each chick, and horizontal bars represent the median PS for each isolate. Strain MG1655 was included as negative control.

Figure 4. Expression of genes found to influence the PS.

Real-time quantitative RT-PCR was used to analyze the expression of kpsMTII, gimB, ibeA and pic in PR001, PR013, PR017 and PR034. ▵ct expresses ct_mean subtracted of ct_mean of tus (housekeeping gene) of the respective isolate. Data represent the average ± SD of two experiments done in triplicates.