Genotypes and Pathogenicity of Cellulitis Isolates Reveal Traits That Modulate APEC Virulence

Nicolle Lima Barbieri,Aline Luísa De Oliveira,Thiago Moreira Tejkowski,Benito Guimarães De Brito,Daniel Brisotto Pavanelo,Fabiana Horn,Letícia Beatriz Matter,Débora Assumpção Rocha,Sidia Maria Callegari-Jacques

doi:10.1371/journal.pone.0072322

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.

Highlights

Extraintestinal infections caused by avian pathogenic Escherichia coli (APEC) include omphalitis in embryos, salpingitis in laying hens, respiratory tract infections, and cellulitis [1]
The frequency of resistance to all the antimicrobials was less than 30% except for tetracycline and sulphonamides (Fig.1)
In an attempt to find out what increases the virulence of APEC isolates in one-day-old chicks, we looked for significant associations between the presence of virulence-associated genes (VAGs) and pathogenicity scores (Table 3)

Summary

Introduction

Extraintestinal infections caused by avian pathogenic Escherichia coli (APEC) include omphalitis in embryos, salpingitis in laying hens, respiratory tract infections, and cellulitis [1]. Cellulitis is one of the most prevalent extraintestinal infections caused by APEC in broiler chickens, and is characterized by the presence of subcutaneous fibrinonecrotic plaques and inflammation of the overlying chicken skin, resulting in rejection of part or all of the carcasses at processing [2,3,4,5]. The virulence genes that permit certain intestinal commensal E. coli to become APEC and infect extraintestinal sites include those encoding for the adhesins type 1 fimbriae and temperaturesensitive haemagglutinin (Tsh), iron-scavenging systems and the protectin Iss [9]. Most of these genes are often carried on Colicin

Methods

Results

Conclusion