The osmoregulated metabolism of trehalose contributes to production of type 1 fimbriae and bladder colonization by extraintestinal Escherichia coli strain BEN2908.

In Escherichia coli, the disaccharide trehalose can be metabolized as a carbon source or be accumulated as an osmoprotectant under osmotic stress. In hypertonic environments, E. coli accumulates trehalose in the cell by synthesis from glucose mediated by the cytosolic enzymes OtsA and OtsB. Trehalose in the periplasm can be hydrolyzed into glucose by the periplasmic trehalase TreA. We have previously shown that a treA mutant of extraintestinal E. coli strain BEN2908 displayed increased resistance to osmotic stress by 0.6 M urea, and reduced production of type 1 fimbriae, reduced invasion of avian fibroblasts, and decreased bladder colonization in a murine model of urinary tract infection. Since loss of TreA likely results in higher periplasmic trehalose concentrations, we wondered if deletion of otsA and otsB genes, which would lead to decreased internal trehalose concentrations, would reduce resistance to stress by 0.6 M urea and promote type 1 fimbriae production. The BEN2908ΔotsBA mutant was sensitive to osmotic stress by urea, but displayed an even more pronounced reduction in production of type 1 fimbriae, with the consequent reduction in adhesion/invasion of avian fibroblasts and reduced bladder colonization in the murine urinary tract. The BEN2908ΔtreAotsBA mutant also showed a reduction in production of type 1 fimbriae, but in contrast to the ΔotsBA mutant, resisted better than the wild type in the presence of urea. We hypothesize that, in BEN2908, resistance to stress by urea would depend on the levels of periplasmic trehalose, but type 1 fimbriae production would be influenced by the levels of cytosolic trehalose.

The Periplasmic Trehalase Affects Type 1 Fimbria Production and Virulence of Extraintestinal Pathogenic Escherichia coli Strain MT78.

Extraintestinal pathogenic Escherichia coli (ExPEC) is responsible for various infections outside the gastrointestinal tract in humans and other animals. ExPEC strain MT78 is invasive to various nonphagocytic cells and highly virulent in vivo To identify genes required for invasion of nonphagocytic cells by this strain, we applied signature-tagged mutagenesis to generate a library of mutants and tested them for invasion of avian fibroblasts. Mutants showing reduced cellular invasion included those with insertions in the fim operon, encoding type 1 fimbriae. Another attenuated mutant showed a disruption in the treA gene, which encodes a periplasmic trehalase. The substrate of TreA, trehalose, can be metabolized and used as a carbon source or can serve as an osmoprotectant under conditions of osmotic stress in E. coli K-12. We generated and characterized mutant MT78ΔtreA In contrast to the wild type, MT78ΔtreA was able to grow under osmotic stress caused by 0.6 M urea but not in minimal M9 medium with trehalose as the only carbon source. It presented decreased association and invasion of avian fibroblasts, decreased yeast agglutination titer, and impaired type 1 fimbria production. In a murine model of urinary tract infection, MT78ΔtreA was less able to colonize the bladder. All phenotypes were rescued in the complemented mutant. Our results show that the treA gene is needed for optimal production of type 1 fimbriae in ExPEC strain MT78 and that loss of treA significantly reduces its cell invasion capacity and colonization of the bladder in a murine model of urinary tract infection.

Prevalence of ColV Plasmid-Linked Genes and In Vivo Pathogenicity of Avian Strains of Escherichia coli.

Avian pathogenic Escherichia coli (APEC) causes extraintestinal infections in birds, leading to an increase in the cost of poultry production. The ColV plasmid-linked genes iroN, ompT, hlyF, iss, and iutA have previously been suggested to be predictors of the virulence of APEC. In this research, we analyzed the frequencies of these genes in a Brazilian collection of E. coli isolated from birds with colibacillosis (APEC) and from apparently healthy birds (avian fecal [A(fecal)]), as well as from the litter of poultry houses of apparently healthy flocks (avian litter [A(litter)]). All the isolates that harbored ompT also harbored hlyF, so they were considered as one trait for statistical analysis. The relationship between in vivo virulence in 1-day-old chicks, expressed as a pathogenicity score, and the number of genes in each isolate showed that isolates with less than two of the four genes were rarely pathogenic, while most pathogenic isolates contained two or more genes. Nevertheless, about half of the nonpathogenic isolates also harbored two or more genes, in agreement with previous observations that commensal E. coli isolates from the birds' microbiota can serve as a reservoir of virulence genes. Thus, the pentaplex polymerase chain reaction can be used to indicate that a strain carrying none or only one gene would be nonpathogenic, but it cannot be used to indicate that a strain with two to four genes would be an APEC. Isolates allocated to phylogenetic group B2, which is frequently associated with extraintestinal infections, had the highest pathogenicity scores, while isolates allocated to group B1 had the lowest.

Molecular characterization and clonal relationships among Escherichia coli strains isolated from broiler chickens with colisepticemia.

This study characterized 52 Escherichia coli isolates from distinct diseased organs of 29 broiler chickens with clinical symptoms of colibacillosis in the Southern Brazilian state of Rio Grande do Sul. Thirty-eight isolates were highly virulent and 14 were virtually avirulent in 1-day-old chicks, yet all isolates harbored virulence factors characteristic of avian pathogenic E. coli (APEC), including those related to adhesion, iron acquisition, and serum resistance. E. coli reference collection phylogenetic typing showed that isolates belonged mostly to group D (39%), followed by group A (29%), group B1 (17%), and group B2 (15%). Phylogenetic analyses using the Amplified Ribosomal DNA Restriction Analysis and pulse-field gel electrophoresis methods were used to discriminate among isolates displaying the same serotype, revealing that five birds were infected with two distinct APEC strains. Among the 52 avian isolates, 2 were members of the pandemic E. coli O25:H4-B2-ST131 clone.

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

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.