Phenotypic features and phylogenetic background of extraintestinal hemolytic Escherichia coli responsible of mortality in puppies

Highlights

• Some E. coli pathogenic strains may cause enteric or extra-intestinal disease.

• We examine high puppy mortality due to extra-intestinal pathogenic E. coli.

• Bitch's milk could be the main source of ExPEC infection.

• Isolated E. coli strains were assigned to phylogroup B2.

• Strains were biofilm producers with ability to colonize and survive in the host.

Abstract

A 10-day-old litter of five puppies of Bracco Italiano dog breed showed weakness and diarrhea and, 2 days later, four of them died. At the same time, the bitch showed high hyperthermia (40 °C) and endometritis. The necropsy of a puppy revealed a severe lobar pneumonia accompanied with a bilateral nephrosis. No gross lesions were detected in other organs. Histopathology of the lung revealed severe multifocal fibrino-suppurative necrotizing bronchiolar-alveolitis associated with rod-shaped bacterial aggregates and diffuse interstitial lymphocytic infiltration. The kidney showed severe multifocal necrosis of the tubular epithelium and diffuse severe congestion of the parenchyma. A pure culture of hemolytic Escherichia coli carrying the Cnf-1 gene was identified, from both the puppy organs and bitch's milk. Moreover, phylo-typing assigned them to the phylogroup B2. Two weeks later, fecal samples from the bitch and the survived puppy were collected for a second microbiological analysis, identifying two hemolytic E. coli strains, Cnf positive and Cdt negative and Cnf and Cdt negative, respectively.

Some E. coli pathogenic strains may cause enteric or extraintestinal disease. In dogs and cats, strains of extraintestinal pathogenic E. coli (ExPEC) produce specific virulent factors such as hemolysis and cytotoxin necrotizing factors (Cnf). In this episode, we hypothesize that the bitch's milk could be the main source of ExPEC infection causing high puppies mortality. The role of the bitch as a carrier could not be excluded: stressful conditions, such as pregnancy and delivery, would change the host–pathogen dynamics possibly increasing the release of the infectious burden.

Introduction

The enterobacterium Escherichia coli is a common inhabitant of the gastrointestinal tract of humans and animals. Besides commensal E. coli, there are pathogenic strains, classified in intestinal (IPEC) or extraintestinal pathotypes (ExPEC) depending on the virulence determinants they show and the type of disease they cause. Extraintestinal diseases may result from infectious strains causing invasive conditions such as uropathogenic (UPEC), mammary pathogenic (MPEC) and septicemic (SEPEC) in both humans and animals (Russo and Johnson, 2003, Köhler and Dobrindt, 2011).

In dogs and cats, ExPEC strains show virulence genes typically distinct from commensal and intestinal pathogenic strains. Reported ExPEC-producing genes involve pili or fimbriae and fimbrial adhesin molecules that promote adherence and tissue colonization, hemolysins that cause tissue necrosis and siderophore receptors for iron acquisition (Russo and Johnson, 2000), all playing different roles also in the biofilm formation (Hancock et al., 2007). Notwithstanding, other ExPEC cannot be unequivocally distinguished from the commensal ones, since they can use different virulence factors, “in a mix-and-match fashion”, with unexpected consequences for their pathogenetic impact (Köhler and Dobrindt, 2011). E. coli adaptable genome can also manipulate basic host cell functions by producing several types of cyclomodulins. Among inhibitory cyclomodulins, two types of cytotoxin necrotizing factors (Cnf-1 and Cnf-2), promoting cellular proliferation, and cytolethal distending toxins (Cdt) are included (Nougayrède et al., 2005).

In human medicine, phylogenetic analysis have shown that E. coli strains can be divided into seven phylogroups (A, B1, B2, C, D, E and F), according to the genetic substructure typical of this bacterial species. ExPEC strains are more likely to belong to phylogroups B2 or D (Johnson and Stell, 2000), whereas most commensal strains belong to group A.

Finally, different authors have pointed out the role of the biofilm formation as a virulent tract in E. coli strains since bacteria embedded in biofilms are less susceptible to host defences and more resistant to both antibiotics and disinfectants (Malcova et al., 2008).

In this study, we report the genetic and phenotypic characterization of E. coli strains isolated in an episode of neonatal mortality in puppies.