Understanding the Genetic Basis for the Emergence of Pathogenic Enterococcus Cecorum

<p>NON-TECHNICAL SUMMARY:<br/> Enterococci are interesting bacteria: While often considered beneficial intestinal bacteria found in many probiotics, enterococci simultaneously function as 1) a major reservoir of antimicrobial resistance and 2) a leading cause of serious infections in hospitalized people. One enterococcus species, Enterococcus cecorum (EC), is carried in the intestinal tracts of chickens and other vertebrates and has recently been responsible for fatal infections of the bones and joints of chickens. This disease first reported in North Carolina has since been reported throughout the United States, Canada and Europe. We have shown that the EC-associated disease in NC flocks is due to genetically related strains which exhibit multidrug resistance. As enterococci are a major food safety and human health concern, there is a critical need to understand the genetic
basis for the recent conversion of EC from beneficial intestinal bacteria to a disease-causing bacterium. Our overall goal is to understand how enterococi cause disease through understanding how EC became a disease-causing bacterium. Understanding the genetic basis behind EC's ability to cause disease is vital to the control of this emerging pathogen of poultry. Outcomes of this work The data generated here will be of interest for future funding from human and poultry health agencies like the USDA, NC Poultry Producers, US Egg and Poultry and the NIH. The outcomes of this work include 1) we will identify 12 targeted genes from other related enterococci in EC which are known to be associated with disease 2) we will assess the ability of EC to attach and invade the gut epithelium and 3) we will sequence pathogenic and non-pathogenic EC strains.
<p>APPROACH:<br/> 1) Work done in the closely related E. faecium and E. faecalis suggests that epithelial attachment is vital to the pathogenesis of virulent enterococci and is mediated by agents of biofilm formation (Esp, Epa) and aggregation substances (Asa1, Asp1, Asc10). These same agents along with secreted proteases (Gelatinase, SprE) also play a role in epithelial invasion. Binding to ECM is also mediated by aggregation substances and microbial surface components recognizing adhesive matrix molecules (MSCRAMMs; Acm, Scm, EcbA, Ace). Resistance to killing via phagocytosis or complement is mediated by capsule (Cps) and aggregation substances. Using published primers or primers developed from the E. faecium and E. faecalis sequence, the prevalence of 12 known virulence genes will be determined for 10 pathogenic (spinal) isolates matched with 10 commensal (cecal)
isolates from epidemiologically diverse outbreaks of ES. Detected genes will be correlated with presence of disease using the Fischer's exact test. 2) Spinal lesion isolates are clonal suggesting intrinsic efficiency in performing one or more of the proposed steps of ES pathogenesis. Epithelial attachment and invasion is the first step in the proposed pathogenesis of ES and will be investigated using epithelial adhesion and invasion assays. Assays will be performed in triplicate, twice (6 technical repeats) using the commercially available chicken epithelial cell line LMH and human colonic epithelial cell line Caco-2. Means of bacterial counts will be compared between pathogenic and commensal isolates using a Student's t-test. 3) The EC genome has not been sequenced. Preliminary data suggest co-acquisition of virulence and antimicrobial resistance determinants. As such,
antimicrobial resistance genes may implicate, by proximity within the genome, potentially important virulence determinants. Additionally, pathogenic isolates are found to be uniformly defective in mannitol metabolism while most commensal isolates can metabolize mannitol. Mapping the mannitol metabolic pathways in the genome may reveal sites of homologous recombination which disrupt these pathways in pathogenic isolates. A representative pathogenic and non-pathogenic isolate of EC has been selected for whole genome sequencing and assembly by the NCSU -GSL sequencing facility. Once the sequencing is completed, we will work closely with Betsy Scholl, Senior Bioinformatics Analyst, Department of Plant Pathology, NCSU to align and annotate the EC genome using sequenced strains of the closely related E. faecium and E. faecalis as scaffold genomes. Comparisons of these genomic sequences will
allow for the detection of putative virulence genes which can be targeted for future studies.