Title: A comparison of BOX-PCR and pulsed-field gel electrophoresis to determine genetic relatedness of enterococci from different environments Authors
Submitted to: Microbial Ecology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 7, 2012
Publication Date: August 20, 2012
Citation: Jackson, C.R., Furtula, V., Farrell, E.G., Barrett, J.B., Hiott, L.M., Chambers, P. 2012. A comparison of BOX-PCR and pulsed-field gel electrophoresis to determine genetic relatedness of enterococci from different environments. Microbial Ecology. 64(2):378-387. Interpretive Summary: The enterococci are commensals of particular interest because they have been implicated in a number of clinical diseases and are known to harbor antimicrobial resistance. The habitat of enterococci not only includes the intestines of many animals, but also food and the environment. Their widespread nature along with their ability to survive in the environment and their propensity for antimicrobial resistance make the enterococci a candidate group for host attribution studies especially in aquatic environments where a variety of hosts may be contributing to fecal contamination. Fraser Valley in the province of British Columbia is considered the poultry capital of Canada. The poultry waste generated from the industry is used as fertilizer and spread onto the fields thus creating a non-point source run-off type of surface and groundwater pollution. Using a new automated approach to separate and visualize the Polymerase Chain Reaction (PCR) products produced from amplification of DNA sequences that repeat (called BOX-PCR), the genetic relationship of enterococci from surface and groundwater to enterococci isolated from poultry sources in Fraser Valley was investigated. Results from BOX-PCR were compared to those from another sub-typing method, Pulsed-Field Gel Electrophoresis (PFGE). Enterococcus (E.) faecium was isolated from layer litter and surface and groundwater, while E. faecalis was isolated from all sources except layer litter. E. faecalis grouped primarily based upon source using BOX-PCR. Isolates from water samples were dispersed more frequently among PFGE groups containing isolates from poultry litter. E. faecium strains had a high degree of genetic diversity as overall clustering was independent of source by both molecular methods. Although enterococci from litter and water sources were grouped together using BOX-PCR and PFGE, water isolates could not be definitively identified as originating from poultry litter. Researchers can use this information for source tracking environmental contamination and in designing strategies to reduce microbial contamination of the environment.
Technical Abstract: Aims: The genetic relatedness of enterococci from poultry litter to enterococci from nearby surface water and groundwater in the Lower Fraser Valley regions of British Columbia, Canada was determined. Methods and Results: BOX-PCR and Pulsed-Field Gel Electrophoresis (PFGE) were used to subtype enterococcal isolates from broiler and layer litter and surface and groundwater. All surface water samples (n=12) were positive for enterococci, as were 11% (3/28) of groundwater samples. Enterococcus (E.) faecium (n=90) was isolated from all sources, while E. faecalis (n=59) was isolated from all sources except layer litter. The majority of E. faecalis originated from broiler litter (28/59; 47.5%) while the majority of E. faecium were isolated from layer litter (29/90; 32.2%). Similarity assignment of BOX-PCR groups was >70% and >50% for PFGE groups for both enterococcal species. E. faecalis grouped primarily by source using BOX-PCR. Isolates from water samples were dispersed more frequently among PFGE groups containing isolates from poultry litter. E. faecium strains had a high degree of genetic diversity as overall clustering was independent of source by both molecular methods. Subgroups of E. faecium isolates based upon source (layer litter) were present in BOX-PCR groups. Three individual E. faecalis groups and two individual E. faecium groups were100% similar using BOX-PCR; only one instance of 100% similarity among isolates using PFGE was observed. Conclusion: Although enterococci from litter and water sources were grouped together using BOX-PCR and PFGE, isolates originating from water could not be definitively identified as originating from poultry litter. Significance and Impact of Study: Automation of BOX-PCR amplicon separation and visualization increases the reproducibility and standardization of subtyping using this procedure.