Title: Application of multiplex PCR, pulsed-field gel electrophoresis (PFGE), and BOX-PCR for molecular analysis of enterococci Authors
Submitted to: Gel Electrophoresis-Principles and Basics
Publication Type: Book / Chapter
Publication Acceptance Date: February 7, 2012
Publication Date: June 1, 2012
Citation: Jackson, C.R., Spicer, L.M., Barrett, J.B., and Hiott, L.M. 2012. Application of multiplex PCR, Pulsed-Field Gel Electrophoresis (PFGE), and BOX-PCR for molecular analysis of enterococci. In: Magdelden, S., editors. Gel Electrophoresis. Rijeka, Croatia: InTech-Open Access. p. 269-298. Available: http://www.intechopen.com/articles/show/title/application-of-multiplex-pcr-pulsed-field-gel-electrophoresis-pfge-and-box-pcr-for-molecular-analysis. Interpretive Summary: Although enterococci are considered opportunistic pathogens, they can be reservoirs of antimicrobial resistance. This is increasingly important considering foodborne illnesses from meat and rising infections from produce. In our previous study, prevalence and antimicrobial resistance of enterococci isolated from retail fruits, vegetables, and meats collected from grocery store chains in the North Georgia USA area were evaluated. E. faecalis isolates from that study were further characterized to determine if any association between antimicrobial resistance and virulence genes existed. Genetic analysis of E. faecalis from retail food items revealed that the isolates did not group according to retail store or year of isolation. The objective of the present study was to use band-based molecular typing methods to determine if genetically related enterococci were found among different stores, food types, or years. Different enterococcal species were prevalent on fruits, vegetables, and meat from retail grocery stores. Specific species, such as Enterococcus casseliflavus from fruits and vegetables, were predominant on certain food products, but were also found in lower numbers on other food items. The majority of enterococcal isolates from the retail food items were resistant primarily to bacitracin, flavomycin, and lincomycin. Resistance of enterococci to penicillin, salinomycin, and nitrofurantoin was low and none of the isolates were resistant to linezolid or vancomycin. Enterococcal isolates with identical banding patterns were identified including isolates from different stores, food types, and of different species. These data suggest that foods commonly purchased and consumed from grocery stores are a source of genetically related antimicrobial resistant enterococci that can be transferred to the human population. This research will be useful to policy makers and researchers who can use this information when studying antimicrobial resistant bacteria that may be found on food.
Technical Abstract: The objective of the study was to use band-based molecular methods including BOX-PCR (Polymerase Chain Reaction) and Pulsed-Field Gel Electrophoresis (PFGE) to determine if genetically related enterococci were found among different stores, food types, or years. Enterococci were also characterized for antimicrobial resistance patterns. From 2000-2001, food items (vegetables, fruits, and meats) were obtained from grocery store chains in the North Georgia USA area and cultured for the presence of enterococci. Of the samples, 47.7% (189/396) were positive for enterococci. Using multiplex PCR to identify the genus Enterococcus and specific enterococcal species, the predominant species identified was Enterococcus faecalis (n=80) from meat followed by Enterococcus casseliflavus (n=66) from fruits and vegetables. While high numbers of isolates were resistant to lincomycin and bacitracin, very few isolates were resistant to salinomycin, penicillin, or nitrofurantoin. None of the isolates were resistant to linezolid or vancomycin. Using BOX-PCR and PFGE, isolates exhibiting 100% similarity in banding pattern were found. These isolates originated from different stores, food types, and in a few cases, species; most banding patterns grouped the isolates by species. These data suggest that foods commonly purchased and consumed from grocery stores in a geographic region are a source of genetically related enterococci; however, overall resistance to antimicrobials is relatively low.