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United States Department of Agriculture

Agricultural Research Service

Research Project: ENHANCING ANIMAL WELL-BEING, IMMUNOCOMPETENCE, AND PERFORMANCE IN SWINE AND BEEF CATTLE Title: Growth of biophotonic Escherichia coli O157:H7 (ATCC #43888) within rumen fluid media

Authors
item Duoss, Heather -
item Donaldson, Janet -
item Callaway, Todd
item Martin, James -
item Carroll, Jeffery
item Ballou, Michael -
item Broadway, Paul -
item Falkenberg, Shollie -
item Ryan, Peter -
item Willard, Scott -
item Schmidt, Ty -

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 22, 2011
Publication Date: N/A

Technical Abstract: The use of biophotonic microbes can allow researchers to gain a better understanding of mechanisms utilized by bacteria to grow and colonize within the ruminant gastrointestinal tract, thus allowing the investigation of how stress management and nutrition impact pathogen shedding in ruminants. However, before biophotonic bacteria can be introduced into an animal model, it is imperative to characterize the growth of the bacterium in relation to its non-transformed wild-type (WT) strain to validate the model. The goal of this study was to characterize the growth of the Escherichia coli O157:H7 (ATCC 43888) strain transformed with the pAK-1 biophotonic plasmid against that of the WT in rumen fluid medium.Rumen contents obtained from a cannulated Holstein steer were filtered (Leyendecker et al., 2004) and added to the rumen medium (Russell and Martin, 1984). Cultures of the ATCC 43888 WT and pAK-1 biophotonic strain were allowed to grow to mid-log phase (OD600 ~0.5) at 37 degrees C in tryptic soy broth. Cultures were then diluted 1:100 in 30 mL of rumen fluid media and incubated at 140 rpm at 37 degrees C for 48 hours. Aliquots of each culture were obtained after 1, 2, 3, 4, 5, 6, 7, 8, 18, 24, and 48 h of growth in the rumen fluid medium. At each time interval, the WT and pAK-1 biophotonic strain were serially diluted in 1X phosphate buffer solution. Aliquots from WT were plated in duplicate on tryptic soy agar (TSA) and aliquots from the pAK-1 biophotonic strain were plated in duplicate on TSA and TSA + ampicillin (amp; 30 ug/mL).Plates were analyzed after 12 h incubation at 37 degrees C. There was no main effect (P>0.05) between the growth kinetics of the WT and the pAK-1 biophotonic strain during the 48 h growth period. Maximum bacterial concentrations were achieved at 18 h post inoculation: 1.1 x109 CFU/mL (WT), 7.4 x 108 CFU/mL (pAK-1 on TSA-amp), and 6.4 x 108 CFU/mL (pAK-1 on TSA). After 48 h of growth in the rumen fluid medium, both the WT and pAK-1 biophotonic strain had significant reductions in growth in comparison to 18 h of growth (P=0.09). No significant differences (P>0.05) were observed for the pAK-1 biophotonic strain between growth on TSA or TSA + amp, indicating that the plasmid was stable for the 48 h growth period in rumen fluid. Additionally, this study indicates that the presence of the pAK-1 plasmid does not impede nor influence the ability of the ATCC 43888 strain to grow in rumen fluid. These results indicate that the concept of biophotonically transformed E. coli O157:H7 can be a viable tool for characterizing the colonization and growth of E. coli in the gut of ruminants in vitro and in vivo.

Last Modified: 10/25/2014
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