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United States Department of Agriculture

Agricultural Research Service

Research Project: PREVENTION OF LOSSES FROM COLIBACILLOSIS AND O157:H7 AND OTHER SHIGA TOXIN-PRODUCING E. COLI (STEC) IN CATTLE AND SWINE Title: Characterization of a 3.3-KB Plasmid of Escherichia Coli O157:h7 and Evaluation of Stability of Genetically Engineered Derivatives of This Plasmid Expressing Green Fluorescence

Authors
item Sharma, Vijay
item Stanton, Thaddeus

Submitted to: Veterinary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 20, 2008
Publication Date: May 24, 2008
Citation: Sharma, V.K., Stanton, T.B. 2008. Characterization of a 3.3-kb plasmid of Escherichia coli O157:H7 and evaluation of stability of genetically engineered derivatives of this plasmid expressing green fluorescence. Veterinary Microbiology. Available: http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD6-4SKB3CT-2&_user=6956098&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000052423&_version=1&_urlVersion=0&_userid=6956098&md5=4acc7d9e43e9b59307dc8e59dc8022c6.

Interpretive Summary: Escherichia coli O157:H7, a Shiga toxin-producing E. coli (STEC), is the most frequent cause of bloody diarrhea in humans. In certain age groups, O157:H7 infections may result in damage to the kidneys and nervous system, a potentially fatal form of the disease. E. coli O157:H7 has been associated with the most important foodborne outbreaks in the United States of America and Canada. Cattle are considered the major reservoir for E. coli O157:H7 and these animals can secrete E. coli O157:H7 in their feces for extended periods of time. Most human infections result from consumption of undercooked ground beef, milk, produce and water that inadvertently become contaminated with cattle feces. Thus, understanding of factors (bacterial and host) responsible for persistence of E. coli O157:H7 in cattle warrants development of biological tools for easy monitoring and enumeration of E. coli O157:H7 in cattle feces. In the present study, we describe that a small plasmid harbored by E. coli O157:H7 can be genetically modified for stable expression of green fluorescence and resistance to antibiotic ampicillin under conditions simulating to some extant the environment of cattle intestine. Practical applications of having this modified plasmid would be the ease with which we will be able to monitor fecal shedding of genetic variants of E. coli O157:H7 (containing this modified plasmid) relative to that of an unmodified parent strain of E. coli O157:H7 (lacking the modified plasmid) in cattle coinfected with a mixture of these strains.

Technical Abstract: Enterohemorrhagic Escherichia coli (EHEC) O157:H7 (strain 86-24) harbors a 3.3 kb, cryptic plasmid (pSP70) that does not encode a selectable phenotype. A transposon (Tn) encoding kanamycin resistance (Kan**r) was inserted by in vitro transposon mutagenesis at a random location on pSP70 to construct pSP70-Kan**r that conferred Kan**r to the host E. coli strain. Oligonucleotides complementary to nucleotide sequences located at 5’ and 3’ ends of Tn were used for initiating nucleotide sequencing from plus and minus strands of pSP70-Kan**r, and thereafter primer walking was used to determine complete nucleotide sequence of pSP70. Analysis of nucleotide sequence revealed that pSM70 contained 3305 base pairs in its genome and this genome was almost 100% homologous to the genome of small plasmids identified in EHEC O157:H7 isolates from Germany and Japan. A DNA cassette encoding a green fluorescent protein (GFP), ampicillin resistance (Amp**r), and a double transcriptional terminator (DT) was cloned in pSP70 either at the BamH1 site (created by deletion of mobA by PCR) or at the Nsi1 site located downstream of mobA. Introduction of pSP70- GFP/Amp**r/DT or pSP70 delta mobA-GFP/Amp**r/DT into EHEC O157:H7 (strain 86-24) resulted in the selection of ampicillin-resistant colonies that glowed green under UV illumination. Repeated cultivation of isolates of EHEC O157:H7 carrying either of these plasmids in LB-broth under conditions simulating the environment of bovine intestine (no selective antibiotic, incubation temperature of 39 degree C, and with or without oxygen) resulted in no significant difference in the recovery of green fluorescence positive isolates on LB-agar medium with or without ampicillin. These findings indicate that pSP70- derivatives carrying a DNA cassette encoding GFP/Amp**r/DT could serve as important markers for tracking EHEC O157:H7 in feces and tissues of bovine animal models commonly used for studying persistence and shedding of this important food-borne pathogen.

Last Modified: 4/19/2014