1520 Journal oj'Food Protection, Vol. 60, No. 12, 1997, Pages 1520-1528 Copyright©, International Association of Milk, Food and Environmental Sanitarians Isolation and Purification of Colicin ECl 12, a Bacteriocin That Inhibits Strains of Escherichia coli 0157:H7t WANDA J. LYONH and DENNIS G. OLSON2 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/12/1520/2301394/0362-028x-60_12_1520.pdf by guest on 01 October 2021 IDepartment of Food Science, Louisiana State University Agricultural Center, Louisiana State University, BalOn Rouge, Louisiana 70803; and 2Department oj'Animal Science, 10wa State University, Ames, 10wa 50011, USA (MS# 96-299: Received 28 October 1996/Accepted 16 March 1997) ABSTRACT tion of raw milk (6, 8). Vegetables, water, and other meat products have also been linked as vehicles for transmission A swine fecal isolate, identified as Escherichia coli ECLl2, of E. coli 0157:H7 infections (17). was found to produce an antimicrobial substance designated as The work reported in this paper was initiated with the colicin ECLl2. Colicin ECLl2 was inhibitory against 20 strains of purpose of finding bacteriocin-producing strains that could E. coli 0157:H7 previously isolated from both human and bovine effectively inhibit E. coli 0157:H7. Since cattle are reser- feces. Identification of the producer strain was determined pheno- voirs for E. coli 0157:H7, bovine feces were examined for typically by biochemical and morphological tests. Colicin ECLl2 was sensitive to several proteolytic enzymes. Adsorption of colicin bacteria that might have developed a selective advantage in ECLl2 to sensitive cells of E. coli 0157:H7 was bactericidal, animals that have not tested positive for shedding of E. coli resulting in a 2 log reduction in viable cell counts. CoLicinECLl2 0157:H7. In this study a bacteriocin produced by a species was purified from strain ECLl2 by cell extraction and ion- of E. coli was isolated, purified, and found to be a protein exchange chromatography. Sodium dodecyl sulfate-polyacryl- with antimicrobial activity against a narrow spectrum of amide gel electrophoresis of colicin ECLl2 resolved a single microorganisms, including strains of E. coli and Shigella protein with a molecular weight of approximately 65,000. sonnei. Key words: Colicin, inhibition, E. coli 0157:H7, food preservative MATERIALS AND METHODS Colicins are antimicrobial proteins synthesized by Esch- Cultures and media erichia coli and active against closely related coliform Escherichia coli ECL 12 was isolated from bovine sewage runoff as described below. The use of colicin ECL 12 in foods and bacteria. Colicin proteins range in molecular weight from the strain that produces it are protected by U.S. patent 5,549,895. 90,000 to 1,500 (12). Colicins are typically encoded on All microorganisms used in this study were grown in media and at plasmids with the exception of colicin Land microcin E966 temperatures indicated in Tables 1 and 2. Bacteriocin-producing (12, 18). A large number of colicins have been isolated and strain E. coli ECLl2 and sensitive E. coli 0157:H7 indicator strains characterized by many investigators, For additional perti- were propagated at 37°C in Trypticase soy broth (TSB) (BBL, nent information the reader is directed to previously pub- Becton Dickinson, Cockeysville, MD) supplemented with 0.5% lished reviews (12, 18). yeast extract (TSYE) (Difco Laboratories, Detroit, MI). Frozen Enterohemorrhagic E. coli (EHEC) strains, which in- stocks were maintained at -80°C in the appropriate medium with clude E. coli 0157:H7, are increasingly becoming recog- 50% glycerol. nized as widespread foodbome pathogens. Enterohemor- rhagic E. coli 0157:H7 causes serious illness in humans in Isolation of bacteriocin-producing bacteria the form of bloody diarrhea and hemolytic uremic syndrome Twenty-five fecal samples were obtained from several Iowa (17). EHEC strains are commonly isolated from the feces of farms. The fecal samples (25 g) were weighed into stomacher bags, clinically normal and diarrheic cattle (22). Cattle have been and 250 ml of phosphate buffer (pH 7.0) was added, the bags implicated as a reservoir for E. coli 0157:H7 (8). Human sealed, the material macerated for 5 min in a stomacher. SampLes were removed and serially diluted in 0.2% peptone, spread-plated infections and zoonotic transmission of E. coli 0157:H7 are onto both TSYE agar and violet red bile agar (VRBA) (Difco thought to occur by ingestion of ground beef and consump- Laboratories), and incubated aerobically at 37°C for 18 h. Indi- vidual colonies were transferred from VRBA plates with sterile toothpicks onto duplicate plates of TSYE and reincubated for 18h. * Author for correspondence. Tel: 504-388-5197; Fax: 504-388-5300; One plate was used for replica plating onto fresh TSYE plates and E-mail: [email protected]. the other was retained as a master plate. After growth, each plate t Joumal paper no. J-96-21-0273 of the Louisiana Agricultural Experiment was overlaid with 5 ml of TSYE containing 0.7% agar that had Station, Baton Rouge, LA. Project: S-263. been previously inoculated with different sensitive strains (20 Ill; COLICIN ECL12 1521 TABLE 1. The inhibitory effect of purified colicin ECLl2 against lated, and all were further characterized and identified as either selected gram-negative bacterial strains using the critical dilution Shigella sonnei, E. coli, or Klebsiella pneumoniae using both the method API 20E analysis as described by the manufacturer (API Analytab Products, Plainview, NY) and phenotypic characteristics listed in Reaction Table 3. Forty-five of the 50 bacteriocin-producing isolates pro- Growth to colicin duced antimicrobial activity that was not effective against E. coli Straina conditions ECL12b 0157:H7. One of the remaining isolates was characterized and Aeromonas hydrophila TSYE,37°C identified as a strain of E. coli and was found to produce an Campylobacter jejuni Thioglycollate, 37°C antimicrobial agent effective against E. coli 0157:H7. This isolate, Escherichia coli JM109 TSYE,37°C ++++ designated E. coli ECLl2, was selected for further characterization, Escherichia coli V517 TSYE,37°C ++++ isolation, and purification of its colicin. Escherichia coli 0157:H7 2903 TSYE,37°C ++++ Escherichia coli 0157:H7 2922 TSYE,37°C ++ Critical dilution assay for colicin activity Escherichia coli 0157:H7 3076 TSYE,37°C +++ Antagonistic activities of colicin ECLl2 preparations were Escherichia coli 0157:H7 2881 TSYE,37°C ++++ confirmed by spotting 5 fll of serially diluted (twofold dilution) Downloaded from http://meridian.allenpress.com/jfp/article-pdf/60/12/1520/2301394/0362-028x-60_12_1520.pdf by guest on 01 October 2021 Escherichia coli 0157:H7 3061 TSYE,37°C +++ colicin solutions onto lawns of indicator organisms as described Escherichia coli 0157:H7 2976 TSYE,37°C +++ previously (13, 14). Approximately 20 fll of an 18-h indicator Escherichia coli 0157:H7 2962 TSYE,37°C +++ culture was added to 5 ml of TSYE soft agar overlays (106 Escherichia coli 0157:H7 2972 TSYE,37°C ++++ CPU/ml), and plates were incubated at 37°C for 18 h. Activity was Escherichia coli 0157:H7 2952 TSYE,37°C +++ defined as the reciprocal of the highest 1:2 dilution causing Escherichia coli 0157:H7 3066 TSYE,37°C +++ complete inhibition of the indicator lawn and was expressed as Escherichia coli 0157: TSYE,37°C ++++ arbitrary units (AU) per m!. All assays were performed in duplicate, H7 NADC 3081 and results presented are the means of duplicate trials. Escherichia coli 0157:H7 2924 TSYE,37°C +++ Escherichia coli 0157:H7 2934 TSYE,37°C ++++ Extraction and purification of colicin ECL12 from producer cells Escherichia coli 0157:H7 2888 TSYE,37°C +++ An 18-h culture of E. coli ECLl2 (3,000 mI) was grown to Escherichia coli 0157:H7 2842 TSYE,37°C ++ early stationary phase (109 cells/ml) in M9 broth (1) at 37°C. The Escherichia coli 0157:H7 FSIS TSYE,37°C +++ cell pellets were washed with 30 ml of 50 mM potassium phosphate 45956-59A buffer (pH 7.2) as described by Herschman and Helinski (9) with Escherichia coli 0157:H7 FSIS TSYE,37°C +++ the following modifications. After the cells had been washed and 45753-57B-31 pelleted by centrifugation at 12,000X g at 4°C, the colicin was Escherichia hermanii TSYE,37°C extracted from the cell pellet by resuspending the pellets in a total ATCC 33650 volume of 100 ml of 50 mM potassium phosphate buffer (pH 7.2) Escherichia fergusonii TSYE,37°C containing 1.0 M NaCl, 1 mM EDTA, 1 mM amidinophenylmeth- ATCC 35469 anesulfonyl fluoride (APMSF), and 1 mM pepstatin (all purchased Pseudomonas fluorescens BRr,32°C from Boehringer Mannheim, Indianapolis, IN). The suspension Pseudomonas aeruginosa BRr,32°C was homogenized at low speed with a Waring blender for 30 min at Salmonella derby TSYE,37°C Salmonella dublin TSYE,37°C Salmonella newington TSYE,37°C TABLE 2. The inhibitory effect of purified colicin ECLl2 against Salmonella typhimurium ATCC TSYE,37°C selected gram-positive bacterial strains using the critical dilution 14028 method Shigella dysenteriae TSYE,37°C Reaction Shigella sonnei TSYE,37°C ++++ Growth to colicin b Shigella sonnei ATCC 9290 TSYE,37°C +++ Selected strainsa conditions ECL12 Vibrio parahaemolyticus BRr,32°C Yersinia enterocolitica ATCC TSYE,37°C Bacillus cereus BRr,37°C 23715 Clostridium perfringens Meat, 37°C Enterococcus faecium TSYE,37°C a Strains with no designation are from the culture collection of the Listeria monocytogenes Scott A TSYE,32°C Department of Microbiology at Iowa State University. E. coli Lactobacillus bulgaricus MRS, 37°C . 0157:H7 strains were obtained from the National Animal Disease Lactococcus casei MRS, 37°C Center, Ames, IA. Pediococcus cerevisiae MRS, 37°C b The reaction was assayed by the critical dilution method de- Propionibacterium shermanii TSYE,32°C scribed in the text.