497 Journal of Food Protection, Vol. 55, No. 7, Pages 497-502 (July 1992) Copyright©, International Association of Milk, Food and Environmental Sanitarians Characterization of Enterocin 1146, a Bacteriocin from Enterococcus faecium Inhibitory to Listeria monocytogenes EUGENIO PARENTE1 and COLIN HILL* The National Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, The Irish Republic Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/7/497/1661676/0362-028x-55_7_497.pdf by guest on 25 September 2021 (Received for publication November 12, 1991) ABSTRACT cultures has been proposed for Cheddar cheese (37), Fontina cheese (6), and water-buffalo Mozzarella cheese (57). Enterococcus faecium DPC 1146 produces a bacteriocin, In this report we describe the characterization of enterocin 1146, which is inhibitory to Listeria monocytogenes. Enterocin 1146 was produced in GM17 and in milk. The bacterio­ enterocin 1146, a bacteriocin produced by E. faecium cin was partially purified by ammonium sulfate precipitation. Its DPC1146, which is relatively specific to Listeria spp. molecular weight, estimated by SDS-PAGE, was 3.0 kDa. It could be stored at -20°C without loss of activity, but pH had a marked MATERIALS AND METHODS effect on enterocin 1146, which was more stable at both high (up to 120°C) and low temperatures (4°C) at pH 5 than at pH 7 and Strains and media 9. The sensitivity of 57 strains belonging to 35 different species Enterocin 1146 is produced by E. faecium DPC1146. Unless was studied using a critical dilution assay. L. monocytogenes and otherwise noted L. innocua DPC 1770 was used as the indicator L. innocua were most sensitive; enterocin 1146 had a bactericidal strain. A complete list of the strains, their growth media, and effect on Listeria. Starter and nonstarter lactic acid bacteria temperature of incubation used in this study is shown in Table 1. (except Lactobacillus sake) were insensitive or relatively resistant All strains were maintained frozen at -80°C in 25% glycerol. to the bacteriocin. Genetic determinants for bacteriocin production Enterococci were routinely propagated in GM17 (M17, Difco and immunity do not appear to be plasmid borne. Laboratories, Detroit, MI + 0.5% glucose). GM17 dialysate (DGM17) was prepared by dissolving M17 in water with the appropriate amount of glucose at 10X concentration. The solution was then dialyzed against sterile distilled water and the permeate Bacteriocins are bacterial proteins which are usually, was sterilized and used as a growth medium. but not always, inhibitory to species and strains closely related to the producer. Several bacteriocins produced by Measurement of bacteriocin activity lactic acid bacteria have been characterized (27). While A critical dilution assay was used. Culture supernatant or most have a narrow inhibitory spectrum, some, like pediocin filter sterilized bacteriocin solutions were serially diluted (two­ A (77), pediocin PA-1 (32), pediocin AcH (8), sakacin A fold) in GM17 broth, and 10 ul aliquots were spotted on GM17 (34), are also active against foodborne pathogens, like plates and dried for 30 min. The plates were overlaid with 3 ml (7 ml for Clostridia) of the appropriate soft (0.7% agar) medium, Listeria monocytogenes, and thus have a potential as natu­ preinoculated with 0.1 ml of an overnight culture of the indicator, ral food preservatives. and incubated at the appropriate temperature. One arbitrary unit The ability of enterococci to produce bacteriocins, (AU) was defined as the reciprocal of the highest dilution of some of which display a broad inhibitory spectrum, is well enterocin 1146 giving a zone of inhibition on the indicator lawn known (9). Enterocin E1A, a well-characterized bacteriocin (32). When a clear inhibition zone was followed by a turbid one, from Enterococcus faecium (22,23), has been shown to be the critical dilution was taken to be the average of the final two active against Listeria (3). According to a number of recent dilutions. reports (2,26), the ability to inhibit L. monocytogenes may be relatively widespread among enterococci. Enterococci Bacterial counts Viable bacterial cells were enumerated using a Spiral Plater have caused foodborne infections only in very rare in­ (Spiral Systems, Inc., Cincinnati, OH). E. faecium DPC1146 was stances and evidence on their role as foodborne pathogens enumerated on GM17 agar after incubation for 24 h at 37°C. L. are scant or lacking (20). They can be isolated from innocua DPC 1770 was enumerated on trypticase soy agar (BBL artisanal cheeses and starters (70), and their use in starter Microbiology Systems, Cockeysville, MD) + 0.6% yeast extract (Oxoid, Ltd., Basingstoke, Hampshire, England) after incubation for 48 h at 30°C. Colonies were enumerated using a Protos ' Present address: Dipartimento di Biologia, Difesa e Biotecnologie Colony Counter (Ai Cambridge, Ltd; Pannisford, Cambridge, Agro-Forestali, Universita' della Basilicata, 85100 Potenia Italy. England). JOURNAL OF FOOD PROTECTION, VOL. 55, JULY 1992 498 PARENTE AND HILL Table 1. Bacterial strains, media, and temperature of incubation used in this study. Relative sensitivity of a strain to partially purified enterocin 1146 (ppE1146) is calculated as the ratio between the activity of the bacteriocin (AU/ml) using that strain as indicator to that obtained on L. innocua DPC 1770 (76800 AU/ml). Strain Medium1 Temp. (°C) Source2 Relative sensitivity Bacillus cereus ATCC9139 TSB 37 TNO 0 B. subtilis BD630 TSB 37 RuG 0 B. subtilis OG1 TSB 37 RuG 0 Clostridium butyricum NCD01713 RCM 30 DPC 0.083 C. sporogenes NCD01789 RCM 37 DPC 0 C. sporogenes C 22/10 RCM 37 TNO 0 C. perfringens NCDO1800 RCM 37 DPC 0.032 C. tyrobutyricum NCD01754 RCM + SL 30 TNO 0 C. tyrobutyricum NCD01756 RCM + SL 30 DPC 0 C. tyrobutyricum NCDO1790 RCM + SL 30 DPC 0 C. tyrobutyricum 3.5 RCM + SL 30 TNO 0 Enterococcus faecium DPC 11463 GM17 37 DPC 0 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/55/7/497/1661676/0362-028x-55_7_497.pdf by guest on 25 September 2021 E. faecium DPC3342" GM17 37 DPC 0.004 E. faecalis NCD0581 GM17 37 DPC 0.063 E. faecalis NCDO610 GM17 37 DPC 0.063 £./aeca/isDPC1142 GM17 37 DPC 0.063 E. faecalis 1 GM17 37 TNO 0.042 Lactobacillus acidophilus ATCC4356 MRS 37 TNO 0 L. casei ATCC334 MRS 37 TNO 0 L. casei ssp. pseudoplantarum DPC2136 MRS 37 DPC 0 L. curvatus NCFB2739 MRS 30 TNO 0.001 L. delbrueckii subsp. bulgaricus ATCC1184 MRS 42 TNO 0.002 L. delbrueckii subp. lactis DPC 1125 MRS 37 DPC 0 L. fermentum ATCC9338 MRS 37 TNO 0 L. helveticus ATCC15009 MRS 42 TNO 0 L. helveticus DPC 1130 MRS 42 DPC 0.005 L. plantarum NCD01193 MRS 37 TNO 0.001 L. reuteri DSM20016 MRS 37 TNO 0 L. sake NCFB2714 MRS 30 TNO 0.500 L. salivarius NCFB2747 MRS 37 TNO 0 Lactococcus lactis subsp. lactis MG1614 GM17 30 DPC 0.063 L. lactis subsp. lactis DPC2612 GM17 30 DPC 0 L. lactis subsp. lactis bv. diacetylactis DPC938 GM17 30 DPC 0 L. lactis subsp. lactis bv. diacetylactis DPC979 GM17 30 DPC 0 L. lactis subsp. lactis bv. diacetylactis DPC990 GM17 30 DPC 0 L. lactis subsp. lactis bv. diacetylactis DRC3 GM17 30 DPC n.t.5 L. lactis subsp. cremoris DPC2645 GM17 30 DPC 0 L. lactis subsp. cremoris CNRZ117 GM17 30 TNO 0 Leuconostoc cremoris DB1275 MRS 25 TNO 0 Leuconostoc subsp. DPC225 MRS 25 TNO 0 Leuconostoc subsp. DPC 1061 MRS 25 DPC 0 Listeria innocua DPC 1770 TSB YE 30 DPC 1 L. innocua BL86/26 TSB YE 30 TNO 0.667 L. monocytogenes 9 TSB YE 30 DPC 0.500 L. monocytogenes DPC 1771 TSBYE 30 DPC 0.667 L. monocytogenes NCTC5348 TSB YE 30 DPC 4.000 L. monocytogenes Scott A TSBYE 30 DPC 1 Pediococcus pentosaceus FBB63 MRS 30 TNO 0.083 P. pentosaceus PCI MRS 30 TNO 0 P. acidilactici NCFB2767 MRS 30 DPC 0.002 Propionibacterium acidipropionici NCFB563 YGL 30 Cranfield 0 Propionibacterium spp. P4 YGL 30 Cranfield 0 Propionibacterium spp. P6 YGL 30 Cranfield 0 Salmonella typhimurium ATCC 14028 TSB 37 DPC 0 Staphylococcus aureus DPC 1772 TSB 37 DPC 0 S. carnosus MCI TSB 37 TNO 0 Streptococcus thermophilus DPC 1780 GM17 37 DPC 0 S. thermophilus DPC2227 GM17 37 DPC 0 RCM = Reinforced clostridial medium (Oxoid Ltd.); RCM + SL = RCM + 0.5% sodium lactate (BDH Chemicals Ltd.); TSB = trypticase soy broth (BBL Microbiology Systems); TSBYE = TSB + 0.6% yeast extract (Oxoid); GM17 = M17 (Difco Laboratories) + 0.5% dextrose (Oxoid); MRS from Difco; YGL = Yeast Glucose Lemco Broth (29). Top and bottom media for the critical dilution assay used for the measurement of sensitivity were obtained adding, respectively, 0.7% and 1.5% agar bacteriological to the broth media listed. TNO: Dr. B. ten Brink, TNO Nutrition and Food Research, Zeist, The Netherlands. RuG: Prof. G. Venema, University of Groningen, The Netherlands. DPC: National Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork. Cranfield: Cranfield Institute of Technology, Cranfield Bedford, England. Producer of enterocin 1146. Bac- derivative of DPC 1146. Not tested. JOURNAL OF FOOD PROTECTION, VOL. 55, JULY 1992 BACTERIOCIN FROM ENTEROCOCCUS FAECIUM 499 Production of enterocin 1146 in different media Adsorption of enterocin 1146 to sensitive and resistant cells An overnight culture of E. faecium DPCl 146 was used to Cells from an overnight culture of L. innocua DPCl770 were inoculate (105 CFU/ml) GM17 broth, 10% skim milk (Oxoid), and collected by centrifugation, washed twice in 50 mM potassium Elliker lactic broth (12). The inoculated media were incubated at phosphate buffer, pH 7, and then diluted 1:10 in the same buffer 37°C, and samples were removed for the measurement of bacte­ (obtaining 2.7 x 10s CFU/ml). Controls were buffer alone or cell riocin activity and viable cell counts.