Bacteriocin-producing Strains Research Articles

FUNDAMENTALS AND PERSPECTIVES FOR THE USE OF BACTERIOCINS PRODUCED BY LACTIC ACID BACTERIA IN MEAT PRODUCTS

Bacteriocins of lactic acid bacteria (LAB) are proteinaceous compounds that may present antimicrobial activity towards important foodborne pathogens and spoilage-related microflora. Due to these properties, bacteriocin-producing strains or purified bacteriocins have a great potential of use in biologically based food preservation systems. Despite the growing number of articles describing the isolation of bacteriocinogenic strains, genetic determinants for production, as well as the purification and biochemical characterization of these inhibitory substances, there are only limited reports of successful application of bacteriocins to meats. This paper presents a critical review of the methods available for screening of bacteriocin-producing LAB strains from meats and also discusses the proposed mechanisms of action for LAB bacteriocins. Additionally, an overview of the Brazilian experience in the application of LAB bacteriocins to meats and meat products is given.

Luminescent method for the detection of antibacterial activities.

A new rapid and sensitive method for the detection of antibacterial activities was based on luminescent indicator strains. Listeria innocua 8811 and Enterococcus faecalis 32 were transformed with plasmid carrying bacterial luciferase genes. Subsequent strains became capable to emit light during the exponential growth phase. The addition of bacteriocin containing culture supernatants to such cultures induced a drop of their light emission which was correlated to the combined antibacterial activity of acid stress and bacteriocin. The detection of antagonistic activity is independent of its mode of action, i.e. bactericidal or bacteriostatic. This method allowed to directly visualize the antagonistic activity of bacteriocin producer strains toward target strains in coculture experiments. However, a control co-culture with non-producing bacteriocin mutant was necessary in order to distinguish between nutrients competition and bacteriocin activity. Finally, five class IIa bacteriocins were purified from culture supernatants of eight strains detected in 3 days from a 120 lactic acid bacteria collection.

Modelling of the growth of populations of Listeria monocytogenes and a bacteriocin-producing strain ofLactobacillus in pure and mixed cultures

The cell growth biokinetics of Listeria monocytogenes and a bacteriocin-producing strain ofLactobacillus sake were studied. The organisms were grown both separately and together in a broth system. It was observed that Lact. sake was better suited to growth at low pH than L. monocytogenes. The presence of bacteriocin impacted on L. monocytogenes, by quickly reducing the number of these microbes in mixed populations. A model for the growth ofL. monocytogenes and Lact. sake populations grown separately and in mixed culture is proposed. The model includes a novel approach for better describing the effect of pH change, the ability of Lact. sake to produce bacteriocin and the effect of bacteriocin on L. monocytogenes in the broth culture. Additionally, terms are introduced that describe the competitive effect of one population on the other. The model is fitted to data collected for the growth of each population, either grown alone or together. There were no significant differences between the parameter estimates for each replicate, supporting the hypothesis that the proposed model is a useful representation of the growth and interaction of the two microbial populations.

Modelling contributes to the understanding of the different behaviour of bacteriocin-producing strains in a meat environment

Bacteriocins from lactic acid bacteria are potent antibacterial agents that enable the producer cells to selectively and efficiently inhibit a part of the competing background flora, including several spoilage bacteria and foodborne pathogens. The use of bacteriocinogenic lactic acid bacterium strains, either as starters or as protective cultures, is very limited in the food industry, possibly because many questions about the in situ functionality of such strains remain unanswered. A modelling approach was developed to study the functionality of lactic acid bacterium bacteriocin producers in a meat environment. The kinetic properties of three potential bacteriocin-producing starter cultures for sausage fermentation, namely Lactobacillus sakei CTC 494, Lactobacillus curvatus LTH 1174, and Lactobacillus amylovorus DCE 471 were compared in MRS, used as a meat simulation medium. Moreover, the application possibilities of the bacteriocin-producing Enterococcus faecium RZS C5 strain were evaluated. It turned out that growth and bacteriocin production by Lb. sakei CTC 494 and Lb. curvatus LTH 1174 are adapted to the meat environment, whereas Lb. amylovorus DCE 471 is kinetically not able to dominate the meat fermentation process. Furthermore, the use of bacteriocin-producing enterococci as functional cocultures seems to be attractive.

Lactococcus lactis subsp. lactis LTM 32, a new bacteriocin-producing strain isolated from Vietnamese fermented milk

Screening for bacteriocin production of 60 strains of lactic acid bacteria (LAB) both isolated from various Vietnamese fermented food and obtained from the culture collection resulted in the detection of a bacteriocin-producing strain, Lactococcus lactis subsp. lactis LTM 32 isolated from Vietnamese fermented milk. This strain was identified and partly characterized. This bacteriocin inhibited not only closely relative strains of LAB but also strains of Listeria monocytogenes, Bacillus sp. including B. cereus, B. stearothermophilus, B. subtilis, B. pumilus and Staphylococcus aureus. It was heat stable at 121°C for 15 min, at 100°C for 120 min and stable during storage at –20°C and 4°C for 3 months. The bacteriocin was inactivated by some proteolytic enzymes, namely by proteinase K and pronase E in concentrations up to 0.5 mg/ml but not by  α-amylase and pepsin. The optimal growth and bacteriocin production were achieved in M17L broth at 30°C and initial pH 6.8. The characteristics of bacteriocin-producing strain Lactococcus lactis subsp. lactis LTM 32 are of interest for food safety.

Antagonistic effect on Listeria monocytogenes and L. innocua of a bacteriocin-like metabolite produced by lactic acid bacteria isolated from sucuk

Two Lactobacilli and four Pediococci strains producing bacteriocin-like metabolities isolated from sucuk were tested with agar spot tests and well diffusion assays for their inhibitory activity against 16 Listeria strains, also isolated from sucuk. The production of organic acids and hydrogen peroxide limited, L. sake Lb 706 (used as a bacteriocin producer strain) and the isolated lactic acid bacteria (LAB) showed inhibitory activity against all of the Listeria strains, while L. sake Lb 706-A (used as a bacteriocin non-producer mutant) had the same effects against only two Listeria monocytogenes strains (51, 52) in agar spot tests. In the well diffusion assays, while L sake Lb 706 and four Pediococci isolates (413, 416, 419, 446) exhibited inhibitory activity against all of the Listeria strains tested, L. sake Lb 706-A and two of the Lactobacilli isolates ( 77, 116) showed no effect on the Listeria strains tested.

Evidence for dual functionality of the operon plnABCD in the regulation of bacteriocin production in Lactobacillus plantarum.

The regulatory operon (plnABCD) involved in bacteriocin production in Lactobacillus plantarum C11 encodes four different proteins: a cationic prepeptide (PlnA); a histidine protein kinase (PlnB); and two highly homologous response regulators (PlnC and PlnD; over 75% sequence similarity). The mature product of PlnA, plantaricin A, serves as an extracellular pheromone that induces bacteriocin production. The exact roles of plnBCD in bacteriocin production have not been established experimentally. A reporter system containing the gusA gene fused with the plnA promoter was used to study plnABCD. We demonstrated that the plnABCD operon codes for an autoregulatory unit capable of activating its own promoter. Deletion analyses, performed in a heterologous expression host to define the roles of the individual genes, confirmed that both the inducer gene (plnA) and the kinase gene (plnB) are required for autoactivation. Apparently, the latter gene encodes a protein that serves as a receptor for the pheromone peptide. It was also demonstrated conclusively that the two regulators PlnC and PlnD, which have been shown previously to bind specifically to the DNA regulatory repeats of the plnA promoter, possess differential activities on the plnA promoter, with PlnC being much more active than PlnD. The functions of the response regulators were investigated further in the bacteriocin producer strain C11 in order to reveal their roles in bacteriocin production. Surprisingly, the two response regulators display totally opposite functions: although overexpression of plnC activated transcription and bacteriocin production, the overexpression of plnD repressed both processes, thus strongly suggesting that PlnD plays a role in the downregulation of bacteriocin synthesis. To our knowledge, this is the first evidence for a protein involved directly in negative regulation of bacteriocin production, and also it was shown for the first time that two highly homologous response regulators, with opposite functions, are encoded by genes located on the same operon.

An analysis of bacteriocins produced by lactic acid bacteria isolated from malted barley.

The aim of this study was to perform a detailed characterization of bacteriocins produced by lactic acid bacteria (LAB) isolated from malted barley. Bacteriocin activities produced by eight LAB, isolated from various types of malted barley, were purified to homogeneity by ammonium sulphate precipitation, cation exchange, hydrophobic interaction and reverse-phase liquid chromatography. Molecular mass analysis and N-terminal amino acid sequencing of the purified bacteriocins showed that four non-identical Lactobacillus sakei strains produced sakacin P, while four Leuconostoc mesenteroides strains were shown to produce bacteriocins highly similar or identical to leucocin A, leucocin C or mesenterocin Y105. Two of these bacteriocin-producing strains, Lb. sakei 5 and Leuc. mesenteroides 6, were shown to produce more than one bacteriocin. Lactobacillus sakei 5 produced sakacin P as well as two novel bacteriocins, which were termed sakacin 5X and sakacin 5T. The inhibitory spectrum of each purified bacteriocin was analysed and demonstrated that sakacin 5X was capable of inhibiting the widest range of beer spoilage organisms. All bacteriocins purified in this study were class II bacteriocins. Two of the bacteriocins have not been described previously in the literature while the remaining purified bacteriocins have been isolated from environments other than malted barley. This study represents a thorough analysis of bacteriocin-producing LAB from malt and demonstrates, for the first time, the variety of previously identified and novel inhibitory peptides produced by isolates from this environment. It also highlights the potential of these LAB cultures to be used as biological controlling agents in the brewing industry.

Determination of the growth stages of Lactobacillus paracasei subsp. paracasei M3 from Bulgarian yellow cheese by electroconductivity

The growth curve and the bacteriocin production by Lactobacillus paracasei subsp. paracasei M3—a strain isolated from Bulgarian yellow cheese, was studied using electroconductivity measurements in the culture medium (MRS). The bacteriocin produced by this bacterium inhibits strains belonging to the Candida species as well as Bacillus subtilis ATCC 6633. The use of the direct impedance technique coupled with statistical treatment of the results for the strain's growth curve allowed the detection of the growth phase-related bacteriocin production. Definition of the average particle size and aggregation estimations for the cells of the bacteriocin-producing strain was also possible. The comparison of results of impedance measurements with results obtained by classic microbiological methods and microscopic observations confirms the good correlation between these methods. In addition, the advantages of an impedance method such as rapidity, simplicity and higher sensitivity are highlighted.

Detection and characterization of a bacteriocin, garviecin L1-5, produced by Lactococcus garvieae isolated from raw cow's milk.

The identification of a bacteriocin-producing lactococcal strain isolated from raw cow's milk is reported, along with production conditions, physical and chemical properties, and mode of action of the bacteriocin. On the basis of resistance to clindamycin, species-specific PCR and amplification of the 16S-23S rDNA spacer region, the strain was identified as Lactococcus garvieae. Its bacteriocin, designated garviecin L1-5, was bactericidal against closely related species and strains of species from different genera, including Listeria monocytogenes and Clostridium spp. Garviecin L1-5 was shown to be proteinaceous by protease inactivation and was unaffected by heat treatments, also at low pH values. When amplifying known lactococcal bacteriocin genes using DNA from strain L1-5 as template, no amplification products were observed on the agarose gel. The molecular weight of garviecin L1-5 was about 2.5 kDa. As far as is known, no bacteriocins have been detected from Lactococcus garvieae. The general properties of garviecin L1-5 are characteristic of the low-molecular-weight bactericidal peptide group. The survey of micro-organisms for novel antimicrobial substances provided valuable information on their physiology, ecology and practical application.

Control of Listeria monocytogenes by bacteriocins and monitoring of bacteriocin-producing lactic acid bacteria by colony hybridization in semi-hard raw milk cheese.

The ability of Listeria monocytogenes to grow at low temperatures and increase to hazardous levels during storage of dairy products is of great concern to the dairy industry. List. monocytogenes was detected in 3·6% of 774 cows' milk samples from 114 farms in Central Spain (Gaya et al. 1998). Ewes' and goats' milks produced in Spain had contamination levels of 2·2 and 2·6% respectively (Rodríguez et al. 1994; Gaya et al. 1996). List. monocytogenes may survive during the manufacture and ripening of most cheese varieties, with higher risk in soft surface-ripened cheeses owing to the rise in pH during ripening (Maisnier-Patin et al. 1992; Giraffa & Carminati, 1997).The efficacy with which actively growing bacteriocin-producing cultures inhibit List. monocytogenes in some cheese varieties has been demonstrated. Nisin-producing starters inhibited List. monocytogenes V7 in Camembert cheese (Maisnier-Patin et al. 1992) and List. innocua BL86/26 in raw ewes' milk Manchego cheese (Rodríguez et al. 1998). A lacticin 3147-producing starter culture (McAuliffe et al. 1999) reduced List. monocytogenes Scott A levels in cottage cheese. Inoculation of milk with enterocin AS-48-producing Enterococcus faecalis INIA 4 inhibited List. monocytogenes strains Ohio and Scott A during the manufacture and ripening of Manchego cheese (Nuñez et al. 1997).Semi-hard cheese varieties from raw or pasteurized cows' milk mixed with different proportions of ewes' and/or goats' milk represent ∼ 50% of Spanish cheese production. The objective of the present work was to evaluate the antilisterial activity of three bacteriocin-producing lactic acid bacteria strains isolated from milk (Rodríguez et al. 2000) during the manufacture and ripening of a semi-hard cheese manufactured from raw milk.

The Effects of Cultivating Lactic Starter Cultures with Bacteriocin-Producing Lactic Acid Bacteria

The effects of bacteriocins produced by six strains of lactic acid bacteria on 9 mesophilic and 11 thermophilic commercial starter cultures were investigated in mixed cultures of commercial starters with bacteriocin-producing strains in milk. The bacteriocins produced by the test organisms were nisin A, nisin Z, lacticin 481, enterocin AS-48, a novel enterocin, and a novel plantaricin. Mesophilic commercial starters were in most cases tolerant of bacteriocins, with only two of the starters being partially inhibited, one by four and the other by two bacteriocins. The aminopeptidase activities of mesophilic starters were generally low, and only one of the combinations of mesophilic starter–bacteriocin producer gave double the aminopeptidase activity of the starter culture without the bacteriocin producer. Thermophilic commercial starters were more sensitive to bacteriocins than mesophilic starters, with six thermophilic starters being partially inhibited by at least one of the bacteriocins. Their aminopeptidase activities were generally higher than those of the mesophilic starters. The aminopeptidase activities of seven thermophilic starters were increased in the presence of bacteriocins, by factors of up to 9.0 as compared with the corresponding starter cultures alone. Bacteriocin-producing strains may be used as adjunct cultures to mesophilic starters for the inhibition of pathogens in soft and semihard cheeses, because mesophilic starters are rather tolerant of bacteriocins. Bacteriocin producers may also be used as adjunct cultures to thermophilic starters of high aminopeptidase activity, more sensitive to lysis by bacteriocins than mesophilic starters, for the acceleration of ripening in semihard and hard cheeses.

Characterization of bacteriocin N15 produced by enterococcus faecium N15 and cloning of the related genes

Enterococcus faecium N15 was isolated from nuka (Japanese rice-bran paste), which is utilized as starter in the fermenting of vegetables, and was found to produce a bacteriocin that exhibited a broad spectrum of activity, including activity against Listeria monocytogenes and Bacillus circulans JCM2504. The bacteriocin was sensitive to proteases (α-chymotrypsin, proteinase K, trypsin, and pepsin) and α-amylase, but it was resistant to lipase. The bacteriocin was resistant to heat treatment at 100°C for 2 h, but its activity was completely lost after autoclaving at 121°C for 15 min. It was active over a wide pH range from 2.0 to 10.0. The bacteriocin showed bactericidal activity against Lactobacillus sake JCM1157 at a concentration of 40 AU/ml. Its molecular weight was estimated by SDS—PAGE to be about 3–5 kDa. PCR primers were designed based on the conserved amino acid sequences of class IIa bacteriocins. A 3-kb DNA fragment was amplified and three open reading frames (ORFs) were found. The first encodes a probable immunity protein of 103 amino acid residues and shows complete homology with the putative immunity protein of E. faecium DPC1146. The second and third ORFs respectively encode a probable transposase gene and an inducing factor. The upstream region of the immunity gene, in which the bacteriocin structural gene is located, was amplified. A homology search revealed that the bacteriocin produced by E. faecium N15 exhibits complete identity to enterocin A, a bacteriocin produced by E. faecium DPC1146. PCR using the primers designed in this study is a rapid and sufficient method of screening for bacteriocin-producing strains.

Characterization of Bacteriocin N15 Produced by Enterococcus faecium N15 and Cloning of the Related Genes.

Enterococcus faecium N15 was isolated from nuka (Japanese rice-bran paste), which is utilized as starter in the fermenting of vegetables, and was found to produce a bacteriocin that exhibited a broad spectrum of activity, including activity against Listeria monocytogenes and Bacillus circulans JCM2504. The bacteriocin was sensitive to proteases (alpha-chymotrypsin, proteinase K, trypsin, and pepsin) and alpha-amylase, but it was resistant to lipase. The bacteriocin was resistant to heat treatment at 100 degrees C for 2 h, but its activity was completely lost after autoclaving at 121 degrees C for 15 min. It was active over a wide pH range from 2.0 to 10.0. The bacteriocin showed bactericidal activity against Lactobacillus sake JCM1157 at a concentration of 40 AU/ml. Its molecular weight was estimated by SDS-PAGE to be about 3-5 kDa. PCR primers were designed based on the conserved amino acid sequences of class IIa bacteriocins. A 3-kb DNA fragment was amplified and three open reading frames (ORFs) were found. The first encodes a probable immunity protein of 103 amino acid residues and shows complete homology with the putative immunity protein of E. faecium DPC1146. The second and third ORFs respectively encode a probable transposase gene and an inducing factor. The upstream region of the immunity gene, in which the bacteriocin structural gene is located, was amplified. A homology search revealed that the bacteriocin produced by E. faecium N15 exhibits complete identity to enterocin A, a bacteriocin produced by E. faecium DPC1146. PCR using the primers designed in this study is a rapid and sufficient method of screening for bacteriocin-producing strains.

Isolation of a bacteriocin-producing Lactococcus lactis subsp. lactis and application to control Listeria monocytogenes in Moroccan jben.

Use of a bacteriocin-producing lactococcal strain to control Listeria monocytogenes in jben. A Lactococcus lactis strain isolated from lben was shown, by the spot technique, to produce a bacteriocin different from nisin. Inhibitory activity of the bacteriocin-producing strain against Listeria monocytogenes was investigated in jben, made from cow's milk fermented with the producer organism and contaminated with 104 or 107 cfu ml-1. Listeria counts were monitored during manufacture, and during conservation at room and at refrigeration temperatures. Results showed that the pathogen was reduced by 2.7 logarithmic units after 30 h of jben processing when the initial inoculum of 107 cfu ml(-1) was used. For the initial inoculum of 104 cfu ml(-1), the bacterium was completely eliminated at 24 h. Furthermore, the use of the bacteriocin-producing starter culture extended the shelf-life of jben by 5 days. In situ production of the lactococcal bacteriocin is an efficient biological means of controlling L. monocytogenes in jben and of allowing shelf-life extension. The proposed technology will essentially benefit minimally processed dairy products and those made with raw milk.

Effect of metronidazole on the growth of vaginal lactobacilli in vitro.

Objective: To determine whether metronidazole has an adverse effect on the growth of Lactobacillus. Methods: Hydrogen peroxide- and bacteriocin-producing strains of Lactobacillus were used as test strains. Concentrations of metronidazole used ranged from 128 to 7000 μg/ml. Susceptibility to metronidazole was conducted by the broth microdilution method recommended by the National Committee for Clinical Laboratory Standards. Results: Growth of Lactobacillus was partially inhibited at concentrations between 1000 and 4000 μg/ml (p = 0.014). Concentrations ≥ 5000 μg/ml completely inhibited growth of Lactobacillus. Concentrations between 128 and 256 μg/ml stimulated growth of Lactobacillus (p = 0.025 and 0.005, respectively). Concentrations of metronidazole between 64 and 128 μg/ml or ≥ 512 μg/ml did not have an inhibitory or a stimulatory effect on the growth of Lactobacillus compared to the control. Conclusions: High concentration of metronidazole, i.e. between 1000 and 4000 μg/ml, partially inhibited the growth of Lactobacillus. Concentrations ≥ 5000 μg/ml completely suppressed the growth of Lactobacillus. Concentrations between ≥ 128 and ≤ 256 μg/ml stimulated the growth of Lactobacillus. Further investigation to determine the ideal concentration of metronidazole is needed in order to use the antimicrobial agent effectively in the treatment of bacterial vaginosis.

Isolation and characterization of a new bacteriocin from Lactobacillus gasseri KT7.

A bacteriocin-producing Lactobacillus gasseri strain, KT7, was isolated from infant faeces. The supernatant fluid showed inhibitory activity not only against some lactic acid bacteria but also, against some pathogenic and food-spoilage species, including Clostridium, Listeria and Enterococcus. An antimicrobial peptide designated gassericin KT7 was isolated from Lactobacillus gasseri KT7. It was purified to homogeneity by a single four-step procedure: a crude supernatant fluid obtained from early stationary-phase culture in MRS medium was subjected to ammonium sulphate fractionation, CM-Sephadex cation-exchange chromatography, Phenyl-Sepharose hydrophobic chromatography and reverse-phase HPLC chromatography. Gassericin KT7 was sensitive to proteolytic enzymes, resistant to heat, active over a wide range of pH, and migrated as a 4.5-5.0 kDa peptide on SDS-PAGE. The bacteriocin was produced constitutively during exponential growth. It was bactericidal to sensitive cells and the bactericidal effect was not produced by cell lysis. The amino acid composition of the bacteriocin was determined and no modified amino acid was found among the residues identified.

A C-Terminal Disulfide Bridge in Pediocin-Like Bacteriocins Renders Bacteriocin Activity Less Temperature Dependent and Is a Major Determinant of the Antimicrobial Spectrum

Several lactic acid bacteria produce so-called pediocin-like bacteriocins that share sequence characteristics, but differ in activity and target cell specificity. The significance of a C-terminal disulfide bridge present in only a few of these bacteriocins was studied by site-directed mutagenesis of pediocin PA-1 (which naturally contains the bridge) and sakacin P (which lacks the bridge). Introduction of the C-terminal bridge into sakacin P broadened the target cell specificity of this bacteriocin, as illustrated by the fact that the mutants were 10 to 20 times more potent than the wild-type toward certain indicator strains, whereas the potency toward other indicator strains remained essentially unchanged. Like pediocin PA-1, disulfide-containing sakacin P mutants had the same potency at 20 and 37 degrees C, whereas wild-type sakacin P was approximately 10 times less potent at 37 degrees C than at 20 degrees C. Reciprocal effects on target cell specificity and the temperature dependence of potency were observed upon studying the effect of removing the C-terminal disulfide bridge from pediocin PA-1 by Cys-->Ser mutations. These results clearly show that a C-terminal disulfide bridge in pediocin-like bacteriocins contributes to widening of the antimicrobial spectrum as well as to higher potency at elevated temperatures. Interestingly, the differences between sakacin P and pediocin PA-1 in terms of the temperature dependency of their activities correlated well with the optimal temperatures for bacteriocin production and growth of the bacteriocin-producing strain.

Inhibition of Listeria monocytogenes by In Situ Produced and Semipurified Bacteriocins of Carnobacterium spp. on Vacuum-Packed, Refrigerated Cold-Smoked Salmon

Listeria monocytogenes inhibition by Carnobacterium strains and crude bacteriocins on sterile and commercial vacuum-packed cold-smoked salmon stored at 4°C and 8°C was investigated. Carnobacterium piscicola V1 was bactericidal against L. monocytogenes at the two temperatures, whereas Carnobacterium divergens V41 presented a bacteriostatic effect. C. piscicola SF668 delayed L. monocytogenes growth at 8°C and had a bacteriostatic effect at 4°C. Listeria growth was not affected by a non–bacteriocin-producing C. piscicola. Crude extracts of piscicocins were bactericidal at 4°C and 8°C. Listeria growth was delayed by divercin V41 at 8°C and was inhibited at 4°C. Nisin delayed Listeria growth at 8°C and was bacteriostatic at 4°C. The present study demonstrates that L. monocytogenes growth could be prevented on vacuum-packed cold-smoked salmon by Carnobacterium and associated bacteriocins at chilled temperatures. Moreover, no product spoilage could be observed with the use of such bacteriocin-producing strains as demonstrated by good sensorial analyses and low biogenic amine production.

Biochemical and genetic evidence of enterocin P production by two Enterococcus faecium-like strains isolated from fermented sausages.

Two bacteriocin-producing Enterococcus faecium-like strains were independently isolated from fermented sausages. Bacteriocins were purified to homogeneity by ammonium sulfate precipitation, gel filtration, cationic exchange, hydrophobic interaction, and reverse-phase liquid chromatography. Two peptide inhibitory fractions were purified from each strain, denominated A and B for E. faecium AA13, and C and D for E. faecium G16. Fraction B was blocked for amino acid sequencing by Edman degradation, while the amino acid sequences obtained from peptides A, C, and D contained the YGNGV consensus motif in positions 5 to 9, and the ATRS sequence in positions 1 to 4. By use of PCR techniques and nucleotide sequencing, the structural gene of enterocin P was found both in E. faecium AA13 and E. faecium G16. Metabolic and genetic features of the two strains suggest that they are slightly different, they may produce more than one bacteriocin, and both produce enterocin P.