Antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. of human and animal origin isolated in Portugal.

The main objective of this study was to detect the antimicrobial activity and the presence of bacteriocin structural genes in 224 enterococcal isolates from fecal origin obtained from humans, pets, wild animals and birds. Direct antimicrobial activity against Listeria monocytogenes CECT4032 was detected in 102 (45.6%) of the tested isolates. From these, only 22 displayed bacteriocin activity against this indicator. The bacteriocinogenic strains contained one or more of the bacteriocin structural genes tested in this study, with those of enterocins P, A and L50 (L50A and L50B) being the most abundant. Our results show a high occurrence of the combination of different bacteriocin structural genes in the enterococcal isolates analyzed, indicating an elevated genetic potential of these strains to produce various bacteriocins.

Molecular analysis of the replication region of the pCIZ2 plasmid from the multiple bacteriocin producer strain Enterococcus faecium L50.

The sequence analysis of the 7383 bp plasmid pCIZ2 from Enterococcus faecium L50 enabled the identification of a DNA region involved in its replication. The structural organization of the pCIZ2 replication region is highly similar to those of well-known theta-replicating plasmids. It contains an untranslated region, the putative replication origin (ori), constituted by two sets of direct repeats of 12 and 22 bp (iterons), and followed by three open-reading frames (orf8 to orf10). orf8 encodes the replication initiation protein (RepE). The transcriptional start site of the replication locus was identified 13 nucleotides upstream of the repE start codon. A two-dimensional agarose gel electrophoresis analysis revealed pCIZ2 intermediates profile typical of the theta-type replication mechanism. Subcloning of different DNA fragments of the pCIZ2 replication region in Escherichia coli and, subsequently, in the plasmidless E. faecium L50/14-2 allowed the determination of the minimal replicon on a 1.2kb DNA fragment containing only the overall ori and repE which also act in trans. The involvement of orf9 in the plasmid copy number and in the plasmid stability was investigated. The pCIZ2 recombinant plasmids constitute narrow-host range shuttle cloning vectors (E. coli-E. faecium) that could be very useful for enterococcal genes studies, allowing an easy identification due to their histochemical recognition.

Antimicrobial and safety aspects, and biotechnological potential of bacteriocinogenic enterococci isolated from mallard ducks (Anas platyrhynchos).

Samples from the intestinal content and carcasses of mallard ducks (Anas platyrhynchos) were evaluated for enterococci with antimicrobial activity, presence of genes coding bacteriocins and their expression, and potential virulence factors. Enterococcus faecalis comprised the largest enterococcal species with antagonistic activity followed by E. faecium, E. hirae, Enterococcus spp., and the non-enterococci. Although all E. faecalis isolates manifested a potent direct antimicrobial activity, no activity was detected in supernatants of most producer cultures. However, all E. faecium isolates showed antimicrobial activity in their supernatants and encoded bacteriocins, although the occurrence in the isolates of several enterocin genes did not always correlate with a higher antagonistic activity in supernatants. The efaAfm determinant was the only virulence gene detected in E. faecium, while E. faecalis showed a larger number of virulence determinants, and E. hirae did not carry any of the virulence genes examined. The rapid identification of genes coding described bacteriocins permits recognition of isolates that are potentially producers of novel bacteriocins. Purification of the antimicrobial activity of E. hirae DCH5 and Lactococcus garvieae DCC43 revealed unique chromatographic fragments after MALDI-TOF mass spectrometry analysis, suggesting the antagonistic peptides were purified to homogeneity. Bacteriocinogenic E. faecium and E. hirae isolates may be considered hygienic for production of bacteriocins, and potentially safe due to their low incidence of potential virulence genes and susceptibility to most clinically relevant antibiotics. However, the presence among the enterococci of E. faecalis strains with a potent antagonistic activity and multiple virulence factors, raises concerns regarding their potential pathogenicity to consumers.

Cloning, production and functional expression of enterocin P, a sec-dependent bacteriocin produced by Enterococcus faecium P13, in Escherichia coli.

The cloning and expression of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, was studied in Escherichia coli. PCR-amplified products of the preenterocin P gene (entP) or entP plus the putative EntP immunity gene (entiP), were cloned in plasmid pETBlue-1 under the control of the inducible T7lac promoter. Although target genes in derivative plasmids pJG01 (entP) and pJG02 (entP plus entiP) did not generate products with antimicrobial activity after an in vitro combined transcription/translation reaction, they were expressed as biologically active products following transformation and induction in the E. coli Tuner(DE3)pLacI host. The use of specific antibodies and an ELISA permitted the detection and quantification of EntP in the supernatant (SN), cellular soluble protein fraction (CSF), and inclusion bodies (IB) of E. coli Tuner(DE3)pLacI cells transformed with either pJG01 or pJG02. Functional EntP from the supernatants of E. coli Tuner(DE3)pLacI (pJG01) cultures grown in a complex medium was recovered, at a high efficiency, by immunoaffinity chromatography in a single step. A purification method based on hydrophobic adsorption and reverse-phase chromatographies also permitted the recovery of active EntP from the supernatants of the same cultures grown in a minimally defined medium. The E. coli Tuner(DE3)pLacI (pJG01) cells would merit consideration as an alternative experimental model for the heterologous production and functional expression of EntP, as well as for the fast and efficient recovery of this bacteriocin from the supernatant of this recombinant producer.

Sakacin M, a bacteriocin-like substance from Lactobacillus sake 148.

The antagonistic activity of Lactobacillus sake 148 was evaluated during its growth on complex broth media and in a semisynthetic defined medium (SDM) with various supplements. The antagonistic activity was a growth-associated property, being detected and quantified when L. sake 148 was grown at either 4, 8, 16, 25 or 32 degrees C. The concentrated culture supernatant of L. sake 148 was subjected to purification by lyophilization and gel filtration. The purification procedure resulted in a small increase in its specific activity (7-fold) and in a low recovery of the original inhibitory activity (8%). Gel filtration analysis of the partially purified activity on Sephadex G-50 revealed an apparent molecular weight of 4640. The partially purified antagonistic activity of L. sake 148 was destroyed by treatment with proteolytic enzymes. However, the antagonistic activity was resistant to heat, having D-values at 121, 135 and 150 degrees C of 23.8, 17.4 and 15.2 min, respectively.

Inhibition of Yersinia enterocolitica by Lactobacillus sake strains of meat origin.

The growth of Yersinia enterocolitica at 4, 8, 15 and 24°C, in mixed cultures with Lactobacillus sake strains previously isolated from Spanish dry fermented sausages was investigated. Growth of Y. enterocolitica was affected by L. sake strains at all temperatures studied. The inhibition was higher as the incubation temperature increased. L. sake 148, a bacteriocinogenic strain, was less inhibitory to Y. enterocolitica growth than L. sake 23, a stronger lactic acid producer strain. The low pH and the lactic acid produced by the lactobacilli seem to be major factors contributing to the inhibition of Y. enterocolitica strains.

Inhibition of Listeria monocytogenes by Lactobacillus sake strains of meat origin.

The ability of two Lactobacillus sake strains of meat origin to inhibit the growth of Listeria monocytogenes at 4, 8, 15, 24 and 32°C in a conventional liquid media was investigated. Growth of L. monocytogenes was affected by Lac. sake strains at all temperatures. The inhibition was higher at 15, 24 and 32°C than at refrigeration temperatures. The inhibitory activity of both lactobacilli was similar perhaps due to the fact that Lac. sake 148 produces a bacteriocin inhibitory to L. monocytogenes, while Lac. sake 23 is a strong lactic acid producer. The antagonism exhibited by the lactobacilli on the L. monocytogenes strains seems to display a bacteriostatic rather than a bacteriocidal effect.

PCR detection of the lactocin S structural gene in bacteriocin-producing lactobacilli from meat.

The lactocin S structural gene (lasA) in seven bacteriocinogenic lactobacilli isolated from fermented sausages was studied. Two degenerate primers were synthesized to amplify a 75-bp fragment of the gene. Three strains amplified the fragment from their plasmid DNA, and hybridization analysis confirmed these results.

Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P13 with a broad antimicrobial spectrum.

Enterocin P is a new bacteriocin produced by Enterococcus faecium P13 isolated from a Spanish dry-fermented sausage. Enterocin P inhibited most of tested spoilage and food-borne gram-positive pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum. Enterocin P is produced during growth in MRS broth from 16 to 45 degrees C; it is heat resistant (60 min at 100 degrees C; 15 min at 121 degrees C) and can withstand exposure to pH between 2.0 and 11.0, freeze-thawing, lyophilization, and long-term storage at 4 and -20 degrees C. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation, gel filtration, cation-exchange, hydrophobic-interaction, and reverse-phase liquid chromatography. The sequence of 43 amino acids of the N terminus was obtained by Edman degradation. DNA sequencing analysis of a 755-bp region revealed the presence of two consecutive open reading frames (ORFs). The first ORF encodes a 71-amino-acid protein containing a hydrophobic N-terminal sec-dependent leader sequence of 27 amino acids followed by the amino acid sequence corresponding to the purified and sequenced enterocin P. The bacteriocin is apparently synthesized as a prepeptide that is cleaved immediately after the Val-Asp-Ala residues (positions -3 to -1), resulting in the mature bacteriocin consisting of 44 amino acids, and with a theoretical molecular weight of 4,493. A second ORF, encoding a putative immunity protein composed of 88 amino acids with a calculated molecular weight of 9,886, was found immediately downstream of the enterocin P structural gene. Enterocin P shows a strong antilisterial activity and has the consensus sequence found in the pediocin-like bacteriocins; however, enterocin P is processed and secreted by the sec-dependent pathway.

Enterocin P causes potassium ion efflux from Enterococcus faecium T136 cells.

Enterocin P is a bacteriocin produced by Enterococcus faecium P13. We studied the mechanism of its bactericidal action using enterocin-P-sensitive E. faecium T136 cells. The bacteriocin is incapable of dissipating the transmembrane pH gradient. On the other hand, depending on the buffer used, enterocin P dissipates the transmembrane potential. Enterocin P efficiently elicits efflux of potassium ions, but not of intracellularly accumulated anions like phosphate and glutamate. Taken together, these data demonstrate that enterocin P forms specific, potassium ion-conducting pores in the cytoplasmic membrane of target cells.

Optimization of enterocin P production by batch fermentation of Enterococcus faecium P13 at constant pH.

The influence of pH on growth, enterocin P production and glucose consumption by Enterococcus faecium P13 was studied during anaerobic batch fermentation in MRS broth at 32 degrees C in a fermentor. Growth and glucose consumption were maximal at pH 7.0. Enterocin P production displayed primary metabolite kinetics and was strongly dependent on pH. A maximum antimicrobial activity of 1,949 bacteriocin units (BU) ml(-1) was obtained at pH 6.0, which represented a four-fold increase compared with the antimicrobial activity obtained without pH regulation. The pH exerted a marked effect on the decrease in bacteriocin activity, with the decrease being maximal at pH 7.0. In this report, we propose models for the growth of E. faecium P13 as well as enterocin P production and inactivation. Enterocin P production decreased when potentially stress-inducing compounds (NaCl or ethanol) were included in the growth medium.

Biochemical and genetic evidence that Enterococcus faecium L50 produces enterocins L50A and L50B, the sec-dependent enterocin P, and a novel bacteriocin secreted without an N-terminal extension termed enterocin Q.

Enterococcus faecium L50 grown at 16 to 32 degrees C produces enterocin L50 (EntL50), consisting of EntL50A and EntL50B, two unmodified non-pediocin-like peptides synthesized without an N-terminal leader sequence or signal peptide. However, the bacteriocin activity found in the cell-free culture supernatants following growth at higher temperatures (37 to 47 degrees C) is not due to EntL50. A purification procedure including cation-exchange, hydrophobic interaction, and reverse-phase liquid chromatography has shown that the antimicrobial activity is due to two different bacteriocins. Amino acid sequences obtained by Edman degradation and DNA sequencing analyses revealed that one is identical to the sec-dependent pediocin-like enterocin P produced by E. faecium P13 (L. M. Cintas, P. Casaus, L. S. Hâvarstein, P. E. Hernández, and I. F. Nes, Appl. Environ. Microbiol. 63:4321-4330, 1997) and the other is a novel unmodified non-pediocin-like bacteriocin termed enterocin Q (EntQ), with a molecular mass of 3,980. DNA sequencing analysis of a 963-bp region of E. faecium L50 containing the enterocin P structural gene (entP) and the putative immunity protein gene (entiP) reveals a genetic organization identical to that previously found in E. faecium P13. DNA sequencing analysis of a 1,448-bp region identified two consecutive but diverging open reading frames (ORFs) of which one, termed entQ, encodes a 34-amino-acid protein whose deduced amino acid sequence was identical to that obtained for EntQ by amino acid sequencing, showing that EntQ, similarly to EntL50A and EntL50B, is synthesized without an N-terminal leader sequence or signal peptide. The second ORF, termed orf2, was located immediately upstream of and in opposite orientation to entQ and encodes a putative immunity protein composed of 221 amino acids. Bacteriocin production by E. faecium L50 showed that EntP and EntQ are produced in the temperature range from 16 to 47 degrees C and maximally detected at 47 and 37 to 47 degrees C, respectively, while EntL50A and EntL50B are maximally synthesized at 16 to 25 degrees C and are not detected at 37 degrees C or above.

Enterocins L50A and L50B, two novel bacteriocins from Enterococcus faecium L50, are related to staphylococcal hemolysins.

Enterocin L50 (EntL50), initially referred to as pediocin L50 (L. M. Cintas, J. M. Rodríguez, M. F. Fernández, K. Sletten, I. F. Nes, P. E. Hernández, and H. Holo, Appl. Environ. Microbiol. 61:2643-2648, 1995), is a plasmid-encoded broad-spectrum bacteriocin produced by Enterococcus faecium L50. It has previously been purified from the culture supernatant and partly sequenced by Edman degradation. In the present work, the nucleotide sequence of the EntL50 locus was determined, and several putative open reading frames (ORFs) were identified. Unexpectedly, two ORFs were found to encode EntL50-like peptides. These peptides, termed enterocin L50A (EntL50A) and enterocin L50B (EntL50B), have 72% sequence identity and consist of 44 and 43 amino acids, respectively. Interestingly, a comparison of the deduced sequences of EntL50A and EntL50B with the corresponding sequences obtained by Edman degradation shows that these bacteriocins, in contrast to other peptide bacteriocins, are secreted without an N-terminal leader sequence or signal peptide. Expression in vivo and in vitro transcription/translation experiments demonstrated that entL50A and entL50B are the only genes required to obtain antimicrobial activity, strongly indicating that their bacteriocin products are not posttranslationally modified. Both bacteriocins possess antimicrobial activity on their own, with EntL50A being the most active. In addition, when the two bacteriocins were combined, a considerable synergism was observed, especially with some indicator strains. Even though the enterocins in some respects are similar to class II bacteriocins, several conserved features common to class II bacteriocins are absent from the EntL50 system. The enterocins have more in common with members of a small group of cytolytic peptides secreted by certain staphylococci. We therefore propose that the enterocins L50A and L50B and the staphylococcal cytolysins together constitute a new family of peptide toxins, unrelated to class II bacteriocins, which possess bactericidal and/or hemolytic activity.

[Utilization of lactic bacteria in the control of pathogenic microorganisms in food]. / Utilización de bacterias lácticas en el control de microorganismos patógenos de los alimentos.

The lactic acid bacteria have the potential to inhibit the growth of pathogenic and spoilage bacteria and the possibility exists of using them to improve the hygienic quality and to extend the shelf-life of different foods. Among the many inhibitory substances produced by the lactic acid bacteria, the bacteriocins are of particular interest. It has been the objective of this work to review the bacteriocins produced by lactic acid bacteria from the genera Lactococcus, Lactobacillus and Pediococcus, as well as Leuconostoc and Carnobacterium to understand their relevant biochemical, immunological and genetic characteristics. The lactic acid bacteria may also express foreign genes codifying metabolites with antimicrobial activities against foodborne pathogens of interest, and this will also permit hypothesize about theoretical and experimental models of microbial antagonism mediated by the lactic acid bacteria.

Isolation of nisin-producing Lactococcus lactis strains from dry fermented sausages.

A total of 4608 lactic acid bacteria (LAB) were isolated from 24 Spanish fermented sausages and screened for bacteriocin production. Two strains, BB24 and G18, produced bacteriocins that inhibited a broad spectrum of Gram-positive bacteria. BB24 and G18 were tentatively identified as Lactococcus lactis by carbohydrate fermentation patterns and other biochemical characteristics. The characterization of their bacteriocins suggested that both could be the well-known lantibiotic nisin. This was confirmed by PCR analysis of their genomic DNA. Nucleotide sequencing revealed that they produced nisin A. The fact that BB24 and G18 were isolated from sausages produced in two different regions of Spain suggests that nisin-producing L. lactis strains may be more widespread in meat products than previously thought. Nisin produced by L. lactis BB24 has been purified to homogeneity by a procedure that included ammonium sulphate precipitation and cation-exchange, hydrophobic-interaction and reverse-phase chromatography. The purification procedure was simple, rapid and reproducible.

Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum.

Lactic acid bacteria were isolated from Spanish dry-fermented sausages and screened for bacteriocin production. About 10% of the isolates produced antimicrobial substances when grown on solid media, but only 2% produced detectable activity in liquid media. Strain L50, identified as Pediococcus acidilactici, showed the strongest inhibitory activity and was active against members of all of the gram-positive genera tested. The strain produced a heat-stable bacteriocin when grown at 8 to 32 degrees C but not at 45 degrees C. The bacteriocin was purified to homogeneity. Its mass was determined to be 5,250.11 +/- 0.30 by electrospray mass spectrometry. The N terminus of the bacteriocin was blocked for sequencing by Edman degradation, but a partial sequence of 42 amino acids was obtained after cleavage of the peptide by cyanogen bromide. The sequence showed no similarity to those of other bacteriocins. Pediocin L50 appears to contain modified amino acids but not lanthionine or methyl-lanthionine.

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.

Generation of polyclonal antibodies of predetermined specificity against pediocin PA-1.

Polyclonal antibodies of predetermined specificity for pediocin PA-1 (pedA1) have been generated by immunization of rabbits with a chemically synthesized C-terminal fragment of this bacteriocin (PH2) conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The sensitivity and specificity of the PH2-KLH-generated antibodies were evaluated by the development of various enzyme-linked immunosorbent assays (ELISAs)-a noncompetitive indirect ELISA (NCI-ELISA), a competitive indirect ELISA (CI-ELISA), and a competitive direct ELISA (CD-ELISA)-and by immunodotting. All immunoassays indicated the existence of pedA1-specific antibodies with high relative affinities and adequate sensitivities in the sera of immunized animals. The limits of detection of pedA1 in MRS medium (Oxoid Ltd., Basingstoke, United Kingdom) were found to be 2.5 microg/ml by immunodotting and 1 microg/ml in the NCI-ELISA. However, the CI-ELISA enhanced the limit of detection of pedA1 to 0.025 microg/ml, while the amount of free pedA1 required for 50% binding inhibition was 10 microg/ml. Moreover, the CD-ELISA increased the affinity of the PH2-KLH-generated antibodies for pedA1; the limit of detection of pedA1 was less than 0.025 microg/ml, and the 50% binding inhibition value was reduced to 0.5 microg of pedA1/ml. All immunoassays and the slot dot assay detected the presence of pedA1 in the supernatant of the producing strain Pediococcus acidilactici 347, with no reactivity or negligible immunoreactivity with the supernatants of other lactic acid bacteria producing or not producing different bacteriocins. The approaches taken for the generation of antibodies and the development of immunoassays could prove useful for the generation and evaluation of antibodies of predetermined specificity for other bacteriocins of interest in the food industry.

Antibodies to a synthetic 1-9-N-terminal amino acid fragment of mature pediocin PA-1: sensitivity and specificity for pediocin PA-1 and cross-reactivity against Class IIa bacteriocins.

Polyclonal antibodies specific for pediocin PA-1 (PedA1) were generated by immunization of rabbits with a chemically synthesized 1-9-N-terminal amino acid fragment of this bacteriocin (PH1) conjugated to the carrier protein keyhole limpet haemocyanin (KLH). The PH1 fragment holds a highly conserved amino acid sequence with closely related Class IIa bacteriocins. The sensitivity and specificity of the PH1-KLH-generated rabbit polyclonal antibodies were evaluated by the development of various ELISAs, such as a non-competitive indirect ELISA (NCI-ELISA), a competitive indirect ELISA (CI-ELISA), a competitive direct ELISA (CD-ELISA) and a sandwich ELISA (S-ELISA), and by protein slot-blotting and Western blotting. NCI- and CI-ELISA were valuable for detecting the existence of PedA1-specific antibodies in the sera of immunized rabbits. The limit of detection of PedA1 in MRS medium was found to be 0.5 microg ml(-1) in NCI-ELISA, while CI-ELISA on plates coated with purified PedA1 increased the affinity of the PH1-KLH-generated antibodies for PedA1; the limit of detection of PedA1 was less than 0.01 microg ml(-1) and 50% binding inhibition was achieved with 0.1 microg PedA1 ml(-1). Similarly, the limits of detection of PedA1 in MRS medium were found to be 5 microg ml(-1) by protein slot-blotting and 0.01 microg ml(-1) by Western blotting. Most importantly, PH1-KLH-generated polyclonal antibodies detected the presence of PedA1 in the supernatants of the producing strains of Pediococcus acidilactici 347, Z102, A172, X13 and P20, with no reactivity or negligible immunoreactivity with the supernatants of other lactic acid bacteria producing or not producing closely related or different bacteriocins. The approaches taken for the selection of the bacteriocin peptide fragment, the generation of antibodies and the development of immunoassays could prove useful for the generation and evaluation of antibodies of adequate specificity for other bacteriocins of interest in the food industry.

Enterocin P selectively dissipates the membrane potential of Enterococcus faecium T136.

Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (DeltaPsi) and the intracellular ATP pool of the indicator organism, the pH gradient (DeltapH) component of the proton motive force (Deltap) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes.