Bacteriocin of Pediococcus acidilactici HW01 Inhibits Biofilm Formation and Virulence Factor Production by Pseudomonas aeruginosa

Probiotics and Antimicrobial Proteins - Pseudomonas aeruginosa is a potential source of food contamination that leads to food spoilage and infections as a result of the generation of biofilm and...     

Influence of Growth Conditions on the Production of a Bacteriocin, Pediocin AcH, by Pediococcus acidilactici H

The influence of growth parameters on the production of pediocin AcH by Pediococcus acidilactici H was studied. This strain produced large quantities of pediocin AcH in TGE broth (Trypticase [1%], glucose [1%], yeast extract [1%], Tween 80 [0.2%], Mn²⁺ [0.033 mM], Mg²⁺ [0.02 mM] [pH 6.5]) within 16 to 18 h at 30 to 37°C (final pH, 3.6 to 3.7). Pediocin AcH production was negligible when the pH of the medium was maintained at 5.0 or above, even in the presence of high cell mass.     

Bacteriocin plasmids ofPediococcus acidilactici

Summary Pediococcus acidilactici strains E, F and H isolated from fermented sausages produced bacteriocins which were protein in nature and inhibitory to a variety of spoilage and pathogenic microorganisms often encountered in foods. These strains harbored two to three plasmids ranging in size from 7.4 to 40.2 megadaltons. Curing experiments and plasmid profile analysis indicated the involvement of plasmid DNA with bacteriocin activity in all three strains. Carbohydrate fermentation and antibiotic resistance phenotypes did not appear to be associated with bacteriocin plasmids. Both bacteriocin activity and resistance determinants were linked in strain H and mediated by a 7.4-megadalton plasmid, whereas in strains E and F these two traits were not linked.     

Catabolism of Serine by Pediococcus acidilactici and Pediococcus pentosaceus

The ability to produce diacetyl from pyruvate and l-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functional dsdA was cloned from P. pentosaceus FAM19132 and expressed in Escherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate from l- and d-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants from dsdA wild-type and dsdA mutant strains of P. pentosaceus did not show differences in serine levels. In contrast, P. acidilactici degraded serine. Moreover, this species also catabolized threonine and produced alanine and α-aminobutyrate.     

Multilocus Hybridization Typing in Pediococcus acidilactici Strains

In previous a study we demonstrated the presence of several genomic subpopulations within a collection of Pediococcus acidilactici strains isolated from different environments, through a multilocus typing analysis taking into consideration housekeeping conserved loci and protein coding genes of the primary metabolism. In this study, representative strains of five genomic subpopulations previously described (I, II, III, V, VII) were analyzed by restriction analysis of chromosomal DNA and subsequent hybridization assays using as probes amplified fragments obtained from five housekeeping genes (16S rDNA, rpoC, ldhD, ldhL, and metS). A computer similarity and clustering analysis of hybridization data showed the subdivision of P. acidilactici strains in five distinct genotypes according to the grouping previously obtained confirming that pediocin AcH/PA-1 producer strains represent one genomic lineage within the species P. acidilactici. Received: 30 January 2001 / Accepted: 16 May 2001     

Autolytic activity and pediocin-induced lysis in Pediococcus acidilactici and Pediococcus pentosaceus strains

AIMS: To evaluate the autolytic phenotype of Pediococcus acidilactici and P. pentosaceus, the peptidoglycan hydrolases content and the effect of pediocin AcH/PA-1 and autolysins on cell lysis. METHODS AND RESULTS: The autolytic phenotype of Pediococcus strains was evaluated under starvation conditions in potassium phosphate buffer. The strains tested showed an extent of autolysis ranging between 40 and 90% after 48 h of starvation at 37 degrees C. Peptidoglycan hydrolase content was evaluated by renaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) using cells of Micrococcus lysodeikticus as a target for the enzymatic activity and a major activity band migrating at about 116 kDa was detected. Additional secondary lytic bands migrating in a range of molecular weight between 45 and 110 kDa were also detected. The lytic activity, evaluated in the presence of different chemicals, was retained in 15 mM CaCl2 and in a range of pH between 5 and 9.5 but was strongly reduced in presence of 8% NaCl and in the presence of protease inhibitors. The substrate specificity of peptidoglycan hydrolases of Pediococcus strains was evaluated in renaturing SDS-PAGE incorporating cells of different bacterial species. Lytic activity was detected against cells of Lactococcus lactis subsp. lactis, L. delbrueckii subsp. bulgaricus, Lactobacillus helveticus and Listeria monocytogenes. The interaction between pediocin AcH/PA-1 and autolysis was evaluated and a relevant effect of bacteriocin in cell-induced lysis was observed. CONCLUSIONS: The autolytic phenotype is widely distributed among P. acidilactici and P. pentosaceus and the rate of autolysis is high in the majority of the analysed strains. Several autolytic bands, detected by renaturing SDS-PAGE, retained their activity against several lactic acid bacteria and L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterization of the autolytic phenotype of Pediococcus acidilactici and P. pentosaceus strains should expand the knowledge of their role in fermentation processes where these species occur as primary or secondary bacterial population.     

Induction and characterization of Pediococcus acidilactici temperate bacteriophage

Mitomycin C was used to induce temperate bacteriophage from three strains of Pediococcus acidilactici. The new bacteriophage, designated pa97, pa40, and pa42, were characterized based on morphology, DNA homology, and major protein profiles. Morphological attributes (small isometric heads with non-contractile tails) place these bacteriophages within the B1 group of the family Siphovirdae. Restriction endonuclease digests suggested that the bacteriophage genomes were linear molecules without cohesive ends, and between 33 and 37 kilobases in length. All three bacteriophages possessed one major protein with an estimated mass of 30 to 35 kilodaltons. Bacteriophage pa42 also contained a second major protein of approximately 47 kilodaltons. DNA-DNA hybridization showed bacteriophages pa40 and pa42 were homologous to each other, but not to pa97, suggesting that Pediococcus acidilactici bacteriophage fall into at least two different species.     

Plasmid transfers by conjugation and electroporation in Pediococcus acidilactici

W.J. KIM, B. RAY AND M.C. JOHNSON. 1992. Plasmid profiles of wild and mutant strains of Pediococcus acidilactici M showed that a 53.7 kb plasmid (pPR72) encodes the sucrose hydrolysis trait ( Suc +) and an 11.1 kb plasmid encodes the bacteriocin production trait ( Pap +). Neither of these plasmids encode traits involving fermentation of other carbohydrates, antibiotic resistance or resistance to bacteriocin. Broad host-range plasmids (pAMβ1 and pIP501) from Enterococcus faecalis and plasmid pPR72 from Ped. acidilactici were conjugally transferred by filter mating into two strains of Ped. acidilactici. Four plasmids, ranging in size from 4.4 to 53.7 kb, were also transferred into Ped. acidilactici strains by electroporation. Optimum transformation of the 4.4 kb plasmid, pGK12, was obtained at a DNA concentration of 1 μg/220 μl. The same amount of DNA gave lower transformation frequencies as the plasmid size increased. Results of these studies indicated that both conjugation and electroporation can be used to transfer plasmid-linked traits in Ped. acidilactici strains.     

Novel extracellular proteolytic activity in Pediococcus acidilactici ATCC 8042

Proteolytic systems are common in lactic acid bacteria, but there are few reports about proteases or peptidases in the genus Pediococcus. To evaluate the presence of these types of enzymes, Pediococcus acidilactici ATCC 8042 was cultured in MRS broth. Supernatants collected during the log phase showed proteolytic activity towards an elastin dispersion when assayed using a spectrophotometer. Zn2+ showed a stimulatory effect, and the proteolytic activity reached its maximum when 200 mmol/L NaCl was included in the reaction buffer. On the other hand, activity was reduced when 5 mmol/L EDTA, 10 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L 1,10-phenanthroline were used or when the sample was heat treated. Zymograms showed two different proteolytic bands when gelatin was used as a substrate (>200 and 107 kDa), but only the higher molecular mass band was detected when casein or elastin was used. The gelatinolytic activity was not detected with zymograms of the 107 kDa band, which was the one inactivated by heat treatment. The use of a renaturing SDS-PAGE gel with embedded Micrococcus lysodeikticus cells allowed for the detection of a band with peptidoglycan hydrolase activity migrating at about 110 kDa. This activity was lost when 10 mmol/L EDTA was added to the renaturing buffer. Therefore, Pediococcus showed at least three different extracellular enzymes that were produced during the logarithmic growth phase and acted on peptide substrates. Each showed different substrate specificity, ion requirements, and thermostability.     

Assessment of Pediococcus acidilactici as a Potential Silage Inoculant

Eighteen Pediococcus strains were screened for their potential as silage inoculants. Pediococcus acidilactici G24 was found to be the most suitable, exhibiting a short lag phase on both glucose and fructose, a rapid rate of acid production, a high sugar-to-lactate conversion efficiency, no detectable breakdown of proteins or lactic acid, and the ability to grow within a broad range of pH and temperature. When tested in laboratory silos using grass with a water-soluble carbohydrate content of 24 g/kg of aqueous extract, P. acidilactici G24 stimulated the natural Lactobacillus plantarum population and accelerated the rates of lactic acid production and pH decrease. After 6 days of fermentation, the inoculated silage exhibited a 12% decrease in ammonia nitrogen and an 11% increase in crude protein levels compared with uninoculated controls. The use of an L. plantarum inoculant at a rate of 10⁴ bacteria per g of grass in conjunction with P. acidilactici G24 produced no additional beneficial effect. Inoculation of grass with a water-soluble carbohydrate level of 8 g/kg of aqueous extract with P. acidilactici G24 led to no acceleration in the rate of L. plantarum growth or pH decrease. However, after 7 days of fermentation the inoculated silage had a 14% lower ammonia nitrogen protein content than did uninoculated controls. The results suggest that P. acidilactici G24 may be useful as a silage inoculant for crops with a sufficiently high water-soluble carbohydrate level.     

Tracing Pediococcus acidilactici in ensiled maize by plasmid-encoded erythromycin resistance

Curing experiments undertaken on Pediococcus acidilactici strain S364, isolated from maize silage, revealed a linkage between a resident 40 MD plasmid and resistance to erythromycin, oleandomycin and tylosin. The possibility of using erythromycin resistance to monitor the fate of this strain in a model ensiling system was also demonstrated.     

Identification of Pediococcus acidilactici and Pediococcus pentosaceus based on 16S rRNA and ldhD gene-targeted multiplex PCR analysis

A multiplex PCR assay that can readily and unambiguously identify Pediococcus acidilactici and Pediococcus pentosaceus strains was developed to give an easy-to-read profile based on the amplification of a 16S rRNA gene fragment, specific for each species, and a d-lactate dehydrogenase gene fragment specific for Pediococcus acidilactici strains.     

Antigenic property of pediocin AcH produced by Pediococcus acidilactici H

Pediocin AcH, a bacteriocin of Pediococcus acidilactici H, inhibits the growth of several food spoilage and pathogenic bacteria. The antigenic property of partially purified pediocin AcH was tested by immunizing mice and a rabbit. Pediocin AcH was not immunogenic in these animals as determined by immunoblotting even after conjugation to bovine serum albumin. The non-immunogenic nature of pediocin AcH, its non-toxicity to laboratory animals and its hydrolysis by gastric proteolytic enzymes may be considered favourably in its possible use as a food preservative.     

Antigenic property of pediocin AcH produced by Pediococcus acidilactici H

Pediocin AcH, a bacteriocin of Pediococcus acidilactici H, inhibits the growth of several food spoilage and pathogenic bacteria. The antigenic property of partially purified pediocin AcH was tested by immunizing mice and a rabbit. Pediocin AcH was not immunogenic in these animals as determined by immunoblotting even after conjugation to bovine serum albumin. The non-immunogenic nature of pediocin AcH, its non-toxicity to laboratory animals and its hydrolysis by gastric proteolytic enzymes may be considered favourably in its possible use as a food preservative.     

Monoclonal antibody-based enzyme immunoassay for pediocins of Pediococcus acidilactici.

Monoclonal antibody (MAb) R2-AR against pediocin RS2 was developed. Mice were immunized for 12 weeks with pediocin RS2 conjugated to a polyacrylamide gel. Two hybridoma fusions yielded an MAb that in Western blots (immunoblots) reacted only with pediocins RS2 and AcH (3 kDa) from Pediococcus acidilactici RS2 and H, respectively, and did not react with any other bacteriocin, including sakacin A from Lactobacillus sake Lb 706, leuconocin LCM1 from Leuconostoc carnosum LM1, nisin from Lactococcus lactis ATCC 11454, and pediocin A from Pediococcus pentosaceus FBB61. Each of the bacteriocin bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was confirmed to be biologically active by a gel overlay test performed with sensitive indicator organisms. In dot immunoblot assays, the MAb could detect a minimum of 32,000 arbitrary units of pediocin RS2 or AcH per ml. In colony immunoblot assays, the MAb was used successfully to differentiate bac+ and bac- variants of P. acidilactici RS2 strains.     

Optimization of Bacteriocin Production by Batch Fermentation of Lactobacillus plantarum LPCO10

Optimization of bacteriocin production by Lactobacillus plantarum LPCO10 was explored by an integral statistical approach. In a prospective series of experiments, glucose and NaCl concentrations in the culture medium, inoculum size, aeration of the culture, and growth temperature were statistically combined using an experimental 2³_5-2 fractional factorial two-level design and tested for their influence on maximal bacteriocin production by L. plantarum LPCO10. After the values for the less-influential variables were fixed, NaCl concentration, inoculum size, and temperature were selected to study their optimal relationship for maximal bacteriocin production. This was achieved by a new experimental 3²_3-1 fractional factorial three-level design which was subsequently used to build response surfaces and analyzed for both linear and quadratic effects. Results obtained indicated that the best conditions for bacteriocin production were shown with temperatures ranging from 22 to 27°C, salt concentration from 2.3 to 2.5%, and L. plantarum LPCO10 inoculum size ranging from 10^7.3 to 10^7.4 CFU/ml, fixing the initial glucose concentration at 2%, with no aeration of the culture. Under these optimal conditions, about 3.2 × 10⁴ times more bacteriocin per liter of culture medium was obtained than that used to initially purify plantaricin S from L. plantarum LPCO10 to homogeneity. These results indicated the importance of this study in obtaining maximal production of bacteriocins from L. plantarum LPCO10 so that bacteriocins can be used as preservatives in canned foods.     

Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici.

A bacteriocin produced by Pediococcus acidilactici has been purified to homogeneity by a rapid and simple four-step purification procedure which includes ammonium sulphate precipitation, chromatography with a cation-exchanger and Octyl Sepharose, and reverse-phase chromatography. The purification resulted in an approximately 80,000-fold increase in the specific activity and about a 6-fold increase in the total activity. The amino acid composition and sequencing data indicated that the bacteriocin contained 43-44 amino acid residues. The predicted M(r) and isolectric point of the bacteriocin are about 4600 and 8.6, respectively. Comparing the amino acid sequence of this bacteriocin with the sequences of leucocin A-UAL 187, sakacin P and curvacin A (bacteriocins produced by Leuconostoc gelidum, Lactobacillus sake and Lactobacillus curvatus, respectively) revealed that all four bacteriocins had in their N-terminal region the sequence Tyr-Gly-Asn-Gly-Val-Xaa-Cys, indicating that this concensus sequence is of fundamental importance for this group of bacteriocins. The bacteriocin from P. acidilactici and sakacin P were very similar, having at least 25 common amino acid residues. The sequence similarity was greatest in the N-terminal half of the molecules--17 of the first 19 residues were common--indicating the fundamental importance of this region. Leucocin A-UAL 187 and curvacin A had, respectively, at least 16 and 13 amino acid residues in common with the bacteriocin from P. acidilactici.     

Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici

An antimicrobial peptide designated pediocin AcH was isolated from Pediococcus acidilactici strain H. The pediocin AcH was purified by ion exchange chromatography. The molecular weight of pediocin AcH was determined by SDS-PAGE to be about 2700 daltons. Pediocin AcH was sensitive to proteolytic enzymes resistant to heat and organic solvents, and active over a wide range of pH. Pediocin AcH exhibited inhibition against several food spoilage bacteria and foodborne pathogens including Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes. It was bactericidal to sensitive cells and acted very rapidly. The bactericidal effect was not produced by either cell lysis or apparent loss of membrane permeability.     

Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici

An antimicrobial peptide designated pediocin AcH was isolated from Pediococcus acidilactici strain H. The pediocin AcH was purified by ion exchange chromatography. The molecular weight of pediocin AcH was determined by SDS-PAGE to be about 2700 daltons. Pediocin AcH was sensitive to proteolytic enzymes, resistant to heat and organic solvents, and active over a wide range of pH. Pediocin AcH exhibited inhibition against several food spoilage bacteria and foodborne pathogens including Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes. It was bactericidal to sensitive cells and acted very rapidly. The bactericidal effect was not produced by either cell lysis or apparent loss of membrane permeability.