Autolytic phenotype of Lactococcus lactis strains isolated from traditional Tunisian dairy products

AIMS: To evaluate the autolytic properties of Lactococcus lactis strains isolated from artisan Tunisian dairy products, their peptidoglycan hydrolase content and their activity spectrum. METHODS AND RESULTS: The autolytic phenotype of Lactococcus strains was evaluated under starvation conditions in potassium phosphate buffer. The results obtained highlighted a high degree of diversity among the strains analysed, allowing the identification of high and low autolytic Lactococcus lactis strains. Peptidoglycan hydrolase content was evaluated by renaturing SDS-PAGE using cells of Micrococcus lysodeikticus as a target for the enzymatic activity. A major activity band migrating at about 45 kDa was observed. The lytic activity, evaluated in the presence of different chemicals, was retained in 8% NaCl, 15 mmol l(-1) CaCl2, and in a pH range between 5 and 9.5. The substrate specificity of peptidoglycan hydrolase from Lactococcus strains was evaluated in renaturing SDS-PAGE incorporating cells of different bacterial species. The major autolysin of Lactococcus lactis was active against cells of Lactococcus lactis subsp. lactis, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus helveticus and Listeria monocytogenes. CONCLUSIONS: Autolytic activity is widely distributed in Lactococcus lactis and the rate of autolysis is strain-dependent. The major peptidoglycan hydrolase showed a wide spectrum of activity against several lactic acid bacteria and bacterial species involved in food-related infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The autolytic phenotype of Lactococcus lactis strains isolated from Tunisian artisan dairy products has been determined, and the data obtained should allow the selection of strains of technological interest in the cheese-ripening process.     

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.     

Purification and identification of the pediocin produced by Pediococcus acidilactici MM33, a new human intestinal strain

Aims:  The aim of this study was to purify and identify the bacteriocin produced by Pediococcus acidilactici MM33, a strain previously isolated from human gut.
Methods and Results:  Purification of the bacteriocin was performed by cationic exchange chromatography followed by a reverse phase step. Biochemical and mass spectrometry analysis showed homology with pediocin PA-1. To verify if P. acidilactici MM33 carried the pediocin PA-1 gene, total DNA was used to amplify the pediocin gene. The PCR product obtained was then sequenced and the nucleotide sequence revealed to be identical to that of pediocin PA-1. Treatment of P. acidilactici MM33 with novobiocin resulted in a plasmid-cured strain without bacteriocin-producing capacity. Antimicrobial assay and molecular analysis demonstrated that this strain was ped ⁻ suggesting that the ped cluster is plasmid encoded. Antimicrobial assay revealed that pediocin was bactericidal against Listeria monocytogenes , showing a minimal inhibitory concentration (MIC) of 200 AU ml⁻¹.
Conclusions:  A two-step purification procedure was elaborated in this study. The bacteriocin secreted by the human strain P. acidilactici MM33 is carried on a plasmid and the amino acid sequence is identical to pediocin PA-1.
Significance and Impact of the Study:  Pediococcus acidilactici MM33 is the first human pediocin-producing strain reported and could be used as probiotic to prevent enteric pathogen colonization.     

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.     

Susceptibility of Pediococcus spp. to antimicrobial agents

AIM: To investigate the susceptibility of Pediococcus species to antimicrobial agents. METHODS AND RESULTS: The susceptibility to 14 antimicrobial agents of 31 genotypically distinct strains of six Pediococcus species was assessed by using Etests on ISO-sensitest agar supplemented with horse blood. The species included were Pediococcus acidilactici, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus. For several antimicrobial agents, some species were more susceptible than others. The two industrially important species, P. acidilactici and P. pentosaceus, differed with respect to erythromycin and trovafloxacin susceptibility, and in general both species had higher minimum inhibitory concentrations than the other species. In an erythromycin-resistant P. acidilactici, an erythromycin resistance methylase B [erm(B)] gene was identified by PCR. Using a plasmid preparation from strain P. acidilactici 6990, a previously erythromycin-sensitive Lactococcus lactis strain was made resistant. Transformants harboured a single plasmid, sized at 11.6 kb through sequence analysis. In addition, the erm(B) gene was identified within the plasmid sequence. CONCLUSIONS: The phenotypic test indicated the absence of acquired antimicrobial resistance genes in 30 of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will help in selection of the best Pediococcus strains for use as starter cultures.     

Plasmid-Associated Bacteriocin Production and Sucrose Fermentation in Pediococcus acidilactici

Production of bacteriocin activity designated pediocin PA-1 was associated with the presence of a 6.2-megadalton plasmid in Pediococcus acidilactici PAC1.0. The bacteriocin exhibited activity against P. acidilactici, P. pentosaceus, Lactobacillus plantarum, L. casei, L. bifermentans, and Leuconostoc mesenteroides subsp. dextranicum. Partial characterization of pediocin PA-1 is described. The molecular weight of pediocin PA-1 was ca. 16,500. Additionally, strain PAC1.0 was found to contain a 23-megadalton plasmid associated with sucrose-fermenting ability.     

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.     

Factors influencing immunity and resistance of Pediococcus acidilactici to the bacteriocin, pediocin AcH

In pediocin AcH producing Pediococcus acidilactici strains the genes for both the production of pediocin and immunity against it are encoded in an 8.9 kb plasmid pSMB74. Following loss of this plasmid, the variants lost the ability to produce pediocin AcH, but some retained the resistance against it. This resistance was a transient trait, acquired while nonproducing cells grew in the presence of pediocin AcH but lost when the cells were grown in the absence of it.     

Isolation and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi

Aims:  Screening and partial characterization of a bacteriocin produced by Pediococcus pentosaceus K23-2 isolated from Kimchi, a traditional Korean fermented vegetable.
Methods and Results:  A total of 1000 lactic acid bacteria were isolated from various Kimchi samples and screened for the production of bacteriocin. Pediocin K23-2, a bacteriocin produced by the Pediococcus pentosaceus K23-2 strain, showed strong inhibitory activity against Listeria monocytogenes . The bacteriocin activity remained unchanged after 15 min of heat treatment at 121°C or exposure to organic solvents; however, it diminished after treatment with proteolytic enzymes. The bacteriocin was maximally produced at 37°C, when the pH of the culture broth was maintained at 5·0 during the fermentation, although the optimum pH for growth was 7·0. The molecular weight of the bacteriocin was about 5 kDa according to a tricine SDS-PAGE analysis.
Conclusions:  Pediococcus pentosaceus K23-2 isolated from Kimchi produces a bacteriocin, which shares similar characteristics to the Class IIa bacteriocins. The bacteriocin is heat stable and shows wide antimicrobial activity against Gram-positive bacteria, especially L. monocytogenes .
Significance and Impact of the Study:  Pediocin K23-2 and pediocin K23-2-producing P. pentosaceus K23-2 could potentially be used in the food and feed industries as natural biopreservatives, and for probiotic application to humans or livestock.     

Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC-1.0.

The plasmid-encoded bacteriocin pediocin PA-1, produced by the gram-positive bacterium Pediococcus acidilactici strain PAC-1.0, was purified to homogeneity. The purified product exhibited antibacterial activity against several gram-positive bacterial strains, including the food pathogen Listeria monocytogenes. Pediocin PA-1 is a 4629-Da peptide with 44 amino acids and two disulfide bonds. The amino acid sequence and arrangement of the disulfide bonds were determined. Sequence data were used to calculate an isoelectric point of 10.0. The small and basic nature of PA-1 is comparable to several other bacteriocins produced by gram-positive bacteria. Reported sequences of other bacteriocins and of other antimicrobial peptides from diverse origins bear no resemblance to the sequence reported here.     

The autolytic phenotype of Bacillus thuringiensis

AIMS: To evaluate the autolytic phenotype of Bacillus thuringiensis. METHODS AND RESULTS: The autolytic rate of 87 strains belonging to different subsp. of B. thuringiensis was examined at pH 6, 6.5 and 8.5 in different buffers under starvation conditions. At pH 6 the extent of autolysis (average in the strain collection 38.3 +/- 21.1) was strain-dependent with wide variability, while at pH 6.5 and 8.5 (averages 72.0 +/- 9.0 and 63.1 +/- 8.2, respectively) it was much more uniform with only a few strains showing low autolytic rates. Forty-one per cent of the strains showed high resistance (>/=80%) to mutanolysin, a commercial muramidase from Streptomyces. The peptidoglycan hydrolase pattern was evaluated by renaturing SDS-PAGE using cells of B. thuringiensis subsp. tolworthi HD125 as indicator. The strain collection showed seven major lytic bands of about 90, 63, 46, 38, 32, 28 and 25 kDa, and in the stationary growth phase (72 h) there was a more intense 25 kDa band in the autolytic pattern. Using Micrococcus lysodeicticus and Listeria monocytogenes as the indicators lytic activity was retained, as seen by the bands of 63, 46, 38, 32 and 25 kDa. Growth in the different media did not affect the autolytic pattern. NaCl abolished the activity of all the peptidoglycan hydrolases in the gel, but in the presence of KCl, MgCl(2), MnCl(2) and EDTA some activity was retained. At basic pH the lytic activity increased. CONCLUSIONS: The autolytic phenotype of B. thuringiensis was found to be strain-dependent, and different proteins exibited peptidoglycan hydrolase activity, particularly at alkaline pH. Several of these proteins retained lytic activity against other bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: The characterisation of the autolytic phenotype of B. thuringiensis should expand the prospects of using this species in bacterial bio-control and field applications.     

Mode of action of pediocin AcH from Pediococcus acidilactici H on sensitive bacterial strains

The peptide, pediocin AcH, from Pediococcus acidilactici H binds to the cell surface of Lactobacillus plantarum NCDO 955, its resistant mutant and several other sensitive and resistant Gram-positive bacteria but not to Gram-negative bacteria. Sensitive cells, following treatment with pediocin AcH, lost intracellular K ions, u.v.-absorbing materials, became more permeable to ONPG and, in some strains, lysed. Binding of pediocin AcH was maximum at pH 6.0. Anions of several salts inhibited binding of pediocin AcH but this was overcome by increased concentrations of pediocin AcH. Treatment of sensitive cells with 1% SDS, 4 mol/1 guanidine-HCl, several organic solvents and enzymes did not reduce subsequent binding of pediocin AcH. Partially purified cell wall from a sensitive strain was also able to bind pediocin AcH. However, treatment of the cell walls to remove lipoteichoic acid prevented binding. These molecules might, therefore, be one of the binding sites of pediocin AcH.     

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.     

Capacity of human nisin- and pediocin-producing lactic Acid bacteria to reduce intestinal colonization by vancomycin-resistant enterococci

This study demonstrated the capacity of bacteriocin-producing lactic acid bacteria (LAB) to reduce intestinal colonization by vancomycin-resistant enterococci (VRE) in a mouse model. Lactococcus lactis MM19 and Pediococcus acidilactici MM33 are bacteriocin producers isolated from human feces. The bacteriocin secreted by P. acidilactici is identical to pediocin PA-1/AcH, while PCR analysis demonstrated that L. lactis harbors the nisin Z gene. LAB were acid and bile tolerant when assayed under simulated gastrointestinal conditions. A well diffusion assay using supernatants from LAB demonstrated strong activity against a clinical isolate of VRE. A first in vivo study was done using C57BL/6 mice that received daily intragastric doses of L. lactis MM19, P. acidilactici MM33, P. acidilactici MM33A (a pediocin mutant that had lost its ability to produce pediocin), or phosphate-buffered saline (PBS) for 18 days. This study showed that L. lactis and P. acidilactici MM33A increased the concentrations of total LAB and anaerobes while P. acidilactici MM33 decreased the Enterobacteriaceae populations. A second in vivo study was done using VRE-colonized mice that received the same inocula as those in the previous study for 16 days. In L. lactis-fed mice, fecal VRE levels 1.73 and 2.50 log(10) CFU/g lower than those in the PBS group were observed at 1 and 3 days postinfection. In the P. acidilactici MM33-fed mice, no reduction was observed at 1 day postinfection but a reduction of 1.85 log(10) CFU/g was measured at 3 days postinfection. Levels of VRE in both groups of mice treated with bacteriocin-producing LAB were undetectable at 6 days postinfection. No significant difference in mice fed the pediocin-negative strain compared to the control group was observed. This is the first demonstration that human L. lactis and P. acidilactici nisin- and pediocin-producing strains can reduce VRE intestinal colonization.     

Expression of lactococcin A and pediocin PA-1 in heterologous hosts

Pediocin PA-1 production, immunity and secretion are specified by a cluster of four genes in Pediococcus acidilactici PAC1.0. The production by, secretion of, and immunity to lactococcin A of Lactococcus lactis are also determined by four genes. Here, expression of the pediocin operon in Lactococcus lactis is reported, which could only be achieved by placing it under control of a lactococcal promoter. Expression of the lactococcin A operon in Pediococcus is also described: recombinant clones of Pediococcus were obtained that produced and secreted both active pediocin PA-1 and lactococcin A.     

Development of molecular RAPD marker for the identification of Pediococcus acidilactici strains

A RAPD analysis performed using a single primer targeted to the pediocin AcH/PA-1 gene was carried out on several P. acidilactici strains and on some related species of lactic acid bacteria. The high degree of genetic variability detected in P. acidilactici strains did not allow the selection of a common RAPD fragment that could be chosen as a potential species-specific DNA marker. Nevertheless a 700 bp fragment, that was found to be peculiar of all potential pediocin producer strains analyzed, was cloned and sequenced with the aim to develop a species specific PCR marker. Sequence analysis of the cloned 700 bp fragment showed one putative small open reading frame (ORF1), with no significant homology with known genes, and a partial putative second coding region (ORF2) with a high degree of similarity with several methionyl tRNA synthesis (metS) genes. The two coding regions were separated by a short spacer region. Primers targeted to ORF2 plus part of the spacer region and primers designed for the amplification of the entire cloned RAPD fragment were found to be species-specific for the detection of P. acidilactici strains. Furthermore primers designed on the ORF1 sequence allowed the amplification of a 439 bp fragment only in some P. acidilactici strains, including pediocin producing strains.     

Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese.

Among 1,962 bacterial isolates from a smear-surface soft cheese (Munster cheese) screened for activity against Listeria monocytogenes, six produced antilisterial compounds other than organic acids. The bacterial strain WHE 92, which displayed the strongest antilisterial effect, was identified at the DNA level as Lactobacillus plantarum. The proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action of the antilisterial compound produced by this bacterium suggested that it was a bacteriocin. Purification to homogeneity and sequencing of this bacteriocin showed that it was a 4.6-kDa, 44-amino-acid peptide, the primary structure of which was identical to that of pediocin AcH produced by different Pediococcus acidilactici strains. We report the first case of the same bacteriocin appearing naturally with bacteria of different genera. Whereas the production of pediocin AcH from P. acidilactici H was considerably reduced when the final pH of the medium exceeded 5.0, no reduction in the production of pediocin AcH from L. plantarum WHE 92 was observed when the pH of the medium was up to 6.0. This fact is important from an industrial angle. As the pH of dairy products is often higher than 5.0, L. plantarum WHE 92, which develops particularly well in cheeses, could constitute an effective means of biological combat against L. monocytogenes in this type of foodstuff.