Plasmid transfer in Pediococcus spp.: intergeneric and intrageneric transfer of pIP501.

Transfer of the broad-host-range resistance plasmid pIP501 from Streptococcus faecalis to Pediococcus pentosaceus and Pediococcus acidilactici occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Expression of the pIP501-linked erythromycin and chloramphenicol resistance determinants was observed in transconjugants. Intrageneric transfer of pIP501 from a P. pentosaceus donor to various pediococcal recipients occurred at frequencies of 10(-4) to 10(-7) transconjugants per input donor cell. Intergeneric transfer of plasmid pIP501 from P. pentosaceus to S. faecalis, Streptococcus sanguis (Challis), and Streptococcus lactis was observed. Similar mating experiments showed no evidence for the transfer of the broad-host-range R-plasmid pAM beta 1 to Pediococcus spp. recipients.     

Plasmid transfer in Pediococcus spp.: intergeneric and intrageneric transfer of pIP501

Transfer of the broad-host-range resistance plasmid pIP501 from Streptococcus faecalis to Pediococcus pentosaceus and Pediococcus acidilactici occurred between cells immobilized on nitrocellulose filters in the presence of DNase. Expression of the pIP501-linked erythromycin and chloramphenicol resistance determinants was observed in transconjugants. Intrageneric transfer of pIP501 from a P. pentosaceus donor to various pediococcal recipients occurred at frequencies of 10(-4) to 10(-7) transconjugants per input donor cell. Intergeneric transfer of plasmid pIP501 from P. pentosaceus to S. faecalis, Streptococcus sanguis (Challis), and Streptococcus lactis was observed. Similar mating experiments showed no evidence for the transfer of the broad-host-range R-plasmid pAM beta 1 to Pediococcus spp. recipients.     

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.     

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.     

Lactic acid bacteria and yeasts associated with gowé production from sorghum in Bénin

AIMS: To identify the dominant micro-organisms involved in the production of gowé, a fermented beverage, and to select the most appropriate species for starter culture development. METHODS AND RESULTS: Samples of sorghum gowé produced twice at three different production sites were taken at different fermentation times. DNA amplification by internal transcribed spacer-polymerase chain reaction of 288 lactic acid bacteria (LAB) isolates and 16S rRNA gene sequencing of selected strains revealed that the dominant LAB responsible for gowé fermentation were Lactobacillus fermentum, Weissella confusa, Lactobacillus mucosae, Pediococcus acidilactici, Pediococcus pentosaceus and Weissella kimchii. DNA from 200 strains of yeasts was amplified and the D1/D2 domain of the 26S rRNA gene was sequenced for selected isolates, revealing that the yeasts species were Kluyveromyces marxianus, Pichia anomala, Candida krusei and Candida tropicalis. CONCLUSIONS: Gowé processing is characterized by a mixed fermentation dominated by Lact. fermentum, W. confusa and Ped. acidilactici for the LAB and by K. marxianus, P. anomala and C. krusei for the yeasts. SIGNIFICANCE AND IMPACT OF THE STUDY: The diversity of the LAB and yeasts identified offers new opportunities for technology upgrading and products development in gowé production. The identified species can be used as possible starter for a controlled fermentation of gowé.     

Identification and cloning of a plasmid-encoded erythromycin resistance determinant from Lactobacillus reuteri

Plasmid analysis, plasmid curing, cloning, and hybridization experiments were used to study four Lactobacillus reuteri strains showing high resistance to erythromycin. Plasmid curing with acriflavine resulted in a loss of erythromycin resistance in a frequency of 1-10%. For three of the strains this was accompanied by a loss of a 6.9-MDa plasmid, which was shown to be identical for the different strains and designated pLUL631. The erythromycin (erm) gene was located on a 5.5-MDa plasmid in the fourth strain. A restriction map of pLUL631 was constructed and the location of the erm gene on the plasmid was identified by cloning in Escherichia coli. By using a Streptococcus lactis-E. coli shuttle vector, the erm gene was also transformed to S. lactis and expressed. The erm gene from L. reuteri was shown to be related to the erm gene from pIP501 (Streptococcus agalactiae) by DNA-DNA hybridization.     

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.     

Development and Characterization of Lactose-Positive Pediococcus Species for Milk Fermentation

Bacteriophages against Streptococcus thermophilus are a growing problem in the Italian cheese industry. One possible control method involves replacing S. thermophilus in mozzarella starter blends with lactic acid bacteria from a different genus or species. In this study, we evaluated lactose-positive pediococci for this application. Because we could not identify any commercially available pediococci with fast acid-producing ability in milk, we transformed Pediococcus pentosaceus ATCC 25744, P. pentosaceus ATCC 25745, and Pediococcus acidilactici ATCC 12697 by electroporation with pPN-1, a 35-kb Lactococcus lactis lactose plasmid. Transformants of P. pentosaceus ATCC 25745 and P. acidilactici ATCC 12697 were then used to examine lactose-positive pediococci for properties related to milk fermentation. Both transformants rapidly produced acid and efficiently retained pPN-1 in lactose broth, and neither bacterium was attacked by bacteriophages in whey collected from commercial cheese facilities. Paired starter combinations of Pediococcus spp. and Lactobacillus helveticus LH100 exhibited synergistic pH reduction in milk, and small-scale cheese trials showed that these cultures could be used to manufacture part-skim mozzarella cheese. Results demonstrate that lactose-positive pediococci have potential as replacement cocci for S. thermophilus in Italian cheese starter blends and may facilitate development of new strain rotation schemes to combat S. thermophilus bacteriophage problems in mozzarella cheese plants.     

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.     

Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococccus acidilactici isolates

AIMS: The aim of this study was to isolate bacteriocin-producing lactic acid bacteria (LAB) from human intestine. METHODS AND RESULTS: A total of 111 LAB were isolated from human adult stool and screened for their bacteriocin production. Neutralized cell-free supernatants from Lactococcus lactis subsp. lactis MM19 and Pediococcus acidilactici MM33 showed antimicrobial activity. The antimicrobials in the supernatant from a culture of L. lactis inhibited Enterococcus faecium, various species of Lactobacillus and Staphylococcus aureus; while those in the supernatant from a culture of P. acidilactici inhibited Enterococcus spp., some lactobacilli and various serotypes of Listeria monocytogenes. The antimicrobial metabolites were heat-stable and were active over a pH range of 2-10. The antimicrobial activities of the supernatants of both bacteria were inhibited by many proteases but not by catalase. The plate overlay assay allowed an approximation of size between 3.5 and 6 kDa for both antimicrobial substances. CONCLUSIONS: As the antagonistic factor(s) produced by L. lactis MM19 and P. acidilactici MM33 were sensitive to proteolytic enzymes, it could be hypothesized that bacteriocins were involved in the inhibitory activities. Inhibition spectrum and biochemical analysis showed that these bacteria produced two distinct bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: We are the first to isolate bacteriocin-producing strains of Pediococcus and Lactococcus from human intestine. These strains might be useful for control of enteric pathogens.     

pEOC01: a plasmid from Pediococcus acidilactici which encodes an identical streptomycin resistance (aadE) gene to that found in Campylobacter jejuni

The complete nucleotide sequence of pEOC01, a plasmid (11,661 bp) from Pediococcus acidilactici NCIMB 6990 encoding resistance to clindamycin, erythromycin, and streptomycin was determined. The plasmid, which also replicates in Lactococcus and Lactobacillus species contains 16 putative open reading frames (ORFs), including regions annotated to encode replication, plasmid maintenance and multidrug resistance functions. Based on an analysis the plasmid replicates via a theta replicating mechanism closely related to those of many larger Streptococcus and Enterococcus plasmids. Interestingly, genes homologous to a toxin/antitoxin plasmid maintenance system are present and are highly similar to the omega-epsilon-zeta operon of Streptococcus plasmids. The plasmid contains two putative antibiotic resistance homologs, an ermB gene encoding erythromycin and clindamycin resistance, and a streptomycin resistance gene, aadE. Of particular note is the aadE gene which holds 100% identity to an aadE gene found in Campylobacter jejuni plasmid but which probably originated from a Gram-positive source. This observation is significant in that it provides evidence for recent horizontal transfer of streptomycin resistance from a lactic acid bacterium to a Gram-negative intestinal pathogen and as such infers a role for such plasmids for dissemination of antibiotic resistance genes possibly in the human gut.     

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.     

Nucleotide sequence, structural organization and host range of pRS4, a small cryptic Pediococcus pentosaceus plasmid that contains two cassettes commonly found in other lactic acid bacteria

The complete nucleotide sequence of the cryptic plasmid pRS4 (3550 bp) from Pediococcus pentosaceus RS4 was determined. Sequence analysis revealed the presence of three open reading frames (ORFs). The putative protein coded by ORF 1 showed 93% identity with the mobilization protein of Lactobacillus casei plasmid pLC88 and 94% identity with a sequenced fragment of the mobilization protein of P. damnosus plasmid pF8801, suggesting a common origin for these three mobilization proteins. The putative protein coded by ORF 2 showed 92% identity with the replication protein of L. plantarum plasmid pWCFS101, a plasmid that replicates via the rolling circle (RC) mechanism, suggesting a similar replication mechanism for pRS4. Supporting this hypothesis, a putative double strand origin (dso) and a region with palindromic sequences that could function as single strand origin (sso), were detected in pRS4. A function could not be assigned to ORF 3. Since ORF 1 exhibits high identity with L. casei plasmid pLC88 but lower identity (58%) with other Lactobacillus plasmids, and ORF 2 exhibits high identity with the L. plantarum plasmid pWCFS101 but lower identity (55-58%) with other Lactobacillus plasmids (including pLC88), two independent cassettes, from different sources, seem to be involved in the structure of pRS4. Plasmids derived from pRS4 containing the chloramphenicol resistance gene were successfully electrotransformed in L. plantarum, L. casei, P. pentosaceus, and Pediococcus acidilactici, suggesting that pRS4 could be used as a cloning vector for lactic acid bacteria. To our knowledge pRS4 is the first RC-replicating plasmid of P. pentosaceus that has been completely sequenced and used as cloning vector.     

Biochemical and genetic characterization of coagulin, a new antilisterial bacteriocin in the pediocin family of bacteriocins, produced by Bacillus coagulans I(4)

A plasmid-linked antimicrobial peptide, named coagulin, produced by Bacillus coagulans I(4) has recently been reported (B. Hyronimus, C. Le Marrec and M. C. Urdaci, J. Appl. Microbiol. 85:42-50, 1998). In the present study, the complete, unambiguous primary amino acid sequence of the peptide was obtained by a combination of both N-terminal sequencing of purified peptide and the complete sequence deduced from the structural gene harbored by plasmid I(4). Data revealed that this peptide of 44 residues has an amino acid sequence similar to that described for pediocins AcH and PA-1, produced by different Pediococcus acidilactici strains and 100% identical. Coagulin and pediocin differed only by a single amino acid at their C terminus. Analysis of the genetic determinants revealed the presence, on the pI(4) DNA, of the entire 3.5-kb operon of four genes described for pediocin AcH and PA-1 production. No extended homology was observed between pSMB74 from P. acidilactici and pI(4) when analyzing the regions upstream and downstream of the operon. An oppositely oriented gene immediately dowstream of the bacteriocin operon specifies a 474-amino-acid protein which shows homology to Mob-Pre (plasmid recombination enzyme) proteins encoded by several small plasmids extracted from gram-positive bacteria. This is the first report of a pediocin-like peptide appearing naturally in a non-lactic acid bacterium genus.     

Identification and characterization of antibiotic resistance genes in Lactobacillus reuteri and Lactobacillus plantarum

Aims:  The study aimed to identify the resistance genes mediating atypical minimum inhibitory concentrations (MICs) for tetracycline, erythromycin, clindamycin and chloramphenicol within two sets of representative strains of the species Lactobacillus reuteri and Lactobacillus plantarum and to characterize identified genes by means of gene location and sequencing of flanking regions.
Methods and Results:  A tet (W) gene was found in 24 of the 28 Lact. reuteri strains with atypical MIC for tetracycline, whereas four of the six strains with atypical MIC for erythromycin were positive for erm (B) and one strain each was positive for erm (C) and erm (T). The two Lact. plantarum strains with atypical MIC for tetracycline harboured a plasmid-encoded tet (M) gene. The majority of the tet (W)-positive Lact. reuteri strains and all erm -positive Lact. reuteri strains carried the genes on plasmids, as determined by Southern blot and a real-time PCR method developed in this study.
Conclusions:  Most of the antibiotic-resistant strains of Lact. reuteri and Lact. plantarum harboured known plasmid-encoded resistance genes. Examples of putative transfer machineries adjacent to both plasmid- and chromosome-located resistance genes were also demonstrated.
Significance and Impact of the Study:  These data provide some of the knowledge required for assessing the possible risk of using Lact. reuteri and Lact. plantarum strains carrying antibiotic resistance genes as starter cultures and probiotics.     

Antibiotic resistance in strains of Listeria spp. from meat products

The susceptibility or resistance to 26 antimicrobial agents was determined for 64 strains of Listeria monocytogenes and 102 strains of L. innocua isolated from Italian meat products. Some strains of L. monocytogenes were found to be resistant to tetracycline, erythromycin, co-trimoxazole and clindamycin. No plasmids were found in any L. monocytogenes strain. Five strains of L. innocua contained a 7.9 kbp plasmid, but these isolates were not resistant to any antibiotic in common and treatment with curing agents could not eliminate resistance to antibiotics. These results suggest that antibiotic resistance was not likely to be plasmid mediated in our strains.     

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.     

DNA-DNA homology, physiological characteristics and distribution of lactic acid bacteria isolated from maize silage

Lactic acid bacteria represent a dynamic bacterial group in maize silages. their establishment, variations and characterization have been studied by investigating 22 samples taken at different times during the ensilage process. After a preliminary screening based on physiological characteristics, 100 of 229 strains isolated were chosen for further taxonomic investigation. Twenty-nine strains of homo-fermentative lactobacilli were identified as Lactobacillus plantarum, L. casei and L. coryniformis subsp. coryniformis ; 24 heterofermentative strains were allotted to the species L. buchneri, L. brevis, L. fermentum and Leuconostoc paramesenteroides ; 22 coccal strains were assigned to Pediococcus pentosaceus and P. acidilactici and 25 coccal strains were identified as Enterococcus faecium, Streptococcus lactis and Strep. bovis. A few strains remained unidentified.     

Genome Sequence of Pediococcus pentosaceus Strain IE-3

We report the 1.8-Mb genome sequence of Pediococcus pentosaceus strain IE-3, isolated from a dairy effluent sample. The whole-genome sequence of this strain will aid in comparative genomics of Pediococcus pentosaceus strains of diverse ecological origins and their biotechnological applications.