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Phylogenetic analysis of the genus Pediococcus, including Pediococcus claussenii sp. nov., a novel lactic acid bacterium isolated from beer.

Pediococci are found in foods and on plants and as beer-spoilage agents. The goal of the present study was to use the DNA sequences of the first three variable regions of the 165 rRNA gene, the 16S-23S rRNA internally transcribed spacer region sequence and approximately a third of the 60 kDa heat-shock protein gene to elucidate phylogenetic groupings within the genus Pediococcus. Phylogenetic trees were created with sequence data from 31 Pediococcus and three Lactobacillus isolates. Complete 16S rRNA gene sequences from selected Pediococcus isolates were also examined. The results were interpreted in relation to the currently accepted Pediococcus species. We found that, where previously done, speciation of many Pediococcus isolates is inaccurate. Also, one grouping of seven isolates did not include any currently recognized Pediococcus species type isolate. Our phylogenetic analyses support the conclusion that these seven isolates, all of brewing spoilage origin, belong to a novel species, for which the name Pediococcus claussenii sp. nov. is proposed (type strain P06(T0 = ATCC BAA-344(T) = DSM 14800(T)). Phylogenetic analysis has therefore helped to resolve problems surrounding species identification of Pediococcus isolates.

Base Composition↗

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.

Bacteriocins↗

Production and characterization of antibacterial compounds produced by Pediococcus damnosus and Pediococcus pentosaceus.

The broad-spectrum antibacterial activity exhibited by three Pediococcus strains isolated from beer was preliminarily characterized. Factors affecting the production rate of bacterial inhibitors were screened and the effects of simultaneous cultivation of Lactococcus and Pediococcus on the production of inhibitory substances were studied. The antibacterial activity against a range of Gram-negative test organisms was not affected by catalase or proteolytic enzymes and was extremely thermotolerant. Production of the inhibitors was maximal between pH 6 and pH 7. A growth medium containing unhopped end-fermented wort was beneficial for the production of inhibitors, particularly by the Pediococcus damnosus strain, and anaerobic growth conditions were preferable. The antagonistic activity against the Gram-negative test organism Salmonella infantis could be demonstrated after an incubation period of only 2 d if the Pediococcus and Lactococcus strains were incubated simultaneously as a mixed population.

Aerobiosis↗

The effect of Pediococcus damnosus and Pediococcus pentosaceus on the growth of pathogens in minced meat.

The antibacterial effects of one strain of Pediococcus damnosus and two strains of Pediococcus pentosacaeus against Clostridium perfringens, Listeria monocytogenes, Salmonella infantis and Yersinia enterocolitica were investigated. Growth inhibition studies were conducted in juice from minced meat incubated at +6 degrees C and +15 degrees C for various periods after the inoculation with pediococci. Inhibitory effects were seen for all bacteria tested.

Animals↗

Resistance of Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. I. Dihydrofolate reductase, thymidylate synthetase and formyltetrahydrofolate synthetase in amethopterin-resistant and -sensitive strains of Pediococcus cerevisiae.

Pediococcus cerevisiae/AMr, resistant to amethopterin, possesses a higher dihydrofolate reductase (5, 6, 7, 8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity than the parent, a folate-permeable and thus amethopterin-susceptible strain and than the wild-type. The properties of dihydrofolate reductase from the three strains have been compared. Temperature, pH optima, heat stability, as well amethopterin binding did not reveal significant differences between the enzymes from the susceptible and resistant strains. The enzyme from the wild-type was 10 times more sensitive to inhibition by amethopterin and more susceptible to heat denaturation. The apparent Km values for dihydrofolate in enzymes from the three strains were in the range of 4.8--7.2 muM and for NADPH 6.5--8.0 muM. The amethopterin-resistant strain exhibited cross-resistance to trimethoprim and was about 40-fold more resistant to the latter than the sensitive parent and the wild-type. The resistance to trimethoprim appears to be a direct result of the increased dihydrofolate reductase activity. Inhibition of dihydrofolate reductase activity by this drug was similar in the three strains. 10--20 nmol caused 50% inhibition of 0.02 enzyme unit. Trimethoprim was about 10 000 times less effective inhibitor of dihydrofolate reductase than amethopterin. The cell extract of the AMr strain possessed a folate reductase activity three times higher than that of the sensitive strain. The activities of other folate-related enzymes like thymidylate synthetase and 10-formyltetrahydrofolate synthetase (formate: tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) were similar in the three strains studied.

Drug Resistance, Microbial↗

Antimicrobial susceptibility of Pediococcus spp. and genetic basis of macrolide resistance in Pediococcus acidilactici HM3020.

We determined the MICs of 28 antimicrobial agents against 36 clinical strains of Pediococcus spp. (25 P. acidilactici, 9 P. pentosaceus, and 2 P. urinaeequi strains). Penicillin G, imipenem, gentamicin, netilmicin, erythromycin, clindamycin, rifampin, chloramphenicol, daptomycin, and ramoplanin were the most active. All strains of P. acidilactici were susceptible to novobiocin, whereas all isolates of P. pentosaceus were resistant. Novobiocin could therefore be helpful for differentiation of these two closely related species. P. acidilactici HM3020 was inducibly resistant to macrolide, lincosamide, and streptogramin B-type (MLS) antibiotics. Resistance was due to a determinant homologous to ermAM and carried by a nontransferable 46-kb plasmid, pVM20. This plasmid was structurally distinct from two enterococcal MLS resistance plasmids, pIP819 and pAM beta 1. The 34 strains of P. acidilactici and P. pentosaceus were resistant to tetracycline, and total DNA of these strains did not hybridize to probes specific for tetK, tetL, tetM, and tetO.

Anti-Bacterial Agents↗

The phylogeny of Aerococcus and Pediococcus as determined by 16S rRNA sequence analysis: description of Tetragenococcus gen. nov.

The phylogenetic interrelationships of the genera Pediococcus and Aerococcus were investigated using reverse transcriptase sequencing of 16S rRNA. The genus Pediococcus was found to be phylogenetically heterogeneous. The four species P. acidilactici, P. damnosus, P. parvulus and P. pentosaceus formed a phylogenetically distinct group. Within this pediococcal cluster, P. acidilactici was closely related to P. pentosaceus whereas P. damnosus showed a specific relationship with P. parvulus. The species P. dextrinicus, although showing significant sequence relatedness with these pediococcal species, was peripheral to the genus. Pediococcus halophilus exhibited low sequence homology with all of the species examined and formed a distinct line of descent. Pediococcus halophilus exhibited a closer affinity with enterococci and carnobacteria than with the other lactic acid bacteria. Pediococcus urinae-equi was phylogenetically very closely related to Aerococcus viridans. The 16S rRNA sequences of the type strains of these species differed by only two nucleotides (99.9% sequence homology) and clearly demonstrate that P. urinae-equi is a member of the genus Aerococcus.

Base Sequence↗

Thermal inactivation of Pediococcus sp. in simulated apple cider during high-temperature short-time pasteurization.

Prompted by concerns regarding outbreaks of food-borne illness which have occurred due to the consumption of commercial, nonpasteurized fruit juices contaminated with Escherichia coli O157:H7, the US Food and Drug Administration and Canadian Food Inspection Agency are considering several new safety standards to apply to fresh juices, including mandatory pasteurization of all apple cider. In support of these initiatives, a study was conducted to evaluate the pasteurization of simulated cider using a heat-resistant nonpathogenic test bacterium, Pediococcus sp. NRRL B-2354. Thermal inactivation of the Pediococcus sp. was determined using a pilot scale high-temperature short-time (HTST) pasteurizer with a plate heat exchanger. The cumulative lethal effect, or pasteurization effect (PE), was obtained by converting times at different temperatures in the various sections of the pasteurizer to the equivalent time at the reference temperature (72 degrees C). PE was then related by a simple linear function to the log(10) of the percentage of viable counts with a power transformation of the PE values to improve linear fit. r(2) values for the four Pediococcus sp. trials varied from 0.921 to 0.981. Intertrial variation was incorporated into the model using @RISK simulation software. Output from simulations confirmed that treatment at 71 degrees C for 16 s can ensure a 5-log reduction of Pediococcus sp.

Beverages↗

Pediocin PD-1, a bactericidal antimicrobial peptide from Pediococcus damnosus NCFB 1832.

Pediocin PD-1, produced by Pediococcus damnosus NCFB 1832, is inhibitory to several food spoilage bacteria and food-borne pathogens. However, pediocin PD-1 is not active against other Pediococcus spp. and differs in this respect to other pediocins produced by Pediococcus acidilactici and Pediococcus pentosaceus. Production of pediocin PD-1 starts during early growth and reaches-a plateau at the end of exponential growth. Pediocin PD-1 was partially purified and its size was determined by tricine-SDS-PAGE as approximately 3.5 kDa. The isoelectric point (pI) of pediocin PD-1 is approximately 3.5, as determined with the Rotofor electrofocusing cell (BioRad). Pediocin PD-1 is heat-resistant (10 min at 121 degrees C) and remains active after 30 min of incubation at pH 2-10. Pediocin PD-1 is resistant to treatment with pepsin, papain, alpha-chemotrypsin and trypsin, but not Proteinase K. Pediocin PD-1 is bactericidal against sensitive cells of Oenococcus oeni (previously Leuconostoc oenos).

Bacteriocins↗

Identification of Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus by rDNA-based techniques.

Ribosomal DNA-based techniques including the analysis of profiles generated by ISR amplification, ISR restriction and ARDRA have been evaluated as molecular tools for identifying Carnobacterium, Lactobacillus, Leuconostoc and Pediococcus. They have been applied for the molecular characterization of 91 strains with the following identities: eight Carnobacterium including the eight type species of the genus; 61 Lactobacillus including 40 type strains out of 45 species, 13 Leuconostoc, out of them 11 are type strains and three are subspecies of Lc. mesenteroides; and nine strains representing the six species of genus Pediococcus. The genetic relationship displayed between these species by rrn-based profiles is sustained by their phylogenetic relationships and can therefore be considered useful for taxonomic purposes. Profiles obtained by ISR amplification allowed identification at genus level of Carnobacterium and Leuconostoc, and even at species level in genus Carnobacterium. Genera Lactobacillus and Pediococcus could not be distinguished from each other by applying this technique. The Lactobacillus species analysed here (45) were differentiated using ARDRA-DdeI and ISR-DdeI profiles, sequentially, and Pediococcus species by ISR-DdeI profiles. It was necessary to combine profiles generated by restriction of ISR-DdeI, ARDRA-DdeI and ARDRA-HaeIII in order to complete the identification of Leuconostoc species.

Bacterial Typing Techniques↗

Riboprinting and 16S rRNA gene sequencing for identification of brewery Pediococcus isolates.

A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI and PvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead of EcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation.

Base Sequence↗

SOME CHARACTERISTICS OF AN OLEATE-REQUIRING, HEMOLYTIC PEDIOCOCCUS.

Deibel, R. H. (St. James Hospital, Chicago Heights, Ill.), J. H. Silliker, and P. T. Fagan. Some characteristics of an oleate-requiring, hemolytic Pediococcus. J. Bacteriol. 88:1078-1083. 1964.-A gram-positive, catalase-negative, tetradforming coccus, isolated from a bronchial aspirate, is described. The organism was classified as a member of the genus Pediococcus on the basis of morphological and physiological characteristics. It differed from previously described members of this genus in that it evidenced a requirement for oleic acid, even in complex culture media. This isolate produced hemolysis on blood-agar formulated with certain specimens of human blood and not with others. It was demonstrated that hemolysis required oleate supplementation and that the level of this fatty acid required for hemolysis is greater than that necessary for growth of the organism. A cell-free hemolysin could not be demonstrated in the culture supernatant fluid of oleate-grown cultures. The organism was isolated from a patient suffering from tuberculosis; however, attempts to isolate similar organisms from other tuberculous patients as well as from routine throat and sputum specimens were negative. No pathogenicity was manifested when laboratory animals were inoculated with the hemolytic Pediococcus.

Catalase↗

A case of septicemia with Pediococcus acidilactici after long-term antibiotic treatment.

Presented here is a case of septicemia caused by an uncommon, multiresistant, gram-positive microorganism (Pediococcus acidilactici) after long-term antibiotic treatment. Pediococcus spp. are rarely cultivated from clinical specimens, and species differentiation is difficult due to the paucity of phenotypic traits. In this case, a polyphasic approach consisting of phenotypic and molecular genetic analyses was used, and the identification of Pediococcus acidilactici was conclusive. Precise identification and antimicrobial susceptibility testing of rarely isolated bacteria are required in order to provide adequate treatment to infected patients and to determine the pathogenic role of these organisms.

Adult↗

[Microbiological and clinical studies of vancomycin resistant Leuconostoc spp. and Pediococcus spp. isolated from septicemia patients].

We described three septicemia cases in which blood cultures yielded gram-positive cocci identified as Leuconostoc spp. and Pediococcus spp. Patients were three male adults aged 63 to 71 years with severe underlying diseases, pancreatic cancer, esophageal cancer and diabetes mellitus with chronic renal failure. They had fever and chills at the onsets of septicemia with acute obstructive suppurative cholangitis, acute pneumonia, and infection complicated with invasion sites of esophageal cancer contagious to bronchus and subcutaneous tissue. Blood cultures yielded catalase and oxidase negative highly vancomycin-resistant (MIC: 1024 micrograms/ml <) gram-positive cocci showing alpha or gamma hemolysis on blood agar plates. Two cases were polymicrobial infections. In one case with esophageal cancer, clinical symptoms persisted after the start of antimicrobial chemotherapy and the patient died 10 days later associated with complications of esophageal cancer. Leuconostoc lactis, Leuconostoc mesenteroides subsp. dextranicum, and Pediococcus acidilactici wee identified by physiological reactions. These strains were also highly resistant to teicoplanin and fosfomycin, and tolerant to all rested beta-lactams such as benzylpenicillin. This is the first report in Japan to our knowledge on the identification of Leuconostoc spp. and Pediococcus spp. isolated from human infectious diseases.

Aged↗

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.

Anti-Bacterial Agents↗

Monoclonal antibody-colony immunoblot method specific for isolation of Pediococcus acidilactici from foods and correlation with pediocin (bacteriocin) production.

BALB/c mice were immunized with broken, heat-killed cells of Pediococcus acidilactici H. After murine cell fusions, one monoclonal antibody (MAb), Ped-2B2, was selected on the basis of its positive reaction with seven of seven strains tested in an enzyme-linked immunosorbent assay with whole cells of P. acidilactici. The MAb Ped-2B2 did not show any cross-reactions with other lactic-acid bacteria or other gram-positive or gram-negative organisms. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis of surface proteins of P. acidilactici indicated that Ped-2B2 reacted with a protein of 116 kDa. MAb Ped-2B2 was used as a probe to isolate Pediococcus species from fermented-meat products by colony immunoblotting. A total of 18 Ped-2B2-reactive Pediococcus spp. isolates were isolated from eight food samples and assayed for bacteriocin production. All of the isolates produced bacteriocins which were heat stable, proteinaceous, and inhibitory to Lactobacillus plantarum NCDO 955. Biochemical characterization of these isolates indicated that they were all P. acidilactici.

Animals↗

Changes in membrane fatty acid composition of Pediococcus sp. strain NRRL B-2354 in response to growth conditions and its effect on thermal resistance.

Membrane fatty acid composition and thermal resistance (D value) of Pediococcus sp. were determined for mid-exponential-phase (ME) and stationary-phase (ST) cells grown in tryptic soy broth (TSB) and tryptone-glucose-yeast extract (TGY) at 28 and 37 degrees C. As the cells entered the stationary phase of growth, the unsaturated fatty acid, C18:1 n11c, produced during the exponential phase of growth was converted to its cyclic form, C19:0 Delta9c. This shift in membrane fatty acid composition was accompanied by an increase in the D values of this bacterium. Data from this study suggest that the membrane fatty acid composition of Pediococcus sp. is dependent on the growth conditions and that membrane fatty acid composition plays a critical role in thermal resistance. Thermal inactivation curves of Pediococcus sp. cells grown in TGY at 28 degrees C indicated the presence of a cell population that is heterogeneous in thermal resistance. The growth of this bacterium in TGY at 37 degrees C and in TSB at 28 and 37 degrees C resulted in cell populations that were uniform in thermal resistance with a lag time for thermal inactivation. Thermal inactivation curves of ME and ST cultures were similar. The data presented here suggest that the cell population's uniformity of thermal inactivation is independent of the growth phase of the culture.

Cell Membrane↗