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Identification of classical Propionibacterium species using 16S rDNA-restriction fragment length polymorphisms.

The phenotypic identification of the classical propionibacteria is essentially still problematic and alternative techniques for the identification of the various species are required. A rapid and sensitive technique for the routine identification of the classical propionibacteria, based on the amplification of 16S rRNA genes using the polymerase chain reaction and the subsequent restriction endonuclease digestion of the PCR products, was previously described. Although this technique enabled differentiation between the various classical species examined it was only evaluated on a limited number of type and reference strains. During this study, the taxonomic relationship between 135 Propionibacterium strains from diverse ecological niches, representing four classical species was investigated using this PCR/RFLP technique. Visual differentiation between the classical Propionibacterium was possible after restriction endonuclease digestion of the PCR products obtained using primers 16sP1-16sP4 and 16sP3-16sP4 with the restriction endonucleases HaeIII, AluI and HpaIII, respectively. With the exception of strains independently identified as "P. rubrum" and "P. sanguineum", the results of this study confirm the consolidation of the "old" species into the various classical species as they currently exist. It was therefore concluded that the PCR/RFLP protocol is suitable for the rapid and routine identification of the classical propionibacteria.

Bacterial Typing Techniques↗

Propionibacterium acnes-metabolites inhibit experimental lung metastasis of murine sarcoma L-1 in BALB/c-mice.

Adhesive interactions between tumor cells and host tissue occur at several stages of metastasis. Such interactions might be inhibited by microbial metabolites resembling the binding regions of matrix molecules. Certain metabolite sequences including Gly, Asp, Arg, and Ser (GAAS) proved to be critical for cell interactions, e.g. with fibronectin. In vitro, the rosette formation of murine pulmonary cells and sarcoma L-1 cells decreased significantly in the presence of Propionibacterium acnes-metabolites rich in GAAS. In vivo, coinjection of Propionibacterium acnes-metabolites and sarcoma L-1 cells significantly inhibited the formation of lung colonies in BALB/c mice. The inhibition of lung colonization by these metabolites appeared to be noncytotoxic and obviously did not result from impairment of cellular tumorigenicity.

Amino Acid Sequence↗

Influence of Propionibacterium avidum KP-40 on the cellular immune system after intensive sport activity.

Intensive sport activities resulted in temporary downregulation of defined immune functions. To check its influence on the cellular immune system, 15 male professional ice hockey players of a German first league club were observed and a decrease of lymphocyte subset counts and activities was found after anaerobic exercise. To stabilize cellular immune functions, the players were orally treated with Propionibacterium avidum KP-40 (10 mg per administration; twice a day; 7 days), a well documented bacterial immunmodifier. After administration of Propionibacterium avidum KP-40, lymphocyte counts and activities after anaerobic exercise resembled normal values. For some subjects, defined lymphocyte subset counts and activities even exceeded normal values.

Exercise Test↗

Identification of critical lysyl residues in the pyrophosphate-dependent phosphofructo-1-kinase of Propionibacterium freudenreichii.

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii was inactivated by low concentrations of the lysine-specific reagent pyridoxal phosphate (PLP) after sodium borohydride reduction. The substrates fructose 6-phosphate and fructose 1,6-bisphosphate protected against inactivation whereas inorganic pyrophosphate had little effect. An HPLC profile of a tryptic digest of PPi-PFK modified at low concentrations of PLP showed a single major peak with only a small number of minor peaks. The major peak peptide was isolated and sequenced to obtain IGAGXTMVQK, where X represents a modified lysine residue, corresponding to Lys-315. Lys-315 was protected from reaction with PLP by fructose 1,6-bisphosphate. As indicated by HPLC maps of PPi-PFK modified with varying concentrations of PLP, a direct correlation was observed between activity loss and the modification of Lys-315. Two of the minor peptide peaks were shown to contain Lys-80 and Lys-85, which were modified in a mutually exclusive manner. Partial protection against modification of these two residues was provided by MgPPi. The data were used to adjust the sequence alignment of the Propionibacterium enzyme with that of ATP-dependent PFK of Escherichia coli to identify homologous residues in the substrate binding site. It is suggested that Lys-315 interacts with the 6-phosphate of fructose 6-phosphate and that Lys-80 and -85 may be located near the pyrophosphate binding site.

Amino Acid Sequence↗

Regulation of an in vivo metal-exchangeable superoxide dismutase from Propionibacterium shermanii exhibiting activity with different metal cofactors.

The anaerobic, but aerotolerant Propionibacterium freudenreichii sp. shermanii contains a single superoxide dismutase [EC 1.15.1.1.] exhibiting comparable activity with iron or manganese as metal cofactor. The formation of superoxide dismutase is not depending on the supplementation of iron or manganese to the culture medium. Even in the absence of these metals the protein is built in comparable amounts. Bacteria grown in the absence of iron and manganese synthesize a superoxide dismutase with very low activity which had incorporated copper. If the medium was also depleted of copper, cobalt was incorporated, leading to an enzymically inactive form. In the absence of cobalt an enzymically inactive superoxide dismutase was built with unknown metal contents. Upon aeration the amount of superoxide dismutase activity increased continuously up to 9 h, due to a de novo synthesis of the protein. This superoxide dismutase had incorporated iron into the active centre. The superoxide dismutase of Propionibacterium shermanii is able to form a much wider variety of complexes with trace metal ions in vivo than previously recognized, leading to the hypothesis that the original function of these proteins was the binding of cytoplasmic trace metals present in excess.

Bacterial Proteins↗

Removal of common Fusarium toxins in vitro by strains of Lactobacillus and Propionibacterium.

This study was conducted to examine the ability of selected strains of Lactobacillus and Propionibacterium to remove common Fusarium toxins, trichothecenes, from liquid media. The trichothecenes studied were deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), nivalenol (NIV), fusarenon (FX), diacetoxyscirpenol (DAS), T-2 toxin (T-2) and HT-2 toxin (HT-2). The Lactobacillus rhamnosus strain GG (LGG), Lactobacillus rhamnosus strain LC-705 (LC-705) and Propionibacterium freudenreichii ssp. shermanii JS (PJS) were incubated in PBS buffer containing 20 microg toxin ml(-1) for 1h at 37 degrees C, and after centrifugation the concentration of the toxins was measured in the supernatant fraction. Both viable and heat-killed forms of LGG and PJS were more efficient than LC-705 in removing the toxins from the liquid media. LGG and PJS removed four of the seven tested toxins (the removal varying from 18 to 93%) and LC-705 two toxins (10-64%). Of the toxins, 3-AcDON was not removed by any of the bacteria; HT-2 was removed by the non-viable LGG and also slightly by non-viable LC-705; DAS was removed by all three bacteria tested. Binding is postulated as the possible mechanism of the removal, since no difference was observed between the ability of viable and heat-killed bacteria in removing the trichothecenes, and no degradation products of the toxins were detected by gas chromatography (GC)-mass spectrometry (MS) analysis. It is concluded that significant differences exist in the ability of the bacteria to bind trichothecenes in vitro.

Decontamination↗

Propionibacterium acnes infection following subdural tap.

The case of an infant who had subdural hematomas that became infected with Propionibacterium acnes is reported. This is the second reported case of intracranial Propionibacterium acnes infection resulting from diagnostic or therapeutic manipulation.

Bacterial Infections↗

Propionibacterium levels in patients with and without acne vulgaris.

Propionibacterium species were quantified on the foreheads and cheeks of persons with and without acne in three age groups: 11 to 15, 16 to 20, and 21 to 25. Propionibacteria were virtually absent in the pubertal non-acne group compared to a geometric mean density of 114,800 per sq cm in the acne group. A similar sharp difference existed between the acne subjects and normals in the age range of 16 to 20 years: 85,800 organisms per sq cm compared to 588 per sq cm. Patients with acne and normal subjects over age 21 showed no difference in Propionibacterium levels. In acne patients, while there was a trend for lower levels, no significant difference was seen as the severity of inflammation increased.

Acne Vulgaris↗

The effect of zinc and erythromycin on the growth of erythromycin-resistant and erythromycin-sensitive isolates of Propionibacterium acnes: an in-vitro study.

The effect of zinc and erythromycin on cultures inoculated with mixtures of different ratios of erythromycin sensitive (ES) and resistant (ER) Propionibacterium acnes cells was studied in vitro. Propionibacterium acnes ES outgrew P. acnes ER in the absence of erythromycin and zinc. At low levels of erythromycin ES outgrew ER, whilst the addition of 600 pg/ml zinc further reduced the growth of ER compared to ES. Growth of ER and ES were similar at levels of erythromycin near the minimum inhibitory concentration (MIC) of ES cells. Concentrations above the MIC for ES cells inhibited ES but not ER cells. At the higher concentrations of erythromycin, the addition of 96 ng/ml zinc delayed the growth of ER cells, whilst the addition of 300 micrograms/ml zinc prevented the growth of ER cells. The combination of erythromycin and zinc, at appropriate concentrations, inhibits both ES and ER.

Bacteriological Techniques↗

PCR detection and 16S rRNA sequence-based phylogeny of a novel Propionibacterium acidipropionici applicable for enhanced fermentation of high moisture corn.

AIMS: The aims of this study were to develop a sensitive and more rapid detection of Propionibacterium acidipropionici DH42 in silage and rumen fluid samples, and to explore its 16S rRNA sequence-based phylogeny. METHODS AND RESULTS: Nested polymerase chain reaction (PCR) was used with DH42-specific primers dhb1 and dhb2 for the secondary amplification of a 1267-bp fragment of 16S rRNA encoding gene. Using the established protocols for PCR amplification, as low as 10(2) and 10(3) CFU ml(-1) of strain DH42 in silage extracts and rumen fluid, respectively, were detected. To determine phylogenetic relationships between DH42 and other representatives of Propionibacterineae, a 1529-bp fragment of its 16S rRNA was amplified by PCR and sequenced. The propionibacterium DH42 formed a cluster with Eubacterium combesii, P. acidipropionici and P. microaerophilus. CONCLUSIONS: 16S rRNA-based PCR detection technique was developed for DH42 in silage and rumen fluid samples. The 16S rRNA sequence confirmed the earlier identification of strain DH42 as P. acidipropionici. However, variable nucleotide positions were revealed. SIGNIFICANCE AND IMPACT OF THE STUDY: Variability of 16S rRNA sequence within the species P. acidipropionici, determined in this study, poses the need of re-sequencing for some species of the suborder Propionibacterineae for a more reliable classification.

Animals↗

Propionibacterium acnes biotypes and susceptibility to minocycline and Keigai-rengyo-to.

BACKGROUND: Propionibacterium acnes is the predominant organism in acne lesions, but the sensitivity of different biotypes of P. acnes to therapeutic agents has seldom been reported. METHODS: To characterize biotypes of P. acnes and to measure the effects of Keigai-rengyo-to (KRT) and minocycline (MINO) on clinical P. acnes isolates. RESULTS: Propionibacterium acnes biotype III (BIII) is the most common form of identified acne lesion, followed by P. acnes biotype I. BIII was isolated from mild, moderate and severe severity and the average lipase activity of BIII was higher than that of Biotypes I, II, IV and V. No significant differences in the decrease of free fatty acid production elicited by KRT or by MINO were found between BIII and the other biotypes. The degree of decreased butyric acid production was greater than that of propionic acid production in the medium supplemented with MINO. The percent decrease of butyric acid production elicited by 1 mg/mL of KRT was the same as that elicited by 0.1 microg/mL of MINO. Among biotypes of P. acnes, the minimal inhibitory concentrations of agents tested were generally higher in erythritol-positive biotypes than in erythritol-negative biotypes. CONCLUSION: The high frequency of BIII might be responsible for the severity of acne in patients. It seems that if the same concentrations of MINO and KRT are used, the antilipase activity of MINO is stronger than that of KRT. Minocycline also has a direct anti-lipase activity against P. acnes. The mechanism underlying the influence of erythritol on the susceptibility of P. acnes to these agents remains unknown.

Acne Vulgaris↗

Propionibacterium acnes.

Propionibacterium acnes, a common skin organism, is most notably recognized for its role in acne vulgaris. It also causes postoperative and device-related infections and has been associated with a number of other conditions such as sarcoidosis and synovitis, acne, pustulosis, hyperostosis and osteitis (SAPHO), although its precise role as a causative agent remains to be determined. Propionibacterium acnes produces a number of virulence factors and is well known for its inflammatory and immunomodulatory properties. Recent publication of the P. acnes genome should provide further insights into the pathogenic capabilities of the organism and potentially lead to the development of new therapies.

Acne Vulgaris↗

Profiling of Propionibacterium acnes recovered from root canal and blood during and after endodontic treatment.

This report describes the first results of an ongoing study of bacteremia after endodontic treatment of teeth with Asymptomatic apical periodontitis. After access cavity preparation, microbiological samples were taken from the root canal under aseptic conditions in 4 single-rooted teeth in 4 patients. In treatment of 2 of the patients, the first 3 reamers (sizes 15-25) were deliberately used to a level 2 mm beyond the apical foramen. In 2 patients the instrumentation ended inside the root canal 1 mm short of the apical foramen. Blood samples were taken from the patients during the endodontic instrumentation and 10 min after the treatment was completed. Using lysis-filtration under anaerobic conditions, the blood was passed through a cellulose membrane filter. The filters as well as the root canal samples were incubated using an anaerobic technique. Anaerobic bacteria were isolated from all root canals. In the 2 patients where overinstrumentation had occurred, Propionibacterium acnes was recovered both from the root canals and from the blood samples taken during and after the treatment had been completed. Biochemical profiles, antibiotic susceptibility tests and electrophoresis of soluble proteins revealed that Propionibacterium acnes isolated from the root canal and blood samples were identical within patients, but varied between patients. Facultative anaerobic bacteria including Streptococcus sanguis were recovered from only one root canal sample and not from the blood samples.

Adult↗

Characterization of Propionibacterium plasmids.

Plasmid DNAs from 15 Propionibacterium strains were characterized by using restriction endonuclease analyses, DNA-DNA hybridizations, and curing experiments. Restriction endonuclease analysis identified seven distinct plasmids (pRGO1 through pRGO7). Detailed restriction maps were constructed for four of these plasmids. DNA-DNA hybridization analysis revealed that plasmids pRGO1 and pRGO2 had extensive sequence homology and that both were homologous to pRGO7 and to similar sequences of pRGO5. Plasmids pRGO4 and pRGO6 did not have any significant sequence homology with any of the other plasmids. Plasmid pRGO3 had partial sequence homology only with pRGO7. Curing of plasmids pRGO1, pRGO2, and pRGO5 was achieved by treatment with acriflavin, but we failed to identify any plasmid-encoded bacteriocin production, carbohydrate fermentation, or antibiotic resistance. However, physical evidence was obtained that tentatively linked the clumping phenotype of Propionibacterium jensenii P38 with plasmid pRGO5.

Base Sequence↗

Jenseniin G, a heat-stable bacteriocin produced by Propionibacterium jensenii P126.

The genus Propionibacterium includes cutaneous species typically found on human skin and the dairy or classical species (Propionibacterium freudenreichii, P. jensenii, P. thoenii, and P. acidipropionici) used industrially for the production of Swiss cheese and propionic acid. Grinstead (1989, M.S. thesis, Iowa State University, Ames) has previously observed that some dairy propionibacteria inhibit other species in the classical grouping. We further investigated the inhibitor(s) produced by P. jensenii P126 (ATCC 4872). An antagonist(s) from anaerobic agar cultures of P126 strongly inhibited two closely related strains of propionibacteria, P. acidipropionici P5 and P. jensenii P54, and Lactobacillus bulgaricus NCDO 1489, Lactobacillus delbrueckii subsp. lactis ATCC 4797, Lactococcus cremoris NCDO 799, and Lactococcus lactis subsp. lactis C2. The inhibitor, designated jenseniin G, was active at pH 7.0; inactivated by treatment with pronase E, proteinase K, and type 14 protease; insensitive to catalase; and stable to freezing, cold storage (4 degrees C, 3 days), and heat (100 degrees C, 15 min). Classification of the inhibitor as a bacteriocin is supported by its proteinaceous nature and its bactericidal activity against L. delbrueckii subsp. lactis ATCC 4797. The lack of detectable plasmids suggests a chromosomal location for the determinant(s) of jenseniin G.

Bacteriocins↗

Cloning and characterization of the gene encoding glutamate 1-semialdehyde 2,1-aminomutase, which is involved in delta-aminolevulinic acid synthesis in Propionibacterium freudenreichii.

The gene from Propionibacterium freudenreichii that encodes glutamate 1-semialdehyde 2,1-aminomutase (EC 5.4.3.8), which is involved in the C5 pathway for synthesis of delta-aminolevulinic acid (ALA), a precursor in heme and cobalamin biosynthesis, was cloned onto a multicopy plasmid, pUC18, via complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of fragments from the initial 3.3-kb chromosomal fragment allowed the isolation of a 1.9-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.9-kb DNA fragment revealed an open reading frame (ORF) that was located downstream from a potential ribosome-binding site. The ORF encoded a polypeptide of 441 amino acid residues, and the deduced molecular mass of this polypeptide is 45,932 Da. A high G+C content (70 mol%) of the codons of the ORF was found and was consistent with the taxonomic features of Propionibacterium species. The amino acid sequence showed a high degree of homology with those of the HemL proteins from other organisms, and a putative binding site for pyridoxal 5'-phosphate was conserved, with the exception of a single substitution of phenylalanine for leucine. These results suggest that ALA is synthesized via the C5 pathway in a producer of vitamin B12, P. freudenreichii.

Amino Acid Sequence↗

Effects of expression of hemA and hemB genes on production of porphyrin in Propionibacterium freudenreichii.

The genus Propionibacterium has a wide range of probiotic activities that are exploited in dairy and fermentation systems such as cheeses, propionic acid, and tetrapyrrole compounds. In order to improve production of tetrapyrrole compounds, we expressed the hemA gene, which encodes delta-aminolevulinic acid (ALA) synthase from Rhodobacter sphaeroides, and the hemB gene, which encodes porphobilinogen (PBG) synthase from Propionibacterium freudenreichii subsp. shermanii IFO12424, either monocistronically or polycistronically in strain IFO12426. The recombinant strains accumulated larger amounts of ALA and PBG, with resultant 28- to 33-fold-higher production of porphyrinogens, such as uroporphyrinogen and coproporphyrinogen, than those observed in strain IFO12426, which harbored the shuttle vector pPK705.

5-Aminolevulinate Synthetase↗

Heterologous production of antimicrobial peptides in Propionibacterium freudenreichii.

Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P4) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P4 and Ppamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.

Antimicrobial Cationic Peptides↗