Effects of pH on biomass, maximum specific growth rate and extracellular enzyme production by three species of cutaneous propionibacteria grown in continuous culture

Three cutaneous propionibacteria, Propionibacterium acnes, Propionibacterium avidum and Propionibacterium granulosum, were grown in chemostats using semi-synthetic medium at various pH values. Growth occurred between pH 4.5 and 7.5 for P. acnes and pH 5.0 and 8.0 for P. avidum and P. granulosum. The highest mumax was at pH 6.0 for the three species. Maximum biomass production was obtained at pH 6.0 for P. acnes and P. avidum and at pH 7.5 for P. granulosum. Extracellular enzyme production occurred over the entire pH growth range when denaturation of the enzymes was taken into account. However, detectable activity of the enzymes was found in a narrower range of pH due to the denaturation of the enzymes at low or high pH values. The highest production of enzymes occurred at pH values between 5.0 and 6.0, apart from the production of hyaluronate lyase of P. granulosum (pH 6.0 to 7.0) and the proteinase of P. acnes and P. avidum (pH 5.0 to 7.5). Propionibacterium acnes produced a lipase, hyaluronate lyase, phosphatase and proteinase activity. Propionibacterium avidum produced a lipase and proteinase activity. Propionibacterium granulosum produced a lipase and hyaluronate lyase.     

Mode of protection of mice against herpes simplex virus type 2 infection by Propionibacterium

We compared various strains of Propionibacterium with regard to protection of young adult mice against lethal infection with herpes simplex virus type 2 (HSV-2). Propionibacterium acnes, P. granulosum, and P. avidum were protective, while P. acidi-propionici and P. lymphophilum were ineffective. The protective effect proved to be in the cell wall fraction. Attempts were made to elucidate possible mechanisms of the protection using both effective and ineffective strains. The results strongly suggest that induction of interferon rather than activation of macrophages and natural killer cells by Propionibacterium pretreatment plays a crucial role, directly or indirectly, in protection against infection by herpes simplex virus. Propionibacterium only moderately protected newborn mice against HSV-2 infection.     

In situ assessment of porphyrin photosensitizers in Propionibacterium acnes

Porphyrins are known to be efficient photosensitizer molecules and the combined action of light and porphyrins in Propionibacterium acnes have a lethal action on the cells. Identification and quantification of in situ porphyrins in P. acnes have been done using an integrating sphere connected to an ordinary absorption spectrophotometer, and the amounts of porphyrins in the cells were quantified by measuring scattering free absorption spectra of the cell suspensions. The concentration of porphyrins in P. acnes cells were increased in either of two ways; by the addition of delta-aminolevulinic acid (ALA), which lead to the formation of coproporphyrin III under the incubation conditions used in these experiments, or by the addition of protoporphyrin IX (PPIX) to the cell suspension. In the latter case, PPIX molecules are taken up by the cells in a membrane-mediated uptake mechanism, and accumulate in the cells either on a monomeric or a particular aggregate form. The fraction of porphyrins on aggregate form increased with increasing PPIX additions. In the case of ALA induced porphyrin production, only monomeric porphyrins were stored in the cells. In both cases, the cells have a limited binding capacity of monomeric porphyrins, which is estimated to be 3 x 10(5) molecules/cell, or one porphyrin molecule to every 100st lipid molecule in the cell membrane.     

Effects of dilution rate on biomass and extracellular enzyme production by three species of cutaneous propionibacteria grown in continuous culture

Propionibacterium acnes, P. avidum and P. granulosum were grown in continuous culture at a range of dilution rates on a semi-synthetic medium. Dilution rates were chosen to allow the bacteria to grow at the same relative growth rates as compared to their respective mumax values. The steady-state levels and production rates of biomass and extracellular enzymes were determined. The lipase and hyaluronate lyase of P. granulosum and the proteolytic activity of P. acnes and P. avidum were growth linked enzymes (i.e. they were produced at constant amounts per unit of biomass). In contrast, the lipase, hyaluronate lyase and acid phosphatase of P. acnes and the lipase of P. avidum were shown to be non-growth linked enzymes.     

Growth of Cutaneous Propionibacteria on Synthetic Medium; Growth Yields and Exoenzyme Production

A synthetic medium containing only low molecular weight components has been developed to grow Propionibacterium acnes (P. acnes), P. avidum and P. granulosum.
The medium supported the growth of 30/32 typed strains, and when solidified with agar supported the growth of these bacteria isolated directly from the skin. The mean overall efficiency of isolation on the synthetic medium was 57% that of reinforced clostridial medium (RCM).
Batch culture experiments with glucose, fructose, glycerol or arginine in the medium showed that the concentrations as well as the type of carbon energy sources used could effect growth and exocellular enzyme production by these organisms.     

Eradication of Propionibacterium acnes by its endogenic porphyrins after illumination with high intensity blue light

Propionibacterium acnes is a Gram-positive, microaerophilic bacterium that causes skin wounds. It is known to naturally produce high amounts of intracellular porphyrins. The results of the present study confirm that the investigated strain of P. acnes is capable of producing endogenic porphyrins with no need for any trigger molecules. Extracts from growing cultures have demonstrated emission peaks around 612 nm when excited at 405 nm, which are characteristic for porphyrins. Endogenic porphyrins were determined and quantified after their extraction from the bacterial cells by fluorescence intensity and by elution retention time on high-performance liquid chromatography (HPLC). The porphyrins produced by P. acnes are mostly coproporphyrin, as shown by the HPLC elution patterns. Addition of delta-aminolevulinic acid (ALA) enhanced intracellular porphyrin synthesis and higher amounts of coproporphyrin have been found. Eradication of P. acnes by its endogenic porphyrins was examined after illumination with intense blue light at 407-420 nm. The viability of 24 h cultures grown anaerobically in liquid medium was reduced by less than two orders of magnitude when illuminated once with a light dose of 75 J cm(-2). Better photodynamic effects were obtained when cultures were illuminated twice or three times consecutively with a light dose of 75 J cm(-2) and an interval of 24 h between illuminations. The viability of the culture under these conditions decreased by four orders of magnitude after two illuminations and by five orders of magnitude after three illuminations. When ALA-triggered cultures were illuminated with intense blue light at a light dose of 75 J cm(-2) the viability of the treated cultures decreased by seven orders of magnitude. This decrease in viability can occur even after a single exposure of illumination for the indicated light intensity. X-ray microanalysis and transmission electron microscopy revealed structural damages to membranes in the illuminated P. acnes. Illumination of the endogenous coproporphyrin with blue light (407-420 nm) apparently plays a major role in P. acnes photoinactivation. A treatment protocol with a series of several illuminations or illumination after application of ALA may be suitable for curing acne. Treatment by both pathways may overcome the resistance of P. acnes to antibiotic treatment.     

The temperature dependence of porphyrin production in Propionibacterium acnes after incubation with 5-aminolevulinic acid (ALA) and its methyl ester (m-ALA).

Topical PDT treatment of the common skin disease acne vulgaris is now in clinical use. Propionibacterium acnes (P. acnes) is known to play an important role in acne. 5-Aminolevulinic acid (ALA) supplementation leads to an enhanced porphyrin production in the bacteria. Subsequent illumination with light of the proper wavelengths can reduce the number of bacteria and this might at least partly explain the PDT effect on acne. We have assessed the effects of temperature on P. acnes washed cell suspensions incubated for 4 h with ALA or ALA methyl ester (m-ALA). The effect on porphyrin production of both the cell suspension incubation temperature as well as the initial growth temperature of the cultivated cells prior to harvesting and use in suspension experiments was investigated. The bacterial porphyrin content was estimated from fluorescence emission spectra. It was found that incubation with ALA or m-ALA at a temperature 42 degrees C resulted in an approx. 100% and 33% increase in the total amount of PDT-relevant porphyrins produced as compared to incubation at 37 degrees C. These results support increasing the skin temperature during incubation with ALA or m-ALA in the clinic. The initial growth temperature, prior to the incubation, had no apparent effect on the ALA or m-ALA induced porphyrins. Activation energy studies indicate slightly higher temperature dependence in the case of ALA produced porphyrins as compared to m-ALA produced porphyrins (77 and 65 kJ mol(-1), respectively).     

Antimicrobial effects of tea-tree oil and its major components on Staphylococcus aureus, Staph. epidermidis and Propionibacterium acnes

Major components of two tea-tree oil samples were identified using thin layer and gas-liquid chromatography (TLC and GLC). Using a TLC-bioautographic technique, the tea-tree oils, terpinen-4-ol, oc-terpineol and α-pinene were found to be active against Staphylococcus aureus, Staph. epidermidis and Propionibacterium acnes whereas cineole was inactive against these organisms. The MIC values of the three active compounds increased in the order α-terpineol < terpinen-4-ol < α-pinene for all three micro-organisms. MIC values of the tea-tree oils and terpinen-4-ol were lower for P. acnes than for the two staphylococci. This study supports the use of tea-tree oil in the treatment of acne, and demonstrates that terpinen-4-ol is not the sole active constituent of the oil.     

Regional variations of cutaneous propionibacteria

Propionibacterium acnes, P. avidum, and P. granulosum were quantitatively measured in 50 young adults. The scalp, forehead, external auditory canal, alae nasi, anterior nares, groin, rectum, and antecubital and popliteal fossa were sampled. These represent various cutaneous microenvironments, differing in moisture, density of sweat, sebaceous glands, and extent of anaerobiosis. These studies show that the propionibacteria are ubiquitous on the skin, with P. acnes predominant in both prevalence and population, especially in areas rich in sebum. P. granulosum recovery paralled that of P. acnes, but the density was significantly lower. P. avidum was found mainly in moist areas and the retum, suggesting an intestinal reservoir.     

Propionibacterium acnes in the etiology of endocarditis]

Propionibacterium acnes is the gram positive anaerobic bacteria belongs to the normal skin and oral microbial flora. The participation of this microorganism in the infective endocarditis is still controversial. The aim of the study was to perform the diagnostic and therapeutic difficulties in 5 patients with infective endocarditis caused by Propionibacterium acnes. In 3 out of 5 patients the infective endocarditis developed after prosthesis valve replacement, in 2 others on the native valves. The inserted prostheses were mechanical ones, propionibacterium acnes was identified as causative organisms in all of the causes (two positive blood and/or valve culture). The bacterial strains were sensitive to the antibiotics as: penicillins, cephalosporins, clindamycin, and vancomycin, however cephalosporins used at the beginning of the treatment in 3 patients and clindamycin in 1 patient had limited clinical efficacy. Later treatment with timentin, augmentin and tienamycin was successful in 3 patients; one patient was cured with vancomycin. One patient died because of septic, embolic complication in early stage of illness. We conclude the effectiveness of penicillins in combination with clavulanic acid and tienamycin in therapy of infective endocarditis due to Propionibacterium acnes. The treatment should be lasted during 4-6 weeks.     

Regional variations of cutaneous propionibacteria.

Propionibacterium acnes, P. avidum, and P. granulosum were quantitatively measured in 50 young adults. The scalp, forehead, external auditory canal, alae nasi, anterior nares, groin, rectum, and antecubital and popliteal fossa were sampled. These represent various cutaneous microenvironments, differing in moisture, density of sweat, sebaceous glands, and extent of anaerobiosis. These studies show that the propionibacteria are ubiquitous on the skin, with P. acnes predominant in both prevalence and population, especially in areas rich in sebum. P. granulosum recovery paralled that of P. acnes, but the density was significantly lower. P. avidum was found mainly in moist areas and the retum, suggesting an intestinal reservoir.     

Electrophoretic protein patterns and enzyme mobilities in anaerobic coryneforms.

The soluble protein patterns and electrophoretic mobilities of malate and succinate dehydrogenases and catalase have been examined in 25 strains of Propionibacterium acnes, P. granulosum, and P. avidum. A distinctive protein pattern for each species was found, and it was possible also to distinguish the serotypes within P. acnes and P. avidum. Strains of P. acnes, P. granulosum, and P. avidum could be differentiated by the mobilities of their malate dehydrogenases. Catalase activity was detected in the soluble fractions of all strains. Catalases from P. acnes and P. avidum strains had the same mobility, whereas that from P. granulosum was slightly slower. Under the conditions used, succinate dehydrogenase activity could be detected, but the patterns were not distinctive.     

Identification of Propionibacterium acnes by polymerase chain reaction for amplification of 16S ribosomal RNA and lipase genes

Propionibacterium acnes belongs to the cutaneous flora and is present in sebaceous follicles. The fatty acids that are released from sebum triglycerides by the action of this bacterial lipase play an important role in the pathogenesis of acne vulgaris. P. acnes is also involved in postoperative disorders and opportunistic infections in immunosuppressed hosts. Recently, it has been proposed that P. acnes causes sarcoidosis. Therefore, rapid isolation and identification of P. acnes is important. This study evaluated the polymerase chain reaction (PCR) for the detection of the 16S rRNA and lipase genes of P. acnes. The PCR used to detect the 16S rRNA gene could amplify the gene of P. acnes, but not the genes of the other tested strains of P. avidum, P. granulosum, P. lymphophilum, P. jensenii, P. acidipropionici and P. thoenii. The PCR to detect the lipase gene of P. acnes, however, could amplify not only the gene of P. acnes but also that of P. avidum. The PCR product of this lipase gene was not found in the strains of the other species tested. Therefore, the organism that has both the 16S rRNA gene and lipase gene was identified as P. acnes, while the strain with the lipase gene but not the 16S rRNA gene of P. acnes was characterized as P. avidum. These findings were confirmed by the conventional biochemical tests including lipase activity. Furthermore, out of the seven clinical isolates from acne vulgaris, four were identified as P. acnes and three as P. avidum by the PCR method and biochemical tests. The combination of two PCR, one for the detection of the 16S rRNA and the other of lipase genes was shown to be an easier, faster and more accurate method to identify P. acnes and P. avidum than conventional methods.     

Purification and partial characterization of hyaluronate lyase (EC 4.2.2.1) from Propionibacterium acnes.

Hyaluronidase from Propionibacterium acnes has been purified 13,000-fold from the culture supernatant to homogeneity (as determined by polyacrylamide disc gel electrophoresis). The molecular weight of the purified enzyme was 85,110 as determined by gel filtration. The purified enzyme had a pH optimum at 6.4, was stable between pH 5 and 5.8 and was completely inactivated after 15 min at 50 degrees C. Preliminary studies suggested that the enzyme is active against chondroitin 4- and 6-sulphates, but not against dermatan sulphate. Analysis by paper chromatography of the reaction products from the degradation of hyaluronic acid by bacterial, testicular and P. acnes enzymes suggested that the P. acnes enzyme is similar in its mode of action to other bacterial hyaluronate lyases. The enzyme from P. acnes may thus be tentatively classified as a hyaluronate lyase.     

Conditions for porphyrin formation by propionic acid bacteria

Porphyrin production by seven species of propionic acid bacteria (Propionibacterium shermanii, its mutant P. shermanii M-82, P. technicum, P. vannielii, P. rubrum, P. thoenii and P. jensenii) was being studied. All the bacteria were cultivated on a glucose-peptone medium. A positive correlation between the amount of the produced porphyrins and the vitamin B12-synthetizing activity was observed for the most of species. Exogenous delta-aminolevulinic acid stimulated the porphyrin accumulation, but the degree of its utilisation decreased as its content in the culture medium increased from 5 to 200 mg/l. A maximum synthesis of porphyrins by P. shermanii M-82 (mainly of coproporpyrin III) was observed at definite concentrations of glucose and cobalt salts.     

Identification of Actinomyces, Arachnia, Bacterionema, Rothia, and Propionibacterium Species by Defined Immunofluorescence

Fractionated fluorescein-isothiocyanate (FITC)-conjugated immunoglobulin G (dye-to-protein ratio <10), produced against whole cells of Actinomyces spp., Arachnia, Bacterionena, Rothia, and Propionibacterium spp., give species-specific conjugates with controlled nonspecific staining reactions when appropriately diluted on the basis of their antibody content (10 mg/ml). Using this standardization in immunofluorescence, serotype-specific conjugates are also available after dilution for all serotypes of these organisms except for Actinomyces viscosus type 2, and Propionibacterium acnes type 1. Adequately adsorbed conjugates could be used to differentiate these serotypes from A. viscosus type 1 and P. acnes type 2, respectively. A serological classification in defined immunofluorescence corresponded to species and serotype designation proposed on the basis of other serological analysis and biochemical characteristics. This includes a separation in immunofluorescence of two serotypes of Propionibacterium acnes. The detection of certain actinomycetes of the family Actinomycetaceae and Propionibacterium species by the defined immunofluorescence in direct smears prepared from clinical specimens agreed to 88% with parallel culturing when including a prereduced (PRAS) medium technique for isolation. Qualitative studies revealed that single cells of these organisms could be specifically identified by immunofluorescence when admixed with morphologically similar bacteria and a large number of other contaminants.     

Dissimilatory nitrate reduction by Propionibacterium acnes

Propionibacterium acnes P13 was isolated from human feces. The bacterium produced a particulate nitrate reductase and a soluble nitrite reductase when grown with nitrate or nitrite. Reduced viologen dyes were the preferred electron donors for both enzymes. Nitrous oxide reductase was never detected. Specific growth rates were increased by nitrate during growth in batch culture. Culture pH strongly influenced the products of dissimilatory nitrate reduction. Nitrate was principally converted to nitrite at alkaline pH, whereas nitrous oxide was the major product of nitrate reduction when the bacteria were grown at pH 6.0. Growth yields were increased by nitrate in electron acceptor-limited chemostats, where nitrate was reduced to nitrite, showing that dissimilatory nitrate reduction was an energetically favorable process in P. acnes. Nitrate had little effect on the amounts of fermentation products formed, but molar ratios of acetate to propionate were higher in the nitrate chemostats. Low concentrations of nitrite (ca. 0.2 mM) inhibited growth of P. acnes in batch culture. The nitrite was slowly reduced to nitrous oxide, enabling growth to occur, suggesting that denitrification functions as a detoxification mechanism.     

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.     

Genome sequence of a novel species, Propionibacterium humerusii

As part of a larger project to sequence multiple clinical isolates of Propionibacterium acnes, we have produced a draft genome sequence of a novel Propionibacterium species that is closely related to, yet distinct (by sequence) from P. acnes. We have tentatively named this new species Propionibacterium humerusii.     

Propionibacterium acnes acts as an adjuvant in in vitro immunization of human peripheral blood mononuclear cells

We have established an in vitro immunization protocol whereby human peripheral blood mononuclear cells (PBMCs) are initially treated with L-leucyl-L-leucine methyl ester (LLME) and subsequently sensitized with antigen in the presence of interleukin (IL)-2, IL-4, and adjuvant. This protocol resulted in the production of antigen-specific antibodies. PBMCs are potentiated to react with exogenous antigens upon treatment with LLME. We are using this system to investigate the immunomodulatory activity of additives. In the present study, we aimed to evaluate the immunomodulatory activity of Propionibacterium acnes (P. acnes), which is known to exhibit various immunomodulatory effects in murine models, using this in vitro immunization protocol. P. acnes was found to augment the production of antigen-specific antibodies by PBMC, possibly through increased production of inflammatory cytokines and/or increased T-B cell interaction. P. acnes hence appears to act as an adjuvant in the antibody response in in vitro immunization.