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.     

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.     

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.     

Nutritional requirements of anaerobic coryneforms.

The nutritional requirements of three species of anaerobic coryneforms and their serotypes (Propionibacterium acnes types I and II, P. avidum types I and II, and P. granulosum) were determined. Strains of P. avidum would consistently grow to a transmittance of 1 to 3% at 560 nm in a basal salts medium supplemented with glucose, pantothenate, biotin, thiamine, and 12 amino acids (alanine, arginine, cysteine, glutamine, glycine, histidine, isoleucine, methionine, phenylalanine, serine, tyrosine, and tryptophan). Strains of P. acnes and P. granulosum, however, failed to grow in this medium unless six additional amino acids were present (asparagine, leucine, lysine, proline, threonine, and valine). All three species grew equally well whether the 18 amino acids were supplied in the form of a casein hydrolysate supplemented with tryptophan or were added separately. Nicotinamide enhanced growth of P. acnes but had no effect on growth of P. avidum and P. granulosum. Other nutrients which were not absolute requirements, but which significantly improved growth of these species, included the purines guanine and/or adenine, Tween 80, which served as a source of oleic acid, sodium L-lactate, alpha-ketoglutarate, and pyruvate. Strains (86) comprising all five groups grew well in the defined medium, except four strains of P. acnes type II (29 tested), which failed to grow unless heme and vitamin K were added to the medium. One strain of P. granulosum (22 tested) failed to grow in any defined medium, suggesting an additional growth factor requirement.     

Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.     

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.     

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.     

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.     

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.     

Membrane enzymes associated with the dissimilation of some citric acid cycle substrates and production of extracellular oxidation products in chemostat cultures of Pseudomonas fluorescens

Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.     

Energy metabolism in Staphylococci containing plasmids of resistance to a number of antibiotics]

Activity patterns of membrane-bound enzymes of an antibiotic sensitive strain of Staphylococcus aureus 8325-4 and its variants containing the plasmids of resistance to penicillin, tetracycline, erythromycin and chloramphenicol were studied. It was shown that acquiring of resistance to the above mentioned antibiotics influenced cellular energy metabolism. The resistant strains exhibited an increase in the specific activity of NADN, malate and succinate oxidases, intensification of the ATP-hydrolyzing activity, changes in the activity of definite dehydrogenases and a decrease in the specific content of cytochromes. No changes in the composition of respiration carriers were detected.     

Effects of tetracyclines on the production of extracellular proteins by members of the propionibacteriaceae

The effect of tetracyclines on the synthesis of proteins in Propionibacterium avidum and P. acnes was examined. Synthesis of an extracellular lipase by P. avidum was slightly more sensitive to inhibition by tetracycline than total (cellular and extracellular) protein synthesis. The effect of tetracycline and other analogues on the synthesis of secreted proteins was also examined in P. avidum and P. acnes by protein radiolabelling experiments. In all cases the synthesis of secreted proteins was only about 2-fold more sensitive to inhibition by tetracyclines than total protein synthesis. These results contrast with previously published findings in Escherichia coli which show that synthesis of secreted proteins is highly susceptible to inhibition by tetracycline. The implications of these findings in relation to inhibition of membrane bound ribosomes by tetracyclines are discussed.     

The effect of benzoyl peroxide on cutaneous micro-organisms in vitro

The survival curves of cutaneous micro-organisms in the presence of benzoyl peroxide were investigated. All the curves exhibited a shoulder prior to exponential cell death. Benzoyl peroxide was lethal to the cutaneous organisms tested and they varied in sensitivity increasing as follows: Propionibacterium acnes, Staphylococcus capitis, Staph. epidermidis, Staph. hominis, Prop, avidum, Prop. granulosum and Pityrosporum ovale.     

Cell wall composition and deoxyribonucleic acid similarities among the anaerobic coryneforms, classical propionibacteria, and strains of Arachnia propionica

Eighty strains of anaerobic coryneforms were compared with 29 strains of classical propionibacteria and 8 strains of Arachnia propionica by cell wall analysis, deoxyribonucleic acid (DNA) base compositions, and nucleotide sequence similarities. The anaerobic coryneforms have DNA base compositions in the range of 58 to 64% guanine + cytosine (GC) and show at least three homology groups. The largest group corresponds to organisms identified as Propionibacterium acnes and shows about 50% homology to strains in the P. avidum homology group. The third group, P. granulosum, shows low levels of similarities to the other two. All strains of anaerobic coryneforms have some combination of galactose, glucose, or mannose as cell wall sugars, and most have alanine (ala), glutamic acid (glu), glycine (gly), and l-alpha-epsilon-diaminopimelic acid (l-DAP) as amino acids of peptidoglycan. However, a few strains in the P. acnes and P. avidum homology groups have meso-DAP and minimal amounts of glycine. Two serological types, based on cell wall antigens, were found in the P. acnes homology group. One type had galactose, glucose, and mannose as cell wall sugars, the other glucose and mannose only. The classical propionibacteria have DNA base compositions in the range of 65 to 68% GC and show four homology groups which correspond closely to van Niel's classification as given in the 7th edition of Bergey's Manual. The P. jensenii group showed about 50% homology to the P. thoenii group and about 30 to 35% to the P. acidi-propionici group. The P. freudenreichii strains showed a rather lower level of similarity (8 to 25%) to the other homology groups. Most of the strains of classical propionibacteria also have some combination of galactose, glucose, or mannose as cell wall sugars and ala, glu, gly, and l-DAP as peptidoglycan amino acids, but P. shermanii and P. freudenreichii strains, which form a single homology group, have galactose, mannose, and rhamnose as cell wall sugars and ala, glu, and meso-DAP in their peptidoglycan. There is a rather low level of DNA homology (10 to 20%) between the anaerobic coryneforms and classical propionibacteria. However, the strains of A. propionica which have a GC content of 64 to 65% and form a single homology group, show no homology to either of the other two major groups.     

New medium for isolating propionibacteria and its application to assay of normal flora of human facial skin.

The conditions for isolation and cultivation of Propionibacterium acnes and related propionibacteria were studied in detail. Triton X-100 added to the diluent inhibited the growth of propionibacteria in concentrations of 0.05 to 0.1%. However, such was not the case with Tween 80; rather, growth of the bacteria was further enhanced by this agent. Consequently, Tween 80 was considered to be a suitable surfactant for addition to the diluent for isolation of propionibacteria. A new medium for isolating propionibacteria from human skin was developed. Comparative studies with colonies of P. acnes, Propionibacterium granulosum, and Staphylococcus epidermidis showed morphological differences among the colonies; thus, the medium was very useful for differentiating and identifying species of the microbes. The new medium was used for studies on the distribution of propionibacteria on the foreheads of 30 Japanese volunteers. Among 447 strains of P. acnes and 86 strains of P. granulosum isolated from the volunteers, all strains of the former were positive for indole, nitrate, milk, and gelatin hydrolysis, whereas all strains of the latter were negative for all of the tests.     

Antibiotic-resistant Propionibacterium acnes on the skin of patients with moderate to severe acne in Stockholm

The objective was to study the prevalence and antibiotic susceptibility patterns of Propionibacterium acnes strains isolated from patients with moderate to severe acne in Stockholm, Sweden and to determine the diversity of pulsed-field gel electrophoresis types among resistant P. acnes strains. One hundred antibiotic-treated patients and 30 non-antibiotic-treated patients with moderate to severe acne participated in the investigation. Facial, neck and trunk skin samples were taken with the agar gel technique. The susceptibility of P. acnes strains to tetracycline, erythromycin, clindamycin and trimethoprim-sulfamethoxazole was determined by the agar dilution method. The genomic profiles of the resistant strains were determined by pulsed-field gel electrophoresis. In the group of patients treated with antibiotics, resistant P. acnes strains were recovered in 37%, while in the non-antibiotic group of patients the incidence of resistant strains was 13%. Thus antibiotic-resistant P. acnes strains were significantly more often isolated from antibiotic-treated patients with moderate to severe acne than from non-antibiotic-treated patients (odds ratio, 3.8; P=0.01). There was a genetic diversity among the P. acnes strains. Forty-four different patterns of SpeI DNA digests were detected and two predominant clones were found. P. acnes strains exhibited different antibiotic susceptibility patterns and identical genotypes or vice versa. A person can be colonized with different strains with varying degrees of antibiotic resistance. The risk of increased resistance of P. acnes must be considered when treating acne patients with antibiotics, and especially long-term therapy should be avoided.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : I. Oxidative Activities with Soluble Substrates

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates: nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and α-ketoglutarate. Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide. Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble. It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E. coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme. The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control.     

Comparative studies of porphyrin production in Propionibacterium acnes and Propionibacterium granulosum.

Porphyrin production by Propionibacterium acnes and that by Propionibacterium granulosum were compared. Porphyrin synthesized by both organisms was identified as coproporphyrin III on the bases of absorption and fluorescence spectra and behavior on paper chromatography and thin-layer chromatography. Quantitative, rather than qualitative, differences in production were found between these organisms. In general, P. granulosum produced significantly greater amounts (P less than 0.001) of porphyrin than did P. acnes. delta-Aminolevulinic acid synthetase appeared to be the rate-limiting enzyme of the heme biosynthetic pathway in both organisms. The increased porphyrin production in P. granulosum is apparently associated with increased delta-aminolevulinic acid synthetase activity.     

Quantitative comparison of three C. parvum strains for immunotherapy of transplanted rat tumours

Summary Preparations of three commercially available killed bacterial suspensions designated Corynebacterium parvum have been compared for immunotherapy of transplanted rat tumours. The Wellcome CN6134 preparation was quantitatively superior to Institut Merieux IM1585 in suppressing local, subcutaneous, growth of tumour when injected in admixture with cells, for the control of thoracic malignant effusions when administered intrapleurally and for active specific immune stimulation where incorporated into vaccines of irradiated cells. The Wellcome CN5888 preparation was virtually ineffective. These observations, together with the recent re-classification of C. parvum into three distinct species of the genus Propionibacterium, i.e., P. acnes (Wellcome CN6134), P. granulosum (Wellcome CN5888), and P. avidum (Institut Merieux IMI1585) emphasize that the simple designation C. parvum is inadequate for the vaccines currently available for experimental or, more importantly, clinical immunotherapy.     

The acid end-products of glucose metabolism of oral and other haemophili

The acids produced in broth culture by various species of oral haemophili and by stock strains of capsulated and other haemophili were identified and measured by gas-liquid chromatography. Succinic acid was the major acid end-product of all strains, with acetic acid also being regularly produced but in smaller amounts. A stock strain, Haemophilus parainfluenzae NCTC 4101, produced less succinic acid than other strains of haemophili. Strain NCTC 4101 possessed all the enzymes of the tricarboxylic acid cycle, as previously reported, but in the other haemophili examined only succinic dehydrogenase, fumarase and malate dehydrogenase could be detected. No other enzymes of the tricarboxylic acid cycle were detected and isocitrate lyase, malate synthase and pyruvate carboxylase were also absent. Phosphoenolpyruvate-carboxylase was present in all strains. A partial tricarboxylic acid cycle and marked malate dehydrogenase activity appear to be characteristic of haemophili. The pathway to succinate in haemophili appears to be via carboxylation of phosphoenolpyruvate to oxalacetate and thence via malate and fumarate. The results of tracer studies on a single oral strain of H. parainfluenzae using various labelled substrates were in keeping with this proposed metabolic pathway.