Biomedical subjects
Publications and source records attributed to C S Cummins.
The cellular fatty acids of Arachnia propionica are largely 13-methyltetradecanoic acid (Ci15:0) and 12-methyltetradecanoic acid (Ca15:0); thus, the fatty acid pattern of this organism resembles the pattern found in propionibacteria. This finding supports the transfer of members of the genus Arachnia to the genus Propionibacterium.
Tumor cells injected into Balb/c mice together with heat-killed 48-h P. acnes cells were rendered nontumorigenic as early as 12 h after injection, as determined by the inability of the tumor cells to give rise to tumors when transferred to a new host. Determination of tumor cell antigen levels by ELISA indicated that the tumour antigens had virtually disappeared by 24 h after injection of tumor cells and P. acnes. In contrast, in control animals injected with tumor cells only, there was an initial drop in tumor antigen levels at 12 h, after which the level rose steadily and tumors developed in 7-10 days. Since the cellular exudate at 12 h was almost entirely composed of polymorphonuclear leukocytes (PMN), we tested the ability of PMN, stimulated by phagocytosis of 48-h P. acnes cells, to produce substances toxic to tumor cells. Results indicated that the supernatant fluid from a phagocytosis mixture of PMN and P. acnes contained material toxic to tumor cells and also to Chinese hamster ovary cells. Tests with scavengers and inhibitors of oxygen-derived radicals suggested that the toxic material is either hydrogen peroxide (H2O2) or hydroxyl radicals (OH). Suspensions of 12-h P. acnes, P. acnes cells walls, P. freudenreichii, or latex beads were ineffective in preventing tumor growth, and induced little toxicity when phagocytosed. We conclude that in this test system 48-h P. acnes prevents tumor growth by stimulating the production of toxic oxygen metabolites during phagocytosis by PMN.
The ability of strains and fractions of killed propionibacteria suspensions to produce chronic rat ear inflammation after intradermal injection of 70-micrograms aliquots was highly correlated with production of splenomegaly in the mouse after i.p. injection of 1.4 mg Propionibacterium acnes strains CN 6134, VPI 0009, ATCC 11828, and UCLA SC and N1 produced a 2- to 3-fold increase in rat ear thickness and a 5- to 7-fold increase in mouse spleen weight 15 days post injection. In contrast P. granulosum CN 5888, P. acnes UCLA 6S and periodated, acetylated, or 12-h cultures of VPI 0009 were inactive or weakly active as stimulators of chronic ear inflammation and splenomegaly. Active strains produced in the rat ear a transepidermal elimination response characterized by follicular encapsulation and the formation of secondary comedones. These effects correlated with persistence of phagocytized bacteria within macrophages. Furthermore, when rats were first immunized and then challenged with active strains of P. acnes, an increased sensitivity to low doses of P. acnes and a chronic exacerbation of inflammation was observed.
A previously undescribed component of the cell wall polysaccharide of Propionibacterium acnes, 2,3-diamino-2,3-dideoxyglucuronic acid, has been identified and synthesized. The component occurs to the extent of about 3 to 5% in the wall polysaccharides of P. acnes types I and II and in Propionibacterium avidum types I and II; it also appears to be present, but in much smaller amounts, in the cell wall of Propionibacterium granulosum.
The addition of chloramphenicol or tetracycline to exponentially growing cells of Propionibacterium acnes, which are not normally able to induce splenomegaly until they reach stationary phase, resulted in a rapid development of ability to induce splenomegaly in mice. However, the activity resulting from chloramphenicol treatment was arrested by the addition of penicillin or vancomycin, which showed that the activity was dependent on cell wall synthesis.
Stationary-phase (48 h) cells of Propionibacterium acnes VPI 0009, a potent stimulator of the reticuloendothelial system, persist unchanged within phagocytes for at least 24 h after ingestion. In contrast, exponential-phase (12 h) cells of the same strain (which do not induce splenomegaly) are extensively degraded within 5 h of phagocytosis. Suspensions of P. granulosum VPI 6500, which fails to induce splenomegaly in mice, also show considerable degradation after phagocytosis. Stationary-phase cells of strain VPI 0009 treated with sodium metaperiodate or with trichloroacetic acid, although without ability to induce splenomegaly, resist destruction almost as well as untreated vaccines. However, bacteria inactivated by acetic anhydride show about 50% breakdown in 24 h.
The surface of Propionibacterium acnes, VPI 0009, was studied using microelectrophoresis following chemical treatments intended to modify specific charged groups. The effect of specific modifications on ability of cells to induce splenomegaly, an indicator of stimulation of the reticuloendothelial system, was also determined. There was little difference between pH mobility curves of P. acnes VPI 0009 and other strains of propionibacteria which were not able to stimulate the reticuloendothelial system. Amino and carboxyl groups were found to be the sole ionizable groups at the cell surface and modification of these groups caused a substantial decrease in the ability of cells to stimulate the reticuloendothelial system. No phosphate groups were detected. Evidence for two types of amino groups was found: one type was present on protein moieties and their modification did not affect ability to stimulate the reticuloendothelial system, whereas modification of the other type, which was present on carbohydrate moieties, caused a loss of ability to stimulate the reticuloendothelial system. Mild oxidation with sodium metaperiodate caused abolition of reticuloendothelial system stimulation, but had no effect on surface charged groups, indicating it was acting on the unsubstituted linkages of sugar residues. Treatments with strong acids caused abolition of ability to stimulate the reticuloendothelial system and this was accompanied by release of polysaccharide material.
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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.
Capsules were detected by the India ink method in cultures of Bacteroides fragilis, B. vulgatus, B. thetaiotaomicron, and B. ovatus. No capsules were found in the five strains of B. distasonis examined.
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.
Strains of serotypes I and II of Propionibacterium were compared for phage sensitivity. The two serotypes could be distinguished by using a typing set consisting of 16 bacteriophages at concentrations that demonstrated selective lysis of serotype I or II bacterial strains. Seven phage types were found; three were composed exclusively of serotype I, and four were exclusively composed of serotype II organisms. Generally, serotype I strains were more sensitive to phage lysis than were serotype II strains. No correlation was found between phage type and site of isolation.
Vaccines prepared from 115 strains of anaerobic coryneforms and other organisms were tested in mice for their reticulostimulating ability as judged by the degree of spleen hypertrophy produced after ip injection. Almost all vaccines caused a statistically significant increase in spleen weight, but the ability to produce spleen ratios (test mean wt:control mean wt) of 4 or more was confined to Propionibacterium acnes and P. avidum strains. P. acnes, type II, gave high spleen ratios more frequently than strains of any other type.
The serological identification of Propionibacterium acnes, P. granulosum, and P. avidum, using trichloroacetic acid extracts, is described. With antisera prepared against reference strains, the method has been tested on 142 strains recently isolated from human skin. All except two of the strains could be identified serologically, and there was excellent agreement between the serological results and the fermentation pattern of the strains. Two serological types of P. acnes and two of P. avidum were identified, but only one of P. granulosum.
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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.
The cell wall compositions of two strains of Corynebacterium bovis were found to differ: one contained lysine, rhamnose, mannose, and glucose, the other meso-alpha, epsilon, diaminopimelic acid (DAP), arabinose, galactose, and mannose. The walls of a strain of C. nephridii were characterized by l-DAP and galactose. Those of a strain of C. paurometabolum and of two strains of "lipophilic diphtheroids" contained meso-DAP, arabinose, galactose, and mannose as did walls of a reference strain of C. xerosis. The results are discussed in relation to the taxonomy of the organisms examined.
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