Interaction of actinomycetes in relation to an intensification of protease biosynthesis

This work was aimed at studying the interactions during the growth of Actinomyces rimosus producing proteases and Actinomyces violocinereus which did not synthesize secreted proteolytic enzymes. The production of proteases in the association of the actinomycetes was shown to be stimulated by metabolic products released by A. violocinereus into the surrounding medium. The stimulating agent from the cultural broth of this culture accelerated differentiation of the mycelium of the first hyphal generation in A. rimosus, decelerated spore formation of the second hyphal generation, inhibited the growth rate, and increased the rate of protease accumulation as well as the productivity of the synthesis.     

Experimental study of the action of the RNAse from Actinomyces rimosus on the variolovaccine virus]

The effect of Actinomyces rimosus RNAase on the variolovaccine virus was studied. The inhibitory effect of the Actinomyces rimosus RNAase on the variolovaccine virus reproduction in the tissue culture cells was shown. In the experiments with the use of chick embryons and rabbits this effect was less pronounced. A 10 time increase in the infection multiplicity both in the tissue culture and the chick embryons had no noticeable effect on the level of the virus inhibition. It was supposed that the inhibitory effect of the RNAase on the variolovaccine was associated with impairment of the cell metabolism.     

Actinomyces rimosus resistance to oxytetracycline

High frequency of spontaneous and UV-and acridine dye-induced variants susceptible to oxytetracycline (OTC) and deprived of the capacity for synthesizing this antibiotic was observed in strain LST-118 of Actinomyces rimosus. The cells of strain LST-118 of Act. rimosus contained extrachromosomal DNA not found in its OTC susceptible variant BS87, which provides evidence in favour of participation of the extrachromosomal genetic elements in control of OTC resistance of the cells of Act. rimosus, LST-118. The OTC resistance in strain LST-118 is of inducable character. The resistance level is increasing from the beginning of the antibiotic synthesis and initially the subinhibitory concentrations of OTC in the medium were the inductors triggering cellular mechanisms ensuring resistance of the cell to the increasing concentrations of OTC in the medium. The capacity for absorption of OTC in Act rimosus is 2--3 times lower than that in E. coli. The experiments with labeled tetracycline showed that the cells of the actinomycete absorbed OTC when it was present in the medium. The absorption of the main amount of the antibiotic was registered during the first 5 minutes. The difference in absorption of OTC by the cells of the antibiotic resistant and sensitive strains was insignificant.     

Mutagenic action of N-nitrosodimethylurea on Actinomyces rimosus and Penicillium chrysogenum]

High mutagenic activity of N-nitrozodimethylurea (NDMU), an agent of the group of the nitrozo compounds not studied in detail was shown with respect to prototrophic and auxotrophic strains of Actinomyces rimosus, an organism producing oxytetracycline and Penicillium chrysogenum, an organism producing penicillin. The rate of direct and back mutations in the auxotrophic strain of Act. rimosus under the effect of NDMU was many times higher than that of spontaneous mutations. NDMU was used at one of the selection stages at which a more active variant of Act. rimosus was obtained. This is evident of a possible use of the mutagen for induction of variation with respect to the quantitative feature of oxytetracycline production. A great number of morphologically changed forms and biochemical mutants of Pen. chrysogenum formed under the effect of this substance. NDMU induced a mutant of Pen. chrysogenum capable of selective synthesis of 6-aminopenicillinic acid without addition of the precursor.     

Effect of Actinomyces rimosus ribonuclease on the reproduction of viruses]

Antiviral activity of RNA-ase isolated from the fermentation broth of Actinomyces rimosus was studied. The effect of the enzyme on multiplication of the viruses of vesicular stomatitis, Newcastle and cariolovaccine diseases was investigated. It was found that the enzyme was capable of suppressing reproduction of the vesicular stomatitis virus (VSV) in the culture of chick fibroblast cells. The suppression level directly depended on the enzyme concentration and decreased with an increase in the infection multiplicity. The enzyme had no effect on multiplication of other viruses tested. RNA-ase decreased the infectious properties of the freshly isolated virus-containing material in concentrations showing the antiviral effect. Preliminary incubation of the cells with the enzyme resulted in suppression of the plaque formation by VSV. The RNA synthesis in such cultures treated with RNA-ase was somewhat lower. It was shown that the antiviral effect of RNA=ase was connected with its enzymic activity. RNA-ase has no antiviral effect in the experiments with mice infected with VSV.     

Effect of metabolic substances of oral Actinomyces on Candida albicans

Actinomyces naeslundii, Actinomyces viscosus and Candida albicans are associated with root cavity. The aim of this study was to determine, in vitro, the effect produced by the metabolic substances elaborated by Actinomyces naeslundii and Actinomyces viscosus on Candida albicans. The strains were isolated of saliva. There were used the double plaque diffusion method (DPDM) and the method of radial diffusion (MRD). The effect of the time of incubation and of different concentrations of metabolic substances elaborated by Actinomyces naeslundii and Actinomyces viscosus on the kinetics of growth of C. albicans were studied. Later, the nature of the substances produced by the two strains of Actinomyces was determined. It was found that there was no inhibition of the growth of C. albicans by A. naeslundii and A. viscosus in the DPDM and the MRD. There was stimulation of the growth of C. albicans by the two strains of Actinomyces when the DPDM was used. In the MRD the results were negative. Metabolic substances produced by both species stimulated the growth of C. albicans in low concentrations but at high concentrations inhibition was observed. The best concentration of the stimulating factor, a protein substance stable to 70 degrees C, corresponds to a dilution of 1/80. The inhibition of the growth of C. albicans was produced by the decrease of the pH, the higher effect being obtained with the dilution 1/5. The metabolic substances produced by A. naeslundii and A. viscosus can have both inhibitory and stimulant effects on C. albicans, according to their concentration. These metabolic interactions would condition the proportion of C. albicans in the oral microbial ecosystems.     

Characterization of Actinomyces species isolated from failed dental implant fixtures

In the oral cavity, Actinomyces form a fundamental component of the indigenous microflora, being among initial colonizers in polymicrobial biofilms. However, some differences may exist between different species in terms of their attachment not only to teeth but also to biomaterials. In this study we investigated the distribution of Actinomyces in 33 dental implant fixtures explanted from 17 patients. The identification was based on comprehensive biochemical testing and gas-liquid chromatography and when needed, 16S rRNA sequencing. Actinomyces was the most prevalent bacterial genus in these failed implants, colonizing 31/33 (94%) of the fixtures. Proportions of Actinomyces growth of the total bacterial growth in the Actinomyces-positive fixtures varied from 0.01% up to 75%. A. odontolyticus was the most common Actinomyces finding, present in 26/31 (84%) Actinomyces-positive fixtures. Actinomyces naeslundii and A. viscosus were both detected in 10/31 (32%) and A. israelii in 7/31 (23%) fixtures. Other Actinomyces species, including A. georgiae, A. gerencseriae and A. graevenitzii, were detected less frequently. Our results suggest that Actinomyces species are frequent colonizers on failed implant surfaces, where A. odontolyticus was the far most prominent Actinomyces species.     

Actinomyces georgiae sp. nov., Actinomyces gerencseriae sp. nov., designation of two genospecies of Actinomyces naeslundii, and inclusion of A. naeslundii serotypes II and III and Actinomyces viscosus serotype II in A. naeslundii genospecies 2

DNAs of type strains and representative members of Actinomyces groups from the human periodontal flora and from other habitats were compared by using the S1 nuclease procedure to determine their genetic relatedness. One rather common group from the human periodontal flora, previously called "Actinomyces D08," is phenotypically distinct from, and genetically unrelated to, previously described species. We propose the name of Actinomyces georgiae for this organism; the type strain is strain ATCC 49285. Another common group from the human periodontal flora is Actinomyces israelii serotype II, which was found genetically distinct from the type strain of A. israelii (serotype I) and from other previously described species of Actinomyces. We propose the name Actinomyces gerencseriae for this organism; the type strain is strain ATCC 23860. A. naeslundii serotype I strains were distinct from the other strains studied. A separate genospecies which included strains of A. naeslundii serotypes II and III and A. viscosus serotype II was delineated. Strains of Actinomyces serotype WVA 963 constitute an additional distinct genospecies. Because there are no reliable phenotypic tests, other than serological analyses, to differentiate Actinomyces serotype WVA 963 and the two genospecies of A. naeslundii, no taxonomic changes are proposed for these three genospecies.     

Assignment of Actinomyces pyogenes-like (CDC coryneform group E) bacteria to the genus Actinomyces as Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov.

In a previous study the authors reported the characterization of some facultatively anaerobic, Gram-positive, non-sporeforming rods which were found in mixed cultures from various infectious processes, including patients with otitis, empyema, perianal abscesses and decubitus ulcers. Phenotypically these organisms closely resembled Actinomyces pyogenes although their precise taxonomic position remained unknown. In the present investigation the authors have determined the 16S rRNA gene sequences of some representative strains of the Actinomyces pyogenes -like bacteria and report the results of a comparative sequence analysis. On the basis of the results of the present and earlier findings two new Actinomyces species, Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov. are proposed. The type strains are DSM 9169^T and DSM 9168^T, respectively.     

Phylogenetic evidence for the transfer of Eubacterium suis to the genus Actinomyces as Actinomyces suis comb. nov.

The 16S rRNA primary structures of Eubacterium suis DSM 20639T (T = type strain) and Bifidobacterium bifidum DSM 20456T were determined by sequencing in vitro amplified rDNA. Sequence comparisons indicated that B. bifidum is moderately related to representatives of the genera Actinomyces and Mobiluncus. The closest relative of E. suis is Actinomyces pyogenes. E. suis and A. pyogenes are more closely related phylogenetically to one another than to the other Actinomyces species that have been investigated by using comparative 16S rRNA analysis. Therefore, we propose that E. suis should be transferred to the genus Actinomyces as Actinomyces suis comb. nov.     

Isolation and Characterization of Actinomyces propionicus

Three cultures of Actinomyces have been identified as Actinomyces propionicus. Two of these strains are recent isolates, one, 427, from a case of cervico-facial actinomycosis, and one, 439, from a case of lacrimal canaliculitis. The third strain, 346, was described by F. Lentze as A. israelii serological type II. These three strains were compared with the type strain of A. propionicus ATTC 14157 and with known strains of five other Actinomyces species. Morphologically and biochemically the three new cultures of A. propionicus were identical with the type strain but closely resembled A. israelii. In serological tests making use of fluorescent antibody, all four A. propionicus strains gave negative results with antisera for A. israelii, A. bovis, A. naeslundii, and A. eriksonii, but gave positive results with antisera for A. propionicus 14157 and strain 346. The A. propionicus antisera did not stain other Actinomyces species. A. propionicus contains diaminopimelic acid (DAP) in its cell wall and produces propionic acid from glucose. All three new isolates were shown to contain DAP and to produce propionic acid. By use of the presence of DAP in the cell wall and serological tests as the differential criteria, the three cultures described in the report were specifically identified as A. propionicus.     

Growth of oral Streptococcus species and Actinomyces viscosus in human saliva.

Microorganisms in dental plaque live in constant association with saliva. The role of saliva in the adherence of bacteria to the teeth and the antibacterial properties of saliva have been well investigated; less interest has been shown in the possible role of saliva as a substrate for oral microorganisms. In this study it was shown that saliva can serve as a growth medium for oral Streptococcus spp. and Actinomyces viscosus. The cell production of these organisms on saliva was carbohydrate limited. The doubling times for growth on glucose-supplemented saliva (4 to 5 mmol/liter) ranged from 1.6 to 4.0 h. The availability of carbohydrate sources for the oral microflora is discussed in relation to microbial growth in the oral cavity.     

Comparative Cell Wall Analyses of Morphological Forms Within the Genus Actinomyces

Comparative cell wall analyses were made of mycelial and smooth forms of Actinomyces bovis and A. israelii to determine the changes which occur in the cell wall composition concurrent with a change in morphology, and to evaluate cell wall analyses as a criterion for taxonomic identification within the genus Actinomyces. Cell walls of the spider forms of A. boyis had little or no aspartic acid and a high hexosamine concentration; cell walls of the smooth forms had a high aspartic acid content and low concentrations of hexosamine. Both forms had large amounts of glutamic acid, alanine, and lysine, as previously reported. A strain of Actinomyces, previously identified as A. naeslundii on the basis of morphology and aerobic growth characteristics, was found to have the basic cell wall composition of A. israelii. When transferred from the Actinomyces maintenance broth to a thioglycolate broth, the cells of this strain passed from a mycelial form through a transient filamentous morphology to become diphtheroidal with continued incubation. Concomitantly, the concentrations of glutamic acid relative to alanine decreased, and the hexosamine content increased. Variation in morphology within the species A. israelii and A. bovis could not be related to any mutual chemical change of their cell walls.     

Kinetics of adherence of Actinomyces viscosus to saliva-coated silica and hydroxyapatite beads

Adherence of 14C-labelled strains of Actinomyces viscosus to uncoated and saliva-coated silica and hydroxyapatite beads had both loose and firm components, probably reflecting different subpopulations of bacteria within a single culture. Adherence was characterized by the proportion of bacteria available for each type of adherence and a constant (Kb) for each combination of bacterial strain and bead surface. Loose adherence, which was greater with silica than with hydroxyapatite beads, always involved many more bacteria than firm adherence. Firm adherence was greater with A. viscosus WVU627 than A. viscosus TF11. The association rate constants (Ka) for loose and firm adherence were similar, indicating simultaneous processes, but the dissociation rate constant (Kd) was lower for loose adherence than for firm adherence. Removal of loosely adhering bacteria by washing may only reflect their distance from the bead surface. Silica beads were convenient for studying bacterial adherence and formed an acceptable coating of salivary glycoprotein.     

Directed biosynthesis of proteolytic enzymes in Actinomyces thermovulgaris]

The biosynthesis of proteolytic enzymes in the thermophilic culture of Actinomyces thermovulgaris, strain T-54, was directed by changing the composition of the medium and the temperature of cultivation. A temperature of 40 degrees C is optimal for the growth and production of neutral and alkaline proteases. The maximum activity of acid proteases was found during the growth on a complex medium at 30 degrees C. An increase of temperature to 50 degrees C during the cultivation of the microorganism on a chemically defined medium resulted in its secondary growth and a sharp rise in the activity of alkaline and neutral proteases.     

Coaggregation between Prevotella nigrescens and Prevotella intermedia with Actinomyces naeslundii strains

Abstract Using a visual coaggregation assay, 43% (6 of 14) of Prevotella nigrescens and 50% (4 of 8) of Prevotella intermedia strains coaggregated with Actinomyces naeslundii strains which represented the six Actinomyces coaggregation groups (A to F). For both species, coaggregation occurred most frequently with A. naeslundii strains from coaggregation groups C, D and E. No coaggregation was observed with Actinomyces israelii , Actinomyces odontolyticus or six oral Streptococcus species. Coaggregation was not inhibited by lactose, saliva or serum. Pretreatment of Prevotella strains with heat, SDS and proteinase K abolished coaggregation when the treated cells were added to untreated Actinomyces strains. The same pretreatment of the Actinomyces strains had no effect on their ability to coaggregate with untreated Prevotella strains. Pretreatment of all coaggregating P. nigrescens strains with trypsin abolished coaggregation, whereas the coaggregation ability of the P. intermedia and Actinomyces strains was resistant to trypsin pretreatment. Pretreatment of the strains of both Prevotella species and the Actinomyces with periodate abolished coaggregation in all cases. These results suggest that the Prevotella strains each possess a protein coaggregation adhesin, which for the P. intermedia strains is resistant to trypsin, that interacts with a non-protein receptor on the A. naeslundii strains.     

一株瘤胃厌氧菌Actinomyces ruminicola的分离鉴定

目的从健康奶牛瘤胃液中分离、筛选出1株以产乙酸为主Actinomyces ruminicola。方法无菌采取装有瘤胃瘘奶牛的瘤胃液,按照厌氧茵分离步骤,通过Actinomyces ruminicola的特异性培养基进行筛选,提取分离菌的基因组DNA,克隆其16SrRNA基因,进行序列测定,分离出1株Actinomyces ruminicola。结果通过形态学观察、生化反应和序列分析证实所分离的1株产乙酸的杆菌为Actinomyces ruminicola。结论从健康牛瘤胃液中成功分离出1株Actinomyces ruminicola,为进一步研究其对瘤胃发酵的影响奠定了基础。     

Comparative Pathogenicity of Actinomyces naeslundii and Actinomyces israelii

Typical actinomycosis has been produced in mice following single intraperitoneal injections of saline suspensions of Actinomyces israelii and A. naeslundii. A. israelii produced infections in 95.8% of the animals inoculated. A. naeslundii, generally considered to be a saprophytic organism, produced lesions in 89.7% of the inoculated animals. The finding that A. naeslundii produced lesions in mice similar to those produced by A. israelii suggests that A. naeslundii has similar pathogenic potential for man. The isolation of A. naeslundii from suppurative lesions of man also supports this conclusion.     

Tetrameric manganese superoxide dismutases from anaerobic Actinomyces

Superoxide dismutase was isolated from each of the anaerobically grown organisms Actinomyces naeslundii, Actinomyces strain E1S.25D, and Actinomyces odontolyticus. The enzymes were 100,000-110,000 mol wt acidic proteins (pI 4.3-4.6) and contained Mn and Zn, but no detectable Fe. The Mn and Zn content varied with the enzyme source. A. naeslundii superoxide dismutase, specific activity 2200 U/mg, contained 2.3 g atoms Mn and 1.4 g atoms Zn per mole tetramer whereas A. odontolyticus SOD, specific activity 700 U/mg, contained 1.4 g atoms Mn and 1.8 g atoms Zn per mole tetramer. Actinomyces strain E1S.25D, specific activity 1300 U/mg, contained 1.8 g atoms Mn and 1.2 g atoms Zn per mole tetramer. The amino acid compositions of the enzymes were comparable except for arginine, lysine, and tryptophan content. The enzymatic activity of each enzyme was stable in 5 mM H2O2 at 23 degrees C for 2 h. The enzymes were only modestly inhibited by 20 mM NaN3. The enzymatic activity was increased at low ionic strength but was markedly decreased at increased ionic strength with each salt tested except sodium perchlorate, which caused marked inhibition even at low ionic strength. Polyclonal antibodies to A. naeslundii and Actinomyces strain E1S.25D precipitated and inactivated their respective antigens whereas the precipitated A. odontolyticus superoxide dismutase-antibody complex retained virtually full catalytic activity. Immunological studies revealed that the native A. naeslundii and Actinomyces strain E1S.25D MnSODs share common epitopes and cross-reacted with precipitin lines of complete identity in Ouchterlony double diffusion gels. Antibody to the A. odontolyticus enzyme displayed only partial cross-reactivity with superoxide dismutase from the two other Actinomyces. Western blotting of the denatured antigens revealed reactivities of the antibodies that differed only slightly from the results of the Ouchterlony gels.