Phenoloxidase activity in organisms isolated from lepromatous and tuberculoid leprosy

Anaerobic corynebacteria (propionibacteria), isolated in pure culture from the plasma of a case of tuberculoid leprosy and from lepromata of cases of lepromatous leprosy, exhibited phenoloxidase activity such as that shown by Prabhakaran to be associated with leprosy bacilli harvested from patients suffering from leprosy. Several corynebacteria, mycobacteria, and nocardias similarly examined did not produce phenoloxidase.     

Production of antibacterial substances by resident corynebacteria isolated from human skin

Coryneform bacteria, especially lipophilic species, form stable but not dominant population on a human skin. This position is probably controlled by secretion of bacteriocin-like substances, which act directly on coexisting bacteria. Among 118 investigated corynebacteria belonging to seven species/taxa and isolated from human skin, 90% possessed an ability to produce such substances. The spectrum of their activity was restricted to killing gram-positive bacteria, but along with corynebacteria it also covered cocci, with Staphylococcus aureus in this group. This feature was revealed better on low pH media (pH 5.6) and media with 1.5% NaCl for cocci, but on pH 7,4 for corynebacteria.     

Structural studies of the cell wall polysaccharide of Nocardia asteroides R 399

As with other bacteria belonging to the corynebacteria, mycobacteria, and nocardia group, Nocardia possess in their cell walls a neutral polysaccharide. Structural analysis of the cell wall polysaccharide of Nocardia asteroides R 399 was undertaken. The carbohydrate polymer contained D-arabinose and D-galactose as in mycobacteria. Besides these two carbohydrates we pointed out the occurrence of two additional components: D-glucose and a polyol. This polyol, because of its small amount and its uneasy detection, had been for a long time ignored. It has been proven to be the 6-deoxy-D-altritol or 1-deoxy-D-talitol. The polymer consists of a main strand composed of----5 Araf 1----and----4Galp1----or----5Galf1----; oligoarabinosyl side chains were localized on C3 of an arabinosyl residue. Other shorter ramifications also occur on some galactosyl units. A characterization of the linkage between polysaccharide and peptidoglycan inside the cell wall has also been carried out. The two polymers are joined by a phosphodiester bond which involves 6-deoxyaltritol. As some corynebacteria previously analyzed were also shown to contain mannose (and sometimes glucose), we can conclude that the main skeleton of cell wall polysaccharides of the corynebacteria, mycobacteria, and nocardia group of bacteria is an arabinogalactan; however, individual structural features of the polysaccharide are varying according to the bacterial species. These results might be connected with variations that were observed in immunological analysis.     

Numerical Classification of Bacteria

Sixty three organisms selected from 12 genera of bacteria were subjected to numerical analysis. The purpose of this work is to examine the relationships among 38 coryneform bacteria included in the test organisms by two coding methods—Sneath’s and Lockhart’s systems—, and to compare the results with conventional classification. In both cases of codification, five groups and one or two single item(s) were found in the resultant classifications. Different codings brought, however, a few distinct differences in some groups, especially in a group of sporogenic bacilli or lactic-acid bacteria. So far as the present work concerns, the result obtained on Lockhart’s coding rather than that obtained on Sneath’s coding resembled the conventional classification. The taxonomic positions of corynebacteria were quite different from those of the conventional classification, regardless of which coding method was applied.

Though animal corynebacteria have conventionally been considered to occupy the taxonomic position neighboring to genera Arthrobacter and Cellulomonas and regarded to be the nucleus of so-called “coryneform bacteria,’ the present work showed that many of the corynebacteria are akin to certain mycobacteria rather than to the organisms belonging to the above two genera.     

Infection et fertilité —Corynebacterium seminale: point de vue du bactériologiste

The authors present two independent studies designed to identify corynebacteria isolated from the semen of patients consulting for infertility. Corynebacteria were identified by conventional biochemical and physiological tests and by determination of volatile fatty acids. In the first study based on 420 patients, the commonest species were Corynebacterium seminale (synonym C. glucuronolyticum) found in 7.4% of specimens, CDC group G (5%) and C. amycolatum (3.8%). Of the 92 semen specimens with more than 103 cfu/ml, 44 were positive for corynebacteria, including 15 C. seminale strains, whereas streptococci, staphylococci and enterobacteriacae were found in 23, 18 and 6 of the 420 specimens, respectively. The presence of C. seminale was more frequently associated with a high bacteria count than the other corynebacteria (p<0.02). In the second study, we compared the presence of corynebacteria in the semen of 1,902 patients with semen indices. C. seminale was present at levels greater than 103 cfu/ml in 2.7% of these specimens, while several other species of corynebacteria were detected in 5.3% of cases. Normal motility was found in only 25.4% of semen specimens with a high C. seminale count in contrast with 45% of specimens containing similar counts of other corynebacteria. These studies demonstrate that the isolation rates from human genital specimens and their clinical implications are different according to the species isolated. Microbiologists should be aware of the need to accurately identify these corynebacteria for further in vitro or in vivo studies on genital infections.     

Characterization and taxonomic implications of the rRNA genes of Mycobacterium leprae.

The number of rRNA genes of Mycobacterium leprae was determined by restriction analysis of M. leprae total chromosomal DNA. A single set of rRNA genes was found. This set was subcloned from a cosmid library of M. leprae DNA into pUC13 and was characterized by restriction analysis and hybridization with Escherichia coli rRNA genes. The 16S, 23S, and 5S genes of M. leprae were clustered on a 5.3-kilobase DNA fragment. On one hand, restriction analysis of the set of rRNA genes showed the uniqueness of M. leprae among mycobacteria, but on the other hand, it suggested that M. leprae strains of several origins are very much alike. Quantitative hybridization studies between M. leprae rDNA and total DNA of various bacteria demonstrated a close relatedness between M. leprae and corynebacteria, nocardia, and mycobacteria, especially Mycobacterium tuberculosis.     

The genome stability in Corynebacterium species due to lack of the recombinational repair system

Corynebacterium species are members of gram-positive bacteria closely related to Mycobacterium species, both of which are classified into the same taxonomic order Actinomycetales. Recently, three corynebacteria, Corynebacterium efficiens, Corynebacterium glutamicum, and Corynebacterium diphtheriae have been sequenced independently. We found that the order of orthologous genes in these species has been highly conserved though it has been disrupted in Mycobacterium species. This synteny suggests that corynebacteria have rarely undergone extensive genome rearrangements and have maintained ancestral genome structures even after the divergence of corynebacteria and mycobacteria. This is the first report that the genome structures have been conserved in free-living bacteria such as C. efficiens and C. glutamicum, although it has been reported that obligate parasites such as Mycoplasma and Chlamydia have the stable genomes. The comparison of recombinational repair systems among the three corynebacteria and Mycobacterium tuberculosis suggested that the absence of recBCD genes in corynebacteria be responsible for the suppression of genome shuffling in the species. The genome stability in Corynebacterium species will give us hints of the speciation mechanism with the non-shuffled genome, particularly the importance of horizontal gene transfer and nucleotide substitution in the genome.     

Distribution of neuraminidase and n-acetylneuraminate lyase activities among corynebacteria, mycobacteria, and nocardias

In Corynebacterium diphtheriae and closely related neuraminidase-producing corynebacteria, we have found an N-acetylneuraminate (NAN) lyase activity which cleaves NAN into N-acetyl-d-mannosamine and, presumably, pyruvate. In vitro, these lyases can be shown to synthesize NAN. A survey of representative corynebacteria, "plant pathogenic corynebacteria," mycobacteria, and nocardias revealed that only those corynebacteria closely related to C. diphtheriae exhibited both neuraminidase and NAN lyase activities.     

Cytopathogenic protein in filtrates from cultures of Propionibacterium acnes isolated from patients with Kawasaki disease.

Propionibacterium acnes may have a role in Kawasaki disease. Filtrates from cultures of P acnes isolated from cervical lymph node biopsy specimens and blood samples from patients with Kawasaki disease were studied and compared with samples from control subjects. After inoculation of human embryo liver cells with filtrates from the patients a cytopathogenic effect and vacuolation were seen. A specific cytopathogenic substance was found in only the filtrates of cultures from patients with Kawasaki disease; it was a protein of about isoelectric point 7.0 with a molecular weight of about 100,000 daltons. The amount of IgG antibody to this cytopathogenic protein was measured by enzyme linked immunosorbent assay (ELISA) in serum samples taken from 63 patients in the acute phase of Kawasaki disease (mean 5.2 (SD 1.1) days after onset of illness), 45 in the subacute phase (mean 23.6 (3.3) days), and 51 in the convalescent phase (mean 18.5 (4.1) months) and from 102 control subjects matched for age. Titres of IgG antibody were significantly raised in patients with Kawasaki disease, particularly in the acute and subacute phases of the illness, compared with in the control subjects. Titres of IgG antibodies to cytopathogenic protein were found to be low in normal children below the age of 4 years but they increased with age thereafter. This may explain why outbreaks of Kawasaki disease, which is most common in children aged under 4, occur every three years.     

Adjuvant Activity of Mycobacterial Fractions

The addition of adjuvant (Bacillus Calmette–Guérin-cell wall skeleton; BCG-CWS) to the culture medium substantially increased the immune response of mouse spleen cells to immunization with heterologous erythrocytes and hapten–protein conjugate in vitro. Cell walls of mycobacteria, nocardia and corynebacteria and their cell wall constituents were used as adjuvants. In the present case, it was found that cell walls of mycobacteria, nocardia and corynebacteria markedly facilitated primary humoral response of mouse spleen cells to heterologous erythrocytes in vitro. The adjuvant effect of BCG-CWS was present only in the formation of 19 S antibody in both primary and secondary responses, but not in that of 7 S antibody in vitro. Primary antihapten response of mouse spleen cells against dinitrophenylated keyhole limpet hemocyanin (DNP-KLH) also succeeded when BCG-CWS was added to the culture medium, and it was found that BCG-CWS increased the helper activity of carrier specific helper T cells in vitro where a double chamber system, separated by a cell-impermeable nucleopore membrane, was used. This result suggested that BCG-CWS acts on T cells, resulting in the release of soluble factor(s) from T cells capable of exerting an adjuvant effect. Furthermore, mucopeptide moiety of BCG-CWS retained some adjuvant activity, but other cell wall constituents, such as mycolic acid, arabinose mycolate, and arabinogalactan, did not show any adjuvant effect in vitro. These results strongly imply that mucopeptide moiety of BCG-CWS plays an important role in the development of adjuvanticity.     

Action of microbe-antagonists on bacilli and corynebacteria of the skin of the breast in nursing women

Five microbes-antagonists are studied for their inhibitory action on 769 strains of bacilli (241) and corynebacteria (528) isolated from the mammary gland skin of 120 nursing women on the 2nd-5th day after labour. Escherichia coli M-17 and Mycococcus krassilinikovi are established to possess the highest antagonistic properties against the spore-bearing aerobic and coryneform bacteria. Bifidobacterium bifidum has exerted a moderate antagonistic action on the culture under study, Bacillus subtilis has exerted a strong inhibitory action on corynebacteria. B. licheniformis has slightly inhibited the both bacterial groups.     

Trehalose-based oligosaccharides isolated from the cytoplasm of Mycobacterium smegmatis. Relation to trehalose-based oligosaccharides attached to lipid.

A series of trehalose-based oligosaccharides were isolated from the cytoplasmic fraction of Mycobacterium smegmatis and purified by gel-filtration and paper chromatography and TLC. Their structures were determined by HPLC and GLC to determine sugar composition and ratios, MALDI-TOF MS to measure molecular mass, methylation analysis to determine linkages, (1)H-NMR to obtain anomeric configurations of glycosidic linkages, and exoglycosidase digestions followed by TLC to determine sequences and anomeric configurations of the monosaccharides. Six different oligosaccharides were identified all with trehalose as the basic structure and additional glucose or galactose residues attached in various linkages. One of these oligosaccharides is the disaccharide trehalose (Glcalpha1-1alphaGlc), which is present in substantial amounts in these cells and also in other mycobacteria. Two other oligosaccharides, the tetrasaccharides Glcalpha1-4Glcalpha1-1alphaGlc6-1alphaGal and Galalpha1-6Galalpha1-6Glcalpha1-1alphaGlc, have not previously been isolated from natural sources or synthesized chemically. The fourth oligosaccharide, Glcbeta1-6Glcbeta1-6Glcalpha1-1alphaGlc, has been isolated from corynebacteria, but not reported in other organisms. Two other oligosaccharides, Glcalpha1-4Glcalpha1-1alphaGlc, which has been synthesized chemically and isolated from insects but not previously reported in mycobacteria, and Glcbeta1-6Glcalpha1-1alphaGlc, which was previously isolated from Mycobacterium fortuitum and yeast, were also characterized. Another trisaccharide found in the cytosol has been partially characterized as arabinosyl-1-4trehalose, but neither the anomeric configuration nor the D or L configuration of the arabinose is known. In analogy with sucrose and its higher homologs, raffinose and stachyose, which may act as protective agents during maturation drying in plants, these trehalose homologs may also have a protective role in mycobacteria, perhaps during latency.     

Isolation and Characterization of Corynebacteria from Burned Children

A total of 221 strains of corynebacteria were isolated and characterized by methods which included tests encompassing five schemes proposed for grouping cutaneous diphtheroids. Seventy-one strains (group I) were isolated from the hospital air in patient areas and from the normal skins of children admitted for reconstructive surgery of old healed burns and from the normal skins of nursing personnel. One hundred and fifty strains (group II) were isolated from various clinical specimens and from normal skins of a population of acutely burned children. The majority of the strains in group I were lipophilic and contained the largest number of fluorescent strains. Among the group II strains, there was a subgroup which was nonsusceptible to oxacillin, lincomycin, erythromycin, and kanamycin and also had in common the fermentation of glucose and galactose, reduction of both nitrate and nitrite, and growth on 40% bile agar. These strains were the most commonly recognized types isolated from acutely burned patients and possibly originated from the patient's intestinal tract. Data indicated that the air was not a means of transmission for these corynebacteria among acute patients. Corynebacteria were isolated from 11% of the burn wound cultures by using a selective medium but were found in 66% of the acute patients. Over 90% of the strains in groups I and II did not conform sufficiently with described characteristics of common human indigenous corynebacteria to be accurately speciated.     

Antagonistic action of corynebacteria and bacilli of a skin ecotype on staphylococci

582 strain of corynebacteria and 235 cultures of bacilli isolated from the skin surface of mammary glands of 120 nursing women were studied for their antagonistic action on staphylococci. It is established that bacilli, especially Bacillus subtilis, B. firmus and B. alvei exert more pronounced inhibitory effect on staphylococci. Representatives of the family Corynebacteriaceae possess a weak antagonistic action both on coagulase-positive and on coagulase-negative species. The ability of coryneform bacteria to retain the colonization resistance is supposed to be due to other factors. Antagonistic properties of B. subtilis are shown expedient to be used for elimination of hospital strains of Staphylococcus aureus from the skin of mammary glands.     

Resistance to antibiotics and histamine production at the bacteria, isolated from the stomatopharynx of the children with bronchial asthma]

1549 strains of bacteria (gram-positive and gram-negative bacteria and Candida) were isolated from the 117 children (65 with bronchial asthma and 52 healthy one) aged from 3 to 14 years old. Susceptibility of 1213 strains to 20 antibiotics was determined by disk-diffusion method. It was shown that 336 strains produce histidine decarboxylase and for 40 strains the quantity of the produced histamine was measured on the Moeller medium. Histamine-modifying activity was investigated at 32 strains in vitro by fluorometric method. Reduced colonization resistance of the pharynx of the children with bronchial asthma was shown. Spectrum of bacteria with histidine decarboxylase activity was more wide at the children with asthma. Histamine production was about 1 mcg/mL at streptococci, staphylococci and hemophilic bacteria, from 1 to 3 mcg/mL - at bacilli, corynebacteria and Candida, from 1 to 10 mcg/mL - at lactobacilli, enterobacteria, pseudomonades, acinetobacteria. Ability for partial histamine destruction in virowas demonstrated for some strains of lactobacilli, enterobacteria and pseudomonades (initial histamine concentration reduced on 19.4-35.8 per cent after 48-hour incubation).     

Polypeptide antibiotic 26a from Bacillus subtilis. III. Physicochemical and biological in vitro properties.

Antibiotic 26a, a weakly basic (pK1 3.85 and pK2 7.1) polypeptide compound, has been recovered from the fermentation fluids of bacillus subtilis cultures as hydrochloride salt easily soluble in water and dimethylsulphoxide, sparingly soluble in lower alcohols and insoluble in several organic solvents. At low concentrations 26a was effective against gram-positive bacteria, mainly micrococci and corynebacteria, moderately active against mycobacteria, and inactive against gram-negative bacteria, yeasts and moulds even at 300 microgram/ml concentration. From the viewpoint of elemental analysis, electrometric titration, optical rotation, UV, IR and NMR spectra, amino acid composition, molecular weight and biological observations, 26a can be considered as an antibiotic, if not identical, then closely related to bacitracin family polypeptides.     

High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria.

We report on the mobilization of shuttle plasmids from gram-negative Escherichia coli to gram-positive corynebacteria mediated by P-type transfer functions. Introduction of plasmids into corynebacteria was markedly enhanced after heat treatment of the recipient cells. High-frequency plasmid transfer was also observed when the restriction system of the recipient was mutated. On the basis of our data, we conclude that efficient DNA transfer from gram-negative to gram-positive bacteria, at least to coryneform bacteria, is conceivable in certain natural ecosystems.     

Pseudomonads as parasites of protozoa]

The experimental study of the interaction of Tetrahymena pyriformis with different microorganisms of the genus Pseudomonas, isolated from the soil, was made. The study revealed that T. pyriformis phagocytosed some Pseudomonas pigment-forming species (P. cepacia, P. putida, P. fluorescens, P. pirkettii). The most pronounced cytopathogenic effect was produced by P. cepacia. The dynamic observations of the ultrastructural features of interaction between P. cepacia and protozoa were made. Even at early stages of this interaction some types of parasitiferous phagosomes containing both intact bacteria capable of multiplication by binary division and Pseudomonas cells exhibiting different degrees of destruction were registered. In several phagosomes morphologically intact bacteria differing in their cell-wall profiles and the density of their cytoplasm and nucleotide were present simultaneously. More dense cells with sinuous cell-wall membranes were more virulent. By hour 18 one giant parasitiferous vacuole was formed by fusion of smaller phagosomes, which subsequently broke up, liberating a new generation of bacteria. In infected cells disturbances in the structure of their mitochondria and macronucleus appeared. During the first 2 days of the joint cultivation of P. cepacia and T. pyriformis the accumulation of bacteria occurred due to the selection and multiplication of digestion-resistant bacterial cells, which ensured the resistance of this Pseudomonas population in association with protozoa.     

Transmissible modification induced in Mycobacterium tuberculosis and Mycobacterium bovis in vitro]

A biologically active material (fraction "S") is isolated from cultures of scotochromogenic mycobacteria. Mycobacterium tuberculosis, or Mycobacterium bovis by disrupting the cells, sedimentation through 2.2 M sucrose, and ultrafiltration. The fraction "S" induces the modification of tubercle bacilli into non acid-fast bacteria forming smooth colonies on nutritive glycerol agar within 24-36 h of incubation. Three new phenotypes are thus obtained; two proved to be stable upon subculturing. Frequently the phenomenon occurs with a very large part of the Koch's bacillus population exposed to the inducing agent effect. It can be reproduced with crude preparations of DNA obtained from the fraction "S." It is inhibited by concanavalin A. The observed modification does not correspond to a transfer of characteristics of the inducing agent from the donor mycobacteria; furthermore it can be manifested even in the strain used for the preparation of the fraction "S."