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[Establishment of gene transduction system in geldanamycin producer - Streptomyces hygroscopicus 17997 and its application for gene disruption experiment].

Streptomyces hygroscopicus 17997 produces the antiviral and antitumor ansamycin antibiotic, geldanamycin. Studies on geldanamycin biosynthetic pathway will provide good tools for genetic manipulation of the antibiotic-producing strain to improve the productivity or to facilitate making novel geldanamycin analogs. The structural similarities between geldanamycin and ansamycins such as rifamycin or ansatrienin suggest that both geldanamycin and ansamycins has a closely related pathways of biosynthesis and that biosynthetic system for geldanamycin is similar to the one of type I polyketide synthase (PKS) enzyme system. To explore the possible PKS genes involved in geldanamycin biosynthesis, the degenerate primers were designed according to the conserved sequence of KS-AT region from erythromycin and oleandomycin type I PKS genes. Cosmids containing multiple PKS genes (pCGBK2,4,6,10,11,18) were obtained by hybridization with the PCR products, which were amplified from S. hygroscopisus 17997 genomic DNA. The designed primers above were used for PCR. Development of a Streptomyces temperate phage phiC31-derivative KC515( tsrR) transduction system was carried out for identification of cosmids containing the PKS gene related to biosynthesis of geldanamycin. Several factors, mainly the Ca2+ and Mg + concentrations in different culture media affecting the frequency of gene transfection, were optimized .Transfection efficiency could reach up to 10(3) /microg DNA on YMG medium supplemented with 10mmol/L MgSO4. Reversely, the transfection efficiency decreased when YMG medium was supplemented with 30mmol/L MgSO4. Gene transfection system based on the integration-defective phage KC515 had been established for S. hygroscopicus17997. Recombinant phages (ph111, 258, 287, 116, 105) were constructed by insertion of the homologous to PKS gene fragments into the KC515 phage vector. Gene disruption experiments were performed by transduction of recombinant phages into S. hygroscopicus 17997 genome, and disruption of geldanamycin production was observed as a result of homologous recombination between the cloned insert in recombinant phage and the S. hygroscopicus 17997 genome by integration. Thiostrepton resistant transductants were selected and integration event was analyzed by Southern hybridization. The fermentation broth extracts from five resistant transductants were analyzed by TLC and HPLC. The results showed that only G16 mutant failed to produce geldanamycin. This result showed that the integration of the insert DNA fragment in recombinant phage phl6 into the chromosome of S. hygroscopicus disrupted the expression of the geldanamycin biosynthetic genes. The original cosmid pCGBK10 containing this cloned insert was predicted to encode PKS genes in the geldanamycin biosynthesis. This study laid the foundation for cloning the PKS genes involved in geldanamycin biosynthetic gene cluster from S. hygroscopicus 17997.

Alcohol Oxidoreductases↗

Antibiotic susceptibility of Streptococcus pneumoniae isolated from clinical materials (author's transl).

We tested the drug-susceptibility of 112 strains of Streptococcus pneumoniae isolated from various clinical materials since January 1975 to May 1976, and observed the changes of susceptibility of this pathogen. All strains tested remained very sensitive to benzylpenicillin and cephaloridine, but 59.8% of strains were resistant to tetracycline and 44.7% to chloramphenicol, and about 90% of chloramphenicol-resistant strains were also resistant to tetracycline. The percentage of resistant strains to tetracycline or chloramphenicol is increasing. Cross-resistance between tetracycline and doxycycline or chloramphenicol and thiamphenicol was observed. On this time we found three strains resistant to macrolides. Two of these strains were resistant to erythromycin, josamycin, oleandomycin, spiarmycin, mydecamycin, lincomycin, clindamycin and tetracycline, but one was resistant to josamycin, spiramycin, mydecamycin and chloramphenicol.

Anti-Bacterial Agents↗

[Sensitivity of pathogenic clostridia to antibiotics].

By the sensitivity levels of the gas infection causative agents, i. e. pathogenic Clostridia to antibiotics, the latter were conditionally divided into 4 groups. The 1st group included the most active antibiotics, such as tetracyclines,, penicillins, cephalosporins, rifampicin, 7-chlor-7-desoxylincomycin. Their minimum inhibitory and bactericidal concentrations did not usually exceed 2 gamma/ml. For most of the strains the inhibitory and bactericidal concentrations amounted to the tenth and hundredth fractions of gamma/ml. The antibiotics of the 2nd group, i. e. erythromycin, lincomycin,ristomycin and levomycetin inhibited multiplication and viability of pathogenic Clostridia in concentrations of 20 gamma/ml. Erythromycin was most active among them The 3rd group consisted of oleandomycin, novobiocin, geliomycin and azalomycin, the minimum inhibitory concentrations of them being 20 to 50 gamma/ml. The antibiotics of the 4th group, i. e. neomycin, monomycin, kanamycin, streptomycin, polymyxin and others affected pathogenic Clostridia at very high concentrations, amounting to the hundrenth and thousandth of gamma/ml.

Anti-Bacterial Agents↗

[Effect of the phosphorus concentration on novobiocin formation by the producer Act. spheroides].

According to the literature data biosynthesis of novobiocin by Act. spheroides unlike other antibiotics does not practically depend on the phosphorus levels in the medium. In the present paper it is shown that production of novobiocin in natural media is sensitive to the concentration of mineral phosphorus in the medium. The optimal concentration of phosphorus for biosynthesis of novobiocin is almost within the same ranges as that for biosynthesis of streptomycin, tetracyclines and oleandomycin.

Carbohydrate Metabolism↗

[The clinical picture of legionnaires' disease].

The results of the clinico-laboratory study of 12 cases of acute pneumonia of Legionella etiology are presented. The laboratory diagnosis of Legionella infection was carried out by the study of paired sera in the passive hemagglutination test with the use of Legionella pneumophila (serotype 1) erythrocyte diagnosticum. The clinical picture of pneumonia was characterized by a severe and moderate course of the disease. Characteristic symptoms indicating the presence of indurations and infiltrations in the lung tissue were registered. Roentgenological examination revealed that the foci of pulmonary tissue infiltration appeared in the segments of the lower lobes of both lungs. In 6 patients neutrophil leukopenia, in 4 patients relative lymphocytopenia, in 5 patients monocytopenia, in 11 patients the increase of the erythrocyte sedimentation rate and in 4 patients normochromic anemia were registered. More seldom changes in the levels of residual nitrogen, urea, fibrinogen and transaminases were observed. In most cases the resolution of pneumonia was observed on weeks 2-3 of treatment. In this treatment erythromycin, rifampicin and oleandomycin, used in combination, used in combination with detoxication and infusion therapy, vitamins, vascular and other symptomatic remedies, proved to be most effective. The cases of Legionella infection under study were sporadic and epidemiologically unrelated. The severity of the course of the disease depended mainly on the general state of the patient prior to infection, age and concomitant diseases.

Acute Disease↗

A new type of macrolide resistance in staphylococci from bovine subclinical mastitis.

Staphylococcal (nine Staphylococcus aureus and 14 coagulase negative) strains, isolated from subclinical bovine mastitis, showed three patterns of macrolide resistance: (i) macrolide-lincosamide constitutive resistance; (ii) macrolide generalised resistance; and (iii) lincosamide generalised resistance. The second pattern of resistance appeared to be a new resistance phenomenon. None of the strains showed dissociated or inducible resistance, neither was there any strain which could inactivate erythromycin, oleandomycin, lincomycin or clindamycin.

Animals↗

[Production of toxic shock exotoxin by Staphylococcus aureus strains resistant and sensitive to antibiotics].

Three hundred and ninety two strains of S. aureus isolated from bacteria carriers and patients with staphylococcal infections in different regions of the Soviet Union were investigated. 55.9 per cent of the isolates were able to produce exotoxin of toxic shock. No regular relation between resistance to definite antibiotics (tetracycline, chloramphenicol, benzylpenicillin, streptomycin, lincomycin, erythromycin, oleandomycin, gentamicin and methicillin) and the polyresistance range on the one hand and the ability to produce toxic shock exotoxin on the other hand was revealed.

Anti-Bacterial Agents↗

[Sensitivity spectrum of Francisella tularensis to antibiotics and synthetic antibacterial drugs].

Sensitivity of 6 F. tularensis strains to 57 antibiotics and synthetic antibacterial drugs was studied. It was shown that the strains were highly sensitive to aminoglycosides, tetracyclines, anzamycins, quinolones, chloramphenicol, nitrofurantoin, nitroxoline, novobiocin and fusidin and resistant to penicillins, cephalosporins, polypeptides, vancomycin and sulfanylamides. The interrace differences in F. tularensis could be detected only by sensitivity to erythromycin, oleandomycin and spiramycin. There was observed no cross resistance to streptomycin and other aminoglycosides in F. tularensis. Assay of F. tularensis sensitivity to antibacterial drugs of various groups with the rapid photometric procedure and the agar diffusion method revealed that the results were highly comparable.

Anti-Bacterial Agents↗

[Molecular cloning of tylosin-producing Streptomyces fradiae B-45 genes determining increased inducible resistance to tylosin in Streptomyces lividans TK64].

DNA of S. fradiae B-45 partially cleaved by Sau3A restrictase was cloned in S. lividans TK64 in the plasmid vector pIJ702. Three recombinant plasmids pVG251, pVG262, and pVG253 with tlr1, tlr2 and tlr3 genes were isolated from the transformed clones of S. lividans TK64 with higher inducible resistance to tylosin as compared to the plasmid-free strain. DNA-DNA blot hybridization was performed between the total DNA cleaved by several restrictases from S. fradiae B-45 and some other strains and the DNA probes containing the tlr genes. It was shown that tlr1 and tlr3 genes were unique in S. fradiae B-45. Sequences homologous to tlr2 gene were present both in DNA of S. fradiae B-45 in 7 copies and in strains of S. antibiotics and S. hygroscopicus producing respectively oleandomycin and turimycin.

Cloning, Molecular↗

[Antibiotic sensitivity of Haemophilus influenzae isolated from patients with inflammatory lung diseases].

In 1980-1986 the sensitivity of 2,045 H. influenzae strains isolated from the bronchial contents of patients with inflammatory lung diseases were studied. This study revealed that 60-80% of H. influenzae cultures circulating in Leningrad were sensitive to tetracyclin, chloramphenicol and erythromycin. During the period of observation the tendency towards the decrease of the number of highly sensitive H. influenzae cultures and the increase of the number of strains resistant to all antibiotic preparations was followed. Most of H. influenzae strains isolated in Leningrad were sensitive to penicillin, oleandomycin and ampicillin. In 1983 the appearance of H. influenzae strains, multiresistant to antibiotics, was noted. In 1986 these strains constituted 4.5% of all isolated cultures.

Anti-Bacterial Agents↗

An in vitro study of the translational attenuation model of ermC regulation.

We have used a Bacillus subtilis in vitro translation system to test the translational attenuation model for ermC regulation. The ermC gene product is known to methylate rRNA, rendering ribosomes unable to bind this antibiotic. We have shown that the induction of ermC methylase in vitro is post-transcriptional and specific for the macrolides erythromycin and oleandomycin. Erythromycin has no significant effect on the stability of the ermC transcript in vitro, and hence the post-transcriptional induction of methylase under these conditions occurs by stimulation of translation. The induction effect requires ribosomes able to bind erythromycin. By adding small proportions of unmethylated to a methylated extract in the presence of erythromycin, methylase synthesis could be induced. Conversely, when small amounts of methylated extracts were mixed with unmethylated extracts, methylase synthesis could be maintained at elevated levels in the presence of a high concentration of erythromycin. These effects were specific for the inducible ermC, were not observed with a constitutive variant, and could be explained satisfactorily by the translational attenuation model. The roles of three segments of the ermC leader in regulation were explored by probing with appropriate complementary synthetic oligodeoxynucleotides. The induction effect of erythromycin was mimicked by using an oligonucleotide that could free the ribosome binding site for methylase.

Bacillus subtilis↗

Demonstration of erythromycin-dependent stalling of ribosomes on the ermC leader transcript.

ermC encodes a methylase that modifies 23 S rRNA, conferring resistance to macrolide-lincosamide-streptogramin B antibiotics. The expression of this gene is induced by erythromycin using a translational mechanism. We have employed the inherent RNase activity of a Bacillus subtilis S-30 extract as a probe for studying the interaction of ribosomes with ermC mRNA in the presence of antibiotics. 5' end-labeled ermC runoff transcript is a substrate for this RNase activity, while the ribosome-bound region of the RNA appears to be protected. Erythromycin- and oleandomycin-dependent protection of fragments of length 79-81 was observed during the translation of end-labeled ermC transcript. This occurs only using unmethylated (erythromycin sensitive) ribosomes. Various other antibiotics including clindomycin, tylosin, and lincomycin do not show this specific protection. These effects parallel the in vivo specificity of ermC induction. The effect of erythromycin can be abolished by using oligonucleotides complementary to regions of the ermC transcript upstream from nucleotide 71 and not by using an oligonucleotide complementary to a region of ermC downstream from that position. These results are interpretable in terms of the translational attenuation model and demonstrate that erythromycin-bound ribosomes initiate translation of the leader peptide, stall upstream from nucleotide 80 on the ermC mRNA, and thus make the ribosome-binding site for methylase message available for ribosome interaction.

Anti-Bacterial Agents↗

[In vitro measurement of the inhibitory effect of macrolides, lincosamides and synergestines on the growth of Toxoplasma gondii].

The in vitro inhibitory effect of macrolides, synergestines and lincosamides on Toxoplasma gondii was evaluated using an enzyme immunoassay performed directly on the infected cultures. Antimicrobial agents were tested at various concentrations, and regression models were used to quantify the relationship between optical density values (correlated to Toxoplasma count in the cultures) and drug concentrations in the cultures. A significant inhibitory effect was observed with the macrolides (erythromycin, roxithromycin, midecamycin, spiramycin, josamycin and oleandomycin) for concentration of 0.1 to 4 micrograms/ml. This effect progressively increased with increasing concentration of drug in the cultures and reached its maximum value for concentrations of 40 to 250 micrograms/ml. Similarly, clindamycin was found to have a significant inhibitory effect for concentration ranging from 0.5 ng/ml to 100 micrograms/ml. Only a partial inhibitory effect was noted with virginiamycin, pristinamycin and lincomycin. The concentrations giving a 50% inhibition of Toxoplasma growth in the culture were determined from regression models and ranged between 0.15 and 12 micrograms/ml according to the drug. This results confirm that macrolides, lincosamides are effective against Toxoplasma gondii in vitro, and can be proposed for treatment of toxoplasmosis because of their large diffusion, their good tolerance and their concentration in tissues such as lung and placenta.

Animals↗

[Various ways of standardizing and unifying biological methods of determining the activity of antibiotics].

It was shown possible to use nutrient media containing only salt components and agar-agar for determination of biological activity of tetracyclines (tetracycline, morphocycline, oxytetracycline and chlortetracycline), erythromycin and oleandomycin. A uniform nutrient medium containing ammonium chloride, trisubstituted sodium citrate, disubstituted sodium phosphate and agar-agar was developed. Glucose is added to this medium simultaneously with the test microbe, B. subtilis ATCC 6633. The medium of the above composition provided readily reproducible results.

Anti-Bacterial Agents↗

[Effect of lytic enzymes on Staphylococcus and the possibilities of their combined use with antibiotics].

Dependence of lytic enzyme preparation activity on temperature and time of Staphylococcus incubation with the preparation was shown. A decrease in the activity with an increase in the ionic strength of the incubation solutions and protective effect of salts on the staphylococcal cells were observed. Possible combined use of the preparation with antibiotics was studied. The enzymatic preparation inactivated penicillins and cephalosporins at the account of the ability of lytic endopeptidases to hydrolyze the peptide bond of the beta-lactam ring. However, its combined use with many other antibiotics such as novobiocin, lincomycin, rifampicin, gramicidin, polymyxin, oleandomycin, streptomycin, kanamycin, tetracycline and levomycetin is quite possible.

Actinomyces↗

[Structure of the antibiotic resistance of the salmonellae found in Transcarpathia].

Resistance of 1280 strains of Salmonella of various serological types isolated in the Zakarpatskaya region within 1967-1976 was studied with respect to benzylpenicillin, streptomycin tetracycline, levomycetin, monomycin, neomycin, erythromycin and oleandomycin. An Increase in the resistance of Salmonella to streptomycin, tetracycline, levomycetin, monomycin and neomycin was shown. During the last years Salmonellae carrying 4-8 resistance determinants were spreading in the region. Among S. typhimurium strains with 7-8 resistance determinants predominated (58.8 per cent). The cases of salmonollosis were mainly due to these strains.

Anti-Bacterial Agents↗

[R-factor distribution and characteristics of the E. coli isolated from pigs].

Studied was the occurrence of the R-factors in Escherichia coli isolated from pigs. It was demonstrated that in 73.3 per cent of the cases drug resistance could be transmitted to Citrobacter. In this instance an occasional drop-out of the markers of resistance to kanamycin, neomycin, ampicillin, tetracycline, and chloramphenicol was observed. In the author's experiments no transmission of the marker of resistance to furazolidon was established. The transmission of the signs of resistance to kanamycin and niomycin as well as to erythromycin and oleandomycin occurred always simultaneously. The possibility of transmitting the drug resistance markers of Escherichia coli to Salmonella typhimurium LT-2 made it reasonable to believe that this is one of the routes for spreading the R-factors among Salmonella organisms. No essential differences were observed between the minimal inhibitory concentration of the donor strains and the level of the acquired resistance with the recombinations. The transmission frequence of the resistance markers proved comparatively high, and ranged from 0.6.10(-3) up to 3.10(-4).

Animals↗

[Proposed neotype Streptomyces ruber (Krainsky, 1914) Waksman et Henrici, 1948].

Culture 78 was proposed as a neotype of Streptomyces ruber. It was isolated from the soils of the Baikal region and was closest, in its taxonomic properties, to the original description of the species [13] whose representative had been lost. Cultures from different microbial collections designated as S. ruber were shown to be unlike the original description. The neotype had the following taxononic properties: the cell wall of type I; spiral sporophores with extended spirals having 2-3 coils; oval spores with a smooth envelope; greyish pink aerial and dark-red substrate mycelia; a red pigment not passing into the medium; slow gelatin liquefaction and milk peptonization; weak starch hydrolysis; assimilation of glucose, xylose, rammose, fructose, and inositol; weak growth on arabinose, raffinose and mannitol, but not on sucrose; no formation of melanoid pigments; synthesis of riboflavin and prodigiosin pigments; inhibition of Gram-positive bacterial and acid-resistant mycobacterial growth; no inhibition of yeast and fungal growth. The culture was sensitive to streptomycin, neomycin, gentamycin, monomycin, tetracycline,erythromycin, oleandomycin, lincomycin, ristomycin, levomycetin, polymyxin and fusidin, but resistant in penicillin. The population was composed of six variants [3]: main, faded, asporogenic red, asporogenic yellow, asporogenic white and nocardia-like. The latter two were not capable of riboflavin and prodigiosin formation. The asporogenic yellow variant was a monosynthetic organism: it formed riboflavin, but could not synthesize prodigiosin. The neotype of S. ruber 78 is deposited withthe national Collection of Microorganisms (the reference number is VKM A-611).

Anti-Bacterial Agents↗