Sensitivity of Mycoplasma hominis cultures isolated from clinical material to certain antibiotics]

Sensitivity to II antibiotics of 80 strains of M. hominis isolated from patients with various inflammatory processes of the urogenital tract was studied by the method of suppressing the metabolic activity. Inhibition of the arginine metabolism of mycoplasma was used as a test for determination of the growth suppression. All the strains tested were highly sensitive to tetracycline and lincomycin. Kanamycin and neomycin were less active against M. hominis. All the strains tested were resistant to erythromycin, oleandomycin, ristomycin, novobiocin and streptomycin. The inhibitory effect of tetracycline and lincomycin on M. hominis decreased by the 5th day.     

Quantitative determination of anhydroleandomycin and glycol in oleandomycin phosphate]

A spectrophotometric method for determination of inactive admixtures in oleandomycin phosphate-anhydrooleandomycin was developed. The method was based on measuring optical density of methanol solutions of oleandomycin and its admixtures at a wave length of 235 nm of UV-spectrum. The minimun detectable amount was 0.5 per cent. A method of thin-layer chromatography in a buffered fixed layer was developed for determination of the 2nd inactive admixture, i.e. glycole. The chromatogrammes were developed with xanthydrol. The minimum detectable amount was 3 mug.     

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.     

Determination of the biological activity of antibiotics by using dry nutrient media

The possibility of using available dry nutrient media Nos. 5 to 12 in assays of antibiotic biological activity with the agar diffusions method was studied with respect to benzylpenicillin, gramicidin S, kanamycin sulfate, kanamycin B, oleandomycin, novobiocin, tetracycline and erythromycin. The dry media were used instead of the respective media prepared with meat hydrolyzate. Optimal conditions of the assays on such media were determined.     

Antibiotic sensitivity of the bacterial microflora from the sputum of children with acute pneumonia]

Bacteriological analysis of sputum of 598 children with acute pneumonia was performed. Sensivity of 1348 cultures belonging to 8 bacterial species with respect to benzylpenicillin, streptomycin, chloramphenicol, tetracycline, oxytetracycline, chlortetracycline, erythromycin, oleandomycin, neomycin and monomycin was determined. It was shown that the sputum microflora was often resistant to the antibiotics widely used in the medical practice for prolonged periods of time, such as benzylpenicillin, streptomycin, chloramphenicol, tetracyclines. However, it usually remained sunsitive to neomycin, monomycin, erythromycin and oleandomycin. It was found that antibioticogrammes defining the antibiotic choice were of great significance for therapy of acute pneumonia.     

Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus

A 5.2 kb region from the oleandomycin gene cluster in Streptomyces antibioticus located between the oleandomycin polyketide synthase gene and sugar biosynthetic genes was cloned. Sequence analysis revealed the presence of three open reading frames (designated oleI , oleN2 and oleR ). The oleI gene product resembled glycosyltransferases involved in macrolide inactivation including the oleD product, a previously described glycosyltransferase from S. antibioticus . The oleN2 gene product showed similarities with different aminotransferases involved in the biosynthesis of 6-deoxyhexoses. The oleR gene product was similar to several glucosidases from different origins. The oleI , oleR and oleD genes were expressed in Streptomyces lividans . OleI and OleD intracellular proteins were partially purified by affinity chromatography in an UDP-glucuronic acid agarose column and OleR was detected as a major band from the culture supernatant. OleI and OleD showed oleandomycin glycosylating activity but they differ in the pattern of substrate specificity: OleI being much more specific for oleandomycin. OleR showed glycosidase activity converting glycosylated oleandomycin into active oleandomycin. A model is proposed integrating these and previously reported results for intracellular inactivation, secretion and extracellular reactivation of oleandomycin.     

Kinetics of thermal inactivation of oleandomycin in aqueous and native alkaline solutions

The process of oleandomycin inactivation in aqueous alkaline solutions with their heating was studied by using the microbiological method of the antibiotic content assay. The initial specific rate of inactivation of crystalline oleandomycin in buffer solutions and oleandomycin in the fermentation broth filtrate was evaluated. It was shown that the inactivation was retarded by the reaction products and the components of the fermentation broth filtrate. The production rate of oleandomycin anhydro derivatives amounting to 3-40 per cent of the total mass of the inactivation product was estimated by UV spectrophotometry.     

Role of glycosylation and deglycosylation in biosynthesis of and resistance to oleandomycin in the producer organism, Streptomyces antibioticus.

Cell extracts of Streptomyces antibioticus, an oleandomycin producer, can inactivate oleandomycin in the presence of UDP-glucose. The inactivation can be detected through the loss of biological activity or by alteration in the chromatographic mobility of the antibiotic. This enzyme activity also inactivates other macrolides (rosaramicin, methymycin, and lankamycin) which contain a free 2'-OH group in a monosaccharide linked to the lactone ring (with the exception of erythromycin), but not those which contain a disaccharide (tylosin, spiramycin, carbomycin, josamycin, niddamycin, and relomycin). Interestingly, the culture supernatant contains another enzyme activity capable of reactivating the glycosylated oleandomycin and regenerating the biological activity through the release of a glucose molecule. It is proposed that these two enzyme activities could be an integral part of the oleandomycin biosynthetic pathway.     

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.     

Sensitivity of the causative agent of tularemia to antibiotics

Sensitivity of the tularemia causative agent of different geographical races to antibiotics such as streptomycin, tetracycline, gentamicin, rifampicin (20 strains), ampicillin, polymyxin M, erythromycin, oleandomycin (361 strains) and lincomycin (294 strains) was studied. High sensitivity of the tularemi a microbe to streptomycin, tetracycline, rifampicin (MIC of 10 gamma/ml), gentamicin (MIC of 1 gamma/ml) and resistance to 50 gamma of ampicillin and 1000 gamma/ml of polymyxin M were found. Combined use of 50 gamma of ampicillin and 100 gamma/ml of polymyxin M added to the nutrient medium for growth inhibition of the foreign flora on isolation of the tularemia causative agent from the infected material including stable laboratory animal carcases was recommended. Marked differences in sensitivity of the strains of different geographical races to the macrolides and lincomycin were observed. The strains of the non-Arctic and Central Asiatic races were of low resistance to the above drugs (the MIC of erythromycin, oleandomycin and lincomycin were 10--50, 50--400 and 25--100 gamma/ml respectively. Within the holarctic race 40 per cent were low resistant and 60 per cent were highly resistant to these drugs. The above drugs should not be used for treatment of tularemia cases.     

The interrelation between the formation of oleandomycin and the resistance to it in different strains of Streptomyces antibioticus]

Strains, producers of oleandomycin, with different level of antibiotic-formation have been studied for their resistance to their own antibiotic. The obtained highly active strain possesses double resistance to oleandomycin and 50% higher activity. Identity of oleandomycin phosphate substances synthesized by initial and produced highly active strains is shown by the HELC method.     

Purification and characterization of macrolide 2'-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics.

The resistance mechanism of Escherichia coli BM2506 to macrolides was found to be due to inactivation. Inactivated oleandomycin was identified as oleandomycin 2'-phosphate by thin-layer chromatography. A new type of macrolide-phosphorylating enzyme, macrolide 2'-phosphotransferase type II (MPH(2')II), was detected, purified 95-fold and its enzymological properties investigated. MPH(2')II was a constitutive intracellular enzyme which showed high levels of activity with both 14-member-ring and 16-member-ring macrolides. The optimum pH for the inactivation of oleandomycin was 8.2 and the optimum temperature of the reaction was 40 degrees C. Enzyme activity was lost by heat treatment at 60 degrees C for 1 min. The isoelectric point and M(r) of the enzyme were 5.3 and 48,000, respectively. Purine nucleotides, such as ITP, GTP and ATP, were effective as cofactors in the inactivation of macrolides. An inhibitory effect of iodine, EDTA, or divalent cations on MPH(2')II activity was observed.     

Assessment of the sensitivity of Neisseria meningitidis strains to benzylpenicillin using the disc diffusion method]

The sensitivity of 235 N. meningitidis strains to 5 antibiotics was estimated by the diameter of growth inhibition zones according to the criteria recommended by a Laboratory (Marseilles, France) collaborating with the WHO. All the strains proved to be sensitive to benzylpenicillin when disks containing 10 and 2 IU of the antibiotic were used. The strains were also shown to be sensitive to chloramphenicol and tetracycline. 95.7 and 7.7 per cent of the strains were sensitive to rifampicin and oleandomycin, respectively. When the strain sensitivity was assayed with the disks containing 10 and 2 IU of benzylpenicillin by the more severe criteria recommended by J. Saez-Nieto et al., significant changes were detected: meningococci with relative resistance to benzylpenicillin were detected in various regions of this country and the number of such strains was found to have a tendency to slightly increase.     

Spectral control of metal admixtures in tetracycline]

Analysis of circular dichroism spectra made it possible to offer a method for estimation of tetracycline solutions contamination with metal ions. By its sensitivity the method is much superior to the spectrophotometric one used at present for determination of the antibiotic purity. In the latter method formation of complexes with metals is traced by batochromic displacement of the absorption spectra. The new method is rapid, relatively selective and requires comparatively small quantities of the substance for the analysis, which provides its use under both laboratory and manufacture conditions. The method is based on identification of the circular dichroism spectra of tetracycline complexes with metals in the long wavelength region. The presence of the circular dichroism concervative bands with strictly defined extremums in the spectra of tetracycline low acid solutions contaminated by multiply charged metal ions allowed vs. the circular dichroism spectra of pure tetracycline sample to conclude that the solution contained admixtures and to suggest their nature. It was shown that the charge, ion radius and tetracycline:metal relation were the factors defining the mark and location of the dichroism band extremums. At lambda(extr)-410-415 nm the tetracycline complexes with light metal ions such as Mg2+, Al3+ and Ca2+ were detected by the circular dichroism negative band in the spectra, while the complexes with heavy metal ions such as Sc3+, Sr3+, Cu3+, Cd3+, Ba2+, Y3+ and the cerium subgroup lanthanides were detected by the circular dichroism positive band. The tetracycline complexes with the lanthanides of the second half of the yttrium subgroup (Ho(3+)-Lu3+) were characterized by the presence of the circular dichroism minimum at lambda(min)-425 nm. When the tetracycline concentration was above 1.5 x 10(-3) M, multiligand complexes with circular dichroism negative extremum at lambda(min)-400 nm formed.     

Biological properties of sherry yeasts in the presence of antimicrobial preparations

The wine yeasts Cheres were not sensitive to high concentration (500 gamma/ml) of benzylpenicillin, streptomycin, tetracycline, oxytetracycline, chlortetracycline, morphocycline, erythromycin, oleandomycin, monomycin, neomycin, kanamycin, polymyxin, ampicillin, oxacillin, methicillin, ceporin, ristomycin, levomycetin, furadonin and furazolidone. In concentrations of 50 to 500 gamma-ml oleadomycin, chlortetracycline, oxytetracycline, kanamycin and ristomycin inhibited synthesis of the Cheres yeast biomass. Benzylpenicillin, polymyxin, neomycin and ampicillin in concentrations of 50 to 100 gamma/ml had a stimulating effect on the yeast biomass synthesis.     

Effect of Pseudomonas aeruginosa melanin on antibiotic activity]

The properties of microbial melanines are very diverse. Melanine of P. aeruginosa is little studied. The pigment was isolated from a strain of P. aeruginosa possessing all characteristic properties of the species. Interaction of P. aeruginosa melanine with various antibiotics was determined by the method of serial dilutions in beaf-peptone broth, using Staph. aureus 209 as a test-microbe, which was added to the medium in an amount of 10(6) cells to each tube. It was found that P. aeruginosa melanine differed from DOPA-melanine in a concentration of 1 mg/ml and did not change the activity of penicillin, tetracycline, oleandomycin, kanamycin and gentamicin with respect to Staph. aureus.     

Effect of hydroacridine derivatives on the sensitivity of polyresistant strains of Staphylococcus aureus and Escherichia coli to antibiotics]

Sensitivity of 4 clinical strains of Staph. aureus and E. coli to 13 hydroacridine derivatives and their combinations with antibiotics, such as benzylpenicillin, ampicillin, semi-synthetic penicillins, streptomycin, chloramphenicol, tetracycline, chlortetracycline, monomycin, oleandomycin and erythromycin was studied. The highest bacteriostatic effect was observed on the use of perhydroactidine derivatives with benzylpenicillin or ampicillin with respect to polyresistant penicillinase-producing strains of Staph. aureus, resistance of which to these antibiotics was decreased 250--1000 times. Under the effect of the above compounds the staphylococcal resistance to chloramphenicol, tetracycline, chlortetracycline, oleandomycine and erythromycin decreased 2--66 times. The combinations of hydroacridine with the antibiotics, except 10-amino-trans-syn-trans-perhydroacridine had no effect on the resistance of the E. coli strains. The results of the combined effect of the above substances were associated with their chemical nature, the bacterial type and possibly the character of the strain resistance.     

Analysis of a Streptomyces antibioticus chromosomal region involved in oleandomycin biosynthesis, which encodes two glycosyltransferases responsible for glycosylation of the macrolactone ring

A 6-kb region from the chromosome of Streptomyces antibioticus, an oleandomycin producer, was cloned and sequenced. This region was located between the 3′ end of the gene encoding the third subunit of the oleandomycin type I polyketide synthase and the oleP and oleB genes, which encode a cytochrome P₄₅₀ monooxygenase and an oleandomycin resistance gene, respectively. Analysis of the nucleotide sequence revealed the presence of five genes encoding a cytochrome P₄₅₀-like protein (oleP1), two glycosyltransferases (oleG1 and oleG2) involved in the transfer of the two 6-deoxysugars (L-oleandrose and D-desosamine) to the oleandomycin macrolactone ring, a methyltransferase (oleM1), and a gene (oleY) of unknown function. Insertional inactivation of this region by gene disruption generated an oleandomycin non-producing mutant which accumulated a compound that, according to mass spectrometry analysis, could correspond to the oleandomycin macrolactone ring (oleandolide), suggesting that the mutation affects oleandrosyl glycosyltransferase.     

Methods of determining admixtures in commercial preparations of tetracycline]

The results and conditions of separation and quantitative determination of the admixtures of epi- and anhydro-derivatives of tetracycline in its market products were compared and analyzed using the following chromatographic methods: chromatography in a thin layer of a sorbent in 2 modifications, circular paper chromatography, chromatography on a column of gel Sephadex G-25. The comparison of the results of the study provided recommendation of the column chromatography with gel Sephadex G-25 for separate or summation quantitative determination of 4-epitetracycline. The method of chromatography in a thin layer of a sorbent may be successfully used for semiquantitative determination of the admixture levels in tetracycline market drugs.     

Characterization of the ATPase activity of the N-terminal nucleotide binding domain of an ABC transporter involved in oleandomycin secretion by Streptomyces antibioticus

Abstract The ole B gene of Streptomyces antibioticus , oleandomycin producer, encodes an ABC transporter containing two putative ATP-binding domains and is involved in oleandomycin resistance and secretion in this organism. We have overexpressed in Escherichia coli the N-terminal nucleotide-binding domain of OleB (OleB') as a fusion protein to a maltose-binding protein and purified the fusion protein by affinity chromatography. The fusion protein showed ATPase activity dependent on the presence of Mg²⁺ ions. ATPase activity was resistant to specific inhibitors of P-, F-, and V-type ATPase whereas sodium azide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole (NBD-C1) were strong inhibitors. The change of Lys71, located within the Walker A motif of the OleB' protein, to Gin or Glu caused a loss of ATPase activity, whereas changing to Gly did not impair the activity. The results suggest that the intrinsic ATPase activity of purified fusion protein can be clearly distinguished from other ATP-hydrolysing enzymes, including ion-translocating ATPases or ABC-traffic ATPases, both on the basis of inhibition by different agents and since it hydrolyzes ATP without interacting with a hydrophobic membrane component.