Comparative study of Streptomyces--producer of aminoglycoside antibiotics, monomycin and kanamycin, and the strain 344 obtained by fusion of their protoplasts by the method of restrictive total DNA fingerprinting]

The method of total DNA restriction finger prints was applied to the study of Streptomyces monomycini INA 1465 producing monomycin, Streptomyces kanamyceticus INA K-13 producing kanamycin and strain 344 isolated after fusion of the protoplasts of strain 1465 and K-13, which produced albofungin and chloralbofungin, aminoglycoside antibiotics. For preparing the finger prints of the strains splitting by endonucleases BamHI, PstI, PvuII, and BgIII was used. The finger prints showed that strain 344 was related to the strain of S. monomycini and markedly differed from the strain of S. kanamyceticus. Strain 344 was likely to result from reconstruction (probably 20-kb deletion) of the genome of S. monomycini INA 1465 induced by the preparation and regeneration of its protoplasts. The reconstruction could affect the genome area with localization of the genes involved in monomycin biosynthesis and monomycin resistance genes.     

Protoplast fusion in Streptomyces fradiae strains producing neomycin and tylosin]

Interspecies fusion of protoplasts of the Streptomyces fradiae strains producing neomycin (an aminoglycoside antibiotic) and tylosin (a macrolide antibiotic) was performed with a view to isolate strains producing novel antibiotics. Fusion of the protoplasts of the neomycin- and tylosin-producing strains labelled by the resistance to monomycin and lincomycin, respectively, caused no formation of stable strains producing antibiotics differing in chromatographic mobility from the antibiotics produced by the initial strains. In fusion of the protoplasts of the unlabelled strains, heat-inactivated protoplasts of the active line of one strain (donor) and native protoplasts of the inactive line of the other strain (recipient) were used. When the neomycin-producing culture was used as a recipient the fusion led to formation of strain 195-34 producing antibiotics of the benzo(a)anthraquinone group. One of these antibiotics, i.e. antibiotic 34-I, proved to be a novel biologically active substance. After regeneration of the protoplasts of the initial strains, no stable strains producing antibiotics differing from neomycin and tylosin were isolated.     

Comparison of the sensitivity of Pseudomonas aeruginosa cultures that synthesize melanin and other pigments to 12 antibiotics and 5-nitro-8-quinolinol]

Sensitivity of 80 Pseudomonas aeruginosa cultures forming melanin and 80 cultures synthesizing other pigments to amikacin, gentamicin, kanamycin, carbenicillin, levomycetin, monomycin, 5-NOK, polymyxin M, rifampicin, streptomycin, tetracycline, tobramycin and erythromycin was determined. It was found that the cultures of Ps. aeruginosa synthesizing melanin were less resistant to most of the antibiotics that the other representatives of this species.     

Antibiotic sensitivity of Shigella isolated from patients from 1974-1985

Antibiotic sensitivity of 3524 Shigella cultures isolated from patients in 1974-1982 and 414 cultures isolated in 1983-1985 was assayed with standard paper disks. The isolates of 1974-1982 were mostly responsive to ampicillin, carbenicillin, kanamycin, gentamicin, monomycin, neomycin and chloramphenicol. Certain differences in the level of the antibiotic resistance were observed in the Shigella isolates belonging to diverse species. Polyresistant cultures of Shigella amounted to 96.5% and ranged from 88.5 to 99.4% in different years. The number of the cultures with multiple resistance among Shigella sonnei was somewhat higher than that among the Flexneri and Newcastle bacilli. The Shigella isolates of 1983-1985 were mostly responsive to gentamicin, carbenicillin, neomycin, kanamycin and monomycin. 55.5% of the Shigella isolates were responsive to chloramphenicol and only 3.1% to tetracycline. Almost all the causative agents of dysentery isolated within that period were polyresistant. Phenotypic characteristics of multiple resistance in the Shigella cultures were studied.     

Metabolic network analysis of Streptomyces tenebrarius, a Streptomyces species with an active entner-doudoroff pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. (13)C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.     

Antibiotic sensitivity of the causative agents of suppurative surgical infection]

Sensitivity of 1492 strains of the causative agents of the surgical purulent infections, i. e. pathogenic Staphylococcus, Proteus, Ps. aeruginosa and E. coli to benzylpenicillin, streptomycin, levomycetin, tetracycline, erythromycin, oleandomycin, monomycin, kanamycin, novobiocin, ampicillin, oxacillin, ceporin, gentamicin and rifampicin was studied. Gentamicin was most active against all the bacterial species tested. The staphylococci were in addition sensitive in a high percentage of the cases to rifampicin, novobiocin, ceporin, monomycin and kanamycin. The isolates of E. coli were in addition sensitive to ceporin, monomycin and kanamycin. Sensitivity of the strains of Ps. aeruginosa and Proteus was low to all of the antibiotics except gentamycin. Most of the strains of the causative agents of the surgical purulent infections were multiresistant to 4 antibiotics. The number of the staphylococcal strains sensitive to benzylpenicillin, streptomycin and levomycetin increased in 1976 as compared to 1975 on the background of a limited use of these antibiotics in clinics.     

Sensitivity of Sherman's propionic acid bacilli to antibacterial preparations and vitamin B12 synthesis

Sherman propionic acid bacilli were sensitive to benzylpenicillin, ampicillin, ceporin, tetracyclines, oleandomycin, oletetrin, tetraolean, sigmamycin, levomycetin and furadonine. Methicillin, oxacillin, monomycin, kanamycin, polymyxin and furazolidone had an insignificant effect on the above organism. The subbacteriostatic concentrations of methicillin, oxacillin, streptomycin, monomycin, kanamycin, neomycin, tetraolean, sigmamycin, polymyxin M and ristomycin increased the biosynthesis of vitamin B12 by Sherman propionic acid bacilli, while benzylpenicillin, ampicillin, tetracyclines, oleandomycin, oletetrin, levomycetin and furadonine in the subbacteriostatic concentrations inhibited this process.     

Resistance of Shigella to antibiotics

Antibiotic sensitivity of 104 Shigella clinical strains and 104 Escherichia coli strains isolated from patients with acute dysentery not treated with antibiotics in 1986-1987 was studied. It was shown that 100 per cent of the dysentery pathogens and colon bacilli were antibiotic resistant. Strains resistant simultaneously to chloramphenicol, ampicillin, streptomycin, tetracycline, monomycin and kanamycin were the most frequent among the dysentery pathogens. Colon bacilli and dysentery pathogens isolated from the same patient had specific sets of antibiotic resistance markers. Pathogenetic therapy of dysentery and exclusion of antibiotic use for several years did not result in lower numbers of Shigella antibiotic resistant strains.     

Antibiotic sensitivity of plague microbe strains from foreign countries]

The method of serial dilutions on the Hottinger agar was applied to comparative assay of antibiotic sensitivity in 50 strains of the plague microbe isolated abroad and in 5 strains isolated in the plague focus in the Central Caucasus. The antibiotics used in the assay were the following: streptomycin, gentamicin, doxycycline, monomycin, kanamycin, tetracycline, erythromycin, ristomycin, lincomycin and polymyxin M. Irrespective of the origin, all the isolates were resistant to erythromycin, lincomycin and polymyxin M. The levels of the sensitivity to the other antibiotics were different. The data serve as a ground for the statement that there is no tendency to development of antibiotic resistance in the plague microbe in patients treated with high doses of the antibiotics and mainly streptomycin. Along with streptomycin, such antibiotics as gentamicin, tetracycline, doxycycline and kanamycin are useful in the therapy of plague and require further investigation.     

Sensitivity to antibacterial preparations of the pseudotuberculosis bacteria isolated on the territory of the Ukraine]

Sensitivity of 92 strains of the causative agent of pseudotuberculosis isolated in the Ukraine was studied with respect to 26 antibacterial drugs. It was found that the strains of the pseudotuberculous bacteria were sensitive to 17 drugs, i.e. benzylpenicillin, ampicillin, carbenicillin, cephaloridin, chloramphenicol, tetracycline, streptomycin, neomycin, monomycin, kanamycin, gentamicin, polymyxin, colistin, furadontin, nalidixic acid, sulfisoxazol and septrin. No differences in the sensitivity of the strains isolated in various districts of the Ukraine and from various sources were found. By their antibiotic sensitivity the strains isolated in the Ukraine did not differ from the cultures isolated in other districts of the USSR and abroad.     

Formation and regeneration of protoplasts of Streptomyces hygroscopicus

Techniques are described for the production and regeneration of protoplasts of Streptomyces hygroscopicus and related strains. No single technique was successful in producing protoplasts from all strains. Regeneration of protoplasts to the mycelial growth form was greatly improved by modifying the physical regeneration environment. Protoplast formation and regeneration was achieved in six out of the seven strains studied.     

Sensitivity of bifidobacteria to antibacterial drugs

Twenty one strains of bifidobacteria belonging to various species were studied with respect to their sensitivity to antibacterial drugs most widely used in medical practice. The strains were isolated from practically healthy persons and from persons in contact with antibiotics in their production. It was found that sensitivity of the strains was the highest with respect to penicillins and tetracyclines. With respect to kanamycin, monomycin and levomycetin the strains were resistant. Strain differences in resistance to separate antibacterial drugs were observed. Increased streptomycin and tetracycline resistance of the strains isolated from the persons being for a prolonged period in contact with these antibiotics in their production, was stated.     

Metabolic Network Analysis of Streptomyces tenebrarius, a Streptomyces Species with an Active Entner-Doudoroff Pathway

Streptomyces tenebrarius is an industrially important microorganism, producing an antibiotic complex that mainly consists of the aminoglycosides apramycin, tobramycin carbamate, and kanamycin B carbamate. When S. tenebrarius is used for industrial tobramycin production, kanamycin B carbamate is an unwanted by-product. The two compounds differ only by one hydroxyl group, which is present in kanamycin carbamate but is reduced during biosynthesis of tobramycin. ¹³C metabolic flux analysis was used for elucidating connections between the primary carbon metabolism and the composition of the antibiotic complex. Metabolic flux maps were constructed for the cells grown on minimal medium with glucose or with a glucose-glycerol mixture as the carbon source. The addition of glycerol, which is more reduced than glucose, led to a three-times-greater reduction of the kanamycin portion of the antibiotic complex. The labeling indicated an active Entner-Doudoroff (ED) pathway, which was previously considered to be nonfunctional in Streptomyces. The activity of the pentose phosphate (PP) pathway was low (10 to 20% of the glucose uptake rate). The fluxes through Embden-Meyerhof-Parnas (EMP) and ED pathways were almost evenly distributed during the exponential growth on glucose. During the transition from growth phase to production phase, a metabolic shift was observed, characterized by a decreased flux through the ED pathway and increased fluxes through the EMP and PP pathways. Higher specific NADH and NADPH production rates were calculated in the cultivation on glucose-glycerol, which was associated with a lower percentage of nonreduced antibiotic kanamycin B carbamate.     

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.     

Reduction of actinomycin biosynthesis during protoplast regeneration in an inactive variant producer

During regeneration of protoplasts in the inactive variant H-2 of the actinomycin-producing organism Streptomyces sp. 26-115 there were detected 1-4 per cent of the colonies synthesizing the antibiotic. The frequency of such colonies (H-2R) did not increase after exposure of the H-2 protoplasts to the fusing agent PEG-1000. The population grown from one colony after three passages on pea agar was sufficiently homogeneous by the antibiotic production property. Variant H-2R was more stable to the effect of streptomycin than the initial variant H-2.     

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus.

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.     

Sensitivity of clinical Proteus strains to antibiotics and their combinations]

In 1976 isolation of Proteus from wounds of patients with various purulent processes amounted to 14.5 per cent. Serotypes 0-10, 0-3 and H-3 predominated among the isolates. Sensitivity of 35 clinical strains of Proteus to 10 antibiotics, furagin and nevigramone was studied by the method of serial dilutions in liquid media. All the isolates were highly resistant to the antibiotics except gentamicin, furagin and nevigramone, the MIC of which for most of the strains was 3.12, 1.6-3.12 and 6.25-12.5 gamma/ml, respectively. The effect of 14 combinations of chemotherapeutics was also studied. The combinations of gentamicin with carbenicillin, gentamicin with ampicillin and monomycin with ampicillin proved to be most effective against the Proteus strains tested. The following combinations may be of practical value: monomycin + carbenicillin, kanamycin + ampicillin, kanamycin + carbenicillin, ampicillin + furagin, gentamicin + nevigramone. The combinations of carbenicillin with furagin and gentamicin with furagin were also rational.     

Flora of the purulent foci of patients at an orthopedic traumatology clinic and its sensitivity to antibiotics

A total of 4664 bacteriological analyses of the wound and purulent discharge from orthopedo-traumatological patients were performed within a 6-year period, i.e. from 1971 to 1976. Staphylococci were the dominating microbes of the purulent-inflammatory foci. Its part in the monoculture amounted to 64.5--82 per cent. The specific weight of the monocultures of various microbes decreased during the last 3 years, while the number of the microbial associations increased from 11.6 to 25.4 per cent Staph. aureus predominated in the inflammatory processes (65.8 to 86.5 per cent). Still, during the last 3 years the number of Staph. epidermidis increased from 16.8 to 26.2 per cent. The number of the so called "intermediate" or dissociated type of Staphylococcus, i.e. Staph. albus usually amounted to 7.5--8.1 per cent. In 1976 its number was 12.5 per cent. The pathogenic microbes of the coccal group were usually sensitive to erythromycin, monomycin, levomycetin and kanamycin. Among these microbes only staphylococci preserved their sensitivity to penicillin. The causative agents of purulent processes, i.e. Escherichia, Proteus, Pseudomonas were resistant to most of the antibiotics. Sensitivity to monomycin was preserved by 50 per cent only in Proteus. The microbial associations were mainly sensitive to monomycin and kanamycin.     

Sensitivity of Shigella to antibiotics]

Three hundred and twenty two Shigella cultures isolated from dysentery patients within 1986-1989 were tested with the use of standard paper disks for their sensitivity to levomycetin, streptomycin, tetracycline, monomycin, neomycin, kanamycin, erythromycin, gentamicin, carbenicillin, ampicillin, oxacillin, methicillin and doxycycline. The number of the cultures belonging to Shigella sonnei amounted to 85.1 per cent of the total number of the strains studied. 91.9, 89.5, 87.3, 87.3, 80.1 and 80.1 per cent of the cultures were sensitive to gentamicin, kanamycin, carbenicillin, neomycin, levomycetin and ampicillin, respectively. 99.4 per cent of the isolates were resistant to streptomycin and 97.2 per cent were resistant to tetracycline. The sensitivity to erythromycin remained rather high (70.2 per cent). The overwhelming majority of the Shigella sonnei isolates had multiple resistance.     

Heterologous expression of the kanamycin biosynthetic gene cluster (pSKC2) in <Emphasis Type="Italic">Streptomyces venezuelae</Emphasis> YJ003

The pSKC2 cosmid, which has 32 kb and 28 open-reading frames, was isolated from Streptomyces kanamyceticus ATCC12853 as the gene cluster of kanamycin. This gene cluster includes the minimal biosynthetic genes of kanamycin with the resistance and regulatory genes. It was heterologously expressed in Streptomyces venezuelae YJ003, which has the advantage of fast growth, good efficiency of the transformation host, and rapid production of the aminoglycosides antibiotic. The isolated compound was analyzed by electrospray ionization–mass spectrometry, liquid chromatography–mass spectrometry, high-performance liquid chromatography, and tandem mass spectrometry and shows a molecular weight of 485 as kanamycin A.