Lethal and mutagenic action of N-nitroso-N-methylbiuret on the producers of levorin, amphotericin and mycoheptin]

The lethal and mutagenic effect of N-nitrozo-N-methylbiuret (NMB) on the organisms producing levorin, amphotericin B and mycoheptin was studied. The mutagen effect depended on the dose, culture and physiological state of the spores. NMB had a low mutagenic effect on the levorin-producing organism characterized by high activity and genetic homogenicity with respect to the colony morphology and antibiotic production. As for the organisms producing amphotericin B and mycoheptin characterized by high genetic heterogenicity, significant variation of all the features studied was observed on their exposure to the mutagen. Inspite of diverse reaction of the organisms producing levorin, amphotericin B and mycoheptin to the effect of NMB mutants with increased antibiotic production were obtained from the three cultures. The lethal and mutagenic effect of NMB on the mycoheptin-producing organism depended on the process of the spore DNA replication. The spores during the DNA replication period were least sensitive to the lethal effect of the mutagen and most mutable with the respect to the colony morphology. For selection of highly active and stable strains exposure to NMB of the spores of the mycoheptin-producing organism during replication of DNA proved to be more effective than that of the spores during the lag-phase.     

Use of acridine yperite in the selection of Act. nodosus, a producer of amphotericin B]

The effect of acridine-yperite (AY) on Act. nodosus LIA 0861 at various physiological stages of development was studied. It was shown that both the spores and the germs were sensitive to AY. The level of the mutagen effect depended on the exposure time and the physiological state of the Act. nodosus cells during the treatment. The treatment of the spore suspension with AY had an insignificant effect on the strain morphological variation, while the number of the morphologically changed colonies markedly decreased when 5-hour germs were subjected to the mutagen effect. A decrease in the average antibiotic activity of Act. nodosus was observed, when its spores were subjected to the treatment with AY, which was associated with a decrease in the number of the plus variants. The treatment of 5-hour germs increased the average antibiotic activity of the culture which correlated with an increase in the number of the plus variants.     

Production of active mutants of a new species of rifamycin producer resistant to specific actinophage]

Selection of a phage-stable strain of a new species of the rifamycin-producing organism was carried out. The phage-stable mutants were selected with respect to the virulent phage 2739 isolated from a lysogenic culture of the rifamycin-producing organism. Spontaneous phage-stable mutants formed at a rate of 0.8 per cent. Most of them belonged to the morphological colony type with a decreased activity level. No shifts in variation with respect to the property of the antibiotic production were noted under the action of phage 2739. 62 per cent of the phage-stable variants isolated from the secondary growth colonies after infection with the phage were lysogenic and liberated phage 2739 to the culture fluid. Induction of mutations with MNNG, UV and gamma(Co30) rays increased the frequency of the phage-stable mutanta by 1.5 times. Active phage-resistant mutants stable to the phage because of its adsorption and liberating no phage 2739 into liquid media during its cultivation were selected.     

The use of the protoplast method for the selection of oleandomycin producers

The use of protoplasting with subsequent reversion to the cellular form in improvement of the oleandomycin-producing organism provided a 110% increase in the range of culture variation with respect to the antibiotic production property. A regenerant with a potency of 12 to 20 per cent higher than that of the initial strain which produced 30 per cent lower amounts of dark pigments of melanin nature was isolated. Repeated protoplasting and regeneration of the regenerant provided a very low regeneration frequency i.e. 0.0002%. The potency of all the secondary regenerants was low.     

Improvement of DNA transfer frequency and transposon mutagenesis of Erwinia carotovora subsp. betavasculorum

The production of antibiotics and their role in microbial competition under natural conditions can be readily studied by the use of transposon mutants. Several antibiotic-producing strains of Erwinia carotovora subsp. betavasculorum were unable to accept foreign DNA. A plasmid delivery system was developed, using ethyl methanesulfonate mutagenesis, which entailed isolating E. carotovora subsp. betavasculorum mutants able to accept foreign DNA and transfer it to other strains. This enabled transposon mutagenesis of a wild-type antibiotic-producing strain of E. carotovora subsp. betavasculorum. Twelve antibiotic-negative mutants were isolated, and one of these showed a reduction in antibiotic production in vitro. Many of these mutants also showed a reduction in their ability to macerate potato tissue. The mutants were classified into four genetic groups on the basis of their genetic and phenotypic characteristics, indicating that several genes are involved in antibiotic biosynthesis by E. carotovora subsp. betavasculorum.     

Action of mitomycin C on the variability of Actinomyces hygroscopicus in forming a proteolytic complex of hygrolytin enzymes and the antibiotic, hygromycin B]

The lethal and mutagenic effect of mitomycin C in doses of 10 and 15 micrograms/ml on the spores and 24-hour culture of Act. hygroscopicus, strain O878 producing hygrolytin, a proteolytic enzyme and hygromycin B, an antibiotic was studied. It was found that mitomycin C had a high lethal effect on the organism. The lethal effect of the antibiotic depended on the stage of the culture development, mitomycin C dose and exposure time. The 24-hour culture was most sensitive to the effect of mitomycin in a dose of 50 micrograms/ml. Exposure to mitomycin increased the actinomycete variation with respect to the colony morphology and induction of new morphological mutations. Exposure of strain O878 to mitomycin C significantly increased the culture variation with respect to the quantitative features of production of the hygrolytin proteolytic enzyme complex and hygromycin B. The character of the strain induced variation with respect to the features studied was different which indicated the absence of correlation between them. The use of mitomycin C proved to be promising in selection of Act. hygroscopicus with a purpose of increasing the culture proteolytic and antibiotic activity.     

Pleiotropic effect of the mutation of streptomycin resistance in Micromonospora purpurea var. violacea]

Variation of different features of populations of streptomycin-sensitive and streptomycin-resistant forms of M. purpurea var. violacea, an organism producing gentamicin was studied. The population of the initial streptomycin-sensitive culture was characterized by high homogeneity with respect to the cultural, morphological and some physiological properties. The variation of the features, such as the colony size, pigment formation, auxotrophic mutations, antibiotic production significantly increased in populations grown on media with streptomycin. Mutants differing from the initial strain by a complex of cultural, morphological and physiological features and in particular the antibiotic production were isolated from populations of the streptomycin-resistant variants.     

Development of high penicillin producing strains for solid state fermentation

Penicillin production with an industrial strain and 4 strains of P. chrysogenum, in solid state fermentation (SSF) and liquid submerged fermentation (LSF), was determined. Their ability to produce the antibiotic in SSF in relation to their capacity to do so in LSF was evaluated. this was done by calculating the ratio PS/PL (production in SSF/production in LSF), which was called relative production. Clones were isolated from each strain and evaluated in a similar way. The strains presented different relative productions (from 1.4 to 2.5). Within the clones, a much wider range of relative productions was observed (0.6 to 16.7). On the other hand, the highest-producing strains in LSF were also the highest producers in SSF. This indicates that the production potential of a strain is an important factor in its production level in SSF. Moreover, the highest penicillin producing ciones (9,500 to 10,500 microg of penicillin/g were generated from high-yielding strains (P2 and ASP-78). However, the higher-producing strains (in LSF) showed lower relative performance, suggesting that higher producing strains tend to express less efficiently their potential in SSF. In this study, several overproducing clones, particularly suited for SSF, were obtained by the procedures followed. Production increases of 500 to 600 %, in this culture system, were achieved.     

Effect of ultraviolet-induced mutants of Trichoderma harzianum with altered antibiotic production on selected pathogens in vitro

Mutants of Trichoderma harzianum with altered antibiotic production were isolated using ultraviolet light mutagenesis. These included strains whose activity in a Fusarium oxysporum spore germination assay was greater than twice that of the parental strain and one that had no detectable antifungal activity. Characterisation of extracellular metabolites of these strains using thin-layer chromatography and gas-liquid chromatography showed that the strains with high activity produced only elevated levels of a 6-n-pentyl pyrone, the antibiotic produced by the parental strain, but two new antifungal compounds. One of these has been identified as an isonitrile antibiotic. The nature of the interactions of the mutants with Fusarium oxysporum, Rhizoctonia solani, and Pythium ultimum was examined in an in vitro dual-plating assay using two media. High antibiotic production by two T. harzianum strains, BC10 and BC63, did increase inhibition of hyphal growth of R. solani and P. ultimum, but there was no correlation between increased antibiotic production and colonisation ability. In some cases the increased antibiotic levels appeared to impede colonisation of F. oxysporum and R. solani by the mutants. Slow growth rate also affected colonising ability. The types of interactions showed great variability depending on the nature of the T. harzianum isolate and on the test fungus.     

The action of its own antibiotic on the producer of imbricin growing on an agarized medium]

The action of imbricin on its own producer Streptomyces imbricatus grown on an agarized medium was studied. Comparatively low concentrations of the antibiotic were shown to have a high lethal action on the streptomycete. The morphological and cultural features of S. imbricatus did not change under the action of imbricin while the variation with respect to the antibiotic production property markedly increased. After the strain exposure to 200 micrograms/ml of imbricin, a stable variant with the antibiotic potency 20 per cent higher than that of the initial organism was isolated.     

Variability of the levorin producer, Actinomyces levoris Krass]

Natural variation of the levorin-producing organism Act. levoris, strain 28 was studied with respect to the colony morphology and production of levorin and levoristatin. The population of strain 28 consisted of 3 morphological colony types, the main type amounting to 99.7 percent. The strain variation with respect to production of levorin and levoristatin ranged from 20 to 180 and from 0 to 300 percent respectively as compared to the control. Mutant M-28 differing from the initial strain by the colony morphology, moderate phage titer and preferable production of levoristatin was isolated as a result of repeated passages of strain 28 onto agarized Chapek media with starch without maintaining selection. Variants differing from the population of strains 28 and M-28 by the ratio of levorin and levoristatin in the culture fluid were selected. No correlation in production of the above antibiotics by strain 28 was noted. Preparations obtained with strain M-28 differed from those obtained with strain 28 in a significant content of levoristatin.     

Streptomyces species producing the streptovaricin complex

Morphological, cultural and physiological-biochemical properties ofStreptomyces sp. strain 1000 and its antibiotic production were investigated. Antibiotics 1011 (identical with the streptovaricin complex) and 1012 (with antibacterial action) were isolated from the cultural broth of this strain. The overproducing natural variant 1011 was isolated from the population of a strain producing antibiotic 1011 at a concentration of 1000 mg/L (activity of the parent strain represents 41 mg/L only). Comparative taxonomical characteristic ofStreptomyces sp. strain 1000 with strains fromS. spectabilis showed that the strain 1000 differed in some properties and antibiotic production being considered as a new variant ofS. spectabilis. The strain shows an expressed antibiotic activity against G+ as well as G− bacterial and yeasts.     

Comparison of Vibrio harveyi strains isolated from shrimp farms and from culture collection in terms of toxicity and antibiotic resistance

Vibrio harveyi strains isolated from shrimp farms (wild strains) were compared with those from culture collections in terms of minimum inhibitory concentration (MIC) and toxicity. Wild strains had higher MIC values for four antibiotics (kanamycin, carbenicillin, oxytetracycline and ampicillin) and also showed higher toxicity compared with culture collection strains. Vibrio harveyi with the lowest antibacterial resistance was chosen to test if a gradual increase in antibiotic concentration and frequent subculture would enhance its antibiotic resistance. Results showed that V. harveyi was able to develop resistance to oxytetracycline. The MIC value was 250 times higher compared with the MIC before subculturing. Moreover, the V. harveyi strain developed slightly higher toxicity. Therefore, it is possible that there is a relationship between antibiotic resistance and toxicity in V. harveyi.     

Study of the effect of protoplast formation on the antibiotic activity of erythromycin producers

The influence of protoplasting and regeneration on the strains of the erythromycin-producing organism differing by their origin was studied with respect to changes in the antibiotic production property. 223 regenerates of the erythromycin-producing culture were tested in several generations and it was shown that there was a marked change in the range of the variation by that property. 40 to 70 per cent of the variants in the IInd generation increased their levels of erythromycin biosynthesis by 20 to 60 per cent as compared to the intact cultures. However, in the subcultures the antibiotic production level decreased and by the IVth generation only 3 to 6 per cent of the variants preserved its increase by 10 to 20 per cent over the control level because of the culture high instability.     

Strain Identification in Rhizobium Using Intrinsic Antibiotic Resistance

The variation in intrinsic resistance to low levels of eight antibiotics was used as an identifying characteristic for 26 Rhizobium leguminosarum strains. The pattern of antibiotic resistance of each strain was a stable property by which rhizobia isolated from root nodules of inoculated Pisum sativum could be recognized. The antibiotic tests for strain identification with R. leguminosarum were applied to R. phaseoli . It was necessary to include reference cultures in tests with this species, as the tests most suitable for the R. leguminosarum strains showed some variability with R. phaseoli .     

Effect of growth conditions on production of rhamnose-containing cell wall and capsular polysaccharides by strains of Lactobacillus casei subsp. rhamnosus.

Strains of Lactobacillus casei subsp. rhamnosus possessing two cell wall polysaccharides, a hexosamine-containing H-polysaccharide and a rhamnose-containing R-polysaccharide, were examined for the effect of growth conditions on the production of these two components. In strain NCTC 6375, R- and H-polysaccharides accounted for an estimated 44 and 20%, respectively, of the cell wall for organisms grown in batch culture with glucose as the carbohydrate source. Growth on fructose-containing media reduced the amount of R-polysaccharide by approximately 50% without affecting the amount of H-polysaccharide. Subculture of fructose-grown organisms in glucose restored the original proportions of the two polysaccharides. Galactose- and sucrose-grown cells behaved similarly to glucose-grown cells with respect to polysaccharide production, whereas growth in rhamnose or ribose showed values close to those for fructose-grown cells. Continuous culture of strain NCTC 6375 for more than 100 generations showed a gradual and irreversible reduction of the R-polysaccharide to less than 5% of the cell wall and an increase of the H-polysaccharide to 40% of the cell wall. Other type culture strains of L. casei subsp. rhamnosus, NCIB 7473 and ATCC 7469, behaved similarly in batch and continuous culture. In contrast, strains of L. casei subsp. rhamnosus isolated at the Institute of Dental Research showed phenotypic stability with respect to the relative proportions of R- and H-polysaccharides in both batch and continuous culture. Changes in polysaccharide composition of type culture strains were also mirrored in changes in the immunogenicity of the two components and resistance to the rate of enzymic lysis of whole organisms. For L. casei subsp. rhamnosus strain NCTC 10302 the R-polysaccharide is present entirely as capsular material. The amount of R-polysaccharide produced was also markedly dependent on the carbohydrate component of the medium in batch culture and both dilution rate and nature of the limiting carbohydrate in continuous culture, varying over a 10-fold range, whereas the cell wall H-polysaccharide remained constant.     

A comparison of aflatoxin B1-induced cytotoxicity,mutagenicity and prophage induction in Salmonella typhimurium mutagen tester strains TA1535, TA1538, TA98 and TA100

Treatment of Ames mutagen tester strains with aflatoxin B1 (AFB1) and S9 mix results not only in the production of a poten mutagen, but induces a pathway that leads to the induction of prophages present in all Ames tester strains.Characterization of the prophage induction and mutagenic response following AFB1 treatment showed that plasmid pKM101 dramatically enhances mutagenesis, but suppressed prophage induction. Spontaneous release of phage by TA98 and TA100 was also lower than in TA1535 and TA1538.In addition to mutagenesis and prophage induction, survival of all 4 tester strains was quantitated after AFB1 treatment. The data show that the frameshift tester strains (TA1538 and TA98) are more sensitive to the bactericidal action of AFB1 than the base-pair tester strains (TA1535 and TA100), survival being significantly affected above 100 ng. One of several hypotheses examined was the difference in the number and types of prophages present in base-pair tester strains that are not detectable in the frame-shift tester strains.These data suggest that prophage induction can detect DNA damage that is non-mutagenic; and that it is important to characterize the lysogenic nature of the Ames strains since it may influence the observed histidine revertant rate and the survival of the tester strain.     

Isolation and characterization of a stable l-arginine producer from continuous culture broth of Corynebacterium acetoacidophilum

In order to produce l-arginine efficiently, continuous culture was attempted using an l-arginine producing strain of Corynebacterium acetoacidophilum, MC-13. l-Arginine production by strain MC-13 decreased after shifting from fed-batch culture to continuous culture because various strains with reduced or no l-arginine productivity, including l-arginine auxotrophs, appeared in addition to the original l-arginine producer. Strain SC-190, isolated from the continuous culture broth of strain MC-13, produced l-arginine on cultivation in a stable fashion for more than 250 h. It is suggested that strain SC-190 acquired a lower susceptibility to the inhibition of growth and l-arginine production by l-arginine than the parental strain MC-13 due to this stabilization.     

Development of the system for protoplast generation in Streptomyces aureofaciens strains--chlortetracycline producers

Conditions for preparation and regeneration of protoplasts in a commercial strain of the culture producing chlortetracycline and its derivatives were determined. The protoplasting level depended on the conditions of the mycelium cultivation and composition of a regeneration medium. Under the optimal conditions it amounted up to 10(6) protoplasts/ml. A mutant able to form regenerating protoplasts at a rate of 10(9) protoplasts/ml was isolated. An autoinhibition effect in regenerating protoplasts was observed. As a result ofprotoplast generationing, the morphological variation increased and the protoplast antibiotic activity changed within wide ranges. Variants with higher productivity in comparison to that of the initial strain were isolated. Stability of the inherited property of antibiotic production in the variants is being studied.     

Antibiotic resistance in Streptococcus pneumoniae and Haemophilus influenzae. Report of a study group on bacterial resistance.

Twenty laboratories in England and Scotland took part in 1977 in a survey of antibiotic resistance in Streptococcus pneumoniae and Haemophilus influenzae. In Str pneumoniae 59 (6.8%) of the 866 strains studied were resistant to tetracycline and three to chloramphenicol, and one strain showed a decreased susceptibility to penicillin. The prevalence of resistance to tetracycline was lower than that found in a similar study performed in 1975. Nine hundred and fifty-two strains of H influenzae were examined: 15 (1.6%) were resistant to ampicillin (all were beta-lactamase producers) and 26 (2.7%) to tetracycline. Only two strains were resistant to chloramphenicol and two to trimethoprim. Sixty-three H influenzae strains were capsulated. Thirty-four of these were of Pittman type b, and antibiotic resistance, particularly to ampicillin, was more common in these than in other serotypes or non-typable strains. Some variation was seen in the resistance rate of both H influenzae and Str pneumoniae to tetracycline in strains from different centres, but too few were isolated to assess whether this represented a true geographical difference.