Streptomycin resistance in chloramphenicol-producing strains of Streptomyces species 3022a.

Resistance to low (5 mug/ml) concentrations of streptomycin in agar media was not inherited by all of the surviving population. Outgrowth of cultures in liquid media supplemented with the antibiotic depended upon inoculum size. Antibiotic titers in the supplemented cultures decreased during incubation, and an inactive radioactive product was detected when [14C] streptomycin was used. This low-level resistance is, therefore, attributed to enzymic inactivation of the antibiotic. Growth 10 mug/ml or higher concentrations of streptomycin on agar media was due to selection of resistant variants present in the parent strain. A range of such variants existed, decreasing in frequency as their degree of resistance increased. Examination of one that was resistant to moderate concentrations of streptomycin, (25 mug/ml) and a second that was resistant to high (100 mug/ml) concentrations of streptomycin suggested that both possed ribosomes which had lower affinity for the antibiotic than those of the parent strain, and that tolerance to high levels of streptomycin was due to a resistant ribosomal system for protein biosynthesis.     

Spatial patterns in antibiotic resistance among stream bacteria: effects of industrial pollution

The spatial distribution of antibiotic resistance to streptomycin and kanamycin was examined in natural bacterial communities of two streams. The proportion of resistant bacteria was substantially higher (P < 0.05) in the midreaches of an industrially perturbed stream, but no such pattern was apparent in an undisturbed reference stream. The highest relative frequency of resistance was found at the confluence of a tributary draining a nuclear reactor and industrial complex. Antibiotic resistance increased with distance upstream from the confluence and was positively correlated (r(2) = 0. 54, P = 0.023) with mercury concentrations in the sediments. When the data for two years were compared, this pattern was stable for streptomycin resistance (paired t test, P < 0.05) but not for kanamycin resistance (P > 0.05). Our results imply that heavy metal pollution may contribute to increased antibiotic resistance through indirect selection.     

Genetic instability of the feature of streptomycin resistance in Streptomyces erythraeus

The feature of streptomycin resistance in S. erythraeus, a culture producing erythromycin, is genetically unstable. Mutants sensitive to 0.03 mg/ml of streptomycin are formed in the initial streptomycin resistant strains at a rate of 0.04 per cent. It was shown possible to perform step-by-step selection for increasing streptomycin resistance (from 0.2 to 15 mg/ml) in the mutants producing and not producing erythromycin. The increase in the streptomycin resistance did not lead to a higher resistance to other aminoglycosides. Restriction analysis of the total DNA with the use of various endonucleases demonstrated that the increase in the resistance was not associated with amplification of DNA nucleotide sequences.     

Induction of streptomycin resistance in the wild tomato Lycopersicon peruvianum

Summary A protoplast mutagenesis and cell selection system was used for the isolation of streptomycin resistant Lycopersicon peruvianum colonies. Protoplasts were treated with the mutagen N-nitroso-methylurea and could be regenerated into fertile plants, carrying the streptomycin resistant character. Several classes of streptomycin resistance could be distinguished. Reciprocal crosses between streptomycin resistant and sensitive plants showed a non-Mendelian transmission of the resistance trait. Streptomycin resistance is the first selectable and maternally inherited cell organelle marker described in tomato.     

Distribution of Streptomycin Resistant Strains of Erwinia amylovora in Egypt during 1988

A survey of the occurrence of strains of Erwinia amylovora resistant to streptomycin in certain Egyptian pear orchards was earned out during April and May 1988. Twenty-two isolates out of 604 isolates collected from 11 orchards showed resistance to streptomycin. All the streptomycin resistant (Str^r) strains isolated in the present work were resistant to high levels of streptomycin with minimal inhibitory concentrations ranging from 1000 to 3000 μg/ml. The occurrence of Str^r strains in Egypt is still limited and the population of resistant strains was at relatively low level. However, such occurrence of E. amylovora with resistance to streptomycin is a potentially serious situation.     

Spatial Patterns in Antibiotic Resistance among Stream Bacteria: Effects of Industrial Pollution

The spatial distribution of antibiotic resistance to streptomycin and kanamycin was examined in natural bacterial communities of two streams. The proportion of resistant bacteria was substantially higher (P < 0.05) in the midreaches of an industrially perturbed stream, but no such pattern was apparent in an undisturbed reference stream. The highest relative frequency of resistance was found at the confluence of a tributary draining a nuclear reactor and industrial complex. Antibiotic resistance increased with distance upstream from the confluence and was positively correlated (r² = 0.54, P = 0.023) with mercury concentrations in the sediments. When the data for two years were compared, this pattern was stable for streptomycin resistance (paired t test, P < 0.05) but not for kanamycin resistance (P > 0.05). Our results imply that heavy metal pollution may contribute to increased antibiotic resistance through indirect selection.     

Streptomycin and lincomycin resistances are selective plastid markers in cultured Nicotiana cells

Summary Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.     

Genetic antagonism and hypermutability in Mycobacterium smegmatis

Multidrug-resistant strains of Mycobacterium tuberculosis are a serious and continuing human health problem. Such strains may contain as many as four or five different mutations, and M. tuberculosis strains that are resistant to both streptomycin and rifampin contain mutations in the rpsL and rpoB genes, respectively. Coexisting mutations of this kind in Escherichia coli have been shown to interact negatively (S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 72:2084-2087, 1975; S. L. Chakrabarti and L. Gorini, Proc. Natl. Acad. Sci. USA 74:1157-1161, 1977). We investigated this possibility in Mycobacterium smegmatis by analyzing the frequency and nature of spontaneous mutants that are resistant to either streptomycin or rifampin or to both antibiotics. Mutants resistant to streptomycin were isolated from characterized rifampin-resistant mutants of M. smegmatis under selection either for one or for both antibiotics. Similarly, mutants resistant to rifampin were isolated from streptomycin-resistant strains. The second antibiotic resistance mutation occurred at a lower frequency in both cases. Surprisingly, in both cases a very high rate of reversion of the initial antibiotic resistance allele was detected when single antibiotic selection was used; the majority of strains resistant to only one antibiotic were isolated by this process. Determinations of rates of mutation to antibiotic resistance in M. smegmatis showed that the frequencies were enhanced up to 10(4)-fold during stationary phase. If such behavior is also typical of slow-growing pathogenic mycobacteria, these studies suggest that the generation of multiply drug-resistant strains by successive mutations may be a more complex genetic phenomenon than suspected.     

Studies on the cross-resistance of Mycobacterium tuberculosis, strain H37Rv, to aminoglycoside- and peptide-antibiotics

Various phenotypes of the resistance to aminoglycoside- and peptide-antibodies of Mycobacterium tuberculosis strain H37Rv were produced by single- and/or two-step selection of the parent strain. Mutants obtained by single-step selection with antibiotics were classified into ten phenotypes; one of single resistance, two of triple resistance, three of quadruple resistance, and four of sextuple resistance. There were two kinds of sextuple resistance (high resistance to enviomycin, viomycin, capreomycin, kanamycin, lividomycin). One was isolated from the parent strain by single-step selection and could be eliminated by mutation to isoniazid resistance, the other was obtained by two-step selections and was not eliminated by mutation to isoniazid resistance. Interaction between mutation to streptomycin resistance and mutation to quadruple resistance (4R phenotype) was observed. Streptomycin resistance interfered with the formation of the 4R phenotype and produced a different phenotype, KR instead of the 4R phenotype. The existence of mutation of the 4R phenotype did not usually interfere with mutation to streptomycin resistance, but a small portion of the mutants with the 4R phenotype were altered in their phenotype from 4R to KR after addition of the mutation to streptomycin resistance. This effect of the mutation to streptomycin resistance was not observed in mutants which already had a mutation to klR phenotype (mutation to low concentrations of kanamycin only).     

Direct selection of cybrids by streptomycin and valine resistance in tobacco

Summary Direct selection of cybrids by simultaneous selection for donor chloroplasts and for the recipient nuclei is described. Mesophyll protoplasts of two tobacco (Nicotiana tabacum) mutants, SR1 (streptomycin resistant) and Val^r-2 (valine resistant), were fused by polyethylene glycol treatment. Streptomycin resistance in the SR1 mutant is a maternally inherited chloroplast trait while valine resistance is a Mendelian (nuclear) digenic recessive character. The fused protoplast population was cultured and colonies were selected for resistance to valine (1 mM) and streptomycin (343 M). The efficiency of selection has been confirmed in three clones by demonstrating seed transmission of both streptomycin and valine resistances. In one subclone both streptomycin resistant and sensitive plants were obtained indicating that the streptomycin sensitive chloroplasts had not been totally eliminated by growth on the selective medium.     

Improvement of antibiotic titers from Streptomyces bacteria by interactive continuous selection

We have applied a technique of interactive continuous selection (ICS) to the isolation of streptomycin-resistant mutants of the streptomycin-producing organism, Streptomyces griseus. A series of mutants, each with a different colonial morphology and expressing successively greater resistance to streptomycin, was isolated during the course of selection. Takeover of the mutants has been correlated with changes in on-line estimates of streptomycin concentration such that these estimates may be used as a real-time measure of the genetic state of the cell population. When grown in the medium employed for ICS, mutants expressed increased antibiotic production titers; the best mutant produced 10 to 20 times more streptomycin than the parent strain. Absolute improvements in the maximum specific growth rate and intrinsic resistance to streptomycin did not account for the observed growth advantage of all mutants. Rather, each mutant exhibited relative increases in specific growth rate at increasing concentrations of streptomycin. (c) 1996 John Wiley & Sons, Inc.     

Genetic study of plasmid-associated high-frequency mutations to streptomycin resistance in Escherichia coli]

Escherichia coli CTR1(RT1)RHfm1) carrying two H-factors and having unusually high frequency of mutation to high level streptomycin resistance is studied. The high frequency of mutation (about 10(-6) to streptomycin resistance is connected with the presence of R factor RHfm1, controlling the resistance to chloramphenicol and low level streptomacin resistance, but not with RT1, controlling the resistance to tetracycline. Spontaneous or ethidium bromide-induced loss of RHfm1 is accompanied by a decrease of the mutation frequency to 10(-9). RHfm1 is efficiently transmissible to other strains at 28 degrees C. The acquisition of RHfm1 by strains of E. coli K-12 ans S. typhimurium LT2 was followed by a 1000--10000-fold increase of the frequejcy of mutation to streptomycin resistance. Some streptomycin resistant mutants were isolated, and chromosome location of the mutations was demonstrated. The streptomycin resistant mutants were unable to transmit high level of resistance to streptomycin with R factor, but only low level one. The loss of RHfm1 by streptomycin resistant mutants was accompanied by the return to the streptomycin sensitivity of the initial R- strans (E. coli K-12 mutants) or by a decrease of the streptomycin resistance to the level, only 2-fold higher than that of R- wild type (E. coli CTR1 mutant). Thus, the mutantions had practically no effect on streptomycin resistance of R- strains, but could lead to high resistance phenotypes in the presence of RHfm1. The mutant loci in all three studied strains were found to be closely linked to the locus "fus" on the genetic map of E. coli.     

Cytoplast-protoplast fusion for interspecific chloroplast transfer in Nicotiana

Summary Protoplasts of Nicotiana tabacum (SR1), carrying a maternally-inherited streptomycin resistance mutation, were enucleated by centrifugation through a Percoll gradient. The resulting cytoplasts containing resistant plastids, were fused with sensitive Nicotiana plumbaginifolia protoplasts. The SR1 cytoplasts, having no nuclei, were unable to form calli. All resistant clones recovered after fusion-induction were therefore supposed to be derived from interspecific cytoplast-protoplast fusion. N. plumbaginifolia plants regenerated in 17 out of the 75 resistant clones studied. Plants obtained from eight of these clones were resistant to streptomycin and inherited the resistance maternally, as expected when transferring SR1 plastids into the N. plumbaginifolia nuclear background. Plastid transfer in these plants has been confirmed by the EcoRI restriction pattern of the chloroplast DNA.In nine clones N. plumbaginifolia plants were sensitive although obtained from initially resistant clones. This phenomenon is explained by the maintenance of plastid heterogeneity on the selective streptomycin medium, and formation of plants from sensitive sectors on the non-selective regeneration medium.SR1 protoplasts, originally present as contaminants in the cytoplast preparation (2–7%) did not form colonies (or very rarely) after polyethylene glycol treatment. The nuclei from such protoplasts were recovered, however, in the interspecific somatic hybrids (56 clones), and in segregants having the SR1 nucleus but some cytoplasm from N. plumbaginifolia (2 clones). The majority (about 80%) of the recovered resistant clones therefore acquired the streptomycin resistance factor from the rare (2–7%) contaminating SR1 protoplasts. This is explained by the protoplasts being more stable during fusion induction.     

Interactions between viomycin resistance and streptomycin resistance on ribosomes of Mycobacterium smegmatis.

We have investigated the mechanism of the expression of resistance to high levels of viomycin and coresistance to streptomycin in a mutant strain of Mycobacterium smegmatis ATCC 14468 (AC-13) which was obtained by serial transfers of parental cells to media containing increasing concentrations of viomycin. It was shown previously that resistance to viomycin by strain AC-13 was due to an alteration in the 50 S ribosomal subunit (20). However, genetic analysis has shown that mutation in 50 S subunits alone gave only low level resistance to viomycin. When a streptomycin resistant mutation (caused by an alteration in the 30 S subunit) was introduced into the low level viomycin resistant recombinant strains, most of them were highly resistant to viomycin. Some recombinants were resistant to intermediate levels of viomycin, and the remainder were not affected by the introduction of the str^r allele. Studies with in vitro cell-free systems have shown that streptomycin resistant 30 S ribosomal subunits obtained from a high level viomycin resistant recombinant were able to modify the levels of resistance to viomycin expressed by the 50 S ribosomal subunit. These findings provide additional evidence concerning the functional relationship between 30 S and 50 S ribosomal components in ribosomes.     

Transformability of Streptomycin-resistant Group H Streptococci

Several resistant mutants of a transformable group H streptococcus, strain Challis, were isolated from media containing high concentrations of streptomycin. Mutants SR5a and SR5 exhibited high and low transformability, respectively, when exposed to deoxyribonucleic acid (DNA) from a novobiocin-resistant Challis strain. With similar exposure, mutant SR30 exhibited loss of transformability. The mutants further differed from the parent strain in time of appearance of optimal competence, and, in the case of SR5 and SR30, total growth was somewhat less than that of the parent. The rapidity with which transformants appeared upon initial exposure to DNA was approximately the same in the mutants and the parent strain. The decrease or loss of transformability of mutants SR5 and SR30 was found to be due to an alteration in capacity to take up DNA. Mutant SR5a (highly transformable) was further differentiated from mutants SR5 and SR30 in that it was somewhat more sensitive to high concentrations of streptomycin. Transformants obtained by treating strain Challis with the three types of mutant DNA, on the other hand, exhibited similar degrees of resistance to increasing concentrations of streptomycin. The additional decrease in transforming ability of mutant SR5a and the loss of transforming ability of mutant SR5 after a second exposure to streptomycin may indicate a stepwise process in the change from transformability to nontransformability. Although streptomycin resistance may not be directly related to inability to transform, results indicate that streptomycin greatly increases the chances of selecting these mutants and also can be of value in serving as a marker in studies of this nature.     

Mitochondrial DNA variation in maize plants regenerated during tissue culture selection

Summary Plants resistant to Helminthosporium maydis race T were obtained following selection for H. maydis pathotoxin resistance in tissue cultures of susceptible, Texas male-sterile (T) cytoplasm maize. The selected lines transmitted H. maydis resistance to their sexual progeny as an extranuclear trait. Of 167 resistant, regenerated plants, 97 were male fertile and 70 were classified male sterile for reasons that included abnormal plant, tassel, anther or pollen development. No progeny were obtained from these male-sterile, resistant plants. Male fertility and resistance to the Phyllosticta maydis pathotoxin that specifically affects T cytoplasm maize were co-transmitted with H. maydis resistance to progeny of male-fertile, resistant plants. These three traits previously were associated only with the normal (N) male-fertile cytoplasm condition in maize. Three generations of progeny testing provided no indication that the cytoplasmic association of male sterility and toxin susceptibility had been broken by this selection and regeneration procedure. Restriction endonuclease analysis of mitochondrial DNA (mtDNA) revealed that three selected, resistant lines had distinct mtDNA organization that distinguished them from each other, from T and from N cytoplasm maize. Restriction patterns of the selected resistant lines were similar to those from T cytoplasm mtDNA; these patterns had not been observed in any previous analyses of various sources of T cytoplasm. The mtDNA analyses indicated that the male-fertile, toxin-resistant lines did not originate from selection of N mitochondrial genomes coexisting previously with T genomes in the T cytoplasm line used for selection.Scientific Journal Series Article no. 11,185 of the Minnesota Agricultural Experiment Station and no. 2295 of the Florida Agricultural Experiment Station. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee of warrantly of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Evaluation of in vitro selection regimes for a mitochondrial trait (methomyl resistance) in cms-T maize

Several factors affecting the success of selection in plant populations were examined for their relevance to in vitro selection. Three in vitro selection schemes and two growth assessment procedures were evaluated for effectiveness in selecting for a mitochondrial trait in maize: resistance to the insecticidal compound methomyl. Regenerable maize callus was derived from immature embryos of the three-way hybrid P39/IL766A2 x W182BN containing Texas male sterile cytoplasm (cms-T). Either low, gradually increasing, or high selection pressures were used to grow callus over a period of 3–5 months. There was no significant difference in recovery of resistant plants using these 3 methods. Growth of callus on medium containing methomyl was assessed by increase in fresh weight during the final month of selection or by increase in number of callus pieces over the course of selection. These quantitative measures of growth were unreliable indicators for gain in resistance within the callus population. A procedure for recovery of methomyl resistant and male-fertile cms-T plants is suggested.     

Expression of R-plasmid functions during anaerobic growth of an Escherichia coli K-12 host.

The normal habitat of enteric bacteria is largely anaerobic. Expression of the three characteristic properties of R-plasmids, drug resistance, vegetative replication, and fertility, was therefore studied in Escherichia coli K-12 during anaerobiosis. Replication and drug resistance functions were not altered in the 45 R-plasmids studies, whereas the expression of fertility varied considerably among different R-plasmids during anaerobiosis. The R-plasmids could be divided into three groups, one showing a strong, the second a moderate, and the third little or no reduction of fertility by anaerobiosis. Plasmid-determined sensitivity to F-, N-, and I-specific phage, respectively, was well, although not absolutely, correlated with each of three groups mentioned. Anaerobiosis-aerobiosis appears to change the fertility of type F R-plasmids by influencing the degree of repression of their fertility functions such as the formation of sex pili. Although the minimum inhibitory concentrations of ampicillin, chloramphenicol, streptomycin, and tetracycline were unaltered by anaerobiosis, sulfonamide was found to be four- to eightfold less active under this condition in both resistant and sensitive strains. A surprisingly high frequency and uniformity of minimal inhibitory concentrations was observed for R-plasmid-mediated resistance to streptomycin and chloramphenicol.     

The in vitro fitness cost of antimicrobial resistance in Escherichia coli varies with the growth conditions

The objective of this study was to investigate the influence of stressful growth conditions on the fitness cost of antimicrobial resistance in Escherichia coli BJ4 caused by chromosomal mutations and plasmid acquisition. The fitness cost of chromosomal streptomycin resistance increased significantly when the bacteria were grown under all stress conditions tested, while the cost in 1/3 Luria–Bertani was not significantly changed in a streptomycin+rifampicin mutant. The increase in the fitness cost depended in a nonregular manner on the strain/stress combination. The fitness cost of plasmid-encoded resistance on R751 did not differ significantly, and was generally less under stressful growth conditions than in rich media. The fitness cost associated with R751 with the multiple drug resistance cassette from Salmonella Typhimurium DT104 increased significantly only under stressful conditions at low pH and at high-salt concentrations. Strains with an impaired rpoS demonstrated a reduced fitness only during growth in a high-salt concentration. In conclusion, it was demonstrated that bacterial fitness cost in association with antimicrobial resistance generally increases under stressful growth conditions. However, the growth potential of bacteria with antimicrobial resistances did not increase in a straightforward manner in these in vitro experiments and is therefore probably even more difficult to predict in vivo .