High Concentrations Of Streptomycin Research Articles

Salmonella antibiotic-mutant strains reduce fecal shedding and organ invasion in broiler chicks

We investigated the exposure to antibiotics in the production of antibiotic-mutant strains of Salmonella. Ten isolates of poultry origin were assayed for antibiotic susceptibilities. One strain of Salmonella Enteritidis, one of Salmonella Heidelberg, and one of Salmonella Typhimurium were selected to induce antimicrobial resistance. Each strain was exposed to high concentrations of streptomycin, rifampicin, and nalidixic acid, respectively. Parent and antibiotic-mutant strains were assayed for antibiotic susceptibilities using a commercial microdilution test and the disk susceptibility test. The strains were assessed for virulence genes and evaluated for fecal shedding, cecal colonization, organ invasion, and mean Salmonella counts after inoculation in 1-day-old chicks. The study revealed that exposure to high concentrations of streptomycin produced the antibiotic-mutant strain SE/LABOR/USP/08 and the exposure to rifampicin produced the antibiotic-mutant SH/LABOR/USP/08. These strains showed significantly reduced fecal shedding (P≤0.05) and organ invasion, persisting less than the parental strains and showing no clinical signs in inoculated chicks. High concentrations of nalidixic acid produced the antibiotic-mutant strain ST/LABOR/USP/08, which did not show any differences compared with the parent strain. Likewise, SE/LABOR/USP/08 did not show the expression of plasmid-encoded fimbriae (pefA) and plasmid virulence protein (spvC), suggesting that after exposure to streptomycin, the parent isolate lost the original gene expression, reducing fecal shedding and organ invasion in inoculated chicks.

The Impact of Environmental Contamination with Antibiotics on Levels of Resistance in Soil Bacteria

This study examined the effects of tetracycline and streptomycin on microorganisms in three different soil habitats: forest soil, agricultural soil, and compost. These antibiotics are commonly used in both medical and veterinary therapy as well as in the production of plant biomass and until quite recently, the production of animal biomass. Microcosms were used as model systems in which the number of microorganisms in environments containing different amounts of antibiotics was analyzed. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of tetracycline and streptomycin were determined. The MIC and MBC values of tetracycline against the tested strains were 20 to 180 microg/ mL and 30 to 300 microg/mL, respectively, and of streptomycin, 360 to 500 microg/mL and > or =500 microg/mL, respectively. Resistant bacterial strains were identified and their physiological profiles assessed. Streptomycin and tetracycline were found to reduce the number of bacteria in the studied soils by between 50 and 80%. Soil bacteria were found to be more resistant to streptomycin than to tetracycline. The bacterial species showing the highest resistance to tetracycline were Rhizobium radiobacter, Burkholderia cepacia, Brevundimonas vesicularis, and Pasteurella multocida. Most soils with high concentrations of streptomycin (5 mg/kg) contained Rhizobium radiobacter, Burkholderia cepacia, and Sphingomonas multivorum, among others. The strains most resistant to tetracycline were isolated from agricultural soil that is constantly subjected to tetracycline pressure from animal manures and biosolids. Among resistant strains, opportunistic pathogens were identified.

Significant Increase in the Isolation of Glycopeptide-Resistant Enterococci From Patients Hospitalized in the Transplant Surgery Ward in 2004–2005

Enterococci despite their low pathogenicity are the third cause of hospital infections. Enterococci resistant to glycopeptides present special risks. The aim of this work was to determine the frequency of isolates of all enterococci versus enterococci resistant to glycopeptides from patients in the Transplant Surgery Ward. Moreover, vancomycin-resistant enterococci (VRE) were characterized with respect to the type of van and ddl genes as well as vancomycin and teicoplanin MIC values. Among 160 enterococcal strains isolated in 2004, only 2 were resistant to glycopeptides (1.3%). In 2005, among 244 enterococci, 44 strains were resistant (18%). All resistant strains were Enterococcus faecium, as confirmed by detection of the ddl gene specific for E. faecium. Moreover, among all enterococci isolated from these patients, E. faecium dominated (over 50% in 5 subsequent years). All examined VRE possessed VanA type of resistance with high vancomycin and teicoplanin MIC values. All of them possessed the vanA ligase gene. The investigated VRE were characterized by high resistance to most antibiotics: penicillin and amoxicillin, rifampicin, ciprofloxacin, and high concentrations of streptomycin, but susceptible to linezolid and quinupristine/dalfopristine. Strains differed in their susceptibility to tetracycline, nitrofurantoin, and high concentrations of gentamicin.

Development of a Novel Genetic System To Create Markerless Deletion Mutants of Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a species of unique obligate predatory bacteria that utilize gram-negative bacteria as prey. Their life cycle alternates between a motile extracellular phase and a growth phase within the prey cell periplasm. The mechanism of prey cell invasion and the genetic networks and regulation during the life cycle have not been elucidated. The obligate predatory nature of the B. bacteriovorus life cycle suggests the use of this bacterium in potential applications involving pathogen control but adds complexity to the development of practical genetic systems that can be used to determine gene function. This work reports the development of a genetic technique for allelic exchange or gene inactivation by construction of in-frame markerless deletion mutants including the use of a counterselectable marker in B. bacteriovorus. A suicide plasmid carrying the sacB gene for counterselection was used to inactivate the strB gene in B. bacteriovorus HD100 by an in-frame deletion. Despite the inactivation of the strB gene, B. bacteriovorus was found to retain resistance to high concentrations of streptomycin. The stability of a plasmid for use in complementation experiments was also investigated, and it was determined that pMMB206 replicates autonomously in B. bacteriovorus. Development of this practical genetic system now facilitates the study of B. bacteriovorus at the molecular level and will aid in understanding the regulatory networks and gene function in this fascinating predatory bacterium.

The ratio of fungi and bacteria in the biomass of different types of soil determined by selective inhibition

Tundra, chernozem (virgin and arable), sod-podzolic (coniferous forest, meadow, and arable), and grey forest (larch forest) soils were used to separate the contributions of fungi and bacteria to substrate-induced respiration (SIR) with the help of antibiotics. For soils with a high content of organic matter (tundra and chernozem: 12 and 8%, respectively), the procedure of selective inhibition of SIR has been optimized. The optimized procedure includes the application of high concentrations of streptomycin (50-120 mg/g of soil) and cycloheximide (50-80 mg/g of soil) and decreasing the weight of the analyzed soil sample. Soils under study have shown the predominant contribution of fungi (63-82%) to the total SIR. The fungal-bacterial ratio in the soils of natural ecosystems (0-5 cm, without litter) was 4.32, 2.19, 1.5, and 1.5 for tundra soil, virgin chernozem, coniferous (sod-podzolic soil), and larch (grey forest soil) forests, respectively. The lower layers of sod-podzolic (5-10 cm) and grey forest (48-58 cm) soils showed a decrease in the fungal and increase in the bacterial component in the total SIR.

Tissue-culture enhanced transposition of the maize transposable element Dissociation in Brassica oleracea var. 'Italica'

To investigate the potential of heterologous transposons as a gene tagging system in broccoli ( Brassica oleracea var. Italica), we have introduced a Ds-based two-element transposon system. Ds has been cloned into a 35S-SPT excision-marker system, with transposition being driven by an independent 35S-transposase gene construct ( Tpase). In three successive selfed generations of plants there was no evidence of germinal-excision events. To overcome this apparent inability to produce B. oleracea plants with germinal excisions, we performed a novel tissue-culture technique to select for fully green shoots from seed with somatic-excision events. The results showed a very high efficiency of regeneration of fully green plants (up to 65%) and molecular analysis indicated that the plants genetically were like plants that contain a germinal-excision event. Further molecular analysis of these plants showed that 69% exhibited reinsertion of Ds back into the plant genome. Sequencing of donor-site footprints after Ds excision, revealed that there is an indication of more-severe deletions and rearrangements when higher concentrations of streptomycin are used in the tissue-culture selection process. Adapted versions of this regeneration technique have a high potential for providing germinal excision-like events in heterologous plants species which show low transposon activity. Alternatively, there is the potential to increase the proportion of 'germinal' plants in earlier generations of more-active plant species.

An improved procedure for protoplast microelectrofusion and culture of Nicotiana tabacum intraspecific somatic hybrids : plant regeneration and initial proofs on organelle segregation

Somatic hybrids were produced between haploid Nicotiana tabacum L. cv. Petite Havana (wild type) and haploid streptomycin resistant (SR1) mutant by an improved version of microelectrofusion of preselected pairs of protoplasts and the culture of fusion products in a nurse culture. Resistance of diploid plants regenerated from 20 somatic hybrid clones was tested at low concentration of streptomycin in the light as well as at high concentrations of streptomycin in the dark. In two independent hybrid lines, plants resistant in the light but sensitive in the dark were found. The existence of this plant type indicates a segregation of chloroplasts and mitocondria in somatic hybrid clones. It is suggested that microelectrofusion of preselected pairs of protoplasts combined with a reliable nurse culture might be a good technique for controlled somatic hybridization, cell reconstitution and partial gene transfer to different plant species. It might also be used to follow and analyse organelle segregation in somatic hybrid clones. The possibility that mitochondria might be resistant to streptomycin in the SR1 mutant is also discussed.

L-type calcium channels may regulate neurite initiation in cultured chick embryo brain neurons and N1E-115 neuroblastoma cells

The intracellular free Ca 2+ concentration, [Ca 2+] i, plays an important role in regulating neurite growth in cultured neurons. Insofar as [Ca 2+] i is partly a function of Ca 2+ influx through voltage-sensitive calcium channels (VSCC), Ca 2+ entry through VSCC should influence neurite growth. Vertebrate neurons may possess several types of VSCC. The most frequently described VSCC types are usually designated L, T and N. In most preparations, these VSCC types respond differently to certain pharmacological agents, including Cd 2+, Ni 2+, the dihydropyridines nifedipine and BAY K8644, and the aminoglycoside antibiotics. We used these agents to study the role of Ca 2+ influx in regulating neurite initiation and length in cultures of chick embryo brain neurons and N1E-115 mouse neuroblastoma cells. In chick neurons, nifedipine and Cd 2+ (<50 μM), which have been reported to inhibit L-type channels, reduced neurite initiation but not mean neurite length. Ni 2+ (<100 μM), reported to inhibit T-type channels, had no effect on either initiation or length. Low concentrations of most aminoglycosides (<300 μM), reported to inhibit N-type channels, had no effect on neurite initiation, but high concentrations of streptomycin (>300 μM), reported to inhibit both L- and N-type channels, reduced neurite initiation. BAY K8644, which enhances current flow through L-type channels, had no effect except at high concentration (50 μM), which inhibited initiation. N1E-115 neuroblastoma cells have been reported to contain L-type and T-type channels, but thus far no channel similar to the N-type has been described. In cultured N1E-115 cells, nifedipine (5 μM), Cd 2+ (5 μM), and streptomycin (200 μM) reduced neurite initiation, while nickel (50 μM) and neomycin (100 μM) did not affect initiation. None of these agents altered neurite length. In N1E-115 cells, whole-cell voltage clamp recordings showed that nifedipine and Cd 2+ inhibited L-type channels but not T-type channels, while Ni 2+ inhibited T-type channels but not L-type channels. Streptomycin slightly inhibited L-type channels but enhanced current flow through T-type channels. Neomycin slightly inhibited both channel types. These data indicate that neurite initiation in these two cell types may be modulated by Ca 2+ influx through L-type channels, but not T- or N-type channels. Neurite length was not significantly influenced by any of the agents tested, suggesting that Ca 2+ influx through VSCC may not affect neurite elongation.

Demonstration of Heterogeneity in a Natural Population of Rhizobium phaseoli using Variation in Intrinsic Antibiotic Resistance

Two strains of Rhizobium phaseoli, one of which (strain 1234) was resistant to high concentrations of streptomycin, were used to inoculate plots of French beans (Phaseolus vulgaris). Bacteria were isolated from nodules and typed using their intrinsic resistance to low concentrations of seven antibiotics. The inoculum strains were a minority of isolates from inoculated plots. The high concentration streptomycin resistance character was used in the case of strain 1234 to confirm the accuracy of identification. The resident population of R. phaseoli was shown to be heterogeneous; 54 different resistance patterns were recorded. Isolates having the same intrinsic resistance pattern, with few exceptions, were uniform in their reaction with antiserum raised against one of the inoculum strains and in their colony morphology.

Streptomycin Resistance in Tobacco: II. Effects of the Drug on the Ultrastructure of Plastids and Mitochondria in Callus Cultures

Summary The effects of streptomycin on the fine structure of chloroplasts and mitochondria were studied in callus cultures of diploid tobacco, and with streptomycin resistant calliclones. Streptomycin has caused a reduction in the amount of thylakoids, prevented the formation of starch grains and lipid droplets in the plastids, caused the development of internal reticulum, and has changed the external shape of the plastids. Also, it brought about the swelling of cristae, and caused degeneration and sometimes elongation, of the mitochondria. These phenomena appear to be concentration related. At high concentrations of streptomycin, only reticulum and no thylakoids were observed within the plastids, and two types of mitochondria were also observed. One type appeared degenerated, while the other appeared functional, but with some swollen cristae. In some cases the plastids and the mitochondria appeared very similar, and the plastid reticulum resembled the cristae in the mitochondria. The streptomycin resistant calliclones exhibited their resistance only by their ability to form well defined grana, starch grains and lipid dropletes in the plastids of cells which were grown at high drug concentrations.

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.

Chromosome transfer in Proteus mirabilis mediated by hybrid plasmid.

A previously-described fused plasmid, P-lacRIdrd19, was found to mediate chromosomal transfer between cells of Proteus mirabilis strain PM5006; PM5006-(P-lacRIdrd19) was usually the donor and various auxotrophs of PM5006 resistant to nalidixic acid and/or streptomycin were recipients. The donor was usually counterselected with nalidixic acid and/or high concentrations of streptomycin. Recombination experiments with single markers indicated a 40-fold variation in recombination frequencies for different markers. Mapping double-auxotrophic markers by their gradient of transmission confirmed this variation and placed each of two independent isolates of eight markers in a linkage group his-ser-ura-pyrB-trp-cys-ade-ilv. Some donor markers did not register. Despite low recombination frequencies, interrupted mating experiments showed a polarity of early marker transfer. The segregation of unselected markers confirmed the order of some markers and showed that genetic material passed from the presumptive donor to the recipient. Recipients with two auxotrophic markers which could not be cotransduced by phage 5006M were converted to prototrophy by conjugation. The plasmid transferred to recipients at high frequency and all recombinants carried it. Recombinants could act as donors in further matings. Recombinants were fully susceptible to phage 5006M, unlike transductants of PM5006 by this phage. Direct involvement of the plasmid was indicated by drastically diminished recombination frequencies in crosses with recipients carrying P-lac as resident. P-lac had previously been shown to reduce the frequency of transfer of the hybrid plasmid to cells harbouring it. The histidine region was the first to register in recipients and recombined at the highest frequency of 5 times 10-6/donor cell. Some temporary association of plasmid and perhaps only the histidine region of the chromosome is favoured as the mechanism of chromosomal transfer. This could explain why not all donor markers could be mapped. Transduction and transformation were excluded as the cause of results.

Streptomycin resistance in Rhizobium japonicum.

Mutants resistant to varying concentrations of streptomycin were recovered from two streptomycin-sensitive, effective nitrogen-fixing strains of Rhizobium japonicum. To determine if there were an upper limit of resistible antibiotic concentration, 3 mutants which were resistant to 10000 μg/ml were challenged by higher concentrations of streptomycin. Only one grew well at 25000 μg/ml, and none grew at 50000 μg/ml. All mutants maintained a smooth colonial morphology, and none exhibited streptomycin-dependence. Streptomycin-resistant mutants of both strains were examined for properties of infectivity and effectiveness. All mutants tested retained the symbiotic properties of the parental strains. The retention of these parental properties by the streptomycin-resistant mutants of R. japonicum is different from the properties described for phenotypically similar mutants in certain other rhizobial species.

Streptomycin lethality and cyclic AMP

The action of low and moderate concentrations of streptomycin has been studied with Escherichia coli B growing in synthetic media with glucose or glycerol as the carbon source. The inception of lethality depended on the carbon source and the presence of cyclic AMP or ATP in the growth medium. At low concentration of streptomycin (2.5 μg/ml), cells in salts-glycerol media lost their viability following two and a half hour lag period. Glucose repressed the bactericidal effect of streptomycin allowing the cells to grow normally although at a slightly reduced rate. The addition of cyclic AMP to the growth medium reversed glucose repression and rendered the cells sensitive to the action of the drug. At higher concentrations of streptomycin (up to 20 μg/ml) glucose and ATP prolonged and glycerol and cyclic AMP shortened the lag before the cells began to lose their viability. At streptomycin concentration of 5 μg/ml, ATP protected the cells against the bactericidal effect of the drug allowing them to grow normally. This effect of ATP could be abolished by the addition of cyclic AMP. At streptomycin concentrations above 20 μg/ml, the effect of glucose, cyclic AMP and ATP was no longer measurable.

Selection of Ribosomal Mutants by Antibiotic Suppression in Yeast

Wild-type Saccharomyces cerevisiae is highly resistant to streptomycin. A histidine auxotroph was found which could grow without histidine in the presence of high concentrations of streptomycin. Selection for derivatives of this strain which could be suppressed by much lower concentrations of streptomycin yielded streptomycin-sensitive mutants which are cold-sensitive and have altered ribosomal profiles.

Phenotypic suppression in Escherichia coli by chloramphenicol and other reversible inhibitors of the ribosome.

Antibiotics that interfere reversibly with various aspects of ribosomal function (chloramphenicol, tetracycline, erythromycin, and spectinomycin) are shown to antagonize, at barely inhibitory concentrations, the inhibitory effect of low concentrations of streptomycin (SM) on the growth of Escherichia coli. Paradoxically, these compounds can also replace SM in supporting the growth of conditionally SM-dependent mutants. Chloramphenicol produced about as much phenotypic suppression as SM in SM-sensitive strains, but less than that attainable with high concentrations of SM in resistant strains. The antagonism to SM inhibition and the phenotypic suppression appear to be specific for those growth inhibitors that act on the ribosome. Since inhibitors of the 50S subunit of the ribosome (chloramphenicol, erythromycin) are as active as inhibitors of the 30S subunit, it is suggested that phenotypic suppression by borderline concentrations of ribosome inhibitors does not necessarily depend on an alteration of the recognition region of the ribosome. Alternatively, partial inhibition of the ribosomes might change the environment in a way that would influence the frequency of misreading. Phenotypic suppression by a low concentration of SM as well as by chloramphenicol was found to depend on the presence of a trace of the required growth factor.

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.

Mutation to high-level streptomycin-resistance in R+ bacteria.

SUMMARY: Bacteria carrying an R factor conferring resistance to low concentrations of streptomycin frequently give highly resistant variants. Resistance to high concentrations of streptomycin can arise in a sensitive bacterium either from a single mutation or as the result of successive mutations in different genes (Demerec, 1948; Hsu & Herriott, 1961). The resistance genes may be either in the chromosome or in an R factor.

Nuclear-transfer studies on induced increases in streptomycin resistance of Amoeba proteus.

SYNOPSIS. The responses of a strain of Amoeba proteus to conditions which could give rise to induced increases in resistance to streptomycin have been investigated. An indirect selection method, based on survival time in high concentrations of streptomycin, was devised. All attempts to obtain substrains with enhanced sensitivity or enhanced resistance by indirect methods were unsuccessful. It was therefore concluded that increases in resistance observed as a result of other treatments were likely to be the result of adaptation of a majority of individuals, rather than selection of pre‐existing resistant variants. Direct treatment with regularly increased concentrations of streptomycin led to a 10‐fold increase in resistance of large cultures. Nuclear transfers were made by the de Fonbrune technique to produce resistant nucleus/normal cytoplasm, and normal cytoplasm/resistant nucleus combinations. Examination of the descendants of these ‘crosses’ showed that the resistance was nuclear dependent and not influenced by the cytoplasm. The resistance proved transitory, disappearing slowly during continued culture without further contact with streptomycin.These results are discussed with reference to possible mechanisms responsible for the increase in streptomycin resistance and its mode of origin.

Effect of streptomycin on greening and biosynthesis in Euglena gracilis

1. 1. An experimental system is described in which etiolated cells of Euglena gracilis, under conditions which do not support growth, synthesise chlorophyll in the light, rapidly and reproducibly, without a lag period. The synthesis of chlorophylls a and b, and of carotenoids, is inhibited by high concentrations of streptomycin. 2. 2. During greening there is little or no fall in the level of the free amino compounds of the cells. Streptomycin sulphate causes an increase in the level of the free amino compounds of the cells. Inorganic sulphates have a similar, although smaller, effect. 3. 3. At concentrations which inhibit greening, streptomycin also inhibits the incorporation of [1- 14C]acetate into the protein and nucleic acid of the cells, both in the light and in the dark. Incorporation of acetate carbon into polysaccharide, however, is markedly stimulated by streptomycin in the light. 4. 4. The various effects of streptomycin on chlorophyll and carotenoid synthesis, and on acetate incorporation, are all annulled if MnCl 2 is added at the same molar concentration as the drug; MgCl 2 is ineffective. 5. 5. It is suggested that streptomycin has a general effect on biosynthesis and that its inhibition of greening is not due to a specific inhibition of chloroplast formation, but is merely one consequence of the effect on general biosynthesis.