Caulobacter crescentus CB15N (8 (link)) and its derivatives were grown in PYE rich or M2G minimal medium (6 (link)) at 28°C. For cloning purposes, plasmids were propagated in Escherichia coli TOP10 (Invitrogen), which was cultivated in Luria-Bertani medium at 37°C. When appropriate, media were supplemented with antibiotics at the following concentrations (liquid/solid media for C. crescentus; liquid/solid media for E. coli; in μg/ml): spectinomycin (25/50; 50/100), kanamycin (5/25; 30/50), rifampicin (2.5/5; 25/50), gentamicin (0.5/5; 15/20), oxytetracycline (1/1; 12/12), chloramphenicol (2/1; 20/30), apramycin (10/60; 30/30). Plasmid transfer into C. crescentus was achieved by electroporation (6 (link)). Escherichia coli was transformed using a chemical method (9 (link)). The CB15N derivatives MT219 (▵vanR) and MT231 (▵vanA) were generated with the help of plasmids pMT422 and pMT487, respectively, following a previously described gene replacement protocol (10 (link)). Strains MT232, MT236 and MT240 were created by transforming strain CB15N with integration plasmids pMT627, pMT704 or pMT760, respectively, and selecting for homologous recombination of the constructs into the chromosomal vanA or xylX locus.
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Apramycin
Apramycin
Apramycin is an aminoglycoside antibiotic used to treat infections caused by gram-negative bacteria.
It is derived from Streptomyces tenaconensis and has a broad spectrum of activity, including against some multidrug-resistant strains.
Apramycin works by binding to the bacterial ribosome, inhibiting protein synthesis and leading to cell death.
It is commonly used in veterinary medicine, but has limited use in human medicine due to its potential for toxicity.
Reserach on Apramycin is ongoing to explore its clinical applications and optimize its safety and efficacy.
It is derived from Streptomyces tenaconensis and has a broad spectrum of activity, including against some multidrug-resistant strains.
Apramycin works by binding to the bacterial ribosome, inhibiting protein synthesis and leading to cell death.
It is commonly used in veterinary medicine, but has limited use in human medicine due to its potential for toxicity.
Reserach on Apramycin is ongoing to explore its clinical applications and optimize its safety and efficacy.
Most cited protocols related to «Apramycin»
Antibiotics, Antitubercular
apramycin
Caulobacter crescentus
Chloramphenicol
Chromosomes
derivatives
Electroporation
Escherichia coli
Genes
Gentamicin
Homologous Recombination
Kanamycin
Oxytetracycline
Plasmids
Rifampin
Spectinomycin
Strains
The strains and plasmids used in this work are listed in Table 2 . Escherichia coli Top10 was used as the cloning host grown on Luria–Bertani (LB) liquid or solid medium. Liquid ATCC172 was used for the vegetative growth of M. echinospora. The conjugal transfer was performed on MS agar. Solid slanting medium was used for M. echinospora sporulation. The previously described media and culture conditions were used for gentamicin production [8 (link)].
Strains and plasmids used in this study
| Strains or plasmids | Relevant characteristic | Reference or source |
|---|---|---|
| Strains | ||
| E. coli TOP10 | F-mcrAΔ(mrr-hsdRMS-mcrBC), φ80lacZΔM15,ΔlacX74, deoR, recA1, araD139Δ(ara-leu)7697, galU, galK, rpsL(StrR), endA1, nupG | Invitrogen |
| E. coli ET12567/pUZ8002 | Methylation defective, strain used in E. coli-streptomyces intergeneric conjugation | [25 (link)] |
| S. kanamyceticus | Kanamycin producing strain | CGMCC4.1441 |
| M. echinospora | Wild-type strain, gentamicin C1a, C2, C2a, and C1 producer | ATCC 15835 |
| M. echinosporaΔP | M. echinospora with disrupted genP | [7 (link)] |
| M. echinosporaΔKΔP | M. echinospora with disrupted genK and genP | This study |
| M. echinospora JK1 | Heterologous, genome-based expression of kanJ and kanK, with replacement of the native promoter by PermE* in M. echinosporaΔKΔP. | This study |
| M. echinospora JK2 | M. echinosporaΔKΔP + heterologous expression of kanJ and kanK under the promoter PermE*, insertion at genP locus in M. echinospora ΔKΔP | This study |
| M. echinospora JK3 | Heterologous expression of kanJ and kanK under promoter PgenP, insertion at genP locus in M. echinospora ΔKΔP | This study |
| M. echinospora JK4 | Heterologous expression of kanJ and kanK under promoter PhrdB, insertion at genP locus in M. echinospora ΔKΔP | This study |
| Plasmids | ||
| pKC1139 | E. coli-streptomyces shuttle vector, AmR | [26 (link)] |
| pSPU241 | pIJ2925 derivative carrying the Streptomyces constitutive promoter PermE* and To terminator, AmpR | [8 (link)] |
| pEAP1 | pSET152 carrying ermE, the apramycin resistance-conferring gene aac(3)IV was replaced by the ampicillin resistance-conferring gene bla, AmpR, ErmR | [8 (link)] |
| pSPU503 | pKC1139 carrying homologous arms of genK (gacD), used in genK disruption | [12 (link)] |
| pJK1 | pEAP1 carrying PermE*-kanJ-kanK, used in generating M. echinospora JK1 | This study |
| pJK2 | pKC1139 carrying homologous arms and kanJK, used in generating M. echinospora JK2 | This study |
| pJK3 | pKC1139 carrying homologous arms and PermE*-kanJ-kanK, used in generating M. echinospora JK3 | This study |
| pJK4 | pKC1139 carrying homologous arms andPhrdB-kanJ-kanK, used in generating M. echinospora JK4 | This study |
AmpR ampicillin resistance, AmR ampramycin resistance, ErmR erythromycin resistance
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Agar
apramycin
Arm, Upper
Erythromycin
Escherichia coli
Genes
Genome
Gentamicin
gentamicin C1a
Plasmids
Shuttle Vectors
Strains
Streptomyces
apramycin
Chromosomes
Cloning Vectors
Codon, Terminator
Genes
Gene Transfer, Horizontal
Genome
Glycoprotein Hormones, alpha Subunit
Oligonucleotide Primers
Pfu DNA polymerase
Plasmids
Protein C
Reading Frames
Regulatory Sequences, Nucleic Acid
Replication Origin
ribosomal A-protein
Tryptophan Synthase
MICs of aminoglycosides (amikacin, gentamicin, tobramycin, apramycin, neomycin, paromomycin, and streptomycin), ciprofloxacin, imipenem, meropenem, piperacillin-tazobactam and trimethoprim-sulfamethoxazole were determined using broth microdilution following the recommendations of the Clinical Laboratory Standards Institute (CLSI) (CLSI, 2017 ). Concentrations of these agents ranged from 0.5 to 256 μg/ml except for trimethoprim-sulfamethoxazole. Escherichia coli ATCC 25922 was used as the quality control and all tests were performed in triplicate. Breakpoints defined by CLST for amikacin, gentamicin and tobramycin (for amikacin, susceptible [S] ≤16 μg/ml, intermediate [I] 32 μg/ml, resistant [R], ≥64 μg/ml; for gentamicin and tobramycin, S ≤4 μg/ml, I 8 μg/ml, R ≥16 μg/ml), ciprofloxacin, imipenem, meropenem, piperacillin-tazobactam and trimethoprim-sulfamethoxazole (CLSI, 2017 ) was used, while no CLSI- or the European Committee on Antimicrobial Susceptibility Testing (EUCAST)-defined breakpoints for the other four agents are available. Breakpoints defined by US Food and Drug Administration (FDA) or the National Antimicrobial Resistance Monitoring System were used for streptomycin (S, ≤32 μg/ml; R, ≥64 μg/ml) and apramycin (S, ≤8 μg/ml; I, 16 or 32 μg/ml; R, ≥64 μg/ml) (Smith and Kirby, 2016 (link)), respectively. Those defined by Comite de L'Antibiogramme de la Société Française de Microbiologie (http://www.sfm-microbiologie.org/ ) were used for neomycin and paromomycin (S, ≤8 μg/ml; R, >16 μg/ml; for both agents).
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Amikacin
Aminoglycosides
apramycin
Ciprofloxacin
Clinical Laboratory Services
Comite
Escherichia coli
Europeans
Gentamicin
Imipenem
Meropenem
Microbicides
Minimum Inhibitory Concentration
Neomycin
Paromomycin
Piperacillin-Tazobactam Combination Product
Streptomycin
Susceptibility, Disease
Tobramycin
Trimethoprim-Sulfamethoxazole Combination
apramycin
Diaminopimelic Acid
Dietary Supplements
DNA-Directed DNA Polymerase
DNA Restriction Enzymes
Escherichia coli
Gifts
Glucose
Ligase
Oligonucleotide Primers
Plasmids
Strains
Streptomyces
Sucrose
Yeast, Dried
Most recents protocols related to «Apramycin»
In vitro interactions of apramycin with colistin, meropenem, minocycline or sulbactam were tested using time–kill methodology. Twenty-one clinical A. baumannii isolates were chosen, exhibiting apramycin MICs of 4–16 mg/L, which were at or below the apramycin preliminary epidemiological cut-off (ECOFF) value of 16 mg/L proposed by Juhas et al.9 (link) These isolates were selected for a range of colistin (4–32 mg/L), meropenem (16–32 mg/L), minocycline (8–32 mg/L) and sulbactam (8–32 mg/L) MICs across the resistant range. Overall, 3, 11 and 7 isolates with apramycin MICs of 16, 8 and 4 mg/L, respectively, were included in time–kill studies, to determine whether synergy was observed with colistin, meropenem, minocycline or sulbactam to which the isolates were resistant. Eight of the 21 isolates exhibiting meropenem MICs of >32 (64–256 mg/L), were also tested for synergy with apramycin/meropenem combination (Table 1 ).
Apramycin was tested at concentrations of 0.5×, 1× and 2× the MIC value. Colistin was tested at a concentration of 2 mg/L (target steady-state colistin concentration when initiating therapy, achieved by 12 h dosing schedule).20 (link) Meropenem was tested at a concentration of 30 mg/L, simulating the Cmax of a prolonged (3 h) infusion regimen of 1 g.21 (link) Minocycline and sulbactam were tested at 3.5 and 24 mg/L, respectively, which represent the Cmax of a 200 mg oral single-dose tablet for minocycline and 1.0 g for three consecutive IV doses for sulbactam.22 (link),23 (link)Tubes containing CAMHB (Becton Dickinson & Co., Sparks, MD, USA) and each antibiotic alone or in combination were inoculated with 105 cfu/mL of the studied strain and were quantitatively subcultured at 0, 1, 3, 5 and 24 h of incubation for viable colony counts. At the above time intervals, an aliquot of 0.1 mL was removed from each tube, serially diluted (seven 10-fold dilutions) and plated on MacConkey agar (Becton Dickinson) plates. Dilution was expected to minimize any probable antibiotic carry-over effect. The results were expressed as log10 cfu/mL. A tube without antibiotic was also included in each experiment as a growth control. The lower limit of detection was 1.6 log10 cfu/mL.
Synergy was defined as a ≥2 log10 decrease in cfu/mL between the combination and the most active single agent at 24 h, with the number of surviving organisms in the presence of the combination being at least 2 log10 cfu/mL below the number of organisms in the starting inoculum. Antagonism was defined as a ≥2 log10 increase in cfu/mL between the combination and the most active single agent. All other interactions were characterized as indifferent. The bactericidal activity of single antibiotics or combinations was defined as a ≥3 log10 reduction in the cfu/mL of the initial inoculum. All studies were conducted in duplicate and the combined data are presented as mean bacterial density (cfu/mL) for all isolates.
Apramycin was tested at concentrations of 0.5×, 1× and 2× the MIC value. Colistin was tested at a concentration of 2 mg/L (target steady-state colistin concentration when initiating therapy, achieved by 12 h dosing schedule).20 (link) Meropenem was tested at a concentration of 30 mg/L, simulating the Cmax of a prolonged (3 h) infusion regimen of 1 g.21 (link) Minocycline and sulbactam were tested at 3.5 and 24 mg/L, respectively, which represent the Cmax of a 200 mg oral single-dose tablet for minocycline and 1.0 g for three consecutive IV doses for sulbactam.22 (link),23 (link)Tubes containing CAMHB (Becton Dickinson & Co., Sparks, MD, USA) and each antibiotic alone or in combination were inoculated with 105 cfu/mL of the studied strain and were quantitatively subcultured at 0, 1, 3, 5 and 24 h of incubation for viable colony counts. At the above time intervals, an aliquot of 0.1 mL was removed from each tube, serially diluted (seven 10-fold dilutions) and plated on MacConkey agar (Becton Dickinson) plates. Dilution was expected to minimize any probable antibiotic carry-over effect. The results were expressed as log10 cfu/mL. A tube without antibiotic was also included in each experiment as a growth control. The lower limit of detection was 1.6 log10 cfu/mL.
Synergy was defined as a ≥2 log10 decrease in cfu/mL between the combination and the most active single agent at 24 h, with the number of surviving organisms in the presence of the combination being at least 2 log10 cfu/mL below the number of organisms in the starting inoculum. Antagonism was defined as a ≥2 log10 increase in cfu/mL between the combination and the most active single agent. All other interactions were characterized as indifferent. The bactericidal activity of single antibiotics or combinations was defined as a ≥3 log10 reduction in the cfu/mL of the initial inoculum. All studies were conducted in duplicate and the combined data are presented as mean bacterial density (cfu/mL) for all isolates.
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Mutations were constructed using the λ Red recombinase system, as previously described [22 (link)]. The mutagenesis plasmid pACBSR carries the genes encoding Red recombinase under the control of the arabinose promoter. Regions (approximately 0.5 kb) flanking the upstream and downstream sequences of the target gene were amplified using specific primers, with KP10042 as the template (Table S1). The apramycin resistance gene was amplified from pCAP03-acc(3)IV, which contains FRT sites to permit subsequent excision of the apramycin cassette. Excision of the apramycin cassette from the chromosome was performed using the helper plasmid pFLP.
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To construct the gene deletion mutants, the upstream and downstream regions (approximately 2 kbp each) of the target genes were amplified using PCR. The amplified DNA fragments were digested with restriction enzymes (see Table S1 for the enzymes used for each fragment) and cloned into pUC19 digested with the same restriction enzymes. The generated plasmids were sequenced to confirm the absence of PCR-derived errors. The cloned fragments were digested with appropriate restriction enzymes and cloned together into pK19mobsacB (19 (link)), whose kanamycin resistance gene had been replaced with the apramycin resistance gene (14 (link)). The generated plasmids were introduced into the wild-type A. missouriensis strain by conjugation, as described previously (20 (link)). Apramycin-resistant colonies resulting from single-crossover recombination were isolated. For each gene, one of them was cultivated in peptone-yeast extract-magnesium liquid broth at 30°C for 48 h, and the mycelia suspended in 0.75% NaCl solution were spread onto Czapek-Dox broth agar medium (BD, NJ, USA) containing extra sucrose (final concentration 5%). After incubation at 30°C for 4–5 days, sucrose-resistant colonies were inoculated onto yeast extract-beef extract-NZ amine-maltose monohydrate (YBNM) agar with or without apramycin to confirm that they were sensitive to apramycin. Apramycin-sensitive and sucrose-resistant colonies resulting from the second single-crossover recombination were isolated as candidates for gene deletion mutants. The deletion of each target gene was analyzed by PCR (data not shown).
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The strategy for creating deletion mutants is based on the unstable multicopy vector pWHM3 (Vara et al., 1989 (link)), as described previously (Świątek et al., 2012 (link)). Briefly, a knock-out construct was generated containing an apramycin resistance cassette that is flanked by the upstream and downstream region of the targeted gene. The about 1 kb upstream and downstream regions of dnrS (Table 2 ) were amplified from S. peucetius ATCC 27952 genomic DNA using primers MH301/MH302 and MH303/MH304. The DNA fragments were subsequently cloned into pWHM3 using EcoRI/HindIII. The apramycin resistance gene aacC4 flanked by two loxP recognition sites was cloned in-between the flanking regions of dnrS using an engineered XbaI restriction site. The resulting knock-out construct, designated as pGWS1431 (Supplementary Figure S1 ), was verified using Sanger sequencing. Subsequently, the construct was introduced to G001 via protoplast transformation (Kieser et al., 2000 (link)). The desired double-crossover mutant was selected by resistance against apramycin (50 μg mL−1) and sensitivity to thiostrepton (20 μg mL−1). The presence of the loxP recognition sites allowed the efficient removal of the apramycin resistance cassette from the chromosome following the introduction of pUWLCRE that expresses the Cre recombinase (Fedoryshyn et al., 2008 (link)). The successful deletion of dnrS and removal of the apramycin resistance cassette was confirmed by gel electrophoresis of the PCR product of primers MH305/MH306. A distinct band was observed at the expected size of 489 (Supplementary Figure S2 ).
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The standard procedures were performed as described in Gust et al. (2003 (link)). The primers were designed to amplify the apramycin resistance cassette by adding 39 nucleotides of the target genes to the ends (Table S2 ). These products were transformed into E. coli BW25113/pIJ790 with the cosmid St1A9 for homologous recombination. The recombinant cosmid was transformed into E. coli ET12567/pUZ8002 and conjugated into S. coelicolor M145 to disrupt the respective genes. Clones were selected with MS plates supplemented with apramycin (50 µg/ml). Two single and double mutant strains were generated: ∆garR, ∆garS, and ∆garR/∆garS. To verify the correct deletion, the mutants were sequenced and probed by PCR with a set of primers (fwd6162shIn, rv6162shOut, fwd6163shIn, and fwd616shOut) that hybridized within the apramycin resistance cassette and an adjacent chromosomal region (Fig. S1 ).
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Top products related to «Apramycin»
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Apramycin is a broad-spectrum aminoglycoside antibiotic. It is used as a laboratory reagent for research purposes.
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Kanamycin is a broad-spectrum antibiotic derived from the bacterium Streptomyces kanamyceticus. It is commonly used as a selective agent in molecular biology and microbiology laboratories for the growth and selection of bacteria that have been genetically modified to express a gene of interest.
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Gentamicin is a laboratory product manufactured by Merck Group. It is an antibiotic used for the detection and identification of Gram-negative bacteria in microbiological analysis and research.
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The Miniprep kit is a laboratory equipment used for the small-scale purification of plasmid DNA from bacterial cultures. It provides a rapid and efficient method to isolate high-quality plasmid DNA for various downstream applications such as sequencing, cloning, and transfection.
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Tobramycin is a laboratory-grade antibiotic used in research and development. It is a broad-spectrum aminoglycoside antibiotic effective against a variety of gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. Tobramycin is commonly utilized in microbiology and molecular biology studies.
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Ampicillin is a broad-spectrum antibiotic used in laboratory settings. It is a penicillin-based compound effective against a variety of gram-positive and gram-negative bacteria. Ampicillin functions by inhibiting cell wall synthesis, leading to bacterial cell lysis and death.
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Spectinomycin is a laboratory equipment product manufactured by Merck Group. It is an antibiotic that inhibits bacterial protein synthesis by binding to the 30S ribosomal subunit, thereby preventing the initiation of translation. The core function of Spectinomycin is to serve as a research tool for studying bacterial physiology and antimicrobial mechanisms.
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Blood Agar Base II is a general-purpose culture medium used for the isolation and cultivation of a variety of microorganisms, including fastidious bacteria, from clinical and non-clinical specimens. It supports the growth of a wide range of microorganisms and provides a platform for further identification and characterization.
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T4 DNA ligase is an enzyme used in molecular biology and genetics to join the ends of DNA fragments. It catalyzes the formation of a phosphodiester bond between the 3' hydroxyl and 5' phosphate groups of adjacent nucleotides, effectively sealing breaks in double-stranded DNA.
More about "Apramycin"
Apramycin is an aminoglycoside antibiotic that is effective against a broad range of gram-negative bacteria, including some multidrug-resistant strains.
Derived from the actinobacterium Streptomyces tenaconensis, apramycin works by binding to the bacterial ribosome and inhibiting protein synthesis, ultimately leading to cell death.
This antibiotic is commonly used in veterinary medicine but has limited applications in human healthcare due to its potential for toxicity.
Ongoing research aims to explore the clinical utility of apramycin and optimize its safety and efficacy.
Scientists are investigating the use of apramycin in combination with other antimicrobial agents, such as kanamycin, gentamicin, and tobramycin, to enhance its effectiveness against resistant pathogens.
Additionally, researchers are exploring the use of apramycin in conjunction with techniques like miniprep kits and Quick-gDNA MiniPrep to facilitate genetic studies and drug development.
Apramycin's broad-spectrum activity and ability to target multidrug-resistant strains make it a valuable tool in the fight against difficult-to-treat bacterial infections.
As research continues, we may see expanded applications of this antibiotic, particularly in the veterinary field and potentially in human medicine if its safety profile can be further improved.
Experence the power of PubCompare.ai today and take your Apramycin research to the next level.
Derived from the actinobacterium Streptomyces tenaconensis, apramycin works by binding to the bacterial ribosome and inhibiting protein synthesis, ultimately leading to cell death.
This antibiotic is commonly used in veterinary medicine but has limited applications in human healthcare due to its potential for toxicity.
Ongoing research aims to explore the clinical utility of apramycin and optimize its safety and efficacy.
Scientists are investigating the use of apramycin in combination with other antimicrobial agents, such as kanamycin, gentamicin, and tobramycin, to enhance its effectiveness against resistant pathogens.
Additionally, researchers are exploring the use of apramycin in conjunction with techniques like miniprep kits and Quick-gDNA MiniPrep to facilitate genetic studies and drug development.
Apramycin's broad-spectrum activity and ability to target multidrug-resistant strains make it a valuable tool in the fight against difficult-to-treat bacterial infections.
As research continues, we may see expanded applications of this antibiotic, particularly in the veterinary field and potentially in human medicine if its safety profile can be further improved.
Experence the power of PubCompare.ai today and take your Apramycin research to the next level.