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Sensitivity and Resistance to Spectinomycin in Escherichia coli.

Inhibition of growth and division of Escherichia coli by spectinomycin is reversible, and the kinetics of its interference with deoxyribonucleic and ribonucleic acid synthesis may be interpreted as secondary effects of inhibition of protein synthesis on the ribosome. Spontaneous mutations to spectinomycin resistance occur in E. coli K-12 at a rate of about 2 x 10(-10). Resistance is transducible with a discrete lag in phenotypic expression, and the kinetics of its development is about the same as that for streptomycin resistance. All spectinomycin-resistant mutants tested contain resistant ribosomes, and all map in a locus (spc) counterclockwise to and 70% cotransducible with the classical str locus. Differences in the residual drug sensitivity of various spectinomycin-resistant mutants, and of their ribosomes, indicate the existence of more than one phenotypic class of resistance.

Journal Article↗

Spectinomycin therapy for gonorrhea in prepubertal children.

Twenty-six episodes of gonococcal urethritis or vulvovaginitis in 24 prepubertal patients were each treated with a single intramuscular (IM) dose of spectinomycin hydrochloride. Twenty-five episodes were treated successfully, and there were no side effects from this therapy. All tested isolates of Neisseria gonorrhoeae were susceptible in vitro to spectinomycin, and all serum levels one hour after therapy were in at least two-fold excess of the observed minimum inhibitory concentrations. Two patients had symptomatic and two had asymptomatic postgonococcal anogenital infections with Chlamydia trachomatis. A single dose of IM spectinomycin is a safe and efficacious regimen for uncomplicated gonococcal infection in prepubertal children who are penicillin-allergic or who have infections unresponsive or resistant to other antimicrobials, but it is not effective against incubating syphilis or chalmydial infection.

Adolescent↗

Aminoglycoside-3''-adenyltransferase confers resistance to spectinomycin and streptomycin in Nicotiana tabacum.

The bacterial gene aadA encodes the enzyme aminoglycoside-3"-adenyltransferase that confers resistance to spectinomycin and streptomycin in Escherichia coli. Chimeric genes have been constructed for expression in plants, and were introduced into Nicotiana tabacum by Agrobacterium binary transformation vectors. Spectinomycin or streptomycin in selective concentrations prevent greening of N. tabacum calli. Transgenic clones, however, formed green calli on selective media containing spectinomycin, streptomycin, or both drugs. Resistance was inherited as a dominant Mendelian trait in the seed progeny. Resistance conferred by the chimeric aadA gene can be used as a color marker similar to the resistance conferred by the streptomycin phosphotransferase gene to streptomycin.

Base Sequence↗

Interaction of the cytoplasmic membrane and ribosomes in Escherichia coli; altered ribosomal proteins in sucrose-dependent spectinomycin-resistant mutants.

Alterations in the ribosomes of sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were studied. Subunit exchange experiments showed that 30S subunits were responsible for the resistance of ribosomes to spectinomycin in all Sucd-Spcr mutants tested. Proteins of 30S ribosomes were analyzed by carboxymethyl cellulose column chromatography based on their elution positions. Mutants YM22 and YM93 had an altered 30S ribosomal protein component, S5, and mutant YM50 had an altered protein, S4. Although a shift of elution position was not detected for all the 30S ribosomal proteins from mutant YM101, the amount of protein S3 was appreciably lowered in the isolated 30S subunits. A partial reconstitution experiment with protein S3 prepared from both the wild-type strain and YM101 revealed that the mutant had altered protein S3 which is responsible for the spectinomycin resistance. These alterations in 30S subunits are discussed in relation to the interaction between ribosomes and the cytoplasmic membrane.

Cell Membrane↗

Amino acid replacement in the protein S5 from a spectinomycin resistant mutant of Bacillus subtilis.

Ribosomal protein S5 was isolated from wild type Bacillus subtilis ATCC 6633 and from a spectinomycin resistant mutant (BSPC 111) derived from spectinomycin sensitive to resistance is accomtrypsin and all the tryptic peptides were isolated by column- and paper-chromatography. By comparative amino acid analyses of the peptides, it was demonstrated that the S5 from the mutant differs from the wild type S5 by a replacement of one amino acid, namely lysine by isoleucine in the peptide T9. The results are compared with E. coli spectinomycin resistant mutants.

Amino Acid Sequence↗

Chloroplast targeting of spectinomycin adenyltransferase provides a cell-autonomous marker for monitoring transposon excision in tomato and tobacco.

Antibiotic resistance genes can act as either cell autonomous or non-cell autonomous genetic markers with which to monitor the excision of plant transposons. To convert spectinomycin resistance from a non-cell autonomous resistance to cell autonomous resistance, a transit peptide for chloroplast localization from a petunia ribulose bisphosphate carboxylase (rbcS) gene was fused in-frame to the aadA gene, which confers spectinomycin and streptomycin resistance. Constructs were generated in which the expression of this chimeric gene was prevented by the presence, in the 5' untranslated leader, of the maize transposons Activator (Ac) or Dissociation (Ds). When progeny of tobacco or tomato plants transformed with these constructs were germinated on spectinomycin-containing medium, germinally revertant and somatically variegated individuals could be distinguished.

Base Sequence↗

Nucleotide sequence of a spectinomycin adenyltransferase AAD(9) determinant from Staphylococcus aureus and its relationship to AAD(3") (9).

The nucleotide sequence of the spc determinant of the Staphylococcus aureus transposon Tn554 has been determined. This gene encodes a spectinomycin adenyltransferase, AAD(9), that mediates resistance to spectinomycin but not to streptomycin. The sequence predicts a 260 amino acid protein of molecular weight 28,943. A spectinomycin-sensitive mutant (spc-1) contains a G----A transition resulting in substitution of threonine (ACA) for alanine (GCA) at residue 165. The predicted amino acid sequence is 36% homologous to that of a widely distributed, gram-negative streptomycin/spectinomycin adenyltransferase, AAD(3") (9), specified by the aadA determinant (Holingshead and Vapnek 1985).

Amino Acid Sequence↗

High-performance liquid chromatographic method for the determination of spectinomycin in turkey plasma.

A selective high-performance liquid chromatographic (HPLC) method with ultraviolet-visible (UV-VIS) detection was developed to measure therapeutic concentrations of spectinomycin in turkey plasma. Treatment of plasma samples with 3% trifluoroacetic acid in acetonitrile facilitated spectinomycin extraction and protein precipitation. After centrifugation, the stable derivatization reagent, 2,4-dinitrophenyl-hydrazine, was added to an aliquot of the supernatant, and the mixture was incubated for 30 min at 70 degrees C. Excess reagent was quenched with acetone and additional heating. The resulting derivative, a proposed spectinomycin-hydrazone, was separated from other compounds by reversed-phase HPLC during a short gradient run. The absorbance of the effluent was monitored spectrophotometrically with the UV-VIS detector set at 205 nm. The detector response was linear through the range of interest, 2-100 micrograms/ml.

Animals↗

Ototoxicity in vitro: effects of neomycin, gentamicin, dihydrostreptomycin, amikacin, spectinomycin, neamine, spermine and poly-L-lysine.

The effects that the aminoglycoside-aminocyclitol antibiotics amikacin, dihydrostreptomycin, gentamicin, neomycin, and spectinomycin, the neomycin fragment neamine, and the polybasic compounds spermine and poly-L-lysine, have on outer hair cells in cochlear cultures prepared from the early post-natal mouse have been assessed using both scanning and transmission electron microscopy (SEM and TEM). The antibiotics were used at concentrations ranging from 0.25-1.0 mM, spermine from 10 microM to 3.0 mM, and poly-L-lysine from 0.05-2 microM. Qualitative assessment of apical surface damage allows the antibiotics to be ranked in the following order: neomycin > gentamicin > dihydrostreptomycin > amikacin > neamine > spectinomycin. At a concentration of 1 mM spectinomycin is essentially non-toxic and the effects of neamine are marginal. Poly-L-lysine and spermine also cause surface damage, with poly-L-lysine being substantially more toxic than any of the antibiotics, and spermine ranking, on the basis of SEM observations, between dihydrostreptomycin and amikacin. TEM indicates that although all toxic compounds cause damage to the apical surface of the hair cell, only neomycin, poly-L-lysine and spermine induce the formation of whorls of tightly packed membrane resembling myelin within the apical surface lesions to any great extent. Apical-surface changes induced by dihydrostreptomycin and amikacin are simply large distensions of the cell filled with cytoplasmic organelles of normal appearance. Although the effects of the aminoglycoside antibiotics are largely limited to the apical surface of the cell, poly-L-lysine induces complete necrosis of the cell, and spermine causes a dramatic increase in cytoplasmic electron density and condensation of the nuclear chromatin.

Amikacin↗

Confirmation of spectinomycin in milk using ion-pair solid-phase extraction and liquid chromatography-electrospray ion trap mass spectrometry.

A confirmation procedure is described for residues of spectinomycin in bovine milk. Spectinomycin is extracted from raw milk using ion-pair reversed-phase solid-phase extraction. The extracts are ion-pair chromatographed on a polymeric reversed-phase column and analyzed on a quadrupole ion trap mass spectrometer equipped with an electrospray interface. MS-MS data are acquired in the scan mode of product ions deriving from m/z 333, the protonated molecular ion. The estimated limit of confirmation is between 0.05 and 0.1 microg/ml. The procedure was validated with control milk, fortified milk (0.1-5.0 microg/ml), and milk from cows dosed with spectinomycin.

Animals↗

HPLC determination of residues of spectinomycin in various tissue types from husbandry animals.

An HPLC method was developed for the determination of bacteriostatic aminocyclitol spectinomycin (SP) in animal tissue products. These products included chicken eggs and edible fat, kidney, liver, muscle tissues from calf, poultry, pig and sheep. Residues of SP were extracted from homogenized tissue and egg-derived material with 25 mM citrate of pH 4.0, trichloroacetic acid and dichloromethane. The extract was purified and concentrated over a carboxylic acid-bonded solid-phase extraction (SPE) column. The SPE-eluate was analysed by cation-exchange HPLC involving a two-column switching system, post-column derivatization and fluorescence detection. Spectinomycin could be successfully determined at levels of 0.05 mg kg-1 and higher. Recoveries from spiked tissue material and from spiked egg material were in excess of 74% and did not show a concentration or tissue-type dependence. Precision of the elution position and signal response was better than 2%. Matrix effects and interference from lincomycin were less than 7 and 2%, respectively, on the signal response. Spectinomycin was shown to be stable at -20 degrees C in combined egg yolk and white over a test period of 12 weeks and in calf and sheep muscle tissue over a test period of 10 days. SP was, however, not stable at this temperature over a period of 12 months in chicken muscle tissue. Incurred SP residues were successfully determined in kidney and muscle tissue at the injection site of pigs administered with two doses of 15 mg kg-1 body weight SP with an intermittent withdrawal period of 15 days. Kidney showed higher concentrations and more persistent residues of SP than muscle tissue at the injection site.

Animals↗

Effects of tetracycline and spectinomycin on the tertiary structure of ribosomal RNA in the Escherichia coli 30 S ribosomal subunit.

Structural analysis of the 16 S rRNA in the 30 S subunit and 70 S ribosome in the presence of ribosome-specific antibiotics was performed to determine whether they produced rRNA structural changes that might provide further insight to their action. An UV cross-linking procedure that determines the pattern and frequency of intramolecular 16 S RNA cross-links was used to detect differences reflecting structural changes. Tetracycline and spectinomycin have specific effects detected by this assay. The presence of tetracycline inhibits the cross-link C967xC1400 completely, increases the frequency of cross-link C1402x1501 twofold, and decreases the cross-link G894xU244 by one-half without affecting other cross-links. Spectinomycin reduces the frequency of the cross-link C934xU1345 by 60% without affecting cross-linking at other sites. The structural changes occur at concentrations at which the antibiotics exert their inhibitory effects. For spectinomycin, the apparent binding site and the affected cross-linking site are distant in the secondary structure but are close in tertiary structure in several recent models, indicating a localized effect. For tetracycline, the apparent binding sites are significantly separated in both the secondary and the three-dimensional structures, suggesting a more regional effect.

Anti-Bacterial Agents↗

A eubacterial gene conferring spectinomycin resistance on Chlamydomonas reinhardtii: integration into the nuclear genome and gene expression.

We have constructed a dominant selectable marker for nuclear transformation of C. reinhardtii, composed of the coding sequence of the eubacterial aadA gene (conferring spectinomycin resistance) fused to the 5' and 3' untranslated regions of the endogenous RbcS2 gene. Spectinomycin-resistant transformants isolated by direct selection (1) contain the chimeric gene(s) stably integrated into the nuclear genome, (2) show cosegregation of the resistance phenotype with the introduced DNA, and (3) synthesize the expected mRNA and protein. Small linearized plasmids appeared to be inserted into the nuclear genome preferentially through their ends, with relatively few large deletions and/or rearrangements. Multiple copy transformants often integrated concatemers of transforming DNA. Our detailed analysis of the complex integration patterns of plasmid DNA in C. reinhardtii nuclear transformants should be useful for improving the technique of insertional mutagenesis. We also found that the spectinomycin-resistance phenotype was unstable in about half of the transformants. When maintained under nonselective conditions, neither the aadA mRNA nor the AadA protein were detected in these subclones. Moreover, since the integrated transforming DNA was not altered or lost expression of the RbcS2::aadA::RbcS2 gene(s) appears to be repressed. Measurements of transcriptional activity, mRNA accumulation, and mRNA stability suggest that expression of this chimeric gene(s) may also be affected by rapid RNA degradation, presumably due to defects in mRNA processing and, or nuclear export. Thus, both gene silencing and transcript instability, rather than biased codon usage, may explain the difficulties encountered in the expression of foreign genes in the nuclear genome of Chlamydomonas.

Animals↗

A comparative study of spectinomycin and erythromycin in the treatment of chancroid.

In a randomized, controlled study, 55 patients with genital ulcerations, culture-positive for Haemophilus ducreyi, were treated with a single intramuscular injection of 2 g spectinomycin. A further 61 patients were treated with a five-day oral course of erythromycin base (500 mg, three times daily). Treatment failure occurred in seven patients who received spectinomycin (13%), whilst no treatment failures were recorded among patients who received erythromycin. This difference in efficacy was statistically significant. In contrast, spectinomycin proved significantly more effective than erythromycin in the treatment of the lymphadenopathy associated with primary chancroid lesions.

Administration, Oral↗

Marking cell layers with spectinomycin provides a new tool for monitoring cell fate during leaf development.

Spectinomycin, an inhibitor of plastid protein synthesis, can be used to mark specific cell layers in the shoot meristem of Brassica napus. Pale yellow-green (YG) plants resulting from spectinomycin-treatment can be propagated indefinitely in vitro. Microscopic examination showed that YG-plants result from inactivation of plastids in the L2 and L3 layers and are composed of a pale green epidermis covering a white mesophyll layer. Epidermal cells of YG and normal green plants are similar and contain 10-20 small pale green plastids. YG plants are equivalent to periclinal chimeras with the important distinction that there is no genotypic difference between the white and green cell layers. Periclinal divisions of epidermal cells take place at all stages of leaf development to produce invaginations of green mesophyll located in sectors of widely varying sizes. A periclinal division rate of 1 in 3000-4000 anticlinal divisions for the adaxial epidermis, was 2-3-fold higher than that estimated for the abaxial epidermis. Analysis of white and green mesophyll showed that chloroplasts are essential for palisade cell differentiation and this requirement is cell-autonomous. Stable marking of cell lineages with spectinomycin is simple, rapid and reveals the requirement for functional plastids in cellular differentiation.

Brassica↗

A comparison of penicillin G plus a beta-lactamase inhibitor (sulbactam) with spectinomycin for treatment of urethritis caused by penicillinase-producing Neisseria gonorrhoeae.

When combined with penicillin, sulbactam, a beta-lactamase inhibitor with weak intrinsic antibacterial activity, produces a marked synergistic effect in vitro against penicillinase-producing Neisseria gonorrhoeae. We compared a regimen of aqueous procaine penicillin G, sulbactam, and probenecid with spectinomycin for the treatment of uncomplicated gonococcal urethritis. Of 101 patients receiving the penicillin-sulbactam regimen, 97 (97%) were cured of their infection, as were 87 (95%) of 92 patients who received spectinomycin. Fifty per cent of patients were infected with penicillinase-producing N. gonorrhoeae; 43 (94%) of 46 treated with the penicillin-sulbactam regimen were cured as compared with 47 (92%) of 51 treated with spectinomycin. Neither regimen was associated with serious adverse effects. The results show that aqueous procaine penicillin G given with sulbactam and probenecid is an effective alternative for single-session therapy of urethritis caused by penicillinase-producing N. gonorrhoeae.

Adult↗

A comparison of single-dose spectinomycin with five days of trimethoprim-sulfamethoxazole for the treatment of chancroid.

In a controlled trial 32 patients with ulcers culture-positive for Haemophilus ducreyi were treated with a single 2-g dose of spectinomycin, and 20 patients were treated with a five-day course of trimethoprim-sulfamethoxazole (TMP-SMZ; 160/800 mg) twice daily. Both regimens rapidly eradicated H. ducreyi from ulcers, with subsequent healing of ulcers and buboes. The cure rates 14 days after initiation of therapy were 94% for spectinomycin and 95% for TMP-SMZ. Patients with H. ducreyi-negative ulcers who were treated with the same regimens also healed and did not show a significantly different efficacy for either regimen. Thus we found that the one-day regimen of spectinomycin was as effective for the treatment of chancroid as was TMP-SMZ given for five days. Further studies on a larger number of patients are needed to confirm our findings.

Adult↗