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Sampling methods for monitoring changes in gonococcal populations.

A total of 160 consecutive isolates of Neisseria gonorrhoeae was collected over a 3-month period. They were tested for their susceptibility to penicillin, erythromycin and spectinomycin and the auxotype and the serotype determined. We have evaluated two sampling methods, the collection of every fifth isolate and the first 20 isolates (10 male and 10 female) each month, to determine whether either is representative of the total population. There was no significant difference between either method of sampling and the total for detecting the predominant auxotypes and serovars or the distributions in antibiotic susceptibility. It is possible to monitor major changes in a gonococcal population, particularly susceptibility to antibiotics, using a sample of the total population.

Bacterial Typing Techniques↗

Interaction of antibiotics with functional sites in 16S ribosomal RNA.

Chemical footprinting shows that several classes of antibiotics (streptomycin, tetracycline, spectinomycin, edeine, hygromycin and the neomycins) protect concise sets of highly conserved nucleotides in 16S ribosomal RNA when bound to ribosomes. These findings have strong implications for the mechanism of action of these antibiotics and for the assignment of functions to specific structural features of 16S rRNA.

Aminoglycosides↗

Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics.

The 30S ribosomal subunit has two primary functions in protein synthesis. It discriminates against aminoacyl transfer RNAs that do not match the codon of messenger RNA, thereby ensuring accuracy in translation of the genetic message in a process called decoding. Also, it works with the 50S subunit to move the tRNAs and associated mRNA by precisely one codon, in a process called translocation. Here we describe the functional implications of the high-resolution 30S crystal structure presented in the accompanying paper, and infer details of the interactions between the 30S subunit and its tRNA and mRNA ligands. We also describe the crystal structure of the 30S subunit complexed with the antibiotics paromomycin, streptomycin and spectinomycin, which interfere with decoding and translocation. This work reveals the structural basis for the action of these antibiotics, and leads to a model for the role of the universally conserved 16S RNA residues A1492 and A1493 in the decoding process.

Anti-Bacterial Agents↗

Removal of antibiotic resistance genes from transgenic tobacco plastids.

Removal of antibiotic resistance genes from genetically modified (GM) crops removes the risk of their transfer to the environment or gut microbes. Integration of foreign genes into plastid DNA enhances containment in crops that inherit their plastids maternally. Efficient plastid transformation requires the aadA marker gene, which confers resistance to the antibiotics spectinomycin and streptomycin. We have exploited plastid DNA recombination and cytoplasmic sorting to remove aadA from transplastomic tobacco plants. A 4.9 kbp insert, composed of aadA flanked by bar and uidA genes, was integrated into plastid DNA and selected to remove wild-type plastid genomes. The bar gene confers tolerance to the herbicide glufosinate despite being GC-rich. Excision of aadA and uidA mediated by two 174 bp direct repeats generated aadA-free T(0) transplastomic plants containing the bar gene. Removal of aadA and bar by three 418 bp direct repeats allowed the isolation of marker-free T(2) plants containing a plastid-located uidA reporter gene.

Aminobutyrates↗

Conjugative, staphylococcal plasmids carrying hitch-hiking transposons similar to Tn554: intra- and interspecies dissemination of erythromycin resistance.

Two staphylococcal plasmids, pWG4 and pWG25, encode production of a diffusible pigment and resistance to erythromycin and spectinomycin. The former was found occurring naturally in a clinical isolate of Staphylococcus aureus and the latter in S. epidermidis. Both plasmids are conjugative, capable of high-frequency, interspecies transfer, only isolated in the open-circular form and identical in molecular weight and pattern of restriction-endonuclease fragments. The only difference between the plasmids is in the expression of resistance, pWG4 encoding inducible and pWG25 constitutive erythromycin resistance. The resistance determinants of both plasmids behave as hitch-hiking transposons in cultural conditions that favour phage-mediated or phage-independent conjugation, always inserting a copy of themselves into the recipient's chromosome, except in S. epidermidis in which the chromosomal insertion site may be absent. The resistance determinants have been cloned and located on a 4 X 7 kbp EcoR1/HindIII restriction fragment which has a restriction map similar to that of the right arm of Tn554 (Murphy and Lofdahl, 1984). The hitch-hiking transposon of plasmid pWG25 has been designated Tn3853.

DNA Restriction Enzymes↗

Antimicrobial resistance profiling and DNA Amplification Fingerprinting (DAF) of thermophilic Campylobacter spp. in human, poultry and porcine samples from the Cork region of Ireland.

Antimicrobial resistance (R) typing and DNA Amplification Fingerprinting (DAF) of a random collection of 84 Irish thermophilic Campylobacter isolates is described. The collection included human, veterinary (porcine) and poultry isolates cultured between 1996 and 1998 in the Cork region of Ireland. Biochemical and molecular methods were used to identify Campylobacter jejuni and Camp. coli. Many of these isolates were simultaneously resistant to several common antimicrobial agents. In particular, resistance to ampicillin, spectinomycin, sulphafurazole and tetracycline was common. A total of 74 DAF profiles was identified among the study collection, showing a high degree of diversity. Dendrogram analysis of the DNA patterns identified three main clusters at the 50% similarity level, which included two clusters of Camp. coli and a third containing a mixture of Camp. jejuni and Camp. coli.

Ampicillin↗

The effect of organic acids on the control of porcine post-weaning diarrhoea.

Post-weaning diarrhoea syndrome (PWDS) of piglets is caused mainly by Enterotoxigenic Escherichia coli (ETEC) strains. Six organic acids were tested for their efficacy in the control of PWDS, using a total of 384 weaned piglets, in eight groups, during a 28-day period. One group (negative control) was offered a diet free of antimicrobials, one group (positive control) was offered the same diet medicated with 44 p.p.m. of lincomycin and 44 p.p.m. spectinomycin (Lincospectin 22 premix, Upjohn), and six groups were offered feed supplemented with either 1.0 per cent propionic acid, 1.6 per cent lactic acid, 1.2 per cent formic acid, 1.2 per cent malic acid, 1.5 per cent citric acid or 1.5 per cent fumaric acid. Groups were compared with regard to the appearance of clinical signs, mortality, weight gain and feed conversion. All groups supplemented with organic acids had reduced incidence and severity of diarrhoea, and performed significantly better than the negative control group (P<0.05). At the end of the trial, ETEC strains were detected in the control group not receiving antibiotics but not in the treated group. Organic acids and especially lactic acid are a useful tool in controlling PWDS.

Animal Feed↗

Treatment of penicillin-resistant Neisseria gonorrhoeae with oral norfloxacin.

Norfloxacin, an orally administered quinoline carboxylic acid that is structurally related to nalidixic acid, has been shown to be highly active in vitro against penicillinase-producing Neisseria gonorrhoeae. Ninety-two men with culture-proved gonococcal urethritis, 46 per cent with penicillinase-producing N. gonorrhoeae, and 27 per cent with non-penicillinase-producing N. gonorrhoeae that was resistant to penicillin were given either 1200 mg of norfloxacin divided into two equal oral doses four hours apart (59 patients) or 2 g of spectinomycin intramuscularly (33 patients). All patients in both treatment groups were cured. No adverse reactions were reported in either group. We conclude that a two-dose, single-day regimen of orally administered norfloxacin is effective therapy for uncomplicated urethritis caused by penicillin-resistant strains of N. gonorrhoeae.

Administration, Oral↗

A community-based outbreak of infection with penicillin-resistant Neisseria gonorrhoeae not producing penicillinase (chromosomally mediated resistance).

From February through November of 1983, 199 cases of penicillin-resistant Neisseria gonorrhoeae infection were identified in a localized epidemic in Durham, North Carolina. The isolates did not produce beta-lactamase but were unusually resistant to penicillin (minimum inhibitory concentration, 2.0 to 4.0 micrograms per milliliter), and 15 of 16 patients treated with 4.8 million units of penicillin G procaine plus 1.0 g of probenecid did not respond to therapy. Recognition of the outbreak was impeded by a lack of routine surveillance for resistance other than that mediated by beta-lactamase. All epidemic isolates had a single serotype, auxotype, and antibiotic-susceptibility profile. The outbreak was halted by changing the treatment for all patients and their contacts to spectinomycin, and by intensive epidemiologic case-finding efforts. The emergence of such resistant strains poses potential major public health problems and indicates a need for reassessment of current surveillance procedures.

Adult↗

Regulation of nuclear DNA replication by thechloroplast in Chlamydomonas.

The experiments described in this paper implicate chloroplast protein synthesis in the regulation of nuclear DNA replication. The inhibition of nuclear DNA replication in the lower eukaryote, Chlamydomonas reinhardi strain 21gr, was examined after growth of cells with a series of antibiotics (streptomycin, neamine, spectinomycin, cleocin, chloramphenicol, and rifampicin) each of which has a known effect upon chloroplast RNA or protein synthesis in this organism. Each antibiotic inhibited nuclear DNA replication at drug concentrations at which there was little or no inhibition of adenine incorporation into chloroplast DNA. That chloroplast DNA was replicating under these conditions rather than merely being repaired, was shown first by the high incorporation rates and second by a (14)N-(15)N density transfer experiment in which chloroplast DNA doubled in the presence of streptomycin, while no incorporation into nuclear DNA was detected. A small DNA peak, Component III, located between nuclear and chloroplast DNA's in CsCl gradients, possibly mitochondrial, was more pronounced in DNA from antibiotic-inhibited cultures than from controls.

Adenine↗

TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.

In Streptomyces coelicolor A3(2) and the related species Streptomyces lividans 66, aerial mycelium formation and antibiotic production are blocked by mutations in bldA, which specifies a tRNA(Leu)-like gene product which would recognize the UUA codon. Here we show that phenotypic expression of three disparate genes (carB, lacZ, and ampC) containing TTA codons depends strongly on bldA. Site-directed mutagenesis of carB, changing its two TTA codons to CTC (leucine) codons, resulted in bldA-independent expression; hence the bldA product is the principal tRNA for the UUA codon. Two other genes (hyg and aad) containing TTA codons show a medium-dependent reduction in phenotypic expression (hygromycin resistance and spectinomycin resistance, respectively) in bldA mutants. For hyg, evidence is presented that the UUA codon is probably being translated by a tRNA with an imperfectly matched anticodon, giving very low levels of gene product but relatively high resistance to hygromycin. It is proposed that TTA codons may be generally absent from genes expressed during vegetative growth and from the structural genes for differentiation and antibiotic production but present in some regulatory and resistance genes associated with the latter processes. The codon may therefore play a role in developmental regulation.

Anti-Bacterial Agents↗

PsbI affects the stability, function, and phosphorylation patterns of photosystem II assemblies in tobacco.

Photosystem II (PSII) core complexes consist of CP47, CP43, D1, D2 proteins and of several low molecular weight integral membrane polypeptides, such as the chloroplast-encoded PsbE, PsbF, and PsbI proteins. To elucidate the function of PsbI in the photosynthetic process as well as in the biogenesis of PSII in higher plants, we generated homoplastomic knock-out plants by replacing most of the tobacco psbI gene with a spectinomycin resistance cartridge. Mutant plants are photoautotrophically viable under green house conditions but sensitive to high light irradiation. Antenna proteins of PSII accumulate to normal amounts, but levels of the PSII core complex are reduced by 50%. Bioenergetic and fluorescence studies uncovered that PsbI is required for the stability but not for the assembly of dimeric PSII and supercomplexes consisting of PSII and the outer antenna (PSII-LHCII). Thermoluminescence emission bands indicate that the presence of PsbI is required for assembly of a fully functional Q(A) binding site. We show that phosphorylation of the reaction center proteins D1 and D2 is light and redox-regulated in the wild type, but phosphorylation is abolished in the mutant, presumably due to structural alterations of PSII when PsbI is deficient. Unlike wild type, phosphorylation of LHCII is strongly increased in the dark due to accumulation of reduced plastoquinone, whereas even upon state II light phosphorylation is decreased in delta psbI. These data attest that phosphorylation of D1/D2, CP43, and LHCII is regulated differently.

Anti-Bacterial Agents↗

Residues of aminoglycoside antibiotics in eggs after medication of laying hens.

1. The transfer of aminoglycoside antibiotics into eggs was determined separately from albumen, yolk or whole egg after oral administration of dihydrostreptomycin (DHS), neomycin and spectinomycin and after an intramuscular injection of DHS. Residues were assayed by an agar plate diffusion method in cylinders with a specific test organism for each antibiotic. 2. Only DHS, administered by the intramuscular route, led to detectable residues in eggs. The total amount of DHS excreted via the eggs represented 1% of the dose administered. 3. Residues in the whole egg were detected for 8 d.

Administration, Oral↗

Heterogeneous expression of fusidic acid resistance in Staphylococcus aureus with plasmid or chromosomally encoded fusidic acid resistance genes.

Fusidic acid resistance expression in a methicillin susceptible Staphylococcus aureus strain (WBG1576), which carries fusidic acid resistance on plasmid pUB101, and a prevalent Western Australian methicillin-fusidic acid resistant strain (WBG8287) were compared. WBG8287 carries fusidic acid resistance on the chromosome and its plasmid content has no effect on the levels of this resistance. WBG1576 and WBG8287 exhibited similar heterogeneous populations in respect to fusidic acid resistance levels in population analyses. A high-level fusidic acid resistant mutant of WBG1576 (BE8) had alterations in Smal chromosomal profiles, but not in plasmid size or resistance expression. Mutations causing increased fusidic acid resistance in WBG1576 are chromosomally located. A high-level fusidic acid resistant mutant of WBG8287 (BE3) had no alterations in Smal chromosomal profiles, or plasmid content and resistances. Comparison of resistance levels to kanamycin and spectinomycin, between high-level resistant colonies of WBG8287 and WBG8287, indicate that mutations in the chromosomal gene fusA, which encodes elongation factor-G, are probably the cause of the increased resistance levels observed in these mutant strains.

Anti-Bacterial Agents↗

The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii.

The sites of synthesis of the major thylakoid membrane polypeptides have been studied in the green alga Chlamydomonas reinhardtii by pulse labeling of cells with [14C]acetate in the presence of inhibitors specific for chloroplast and cytoplasmic protein synthesis. The labeled membrane polypeptides were separated by an improved method of sodium dodecyl sulfate (SDS) gradient gel electrophoresis, and autoradiographs were made of the dried gels. The results demonstrate that of the 33 polypeptides resolved in the gels, at least nine are made on chloroplast ribosomes. Two of these (polypeptides 2 and 6) are associated with the reaction centers of photosystems I and II. Another polypeptide (polypeptide 5) appears from genetic data to be coded by chloroplast DNA. Experiments with a mutant whose chloroplast ribosomes are resistant to spectinomycyn (spr-u-1-6-2) show that polypeptides whose synthesis takes place on chloroplast ribosomes are made in the presence of spectinomycin in the mutant although their synthesis is blocked by this antibiotic in wild type cells.

Acetates↗

Role of emm and mrp genes in the virulence of group A streptococcal isolate 64/14 in a mouse model of skin infection.

The virulence of group A streptococcal isolate 64/14 and paired isogenic mutants in which either the emm or mrp gene had been insertionally inactivated was compared in mice. Loss of expression of the emm gene product resulted in a significant loss of virulence when the isolate was injected into the skin but had no significant difference when injected intraperitoneally. By contrast, inactivation of the mrp gene caused the organism to be more virulent in the skin, while having no significant effect intraperitoneally. An isogenic mutant, in which the mga gene was inactivated and neither the emm gene nor the mrp gene was expressed, demonstrated no significant difference in virulence from the wild type organism. Organisms recovered from the spleen of mice lethally infected with the mga mutant expressed all Mga-regulated IgG-binding gene products despite the presence of the spectinomycin-resistance cassette, which was used to inactivate the mga gene, in its original position.

Animals↗

The value of plasmid studies in the epidemiology of infections due to drug-resistant Salmonella wien.

Since 1969 strains of Salmonella wien that are resistant to multiple antibacterial drugs have caused widespread epidemics of enteritis in Europe and North Africa. Of 113 British strains examined between January 1970 and January 1977, 67 were multi-resistant. These strains and 22 strains from six other countries were examined to determine their plasmid content. Two plasmids were found in the vast majority of strains: an FIme factor, conferring resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, spectinomycin, sulfonamides, and tetracyclines (ACKSSpSuT), and a nonautotransferring plasmid of resistance type ASSu. The FIme plasmids have dual incompatibility: they are incompatible with group FI factors and with the MP10 plasmid of Salmonella typhimurium, which belongs to a separate group. Other plasmids found in S. wien included principally a ColIa factor and an autotransferring plasmid that codes for ampicillin resistance and belongs to compatibility group I2. The similarity in plasmid content of strains isolated in widely separated areas suggests that they have a clonal origin.

Ampicillin↗

Serogroup specificity and antimicrobial susceptibilities of Neisseria gonorrhoeae isolated in Mexico City.

The serogroup pattern of 87 clinical isolates of Neisseria gonorrhoeae was determined by monoclonal coagglutination and the in-vitro activity of seven antimicrobial agents against the same strains was tested by an agar dilution method. The frequency of resistance to spectinomycin, ampicillin, penicillin, erythromycin, chloramphenicol and tetracycline was 14.9%, 33.3%, 34.4%, 30%, 40.2% and 41.3%, respectively. All strains were susceptible to cefotaxime. Out of 87 strains tested, 29.8% produced beta-lactamases and 4.5% were chromosomally resistant to penicillin. In all instances resistance to a drug was associated with serogroup 1-B except for erythromycin. The results presented here correlate with observations made worldwide.

Ampicillin↗