Comparative evaluation of 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)2H-tetrazolium, monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide(XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the 1092 dilution MIC results revealed overall agreement, within the accuracy limits of the standard test ( 1 1092 dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test. (c) 2007 Elsevier B.V. All rights reserved.

Rapid identification of precursor cDNAs encoding five structural classes of antimicrobial peptides from pickerel frog (Rana palustris) skin secretion by single step “shotgun” cloning.

The skin secretion of the North American pickerel frog (Rana palustris) has long been known to have pronounced noxious/toxic properties and to be highly effective in defence against predators and against other sympatric amphibians. As it consists largely of a complex mixture of peptides, it has been subjected to systematic peptidomic study but there has been little focus on molecular cloning of peptide-encoding cDNAs and by deduction, the biosynthetic precursors that they encode. Here, we demonstrate that the cDNAs encoding the five major structural families of antimicrobial peptides can be elucidated by a single step “shotgun” cloning approach using a cDNA library constructed from the source material of the peptidomic studies—the defensive skin secretion itself. Using a degenerate primer pool designed to a highly conserved nucleic acid sequence 5' to the initiation codon of known antimicrobial peptide precursor transcripts, we amplified cDNA sequences representing five major classes of antimicrobial peptides, such as esculentins, brevinins, ranatuerins, palustrins and temporins. Bioinformatic comparisons of precursor open-reading frames and nucleic acid sequences revealed high degrees of structural similarities between analogous peptides of R. palustris and the Chinese bamboo odorous frog, Rana versabilis. This approach thus constitutes a robust technique that can be used either alone or ideally, in parallel with peptidomic analysis of skin secretion, to rapidly extract primary structural information on amphibian skin secretion peptides and their biosynthetic precursors.

High-frequency oscillations in a solar active region observed with the RAPID DUAL IMAGER

High-cadence, synchronized, multiwavelength optical observations of a solar active region (NOAA 10794) are presented. The data were obtained with the Dunn Solar Telescope at the National Solar Observatory/Sacramento Peak using a newly developed camera system: the rapid dual imager. Wavelet analysis is undertaken to search for intensity related oscillatory signatures, and periodicities ranging from 20 to 370 s are found with significance levels exceeding 95%. Observations in the H-α blue wing show more penumbral oscillatory phenomena when compared to simultaneous G-band observations. The H-α oscillations are interpreted as the signatures of plasma motions with a mean velocity of 20 km s-1. The strong oscillatory power over H-α blue-wing and G-band penumbral bright grains is an indication of the Evershed flow with frequencies higher than previously reported.

Rapid change in energy status in fighting animals: causes and effects of strategic decisions

Energetic costs of fighting, such as high lactate or low glucose, have been shown in a range of species to correlate with the decisions made by each opponent, particularly the decision by one opponent, the 'loser', to end the fight by 'giving up'. Studies based on complete fights of differing duration, however, do not provide information on the changes in the physiological correlates of fighting that may take place during the course of the encounter, or how these changes may influence the capability and decisions of the contestants. We interrupted fights between hermit crabs, Pagurus bernhardus, at specific points, and related energy status to the preceding activities. Costs rose quickly with a rapid accumulation of lactic acid in attackers and declining muscular glycogen in defenders. Changes in physiological status appeared much earlier than the changes in behaviour that they may have caused. Furthermore, some physiological changes might have been an effect, rather than the cause, of fight decisions. (c) 2005 The Association for the Study of Animal Behaviour Published by Elsevier Ltd. All rights reserved.

Quantitative surface-enhanced Raman spectroscopy of dipicolinic acid - towards rapid anthrax endospore detection

Dipicolinic acid (DPA) is an excellent marker compound for bacterial spores, including those of Bacillus anthracis ( anthrax). Surface-enhanced Raman spectroscopy (SERS) potentially has the sensitivity and discrimination needed for trace DPA analysis, but mixing DPA solutions with citrate-reduced silver colloid only yielded measurable SERS spectra at much higher (> 80 ppm) concentrations than would be desirable for anthrax detection. Aggregation of the colloid with halide salts eliminated even these small DPA bands but aggregation with Na2SO4(aq) resulted in a remarkable increase in the DPA signals. With sulfate aggregation even 1 ppm solutions gave detectable signals with 10 s accumulation times, which is in the sensitivity range required. Addition of CNS- as an internal standard allowed quantitative DPA analysis, plotting the intensity of the strong DPA 1010 cm(-1) band (normalised to the ca. 2120 cm(-1) CNS- band) against DPA concentration gave a linear calibration (R-2 = 0.986) over the range 0 - 50 ppm DPA. The inclusion of thiocyanate also allows false negatives due to accidental deactivation of the enhancing medium to be detected.