Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a multicenter study

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five rel

Strongyloides ratti: Mitochondrial enzyme activities of the classical electron transport pathway in the infective (L3) larvae

Submitochondrial particles prepared from S. ratti L3 larvae exhibited NADH-oxidase (NOX), NADH-ferricyanide reductase (NFR), NADH-cytochrome-c-reductase (NCR), succinate-cytochrome-c-reductase (SCR), and cytochrome-aa3-oxidase activities of 2.1 ± 0.3, 8.9 ± 1.3, 0.6 ± 0.1, 1.0 ± 0.2 and 1.2 ± 0.3 nm min−1 mg protein−1 respectively, at 37°C. The NCR and NOX activities were 39.3% and 23.5% of the NFR activity, suggesting the occurrence of a rate-limiting step or bifurcation of the respiratory electron transport (RET) pathway on the oxygen-side of RET-Complex I. The NCR activity was 50% that of cytochrome-aa3-oxidase activity which suggests partitioning of electron flow at the level of RET-Complex III and/or the quinone-function. Antimycin A and rotesone but not 2-thenoyl trifluoreacetone (TTFA) inhibited NCR activity, the EC50 values were 3.6 × 10−6m, 3.7 × 10−7m, respectively. SCR activity was inhibited by antimycin A (EC50 = 3.8 × 10−6m) and TTFA (EC50 = 2.8 × 10−5m) but not by rotenone. The results suggest that presence of classical and alternate RET-pathways in S. ratti L3 larvae

Strongyloides ratti: Fumarate reductase and succinate dehydrogenase activities of infective larvae

Submitochondrial particles prepared from axenised infective (L3) larvae of S. ratti (homogonic-strain) were assayed spectrophotometrically for fumarate reductase (FR) and succinate dehydrogenase (SDH) and their kinetic properties characterised. The S. ratti FR (pH 8.2; 37°C) exhibited a maximum specific activity of 3.45 nmol (min)−1 (mg protein)−1 at a sodium fumarate concentration of 0.3 mm. Interestingly, the FR activity declined at fumarate concentrations greater than 0.3 mm. The mechanism of this unusual inhibitory effect requires further study. The S. ratti SDH (pH 8.2; 37°C) showed a Vmax of 17.4 nmol (min)−1 (mg protein)−1; the Kmsucc was 0.5 mm. Although the SDH:FR ratio cannot predicate vectorial electron flow as would occur in vivo, an in vitro ratio of 5.04:1 was observed for SMPs derived from S. ratti L3 larvae

The effect of electron transport (ET) inhibitors and thiabendazole on the fumarate reductase (FR) and succinate dehydrogenase (SDH) of Strongyloides ratti infective (L3) larvae

The fumarate reductase (FR) and succinate dehydrogenase (SDH) activities of isolated submitochondrial particles (SMPs) prepared from axenised L3 larvae of S. ratti were characterised with respect to their response to a selected range of inhibitors. Rotenone (a specific inhibitor of electron transport Complex I) inhibited the S. ratti FR (EC50 = 3.0 × 10−7m) but not SDH. This strongly suggests that the S. ratti FR is functionally linked with the S. ratti ET-Complex I. 2-Thenoyltrifluoroacetone (TTFA, an inhibitor of ET-Complex II) inhibited FR (EC50 = 2.6 × 10−5m) and SDH (EC50 = 2.8 × 10−5m) with similar effectiveness. Sodium malouate (substrate analogue of succinate) had a greater affinity for SDH (EC50 = 6.8 × 10−4m) than FR (EC50 = 1.9 × 10−2m). Sodium fumarate was ca. 8-fold more effective in inhibiting the S. ratti FR (EC50 = 6.0 × 10−4m) than SDH (EC50 = 4.8 × 10−3m). The S. ratti FR was more sensitive to inhibition by thiabendazole (TBZ; EC50 = 4.6 × 10−4m) than SDH (EC50 > 1.0 × 10−3m), suggesting that one of the sites-of-action of TBZ to be the FR of S. ratti mitochondria. More potent inhibitors of S. ratti FR, if developed, may prove to be effective chemotherapeutic agents in the management of human strongyloidiasis

Genetic organization of mecA and mecA-regulatory genes in epidemic methicillin-resistant Staphylococcus aureus from Australia and England

The mecA gene that encodes methicillin resistance in Staphylococcus aureus may be regulated by the mecR1 and mecl genes, and this region has been referred to as the mec complex. An analysis of these regulatory genes in 35 epidemic methicillin-resistant S. aureus (MRSA) isolated in England and Australia has found that they contain three classes of mec complex. Firstly, the Class A mec complex with complete mecR1 and mecl genes. Secondly, a new variant of Class A, the Class A1 mec complex, with a 166 bp deletion in the membrane-spanning domain of mecR1 and a complete mecl gene. Thirdly, the Class B mec complex, in which the penicillin-binding domain of mecR1 and the whole mecl gene are deleted by the insertion of a partial sequence of IS1272. Seven MRSA isolated in England and Australia over different time periods had the Class A mec complex. However, the isolates did not have closely related pulsed-field gel electrophoresis (PFGE) patterns. The Class A1 mec complex was found in 12 Australian isolates and the English epidemic MRSA, EMRSA-1. All these organisms were isolated in the early 1980s and had closely related PFGE patterns. The Class B mec complex region was found in nine EMRSA and seven Australian MRSA isolated over the period from the 1970s to 2000. These isolates had related PFGE patterns. The mecA region was also compared in the isolates and all but two of the isolates had an Xbal restriction site. These results support the global spread of epidemic clones and confirm the close relationship between the Australian and English MRSA strains

Difference spectroscopic characterisation of cytochrome complement in Acanthocheilonema viteae

No abstract availabl

Difference spectroscopic characterisation of the cytochrome complement in Acanthocheilonema viteae

(Dithionite-reduced) minus (ferricyanide-oxidised) difference spectra of 600 × g and 12 000 × g subcellular pellet fractions of adult male Acanthocheilonema viteae exhibited α-absorption maxima (296 K) attributable to Cyt c555, Cyt b562 and aa3 (600–605 nm). The γ(Soret) maximum of both fractions was evident at 427 nm, with a shoulder at 432–434 nm. 600 × g and 12 000 × g pellet fractions of adult female and mixed-sex adult A. viteae exhibited similar absorption maxima. (Succinate-reduced) — (ferricyanide-oxidised) difference spectra of the 12 000 × g pellet fraction of mixed-sex adult A. viteae showed absorption maxima at 555 and 562 nm, 600 and 630 nm, suggesting the reduction of Cyt c555, Cyt b562, Cyt aa3 (600 nm) and an unidentified species (630 nm peak) Antimycin A (10′-6 M) induced the disappearance of the maxima at the maxima at 555, 600 and 630 nm corresponding to Cyt c555, Cyt aa3 and the unidentified species; the maximum at 562 nm prevailed in the presence of antimycin A. These antimycin A induced changes can be cited as classical evidence for the functional involvement of these a, b and c type cytochromes in respiratory electron transport. (Dithionite reduced + CO) — (dithionite reduced) difference spectra suggest that adult A. viteae may have one or more CO-binding-species, one of which appears to be a low-spin-haemoprotein with a b-type or c-type haem, which has essentially an electron carrier function rather than a ligand binding function

An alternative protocol for the serial passage and maintenance of a homogonic strain of Strongyloides ratti in female Sprague-Dawley rats

A method which does not involve the tedious use of watch glass coprocultures for obtaining filariform infective (L3) larvae of Strongyloides ratti from faecal pellets of infected Sprague-Dawley rats is described. The alternative method utilises Baermannization (18 h) of faecal pellets to yield rhabditiform (L1) larvae of S. ratti and their subsequent culture for 72 h at 19 degrees C in tissue-culture-flasks containing only dechlorinated tap water to yield infective filariform (L3) larvae. The yields and infectivity of the L3 larvae obtained from the two methods were essentially similar

Murine strongyloidiasis: The effects of Cyclosporin A and Thiabendazole administered singly and in combination

A single Cyclosporin A (CsA) dose of 30 mg kg−1 given orally at day 4 post-infection (p.i.) to Sprague-Dawley rats infected with Strongyloides ratti, reduced the faecal larval count by 46.8 ± 1.2%. CsA was equally effective when the same dose rate was administered subcutaneously at day 4 p.i., reducing the faecal larval count by 41.6 ± 8.6%. Thiabendazole (TBZ) given orally at 5 or 10 mg kg−1 (single dose at day 4 p.i.) reduced the faecal larval counts by 57.1 ± 4.1% and 69.0 ± 9.6%, respectively. Orally administrated CsA was less effective than 5 mg TBZ kg−1 (at day 4 p.i.) Co-administration of 5 mg TBZ kg−1 and CsA did not elicit synergy or additive efficacy, indicating that CsA did not antagonise the anti-strongyloides activity of TBZ. The data suggests that for patients with current, historical or serological evidence of strongyloidiasis, CsA may be used where immunosuppressive therapy is required for other concurrent reasons or when TBZ is contraindicated

A British epidemic strain of methicillin-resistant Staphylococcus aureus (UK EMRSA-15) in Western Australia

Letter [No abstract available