Cysteine residues play a unique role in structural analysis. We examined endogenous cysteine residues in the cholecystokinin receptor to determine participation in disulfide bonds and accessibility to methanethiosulfonate (MTS) reagents. Bonds linking Cys114 to Cys196 and Cys18 to Cys29 were demonstrated, with the first functionally important and the amino-terminal bond having no apparent function. Cys94, in the second transmembrane segment, was also accessible. Mutation of this residue to serine (C94S) was key for establishing a null cysteine-reactive pseudo-wild type receptor that could act as a template for insertion of a reactive cysteine (N102C, A204C, and T341C). Modification of T341C with a negatively charged MTS reagent reduced CCK agonist binding, while this binding was enhanced by a positively charged MTS reagent. This pattern was repeated in mutants having the same residue directly replaced with a charged residue.

To improve the clinical outcome of Staphylococcus aureus septicaemia, early selection of appropriate antibiotic treatment is crucial. Molecular diagnostics represent an attractive approach for rapid identification of S. aureus and determination of its methicillin resistance. In direct comparison with other molecular assays (sa442 and mecA real time PCRs) and standard laboratory procedures, we evaluated the BD GeneOhm TM StaphSR assay for its use in the detection of S. aureus and methicillin-resistant S. aureus (MRSA) staphylococci from spiked blood culture bottles (n=134). In case of detecting S. aureus (n=90; MSSA: n=45, MRSA: n=45), the BD GeneOhm TM StaphSR assay had a sensitivity and a specificity of 100% each [95%-confidence interval (CI): 96.0%-100% and 82.4%-100%, respectively]. For MRSA (n=45), the test was 95.6% (95%-CI: 84.9%-99.5%) sensitive and 95.3% (95%-CI: 86.9%-99.0%) specific. Overall, 5 discrepant results arose with this assay due to the presence of methicillin-susceptible, revertant MRSA strains (3/45) and MRSA strains undetected with the BD GeneOhm TM StaphSR assay (2/45). Compared to other real-time-PCR based molecular approaches and to conventional standard laboratory methods, the BD GeneOhm TM StaphSR turned out to be an appropriate diagnostic tool for a rapid (~1.5 hours), preliminary identification of S. aureus and MRSA from blood cultures.Bacterial contamination is currently the major infectious hazard of platelet transfusion. The detection of bacterial contamination in platelet concentrates (PCs) has been implemented in several blood services as a routine quality control testing, but to date transfusion-transmitted bacterial sepsis has not been completely prevented. In the present study, a novel flow cytometry-based method was developed for the rapid point-of-issue screening of PCs for bacterial contamination. The BactiFlow flow cytometer was used to detect and count bacteria based on esterase activity in viable cells. A protocol for bacterial screening of PCs was established by enzymatic digestion and centrifiltration for the elimination of viable platelets and selective labeling of bacteria with a fluorescent esterase substrate. Results from the BactiFlow (Counts/mL) showed an excellent correlation to plate count results (CFU/mL). The lower detection limit of the assay was determined to 150 CFU/mL, whereas the time-to result was less than 1 hour. We investigated this new application in comparison to incubation (BacT/Alert culture system) and rapid nucleic acid-based or immunoassay detection methods (RT-PCR, Pan Genera Detection Technology). Our study demonstrates that the BactiFlow assay is most suitable for rapid bacterial screening of PCs and fulfils the requirements for a point-of-issuetesting of PCs with acceptable time-to-result, specificity, sensitivity and costs.

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