Development of an in situ culture-free screening test for the rapid detection of Staphylococcus aureus within healthcare environments (original) (raw)

2013, Organic & Biomolecular Chemistry

infection, and were part of a cohort of bacteria taken from UK hospitals. E.coli samples were a combination of clinical isolates and wild type strains (E.coli 1-10), and coagulase negative Staphylococci include strains of S.epidermidis, S. warneri, S.hominus and M. luteus, and were obtained from clinical isolates from Sutton hospital. All bacteria were cultured initially on Brain Heart infusion agar, after which they were subcultured twice onto nutrient agar overnight at 37 °C, under aerobic conditions. Each bacterial isolate possesses individual characteristics, and thus displays a variance in the amount of coagulase produced, and therefore there is a degree of variance in results, lending to a greater amount of error in the results depicted. Preparation of the LGX solution A 100 μM and 50 μM solution of compound 3 was prepared by dissolving it in 2.5 % methanol followed by dilution in 1 x PBS. Solutions of Tris Base (0.05 M) and NaCl (0.1 M) in deionised H 2 O were added to the solution until an optimum pH of 8.5 was achieved. The appropriate amount of human prothrombin in 1 x PBS was then added to produce final prothrombin concentrations of 83.6 nM and 41.8 nM, respectively. The resulting solution was termed "LGX". The concentrations of LGX, subsequently discussed, refer to the concentration of the active constituent compound 3. Testing procedure the efficacy of LGX as a selective and sensitive means of detecting S. aureus In order to detect the presence of staphylocoagulase and thus the efficacy of LGX, varying cell concentrations (10 6 , 10 5 , 10 3 , 10 2 CFU/mL (50 μM LGX) and 10 4 , 10 3 , 10 2 , 10 0 CFU/mL (100 μM LGX) were added to a microtitre plate (Nunclon 96 well plates) in a 1:1 ratio with either 50 μM or 100 μM of prepared LGX solution. This provided a final LGX concentration of 25 μM or 50 μM, respectively. The relative fluorescence was then recorded every fifteen minutes over a six hour time period (λ ex. = 488 nm and λ em. = 525 nm). Positive controls used in each experiment were a control strain of MRSA (NCTC 12493), and the negative control was a clinical isolate of E. coli. A 1:1 ratio of both the 100 μM and 50 μΜ of LGX solution with 1 x PBS was used, and termed "LGX alone". Fluorescence intensity was determined at 15 minute intervals over a 6 hour time period and n=3 samples were assayed in each case. We assayed 15 Strains of MRSA at varying cell concentration (10 6 , 10 5 , 10 3 , 10 2 CFU/mL (50 μM LGX) and 10 4 , 10 3 , 10 2 , 10 0 CFU/mL (100 μM) LGX)) with equal volume of both 50 μM and 100 μM LGX, respectively, to give a final LGX concentration of 25 μM or 50 μM, respectively, in addition to a strain of E-coli, which served as a negative control as described above.