Development of a Novel Assay for the Simultaneous Identification of Deinococcus radiodurans and Determination of Susceptibility to a Selected Antibiotic
J. Faro, G. Riddle, S. Faro. The Woman’s Hospital of Texas, Houston, TX
Objective: To examine the ability of a novel diagnostic ELISA to identify Deinococcus radiodurans in a reduced time when compared to culture. D. radiodurans is a relatively new bacteria, first discovered in spoiled food which had first been irradiated. Although slow growing, the bacterium has been shown to be very resistant to radiation, extremes in temperature, and dehydration. As culture of this organism may take up to two weeks, food and beverage handlers, as well as medical device manufactures, would benefit greatly from an assay that allows for more rapid detection of D. radiodurans.
Methods: Microtiter wells were coated with polyclonal IgG rabbit antibody directed against D. radiodurans at a dilution of 1:50 in coating buffer. After 120 minutes, wells were emptied and then blocked with StartingBockTM. After blocking for 30 min, wells were emptied, and then bacterial isolates were added. For the 30-minute test, a series of dilutions of D. radiodurans were prepared in PBS, starting at 107 bacteria/ml, and diluting out ten-fold to 100 bacteria/ml. Samples were either run with D. radiodurans alone, or in the presence of other selected bacteria (Staphylococcus aureus, Enterococcus faecalis, Gordonia, Streptococcus agalactieae, Kocuria, Roseamonas) all at 107 bacteria/ml. In order to increase the limit of detection, D. radiodurans dilutions were prepared in FastidiousBrothTM, and cultured for predetermined lengths of time. After this culture period, samples were added to microtiter wells diluted 1:1 in phosphate buffered saline (PBS), and allowed to stand at room temperature for 30 minutes. Following this incubation step, wells were washed, and then bound bacteria was detected with HRP-conjugated anti-D. radiodurans antibody at 1:50 dilution for 20 minutes at room temperature. Wells were then washed, and signal was generated with TMB solution. Optical density was read at 450 nm. In order to determine antibiotic susceptibility, D. radiodurans dilutions were incubated for 72 hours in increasing concentrations of Cefazolin, starting at 0.08 ?g/ ml, and increasing up to 32 µg/ml. Cefazolin was selected from a panel of antibiotics which were shown to promote inhibition of growth by in-house turbidity assays.
Results: Following a 30-minute incubation in PBS, D. radiodurans was detected at a limit of 106 bacteria/ml. No interference was observed with any of the other bacteria tested. By increasing the incubation time in broth, the limit of detection increased to 105 bacteria/ml at 24 hours, 104 bacteria/ml at 48 hours, and 101 bacteria/ml following 72 hours. When dilutions of D. radiodurans were prepared in the presence of Cefazolin, the limit of detection (LoD) decreased as the antibiotic concentration increased: with 0.08 µg/ml Cefazolin, the LoD was 102 bacteria/ml; with 0.8 µg/ml Cefazolin, the LoD was 104 bacteria/ml, and with 8 µg/ml Cefazolin, the LoD was 106 bacteria/ml; following 72-hour incubation.
Conclusion: This novel assay allows for the simultaneous identification D. radiodurans and determination of antibiotic susceptibility in as little as 72 hours.
----------------------------------------------------------------------------------------------------------------------------------
H/T to mitstevfel at another board.
