Objective: To develop a fast and widely applicable, culture-based antibiotic sensitivity testing (AST) assay based around the growth and in situ imaging of micro-organisms on a porous ceramic support. Methods: Sterile Anopore strips (an inert nanoporous ceramic derived from aluminium oxide, see figure) were placed upon appropriate agar plates (generally Mueller-Hinton or Sabouraud media) containing defined concentrations of test antibiotics. The strips were inoculated with the bacteria or fungi to be tested and were incubated in a CO2 incubator at 37Â°C (for 40 minutes to a period of several hours). Subsequent staining of cells on the Anopore surface was with fluorescent dyes (Fun-1 for fungi, Syto 9 for bacteria: Invitrogen) through the pores from beneath. Stained microcolonies were imaged directly using an Olympus BX41 epifluorescence microscope. Image capture used an 8-bit Kappa CCD camera and TIFF files were converted to a binary image and then quantified using ImageJ software (http://rsb.info.nih.gov/ij/). Comparisons were made between the Vitek 2 and E -Test methods and this approach for 24 clinical isolates of the Enterobacteriaceae. Fungal pathogens, including Candida albicans, were also tested. Results: Growth of Enterobacter aerogenes could be detected on anopore within 25 minutes and the earliest effects of the trimethoprim were noticed after 40 minutes (Figure: Scanning electron micrograph of a dividing Enterobacter aerogenes cell cultured on Anopore). AST testing against trimethoprim was performed with 2-3 hours culture and gave MIC values that were comparable with the Vitek 2 and E-Test methods. By the Anopore method, 5/5 trimethoprim-resistant and 19/19 trimethoprim-sensitive strains were assigned correctly. Additionally, AST testing of Candida was demonstrated with detection of sensitivity with 90 minutes culture and MIC values could be derived with 3 hours culture. Conclusions: Microcolony imaging appears to be a rapid and effective approach to AST testing. Performing these tests on a porous ceramic facilitates staining and imaging in situ and has allowed rapid assessment of whether an antibiotic was inhibitory to growth.
Full conference title:
16th European Congress of Clinical Microbiology and Infectious Diseases
- ECCMID 16th (2006)