Abstract:
Introduction Antibiotic resistance is a serious threat to public health. The empiric use of the wrong antibiotic occurs due to urgency in treatment combined with slow, culture-based diagnostic techniques. Inappropriate antibiotic choice can promote the development of antibiotic resistance. Methods We have developed a spectroscopic device (Optrode) to measure fluorescence from SYTO 9 and propidium iodide (PI) stained cells that can be used to enumerate the bacterial load. We propose a procedure using the Optrode that will take bacteria in a clinical sample, challenge with a panel of antibiotics and measure live/dead ratios to determine the best bactericidal choice. To this end, we have applied, and optimised, the methodology to detect live and dead E. coli in populations challenged with a range of antibiotics. We investigated the parameters required for lytic and non-lytic bactericidal antibiotics, and examined the influence of washing the sample before staining. Results We show that samples of antibiotic challenged culture do not require washing and that washing may cause loss of DNA from solution. Knockdown of E. coli treated with ampicillin and polymyxin B stained with SYTO 9 and PI can be detected by the Optrode in near real-time. For the non-lytic antibiotics, kanamycin and ciprofloxacin, the current analysis of live/dead staining was unable to detect the knockdown as indicated by plate counts. Conclusion The live/dead based Optrode assays can detect knockdown of E. coli challenged with lytic antibiotics in near real-time; however the assays are not very sensitive to the bactericidal action of non-lytic antibiotics.
