A study on resistance loss of multidrug resistant (MDR) Pseudomonas aeruginosa strains after treatment with dilutions of acridine orange

Abstract

Otajevwo F. D. and Okungbowa A.

This study investigated the loss of resistance of multidrug resistant (MDR) Pseudomonas aeruginosa strains after exposure to dilutions of acridine orange. Five pure axenic strains of P. aeruginosa coded PA1 to PA5 obtained from five infected human sources which included middle ear, urethra, trachea, wound and urine were obtained from the Medical Microbiology Department of the University of Benin Teaching Hospital, Nigeria and stocked on sterile Nutrient agar slants. Slant cultures were sub-cultured aseptically on sterile MacConkey and Blood agar plates and incubated aerobically at 37°C for 24 h to confirm for P. aeruginosa. Gram staining and oxidase test were carried out on resulting colonies. Antibiotic sensitivity test was done by agar disc diffusion method on all confirmed strains on sterile Mueller-Hinton agar plates before and after treatment with acridine orange (AO). P. aeruginosa strains that showed ≤50.0% reduction in resistance markers (RM) after treatment with 0.35, 0.55, 0.75 and 0.95 µg/ml dilutions of AO were noted. Minimum inhibitory concentration (MIC) assay was done using gentamicin on PA5 strain with all four dilutions. All five strains showed 100% resistance against augmentin, nalidixic acid, nitrofurantoin, cotrimoxazole, amoxicillin and tetracycline. Sensitivity was recorded for ofloxacin and gentamicin with 14.6±9.5 and 8.4±4.4 mm zones of inhibition, respectively for all the strains except strain PA3 which was resistant to 8 (100.0%) of antibiotics used. Strains PA1, PA2, PA4 and PA5 were each resistant to 6 (75.0%) of the antibiotics tested. There was loss of RM of 52.1±18.6 and 54.7±37.6% to ofloxacin after treatment with 0.35 and 0.55 µg/ml dilutions, respectively by all MDR P. aeruginosa strains. Loss of RM to gentamicin by strains PA1, PA2, PA4 and PA5 after 0.35 µg/ml acridine orange treatment was recorded as 0.0, 61.5, 58.3 and 60.0%, respectively with a mean±standard error (SE) of 45.0±15.0%. With 0.55 µg/ml dilution, 97.6±28.3% loss of RM was recorded while less than 45.0 and 35.0% loss of RM were recorded for 0.75 and 0.95 µg/ml dilutions, respectively. Acridine orange dilutions of 0.35 and 0.55 µg/ml recorded two-fold (5 µg) and four-fold (2.5 µg) reduction in MIC of gentamicin, respectively. The implications of these findings are discussed.