Abstract

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. Key words: Resistance loss, Pseudomonas aeruginosa, treatment, dilutions, acridine orange.

Highlights

  • Pseudomonas aeruginosa is a highly invasive and toxigenic aerobic Gram negative bacterium

  • Sensitivity was recorded for oflaxacin and gentamicin with mean±standard error (SE) zones of inhibition of 14.6±9.54 and 8.4±4.40 mm, respectively for all P. aeruginosa strains with exception of PA3 which did not respond to the tested drugs at all

  • The antibiotics susceptibility profile of all five strains of P. aeruginosa before acridine orange treatment in this study showed that the five strains were sensitive to ofloxacin and gentamicin with mean±SE zones of inhibition of 14.6±9.5 and 8.4±4.4 mm, respectively

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Summary

Introduction

Pseudomonas aeruginosa is a highly invasive and toxigenic aerobic Gram negative bacterium. It is nonsporing, non-capsulated and usually motile with the help of one or two flagella. The reasons for the preeminence of this microorganism as a human pathogen range from its adaptability, its innate resistance to many antibiotics, disinfectants and its virulence factors (Aendekerk et al, 2005). P. aeruginosa is an opportunistic pathogen with innate resistance to many antibiotics and disinfectants (Shahid et al, 2003). P. aeruginosa is notorious for its resistance to antibiotics as it maintains antibiotic resistance plasmid (R) factor (Radi and Rahman, 2010) These plasmids are transmissible to sensitive bacteria which make them acquire resistance to antibiotics and have the ability to undergo recombination through conjugation, transformation and transduction. Multidrug active efflux systems have recently been recognized in a number of bacteria as efficient mechanisms of resistances in P. aeruginosa by which antibiotics are expelled from the cells by membrane transporter proteins, the so called drug efflux pumps (Lomovskaya et al, 2001)

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