Interactions of Antimicrobial Compounds with Selected Drugs Used in the Clinical Management of Sickle Cell Disease (SCD)

Abstract

Though the promotion of resistant bacteria worldwide has mainly been attributed to the injudicious use of many antibiotics, the role of non-antimicrobial drugs used in the clinical management of pathological conditions of non-infectious diseases in the modulation of microbial drug resistance or susceptibility needs to be assessed. The effects of the antibioticnon- antibiotic drug-drug interactions against bacteria in SCD patients and in other chronic diseases cannot be overemphasized. The interactions of nine SCD management drugs and fifteen phenotype modulating compounds on the activities of 19 standard antimicrobials against clinical isolates of methicillin-resistant S. aureus (MRSA GGP E120) and E. coli (E. coli GGP E100) were investigated by pairwise drug combinations between SCD drugs/phenotypic compounds and antimicrobial agents using the agar plate disc diffusion method. The comparative effects of the SCD drugs/PMCs on ethidium bromide (EtBr) uptake and efflux in the bacterial cells relative to verapamil were detected by measuring fluorescence of intracellular EtBr. SCD drugs and PMCs biofilm inhibitory and disrupting abilities were analyzed by reading absorbencies of crystal violet stained biofilms. Bacterial strains were obtained from the bacteriology departments of the Noguchi Memorial Institute for Medical Research, University of Ghana, Accra. In the preliminary pairwise combination screen by disc diffusion, SCD drugs produced mostly resistance-inducing interactions with standard antibiotics particularly the cell wall inhibitors, amoxicillin and ampicillin and some few weak resistance-breaking features mostly with gentamicin. The opioid medication, morphine emerged as a key resistance-breaking (antibiotic enhancing) SCD drug. Methotrexate, an anti-inflammatory and anti-folate compound generated significant interactions with antibiotics against both organisms. Resistance breaking and resistance inducing effects were characterized as increase and decrease in zones of inhibition by four millimetres or more respectively, compared to zones produced in the absence of SCD drug. In E. coli, no SCD drug exceeded the accumulated intracellular ethidium bromide (EtBr)

level achieved by verapamil, the control for maximum accumulation. Hydroxyurea and paracetamol induced higher uptake of EtBr within MRSA relative to verapamil. Results were analyzed using the fluorescence value of verapamil control as standard to assess and compare the effects of the SCD drugs, taking into consideration the overlapping of error bars. Generally, all SCD drugs showed higher rates of efflux inhibition (low efflux activity) relative to the no drug treatment control (bacterial cells and EtBr only) in both organisms, following the course of the line curves. Efflux activities of SCD drugs were not significantly different from verapamil with the exception of deferasirox and methotrexate in E. coli. In MRSA, efflux activities of methotrexate, morphine, ibuprofen and paracetamol treated cells were not significantly distinct relative to efflux activity of verapamil, the control for minimum rate of efflux. Using a 50% value of biofilm formed in the compound free medium as a cut-off to analyze the effects of SCD drugs, the most effective biofilm inhibiting abilities (approximately greater than 60%) was observed for methotrexate and pethidine against E. coli and none (greater than 50%) against MRSA. Deferasirox showed above 50% biofilm disrupting activity against E. coli biofilm and again none against MRSA biofilm. Overlapping of error bars representing standard error of the mean was carefully considered in the analyses. Biofilm formation and integrity tended to be more robust in MRSA than in E. coli. Extracts from four fungal isolates – SBF 052, SBF 062, TEF 246 and TEF 256 showed antibacterial activities against both E. coli and MRSA in a preliminary disc diffusion assay. The resistance-inducing interactions and few resistance-breaking interactions generated by SCD drugs to the antibiotics against both bacterial strains could not be attributed to their effects on efflux or biofilm formation and thus, the need for further investigations to establish the molecular mechanisms causing such interactions. Further investigations are also necessary to provide healthcare providers with information on what antibiotics to prescribe for certain chronic disease patients who are continually taking particular management medications. Analyses of microbial stress responses to the unique antibiotic-non-antibiotic interactions in further studies would reveal a number of endogenous resistance-promoting genes that represent possible therapeutic targets in countering antimicrobial resistance.