ABSTRACT
Enterococcus faecalis is recognized as a multidrug-resistant nosocomial pathogen. The phenotypic basis for this is largely uncharacterized. The intrinsic efflux system of the antibiotic-susceptible E. faecalis ATCC29212 strain was studied using a semi-automated method that assesses accumulation and efflux of the universal efflux pump substrate ethidium bromide (EB). The results show that the intrinsic efflux system of this Enterococcus strain is controlled by energy derived from the catabolism of glucose and the proton concentration of the medium. At pH 5, agents that inhibit efflux pumps in Gram-positive organisms and the proton gradient un-coupler CCCP do not increase accumulation nor inhibit efflux of EB. In contrast, at pH 8, where the proton concentration is 1,000-fold lower, these agents increase accumulation and efflux of EB. These results are relevant to infections produced by E. faecalis and subsequent antibiotic therapy with antibiotics to which the organism is known to be intrinsically resistant.
Subject(s)
Drug Resistance, Multiple, Bacterial/drug effects , Enterococcus faecalis/drug effects , Ethidium/pharmacology , Membrane Transport Proteins/metabolism , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cell Survival/drug effects , Drug Resistance, Multiple, Bacterial/physiology , Enterococcus faecalis/metabolism , Ethidium/metabolism , Glucose/metabolism , Microbial Sensitivity Tests , Reserpine/pharmacology , Thioridazine/pharmacology , Uncoupling Agents/pharmacologyABSTRACT
Intracellular efflux pumps have been largely the research focus in multidrug-resistant (MDR) Gram-positive and Gram-negative microorganisms and parasites including cancers. However, drug efflux mechanisms other than pumps per se have been observed, supported by the effects of isomeric, non-antibiotic depressant (DPR), phenothiazines and thixenes, and antidepressant (ADPR) phenylpiperidine neurotropic drugs, alone or in combination with classical antimicrobials on MDR Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes and Streptococcus pneumoniae. Of the non-antibiotics we investigated, the DPR l-thioridazine, trans-clopenthixol and isomers of phenylpiperidines NNC 20-4962 (isomer of femoxetine) and NNC 20-7052 (isomer of paroxetine) were potent antimicrobials with the least neurotropic activity, pointing to a possible general isomeric structure-activity relationship. These compounds may be regarded as new efflux inhibitors. Moreover, these isomers have considerably reduced, in some cases absent, neurotropism and reduced mammalian toxicity. This may alleviate concerns about adverse effects and therapeutic safety for infected patients in life-threatening situations where the non-antibiotic dosage would be in the lower, non-chronic dosage ranges generally prescribed for individuals with mild mental health problems. The results point to the prokaryotic and eukaryotic microorganisms' phospholipid/protein domain involvement of the cationic, amphiphilic, non-antibiotic DPR and ADPR, with the phospholipids being the initial sites attracting and concentrating the neurotropes to induce a form of suspended animation, followed by gross changes of cell wall and membrane structure, and thereby potentiating their destructive or immobilizing effects on various as yet only hinted at resistance and efflux mechanisms. Combination of appropriate isomeric non-antibiotic DPR and ADPR of low neurotropism and toxicity with conventional and classical antimicrobials promises early, new therapeutic strategies salutary against microbial resistance, resistance development, pathogenicity and virulence.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/drug effects , Phenothiazines/pharmacology , Animals , Bacterial Infections/drug therapy , Bacterial Infections/microbiology , Gram-Negative Bacteria/drug effects , Gram-Negative Bacterial Infections/drug therapy , Gram-Negative Bacterial Infections/microbiology , Gram-Positive Bacteria/drug effects , Gram-Positive Bacterial Infections/drug therapy , Gram-Positive Bacterial Infections/microbiology , Humans , Mycobacterium/drug effects , Phenothiazines/adverse effects , Phenothiazines/therapeutic use , Phospholipids/metabolismABSTRACT
Our previous studies demonstrated that exposure of a bacterium to increasing concentrations of an antibiotic would increase resistance to that antibiotic as a consequence of activating efflux pumps. This study utilises the same approach; however, it employs the methicillin-resistant Staphylococcus aureus (MRSA) COL strain, which is highly resistant to oxacillin (OXA). MRSA COL was adapted to 3200 mg/L of OXA. Changes in resistance to other antibiotics were evaluated and efflux pump activity during the adaptation process was determined. MRSA COL was exposed to stepwise two-fold increases of OXA. At the end of each step, minimum inhibitory concentration determination for erythromycin (ERY) and other antibiotics was conducted. Reserpine (RES) was employed to evaluate whether resistance to ERY was dependent on efflux pump activity. Efflux pump activity was also evaluated using the ethidium bromide (EB) assay. DNA typing of the products of each culture step was conducted to assess purity. Serial exposure of MRSA COL to increasing concentrations of OXA resulted in increased resistance to ERY, which could be eliminated with RES. Evaluation of efflux pump activity by the EB method indicated increased efflux activity. Resistance to ERY was accompanied by resistance to kanamycin, amikacin, ofloxacin, norfloxacin, ciprofloxacin and rifampicin. This is the first time that a multidrug-resistant phenotype has been experimentally produced as a consequence of exposure of the organism to an antibiotic to which it is initially highly resistant.
