Leonard Amaral

Geraniol Restores Antibiotic Activities against Multidrug-Resistant Isolates from Gram-Negative Species

ABSTRACT The essential oil of Helichrysum italicum significantly reduces the multidrug resistance of Enterobacter aerogenes, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Combinations of the two most active fractions of the essential oil with each other or with phenylalanine arginine β-naphthylamide yield synergistic activity. Geraniol, a component of one fraction, significantly increased the efficacy of β-lactams, quinolones, and chloramphenicol.

Inhibition of quorum‐sensing signals by essential oils

The role of quorum sensing (QS) is well known in microbial pathogenicity and antibiotic resistance. QS is responsible for motility, swarming, and biofilm production based on the signal molecules, e.g., acylated homoserine lactones (AHLs) produced by micro‐organisms above certain population density. The inhibition of QS may reduce pathogenicity, antibiotic resistance and biofilm formation in systemic and local infections. The homoserine lactones and other transmitters contribute to antibiotic resistance and pathogenicity of several bacteria; consequently the inhibition of QS signals reduces the problem of resistance and virulence. Due to the increasing number of persistent non‐treatable infections, there is an urgent need to develop new strategies to combat infections that destabilize bacterial communities in the host.

pH modulation of efflux pump activity of multi-drug resistant Escherichia coli: Protection during its passage and eventual colonization of the colon

Background Resistance Nodulation Division (RND) efflux pumps of Escherichia coli extrude antibiotics and toxic substances before they reach their intended targets. Whereas these pumps obtain their energy directly from the proton motive force (PMF), ATP-Binding Cassette (ABC) transporters, which can also extrude antibiotics, obtain energy from the hydrolysis of ATP. Because E. coli must pass through two pH distinct environments of the gastrointestinal system of the host, it must be able to extrude toxic agents at very acidic and at near neutral pH (bile salts in duodenum and colon for example). The herein described study examines the effect of pH on the extrusion of ethidium bromide (EB). Methodology/Principal Findings E. coli AG100 and its tetracycline induced progeny AG100TET that over-expresses the acrAB efflux pump were evaluated for their ability to extrude EB at pH 5 and 8, by our recently developed semi-automated fluorometric method. At pH 5 the organism extrudes EB without the need for metabolic energy (glucose), whereas at pH 8 extrusion of EB is dependent upon metabolic energy. Phe-Arg β-naphtylamide (PAβN), a commonly assumed inhibitor of RND efflux pumps has no effect on the extrusion of EB as others claim. However, it does cause accumulation of EB. Competition between EB and PAβN was demonstrated and suggested that PAβN was preferentially extruded. A Km representing competition between PAβN and EB has been calculated. Conclusions/Significance The results suggest that E. coli has two general efflux systems (not to be confused with a distinct efflux pump) that are activated at low and high pH, respectively, and that the one at high pH is probably a putative ABC transporter coded by msbA, which has significant homology to the ABC transporter coded by efrAB of Enterococcus faecalis, an organism that faces similar challenges as it makes its way through the toxic intestinal system of the host.

The role of efflux pumps in macrolide resistance in Mycobacterium avium complex

Mycobacteriumavium complex (MAC) is clinically important since it can cause severe infections in acquired immune deficiency syndrome (AIDS) patients and other immunocompromised individuals. Use of the macrolides clarithromycin and azithromycin has improved the outcome of MAC infections, but therapeutic failure is still a major problem. In this work, we studied efflux pump activity in MAC clinical strains and evaluated the contribution of active efflux to macrolide resistance. Eighteen clinical strains isolated from AIDS patients were evaluated for macrolide resistance in the presence and absence of the efflux pump inhibitors (EPIs) thioridazine, chlorpromazine and verapamil. The efflux activity of these strains was then assessed by a semi-automated fluorometric method that detects extrusion of ethidium bromide (EtBr), a known efflux pump substrate. Resistance to clarithromycin was significantly reduced in the presence of thioridazine, chlorpromazine and verapamil. The same EPIs were effective in decreasing the efflux of EtBr from MAC cells. Moreover, increased retention of [(14)C]-erythromycin in the presence of these EPIs further demonstrated that active efflux contributes to MAC resistance to macrolides. This study demonstrates that efflux pumps play an important role in MAC resistance to antibiotics.

Identification of Efflux-Mediated Multi-drug Resistance in Bacterial Clinical Isolates by Two Simple Methods

Two simple, instrument-free, user-friendly methods that can readily be implemented by a routine microbiology laboratory are described for the detection of multi-drug-resistant (MDR) isolates that overexpress efflux pump (EP) systems responsible for the MDR phenotype. The first method employs the universal EP substrate ethidium bromide (EB) at varying concentrations in agar-containing plates upon which the contents of an overnight culture are swabbed as spokes of a wheel. In this method, named the EB-agar cartwheel method, it is assumed that the smallest concentration of EB that produces fluorescence of the bacterial mass represents the highest concentration of EB that the bacteria can exclude. Consequently, as the efflux system(s) of a given MDR clinical bacterial isolate is overexpressed relative to that of a reference strain, the minimal concentration of EB producing fluorescence is significantly greater. A simple formula is provided which affords the ranking of MDR clinical isolates with respect to the degree of their efflux capacity. The second method, which can be used after the first one, determines whether the MDR phenotype is based upon an overexpressed efflux system. This method employs a 24-well microplate with separate wells containing or lacking an efflux pump inhibitor (EPI) and Kirby-Bauer discs that correspond to the antibiotics to which the MDR strain is resistant. After the wells are inoculated with the MDR clinical isolate, the plate is incubated overnight and each well is evaluated by eye for evidence of growth. Comparison of growth to the relevant control enables the observer to determine the following outcomes: no growth produced by the EPI-antibiotic combination (i.e., reversal of antibiotic resistance); reduced growth produced by the EPI-antibiotic combination; no difference in growth, i.e., EPI does not affect the resistance to the given antibiotic. If the first method showed that there was a significant difference between the minimum concentrations of EB in agar that produced fluorescence for the clinical isolate and its reference strain, then one can conclude that if the EPI had no effect on reducing antibiotic resistance, the differences in the EB concentrations that produced fluorescence are probably due to differences in the permeability of the strain to EB, reflecting a downregulation of porins if the clinical isolate is a Gram-negative bacterium.