Random mutagenesis of the multidrug transporter AcrB from Escherichia coli for identification of putative target residues of efflux pump inhibitors.

Efflux is an important mechanism of bacterial multidrug resistance (MDR), and the inhibition of MDR pumps by efflux pump inhibitors (EPIs) could be a promising strategy to overcome MDR. 1-(1-Naphthylmethyl)-piperazine (NMP) and phenylalanine-arginine-ß-naphthylamide (PAßN) are model EPIs with activity in various Gram-negative bacteria expressing AcrB, the major efflux pump of Escherichia coli, or similar homologous pumps of the resistance-nodulation-cell division class. The aim of the present study was to generate E. coli AcrB mutants resistant to the inhibitory action of the two model EPIs and to identify putative EPI target residues in order to better understand mechanisms of pump inhibition. Using an in vitro random mutagenesis approach focusing on the periplasmic domain of AcrB, we identified the double mutation G141D N282Y, which substantially compromised the synergistic activity of NMP with linezolid, was associated with similar intracellular linezolid concentrations in the presence and absence of NMP, and did not impair the intrinsic MICs of various pump substrates and dye accumulation. We propose that these mutations near the outer face of the distal substrate binding pocket reduce NMP trapping. Other residues found to be relevant for efflux inhibition by NMP were G288 and A279, but mutations at these sites also changed the susceptibility to several pump substrates. Unlike with NMP, we were unable to generate AcrB periplasmic domain mutants with resistance or partial resistance to the EPI activity of PAßN, which is consistent with the modes of action of PAßN differing from those of NMP.

Organochlorine pesticides and polychlorinated biphenyls in common kestrel eggs from the Canary Islands: Spatiotemporal variations and effects on eggshell and reproduction.

Organochlorine (OC) pesticides were widely used on the Canary Islands (Spain) for intensive crop production and against plagues of African locust. A previous study performed in 1988-1994 showed a high concentration of p,p'-DDE in the eggs of common kestrels (Falco tinnunculus) from the island of Tenerife. The present study shows OC pesticide and polychlorinated biphenyl (PCBs) levels in 40 unhatched common kestrel eggs collected from southern Tenerife between 2009 and 2016. The protoporphyrin IX in eggshells has also been analysed in order to explore the use of this pigment as a biomarker. Egg biometry, status of embryo development, eggshell thickness and mass of extractable lipids of each egg were recorded. Surrounding land use and reproductive parameters (hatching and fledging rates) were obtained for each nest. The most abundant compound was p,p'-DDE (15.0 µg/g d.w), followed by PCBs (0.46 µg/g d.w.). The decline in p,p'-DDE levels in southern Tenerife (with 23.6 µg/g d.w. in 1988-1994) was 36.4%. p,p'-DDE levels were positively associated with the surface of active and abandoned cropland in a 200 m-radius around the nest and with proximity to urban areas. PCB levels were associated with proximity to roads. Shell thickness was negatively affected by the p,p'-DDE concentration. The concentration of protoporphyrin IX in the eggshell was negatively associated with the concentration of hexachlorobenzene in the egg content. Despite the total ban on the use of p,p'-DDT in Spain since 1986, p,p'-DDE levels remain elevated in those areas in which that use was formerly intensive.