The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre-and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrABTolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies.efflux | antimicrobial resistance | AcrB | whole genome sequencing

Coccolithophores are an important component of the Earth system, and, as calcifiers, their possible susceptibility to ocean acidification is of major concern. Laboratory studies at enhanced pCO 2 levels have produced divergent results without overall consensus. However, it has been predicted from these studies that, although calcification may not be depressed in all species, acidification will produce "a transition in dominance from more to less heavily calcified coccolithophores" [Ridgwell A, et al., (2009) Biogeosciences 6:2611-2623. A recent observational study [Beaufort L, et al., (2011) Nature 476:80-83] also suggested that coccolithophores are less calcified in more acidic conditions. We present the results of a large observational study of coccolithophore morphology in the Bay of Biscay. Samples were collected once a month for over a year, along a 1,000-km-long transect. Our data clearly show that there is a pronounced seasonality in the morphotypes of Emiliania huxleyi, the most abundant coccolithophore species. Whereas pH and CaCO 3 saturation are lowest in winter, the E. huxleyi population shifts from <10% (summer) to >90% (winter) of the heavily calcified form. However, it is unlikely that the shifts in carbonate chemistry alone caused the morphotype shift. Our finding that the most heavily calcified morphotype dominates when conditions are most acidic is contrary to the earlier predictions and raises further questions about the fate of coccolithophores in a high-CO 2 world.phytoplankton | North Atlantic | climate change C occolithophores contribute between ∼1% and 10% of marine primary production (1), dominate the pelagic calcium carbonate flux (2), and alter ocean albedo (3). Model predictions suggest that, if CO 2 emissions continue unabated, global surface ocean pH will decrease by 0.3-0.5 units by 2100, leading to a halving of the carbonate ion concentration (4). Along with other calcifiers, coccolithophores such as Emiliania huxleyi are considered susceptible to this ocean acidification (OA). This hypothesis is contentious, however, with diverse calcification responses reported for culture experiments. Many experiments on E. huxleyi (the most common coccolithophore) have found depressed calcification at elevated CO 2 concentration and the associated low pH and low CaCO 3 saturation state (Ω) (5-11), whereas others have found elevated calcification (12, 13) or no trend (10). An in-depth discussion on the reasons behind the contrasting results of Riebesell et al. (5) and can be found in refs. 14 and 15. In a recent study, four different strains of E. huxleyi cultured under identical environmental conditions exhibited varying responses to elevated CO 2 (16), as was also found between coccolithophore species (17).Laboratory studies are unrealistic in many respects and, because of their typically short timescales, preclude the possibility of evolutionary adaptation to the imposed change, a key uncertainty in OA research (17-19). It is therefore vital to complement laboratory experiments with observ...

ObjectivesEnterobacteriaceae have multiple efflux pumps that confer intrinsic resistance to antibiotics. AcrB mediates clinically relevant multidrug resistance and is required for virulence and biofilm formation, making it an attractive target for the design of inhibitors. The aim of this study was to assess the viability of single transporters as a target for efflux inhibition using Salmonella Typhimurium as the model pathogen.MethodsThe expression of resistance–nodulation–division (RND) efflux pump genes in response to the inactivation of single or multiple homologues was measured using real-time RT–PCR. Phenotypes of mutants were characterized by measuring antimicrobial susceptibility, dye accumulation and the ability to cause infection in vitro.ResultsThe expression of all RND efflux pump genes was increased when single or multiple acr genes were inactivated, suggesting a feedback mechanism that activates the transcription of homologous efflux pump genes. When two or three acr genes were inactivated, the mutants had further reduced efflux, altered susceptibility to antimicrobials (including increased susceptibility to some, but conversely and counterintuitively, decreased susceptibility to some others) and were more attenuated in the tissue culture model than mutants lacking single pumps were.ConclusionsThese data indicate that it is critical to understand which pumps an inhibitor is active against and the effect of this on the expression of homologous systems. For some antimicrobials, an inhibitor with activity against multiple pumps will have a greater impact on susceptibility, but an unintended consequence of this may be decreased susceptibility to other drugs, such as aminoglycosides.

A systematic review following PRISMA guidelines was conducted to answer the question: What epigenetic, telomeric and associated biological changes are associated with exposure to adverse childhood experiences (ACEs) in the under 12s? Using PRISMA guidelines, appropriate databases were searched. 190 papers were returned with 38 articles fully reviewed. Articles were each independently quality rated by two authors using the Crowe Critical Appraisal Tool and data were extracted. Of the 38 articles, 23 were rated as very high quality. Most study participants were adults (n = 7769) with n = 727 child participants. Only seven of the very/high-quality studies were prospective and involved children. Methylation was the most studied method of epigenetic modification. There is some evidence supporting epigenetic modification of certain markers in participants exposed to ACEs measured in adulthood. Research is lacking on non-coding aspects of the epigenome and on coding aspects other than DNA methylation. There is some evidence of a more powerful effect on telomere length if physical neglect was involved. Much further work is required to model biological and psychological effects of epigenetic changes during childhood using prospective study designs. The effect of ACEs on the cellular ageing process during childhood is inadequately investigated and relies solely on measure of telomere length. Future research suggestions are proposed.