Gradient microfluidics enables rapid bacterial growth inhibition testing

Bacterial growth inhibition tests have become a standard measure of the adverse effects of inhibitors for a wide range of applications, such as toxicity testing in the medical and environmental sciences. However, conventional well-plate formats for these tests are laborious and provide limited information (often being restricted to an end-point assay). In this study, we have developed a microfluidic system that enables fast quantification of the effect of an inhibitor on bacteria growth and survival, within a single experiment. This format offers a unique combination of advantages, including long-term continuous flow culture, generation of concentration gradients, and single cell morphology tracking. Using Escherichia coli and the inhibitor amoxicillin as one model system, we show excellent agreement between an on-chip single cell-based assay and conventional methods to obtain quantitative measures of antibiotic inhibition (for example, minimum inhibition concentration). Furthermore, we show that our methods can provide additional information, over and above that of the standard well-plate assay, including kinetic information on growth inhibition and measurements of bacterial morphological dynamics over a wide range of inhibitor concentrations. Finally, using a second model system, we show that this chip-based systems does not require the bacteria to be labeled and is well suited for the study of naturally occurring species. We illustrate this using Nitrosomonas europaea, an environmentally important bacteria, and show that the chip system can lead to a significant reduction in the period required for growth and inhibition measurements (<4 days, compared to weeks in a culture flask)

Selection for antimicrobial resistance is reduced when embedded in a natural microbial community

This is the final version. Available from Springer Nature via the DOI in this record.Antibiotic resistance has emerged as one of the most pressing, global threats to public health. In single-species experiments selection for antibiotic resistance occurs at very low antibiotic concentrations. However, it is unclear how far these findings can be extrapolated to natural environments, where species are embedded within complex communities. We competed isogenic strains of Escherichia coli, differing exclusively in a single chromosomal resistance determinant, in the presence and absence of a pig faecal microbial community across a gradient of antibiotic concentration for two relevant antibiotics: gentamicin and kanamycin. We show that the minimal selective concentration was increased by more than one order of magnitude for both antibiotics when embedded in the community. We identified two general mechanisms were responsible for the increase in minimal selective concentration: an increase in the cost of resistance and a protective effect of the community for the susceptible phenotype. These findings have implications for our understanding of the evolution and selection of antibiotic resistance, and can inform future risk assessment efforts on antibiotic concentrations.Medical Research Council (MRC)European Commissio

Safe for saplings not safe for seeds:Quercus robur recruitment in relation to coarse woody debris in Bialowieza Primeval Forest, Poland

<p>In forested ecosystems, oak saplings can be found in association with coarse woody debris (CWD) that offers protection against herbivore browsing. In this study we investigated whether CWD is already a safe site during the earlier stages of oak recruitment, i.e. at the seed and seedling phase, or whether these phases require different micro-environments reflecting spatial discordance. We tested our hypothesis in the Bialowieza Primeval Forest in Poland, one of the last remaining examples of natural, lowland forest in Europe. We performed a seed removal experiment in two contrasting forest types (deciduous and coniferous), where we followed the fate of 576 acorns cached in plots with and without CWD. At all locations camera traps and rodent live traps were placed to assess species and abundance of responsible seed removers. To determine the spatial distribution of seedlings we surveyed transects (500 m x 2 m) in deciduous and coniferous forest (total 0.6 ha) and recorded for each sapling its association with CWD. Our experiment clearly showed that, despite a small sample size, acorn removal is higher when associated with CWD and higher in deciduous forest (100%) than in coniferous forest (58.2%). Both wild boar and rodents were responsible for the acorn removal, but wild boar had more impact in deciduous forest and rodents had more impact in coniferous forest. In line with our observed acorn removal, established seedlings were only found in coniferous forest, away from CWD. Our study suggests that spatial discordance occurs during the multiple stages of oak recruitment. The best place to survive for seed and seedling is in coniferous forest away from CWD, while the best place to survive for saplings is near CWD. Our findings may well explain the mechanisms behind recruitment limitation in our studied system, as well as in other forested ecosystems. (C) 2013 Elsevier B.V. All rights reserved.</p>