Fitness trade-offs of multidrug efflux pumps in Escherichia coli K-12 in acid or base, and with aromatic phytochemicals.

Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness trade-offs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-cultured Escherichia coli K-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with carbonyl cyanide 3-chlorophenylhydrazone or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemical's lipophilicity (logP). Methyl salicylate and salicylamide selected strongly against AcrA, without genetic induction of drug resistance regulons. MdtEF-TolC and EmrAB-TolC each had a fitness cost at pH 5.5, but salicylate or benzoate made the fitness contribution positive. Pump fitness effects were not explained by gene expression (measured by digital PCR). Between pH 5.5 and 8.0, acrA and emrA were upregulated in the log phase, whereas mdtE expression was upregulated in the transition-to-stationary phase and at pH 5.5 in the log phase. Methyl salicylate did not affect pump gene expression. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without inducing antibiotic resistance regulons.IMPORTANCEFor drugs that are administered orally, we need to understand how ingested phytochemicals modulate drug resistance in our gut microbiome. Bacteria maintain low-level resistance by proton-motive force (PMF)-driven pumps that efflux many different antibiotics and cell waste products. These pumps play a key role in bacterial defense by conferring resistance to antimicrobial agents at first exposure while providing time for a pathogen to evolve resistance to higher levels of the antibiotic exposed. Nevertheless, efflux pumps confer energetic costs due to gene expression and pump energy expense. The bacterial PMF includes the transmembrane pH difference (ΔpH), which may be depleted by permeant acids and membrane disruptors. Understanding the fitness costs of efflux pumps may enable us to develop resistance breakers, that is, molecules that work together with antibiotics to potentiate their effect. Non-acidic aromatic molecules have the advantage that they avoid the Mar-dependent induction of regulons conferring other forms of drug resistance. We show that different pumps have distinct selection criteria, and we identified non-acidic aromatic molecules as promising candidates for drug resistance breakers.

Depth-dependent intracortical myelin organization in the living human brain determined by in vivo ultra-high field magnetic resonance imaging.

BACKGROUND: Intracortical myelin is a key determinant of neuronal synchrony and plasticity that underpin optimal brain function. Magnetic resonance imaging (MRI) facilitates the examination of intracortical myelin but presents with methodological challenges. Here we describe a whole-brain approach for the in vivo investigation of intracortical myelin in the human brain using ultra-high field MRI. METHODS: Twenty-five healthy adults were imaged in a 7 Tesla MRI scanner using diffusion-weighted imaging and a T1-weighted sequence optimized for intracortical myelin contrast. Using an automated pipeline, T1 values were extracted at 20 depth-levels from each of 148 cortical regions. In each cortical region, T1 values were used to infer myelin concentration and to construct a non-linearity index as a measure the spatial distribution of myelin across the cortical ribbon. The relationship of myelin concentration and the non-linearity index with other neuroanatomical properties were investigated. Five patients with multiple sclerosis were also assessed using the same protocol as positive controls. RESULTS: Intracortical T1 values decreased between the outer brain surface and the gray-white matter boundary following a slope that showed a slight leveling between 50% and 75% of cortical depth. Higher-order regions in the prefrontal, cingulate and insular cortices, displayed higher non-linearity indices than sensorimotor regions. Across all regions, there was a positive association between T1 values and non-linearity indices (P < 10-5). Both T1 values (P < 10-5) and non-linearity indices (P < 10-15) were associated with cortical thickness. Higher myelin concentration but only in the deepest cortical levels was associated with increased subcortical fractional anisotropy (P = 0.05). CONCLUSIONS: We demonstrate the usefulness of an automatic, whole-brain method to perform depth-dependent examination of intracortical myelin organization. The extracted metrics, T1 values and the non-linearity index, have characteristic patterns across cortical regions, and are associated with thickness and underlying white matter microstructure.

Reproducibility of myelin content-based human habenula segmentation at 3 Tesla.

In vivo morphological study of the human habenula, a pair of small epithalamic nuclei adjacent to the dorsomedial thalamus, has recently gained significant interest for its role in reward and aversion processing. However, segmenting the habenula from in vivo magnetic resonance imaging (MRI) is challenging due to the habenula's small size and low anatomical contrast. Although manual and semi-automated habenula segmentation methods have been reported, the test-retest reproducibility of the segmented habenula volume and the consistency of the boundaries of habenula segmentation have not been investigated. In this study, we evaluated the intra- and inter-site reproducibility of in vivo human habenula segmentation from 3T MRI (0.7-0.8 mm isotropic resolution) using our previously proposed semi-automated myelin contrast-based method and its fully-automated version, as well as a previously published manual geometry-based method. The habenula segmentation using our semi-automated method showed consistent boundary definition (high Dice coefficient, low mean distance, and moderate Hausdorff distance) and reproducible volume measurement (low coefficient of variation). Furthermore, the habenula boundary in our semi-automated segmentation from 3T MRI agreed well with that in the manual segmentation from 7T MRI (0.5 mm isotropic resolution) of the same subjects. Overall, our proposed semi-automated habenula segmentation showed reliable and reproducible habenula localization, while its fully-automated version offers an efficient way for large sample analysis.

Selective vulnerability of the cerebral vasculature to blast injury in a rat model of mild traumatic brain injury.

BACKGROUND: Blast-related traumatic brain injury (TBI) is a common cause of injury in the military operations in Iraq and Afghanistan. How the primary blast wave affects the brain is not well understood. The aim of the present study was to examine whether blast exposure affects the cerebral vasculature in a rodent model. We analyzed the brains of rats exposed to single or multiple (three) 74.5 kPa blast exposures, conditions that mimic a mild TBI. Rats were sacrificed 24 hours or between 6 and 10 months after exposure. Blast-induced cerebral vascular pathology was examined by a combination of light microscopy, immunohistochemistry, and electron microscopy. RESULTS: We describe a selective vascular pathology that is present acutely at 24 hours after injury. The vascular pathology is found at the margins of focal shear-related injuries that, as we previously showed, typically follow the patterns of penetrating cortical vessels. However, changes in the microvasculature extend beyond the margins of such lesions. Electron microscopy revealed that microvascular pathology is found in regions of the brain with an otherwise normal neuropil. This initial injury leads to chronic changes in the microvasculature that are still evident many months after the initial blast exposure. CONCLUSIONS: These studies suggest that vascular pathology may be a central mechanism in the induction of chronic blast-related injury.

Blast overpressure induces shear-related injuries in the brain of rats exposed to a mild traumatic brain injury.

BACKGROUND: Blast-related traumatic brain injury (TBI) has been a significant cause of injury in the military operations of Iraq and Afghanistan, affecting as many as 10-20% of returning veterans. However, how blast waves affect the brain is poorly understood. To understand their effects, we analyzed the brains of rats exposed to single or multiple (three) 74.5 kPa blast exposures, conditions that mimic a mild TBI. RESULTS: Rats were sacrificed 24 hours or between 4 and 10 months after exposure. Intraventricular hemorrhages were commonly observed after 24 hrs. A screen for neuropathology did not reveal any generalized histopathology. However, focal lesions resembling rips or tears in the tissue were found in many brains. These lesions disrupted cortical organization resulting in some cases in unusual tissue realignments. The lesions frequently appeared to follow the lines of penetrating cortical vessels and microhemorrhages were found within some but not most acute lesions. CONCLUSIONS: These lesions likely represent a type of shear injury that is unique to blast trauma. The observation that lesions often appeared to follow penetrating cortical vessels suggests a vascular mechanism of injury and that blood vessels may represent the fault lines along which the most damaging effect of the blast pressure is transmitted.