A genetic platform to investigate the functions of bacterial drug efflux pumps.

Efflux pumps are a serious challenge for the development of antibacterial agents. Overcoming efflux requires an in-depth understanding of efflux pump functions, specificities and the development of inhibitors. However, the complexities of efflux networks have limited such studies. To address these challenges, we generated Efflux KnockOut-35 (EKO-35), a highly susceptible Escherichia coli strain lacking 35 efflux pumps. We demonstrate the use of this strain by constructing an efflux platform comprising EKO-35 strains individually producing efflux pumps forming tripartite complexes with TolC. This platform was profiled against a curated diverse compound collection, which enabled us to define physicochemical properties that contribute to transport. We also show the E. coli drug efflux network is conditionally essential for growth, and that the platform can be used to investigate efflux pump inhibitor specificities and efflux pump interplay. We believe EKO-35 and the efflux platform will have widespread application for the study of drug efflux.

Exploring functional interplay amongst Escherichia coli efflux pumps.

Bacterial efflux pumps exhibit functional interplay that can translate to additive or multiplicative effects on resistance to antimicrobial compounds. In diderm bacteria, two different efflux pump structural types - single-component inner membrane efflux pumps and cell envelope-spanning multicomponent systems - cooperatively export antimicrobials with cytoplasmic targets from the cell. Harnessing our recently developed efflux platform, which is built upon an extensively efflux-deficient strain of Escherichia coli, here we explore interplay amongst a panel of diverse E. coli efflux pumps. Specifically, we assessed the effect of simultaneously expressing two efflux pump-encoding genes on drug resistance, including single-component inner membrane efflux pumps (MdfA, MdtK and EmrE), tripartite complexes (AcrAB, AcrAD, MdtEF and AcrEF), and the acquired TetA(C) tetracycline resistance pump. Overall, the expression of two efflux pump-encoding genes from the same structural type did not enhance resistance levels regardless of the antimicrobial compound or efflux pump under investigation. In contrast, a combination of the tripartite efflux systems with single-component pumps sharing common substrates provided multiplicative increases to antimicrobial resistance levels. In some instances, resistance was increased beyond the product of resistance provided by the two pumps individually. In summary, the developed efflux platform enables the isolation of efflux pump function, facilitating the identification of interactions between efflux pumps.

Evaluation of first ray instability using the double dorsiflexion test: A prospective observational case-controlled study.

BACKGROUND: First-Ray (FR) stability allows for foot propulsion in-stance, taking 60% weight. First-ray instability (FRI) is associated with middle column overload, synovitis, deformity and osteoarthritis. Clinical detection can still be challenging. We propose to develop a clinical test that helps identify FRI using two simple manual manoeuvres. METHODS: 10 patients with unilateral FRI were recruited. Unaffected contralateral feet were used as controls. Stringent exclusion criteria were applied including hallux MTP pain, laxity, inflammatory arthropathy and collagen disorders. A Klauemeter directly measured the sagittal plane dorsal first metatarsal head translation of affected vs unaffected feet. Maximum passive proximal phalanx 1st MTP joint dorsiflexion was measured using a video capture and Tracker motion software analysis with and without applying a dorsal force at the 1st metatarsal head using a Newton meter. Proximal phalanx motion was compared in affected vs unaffected feet with and without dorsal metatarsal head force application and compared to direct measurements using the Klaumeter. P value of < 0.05 was considered significant. RESULTS: FRI feet had dorsal translation greater than 8 mm (median, 11.94; interquartile range [IQR], 10.23-13.81) vs 1.77 for unaffected control feet was (median, 1.77; interquartile range [IQR], 1.23-2.96) using the Klauemeter. The percentage reduction in 1st MTP joint dorsiflexion ROM when applying the double dorsiflexion test FRI (mean reduction of 67.98%) when compared to control feet (mean reduction of 28.44%)(P < 0.01). Receiver operating characteristic (ROC) analysis showed that a 50% reduction in dorsiflexion ROM of 1st MTPJ when performing the double dorsiflexion test achieved a specificity of 100% and sensitivity of 90% (AUC =0.990, 95%CI [0.958-1.000], P > 0.0001). CONCLUSION: The double dorsiflexion (DDF) is easy to perform with two relatively simple manual manoeuvres that avoids the need for complex instrumented and radiation-based assessment. Greater than 50% decrease in proximal phalanx motion has an over 90% sensitivity in identifying feet with FRI. LEVEL OF EVIDENCE: This was a prospective case-controlled study of consecutive cases of a level II evidence.

Evidence for Roux-en-Y Pancreatic Duct Drainage Versus Standard Anastomosis in Pancreatic Transplantation.

OBJECTIVES: Pancreatic transplantation is usually performed simultaneously with renal transplantation in the setting of end-stage nephropathy and type 1 diabetes. Surgical methods for dealing with exocrine secretions include bladder drainage, direct duodenojejunostomy and Roux-en-Y (ReY) enteric drainage. Roux-en-Y may confer an advantage over duodenojejunostomy because it distances enteric content from the transplant duodenal anastomosis. We examined the effect of enteric drainage method on transplant outcomes. METHODS: Data were obtained from the UK transplant registry on 2172 consecutive pancreatic transplants. Early graft loss was the primary endpoint. Secondary endpoints included return to theater, length of inpatient stay, readmission with pancreatitis, graft survival, and patient survival. RESULTS: There was no protective effect of ReY drainage (early graft loss, 4.6% vs 3.1%, P = 0.30; hazard ratio, 0.98; 95% confidence interval, 0.63-1.52; P = 0.91). There was a significant association between ReY and return to theater, reflecting either the technique or indication for ReY (multivariate odds ratio, 2.05; 95% confidence interval, 1.38-3.06; P < 0.01). The effect of transplant center on graft survival was assessed and adjusted for. CONCLUSIONS: There was no evidence of a protective benefit of ReY drainage over duodenojejunostomy, but there was an increased risk of return to theater.

Non-contact infrared thermometers compared with current approaches in primary care for children aged 5 years and under: a method comparison study.

BACKGROUND: Current options for temperature measurement in children presenting to primary care include either electronic axillary or infrared tympanic thermometers. Non-contact infrared thermometers could reduce both the distress of the child and the risk of cross-infection. OBJECTIVES: The objective of this study was to compare the use of non-contact thermometers with the use of electronic axillary and infrared tympanic thermometers in children presenting to primary care. DESIGN: Method comparison study with a nested qualitative study. SETTING: Primary care in Oxfordshire. PARTICIPANTS: Children aged ≤ 5 years attending with an acute illness. INTERVENTIONS: Two types of non-contact infrared thermometers [i.e. Thermofocus (Tecnimed, Varese, Italy) and Firhealth (Firhealth, Shenzhen, China)] were compared with an electronic axillary thermometer and an infrared tympanic thermometer. MAIN OUTCOME MEASURES: The primary outcome was agreement between the Thermofocus non-contact infrared thermometer and the axillary thermometer. Secondary outcomes included agreement between all other sets of thermometers, diagnostic accuracy for detecting fever, parental and child ratings of acceptability and discomfort, and themes arising from our qualitative interviews with parents. RESULTS: A total of 401 children (203 boys) were recruited, with a median age of 1.6 years (interquartile range 0.79-3.38 years). The readings of the Thermofocus non-contact infrared thermometer differed from those of the axillary thermometer by -0.14 °C (95% confidence interval -0.21 to -0.06 °C) on average with the lower limit of agreement being -1.57 °C (95% confidence interval -1.69 to -1.44 °C) and the upper limit being 1.29 °C (95% confidence interval 1.16 to 1.42 °C). The readings of the Firhealth non-contact infrared thermometer differed from those of the axillary thermometer by -0.16 °C (95% confidence interval -0.23 to -0.09 °C) on average, with the lower limit of agreement being -1.54 °C (95% confidence interval -1.66 to -1.41 °C) and the upper limit being 1.22 °C (95% confidence interval 1.10 to 1.34 °C). The difference between the first and second readings of the Thermofocus was -0.04 °C (95% confidence interval -0.07 to -0.01 °C); the lower limit was -0.56 °C (95% confidence interval -0.60 to -0.51 °C) and the upper limit was 0.47 °C (95% confidence interval 0.43 to 0.52 °C). The difference between the first and second readings of the Firhealth thermometer was 0.01 °C (95% confidence interval -0.02 to 0.04 °C); the lower limit was -0.60 °C (95% confidence interval -0.65 to -0.54 °C) and the upper limit was 0.61 °C (95% confidence interval 0.56 to 0.67 °C). Sensitivity and specificity for the Thermofocus non-contact infrared thermometer were 66.7% (95% confidence interval 38.4% to 88.2%) and 98.0% (95% confidence interval 96.0% to 99.2%), respectively. For the Firhealth non-contact infrared thermometer, sensitivity was 12.5% (95% confidence interval 1.6% to 38.3%) and specificity was 99.4% (95% confidence interval 98.0% to 99.9%). The majority of parents found all methods to be acceptable, although discomfort ratings were highest for the axillary thermometer. The non-contact thermometers required fewer readings than the comparator thermometers. LIMITATIONS: A method comparison study does not compare new methods against a reference standard, which in this case would be central thermometry requiring the placement of a central line, which is not feasible or acceptable in primary care. Electronic axillary and infrared tympanic thermometers have been found to have moderate agreement themselves with central temperature measurements. CONCLUSIONS: The 95% limits of agreement are > 1 °C for both non-contact infrared thermometers compared with electronic axillary and infrared tympanic thermometers, which could affect clinical decision-making. Sensitivity for fever was low to moderate for both non-contact thermometers. FUTURE WORK: Better methods for peripheral temperature measurement that agree well with central thermometry are needed. TRIAL REGISTRATION: Current Controlled Trials ISRCTN15413321. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 53. See the NIHR Journals Library website for further project information.

General practitioners commonly measure children's temperature using a thermometer placed in the armpit or ear canal. New 'non-contact' thermometers use infrared light to measure temperature without touching the child. They are easy to use and there is no risk of passing on infections. However, we do not know how well they measure temperature compared with thermometers that use the armpit or the ear. This study aimed to compare two non-contact thermometers with current thermometers. We measured children's temperature with all thermometer types, and asked children and their parents about their views. The study was performed in general practices in Oxfordshire with children aged ≤ 5 years who had come to see their general practitioner because they had recently become unwell. Both the cheaper and more expensive non-contact thermometers gave slightly lower temperature readings on average than current thermometers. The vast majority of readings ranged from 1.6 °C lower to 1.3 °C higher than current thermometers. The detection of fever of at least 38 °C was low to moderate for both non-contact thermometers. Most parents did not think that their child was distressed by having their temperature taken using any of the thermometers, but the armpit thermometer was rated as the least comfortable. When interviewed, parents were more negative about the armpit thermometers, although still willing to use them if they were recommended by doctors. Although we found that the readings from the different thermometers did not match, we do not know whether the non-contact or the current thermometers were giving readings that were closer to the real temperature of the child. To understand this, we would need to do a study that included a more invasive procedure for temperature assessment.

Spray-deposition and photopolymerization of organic-inorganic thiol-ene resins for fabrication of superamphiphobic surfaces.

Superamphiphobic surfaces, exhibiting high contact angles and low contact angle hysteresis to both water and low surface tension liquids, have attracted a great deal attention in recent years because of the potential of these materials in practical applications such as liquid-resistant textiles, self-cleaning surfaces, and antifouling/anticorrosion coatings. In this work, we present a simple strategy for fabricating of superamphiphobic coatings based on photopolymerization of hybrid thiol-ene resins. Spray-deposition and UV photopolymerization of thiol-ene resins containing hydrophobic silica nanoparticles and perfluorinated thiols provide a multiscale topography and low-energy surface that endows the surface with superamphiphobicity. The wettability and chemical composition of the surfaces were characterized by contact-angle goniometry and X-ray photoelectron spectroscopy, respectively. The hierarchical roughness features of the thiol-ene surfaces were investigated with field-emission scanning electron microscopy. Droplet impact and sandpaper abrasion tests indicate the coatings respectively possess a robust antiwetting behavior and good mechanical durability.

Superhydrophobic hybrid inorganic-organic thiol-ene surfaces fabricated via spray-deposition and photopolymerization.

We report a simple and versatile method for the fabrication of superhydrophobic inorganic-organic thiol-ene coatings via sequential spray-deposition and photopolymerization under ambient conditions. The coatings are obtained by spray-deposition of UV-curable hybrid inorganic-organic thiol-ene resins consisting of pentaerythritol tetra(3-mercaptopropionate) (PETMP), triallyl isocyanurate (TTT), 2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane (TMTVSi), and hydrophobic fumed silica nanoparticles. The spray-deposition process and nanoparticle agglomeration/dispersion provide surfaces with hierarchical morphologies exhibiting both micro- and nanoscale roughness. The wetting behavior, dependent on the concentration of TMTVSi and hydrophobic silica nanoparticles, can be varied over a broad range to ultimately provide coatings with high static water contact angles (>150°), low contact angle hysteresis, and low roll off angles (<5°). The cross-linked thiol-ene coatings are solvent resistant, stable at low and high pH, and maintain superhydrophobic wetting behavior after extended exposure to elevated temperatures. We demonstrate the versatility of the spray-deposition and UV-cure process on a variety of substrate surfaces including glass, paper, stone, and cotton fabric.

A winter distribution model for Bicknell's Thrush (Catharus bicknelli), a conservation tool for a threatened migratory songbird.

Conservation planning and implementation require identifying pertinent habitats and locations where protection and management may improve viability of targeted species. The winter range of Bicknell's Thrush (Catharus bicknelli), a threatened Nearctic-Neotropical migratory songbird, is restricted to the Greater Antilles. We analyzed winter records from the mid-1970s to 2009 to quantitatively evaluate winter distribution and habitat selection. Additionally, we conducted targeted surveys in Jamaica (n = 433), Cuba (n = 363), Dominican Republic (n = 1,000), Haiti (n = 131) and Puerto Rico (n = 242) yielding 179 sites with thrush presence. We modeled Bicknell's Thrush winter habitat selection and distribution in the Greater Antilles in Maxent version 3.3.1. using environmental predictors represented in 30 arc second study area rasters. These included nine landform, land cover and climatic variables that were thought a priori to have potentially high predictive power. We used the average training gain from ten model runs to select the best subset of predictors. Total winter precipitation, aspect and land cover, particularly broadleaf forests, emerged as important variables. A five-variable model that contained land cover, winter precipitation, aspect, slope, and elevation was the most parsimonious and not significantly different than the models with more variables. We used the best fitting model to depict potential winter habitat. Using the 10 percentile threshold (>0.25), we estimated winter habitat to cover 33,170 km(2), nearly 10% of the study area. The Dominican Republic contained half of all potential habitat (51%), followed by Cuba (15.1%), Jamaica (13.5%), Haiti (10.6%), and Puerto Rico (9.9%). Nearly one-third of the range was found to be in protected areas. By providing the first detailed predictive map of Bicknell's Thrush winter distribution, our study provides a useful tool to prioritize and direct conservation planning for this and other wet, broadleaf forest specialists in the Greater Antilles.