Rapid determination of antimicrobial susceptibility of Gram-negative bacteria from clinical blood cultures using a scattered light-integrated collection device

Background. A bloodstream infection (BSI) presents a complex and serious health problem, a problem that is being exacerbated by increasing antimicrobial resistance (AMR). Gap Statement. The current turnaround times (TATs) for most antimicrobial susceptibility testing (AST) methods offer results retrospective of treatment decisions, and this limits the impact AST can have on antibiotic prescribing and patient care. Progress must be made towards rapid BSI diagnosis and AST to improve antimicrobial stewardship and reduce preventable deaths from BSIs. To support the successful implementation of rapid AST (rAST) in hospital settings, a rAST method that is affordable, is sustainable and offers comprehensive AMR detection is needed. Aim. To evaluate a scattered light-integrated collection (SLIC) device against standard of care (SOC) to determine whether SLIC could accelerate the current TATs with actionable, accurate rAST results for Gram-negative BSIs. Methods. Positive blood cultures from a tertiary referral hospital were studied prospectively. Flagged positive Gram-negative blood cultures were confirmed by Gram staining and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Vitek 2, disc diffusion (ceftriaxone susceptibility only) and an SLIC device. Susceptibility to a panel of five antibiotics, as defined by European Committee on Antimicrobial Susceptibility Testing breakpoints, was examined using SLIC. Results. A total of 505 bacterial-antimicrobial combinations were analysed. A categorical agreement of 95.5% (482/505) was achieved between SLIC and SOC. The 23 discrepancies that occurred were further investigated by the broth microdilution method, with 10 AST results in agreement with SLIC and 13 in agreement with SOC. The mean time for AST was 10.53±0.46 h and 1.94±0.02 h for Vitek 2 and SLIC, respectively. SLIC saved 23.96±1.47 h from positive blood culture to AST result. Conclusion. SLIC has the capacity to provide accurate AST 1 day earlier from flagged positive blood cultures than SOC. This significant time saving could accelerate time to optimal antimicrobial therapy, improving antimicrobial stewardship and management of BSIs.</p

Real-time monitoring of live mycobacteria with a microfluidic acoustic-Raman platform

Funding: UK Engineering and Physical Sciences Research Council for funding through grant EP/P030017/1 and fellowship EP/L025035/1. This work was also supported by the PreDiCT-TB consortium [IMI Joint undertaking grant agreement number 115337, resources of which are composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA companies’ in kind contribution (www.imi.europa.eu)] and the PanACEA consortium [funded by the European & Developing Countries Clinical Trials Partnership (EDCTP); grant agreement: TRIA-2015-1102]Tuberculosis (TB) remains a leading cause of death worldwide. Lipid rich, phenotypically antibiotic tolerant, bacteria are more resistant to antibiotics and may be responsible for relapse and the need for long-term TB treatment. We present a microfluidic system that acoustically traps live mycobacteria, M. smegmatis, a model organism for M. tuberculosis. We then perform optical analysis in the form of wavelength modulated Raman spectroscopy (WMRS) on the trapped M. smegmatis for up to eight hours, and also in the presence of isoniazid (INH). The Raman fingerprints of M. smegmatis exposed to INH change substantially in comparison to the unstressed condition. Our work provides a real-time assessment of the impact of INH on the increase of lipids in these mycobacteria, which could render the cells more tolerant to antibiotics. This microfluidic platform may be used to study any microorganism and to dynamically monitor its response to different conditions and stimuli.Publisher PDFPeer reviewe

Enhanced methodologies for detecting phenotypic resistance in mycobacteria

Lipid droplets found in algae and other microscopic organisms have become of interest to many researchers partially because they carry the capacity to produce bio-oil for the mass market. They are of importance in biology and clinical practice because their presence can be a phenotypic marker of an altered metabolism, including reversible resistance to antibiotics, prompting intense research.A useful stain for detecting lipid bodies in the lab is Nile red. It is a dye that exhibits solvatochromism; its absorption band varies in spectral position, shape and intensity with the nature of its solvent environment, it will fluoresce intensely red in polar environment and blue shift with the changing polarity of its solvent. This makes it ideal for the detection of lipid bodies within Mycobacterium spp. This is because mycobacterial lipid bodies' primary constituents are nonpolar lipids such as triacylglycerols but bacterial cell membranes are primarily polar lipid species. In this chapter we describe an optimal method for using Nile red to distinguish lipid containing (Lipid rich or LR cells) from those without lipid bodies (Lipid Poor or LP). As part of the process we have optimized a method for separating LP and LR cells that does not require the use of an ultracentrifuge or complex separation media. We believe that these methods will facilitate further research in these enigmatic, transient and important cell states.Postprin

Effect of the dietary supplement Meltdown on catecholamine secretion, markers of lipolysis, and metabolic rate in men and women: a randomized, placebo controlled, cross-over study

<p>Abstract</p> <p>Background</p> <p>We have recently reported that the dietary supplement Meltdown^®increases plasma norepinephrine (NE), epinephrine (EPI), glycerol, free fatty acids (FFA), and metabolic rate in men. However, in that investigation measurements ceased at 90 minutes post ingestion, with values for blood borne variables peaking at this time. It was the purpose of the present investigation to extend the time course of measurement to 6 hours, and to include women within the design to determine if sex differences to treatment exist.</p> <p>Methods</p> <p>Ten men (24 ± 4 yrs) and 10 women (22 ± 2 yrs) ingested Meltdown^®or a placebo, using a randomized, cross-over design with one week separating conditions. Blood samples were collected immediately before supplementation and at one hour intervals through 6 hours post ingestion. A standard meal was provided after the hour 3 collection. Samples were assayed for EPI, NE, glycerol, and FFA. Five minute breath samples were collected at each time for measurement of metabolic rate and substrate utilization. Area under the curve (AUC) was calculated. Heart rate and blood pressure were recorded at all times. Data were also analyzed using a 2 (sex) × 2 (condition) × 7 (time) repeated measures analysis of variance, with Tukey <it>post hoc </it>testing.</p> <p>Results</p> <p>No sex × condition interactions were noted for AUC for any variable (p > 0.05). Hence, AUC data are collapsed across men and women. AUC was greater for Meltdown^®compared to placebo for EPI (367 ± 58 pg·mL^-1·6 hr^-1vs. 183 ± 27 pg·mL^-1·6 hr^-1; p = 0.01), NE (2345 ± 205 pg·mL^-1·6 hr^-1vs. 1659 ± 184 pg·mL^-1·6 hr^-1; p = 0.02), glycerol (79 ± 8 μg·mL^-1·6 hr^-1vs. 59 ± 6 μg·mL^-1·6 hr^-1; p = 0.03), FFA (2.46 ± 0.64 mmol·L^-1·6 hr^-1vs. 1.57 ± 0.42 mmol·L^-1·6 hr^-1; p = 0.05), and kilocalorie expenditure (439 ± 26 kcal·6 hrs^-1vs. 380 ± 14 kcal·6 hrs^-1; p = 0.02). No effect was noted for substrate utilization (p = 0.39). Both systolic and diastolic blood pressure (p < 0.0001; 1–16 mmHg), as well as heart rate (p = 0.01; 1–9 bpm) were higher for Meltdown^®. No sex × condition × time interactions were noted for any variable (p > 0.05).</p> <p>Conclusion</p> <p>Ingestion of Meltdown^®results in an increase in catecholamine secretion, lipolysis, and metabolic rate in young men and women, with a similar response for both sexes. Meltdown^®may prove to be an effective intervention strategy for fat loss, assuming individuals are normotensive and their treatment is monitored by a qualified health care professional.</p

Centrifugation and decontamination procedures selectively impair recovery of important populations in Mycobacterium smegmatis

This work was supported by PreDiCT-TB (SMDO XEU-07).Diagnosis and treatment monitoring of patients with tuberculosis (TB) requires detection of all viable mycobacteria in clinical samples. Quantitation of Mycobacterium tuberculosis (Mtb) in sputum is commonly performed by culture after sample decontamination to prevent overgrowth by contaminant organisms. Exponentially growing cultures have cells that predominately lack non-polar lipid bodies whereas stationary cultures have a predominance of cells with non-polar lipid bodies. This may reflect rapidly growing ‘active’ and non-replicating ‘persister’ sub-populations respectively in sputum from TB patients. We investigated the effect of decontamination on culture-based quantitation of exponential and stationary phase cultures of Mycobacterium smegmatis in an artificial sputum model. Exponentially growing populations were between 89 and 50 times more susceptible to decontamination than stationary phase cultures when quantified by most probable number and colony forming units. These findings suggest that decontamination selectively eliminates the ‘active’ population. This may impair diagnostic sensitivity, treatment monitoring, and compromise clinical trials designed to identify new antibiotic combinations with activity against all mycobacterial cell states.PostprintPeer reviewe

Atom Interferometers

Interference with atomic and molecular matter waves is a rich branch of atomic physics and quantum optics. It started with atom diffraction from crystal surfaces and the separated oscillatory fields technique used in atomic clocks. Atom interferometry is now reaching maturity as a powerful art with many applications in modern science. In this review we first describe the basic tools for coherent atom optics including diffraction by nanostructures and laser light, three-grating interferometers, and double wells on AtomChips. Then we review scientific advances in a broad range of fields that have resulted from the application of atom interferometers. These are grouped in three categories: (1) fundamental quantum science, (2) precision metrology and (3) atomic and molecular physics. Although some experiments with Bose Einstein condensates are included, the focus of the review is on linear matter wave optics, i.e. phenomena where each single atom interferes with itself.Comment: submitted to Reviews of Modern Physic

Pelvic mass associated with raised CA 125 for benign condition: a case report

<p>Abstract</p> <p>Background</p> <p>Raised CA 125 with associated pelvic mass is highly suggestive of ovarian malignancy, but there are various other benign conditions that can be associated with pelvic mass and a raised CA 125.</p> <p>Case presentation</p> <p>We present a case of 19 year old, Caucasian British woman who presented initially with sudden onset right sided iliac fossa pain and on imaging was found to have 9.8 × 4.5 cm complex cystic mass in right adnexa with a raised CA 125 of 657, which was initially thought to be highly suspicious of cancer but was subsequently found to be due to pelvic inflammatory disease on histology.</p> <p>Conclusion</p> <p>This case highlights the fact that though a pelvic mass with raised CA 125 is highly suggestive of malignancy, pelvic inflammatory disease should always be considered as a differential diagnosis especially in a young patient and a thorough sexual history and screening for pelvic infection should always be carried out in these patients.</p

RASSF1A–LATS1 signalling stabilizes replication forks by restricting CDK2-mediated phosphorylation of BRCA2

Genomic instability is a key hallmark of cancer leading to tumour heterogeneity and therapeutic resistance. ​BRCA2 has a fundamental role in error-free DNA repair but also sustains genome integrity by promoting ​RAD51 nucleofilament formation at stalled replication forks. ​CDK2 phosphorylates ​BRCA2 (pS3291-​BRCA2) to limit stabilizing contacts with polymerized ​RAD51; however, how replication stress modulates ​CDK2 activity and whether loss of pS3291-​BRCA2 regulation results in genomic instability of tumours are not known. Here we demonstrate that the Hippo pathway kinase ​LATS1 interacts with ​CDK2 in response to genotoxic stress to constrain pS3291-​BRCA2 and support ​RAD51 nucleofilaments, thereby maintaining genomic fidelity during replication stalling. We also show that ​LATS1 forms part of an ​ATR-mediated response to replication stress that requires the tumour suppressor ​RASSF1A. Importantly, perturbation of the ​ATR–​RASSF1A–​LATS1 signalling axis leads to genomic defects associated with loss of ​BRCA2 function and contributes to genomic instability and ‘BRCA-ness’ in lung cancers

The prevalence of disordered eating in elite male and female soccer players

PurposeTo examine the prevalence of disordered eating (DE) in elite male and female soccer players and the influence of perfectionism.MethodsUsing a cross-sectional design, elite male (n = 137) and female (n = 70) soccer players and non-athlete controls (n = 179) completed the clinical perfectionism questionnaire (CPQ-12) and the eating attitudes test (EAT-26) to assess perfectionism and DE risk, respectively.ResultsMale soccer players had higher EAT-26 scores than controls (10.4 ± 9.9 vs. 6.8 ± 6.7; P = 0.001), but there were no differences in the prevalence of clinical levels of DE (EAT-26 score ≥ 20) (15 vs. 5%, respectively; X2 = 0.079) The proportion of females with DE risk was higher in controls [EAT-26: 13.9 ± 11.6 (25% of population)] than female players [EAT-26: 10.0 ± 9.0% (11% of population)] (X2 = 0.001). With linear regression, perfectionism explained 20% of the variation in DE risk in males (P = 0.001); in females, athletic status (player vs. control) and perfectionism were significant predictors of DE risk, explaining 21% of the variation (P = 0.001). Male reserve team players had higher EAT-26 (+ 3.5) and perfectionism (+ 2.7) scores than first-team players (P ConclusionsThe prevalence of DE risk was not different in elite male and female soccer players; in fact, the prevalence was greatest in non-athlete female controls. Perfectionism is a significant predictor of DE risk in males and females.Level of evidenceIII, case–control study.</div

Formulation Pre-screening of Inhalation Powders Using Computational Atom–Atom Systematic Search Method

The synthonic modeling approach provides a molecule-centered understanding of the surface properties of crystals. It has been applied extensively to understand crystallization processes. This study aimed to investigate the functional relevance of synthonic modeling to the formulation of inhalation powders by assessing cohesivity of three active pharmaceutical ingredients (APIs, fluticasone propionate (FP), budesonide (Bud), and salbutamol base (SB)) and the commonly used excipient, α-lactose monohydrate (LMH). It is found that FP (−11.5 kcal/mol) has a higher cohesive strength than Bud (−9.9 kcal/mol) or SB (−7.8 kcal/mol). The prediction correlated directly to cohesive strength measurements using laser diffraction, where the airflow pressure required for complete dispersion (CPP) was 3.5, 2.0, and 1.0 bar for FP, Bud, and SB, respectively. The highest cohesive strength was predicted for LMH (−15.9 kcal/mol), which did not correlate with the CPP value of 2.0 bar (i.e., ranking lower than FP). High FP–LMH adhesive forces (−11.7 kcal/mol) were predicted. However, aerosolization studies revealed that the FP–LMH blends consisted of agglomerated FP particles with a large median diameter (∼4–5 μm) that were not disrupted by LMH. Modeling of the crystal and surface chemistry of LMH identified high electrostatic and H-bond components of its cohesive energy due to the presence of water and hydroxyl groups in lactose, unlike the APIs. A direct comparison of the predicted and measured cohesive balance of LMH with APIs will require a more in-depth understanding of highly hydrogen-bonded systems with respect to the synthonic engineering modeling tool, as well as the influence of agglomerate structure on surface–surface contact geometry. Overall, this research has demonstrated the possible application and relevance of synthonic engineering tools for rapid pre-screening in drug formulation and design