A Genetic and Physiological Study of the Role of Extracellular Copper-Binding Proteins in Copper Detoxification by the Marine Bacterium \u3ci\u3eVibrio alginolyticus\u3c/i\u3e

Supernatant proteins in Vibrio alginolyticus batch cultures were analyzed by SDS-PAGE before copper was added, 24 and 48 hours after the addition of copper, and in 24 hour control (no Cu) cultures. Two proteins, one 21 kilodalton (kDa) and one 19 kDa, were found to be copper-induced, and were designated copper-binding protein 1 (CuBP1) and CuBP2. CuBP1 and CuBP2 became detectable in supernatants during the Cu-induced lag phase, and increased in concentration over the following 48 hours. Chloramphenicol inhibited production of these proteins. Gel-to-gel variability was implicated as the dominant factor determining whether one or two Cu-induced proteins were detected in Vibrio alginolyticus supernatants, and ca. 20 kDa Cu-induced proteins were quantitated together in subsequent analyses. Experiments in continuous (chemostat) cultures of Vibrio alginolyticus demonstrated that the bacteria could survive copper stress in an open system. Copper stress reversibly inhibited swarming in most colonies from long-term copper-stressed cultures, and permanent inhibition of swarming was observed in some isolates. Mutation to an oxidase negative phenotype, which was not reversible, occurred at high frequency in copper-stressed continuous cultures. The stability of two Cur mutants isolated from continuous culture was demonstrated by subculturing each isolate ten times on nonselective marine agar (10° MA), and comparing plate counts on unamended and 40μM Cu-amended agar to corresponding plate counts of isolates freshly passed on Cu-amended agar. One Cur isolate, Cu40B3, constitutively produced a ca. 21 kDa protein which displayed the same chromatographic behavior (immobilized metal ion affinity chromatography followed by reverse phase high performance liquid chromatography) as CuBP. After fifteen nonselective subcultures, a revertant Cus derivative of Cu40B3 (Cu40B3(SW)) was isolated. Cu40B3(SW) lost the mutation to constitutive CuBP production and copper resistance simultaneously, indicating that constitutive CuBP production in Cu40B3 is necessary for maintenance of its copper-resistant phenotype. Copper-sensitive Vibrio alginolyticus mutants displayed a range of alterations in supernatant protein profiles, and two of the seven mutants were indistinguishable from the wild-type in terms of supernatant proteins with and without copper stress. One Cus mutant was isolated which contained no CuaP in supernatants from 50 μM copper-stressed cultures

To what extent do water reuse treatments reduce antibiotic resistance indicators? A comparison of two full-scale systems

Water reuse is an essential strategy for reducing water demand from conventional sources, alleviating water stress, and promoting sustainability, but understanding the effectiveness of associated treatment processes as barriers to the spread of antibiotic resistance is an important consideration to protecting human health. We comprehensively evaluated the reduction of antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB) in two field-operational water reuse systems with distinct treatment trains, one producing water for indirect potable reuse (ozone/biologically-active carbon/granular activated carbon) and the other for non-potable reuse (denitrification-filtration/chlorination) using metagenomic sequencing and culture. Relative abundances of total ARGs/clinically-relevant ARGs and cultured ARB were reduced by several logs during primary and secondary stages of wastewater treatment, but to a lesser extent during the tertiary water reuse treatments. In particular, ozonation tended to enrich multi-drug ARGs. The effect of chlorination was facility-dependent, increasing the relative abundance of ARGs when following biologically-active carbon filters, but generally providing a benefit in reduced bacterial numbers and ecological and human health resistome risk scores. Relative abundances of total ARGs and resistome risk scores were lowest in aquifer samples, although resistant Escherichia coli and Klebsiella pneumoniae were occasionally detected in the monitoring well 3-days downgradient from injection, but not 6-months downgradient. Resistant E. coli and Pseudomonas aeruginosa were occasionally detected in the nonpotable reuse distribution system, along with increased levels of multidrug, sulfonamide, phenicol, and aminoglycoside ARGs. This study illuminates specific vulnerabilities of water reuse systems to persistence, selection, and growth of ARGs and ARB and emphasizes the role of multiple treatment barriers, including aquifers and distribution systems

Individual-Based Measure of Socio-Economic Disadvantage: Making Identification "Agile"

This project aimed to develop an individual-based measure of socio-economic disadvantage that can assist Australian universities to identify socio-economic disadvantage at the level of the individual student. A mixed-methods design was used. Phase 1 comprised an online questionnaire distributed to staff working in the area of widening participation and student support at each of the 42 universities in Australia (n = 256) and an online questionnaire for undergraduate students at three universities (one a member of the Group of Eight and the other two members of the Innovative Research Universities group – one a multi-campus metropolitan university and one a rural university) (n = 4,114). In Phase 2, five focus groups were conducted with staff and six with students to further explore the issues raised in Phase 1. Phase 3 consisted of a desk audit of university websites to provide data on the information publicly available to students experiencing socio-economic disadvantage. In the final phase, Phase 4, a possible approach to measuring disadvantage was evaluated through its presentation to students at two universities which had been part of Phase 1 (n = 91). The following factors were found to be indicators of socio-economic disadvantage. Whether the student: provides financially for their family; is the first in family to attend university; is experiencing financial hardship; and/or is in receipt of Youth Allowance, Austudy or ABSTUDY during Years 11 and/or 12 (or another Centrelink income and asset-tested entitlement) for a period of at least three months during Years 11 and/or 12 or equivalent. These were found to be questions that students were willing to answer and which would lead to effective and efficient identification

Beach sand and the potential for infectious disease transmission: observations and recommendations

Recent studies suggest that sand can serve as a vehicle for exposure of humans to pathogens at beach sites, resulting in increased health risks. Sampling for microorganisms in sand should therefore be considered for inclusion in regulatory programmes aimed at protecting recreational beach users from infectious disease. Here, we review the literature on pathogen levels in beach sand, and their potential for affecting human health. In an effort to provide specific recommendations for sand sampling programmes, we outline published guidelines for beach monitoring programmes, which are currently focused exclusively on measuring microbial levels in water. We also provide background on spatial distribution and temporal characteristics of microbes in sand, as these factors influence sampling programmes. First steps toward establishing a sand sampling programme include identifying appropriate beach sites and use of initial sanitary assessments to refine site selection. A tiered approach is recommended for monitoring. This approach would include the analysis of samples from many sites for faecal indicator organisms and other conventional analytes, while testing for specific pathogens and unconventional indicators is reserved for high-risk sites. Given the diversity of microbes found in sand, studies are urgently needed to identify the most significant aetiological agent of disease and to relate microbial measurements in sand to human health risk

Microbes in beach sands : integrating environment, ecology and public health

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Reviews in Environmental Science and Bio/Technology 13 (2014): 329-368, doi:10.1007/s11157-014-9340-8.Beach sand is a habitat that supports many microbes, including viruses, bacteria, fungi and protozoa (micropsammon). The apparently inhospitable conditions of beach sand environments belie the thriving communities found there. Physical factors, such as water availability and protection from insolation; biological factors, such as competition, predation, and biofilm formation; and nutrient availability all contribute to the characteristics of the micropsammon. Sand microbial communities include autochthonous species/phylotypes indigenous to the environment. Allochthonous microbes, including fecal indicator bacteria (FIB) and waterborne pathogens, are deposited via waves, runoff, air, or animals. The fate of these microbes ranges from death, to transient persistence and/or replication, to establishment of thriving populations (naturalization) and integration in the autochthonous community. Transport of the micropsammon within the habitat occurs both horizontally across the beach, and vertically from the sand surface and ground water table, as well as at various scales including interstitial flow within sand pores, sediment transport for particle-associated microbes, and the large-scale processes of wave action and terrestrial runoff. The concept of beach sand as a microbial habitat and reservoir of FIB and pathogens has begun to influence our thinking about human health effects associated with sand exposure and recreational water use. A variety of pathogens have been reported from beach sands, and recent epidemiology studies have found some evidence of health risks associated with sand exposure. Persistent or replicating populations of FIB and enteric pathogens have consequences for watershed/beach management strategies and regulatory standards for safe beaches. This review summarizes our understanding of the community structure, ecology, fate, transport, and public health implications of microbes in beach sand. It concludes with recommendations for future work in this vastly under-studied area.2015-05-0

Dihimo-γ-linolenic acid inhibits several key cellular processes associated with atherosclerosis

Atherosclerosis and its complications are responsible for one in three global deaths. Nutraceuticals show promise in the prevention and treatment of atherosclerosis but require an indepth understanding of the mechanisms underlying their actions. A previous study showed that the omega-6 fatty acid, dihomo-γ-linolenic acid (DGLA), attenuated atherosclerosis in the apolipoprotein E deficient mouse model system. However, the mechanisms underlying such protective effects of DGLA are poorly understood and were therefore investigated. We show that DGLA attenuates chemokine-driven monocytic migration together with foam cell formation and the expression of key pro-atherogenic genes induced by three pro-inflammatory cytokines in human macrophages. The effect of DGLA on interferon-γ signaling was mediated via inhibition of signal transducer and activator of transcription-1 phosphorylation on serine 727. In relation to anti-foam cell action, DGLA inhibits modified LDL uptake by both macropinocytosis and receptor-mediated endocytosis, the latter by reduction in expression of two key scavenger receptors (SR-A and CD36), and stimulates cholesterol efflux from foam cells. DGLA also improves macrophage mitochondrial bioenergetic profile by decreasing proton leak. Gamma-linolenic acid and prostaglandin E1, upstream precursor and key metabolite respectively of DGLA, also acted in an anti-atherogenic manner. The actions of DGLA extended to other key atherosclerosis-associated cell types with attenuation of endothelial cell proliferation and migration of smooth muscle cells in response to platelet-derived growth factor. This study provides novel insights into the molecular mechanisms underlying the anti-atherogenic actions of DGLA and supports further assessments on its protective effects on plaque regression in vivo and in human trials

Lipid Classes and Fatty Acid Patterns are Altered in the Brain of γ-Synuclein Null Mutant Mice

The well-documented link between α-synuclein and the pathology of common human neurodegenerative diseases has increased attention to the synuclein protein family. The involvement of α-synuclein in lipid metabolism in both normal and diseased nervous system has been shown by many research groups. However, the possible involvement of γ-synuclein, a closely-related member of the synuclein family, in these processes has hardly been addressed. In this study, the effect of γ-synuclein deficiency on the lipid composition and fatty acid patterns of individual lipids from two brain regions has been studied using a mouse model. The level of phosphatidylserine (PtdSer) was increased in the midbrain whereas no changes in the relative proportions of membrane polar lipids were observed in the cortex of γ-synuclein-deficient compared to wild-type (WT) mice. In addition, higher levels of docosahexaenoic acid were found in PtdSer and phosphatidylethanolamine (PtdEtn) from the cerebral cortex of γ-synuclein null mutant mice. These findings show that γ-synuclein deficiency leads to alterations in the lipid profile in brain tissues and suggest that this protein, like α-synuclein, might affect neuronal function via modulation of lipid metabolism

Effectiveness of a national quality improvement programme to improve survival after emergency abdominal surgery (EPOCH): a stepped-wedge cluster-randomised trial

Background: Emergency abdominal surgery is associated with poor patient outcomes. We studied the effectiveness of a national quality improvement (QI) programme to implement a care pathway to improve survival for these patients. Methods: We did a stepped-wedge cluster-randomised trial of patients aged 40 years or older undergoing emergency open major abdominal surgery. Eligible UK National Health Service (NHS) hospitals (those that had an emergency general surgical service, a substantial volume of emergency abdominal surgery cases, and contributed data to the National Emergency Laparotomy Audit) were organised into 15 geographical clusters and commenced the QI programme in a random order, based on a computer-generated random sequence, over an 85-week period with one geographical cluster commencing the intervention every 5 weeks from the second to the 16th time period. Patients were masked to the study group, but it was not possible to mask hospital staff or investigators. The primary outcome measure was mortality within 90 days of surgery. Analyses were done on an intention-to-treat basis. This study is registered with the ISRCTN registry, number ISRCTN80682973. Findings: Treatment took place between March 3, 2014, and Oct 19, 2015. 22 754 patients were assessed for elegibility. Of 15 873 eligible patients from 93 NHS hospitals, primary outcome data were analysed for 8482 patients in the usual care group and 7374 in the QI group. Eight patients in the usual care group and nine patients in the QI group were not included in the analysis because of missing primary outcome data. The primary outcome of 90-day mortality occurred in 1210 (16%) patients in the QI group compared with 1393 (16%) patients in the usual care group (HR 1·11, 0·96–1·28). Interpretation: No survival benefit was observed from this QI programme to implement a care pathway for patients undergoing emergency abdominal surgery. Future QI programmes should ensure that teams have both the time and resources needed to improve patient care. Funding: National Institute for Health Research Health Services and Delivery Research Programme