Effect of hydrogen peroxide production and the Fenton reaction on membrane composition of Streptococcus pneumoniae

AbstractAs part of its aerobic metabolism, Streptococcus pneumoniae generates high levels of H2O2 by pyruvate oxidase (SpxB), which can be further reduced to yield the damaging hydroxyl radicals via the Fenton reaction. A universal conserved adaptation response observed among bacteria is the adjustment of the membrane fatty acids to various growth conditions. The aim of the present study was to reveal the effect of endogenous reactive oxygen species (ROS) formation on membrane composition of S. pneumoniae. Blocking carbon aerobic metabolism, by growing the bacteria at anaerobic conditions or by the truncation of the spxB gene, resulted in a significant enhancement in fatty acid unsaturation, mainly cis-vaccenic acid. Moreover, reducing the level of OH· by growing the bacteria at acidic pH, or in the presence of an OH· scavenger (salicylate), resulted in increased fatty acid unsaturation, similar to that obtained under anaerobic conditions. RT-PCR results demonstrated that this change does not originate from a change in mRNA expression level of the fatty acid synthase II genes. We suggest that endogenous ROS play an important regulatory role in membrane adaptation, allowing the survival of this anaerobic organism at aerobic environments of the host

Support and Assessment for Fall Emergency Referrals (SAFER 1) trial protocol. Computerised on-scene decision support for emergency ambulance staff to assess and plan care for older people who have fallen: evaluation of costs and benefits using a pragmatic cluster randomised trial

Background: Many emergency ambulance calls are for older people who have fallen. As half of them are left at home, a community-based response may often be more appropriate than hospital attendance. The SAFER 1 trial will assess the costs and benefits of a new healthcare technology - hand-held computers with computerised clinical decision support (CCDS) software - to help paramedics decide who needs hospital attendance, and who can be safely left at home with referral to community falls services. Methods/Design: Pragmatic cluster randomised trial with a qualitative component. We shall allocate 72 paramedics ('clusters') at random between receiving the intervention and a control group delivering care as usual, of whom we expect 60 to complete the trial. Patients are eligible if they are aged 65 or older, live in the study area but not in residential care, and are attended by a study paramedic following an emergency call for a fall. Seven to 10 days after the index fall we shall offer patients the opportunity to opt out of further follow up. Continuing participants will receive questionnaires after one and 6 months, and we shall monitor their routine clinical data for 6 months. We shall interview 20 of these patients in depth. We shall conduct focus groups or semi-structured interviews with paramedics and other stakeholders. The primary outcome is the interval to the first subsequent reported fall (or death). We shall analyse this and other measures of outcome, process and cost by 'intention to treat'. We shall analyse qualitative data thematically. Discussion: Since the SAFER 1 trial received funding in August 2006, implementation has come to terms with ambulance service reorganisation and a new national electronic patient record in England. In response to these hurdles the research team has adapted the research design, including aspects of the intervention, to meet the needs of the ambulance services. In conclusion this complex emergency care trial will provide rigorous evidence on the clinical and cost effectiveness of CCDS for paramedics in the care of older people who have fallen

Paramedic assessment of older adults after falls, including community care referral pathway : cluster randomized trial

Study objective We aim to determine clinical and cost-effectiveness of a paramedic protocol for the care of older people who fall. Methods We undertook a cluster randomized trial in 3 UK ambulance services between March 2011 and June 2012. We included patients aged 65 years or older after an emergency call for a fall, attended by paramedics based at trial stations. Intervention paramedics could refer the patient to a community-based falls service instead of transporting the patient to the emergency department. Control paramedics provided care as usual. The primary outcome was subsequent emergency contacts or death. Results One hundred five paramedics based at 14 intervention stations attended 3,073 eligible patients; 110 paramedics based at 11 control stations attended 2,841 eligible patients. We analyzed primary outcomes for 2,391 intervention and 2,264 control patients. One third of patients made further emergency contacts or died within 1 month, and two thirds within 6 months, with no difference between groups. Subsequent 999 call rates within 6 months were lower in the intervention arm (0.0125 versus 0.0172; adjusted difference –0.0045; 95% confidence interval –0.0073 to –0.0017). Intervention paramedics referred 8% of patients (204/2,420) to falls services and left fewer patients at the scene without any ongoing care. Intervention patients reported higher satisfaction with interpersonal aspects of care. There were no other differences between groups. Mean intervention cost was $23 per patient, with no difference in overall resource use between groups at 1 or 6 months. Conclusion A clinical protocol for paramedics reduced emergency ambulance calls for patients attended for a fall safely and at modest cost

Differences in Membrane Fluidity and Fatty Acid Composition between Phenotypic Variants of Streptococcus pneumoniae

Phase variation in the colonial opacity of Streptococcus pneumoniae has been implicated as a factor in the pathogenesis of pneumococcal disease. This study examined the relationship between membrane characteristics and colony morphology in a few selected opaque-transparent couples of S. pneumoniae strains carrying different capsular types. Membrane fluidity was determined on the basis of intermolecular excimerization of pyrene and fluorescence polarization of 1,6-diphenyl 1,3,5-hexatriene (DPH). A significant decrease, 16 to 26% (P ≤ 0.05), in the excimerization rate constant of the opaque variants compared with that of the transparent variants was observed, indicating higher microviscosity of the membrane of bacterial cells in the opaque variants. Liposomes prepared from phospholipids of the opaque phenotype showed an even greater decrease, 27 to 38% (P ≤ 0.05), in the pyrene excimerization rate constant compared with that of liposomes prepared from phospholipids of bacteria with the transparent phenotype. These findings agree with the results obtained with DPH fluorescence anisotropy, which showed a 9 to 21% increase (P ≤ 0.001) in the opaque variants compared with the transparent variants. Membrane fatty acid composition, determined by gas chromatography, revealed that the two variants carry the same types of fatty acids but in different proportions. The trend of modification points to the presence of a lower degree of unsaturated fatty acids in the opaque variants compared with their transparent counterparts. The data presented here show a distinct correlation between phase variation and membrane fluidity in S. pneumoniae. The changes in membrane fluidity most probably stem from the observed differences in fatty acid composition

Metabolite Profiling and Integrative Modeling Reveal Metabolic Constraints for Carbon Partitioning under Nitrogen-Starvation in the Green Alga Haematococcus pluvialis.

The green alga Haematococcus pluvialis accumulates large amounts of the antioxidant astaxanthin under inductive stress conditions, such as nitrogen starvation. The response to nitrogen starvation and high-light leads to the accumulation of carbohydrates and fatty acids, as well as increased activity of the tricarboxylic acid cycle. Although the behavior of individual pathways is well-investigated, little is known about the systemic effects of the stress-response mechanism. Here we present time-resolved metabolite, enzyme activity, and physiological data that capture the metabolic response of H. pluvialis under nitrogen starvation and high-light. The data were integrated into a putative genome-scale model of the green alga to in silico test the hypothesis of underlying carbon partitioning. The model-based hypothesis testing reinforces the involvement of starch degradation to support fatty acid synthesis in the later stages of the stress response. In addition, our findings support a possible mechanism for the involvement of the increased activity of the tricarboxylic acid cycle in carbon repartitioning. Finally, the in vitro experiments and the in silico modeling presented here emphasize the predictive power of large-scale integrative approaches to pinpoint metabolic adjustment to changing environments

Metabolite Profiling and Integrative Modeling Reveal Metabolic Constraints for Carbon Partitioning under Nitrogen Starvation in the Green Algae Haematococcus pluvialis

The green alga Haematococcus pluvialis accumulates large amounts of the antioxidant astaxanthin under inductive stress conditions, such as nitrogen starvation. The response to nitrogen starvation and high-light leads to the accumulation of carbohydrates and fatty acids, as well as increased activity of the tricarboxylic acid cycle. Although the behavior of individual pathways is well-investigated, little is known about the systemic effects of the stress-response mechanism. Here we present time-resolved metabolite, enzyme activity, and physiological data that capture the metabolic response of H. pluvialis under nitrogen starvation and high-light. The data were integrated into a putative genome-scale model of the green alga to in silico test the hypothesis of underlying carbon partitioning. The model-based hypothesis testing reinforces the involvement of starch degradation to support fatty acid synthesis in the later stages of the stress response. In addition, our findings support a possible mechanism for the involvement of the increased activity of the tricarboxylic acid cycle in carbon repartitioning. Finally, the in vitro experiments and the in silico modeling presented here emphasize the predictive power of large-scale integrative approaches to pinpoint metabolic adjustment to changing environments