Mutation bias and adaptation in bacteria.

Genetic mutation, which provides the raw material for evolutionary adaptation, is largely a stochastic force. However, there is ample evidence showing that mutations can also exhibit strong biases, with some mutation types and certain genomic positions mutating more often than others. It is becoming increasingly clear that mutational bias can play a role in determining adaptive outcomes in bacteria in both the laboratory and the clinic. As such, understanding the causes and consequences of mutation bias can help microbiologists to anticipate and predict adaptive outcomes. In this review, we provide an overview of the mechanisms and features of the bacterial genome that cause mutational biases to occur. We then describe the environmental triggers that drive these mechanisms to be more potent and outline the adaptive scenarios where mutation bias can synergize with natural selection to define evolutionary outcomes. We conclude by describing how understanding mutagenic genomic features can help microbiologists predict areas sensitive to mutational bias, and finish by outlining future work that will help us achieve more accurate evolutionary forecasts.

Mutational hotspots lead to robust but suboptimal adaptive outcomes in certain environments.

The observed mutational spectrum of adaptive outcomes can be constrained by many factors. For example, mutational biases can narrow the observed spectrum by increasing the rate of mutation at isolated sites in the genome. In contrast, complex environments can shift the observed spectrum by defining fitness consequences of mutational routes. We investigate the impact of different nutrient environments on the evolution of motility in Pseudomonas fluorescens Pf0-2x (an engineered non-motile derivative of Pf0-1) in the presence and absence of a strong mutational hotspot. Previous work has shown that this mutational hotspot can be built and broken via six silent mutations, which provide rapid access to a mutation that rescues swimming motility and confers the strongest swimming phenotype in specific environments. Here, we evolved a hotspot and non-hotspot variant strain of Pf0-2x for motility under nutrient-rich (LB) and nutrient-limiting (M9) environmental conditions. We observed the hotspot strain consistently evolved faster across all environmental conditions and its mutational spectrum was robust to environmental differences. However, the non-hotspot strain had a distinct mutational spectrum that changed depending on the nutrient environment. Interestingly, while alternative adaptive mutations in nutrient-rich environments were equal to, or less effective than, the hotspot mutation, the majority of these mutations in nutrient-limited conditions produced superior swimmers. Our competition experiments mirrored these findings, underscoring the role of environment in defining both the mutational spectrum and the associated phenotype strength. This indicates that while mutational hotspots working in concert with natural selection can speed up access to robust adaptive mutations (which can provide a competitive advantage in evolving populations), they can limit exploration of the mutational landscape, restricting access to potentially stronger phenotypes in specific environments.

Predicting Delayed Shock in Multisystem Inflammatory Disease in Children: A Multicenter Analysis From the New York City Tri-State Region.

OBJECTIVES: Patients with multisystem inflammatory disease in children (MIS-C) are at risk of developing shock. Our objectives were to determine independent predictors associated with development of delayed shock (≥3 hours from emergency department [ED] arrival) in patients with MIS-C and to derive a model predicting those at low risk for delayed shock. METHODS: We conducted a retrospective cross-sectional study of 22 pediatric EDs in the New York City tri-state area. We included patients meeting World Health Organization criteria for MIS-C and presented April 1 to June 30, 2020. Our main outcomes were to determine the association between clinical and laboratory factors to the development of delayed shock and to derive a laboratory-based prediction model based on identified independent predictors. RESULTS: Of 248 children with MIS-C, 87 (35%) had shock and 58 (66%) had delayed shock. A C-reactive protein (CRP) level greater than 20 mg/dL (adjusted odds ratio [aOR], 5.3; 95% confidence interval [CI], 2.4-12.1), lymphocyte percent less than 11% (aOR, 3.8; 95% CI, 1.7-8.6), and platelet count less than 220,000/uL (aOR, 4.2; 95% CI, 1.8-9.8) were independently associated with delayed shock. A prediction model including a CRP level less than 6 mg/dL, lymphocyte percent more than 20%, and platelet count more than 260,000/uL, categorized patients with MIS-C at low risk of developing delayed shock (sensitivity 93% [95% CI, 66-100], specificity 38% [95% CI, 22-55]). CONCLUSIONS: Serum CRP, lymphocyte percent, and platelet count differentiated children at higher and lower risk for developing delayed shock. Use of these data can stratify the risk of progression to shock in patients with MIS-C, providing situational awareness and helping guide their level of care.

The association of penicillin allergy with outcomes after open ventral hernia repair.

BACKGROUND: Up to 11% of patients report a penicillin allergy (PA), with 1-2% demonstrating a true IgE mediated allergy upon testing. PA patients often receive non-beta-lactam antibiotic surgical prophylaxis (non-BLP). This study evaluates the relationship of PA to outcomes after open ventral hernia repair (OVHR). METHODS: A prospective institutional database was queried for patients undergoing OVHR. Demographics, operative characteristics, and outcomes were evaluated by the reported PA and the administration of beta-lactam prophylaxis (BLP). RESULTS: Allergy histories were reviewed in 1178 patients. PA was reported in 21.6% of patients, with 55.5% reporting rash or hives, 15.0% airway compromise or anaphylaxis, and 29.5% no specific reaction. BLP was administered to 76.3% of patients, including 22.1% of PA patients and 89.9% of patients without PA. PA patients were more often female (64.6% PA patients vs. 56% non-PA, p = 0.01), with higher rates of chronic steroids, MRSA, anxiety, asthma, COPD, chronic pain, and sleep apnea (p < 0.03 all values). PA patients had higher rates of contaminated cases, including mesh infection and fistula. Of the 683 clean cases, 82.1% received BLP. Of the 117 clean contaminated cases (CDC wound class 2), 82.9% received BLP, which was associated with reduced long-term readmission for hernia complications (21.5 vs. 55%, p = 0.002, OR 0.27, CI 0.09-0.83). In the 120 CDC wound class 3 and 4 patients, 65.8% received BLP. In multivariate analysis, BLP was associated with lower rates of reoperation (OR 0.31, CI 0.12-0.76) and recurrence (OR 0.32, CI 0.11-0.86). BLP was given to 22.1% of the PA patients with no adverse reactions noted. CONCLUSION: PA patients had more comorbidities and complex ventral hernias. When controlling for contamination and MRSA history, BLP is associated with improved outcomes particularly in contaminated cases. PA may be a risk factor for patient complexity, and further studies are warranted to determine if allergy testing can be warranted in known or anticipated contaminated cases.

Effect of Algorithm-Based Therapy vs Usual Care on Clinical Success and Serious Adverse Events in Patients with Staphylococcal Bacteremia: A Randomized Clinical Trial.

Importance: The appropriate duration of antibiotics for staphylococcal bacteremia is unknown. Objective: To test whether an algorithm that defines treatment duration for staphylococcal bacteremia vs standard of care provides noninferior efficacy without increasing severe adverse events. Design, Setting, and Participants: A randomized trial involving adults with staphylococcal bacteremia was conducted at 16 academic medical centers in the United States (n = 15) and Spain (n = 1) from April 2011 to March 2017. Patients were followed up for 42 days beyond end of therapy for those with Staphylococcus aureus and 28 days for those with coagulase-negative staphylococcal bacteremia. Eligible patients were 18 years or older and had 1 or more blood cultures positive for S aureus or coagulase-negative staphylococci. Patients were excluded if they had known or suspected complicated infection at the time of randomization. Interventions: Patients were randomized to algorithm-based therapy (n = 255) or usual practice (n = 254). Diagnostic evaluation, antibiotic selection, and duration of therapy were predefined for the algorithm group, whereas clinicians caring for patients in the usual practice group had unrestricted choice of antibiotics, duration, and other aspects of clinical care. Main Outcomes and Measures: Coprimary outcomes were (1) clinical success, as determined by a blinded adjudication committee and tested for noninferiority within a 15% margin; and (2) serious adverse event rates in the intention-to-treat population, tested for superiority. The prespecified secondary outcome measure, tested for superiority, was antibiotic days among per-protocol patients with simple or uncomplicated bacteremia. Results: Among the 509 patients randomized (mean age, 56.6 [SD, 16.8] years; 226 [44.4%] women), 480 (94.3%) completed the trial. Clinical success was documented in 209 of 255 patients assigned to algorithm-based therapy and 207 of 254 randomized to usual practice (82.0% vs 81.5%; difference, 0.5% [1-sided 97.5% CI, -6.2% to ∞]). Serious adverse events were reported in 32.5% of algorithm-based therapy patients and 28.3% of usual practice patients (difference, 4.2% [95% CI, -3.8% to 12.2%]). Among per-protocol patients with simple or uncomplicated bacteremia, mean duration of therapy was 4.4 days for algorithm-based therapy vs 6.2 days for usual practice (difference, -1.8 days [95% CI, -3.1 to -0.6]). Conclusions and Relevance: Among patients with staphylococcal bacteremia, the use of an algorithm to guide testing and treatment compared with usual care resulted in a noninferior rate of clinical success. Rates of serious adverse events were not significantly different, but interpretation is limited by wide confidence intervals. Further research is needed to assess the utility of the algorithm. Trial Registration: ClinicalTrials.gov Identifier: NCT01191840.

The cutaneous microbiome in outpatients presenting with acute skin abscesses.

BACKGROUND: Previous studies have demonstrated an association between antibiotic use and the development of skin abscesses. We tested the hypothesis that alterations in the composition of the cutaneous microbiota may predispose individuals to skin abscesses. METHODS: We studied 25 patients with skin abscesses and 25 age-matched controls, who each completed a questionnaire. Skin swab samples were obtained for DNA analysis from 4 sites around the abscess site (hereafter, "peri-abscess specimens") and from similar sites on the patient's contralateral side and on healthy control subjects. DNA was extracted and analyzed by quantitative polymerase chain reaction (qPCR) and high-throughput sequencing. The purulent abscess drainage was sent for culture. RESULTS: Fifteen patients with abscess were infected with Staphylococcus aureus. Use of nuc qPCR to quantitate S. aureus revealed a significantly greater frequency of positive results for peri-abscess and contralateral skin samples, compared with control skin specimens. Analysis of community structure showed greater heterogeneity in the control samples than in the peri-abscess and contralateral samples. Metagenomic analysis detected significantly more predicted genes related to metabolic activity in the peri-abscess specimens than in the control samples. CONCLUSIONS: The peri-abscess microbiome was similar to the contralateral microbiome, but both microbiomes differed from that for control patients. Host characteristics affecting microbial populations might be important determinants of abscess risk.

Comparison of culture and molecular techniques for microbial community characterization in infected necrotizing pancreatitis.

BACKGROUND: Infected necrotizing pancreatitis is associated with significant morbidity and mortality. Peripancreatic fluid cultures may fail to identify all the infecting organisms. The aim of this study was to compare the bacterial biome of peripancreatic fluid from infected necrotizing pancreatitis patients using 16S ribosomal RNA (rRNA) DNA deep sequencing and quantitative polymerase chain reaction (qPCR) targeting the 16S rRNA gene versus standard laboratory culture. MATERIALS AND METHODS: Peripancreatic fluid was collected during operative or radiologic intervention and samples sent for culture. In parallel, microbial DNA was extracted, qPCR targeting the 16S rRNA gene and 16S rRNA PCR amplification followed by Illumina deep sequencing were performed. RESULTS: Using culture techniques, the bacterial strains most frequently identified were gram-negative rods (Escherichia coli, Klebsiella pneumoniae) and Enterococcus. Samples in which culture results were negative had copy numbers of the 16S rRNA gene close to background in qPCR analysis. For samples with high bacterial load, sequencing results were in some cases in good agreement with culture data, whereas in others there were disagreements, likely due to differences in taxonomic classification, cultivability, and differing susceptibility to background contamination. Sequencing results appeared generally unreliable in cases of negative culture where little microbial DNA was input into qPCR sequencing reactions. CONCLUSIONS: Both sequencing and culture data display their own sources of bias and potential error. Consideration of data from multiple techniques will yield a more accurate view of bacterial infections than can be achieved by any single technique.

Impact of the establishment of a specialty hernia referral center.

BACKGROUND: Creating a surgical specialty referral center requires a strong interest, expertise, and a market demand in that particular field, as well as some form of promotion. In 2004, we established a tertiary hernia referral center. Our goal in this study was to examine its impact on institutional volume and economics. MATERIALS AND METHODS: The database of all hernia repairs (2004-2011) was reviewed comparing hernia repair type and volume and center financial performance. The ventral hernia repair (VHR) patient subset was further analyzed with particular attention paid to previous repairs, comorbidities, referral patterns, and the concomitant involvement of plastic surgery. RESULTS: From 2004 to 2011, 4927 hernia repairs were performed: 39.3% inguinal, 35.5% ventral or incisional, 16.2% umbilical, 5.8% diaphragmatic, 1.6% femoral, and 1.5% other. Annual billing increased yearly from 7% to 85% and averaged 37% per year. Comparing 2004 with 2011, procedural volume increased 234%, and billing increased 713%. During that period, there was a 2.5-fold increase in open VHRs, and plastic surgeon involvement increased almost 8-fold, (P = .004). In 2005, 51 VHR patients had a previous repair, 27.0% with mesh, versus 114 previous VHR in 2011, 58.3% with mesh (P < .0001). For VHR, in-state referrals from 2004 to 2011 increased 340% while out-of-state referrals jumped 580%. In 2011, 21% of all patients had more than 4 comorbidities, significantly increased from 2004 (P = .02). CONCLUSION: The establishment of a tertiary, regional referral center for hernia repair has led to a substantial increase in surgical volume, complexity, referral geography, and financial benefit to the institution.

Pseudomonas aeruginosa's adaptive trajectory: diverse origins, convergent paths.

Does genetic background contribute to populations following the same or divergent adaptive trajectories? A recent study by Filipow et al. evolved multiple genetically distinct Pseudomonas aeruginosa strains to an artificial cystic fibrosis lung sputum media. The strains adapted at different rates but converged on similar phenotypes despite their initial diversity.

Transient mutation bias increases the predictability of evolution on an empirical genotype-phenotype landscape.

Predicting how a population will likely navigate a genotype-phenotype landscape requires consideration of selection in combination with mutation bias, which can skew the likelihood of following a particular trajectory. Strong and persistent directional selection can drive populations to ascend toward a peak. However, with a greater number of peaks and more routes to reach them, adaptation inevitably becomes less predictable. Transient mutation bias, which operates only on one mutational step, can influence landscape navigability by biasing the mutational trajectory early in the adaptive walk. This sets an evolving population upon a particular path, constraining the number of accessible routes and making certain peaks and routes more likely to be realized than others. In this work, we employ a model system to investigate whether such transient mutation bias can reliably and predictably place populations on a mutational trajectory to the strongest selective phenotype or usher populations to realize inferior phenotypic outcomes. For this we use motile mutants evolved from ancestrally non-motile variants of the microbe Pseudomonas fluorescens SBW25, of which one trajectory exhibits significant mutation bias. Using this system, we elucidate an empirical genotype-phenotype landscape, where the hill-climbing process represents increasing strength of the motility phenotype, to reveal that transient mutation bias can facilitate rapid and predictable ascension to the strongest observed phenotype in place of equivalent and inferior trajectories. This article is part of the theme issue 'Interdisciplinary approaches to predicting evolutionary biology'.

Outcomes of total hip and knee arthroplasty in special populations: a synopsis and critical appraisal of systematic reviews.

INTRODUCTION: This study aimed to present and critically appraise the best available evidence investigating associations between some pre-defined patient-related characteristics and perioperative complications or other outcomes in THA and TKA. METHODS: Electronic databases were searched (Medline, EMBASE, Scopus, CENTRAL) for systematic reviews assessing the following pre-defined patient-related characteristics as possible risk factors for worse peri-operative outcomes in THA and TKA: smoking, alcohol excess, rheumatoid arthritis, human immunodeficiency virus infection, hepatitis C virus infection, mental health conditions, and solid organ transplantation. Our primary outcome was periprosthetic joint infection. Results were analysed separately for THA, TKA and THA/TKA (mixed data). RESULTS: Based on at least two systematic reviews being in agreement, the following patient-related characteristics were associated with increased incidence of complications as follows: a) Smoking for all-cause revision in THA, for periprosthetic joint infection in TKA and THA/TKA; b) alcohol excess for periprosthetic joint infection in THA/TKA; c) human immunodeficiency virus for periprosthetic joint infection in THA/TKA; d) hepatitis C virus for overall complications, periprosthetic joint infection and all-cause revision in THA and THA/TKA, and for overall complications in TKA. Our study found conflicting evidence for a) smoking as a risk factor for periprosthetic joint infection and aseptic loosening in THA; b) human immunodeficiency virus as a risk factor for all-cause revision for THA/TKA; c) hepatitis C virus as a risk factor for periprosthetic joint infection and all-cause revision in TKA. No certainty of evidence was assigned to these results as this was not assessed by the authors of the majority of the included systematic reviews. CONCLUSION: We found that smoking, excess alcohol consumption, RA, and infection with HIV and HCV were associated with a higher incidence of periprosthetic joint infection in one or both of THA and TKA or mixed THA/TKA data. All our results should be interpreted and communicated to patients with caution as the quality of the included systematic reviews was generally poor.

Cutaneous Microbiome Profiles Following Chlorhexidine Treatment in a 72-Hour Daily Follow-Up Paired Design: a Pilot Study.

Venous catheter-related bloodstream infections represent a significant problem in the United States. Our objective was to determine daily changes in skin microbiome profiles up to 72h postchlorhexidine treatment. Left and right forearm skin swab samples were obtained from 10 healthy volunteers over 72h at 24h intervals. Dorsal surface of left arm was treated with chlorohexidine gluconate (CHG) at initial time point (T = 0), while the right arm remained untreated (control). Swab samples were obtained shortly before (T = 0) and after CHG treatment (T = 24-48-72h). Bacterial DNA extraction, 16S rRNA gene V1-V3 sequencing and taxonomic annotation were performed using ZymoBIOMICS pipeline. PERMANOVA, linear discriminant and bacterial interaction network analyses were performed. A total of 13 total phyla, 273 genera, and 950 total species were detected across all time points, CHG-treated or CHG-untreated. Most abundant species included Cutibacterium acnes, Staphylococcus epidermidis, and Rothia Mucilaginosa. Low biomass-related inconsistent taxa detection was observed. PERMANOVA suggested a marginal difference between CHG-treated and CHG-untreated microbiome profiles (Genera: P(perm) = 0.0531; Species: P(perm) = 0.0450). Bacterial interaction network guided PERMANOVA analyses detected a microbiome change over time, suggesting a consistent CHG treatment-specific change. LEfSe identified Finegoldia magna, Bacillus pumilus, Bacillus thermoamylovorans as the only distinctive species. These species were more abundant and/or present post-CHG treatment in the CHG-treated group. These findings suggest that the skin microbiome was not significantly different 24, 48, or 72h after CHG treatment. Previous culture-based studies have found similar results after 24h. Future studies will be needed to determine the mechanisms of bacterial regrowth after CHG treatment. IMPORTANCE Annually, over 80,000 central line infections occur in the United States. Understanding the pathogenesis of these infections is crucial. Chlorhexidine is the most commonly used skin preparation before line placement. We hypothesized that the use of chlorhexidine and dressings will alter the normal arm skin microbiome over a period of 72h. We used 16S-rRNA gene next generation sequencing (NGS) to determine the forearm skin microbiome of volunteers. The left arm was swabbed with chlorhexidine and the right arm served as control. The skin microbiome returned to normal after 24h. Our NGS results confirm findings of two previous culture-based studies. Relative abundance of Bacillus spp. in the chlorhexidine-treated samples was increased, consistent with one previous study. Based on the results of this pilot study, we will need to measure viable bacteria during a 24h time course following chlorhexidine treatment to understand the source of skin microbiome replenishment.

Association between timing of antibiotic administration and mortality from septic shock in patients treated with a quantitative resuscitation protocol.

OBJECTIVE: We sought to determine the association between time to initial antibiotics and mortality of patients with septic shock treated with an emergency department-based early resuscitation protocol. DESIGN: Preplanned analysis of a multicenter randomized controlled trial of early sepsis resuscitation. SETTING: Three urban U.S. emergency departments. PATIENTS: Adult patients with septic shock. INTERVENTIONS: A quantitative resuscitation protocol in the emergency department targeting three physiological variables: central venous pressure, mean arterial pressure, and either central venous oxygen saturation or lactate clearance. The study protocol was continued until all end points were achieved or a maximum of 6 hrs. MEASUREMENTS AND MAIN RESULTS: Data on patients who received an initial dose of antibiotics after presentation to the emergency department were categorized based on both time from triage and time from shock recognition to initiation of antibiotics. The primary outcome was inhospital mortality. Of 291 included patients, mortality did not change with hourly delays in antibiotic administration up to 6 hrs after triage: 1 hr (odds ratio [OR], 1.2; 0.6-2.5), 2 hrs (OR, 0.71; 0.4-1.3), 3 hrs (OR, 0.59; 0.3-1.3). Mortality was significantly increased in patients who received initial antibiotics after shock recognition (n = 172 [59%]) compared with before shock recognition (OR, 2.4; 1.1-4.5); however, among patients who received antibiotics after shock recognition, mortality did not change with hourly delays in antibiotic administration. CONCLUSION: In this large, prospective study of emergency department patients with septic shock, we found no increase in mortality with each hour delay to administration of antibiotics after triage. However, delay in antibiotics until after shock recognition was associated with increased mortality.

A mutational hotspot that determines highly repeatable evolution can be built and broken by silent genetic changes.

Mutational hotspots can determine evolutionary outcomes and make evolution repeatable. Hotspots are products of multiple evolutionary forces including mutation rate heterogeneity, but this variable is often hard to identify. In this work, we reveal that a near-deterministic genetic hotspot can be built and broken by a handful of silent mutations. We observe this when studying homologous immotile variants of the bacteria Pseudomonas fluorescens, AR2 and Pf0-2x. AR2 resurrects motility through highly repeatable de novo mutation of the same nucleotide in >95% lines in minimal media (ntrB A289C). Pf0-2x, however, evolves via a number of mutations meaning the two strains diverge significantly during adaptation. We determine that this evolutionary disparity is owed to just 6 synonymous variations within the ntrB locus, which we demonstrate by swapping the sites and observing that we are able to both break (>95% to 0%) and build (0% to 80%) a deterministic mutational hotspot. Our work reveals a key role for silent genetic variation in determining adaptive outcomes.

Etiology of illness in patients with severe sepsis admitted to the hospital from the emergency department.

BACKGROUND: Patients identified with sepsis in the emergency department often are treated on the basis of the presumption of infection; however, various noninfectious conditions that require specific treatments have clinical presentations very similar to that of sepsis. Our aim was to describe the etiology of illness in patients identified and treated for severe sepsis in the emergency department. METHODS: We conducted a prospective observational study of patients treated with goal-directed resuscitation for severe sepsis in the emergency department. Inclusion criteria were suspected infection, 2 or more criteria for systemic inflammation, and evidence of hypoperfusion. Exclusion criteria were age of <18 years and the need for immediate surgery. Clinical data on eligible patients were prospectively collected for 2 years. Blinded observers used a priori definitions to determine the final cause of hospitalization. RESULTS: In total, 211 patients were enrolled; 95 (45%) had positive culture results, and 116 (55%) had negative culture results. The overall mortality rate was 19%. Patients with positive culture results were more likely to have indwelling vascular lines (P = .03), be residents of nursing homes (P = .04), and have a shorter time to administration of antibiotics in the emergency department (83 vs 97 min; P = .03). Of patients with negative culture results, 44% had clinical infections, 8% had atypical infections, 32% had noninfectious mimics, and 16% had an illness of indeterminate etiology. CONCLUSION: In this study, we found that >50% of patients identified and treated for severe sepsis in the emergency department had negative culture results. Of patients identified with a sepsis syndrome at presentation, 18% had a noninfectious diagnosis that mimicked sepsis, and the clinical characteristics of these patients were similar to those of patients with culture-positive sepsis.

Prospective external validation of the clinical effectiveness of an emergency department-based early goal-directed therapy protocol for severe sepsis and septic shock.

OBJECTIVE: To determine the clinical effectiveness of implementing early goal-directed therapy (EGDT) as a routine protocol in the emergency department (ED). METHODS: Prospective interventional study conducted over 2 years at an urban ED. Inclusion criteria included suspected infection, criteria for systemic inflammation, and either systolic BP < 90 mm Hg after a fluid bolus or lactate concentration >/= 4 mol/L. Exclusion criteria were age < 18 years, contraindication to a chest central venous catheter, and need for immediate surgery. We prospectively recorded preintervention clinical and mortality data on consecutive, eligible patients for 1 year when treatment was at the discretion of board-certified emergency physicians. We then implemented an EGDT protocol (the intervention) and recorded clinical data and mortality rates for 1 year. Prior to the first year, we defined a 33% relative reduction in mortality (relative mortality reduction that was found in the original EGDT trial) to indicate clinical effectiveness of the intervention. RESULTS: We enrolled 79 patients in the preintervention year and 77 patients in the postintervention year. Compared with the preintervention year, patients in the postintervention year received significantly greater crystalloid volume (2.54 L vs 4.66 L, p < 0.001) and frequency of vasopressor infusion (34% vs 69%, p < 0.001) during the initial resuscitation. In-hospital mortality was 21 of 79 patients (27%) before intervention, compared with 14 of 77 patients (18%) after intervention (absolute difference, - 9%; 95% confidence interval, + 5 to - 21%). CONCLUSIONS: Implementation of EGDT in our ED was associated with a 9% absolute (33% relative) mortality reduction. Our data provide external validation of the clinical effectiveness of EGDT to treat sepsis and septic shock in the ED.

Increased anterior corpus callosum size associated positively with hypnotizability and the ability to control pain.

This is the first MRI study to report differences in brain structure size between low and highly hypnotizable, healthy, right-handed young adults. Participants were stringently screened for hypnotic susceptibility with two standardized scales, and then exposed to hypnotic analgesia training to control cold pressor pain. Only the highly hypnotizable subjects (HHs) who eliminated pain perception were included in the present study. These HHs, who demonstrated more effective attentional and inhibitory capabilities, had a significantly (P < 0.003) larger (31.8%) rostrum, a corpus callosum area involved in the allocation of attention and transfer of information between prefrontal cortices, than low hypnotizable subjects (LHs). These results provide support to the neuropsychophysiological model that HHs have more effective frontal attentional systems implementing control, monitoring performance and inhibiting unwanted stimuli from conscious awareness, than LHs.

Metabolic fate of [14C]-labeled meal protein amino acids in Aedes aegypti mosquitoes.

We developed a method to follow the metabolic fate of [(14)C]-labeled Euglena gracilis protein amino acids in Aedes aegypti mosquitoes under three different adult nutritional regimes. Quantitative analysis of blood meal protein amino acid metabolism showed that most of the carbon of the amino acids was either oxidized to CO(2) or excreted as waste. Under the three different adult nutritional regimes, no significant differences in the metabolism of amino acids were found, which indicated that the female A. aegypti mosquitoes possess a substantial capacity of maintaining metabolic homeostasis during a gonotrophic cycle. The amount of maternal glycogen and lipid after egg laying were significantly lower in the mosquitoes that underwent a partial starvation before a blood meal and/or starvation after the blood meal. The content of egg lipid or protein or the number of eggs laid did not show a significant difference among the three different regimes, which indicates that stable fecundity of A. aegypti under the partial starvation before a blood meal and/or starvation after the blood meal seemed to result from a trade-off between current fecundity and future survival after the eggs laid. The methods described in this paper can be applied to a wide range of questions about the effects of environmental conditions on the utilization of blood meal amino acids.

On-line monitoring system for chemical warfare agents using automated capillary micellar electrokinetic chromatography.

We present an automated analysis system for the detection of the chemical warfare blister agents, sulfur mustard (HD) and lewisite (L), in aqueous samples without any chemical derivatization. The system is compact in size and designed to operate in the field in a safe, autonomous manner for near real-time monitoring applications. It uses anionic surfactant-based capillary micellar electrokinetic chromatography (MEKC) to separate the sample followed by UV detection. The analysis time is sufficiently fast to allow direct detection of HD which enabled the estimation of effective hydrolysis rates in the aqueous sample matrix. The estimated hydrolysis half-life of HD in our system was 4.85 ± 0.05 min. The detection limit of HD was determined to be 10 ppm with a signal to noise ratio of 5. By contrast, L hydrolyzed too rapidly in aqueous samples to enable direct detection. Instead the first hydrolysis product 2-chlorovinyl arsonous acid (CVAA), also considered a blister agent, was detected with a detection limit of 0.7 ppm with a signal to noise ratio of 5.