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Antibiotic therapy is expected to impact host microbial communities considerably, yet many studies focused on microbiome and health are often confounded by limited information about antibiotic exposure. Given that antibiotics have diverse pharmacokinetic and antimicrobial properties, investigating the type and concentration of these agents in specific host specimens would provide much needed insight into their impact on the microbes therein. Here, we developed liquid chromatography mass spectrometry (LC-MS) methods to detect 18 antibiotic agents in sputum from persons with cystic fibrosis. Antibiotic spike-in control samples were used to compare three liquid extraction methods on the Waters Acquity Quattro Premier XE. Extraction with dithiothreitol captured the most antibiotics and was used to detect antibiotics in sputum samples from 11 people with cystic fibrosis, with results being compared to the individuals' self-reported antibiotic use. For the sputum samples, two LC-MS assays were used; the Quattro Premier detected nanomolar or micromolar concentrations of 16 antibiotics, whereas the Xevo TQ-XS detected all 18 antibiotics, most at subnanomolar levels. In 45% of tested sputum samples (71/158), at least one antibiotic that was not reported by the subject was detected by both LC-MS methods, a discordance largely explained by the thrice weekly administration and long half-life of azithromycin. For â¼37% of samples, antibiotics reported as taken by the individual were not detected by either instrument. Our results provide an approach for detecting a variety of antibiotics at the site of infection, thereby providing a means to include antibiotic usage data into microbiome studies.
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Fibrosis Quística , Antibacterianos/uso terapéutico , Cromatografía Liquida , Fibrosis Quística/tratamiento farmacológico , Humanos , Espectrometría de Masas , EsputoRESUMEN
Stable isotope tracers are applied for in vivo and in vitro studies to reveal the activity of enzymes and intracellular metabolic pathways. Most often, such tracers are used with gas chromatography coupled to mass spectrometry (GC-MS) owing to its ease of operation and reproducible mass spectral databases. Differences in isotope tracer performance of the classic GC-quadrupole MS instrument and newer time-of-flight instruments are not well studied. Here, we used three commercially available instruments for the analysis of identical samples from a stable isotope labeling study that used [U-13C6] d-glucose to investigate the metabolism of the bacterium Rothia mucilaginosa with respect to 29 amino acids and hydroxyl acids involved in primary metabolism. The prokaryote R. mucilaginosa belongs to the family of Micrococcaceae and is present and metabolically active in the airways and sputum of cystic fibrosis patients. Overall, all three GC-MS instruments (low-resolution GC-SQ MS, low-resolution GC-TOF MS, and high-resolution GC-QTOF MS) can be used to perform stable isotope tracing studies for glycolytic intermediates, tricarboxylic acid (TCA) metabolites, and amino acids, yielding similar biological results, with high-resolution GC-QTOF MS offering additional capabilities to identify the chemical structures of unknown compounds that might show significant isotope enrichments in biological studies.
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Cromatografía de Gases y Espectrometría de Masas/métodos , Marcaje Isotópico/métodos , Micrococcaceae/metabolismoRESUMEN
BACKGROUND: Clostridium perfringens is a Gram-positive anaerobic pathogen that causes multiple diseases in humans and animals. C. perfringens lack flagella but have type IV pili (TFP) and can glide on agar surfaces. When C. perfringens bacteria are placed on surfaces, they become elongated, flexible and have TFP on their surface, traits not seen in liquid-grown cells. In addition, the main pilin in C. perfringens TFP, PilA2, undergoes differential post-translational modification when grown in liquid or on plates. To understand the mechanisms underlying these phenotypes, bacteria were grown in three types of liquid media and on agar plates with the same medium to compare gene expression using RNA-Seq. RESULTS: Hundreds of genes were differentially expressed, including transcriptional regulatory protein-encoding genes and genes associated with TFP functions, which were higher on plates than in liquid. Transcript levels of TFP genes reflected the proportion of each protein predicted to reside in a TFP assembly complex. To measure differences in rates of translation, the Escherichia coli reporter gene gusA gene (encoding ß-glucuronidase) was inserted into the chromosome downstream of TFP promoters and in-frame with the first gene of the operon. ß-glucuronidase expression was then measured in cells grown in liquid or on plates. ß-glucuronidase activity was proportional to mRNA levels in liquid-grown cells, but not plate-grown cells, suggesting significant levels of post-transcriptional regulation of these TFP-associated genes occurs when cells are grown on surfaces. CONCLUSIONS: This study reveals insights into how a non-flagellated pathogenic rod-shaped bacterium senses and responds to growth on surfaces, including inducing transcriptional regulators and activating multiple post-transcriptional regulatory mechanisms associated with TFP functions.
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Clostridium perfringens/fisiología , Proteínas Fimbrias/genética , Regulación Bacteriana de la Expresión Génica , Animales , Adhesión Bacteriana , Toxinas Bacterianas/genética , Secuencia de Bases , Perfilación de la Expresión Génica , Orden Génico , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Mioblastos/virología , Operón , Regiones Promotoras Genéticas , Temperatura , TranscriptomaRESUMEN
The airway fluids of cystic fibrosis (CF) patients contain local pH gradients and are more acidic than those of healthy individuals. pH is a critical factor that is often overlooked in studies seeking to recapitulate the infection microenvironment. We sought to determine the impact of pH on the physiology of a ubiqituous yet understudied microbe, Stenotrophomonas maltophilia Phylogenomics was first used to reconstruct evolutionary relationships between 74 strains of S. maltophilia (59 from CF patients). Neither the core genome (2,158 genes) nor the accessory genome (11,978 genes) distinguish the CF and non-CF isolates; however, strains from similar isolation sources grouped into the same subclades. We grew two human and six CF S. maltophilia isolates from different subclades at a range of pH values and observed impaired growth and altered antibiotic tolerances at pH 5. Transcriptomes revealed increased expression of both antibiotic resistance and DNA repair genes in acidic conditions. Although the gene expression profiles of S. maltophilia in lab cultures and CF sputum were distinct, we found that the same genes associated with low pH were also expressed during infection, and the higher pH cultures were more similar to sputum metatranscriptomes. Our findings suggest that S. maltophilia is not well adapted to acidity and may cope with low pH by expressing stress response genes and colonizing less acidic microenvironments. As a whole, our study underlines the impact of microenvironments on bacterial colonization and adaptation in CF infections.IMPORTANCE Understanding bacterial responses to physiological conditions is an important priority for combating opportunistic infections. The majority of CF patients succumb to inflammation and necrosis in the airways, arising from chronic infection due to ineffective mucociliary clearance. Steep pH gradients characterize the CF airways but are not often incorporated in standard microbiology culture conditions. Stenotrophomonas maltophilia is a prevalent CF opportunistic pathogen also found in many disparate environments, yet this bacterium's contribution to CF lung damage and its response to changing environmental factors remain largely understudied. Here, we show that pH impacts the physiology and antibiotic susceptibility of S. maltophilia, with implications for the development of relevant in vitro models and assessment of antibiotic sensitivity.
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Adaptación Fisiológica , Fibrosis Quística/complicaciones , Infecciones por Bacterias Gramnegativas/microbiología , Stenotrophomonas maltophilia/efectos de los fármacos , Stenotrophomonas maltophilia/fisiología , Perfilación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Stenotrophomonas maltophilia/genética , Stenotrophomonas maltophilia/aislamiento & purificaciónRESUMEN
We searched the PubMed database for clinical trials and observational human studies about postexposure vaccination effects, targeting infections with approved vaccines and vaccines licensed outside the United States against dengue, hepatitis E, malaria, and tick-borne encephalitis. Studies of animal models, serologic testing, and pipeline vaccines were excluded. Eligible studies were evaluated by definition of exposure; attempts to distinguish pre- and postexposure effects were rated on a scale of 1 to 4. We screened 4,518 articles and ultimately identified for this review 14 clinical trials and 31 observational studies spanning 7 of the 28 vaccine-preventable diseases. For secondary attack rate, the following medians were found for postexposure vaccination effectiveness: hepatitis A, 85% (interquartile range (IQR), 28; n = 5 sources); hepatitis B, 85% (IQR, 22; n = 5 sources); measles, 83% (IQR, 21; n = 8 sources); varicella, 67% (IQR: 48; n = 9 sources); smallpox, 45% (IQR, 39; n = 4 sources); and mumps, 38% (IQR, 7; n = 2 sources). For case fatality proportions resulting from rabies and smallpox, the median vaccine postexposure efficacies were 100% (IQR, 0; n = 6 sources) and 63% (IQR, 50; n = 8 sources), respectively. Many available vaccines can modify or preclude disease if administered after exposure. This postexposure effectiveness could be important to consider during vaccine trials and while developing new vaccines.
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Enfermedades Transmisibles , Profilaxis Posexposición , Vacunación , Adolescente , Adulto , Niño , Preescolar , Humanos , Lactante , Resultado del Tratamiento , Adulto JovenRESUMEN
Chronic infections with slow-growing pathogens have plagued humans throughout history. However, assessing the identities and growth rates of bacteria in an infection has remained an elusive goal. Neubauer et al. (J. Bacteriol. 200:e00365-18, 2018, https://doi.org/10.1128/JB.00365-18) combine two cutting-edge approaches to make progress on both fronts: probing specific RNA molecules to assess the identity of actively transcribing microbes and measuring growth rates through incorporation of stable isotope labels. They found that growth rates of pathogens were relatively stable during antibacterial therapy. The article delves into a basic and unanswered question that gets to the heart of understanding infection: what are the microbial growth rates?
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Fibrosis Quística , Infecciones Estafilocócicas , Humanos , Lípidos , Esputo , Staphylococcus aureusRESUMEN
Acetaminophen (APAP) overdose results in acute liver failure and has limited treatment options. Previous studies show that stimulating liver regeneration is critical for survival after APAP overdose, but the mechanisms remain unclear. In this study, we identified major signaling pathways involved in liver regeneration after APAP-induced acute liver injury using a novel incremental dose model. Liver injury and regeneration were studied in C57BL/6 mice treated with either 300 mg/kg (APAP300) or 600 mg/kg (APAP600) APAP. Mice treated with APAP300 developed extensive liver injury and robust liver regeneration. In contrast, APAP600-treated mice exhibited significant liver injury but substantial inhibition of liver regeneration, resulting in sustained injury and decreased survival. The inhibition of liver regeneration in the APAP600 group was associated with cell cycle arrest and decreased cyclin D1 expression. Several known regenerative pathways, including the IL-6/STAT-3 and epidermal growth factor receptor/c-Met/mitogen-activated protein kinase pathways, were activated, even at APAP600, where regeneration was inhibited. However, canonical Wnt/ß-catenin and NF-κB pathways were activated only in APAP300-treated mice, where liver regeneration was stimulated. Furthermore, overexpression of a stable form of ß-catenin, where serine 45 is mutated to aspartic acid, in mice resulted in improved liver regeneration after APAP overdose. Taken together, our incremental dose model has identified a differential role of several signaling pathways in liver regeneration after APAP overdose and highlighted canonical Wnt signaling as a potential target for regenerative therapies for APAP-induced acute liver failure.
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Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Regeneración Hepática/fisiología , Transducción de Señal/fisiología , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/efectos de los fármacosRESUMEN
Two novel series of tris-cationic, tripled-headed, double-tailed amphiphiles were synthesized and the effects of tail length and head group composition on the critical aggregation concentration (CAC), thermodynamic parameters, and minimum inhibitory concentration (MIC) against six bacterial strains were investigated. Synergistic antibacterial combinations of these amphiphiles were also identified. Amphiphiles in this study are composed of a benzene core with three benzylic ammonium bromide groups, two of which have alkyl chains, each 8-16 carbons in length. The third head group is a trimethylammonium or pyridinium. Log of critical aggregation concentration (log[CAC]) and heat of aggregation (ΔHagg) were both inversely proportional to the length of the linear hydrocarbon chains. Antibacterial activity increases with tail length until an optimal tail length of 12 carbons per chain, above which, activity decreased. The derivatives with two 12 carbon chains had the best antibacterial activity, killing all tested strains at concentrations of 1-2µM for Gram-positive and 4-16µM for Gram-negative bacteria. The identity of the third head group (trimethylammonium or pyridinium) had minimal effect on colloidal and antibacterial activity. The antibacterial activity of several binary combinations of amphiphiles from this study was higher than activity of individual amphiphiles, indicating that these combinations are synergistic. These amphiphiles show promise as novel antibacterial agents that could be used in a variety of applications.
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Antibacterianos/farmacología , Derivados del Benceno/farmacología , Compuestos de Amonio Cuaternario/farmacología , Tensoactivos/farmacología , Antibacterianos/síntesis química , Bacillus cereus/efectos de los fármacos , Bacillus cereus/crecimiento & desarrollo , Derivados del Benceno/síntesis química , Bromuros/química , Coloides , Sinergismo Farmacológico , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/crecimiento & desarrollo , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Floculación , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/crecimiento & desarrollo , Compuestos de Amonio Cuaternario/síntesis química , Compuestos de Amonio Cuaternario/química , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/crecimiento & desarrollo , Streptococcus agalactiae/efectos de los fármacos , Streptococcus agalactiae/crecimiento & desarrollo , Relación Estructura-Actividad , Tensoactivos/síntesis química , Temperatura , TermodinámicaRESUMEN
Introduction: The unmet need for remote monitoring of visual function with home-based, patient-centric technologies became increasingly palpable during the COVID-19 pandemic. Many patients with chronic eye conditions lack access to office-based examinations. Here, we evaluate the efficacy of the Accustat® test, a virtual application for measuring near visual acuity on any portable electronic device via telehealth. Materials and methods: Thirty-three adult subjects from the telehealth remote monitoring service of a retina practice performed the Accustat® acuity testing at home. All patients underwent in-office general eye examination with additional fundoscopic examination and optical coherence tomography retina imaging. Best corrected visual acuity assessment using a Snellen chart was compared with remote visual acuity assessment with the Accustat® test. Visual acuity was analyzed and compared between the best-corrected near visual acuity potential achieved on the Accustat® and in-office distance best-corrected Snellen visual acuity. Results: The mean logarithm of the minimum angle of resolution (logMAR) visual acuities of all eyes tested using the Accustat test was 0.19 ± 024 and for the office Snellen test 0.21 ± 0.21. A linear regression model with 95% confidence intervals reveals that there is a strong linear relationship between Accustat logMAR and office Snellen logMAR. Bland-Altman analysis demonstrated 95.2% significant agreement between Accustat and Office Snellen's best corrected visual acuity. Intraclass correlation coefficient (ICC = 0.94) demonstrated a strong positive correlation between at home versus office visual acuity. Conclusion: There was a high correlation between the visual acuity measured with the Accustat near vision digital self-test and the office Snellen acuity test, suggesting the potential utility of scalable remote monitoring of central retinal function via telehealth.
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Introduction: There is significant unmet need for patient-centric remote monitoring of visual function for chronic retinal diseases, as demonstrated by the COVID-19 pandemic. The Macustat® central retinal function scan is a novel cloud-based digital health application for remote monitoring. The aim of this study is to assess the efficacy of the Macustat® compared to traditional in-office retinal evaluations. Materials and methods: Patients with underlying macular pathology underwent office-based retinal and visual acuity examinations and OCT macula imaging followed by remote tele-monitoring assessment with the Macustat. Central visual function was assessed with the multi-modal Macustat test using dynamic virtual Amsler grid testing, hyperacuity perimetry and visual acuity testing. The results were compared to the findings of the in-office comprehensive retina exam and OCT evaluation. Results: The foveal acuity potential registered with the Macustat test showed high correlation with the office Snellen acuity potential 96% of eyes registered Macustat acuity within 0.2 LogMAR of office acuity measurement. In Wet AMD eyes with CNV pathology documented on OCT, the Macustat foveal function scan showed a corresponding abnormality in 89% of any CNV eyes and 100% of all visually significant CNV. In normal eyes without any visually significant edema or CNV, more than 92% showed corresponding normal retinal function scan. Conclusion: The Macustat demonstrates high concordance with clinical findings using traditional diagnostic devices. Home monitoring with the Macustat® may offer complementary clinical utility as a telehealth tool for the assessment of visual acuity and macular function in patients at high risk for macular disease.
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BACKGROUND: The specificity of gradient echo (GE)-BOLD laminar fMRI activation profiles is degraded by intracortical veins that drain blood from lower to upper cortical layers, propagating activation signal in the same direction. This work describes an approach to obtain layer specific profiles by deconvolving the measured profiles with a physiological Point Spread Function (PSF). NEW METHOD: It is shown that the PSF can be characterised by a TE-dependent peak to tail (p2t) value that is independent of cortical depth and can be estimated by simulation. An experimental estimation of individual p2t values and the sensitivity of the deconvolved profiles to variations in p2t is obtained using laminar data measured with a multi-echo 3D-FLASH sequence. These profiles are echo time dependent, but the underlying neuronal response is the same, allowing a data-based estimation of the PSF. RESULTS: The deconvolved profiles are highly similar to the gold-standard obtained from extremely high resolution 3D-EPI data, for a range of p2t values of 5-9, which covers both the empirically determined value (6.8) and the value obtained by simulation (6.3). -Comparison with Existing Method(s) Corrected profiles show a flatter shape across the cortex and a high level of similarity with the gold-standard, defined as a subset of profiles that are unaffected by intracortical veins. CONCLUSIONS: We conclude that deconvolution is a robust approach for removing the effect of signal propagation through intracortical veins. This makes it possible to obtain profiles with high laminar specificity while benefitting from the higher efficiency of GE-BOLD sequences.
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Mapeo Encefálico , Imagen por Resonancia Magnética , Simulación por ComputadorRESUMEN
Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.
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Fibrosis Quística , Microbiota , Fibrosis Quística/terapia , Humanos , Metagenoma , Oxígeno , EsputoRESUMEN
OBJECTIVE: Despite common administration of intralesional triamcinolone to acne lesions, there is little published data or consensus on best practices. This study aimed to evaluate specific characteristics of intralesional triamcinolone for acne among various dermatology healthcare professionals. DESIGN: One hundred participants (82 attending physicians, 9 physician assistants, 8 other healthcare professionals, and 1 unidentified) from private practices and academic centers completed a 10-question survey to assess specific characteristics of intralesional triamcinolone injections, including frequency, indication, depth of injection, concentration, volume, as development of adverse events. RESULTS: The most common reported concentration of intralesional triamcinolone was 2.5mg/mL (52.5%). The most frequently used volume injected was 0.05mL (42.3%). In total, 61.6 percent of those surveyed answered that they inject into the center of the lesion. Additionally, 50.5 percent of respondents counsel patients on potential adverse effects of hypopigmentation and atrophy before every injection. The majority of respondents (88.8%) reported that less than one percent of their patients returned for adverse events resulting from triamcinolone usage, and 48.4 percent reported that atrophy lasted over six months (48.4%). CONCLUSION: The data collected from this study can offer guidance on best practices in administering intralesional kenalog to patients. While consistency exists for the concentration of triamcinolone used, there was significant discordance in the volumes and depth of triamcinolone injection. Observed skin atrophy rates are extremely low, but they are long lasting when it occurred. We can use these data to refine our treatment techniques as well as improve treatment outcomes and patient satisfaction.
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Pseudomonas aeruginosa is a well-known dominant opportunistic pathogen in cystic fibrosis (CF) with a wide range of metabolic capacities. However, P. aeruginosa does not colonize the airways alone, and benefits from the metabolic products of neighboring cells-especially volatile molecules that can travel between different parts of the airways easily. Here, we present a study that investigates the metabolic, gene expression profiles and phenotypic responses of a P. aeruginosa clinical isolate to fermentation products lactic acid and 2,3-butanediol, metabolites that are produced by facultative anaerobic members of the CF polymicrobial community and potential biomarkers of disease progression. Although previous studies have successfully investigated the metabolic and transcriptional profiles of P. aeruginosa, most have used common lab reference strains that may differ in important ways from clinical isolates. Using transcriptomics and metabolomics with gas chromatography time of flight mass spectrometry, we observe that fermentation products induce pyocyanin production along with the expression of genes involved in P. aeruginosa amino acid utilization, dormancy and aggregative or biofilm modes of growth. These findings have important implications for how interactions within the diverse CF microbial community influence microbial physiology, with potential clinical consequences.
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Biopelículas , Fibrosis Quística/microbiología , Pulmón/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Butileno Glicoles/metabolismo , Fibrosis Quística/metabolismo , Fermentación , Humanos , Ácido Láctico/metabolismo , Pulmón/microbiología , Filogenia , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/aislamiento & purificación , Pseudomonas aeruginosa/fisiología , Piocianina/metabolismo , Esputo/microbiologíaRESUMEN
Bacteriophages are highly abundant in human microbiota where they coevolve with resident bacteria. Phage predation can drive the evolution of bacterial resistance, which can then drive reciprocal evolution in the phage to overcome that resistance. Such coevolutionary dynamics have not been extensively studied in human gut bacteria, and are of particular interest for both understanding and eventually manipulating the human gut microbiome. We performed experimental evolution of an Enterococcus faecium isolate from healthy human stool in the absence and presence of a single infecting Myoviridae bacteriophage, EfV12-phi1. Four replicates of E. faecium and phage were grown with twice daily serial transfers for 8 days. Genome sequencing revealed that E. faecium evolved resistance to phage through mutations in the yqwD2 gene involved in exopolysaccharide biogenesis and export, and the rpoC gene which encodes the RNA polymerase ß' subunit. In response to bacterial resistance, phage EfV12-phi1 evolved varying numbers of 1.8 kb tandem duplications within a putative tail fiber gene. Host range assays indicated that coevolution of this phage-host pair resulted in arms race dynamics in which bacterial resistance and phage infectivity increased over time. Tracking mutations from population sequencing of experimental coevolution can quickly illuminate phage entry points along with resistance strategies in both phage and host - critical information for using phage to manipulate microbial communities.
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Due to a lack of effective immune clearance, the airways of cystic fibrosis patients are colonized by polymicrobial communities. One of the most widespread and destructive opportunistic pathogens is Pseudomonas aeruginosa; however, P. aeruginosa does not colonize the airways alone. Microbes that are common in the oral cavity, such as Rothia mucilaginosa, are also present in cystic fibrosis patient sputum and have metabolic capacities different from those of P. aeruginosa Here we examine the metabolic interactions of P. aeruginosa and R. mucilaginosa using stable-isotope-assisted metabolomics. Glucose-derived 13C was incorporated into glycolysis metabolites, namely, lactate and acetate, and some amino acids in R. mucilaginosa grown aerobically and anaerobically. The amino acid glutamate was unlabeled in the R. mucilaginosa supernatant but incorporated the 13C label after P. aeruginosa was cross-fed the R. mucilaginosa supernatant in minimal medium and artificial-sputum medium. We provide evidence that P. aeruginosa utilizes R. mucilaginosa-produced metabolites as precursors for generation of primary metabolites, including glutamate.IMPORTANCEPseudomonas aeruginosa is a dominant and persistent cystic fibrosis pathogen. Although P. aeruginosa is accompanied by other microbes in the airways of cystic fibrosis patients, few cystic fibrosis studies show how P. aeruginosa is affected by the metabolism of other bacteria. Here, we demonstrate that P. aeruginosa generates primary metabolites using substrates produced by another microbe that is prevalent in the airways of cystic fibrosis patients, Rothia mucilaginosa These results indicate that P. aeruginosa may get a metabolic boost from its microbial neighbor, which might contribute to its pathogenesis in the airways of cystic fibrosis patients.
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Coinfección/metabolismo , Micrococcaceae/metabolismo , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiología , Acetatos/metabolismo , Radioisótopos de Carbono , Medios de Cultivo/farmacología , Fibrosis Quística/microbiología , Ácido Glutámico/metabolismo , Humanos , Ácido Láctico/metabolismo , Metabolómica , Interacciones Microbianas/fisiología , Micrococcaceae/crecimiento & desarrollo , Pseudomonas aeruginosa/efectos de los fármacos , Sistema Respiratorio/microbiologíaRESUMEN
Detection of bacterial metabolites at low concentrations in fluids with complex background allows for applications ranging from detecting biomarkers of respiratory infections to identifying contaminated medical instruments. Surface-enhanced Raman scattering (SERS) spectroscopy, when utilizing plasmonic nanogaps, has the relatively unique capacity to reach trace molecular detection limits in a label-free format, yet large-area device fabrication incorporating nanogaps with this level of performance has proven difficult. Here, we demonstrate the advantages of using chemical assembly to fabricate SERS surfaces with controlled nanometer gap spacings between plasmonic nanospheres. Control of nanogap spacings via the length of the chemical crosslinker provides uniform SERS signals, exhibiting detection of pyocyanin, a secondary metabolite of Pseudomonas aeruginosa, in aqueous media at concentration of 100 pg·mL-1. When using machine learning algorithms to analyze the SERS data of the conditioned medium from a bacterial culture, having a more complex background, we achieve 1 ng·mL-1 limit of detection of pyocyanin and robust quantification of concentration spanning 5 orders of magnitude. Nanogaps are also incorporated in an in-line microfluidic device, enabling longitudinal monitoring of P. aeruginosa biofilm formation via rapid pyocyanin detection in a medium effluent as early as 3 h after inoculation and quantification in under 9 h. Surface-attached bacteria exposed to a bactericidal antibiotic were differentially less susceptible after 10 h of growth, indicating that these devices may be useful for early intervention of bacterial infections.
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Biopelículas , Antibacterianos , Límite de Detección , Pseudomonas aeruginosa , Espectrometría RamanRESUMEN
Metabolites of human or microbial origin have the potential to be important biomarkers of the disease state in cystic fibrosis (CF). Clinical sample collection and storage conditions may impact metabolite abundances with clinical relevance. We measured the change in metabolite composition based on untargeted gas chromatography-mass spectrometry (GC-MS) when CF sputum samples were stored at 4°C, -20°C, or -80°C with one or two freeze-thaw cycles. Daily measurements were taken for 1 week and then weekly for 4 weeks (4°C) and 8 weeks (-20°C). The metabolites in samples stored at -20°C maintained abundances similar to those found at-80°C over the course of 8 weeks (average change in Bray-Curtis distance, 0.06 ± 0.04) and were also stable after one or two freeze-thaw cycles. However, the metabolite profiles of samples stored at 4°C shifted after 1 day and continued to change over the course of 4 weeks (average change in Bray-Curtis distance, 0.31 ± 0.12). The abundances of several amino acids and other metabolites increased with time of storage at 4°C but remained constant at -20°C. Storage temperature was a significant factor driving the metabolite composition (permutational multivariate analysis of variance: r2 = 0.32 to 0.49, P < 0.001). CF sputum samples stored at -20°C at the time of sampling maintain a relatively stable untargeted GC-MS profile. Samples should be frozen on the day of collection, as more than 1 day at 4°C impacts the global composition of the metabolites in the sample. IMPORTANCE Metabolomics has great potential for uncovering biomarkers of the disease state in CF and many other contexts. However, sample storage timing and temperature may alter the abundance of clinically relevant metabolites. To assess whether existing samples are stable and to direct future study design, we conducted untargeted GC-MS metabolomic analysis of CF sputum samples after one or two freeze-thaw cycles and storage at 4°C and -20°C for 4 to 8 weeks. Overall, storage at -20°C and freeze-thaw cycles had little impact on metabolite profiles; however, storage at 4°C shifted metabolite abundances significantly. GC-MS profiling will aid in our understanding of the CF lung, but care should be taken in studies using sputum samples to ensure that samples are properly stored.
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Hydra amphiphiles mimic the morphology of the mythical multi-headed creatures for which they are named. Likewise, when faced with a pathogenic bacterium, some hydra derivatives are as destructive as their fabled counterparts were to their adversaries. This report focuses on eight new tricephalic (triple-headed), single-tailed amphiphiles. Each amphiphile has a mesitylene (1,3,5-trimethylbenzene) core, two benzylic trimethylammonium groups and one dimethylalkylammonium group with a linear hydrophobe ranging from short (C8H17) to ultralong (C22H45). The logarithm of the critical aggregation concentration, log(CAC), decreases linearly with increasing tail length, but with a smaller dependence than that of ionic amphiphiles with fewer head groups. Tail length also affects antibacterial activity; amphiphiles with a linear 18 or 20 carbon atom hydrophobic chain are more effective at killing bacteria than those with shorter or longer chains. Comparison to a recently reported amphiphilic series with three heads and two tails allows for the development of an understanding of the relationship between number of tails and both colloidal and antibacterial properties.