RESUMEN
Noncarbapenemase-producing carbapenem-resistant Enterobacterales (non-CP-CRE) are increasingly recognized as important contributors to prevalent carbapenem-resistant Enterobacterales (CRE) infections. However, there is limited understanding of mechanisms underlying non-CP-CRE causing invasive disease. Long- and short-read whole-genome sequencing was used to elucidate carbapenem nonsusceptibility determinants in Enterobacterales bloodstream isolates at MD Anderson Cancer Center in Houston, Texas. We investigated carbapenem nonsusceptible Enterobacterales (CNSE) mechanisms (i.e., isolates with carbapenem intermediate resistance phenotypes or greater) through a combination of phylogenetic analysis, antimicrobial resistance gene detection/copy number quantification, porin assessment, and mobile genetic element (MGE) characterization. Most CNSE isolates sequenced were non-CP-CRE (41/79; 51.9%), whereas 25.3% (20/79) were Enterobacterales with intermediate susceptibility to carbapenems (CIE), and 22.8% (18/79) were carbapenemase-producing Enterobacterales (CPE). Statistically significant copy number variants (CNVs) of extended-spectrum ß-lactamase (ESBL) genes (Wilcoxon Test; P-value < 0.001) were present in both non-CP-CR E. coli (median CNV = 2.6×; n = 17) and K. pneumoniae (median CNV = 3.2×, n = 17). All non-CP-CR E. coli and K. pneumoniae had predicted reduced expression of at least one outer membrane porin gene (i.e., ompC/ompF or ompK36/ompK35). Completely resolved CNSE genomes revealed that IS26 and ISEcp1 structures harboring blaCTX-M variants along with other antimicrobial resistance elements were associated with gene amplification, occurring in mostly IncFIB/IncFII plasmid contexts. MGE-mediated ß-lactamase gene amplifications resulted in either tandem arrays, primarily mediated by IS26 translocatable units, or segmental duplication, typically due to ISEcp1 transposition units. Non-CP-CRE strains were the most common cause of CRE bacteremia with carbapenem nonsusceptibility driven by concurrent porin loss and MGE-mediated amplification of blaCTX-M genes. IMPORTANCE Carbapenem-resistant Enterobacterales (CRE) are considered urgent antimicrobial resistance (AMR) threats. The vast majority of CRE research has focused on carbapenemase-producing Enterobacterales (CPE) even though noncarbapenemase-producing CRE (non-CP-CRE) comprise 50% or more of isolates in some surveillance studies. Thus, carbapenem resistance mechanisms in non-CP-CRE remain poorly characterized. To address this problem, we applied a combination of short- and long-read sequencing technologies to a cohort of CRE bacteremia isolates and used these data to unravel complex mobile genetic element structures mediating ß-lactamase gene amplification. By generating complete genomes of 65 carbapenem nonsusceptible Enterobacterales (CNSE) covering a genetically diverse array of isolates, our findings both generate novel insights into how non-CP-CRE overcome carbapenem treatments and provide researchers scaffolds for characterization of their own non-CP-CRE isolates. Improved recognition of mechanisms driving development of non-CP-CRE could assist with design and implementation of future strategies to mitigate the impact of these increasingly recognized AMR pathogens.
Asunto(s)
Bacteriemia , Sepsis , Humanos , Carbapenémicos/farmacología , Antibacterianos/farmacología , Escherichia coli/genética , Amplificación de Genes , Filogenia , beta-Lactamasas/genética , Klebsiella pneumoniae/genética , Sepsis/genética , Bacteriemia/tratamiento farmacológico , Porinas/genética , Secuencias Repetitivas EsparcidasRESUMEN
The physical characteristics which govern the water relations of the giant-celled sporangiophore of Phycomyces blakesleeanus were measured with the pressure probe technique and with nanoliter osmometry. These properties are important because they govern water uptake associated with cell growth and because they may influence expansion of the sporangiophore wall. Turgor pressure ranged from 1.1 to 6.6 bars (mean = 4.1 bars), and was the same for stage I and stage IV sporangiophores. Sporangiophore osmotic pressure averaged 11.5 bars. From the difference between cell osmotic pressure and turgor pressure, the average water potential of the sporangiophore was calculated to be about -7.4 bars. When sporangiophores were submerged under water, turgor remained nearly constant. We propose that the low cell turgor pressure is due to solutes in the cell wall solution, i.e., between the cuticle and the plasma membrane. Membrane hydraulic conductivity averaged 4.6 x 10(-6) cm s-1 bar-1, and was significantly greater in stage I sporangiophores than in stage IV sporangiophores. Contrary to previous reports, the sporangiophore is separated from the supporting mycelium by septa which prevent bulk volume flow between the two regions. The presence of a wall compartment between the cuticle and the plasma membrane results in anomalous osmosis during pressure clamp measurements. This behavior arises because of changes in solute concentration as water moves into or out of the wall compartment surrounding the sporangiophore. Theoretical analysis shows how the equations governing transient water flow are altered by the characteristics of the cell wall compartment.
Asunto(s)
Compartimento Celular/fisiología , Presión Osmótica , Phycomyces/citología , Phycomyces/fisiología , Agua , Membrana Celular/fisiología , Permeabilidad de la Membrana Celular , Pared Celular/fisiología , Phycomyces/metabolismo , Presión , Aceites de Silicona/farmacocinéticaRESUMEN
The gravitropism of a mature stage IV Phycomyces sporangiophore has a shorter and more uniform latency if the sporangiophore is exposed horizontally to gravity during its earlier development (stage II and stage III). This early exposure to an altered gravitational orientation causes the sporangiophore to develop a gravireceptor as it matures to stage IV and resumes elongation. A technique has been developed to observe the spatial relationship between the vacuole and the protoplasm of a living sporangiophore and to show the reorganization caused by this exposure to altered gravity. Possible gravireceptor mechanisms are discussed.
Asunto(s)
Hongos/crecimiento & desarrollo , Phycomyces/crecimiento & desarrollo , Citoplasma/fisiología , Citoplasma/ultraestructura , Gravitación , Luz , Oscilometría , Phycomyces/fisiología , Phycomyces/ultraestructura , Vacuolas/fisiología , Vacuolas/ultraestructuraRESUMEN
Aggregation and fruiting body formation by Stigmatella aurantiaca were stimulated most effectively by low irradiances of blue light between 400 and 500 nm. At higher irradiances, other wavelengths of light, including those in the far-red region of the spectrum, were also effective.
Asunto(s)
Luz , Myxococcales/crecimiento & desarrollo , Movimiento , Myxococcales/fisiología , Myxococcales/efectos de la radiaciónRESUMEN
An action spectrum for light-induced carotenoid biosynthesis in Neurospora crassa was determined in 4 to 20 nm steps from 260 to 800 nm. Four-day, dark-grown mycelial pads of N. crassa were exposed to varying amounts of monochromatic radiant energy and time. After a 48-hour incubation period at 6 C, carotenoid content was assayed spectrophotometrically in vivo. The action spectrum has maxima at 450 and 481 nm in the visible range and at 280 and 370 nm in the ultraviolet. A pigment synthesized by Neurospora whose absorption spectrum resembles the action spectrum is beta-carotene.A model for the regulation of carotenoid biosynthesis in N. crassa is proposed which describes a mechanism by which beta-carotene could act as a photoregulator. This carotenoid is suggested to be both photoreceptor for and regulator of carotenoid biosynthesis.
RESUMEN
Ethylene production was induced in sections of dark-grown Sorghum vulgare L. seedlings by treatment with light in the blue and far red regions of the light spectrum. The action spectrum closely resembled the previously reported spectra for high irradiance response; thus, light-induced ethylene production is probably a high irradiance response with phytochrome as the initial photoreceptor.
Asunto(s)
Pigmentos Biliares , Fotosíntesis , Fitocromo , Proteínas de Plantas , Permeabilidad de la Membrana Celular , Fenómenos Químicos , Química , AMP Cíclico/fisiología , Oscuridad , Ecología , Inducción Enzimática , Represión Enzimática , Fluorescencia , Genes , Luz , Potenciales de la Membrana , Microscopía Electrónica , Conformación Molecular , Peso Molecular , Fitocromo/biosíntesis , Fitocromo/metabolismo , Fitocromo/farmacología , Pigmentos Biológicos/metabolismo , Biosíntesis de Proteínas , Rodopsina/fisiología , Espectrofotometría , Transcripción GenéticaRESUMEN
Phytochrome (far red form) alone can mediate anthocyanin synthesis in the mustard seedling (Sinapis alba L.). Complete photoreversibility and reciprocity, for both red and far red light exposures over a period of at least 5 minutes, demonstrate this phytochrome involvement.The duration of the initial lag-phase is constant (about 3 hours at 25 C) for seedlings more than 30 hours old and is specific for the system, being independent of the dose or quality of light. Since a complete reversal by far red of a red light induction is possible only during a 5 minute period, phytochrome (far red form) obviously mediates anthocyanin synthesis during the lag-phase although the actual synthesis of pigment can proceed only after the lag-phase is overcome. We suggest that phytochrome (far red form) exerts a double function during the initial lag-phase. It mediates both the build up of a biosynthetic potential ("capacity") and anthocyanin synthesis. However, the sequence of events leading to anthocyanin is arrested at some intermediate stage until this "capacity" is built up after 3 hours. Once "capacity" is achieved it does not decay readily. Therefore, no significant "secondary lag-phase" occurs if the seedling, under appropriate conditions, is reirradiated after an intervening dark period. The rate or extent of synthesis for both anthocyanin and lipoxygenase, previously reported (32), are functions of the amount of phytochrome (far red form). No "phytochrome paradoxes," i.e., nonrational relationships between the amount of phytochrome (far red form) and rate or extent of response, were detected. This fact suggests that the mustard seedling is especially well suited for investigating the biophysical and molecular mechanisms of phytochrome action.
Asunto(s)
Grano Comestible , Reguladores del Crecimiento de las Plantas/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Cromatografía por Intercambio Iónico , Frío , Oscuridad , Diálisis , Estabilidad de Medicamentos , Concentración de Iones de Hidrógeno , Luz , Métodos , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/metabolismo , Semillas , Espectrofotometría , TritioAsunto(s)
Grano Comestible , Reguladores del Crecimiento de las Plantas , Proteínas de Plantas , Alquilación , Aminoácidos/análisis , Fenómenos Químicos , Química , Cromatografía en Gel , Oscuridad , Electroforesis , Fluorescencia , Luz , Microscopía Electrónica , Peso Molecular , Péptidos , Reguladores del Crecimiento de las Plantas/análisis , Proteínas de Plantas/análisis , Hidrolisados de Proteína , Espectrofotometría , Tripsina , UltracentrifugaciónRESUMEN
During a seven-fold increase in length the content of the coleoptile in photoreversible phytochrome increased four-fold and that of the primary leaf nine-fold. The phytochrome content, during growth, expressed on a fresh- or dry-weight basis did not vary greatly for either organ. Phytochrome per mg dry weight (ΔOD(730)/mg=0.5) was nearly the same in the leaf as in the coleoptile. Coleoptiles studied had a constant DNA content of 4.1 µg per organ. DNA content of the leaf increased with age. Phytochrome per DNA was much higher in the coleoptile than in the primary leaf and increased with growth in each of these organs. Thus, there was not a constant amount of phytochrome per cell in either tissue with increasing age and there was not the same amount of phytochrome per cell in the coleoptile as in the primary leaf at any age.
RESUMEN
Direct in vivo measurements of phytochrome have been made in Phaseolus vulgaris by 2-filter difference spectrophotometry (Ratiospect). All measurements were made at 730 versus 800 nm and it is assumed that the Delta (DeltaOD) is directly proportional to the PFR concentration of phytochrome present. Dose response curves were determined for both physiological and spectrophotometric responses for red induction and far-red photoinactivation. For induction, saturation occurs at 100 mj/cm(2) and for inactivation at 30 mj/cm(2). The rate of hook opening and the physiological response measured 20 hours after induction are both shown to be directly proportional to the initial amount of PFR present spectrophotometrically. The sensitivity of the tissue correlates well with the absolute amount of phytochrome present, the inner portion of the hook having the maximum concentration of 0.042 Delta (DeltaOD)/g fresh weight. If the total reversible phytochrome concentration is reduced by exposure to red light and allowing PFR to decay out of the system the remaining sensitivity of the tissue is shown to be directly correlated with the amount of PR remaining in the tissue. PFR disappears rapidly in the dark at 25 degrees , and is not detectable after 6 hours. There is no indication that PFR reverts in the system to PR. At 4 degrees , PFR does not disappear measurably up to 1 hour and is nearly totally reversible to PR.
RESUMEN
The concentrations of extractable adenosine triphosphate (ATP) following the induction of positive light-growth responses in Phycomyces sporangiophores by blue light stimuli have been measured by means of the luciferin-luciferase assay. The ATP concentration in the light-sensitive growing zone increases 30 to 50% within 30 seconds after the start of a light stimulus and returns to the normal adapted level within 1 minute after stimulation. The ATP concentration is constant for any level of light adaptation and is uniform along the length of sporangiophores even though the light sensitivity is confined to a growing zone less than 5 mm long. These results suggest that one of the initial biochemical steps after a light stimulus is the production of extractable ATP.