RESUMEN
BACKGROUND: Central venous catheter-related bloodstream infection (CRBSI) is a huge public health concern with considerable impact on mortality and health costs. AIM: A three-year observational study enrolling three tertiary hospitals located in Lisbon, Portugal, was designed to identify the major aetiological agents of CRBSI, their ability to colonize central venous catheters and their antimicrobial resistance profiles. METHODS: Aetiological agents of CRBSI were identified by Vitek 2. Whole-genome sequencing was used to confirm CRBSI by the most prevalent aetiological agents and characterize their resistome. Central venous catheter colonization (namely by biofilm assembly) was monitored by scanning electron microscopy. FINDINGS: Staphylococci were the most prevalent causative agent (36/58, 62.0%), with S. aureus and coagulase-negative S. epidermidis accounting for 24.1% and 36.2% of CRBSIs, respectively. Fifty-nine of 72 staphylococci isolates were meticillin resistant. Comparative genomic analysis of central venous catheters/haemoculture pairs of isolates revealed genomic matches for 35 of 36 pairs and a good correlation between antibiotic susceptibility phenotype and the presence of antimicrobial resistance genetic determinants. Biofilms were present on 48.6% of the central venous catheters; nevertheless, no statistically significant association was established between biofilm assembly and CRBSI, and the presence/absence of ica operon and agr groups did not correlate with biofilm phenotypes, highlighting the need for further studies to elucidate biofilms' role on this healthcare-associated infection. CONCLUSION: Whole-genome sequencing was shown to be a valuable tool to confirm CRBSI. Although more than 42.3% of the central venous catheters were colonized by staphylococci, no statistically significant association was found between CRBSI and biofilms.
Asunto(s)
Bacteriemia , Infecciones Relacionadas con Catéteres , Catéteres Venosos Centrales , Bacteriemia/epidemiología , Infecciones Relacionadas con Catéteres/complicaciones , Infecciones Relacionadas con Catéteres/epidemiología , Catéteres Venosos Centrales/efectos adversos , Resistencia a Múltiples Medicamentos , Humanos , Staphylococcus , Staphylococcus aureusRESUMEN
Multiheme cytochromes c are important in electron transfer pathways in reduction of both soluble and insoluble Fe(III) by Geobacter sulfurreducens. We determined the crystal structure at 3.2Å resolution of the first dodecaheme cytochrome c (GSU1996) along with its N-terminal and C-terminal hexaheme fragments at 2.6 and 2.15Å resolution, respectively. The macroscopic reduction potentials of the full-length protein and its fragments were measured. The sequence of GSU1996 can be divided into four c(7)-type domains (A, B, C and D) with homology to triheme cytochromes c(7). In cytochromes c(7) all three hemes are bis-His coordinated, whereas in c(7)-type domains the last heme is His-Met coordinated. The full-length GSU1996 has a 12nm long crescent shaped structure with the 12 hemes arranged along a polypeptide to form a "nanowire" of hemes; it has a modular structure. Surprisingly, while the C-terminal half of the protein consists of two separate c(7)-type domains (C and D) connected by a small linker, the N-terminal half of the protein has two c(7)-type domains (A and B) that form one structural unit. This is also observed in the AB fragment. There is an unexpected interaction between the hemes at the interface of domains A and B, which form a heme-pair with nearly parallel stacking of their porphyrin rings. The hemes adjacent to each other throughout the protein are within van der Waals distance which enables efficient electron exchange between them. For the first time, the structural details of c(7)-type domains from one multiheme protein were compared.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Citocromos c/química , Citocromos c/metabolismo , Geobacter/metabolismo , Hemo/metabolismo , Hemo/química , Estructura Secundaria de ProteínaRESUMEN
Periplasmic sensor domains from two methyl-accepting chemotaxis proteins from Geobacter sulfurreducens (encoded by genes GSU0935 and GSU0582) were expressed in Escherichia coli. The sensor domains were isolated, purified, characterized in solution, and their crystal structures were determined. In the crystal, both sensor domains form swapped dimers and show a PAS-type fold. The swapped segment consists of two helices of about 45 residues at the N terminus with the hemes located between the two monomers. In the case of the GSU0582 sensor, the dimer contains a crystallographic 2-fold symmetry and the heme is coordinated by an axial His and a water molecule. In the case of the GSU0935 sensor, the crystals contain a non-crystallographic dimer, and surprisingly, the coordination of the heme in each monomer is different; monomer A heme has His-Met ligation and monomer B heme has His-water ligation as found in the GSU0582 sensor. The structures of these sensor domains are the first structures of PAS domains containing covalently bound heme. Optical absorption, electron paramagnetic resonance and NMR spectroscopy have revealed that the heme groups of both sensor domains are high-spin and low-spin in the oxidized and reduced forms, respectively, and that the spin-state interconversion involves a heme axial ligand replacement. Both sensor domains bind NO in their ferric and ferrous forms but bind CO only in the reduced form. The binding of both NO and CO occurs via an axial ligand exchange process, and is fully reversible. The reduction potentials of the sensor domains differ by 95 mV (-156 mV and -251 mV for sensors GSU0582 and GSU0935, respectively). The swapped dimerization of these sensor domains and redox-linked ligand switch might be related to the mechanism of signal transduction by these chemotaxis proteins.
Asunto(s)
Proteínas Bacterianas/química , Geobacter/metabolismo , Hemo/metabolismo , Transducción de Señal , Proteínas Bacterianas/metabolismo , Monóxido de Carbono/metabolismo , Quimiotaxis , Cromatografía en Gel , Dicroismo Circular , Cristalografía por Rayos X , Dimerización , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Imidazoles/metabolismo , Espectroscopía de Resonancia Magnética , Óxido Nítrico/metabolismo , Oxidación-Reducción , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The populations of group B streptococcus (GBS) associated with vaginal carriage in pregnant women and invasive neonatal infections in Portugal were compared. GBS isolates were characterized by serotyping, pulsed-field gel electrophoresis (PFGE) profiling, and multilocus sequence typing (MLST). Serotypes III and V accounted for 44% of all colonization isolates (n = 269), whereas serotypes III and Ia amounted to 69% of all invasive isolates (n = 64). Whereas serotype Ia was associated with early-onset disease (EOD), serotype III was associated with late-onset disease (LOD). Characterization by PFGE and MLST identified very diverse populations in carriage and invasive disease. Serotype Ia was represented mainly by a single PFGE cluster defined by sequence type 23 (ST23) and the infrequent ST24. In contrast, serotype III was found in a large number of PFGE clusters and STs, but a single PFGE cluster defined by ST17 was found to be associated with invasive disease. Although serotype III was associated only with LOD, ST17 showed an enhanced capacity to cause both EOD and LOD. Our data reinforce the evidence for enhanced invasiveness of ST17 and identify a lineage expressing serotype Ia capsule and represented by ST23 and ST24 as having enhanced potential to cause EOD.
Asunto(s)
Portador Sano/microbiología , Transmisión Vertical de Enfermedad Infecciosa , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/clasificación , Vagina/microbiología , Electroforesis en Gel de Campo Pulsado , Femenino , Humanos , Recién Nacido , Embarazo , Análisis de Secuencia de ADN , Serotipificación , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidadRESUMEN
The tetrahaem cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 is a small protein (86 residues) involved in electron transfer to Fe(III), which can be used as a terminal respiratory oxidant by this bacterium. A 3D solution structure model of the reduced form of the cytochrome has been determined using NMR data in order to determine the relative orientation of the haems. The haem core architecture of S. frigidimarina tetrahaem cytochrome differs from that found in all small tetrahaem cytochromes c(3) so far isolated from strict anaerobes, but has some similarity to the N-terminal cytochrome domain of flavocytochrome c(3) isolated from the same bacterium. NMR signals obtained for the four haems of S. frigidimarina tetrahaem cytochrome at all stages of oxidation were cross-assigned to the solution structure using the complete network of chemical exchange connectivities. Thus, the order in which each haem in the structure becomes oxidised was determined.
Asunto(s)
Grupo Citocromo c/química , Hemo/química , Shewanella/química , Secuencia de Aminoácidos , Grupo Citocromo c/genética , Hemo/metabolismo , Histidina/metabolismo , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Homología de Secuencia de Aminoácido , Shewanella/enzimología , TermodinámicaRESUMEN
This work analyzes the relationship between the number of viable cells and alteration of the cardiomyocytes growth response capacity of the hypertensive rat myocardium. Hypertension was induced in Wistar rats by means of nitric oxide synthesis blockade using NG-nitro-L-arginine methyl ester (L-NAME). L-NAME (12 mg/kg per day) was given to animals in drinking water ad lib for 15 weeks. Proliferating cell nuclear antigen (PCNA) protein expression and the disector method were used to evaluate the proliferation capacity of the cardiomyocytes and its numerical density alteration (Nv[m]), respectively. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) and monoclonal antibody to single-stranded DNA were two methods that detected the process of the apoptotic cell death. The association of the p53 expression with the apoptosis was investigated using anti-p53 antibody. The heart weight, body weight, and heart weight/body weight ratio of the control rats increased 114%, 77%, and 22%, respectively, and the Nv[m] decreased 60% (P<0.0001) relative to the L-NAME rats. The cardiomyocytes did not present PCNA labeling, indicating the absence of cellular proliferation. The decline of the Nv[m] was also associated with apoptotic cell death in the myocardium of the hypertensive rats. A p53-dependent pathway seems to mediate the programmed cell death in this model of hypertension.
Asunto(s)
Recuento de Células , Inhibidores Enzimáticos/farmacología , Hipertensión/inducido químicamente , Miocardio/patología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales , Apoptosis , Femenino , Hipertensión/enzimología , Hipertensión/patología , Etiquetado Corte-Fin in Situ , Masculino , Miocardio/química , Miocardio/enzimología , Antígeno Nuclear de Célula en Proliferación/análisis , Ratas , Ratas Wistar , Proteína p53 Supresora de Tumor/análisisRESUMEN
Morphological changes in the myocardium after left ventricular hypertrophy, due to chronic experimental hypertension, require an understanding of the quantitative relationship between myocyte and nonmyocyte compartments forming the structural framework of the myocardium. Hypertension was induced by long-term low-dosage inhibition of nitric oxide synthesis by NG-nitro-L-arginine methyl ester (L-NAME) in rats. L-NAME (12 mg/kg) was given to animals in water ad libitum during 15 weeks. After this period, systolic blood pressure increased almost 50% as compared with that in the control group. Morphological changes in control and L-NAME animals were investigated with stereology and immunohistochemistry. Comparing control and L-NAME animals, the surface density of myocytes decreased 73.7% while the mean cross-sectional area increased 97.6% in L-NAME rats. The volume density of myocytes decreased 45.9% and the volume density of the interstitium increased 71.7% in L-NAME rats. No stereological difference was found in blood vessels comparing the two groups. Remodeling of the cardiac interstitium occurred with increased deposition of both fibronectin and type III collagen. Fibronectin was seen in both early and latter responses to infarction while type III collagen was seen mainly in areas of incomplete healing among myocytes and around intramyocardial branches of the coronary arteries. The long-term low-dosage administration of an inhibitor of the NO synthase such as L-NAME causes myocyte hypertrophy and early interstitial and perivascular fibrosis without important quantitative changes in microcirculation.
Asunto(s)
Hipertensión/patología , Miocardio/química , Miocardio/patología , Animales , Recuento de Células/efectos de los fármacos , Colágeno/análisis , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/farmacología , Fibronectinas/análisis , Hipertensión/inducido químicamente , Hipertensión/complicaciones , Inmunohistoquímica , NG-Nitroarginina Metil Éster/administración & dosificación , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
OBJECTIVE: To study the healing process of the myocardium in hypertensive rats undergoing inhibition of nitric oxide synthesis. METHODS: Two groups of animals were studied: one received L-NAME, 12 mg/kg/day, and the other was a control group. The presence of type III collagen, fibronectin, and alpha-smooth muscle actin-positive cells was assessed by immunohistochemistry. RESULTS: Fibronectin was seen in both early and late lesions, while type III collagen was seen mainly in areas of incomplete healing, situated among myocytes and around the intramyocardial branches of the coronary arteries. Areas representing early and late lesions showed a population of spindle-shaped cells. Immunohistochemistry showed that these cells were positive for alpha-smooth muscle actin. CONCLUSION: In the myocardium of hypertensive rats, the alpha-smooth muscle actin-positive cells are related to the accumulation of type III collagen and fibronectin in the areas of myocardial damage.
Asunto(s)
Actinas/análisis , Colágeno/análisis , Fibronectinas/análisis , Miocardio/química , Óxido Nítrico Sintasa/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos , Femenino , Fibroblastos , Hipertensión/inducido químicamente , Masculino , Miocardio/enzimología , Ratas , Ratas WistarRESUMEN
We studied the sensitivity of H. pylori to clarithromycin and metronidazole, as well as the sensitivity and specificity of H. pylori culture, urease test and histology on a sample of 166 Portuguese patients. We observed a prevalence of 5.8% resistance to clarithromycin and 60% resistance to metronidazole. The sensitivity and specificity for the diagnostic tests were: culture 98% and 100%, urease test 98% and 91%, histology 50% and 95%, respectively.
Asunto(s)
Antibacterianos/antagonistas & inhibidores , Claritromicina/antagonistas & inhibidores , Helicobacter pylori/efectos de los fármacos , Metronidazol/antagonistas & inhibidores , Biopsia , Farmacorresistencia Microbiana , Gastroscopía , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/aislamiento & purificación , Humanos , Pruebas de Sensibilidad Microbiana , Sensibilidad y Especificidad , Estómago/microbiología , Estómago/patologíaRESUMEN
We studied six human embryos of the second gestational month (postsomitic period, from stages 15 to 23). They were fixed in 4% formaldehyde and serially sectioned. Stereological determinations were made from the compact layer of the ventricular myocardium: a) volume density of the myocardial parts: myocytes (Vv[myocyte]) and interstitium (Vv[interstitium]), b) numerical density of the myocytes (Nv[myocyte] mm3) calculated from six optical disector pairs per embryo, c) total number of myocytes (N[myocyte]), d) volume of the myocytes (V[myocyte] micron 3). In embryos from stages 15 to 19 the quantities of the myocytes and interstitium remained practically unchanged (no statistical difference was found). However, the volume of the ventricular myocardium mass increased more than 5 times during this period. Comparing embryos of stages 15 and 23, the mean value of the Nv[myocyte] decreased about 30 per cent, while N[myocyte] increased almost 2,000 per cent in the same period. Simultaneously, the volume of the ventricular myocardial mass increased almost 30 times, and Vv[myocyte] and Vv[interstitium] showed a small increase in the myocyte component (about 20 per cent), with a decrease of the interstitial component (about 70 per cent). In the early post-somitic period the human myocardium has a relatively small number of small myocytes, in the late post-somitic period it is composed of large and relatively abundant cardiac myocytes. The conspicuous increase in the ventricular myocardial volume observed in stage 23 seems not to be related to the increase in the interstitial portion of the myocardium. These arguments suggest both the enlargement and the division of the cardiac myocytes during the post-somitic period.
Asunto(s)
Corazón/embriología , Miocardio/química , Recuento de Células , Embrión de Mamíferos , Edad Gestacional , HumanosRESUMEN
We studied with quantitative methods the myocardium of 32 specimens of rats divided into three age groups: embryos, fetuses and neonates. Days of gestation were counted from the day following an overnight mating that was considered day 1 of gestation and only one animal per litter was used. The hearts were fixed in Bouin's fixative, sectioned and stained by routine methods. Stereological determinations were made on ventricular myocardium: (1) volume density of the myocytes [Vv(myocyte)] and interstitium [Vv(interstitium)], and (2) numerical density of the myocytes [Nv(myocyte) 1/mm3] calculated from fifteen optical dissector pairs per specimen. The total number of cardiac myocytes [N(myocyte)] was calculated as the product of Nv(myocyte) and the cardiac volume. The Nv(myocyte) increased from embryos to neonates, differences between embryos and fetuses and between embryos and neonates were statistically significant. The Vv(myocyte) increased from embryos to neonates (from 80.0 to 94.0%). During this period the Vv(interstitium) decreased from 21.0 to 5.5%. Differences of the Vv(myocyte) and Vv(interstitium) were significant comparing embryos with neonates and comparing fetuses with neonates. The N(myocyte) is roughly (mean +/- standard error of the mean) 9,297 +/- 487 in fetuses and 38,438 +/- 612 in neonates. This represents an increase of about 3.1 times from fetuses to neonates while the cardiac weight increased about 2.2 times in the same period. Coefficients of error for the Nv(myocyte) estimates averaged about 7.3%, for the Vv(myocyte) estimates averaged about 1.8%, and for the Vv(interstitium) estimates averaged about 9.1%. These results suggest a high mitotic activity in the rat myocardium during prenatal life and after birth.
Asunto(s)
Corazón/embriología , Miocardio/citología , Animales , Recuento de Células , División Celular , Desarrollo Embrionario y Fetal , Femenino , Tamaño de los Órganos , Embarazo , Ratas , Ratas WistarRESUMEN
We present a three-dermal pedicle technique for nipple-areola complex migration in cases of reduction of gigantomastia . The dermal flap offers a better blood supply for the areola even in cases with long transposition and excessive reduction of large hypertrophies.