Rose Bengal and Riboflavin Mediated Photodynamic Antimicrobial Therapy Against Selected South Florida Nocardia Keratitis Isolates.

Purpose: To examine and compare the efficacy of in vitro growth inhibition using rose bengal and riboflavin photodynamic antimicrobial therapy (PDAT) for Nocardia keratitis isolates. Methods: Nocardia asteroides complex, Nocardia amikacinitolerans, and Nocardia farcinica species were isolated from patients with confirmed Nocardia keratitis. Isolates were tested against three experimental groups: (1) no photosensitizer/no irradiation, (2) photosensitizer/no irradiation, and (3) photosensitizer/irradiation. Each isolate was prepared in suspension to a concentration of 1.5 × 108 CFU/mL. Bacterial suspensions were mixed with water or prepared 0.1% photosensitizer solution for a final bacterial concentration of 1.5 × 107 CFU/mL. Aliquots of 1 mL were plated on 5% sheep blood agar. Rose bengal and riboflavin PDAT plates were irradiated for 15 minutes with a 525- or 375-nm custom 6-mW/cm2 powered light source for a total fluence of 5.4 J/cm2. All experimental groups were repeated in triplicate. Plates were incubated in a 35°C non-CO2 incubator for 96 hours and photographed. Percent inhibition was evaluated using LabVIEW-based software. Results: All strains of Nocardia tested with 0.1% rose bengal and irradiated for 15 minutes demonstrated statistically significant inhibition of growth (P < 0.05). No other experimental groups displayed any bacterial inhibition. Conclusions: Rose bengal is superior to riboflavin PDAT against selected Nocardia isolates. In vivo testing is warranted to investigate the utility of rose bengal PDAT for severe Nocardia keratitis. Translational Relevance: In vitro results for three clinical strains of Nocardia support the possible use of rose bengal PDAT as a complementary treatment of Nocardia keratitis.

Characterization of Pseudomonas aeruginosa isolates from patients with endophthalmitis using conventional microbiologic techniques and whole genome sequencing.

PURPOSE: To demonstrate antibiotic susceptibility and genomic virulence factor profiles of Pseudomonas aeruginosa isolates from patients with culture-confirmed endophthalmitis. METHODS: Clinical isolates from patients diagnosed with pseudomonas endophthalmitis were included. Laboratory antibiotic susceptibility testing and whole genome sequencing was performed on all isolates. RESULTS: In the current study, 8 patients had vitreous culture-confirmed endophthalmitis due to P. aeruginosa. All isolates were multi-drug resistant but sensitive to ceftazidime and each fluoroquinolone tested. Whole genome sequencing revealed a total of 179 unique genes. The most common type of virulence genes included those involved in adherence and the secretion system. Seven of 8 (88%) isolates were of the cytoinvasive phenotype (exoST) and no isolates contained exoU. CONCLUSIONS: P. aeruginosa associated endophthalmitis is often multi-drug resistant and demonstrates a variety of virulence factors with those involved in adherence and the secretion system being the most common.

Dynamics of a capsule flowing in a tube under pulsatile flow.

We analyze numerically the behavior of a deformable micro-capsule confined in a pipe under a pulsatile flow. The capsule moves and is deformed by the action of a pulsatile flow inside the tube with a non-null mean velocity. This configuration can be found in the nature and in many bioengineering systems where artificial capsules are driven by micro-pumps through micro-channels. The capsule is considered as a thin hyperelastic membrane, which encloses an internal fluid. As it has been demonstrated in the literature, this model represents a wide range of artificial capsules, for example, the alginate-based capsules, typically used in bioengineering applications. A hybrid isogeometric finite element method and boundary element method based on a T-spline discretization and formulated in the time domain is used to solve the mechanical and hydrodynamical equations. The influence of the relative rigidity of the membrane, frequency and amplitude of the pulsatile flow is studied. Results show that the behavior of the capsule differs from steady flows and it depends strongly on the frequency of the flow and mechanical characteristic of the capsule.

In situ RT-PCR Optimized for Electron Microscopy Allows Description of Subcellular Morphology of Target mRNA-Expressing Cells in the Brain.

In situ RT-PCR detects and amplifies mRNA (cDNA) while obtaining spatial information of gene expression. When the intended use is an ultrastructural analysis of morphology, the procedure may be technically challenging and quality of tissue dramatically altered by proteolytic digestion and extreme astringency and temperature conditions. We describe a low-damaging protocol of in situ RT-PCR combined to conventional electron microscopy that preserves fine morphology, increases sensitivity, and decreases costs and complexity associated to RNA probes.

Age-Related Lipid Metabolic Signature in Human LMNA-Lipodystrophic Stem Cell-Derived Adipocytes.

CONTEXT: Lamin A (LMNA)-linked lipodystrophies belong to a group of clinical disorders characterized by a redistribution of adipose tissue with a variable range of metabolic complications. The leading cause of these disorders is the nonphysiological accumulation of the lamin A precursor, prelamin A. However, the molecular mechanisms by which prelamin A induces the pathology remain unclear. OBJECTIVE: The aim of this study is to use an experimental LMNA-lipodystrophy model based on human mesenchymal stem cell (hMSC)-derived adipocytes that accumulate prelamin A to gain deeper insights into the mechanisms governing these diseases. DESIGN/SETTING/PARTICIPANTS: Prelamin A-induced or -noninduced hMSC-derived adipocytes were obtained from healthy donors. The study was performed at the Biocruces Health Research Institute. MAIN OUTCOME MEASURES: Lipolytic activity was determined by the measurement of glycerol and free fatty acids. Ultrastructural analysis was performed by electron microscopy. Flow cytometry was used to assess mitochondrial membrane potential, and ultra-performance liquid chromatography coupled to mass spectrometry was used to explore lipid profiles. RESULTS: Prelamin A accumulating hMSC-derived adipocytes revealed increased lipolysis, mitochondrial dysfunction, and endoplasmic reticulum stress. Accumulation of prelamin A induces an altered lipid profile characterized by reduced diacylglyceride content, a higher ratio of monounsaturated over polyunsaturated fatty acids, and decreased stearoyl-coenzyme A desaturase-1 activity. In contrast, the ratio of diacylglycerophosphatidylcholine over diacylglycerophosphatidylethanolamine and the activity of phosphatidylethanolamine-methyltransferase were increased. CONCLUSIONS: Prelamin A accumulation causes mitochondrial dysfunction, endoplasmic reticulum stress, and altered lipid metabolism resembling a premature aging phenotype.

Selección de cepas atenuadas de Vibrio cholerae para la obtención de candidatos vacunales atenuados orales contra el cólera / Selection of attenuated Vibrio cholerae strains to obtain oral attenuated candidate vaccines against cholera

Se desarrolló una metodología para la selección de cepas de Vibrio cholerae O1 y O139 modificadas genéticamente, con el objetivo de obtener candidatos vacunales atenuados orales contra el cólera. A las cepas modificadas se les realizó caracterización microbiológica, susceptibilidad bacteriana y diferentes pruebas biológicas (dosis letal media, capacidad colonizadora, adherencia en ratones, intestino ligado e inoculación intraduodenal en conejos como pruebas de virulencia y potencia). Las cepas 81, 638, 638T y 1333 fueron evaluadas en ensayos clínicos para determinar su reactogenicidad e inmunogenicidad. Todas las cepas fueron sensibles a la tetraciclina y doxiclicina y mostraron su atenuación e inmunogenicidad en modelos animales, resultando las cepas 638 y 1333 inmunogénicas y no reactogénicas en voluntarios

The vaccine candidate Vibrio cholerae 638 is protective against cholera in healthy volunteers.

Vibrio cholerae 638 is a living candidate cholera vaccine strain attenuated by deletion of the CTXPhi prophage from C7258 (O1, El Tor Ogawa) and by insertion of the Clostridium thermocellum endoglucanase A gene into the hemagglutinin/protease coding sequence. This vaccine candidate was previously found to be well tolerated and immunogenic in volunteers. This article reports a randomized, double-blind, placebo-controlled trial conducted to test short-term protection conferred by 638 against subsequent V. cholerae infection and disease in volunteers in Cuba. A total of 45 subjects were enrolled and assigned to receive vaccine or placebo. The vaccine contained 10(9) CFU of freshly harvested 638 buffered with 1.3% NaHCO(3), while the placebo was buffer alone. After vaccine but not after placebo intake, 96% of volunteers had at least a fourfold increase in vibriocidal antibody titers, and 50% showed a doubling of at least the lipopolysaccharide-specific immunoglobulin A titers in serum. At 1 month after vaccination, five volunteers from the vaccine group and five from the placebo group underwent an exploratory challenge study with 10(9) CFU of DeltaCTXPhi attenuated mutant strain V. cholerae 81. Only two volunteers from the vaccine group shed strain 81 in their feces, but none of them experienced diarrhea; in the placebo group, all volunteers excreted the challenge strain, and three had reactogenic diarrhea. An additional 12 vaccinees and 9 placebo recipients underwent challenge with 7 x 10(5) CFU of virulent strain V. cholerae 3008 freshly harvested from a brain heart infusion agar plate and buffered with 1.3% NaHCO(3). Three volunteers (25%) from the vaccine group and all from the placebo group shed the challenge agent in their feces. None of the 12 vaccinees but 7 volunteers from the placebo group had diarrhea, and 2 of the latter exhibited severe cholera (>5,000 g of diarrheal stool). These results indicate that at 1 month after ingestion of a single oral dose (10(9) CFU) of strain 638, volunteers remained protected against cholera infection and disease provoked by the wild-type challenge agent V. cholerae 3008. We recommend that additional vaccine lots of 638 be prepared under good manufacturing practices for further evaluation.

[Selection of attenuated Vibrio cholerae strains to obtain oral attenuated candidate vaccines against cholera]. / Selección de cepas atenuadas de Vibrio cholerae para la obtención de candidatos vacunales atenuados orales contra el cólera.

A methodology was developed for the selection of genetically modified strains of Vibrio cholerae 01 and 0139 aimed at obtaining oral attenuated candidate vaccines against cholera. The modified strains underwent microbiological characterization, bacterial susceptibility and different biological tests (mean lethal dose, colonizing capacity, adherence in mice, ligated intestine and intraduodenal inoculation in rabbits as virulence and potency tests. The strains 81, 638, 638T and 1333 were evaluated in clinical trials to determine their reactogenicity and immunogenicity. All the strains were sensitive to tetracycline and doxoclycine. They showed their attenuation and immunogenicity in animal models. The strains 638 and 1333 proved to be immunogenic and non reactogenic in volunteers.

Detección de micobacterias en muestras clínicas mediante la Reacción en Cadena de la Polimerasa / Detection by polimerase chain reaction of mycobacteria in clinical samples

Introducción. Las micobacteriosis constituyen un importante problema de salud a nivel mundial, siendo aún mayor en pacientes con el Síndrome de Inmunodeficiencia Adquirida (SIDA). En este trabajo utilizamos la Reacción en Cadena de la Polimerasa (RCP) para el diagnóstico de las principales micobacteriosis, y comparamos los resultados obtenidos con los del diagnóstico convencional.Material y métodos. Se procesaron cepas controles y 15 muestras: 14 esputos y 1 líquido cefalorraquídeo provenientes de pacientes con sospecha clínica de tuberculosis. El procesamiento de las muestras para la RCP se realizó por calentamiento. Se emplearon oligonucleótidos que amplifican un fragmento del gen de la proteína de 65 kDa de los complejos Mycobacterium tuberculosis y Mycobecterium avium intracellulare (MAI).Resultados. Ocho muestras resultaron positivas a la RCP revelada por electroforesis sobre gel de agarosa, y 7 resultaron negativas. Los resultados de la RCP y del cultivo micobacteriológico coincidieron en 10 casos.Discusión. La RCP es una técnica que reduce a horas el tiempo de diagnóstico de micobacteriosis.

Vibrio cholerae No-01 toxigénico / Toxigenic Vibrio cholerae non-01

The antimicrobial susceptibility and the presence of a heat-stable toxin were researched into 100 non-01 Vibrio cholerae strains sent by 7 different health centers to the National Reference Laboratory of Acute Diarrheal Diseases in "Pedro KourÝ" Tropical Medicine Institute. The presence of 20 toxigenic non-01 Vibrio cholerae was detected, a figure substantially higher than that reported in other geographic areas, except for endemic areas. This result will make it possible to set epidemiological alert in Cuba because these strains can be infected by CTX phages (element transporting genes that encode for choleric toxin) which will give such strains an epidemic potential similar to that of the etiologic agent of cholera. The identified strains could be studied as possible cholera vaccine candidates.

Detección de Escherichia coli toxigénica (LT) mediante la reacción en cadena de la polimerasa

Se describe en este trabajo la detección de Escherichia coli enterotoxigénica LT(+). Este método está basado en la amplificación de un fragmento de DNA de 400 pares de bases mediante la reacción en cadena de la polimerasa (PCR). Los oligonucleótidos fueron diseñados por los autores y los patrones característicos se observaron cuando las muestras fueron sometidas a una electroforesis en gel de agarosa al 2 %. La PCR tuvo resultados positivos con las cepa de colección Escherichia coli O:149 K: 88 (LT+) y con 20 cepas aisladas de pacientes con diarreas agudas. Se observaron resultados negativos con Escherichia coli O:101 K:99 NM (ST+), Vibrio cholerae 01 y Acromons hydrophila.

In this paper it is described the detection enteroxigenic Escherichia coli LT (+). This method is based on the amplification of a DNA fragment of 400 pairs of bases by polymerase chain reaction (PRC). The oligonuclotides were designed by the authors and the characteristic patterns were observed when the samples were submitted to an electrophoresis in an Agarose gel at 2 %. The PCR had positive results with the strains of Escherichia coli 0:149 K; 88 (LT+) collection and with 20 strains isolated from patients with acute diarrhea. Negative results were found in Escherichia coli 0:101 K:99 NM (ST+), Vibrio cholerae 01 and Aeromonas hydrophilia.