Effective Treatment against ESBL-Producing Klebsiella pneumoniae through Synergism of the Photodynamic Activity of Re (I) Compounds with Beta-Lactams.

BACKGROUND: Extended-spectrum beta-lactamase (ESBL) and carbapenemase (KPC+) producing Klebsiella pneumoniae are multidrug-resistant bacteria (MDR) with the highest risk to human health. The significant reduction of new antibiotics development can be overcome by complementing with alternative therapies, such as antimicrobial photodynamic therapy (aPDI). Through photosensitizer (PS) compounds, aPDI produces local oxidative stress-activated by light (photooxidative stress), nonspecifically killing bacteria. METHODOLOGY: Bimetallic Re(I)-based compounds, PSRe-µL1 and PSRe-µL2, were tested in aPDI and compared with a Ru(II)-based PS positive control. The ability of PSRe-µL1 and PSRe-µL2 to inhibit K. pneumoniae was evaluated under a photon flux of 17 µW/cm2. In addition, an improved aPDI effect with imipenem on KPC+ bacteria and a synergistic effect with cefotaxime on ESBL producers of a collection of 118 clinical isolates of K. pneumoniae was determined. Furthermore, trypan blue exclusion assays determined the PS cytotoxicity on mammalian cells. RESULTS: At a minimum dose of 4 µg/mL, both the PSRe-µL1 and PSRe-µL2 significantly inhibited in 3log10 (>99.9%) the bacterial growth and showed a lethality of 60 and 30 min of light exposure, respectively. Furthermore, they were active on clinical isolates of K. pneumoniae at 3-6 log10. Additionally, a remarkably increased effectiveness of aPDI was observed over KPC+ bacteria when mixed with imipenem, and a synergistic effect from 3 to 6log10 over ESBL producers of K. pneumoniae clinic isolates when mixed with cefotaxime was determined for both PSs. Furthermore, the compounds show no dark toxicity and low light-dependent toxicity in vitro to mammalian HEp-2 and HEK293 cells. CONCLUSION: Compounds PSRe-µL1 and PSRe-µL2 produce an effective and synergistic aPDI effect on KPC+, ESBL, and clinical isolates of K. pneumoniae and have low cytotoxicity in mammalian cells.

Effective Photodynamic Therapy with Ir(III) for Virulent Clinical Isolates of Extended-Spectrum Beta-Lactamase Klebsiella pneumoniae.

BACKGROUND: The extended-spectrum beta-lactamase (ESBL) Klebsiella pneumoniae is one of the leading causes of health-associated infections (HAIs), whose antibiotic treatments have been severely reduced. Moreover, HAI bacteria may harbor pathogenic factors such as siderophores, enzymes, or capsules, which increase the virulence of these strains. Thus, new therapies, such as antimicrobial photodynamic inactivation (aPDI), are needed. METHOD: A collection of 118 clinical isolates of K. pneumoniae was characterized by susceptibility and virulence through the determination of the minimum inhibitory concentration (MIC) of amikacin (Amk), cefotaxime (Cfx), ceftazidime (Cfz), imipenem (Imp), meropenem (Mer), and piperacillin-tazobactam (Pip-Taz); and, by PCR, the frequency of the virulence genes K2, magA, rmpA, entB, ybtS, and allS. Susceptibility to innate immunity, such as human serum, macrophages, and polymorphonuclear cells, was tested. All the strains were tested for sensitivity to the photosensitizer PSIR-3 (4 µg/mL) in a 17 µW/cm2 for 30 min aPDI. RESULTS: A significantly higher frequency of virulence genes in ESBL than non-ESBL bacteria was observed. The isolates of the genotype K2+, ybtS+, and allS+ display enhanced virulence, since they showed higher resistance to human serum, as well as to phagocytosis. All strains are susceptible to the aPDI with PSIR-3 decreasing viability in 3log10. The combined treatment with Cfx improved the aPDI to 6log10 for the ESBL strains. The combined treatment is synergistic, as it showed a fractional inhibitory concentration (FIC) index value of 0.15. CONCLUSIONS: The aPDI effectively inhibits clinical isolates of K. pneumoniae, including the riskier strains of ESBL-producing bacteria and the K2+, ybtS+, and allS+ genotype. The aPDI with PSIR-3 is synergistic with Cfx.

Genetic Rescue of Mitochondrial and Skeletal Muscle Impairment in an Induced Pluripotent Stem Cells Model of Coenzyme Q10 Deficiency.

Coenzyme Q10 (CoQ10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ10 ], CoQ10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4ed -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4ed -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687-1703.

Grapevine Pathogenic Microorganisms: Understanding Infection Strategies and Host Response Scenarios.

Grapevine (Vitis vinifera L.) is one of the most important fruit crop worldwide. Commercial cultivars are greatly affected by a large number of pathogenic microorganisms that cause diseases during pre- and/or post-harvest periods, affecting production, processing and export, along with fruit quality. Among the potential threats, we can find bacteria, fungi, oomycete, or viruses with different life cycles, infection mechanisms and evasion strategies. While plant-pathogen interactions are cycles of resistance and susceptibility, resistance traits from natural resources are selected and may be used for breeding purposes and for a sustainable agriculture. In this context, here we summarize some of the most important diseases affecting V. vinifera together with their causal agents. The aim of this work is to bring a comprehensive review of the infection strategies deployed by significant types of pathogens while understanding the host response in both resistance and susceptibility scenarios. New approaches being used to uncover grapevine status during biotic stresses and scientific-based procedures needed to control plant diseases and crop protection are also addressed.

Transmisión del virus de la rabia entre murciélagos urbanos del departamento del Valle del Cauca, Colombia, 1999-2008 / Rabies virus transmission among urban bats in the department of Valle del Cauca, Colombia, 1999-2008

Introducción. La intromisión humana en ecosistemas silvestres ha provocado cambios de comportamiento en los murciélagos, con la consecuente invasión a viviendas, convirtiéndolos en un factor de riesgo para la transmisión de la rabia a humanos y mascotas. Objetivos. Determinar en el departamento del Valle del Cauca, las asociaciones entre hábitos de comportamiento y transmisión de rabia entre murciélagos y su significado epidemiológico con énfasis en riesgo para la población humana. Materiales y métodos. Durante el periodo de diciembre 1999 a junio 2008, fueron capturados 1.321 murciélagos por el programa de vigilancia de rabia en el departamento del Valle del Cauca. El diagnóstico de rabia se hizo por inmunofluorescencia directa e inoculación en ratones, utilizando tejido encefálico de los murciélagos capturados. La tipificación viral se hizo por inmunofluorescencia indirecta usando anticuerpos monoclonales. Resultados. Se detectaron dos ejemplares de Eptesicus brasiliensis positivos para rabia en los años 2000 y 2002, y dos casos más en especímenes de E. brasiliensis y Molossus molossus, en el 2008. No se detectó el virus de la rabia en E. brasiliensis ni en M. molossus, ni en ninguna otra especie durante los años 1999, 2001, 2003, 2004, 2006 y 2007. Se encontraron distintas especies de murciélagos, como E. brasiliensis, M. molossus, Myotis nigricans, Glossophaga soricina, Noctiliio albiventris y Carollia perspicillata, compartiendo refugios en casas. Se detectaron virus rábicos de las variantes antigénicas 3 y 4, en murciélagos M. molossus y E. brasiliensis. Conclusiones. La presencia en el Departamento del Valle del Cauca de las variantes antigénicas 3 y 4 del virus rábico en murciélagos no hematófagos de hábitos caseros, probablemente, ha sido facilitada por la deforestación de los hábitats naturales de estas especies; además, el estilo de arquitectura urbana provee un hábitat artificial que posibilita el contacto físico entre las especies y la transmisión de rabia entre ellas. Ante las dificultades para controlar la rabia en murciélagos y la falta de herramientas adecuadas, la vigilancia continua de la enfermedad en los murciélagos, basada en el diagnóstico y la tipificación de los virus rábicos por laboratorio, en de los asentamientos humanos y alrededor de ellos, la vacunación preventiva en animales domésticos y de producción, así como la educación de la comunidad (para la concientización del riesgo y la recolección pasiva de muestras para su análisis), se convierten en las mejores herramientas para prevenir la transmisión a humanos.

Introduction: Human activities in the wild have recently increased the changes in bat behavior and invasion of houses, turning these animals into a health threat for humans and pets. Objectives: To determine the associations between behavioral habits and rabies transmission among bats in the department of Valle del Cauca and to assess the risk the existence of rabies in house dwelling bats presents for human health. Material and methods: In the period from December 1999 to June 2008, 1,321 hematophagous and non-hematophagous bats were captured for rabies epidemiological surveillance in the department of Valle del Cauca, Colombia. Rabies was diagnosed by direct immunofluorescence test on the brain tissue of collected animals. Viral typification was achieved by indirect immunofluorecense using rabies specific monoclonal antibodies. Results: Four bats were positive for rabies: two Eptesicus brasiliensis in 2000 and 2002, and an Eptesicus brasiliensis and a Molossus molossus in 2008. During the years 1999, 2001, 2003, 2004, 2006 and 2007 no rabies virus was found in E. brasiliensis and M. molossus or in any other bat species. Various species including E. brasiliensis, M. molossus, Myotis nigricans, Glossophaga soricina, Noctiliio albiventris and Carollia perspicillata were found sharing shelters in houses. Rabies virus antigenic variants 3 and 4 were found in M. molossus and E. brasiliensis bats only. Conclusions: The presence and potential spread of rabies antigenic variants 3 and 4 to cities have very likely been facilitated by the perturbation of the natural habitats of non-hematophagous bats in the department of Valle del Cauca; the urban architecture style also provides an artificial habitat which allows for physical contact and rabies transmission among the species. Seeing the difficulty for controlling rabies in bats and the lack of adequate tools, intensive laboratory based rabies surveillance in and around human settlements, preventive vaccination for house and production animals, and education for the community (in relation to increasing the awareness concerning the risk, and passive recollection of samples for analysis) represent major preventive strategies against bat rabies transmission to humans and pets.

Brote de rabia humana transmitida por gato en el municipio de Santander de Quilichao, Colombia, 2008 / An outbreak of human rabies transmitted by a cat in the town of Santander de Quilichao, Colombia, 2008

Objetivos En marzo de 2008 ocurrió en el municipio de Santander de Quilichao- Cauca, Colombia, un brote de rabia de origen silvestre con 2 víctimas humanas. El presente artículo apunta a describir las técnicas diagnósticas de laboratorio, las acciones de investigación de campo y control de foco empleadas, y su significado epidemiológico e implicaciones en salud pública. Métodos La rabia se diagnosticó por inmunofluorescencia directa, prueba biológica en ratón, histopatología e inmunohistoquímica, y se tipificó utilizando anticuerpos monoclonales. La investigación de campo se enfocó en la búsqueda de contactos humanos y animales, identificación de casos sospechosos y búsqueda institucional de accidentes rábicos. El control de foco consistió en aplicación de tratamiento post exposición a la población expuesta, vacunación canina y felina, recolección y eliminación de animales callejeros y educación a la comunidad. Resultados Dos casos de rabia en humanos fueron diagnosticados y uno en gato por nexo epidemiológico. La variante antigénica 3 fue aislada de los casos humanos. Se vacunaron en total 11 369 caninos, 3 325 felinos y 217 humanos. Conclusiones Se confirma la amenaza para los humanos que representa la rabia en el ecosistema silvestre. El brote tuvo origen en vampiro y el transmisor a los humanos fue un gato confirmando a esta especie doméstica como vínculo entre la rabia de origen silvestre y el ecosistema urbano, por ende su importancia en el enfoque de las acciones de prevención y control de la rabia. Se resalta la necesidad de implementar y mantener acciones para el control de la rabia silvestre que permitan minimizar su impacto en humanos.

Objectives A sylvatic rabies outbreak during March 2008 caused two human deaths in the town of Santander de Quilichao in Cauca, Colombia. This article describes the diagnostic laboratory techniques used, the field investigation and focus control used, as well as this outbreak's epidemiological significance and implications for public health. Methods Rabies was diagnosed by direct immunofluorescence, biological tests involving inoculating mice, histopathology and immunohistochemistry and then typed by using monoclonal antibodies. Field investigation focused on searching for human and animal contacts, identifying suspicious cases and conducting an institutional search for rabid accidents. Focus control consisted of post-exposure treatment of the exposed population, vaccinating dogs and cats, collecting and eliminating stray animals and educating the community. Results Two human rabies cases were confirmed in the laboratory and another was inferred in a cat by epidemiological nexus. Antigenic variant 3 was isolated from the human cases. 11,369 dogs, 3,325 cats and 217 humans were vaccinated. Conclusions This study confirmed that rabies in the wild represents a threat for humans. The outbreak described here originated in vampire bats and was transmitted to humans by a cat, pointing out the nexus between wild rabies and the urban ecosystem which cats represent, thereby becoming a target for rabies' control and prevention activities. This study underlines the urgency of implementing and maintaining rabies control and prevention activities in the wild to minimise its impact on humans.

[An outbreak of human rabies transmitted by a cat in the town of Santander de Quilichao, Colombia, 2008]. / Brote de rabia humana transmitida por gato en el municipio de Santander de Quilichao, Colombia, 2008.

OBJECTIVES: A sylvatic rabies outbreak during March 2008 caused two human deaths in the town of Santander de Quilichao in Cauca, Colombia. This article describes the diagnostic laboratory techniques used, the field investigation and focus control used, as well as this outbreak's epidemiological significance and implications for public health. METHODS: Rabies was diagnosed by direct immunofluorescence, biological tests involving inoculating mice, histopathology and immunohistochemistry and then typed by using monoclonal antibodies. Field investigation focused on searching for human and animal contacts, identifying suspicious cases and conducting an institutional search for rabid accidents. Focus control consisted of post-exposure treatment of the exposed population, vaccinating dogs and cats, collecting and eliminating stray animals and educating the community. RESULTS: Two human rabies cases were confirmed in the laboratory and another was inferred in a cat by epidemiological nexus. Antigenic variant 3 was isolated from the human cases. 11,369 dogs, 3,325 cats and 217 humans were vaccinated. CONCLUSIONS: This study confirmed that rabies in the wild represents a threat for humans. The outbreak described here originated in vampire bats and was transmitted to humans by a cat, pointing out the nexus between wild rabies and the urban ecosystem which cats represent, thereby becoming a target for rabies' control and prevention activities. This study underlines the urgency of implementing and maintaining rabies control and prevention activities in the wild to minimise its impact on humans.

[Molecular epidemiology of rabies epizootics in Colombia, 1994-2002: evidence of human and canine rabies associated with chiroptera]. / Epidemiología molecular de epizootias de rabia en Colombia, 1994-2002: evidencia de rabia humana y canina asociada a quirópteros.

Three urban rabies outbreaks have been reported in Colombia during the last two decades, one of which is ongoing in the Caribbean region (northern Colombia). The earlier outbreaks occurred almost simultaneously in Arauca (eastern Colombia) and in the Central region, ending in 1997. Phylogenetic relationships among rabies viruses isolated from the three areas were based on a comparison of cDNA fragments coding for the endodomain of protein G and a fragment of L protein obtained by RT-PCR. The sequenced amplicons which included the G-L intergenic region contained 902 base pairs. Phylogenetic analysis showed three distinct groups of viruses. Colombian genetic variant I viruses were isolated only from Arauca and the Central region, but are now apparently extinct. Colombian genetic variant II viruses were isolated in the Caribbean region and are still being transmitted in that area. The third group of bat rabies variants were isolated from two insectivorous bats, three domestic dogs and a human. This associates bat rabies virus with rabies in Colombian dogs and humans, and indicates bats to be a rabies reservoir of public health significance.

Molecular epidemiology of rabies epizootics in Colombia: evidence for human and dog rabies associated with bats.

Three urban rabies outbreaks have been reported in Colombia during the last two decades, one of these is occurring in the Caribbean Region (northern Colombia), while the other two occurred almost simultaneously in Arauca (eastern Colombia) and in the Central Region and ended in 1997. In order to derive phylogenetic relationships between rabies viruses isolated in these three areas, 902 nt cDNA fragments encoding the cytoplasmic domain of protein G and a fragment of protein L were obtained by RT-PCR. These amplicons contained the G-L intergenic region and were sequenced to draw phylogenetic trees. Phylogenetic analysis showed three distinct groups of viruses in the study sample. Colombian genetic variant I viruses were isolated in both Arauca and the Central Region. These viruses are apparently extinct in Colombia. Colombian genetic variant II viruses were isolated in the Caribbean Region and are still being transmitted in that area. The third group of viruses consists of viruses isolated from two insectivorous bats, three domestic dogs and a human. According to sequence analysis, the data here indicate that the isolates in this third group are bat rabies virus variants. This finding is the first that associates bats to rabies in Colombian dogs and humans, showing an unsuspected vector threatening animal and public health.

Epidemiología molecular de epizootias de rabia en Colombia, 1994-2002: evidencia de rabia humana y canina asociada a quirópteros / Molecular epidemiology of rabies epizooties in Colombia, 1994 - 2002. Evidence of human and canine rabies associated with bats

Tres brotes de rabia canina han sido informados en Colombia durante las pasadas dos décadas, uno de los cuales aún ocurre en la Región Caribe. Los otros dos ocurrieron en el departamento de Arauca y en la Región Central (departamentos de Boyacá y Cundinamarca) hasta 1997. Con la finalidad de investigar las relaciones filogenéticas existentes entre los virus r bicos aislados en las regiones mencionadas, se empleó la técnica de RT-PCR para obtener fragmentos de ADN de 902 nucleótidos complementarios a una región del ARN rábico codificante para el endodominio de la proteína G, para una parte de la proteína L, fragmentos que, además, contienen la región intergénica no codificante G-L. Los amplificados fueron secuenciados y agrupados en árboles filogenéticos. Los resultados mostraron la existencia de tres grupos de virus. Los virus r bicos pertenecientes a la variante genética colombiana I fueron aislados exclusivamente en el departamento de Arauca y la Región Central colombiana hasta 1997 y, aparentemente, se encuentran extintos. Los virus rábicos pertenecientes a la variante genética colombiana II fueron aislados exclusivamente en la Región Caribe, en donde actualmente su transmisión continúa. Un tercer grupo se compone de variantes rábicas originarias de quirópteros, que fueron aisladas de dos murciélagos insectívoros, tres perros y un humano. Con este trabajo se estableció una asociación entre los quirópteros y la rabia en perros y humanos en Colombia, lo cual los muestra como reservorios de importancia en salud pública.