Caracterización de Candida spp. aisladas a partir de urocultivos en la ciudad de Medellín / Characterization of Candida spp isolated from urine cultures of Medellín

Resumen Candida spp. es un agente etiológico importante en infecciones del tracto urinario, principalmente en población con terapia antimicótica de amplio espectro y con catéteres urinarios. Candida albicans es la especie más frecuente, pero otras especies han surgido como patógenos emergentes. En este trabajo se recolectaron aislamientos de Candida spp. de urocultivos de pacientes que consultaron en Dinamica IPS entre enero 2016 y noviembre 2017. Para estimar la frecuencia de las especies y observar los patrones de sensibilidad, se realizó la identificación fenotípica y su perfil de sensibilidad con el sistema comercial Vitek 2® (BioMérieux, Inc.), adicionalmente se evaluaron mediante análisis de las secuencia y filogenética ITS1-5.8S-ITS2. En el estudio se incluyeron 78 aislamientos de Candida spp. Las frecuencias de especies de Candida identificadas empleando las herramientas moleculares fueron: C. albicans (38,5%), C. tropicalis (23,1%), C. glabrata (21,8%), C. parapsilosis (10,3%), C. metapsilosis y C. krusei (2,5%) y C. guillermondi (1,3%). La identificación por métodos moleculares y por el sistema Vitek 2 fue: C. albicans (93,3%), C. glabrata (94,1%), C. tropicalis (83,3%), C. parapsilosis (75%) C. guilliermondii y C. krusei (100%). La sensibilidad de todos los aislamientos al fluconazol fue 93,6%.

Abstract Candida spp is an important etiologic agent in urinary tract infections, mainly in patients in broad-spectrum antifungal therapy, with urinary catheters. Candida albicans is the most frequent specie; but other species have arised as emerging pathogens. In this study, isolates of Candida spp. of urine cultures from patients who consulted in Dinamica IPS between January 2016 and November 2017 were evaluated. To estimate the frequency of the species and to observe the sensitivity patterns, the phenotypic identification and its sensitivity profile was performed employed the Vitek 2® commercial system. (BioMérieux, Inc) In addition the isolates were evaluated by sequence analysis and phylogenetics ITS1-5.8S-ITS2. This study included 78 isolates of Candida spp. The frequencies of Candida species identified using the molecular tools were: C. albicans (38.5%), C. tropicalis (23.1%), C. glabrata (21.8%), C. parapsilosis (10.3%), C. guillermondi (1.3%) and C. metapsilosis and C. krusei (2.5%). The identification by molecular methods and by Vitek 2 system were: C. albicans (93.3%), for C. glabrata (94.1%), C. tropicalis (83.3%), C. parapsilosis (75%) and 100% for C. guilliermondii and C. krusei.. fluconazole sensitivity of all isolates was 93.6%

Evaluación de una técnica de PCR en tiempo real para determinar colonización por Streptococcusagalactiae en mujeres gestantes de Medellín que consultan en Dinamica IPS / Evaluation of a real-time PCR technique to determine colonization by Streptococcus agalactiae in pregnant women of Medellín who consult in Dinamica IPS

Objetivo: Evaluar una técnica de PCR en tiempo real para determinar colonización por Streptococcus agalactiae en mujeres gestantes de Medellín. Materiales Y Métodos: Se realizó un estudio descriptivo prospectivo, en 150 mujeres gestantes, seleccionadas de forma aleatoria, en una IPS en el periodo comprendido entre Enero-Julio 2016. Criterio de inclusión: Ser gestante entre la semana 35-37, declaración voluntaria de participación en el estudio y de exclusión el uso de antibióticos. A las pacientes, se les tomó muestra con hisopo de lintroito vaginal y de la región anal. Las muestras se procesaron para qPCR, cultivo en caldo selectivo con posterior siembra en agar sangre de carnero y medio cromogénico para S. agalactiae STRB (ChromIDTMStrepto,BioMérieuxSA.). Resultados: La prevalencia de colonización por S. agalactiae en las gestantes fue de 20,9% y 22,3% en agar sangre y agar cromogénico STRB respectivamente, mientras que mediante PCR en tiempo real la prevalencia fue de 36%. Al comparar la qPCR con la prueba de oro se encontró: sensibilidad 79,31% (ICdel95%:0,61-0,90), especificidad 75,45% (IC del 95%: 0,66-0,82), valor predictivo positivo 46% (IC del 95%:0,32-0,59) y negativo 93,2% (IC del 95%: 0,86-0,96). Discusión: Elempleo de la qPCR permitió aumentar la sensibilidad y la oportunidad diagnostica (Eltiempo requerido empleando elcultivo fue de 24-48 Horas y por qPCR 6 horas) ,impactando la reducción de riesgos de transmisión neonata lde S.agalactiae, lo cualpodría representar una Disminución en días de estancia y costos hospitalarios por una infección prevenible.

Materials and Methods: A prospective and descriptive study was conducted in 150 pregnant women, randomly selected, at an IPS between January and July 2016. Inclusion criteria: gestation period week 35-37, voluntary declaration of participation in the study. Exclusion criteria: the use of antibiotics. Samples were taken from the vaginal introitus and the anal region using a hyssop and processed for qPCR as well as the gold standard test [selective broth culture with subsequent culture in blood agar and chromogenic medium for S. agalactiae STRB (ChromIDTMStrepto, BioMérieux SA)]. Results: The prevalence of colonization by S. agalactiae in pregnant women was 20.9% and 22.3% in blood agar and chromogenic agar STRB respectively, where as using qPCR the prevalence was 36.0%. The time required using the culture was 24-48h compared to 6h for qPCR. Our data comparing qPCR with the gold standard test showed: sensitivity 79.31% (95% CI: 0.61-0.90), specificity 75.45% (95% CI: 0.66-0.82), positive predictive value 46.0% (95% CI: 0.32-0.59) and negative 93.2%(95% CI: 0.86-0.96). Discussion: The use of the qPCR increased the sensitivity and the diagnostic opportunity (4x to 8x faster using qPCR), which can lead to a decrease in the risk of neonatal transmission of S. agalactiae and result in a reduction in the length of hospital stay and costs induced by a preventable infection.

Paracoccidioides spp. catalases and their role in antioxidant defense against host defense responses.

Dimorphic human pathogenic fungi interact with host effector cells resisting their microbicidal mechanisms. Yeast cells are able of surviving within the tough environment of the phagolysosome by expressing an antioxidant defense system that provides protection against host-derived reactive oxygen species (ROS). This includes the production of catalases (CATs). Here we identified and analyzed the role of CAT isoforms in Paracoccidioides, the etiological agent of paracoccidioidomycosis. Firstly, we found that one of these isoforms was absent in the closely related dimorphic pathogen Coccidioides and dermatophytes, but all of them were conserved in Paracoccidioides, Histoplasma and Blastomyces species. We probed the contribution of CATs in Paracoccidioides by determining the gene expression levels of each isoform through quantitative RT-qPCR, in both the yeast and mycelia phases, and during the morphological switch (transition and germination), as well as in response to oxidative agents and during interaction with neutrophils. PbCATP was preferentially expressed in the pathogenic yeast phase, and was associated to the response against exogenous H2O2. Therefore, we created and analyzed the virulence defects of a knockdown strain for this isoform, and found that CATP protects yeast cells from H2O2 generated in vitro and is relevant during lung infection. On the other hand, CATA and CATB seem to contribute to ROS homeostasis in Paracoccidioides cells, during endogenous oxidative stress. CAT isoforms in Paracoccidioides might be coordinately regulated during development and dimorphism, and differentially expressed in response to different stresses to control ROS homeostasis during the infectious process, contributing to the virulence of Paracoccidioides.

Identification and Analysis of the Role of Superoxide Dismutases Isoforms in the Pathogenesis of Paracoccidioides spp.

The ability of Paracoccidioides to defend itself against reactive oxygen species (ROS) produced by host effector cells is a prerequisite to survive. To counteract these radicals, Paracoccidioides expresses, among different antioxidant enzymes, superoxide dismutases (SODs). In this study, we identified six SODs isoforms encoded by the Paracoccidioides genome. We determined gene expression levels of representative isolates of the phylogenetic lineages of Paracoccidioides spp. (S1, PS2, PS3 and Pb01-like) using quantitative RT-PCR. Assays were carried out to analyze SOD gene expression of yeast cells, mycelia cells, the mycelia-to-yeast transition and the yeast-to-mycelia germination, as well as under treatment with oxidative agents and during interaction with phagocytic cells. We observed an increased expression of PbSOD1 and PbSOD3 during the transition process, exposure to oxidative agents and interaction with phagocytic cells, suggesting that these proteins could assist in combating the superoxide radicals generated during the host-pathogen interaction. Using PbSOD1 and PbSOD3 knockdown strains we showed these genes are involved in the response of the fungus against host effector cells, particularly the oxidative stress response, and in a mouse model of infection. Protein sequence analysis together with functional analysis of knockdown strains seem to suggest that PbSOD3 expression is linked with a pronounced extracellular activity while PbSOD1 seems more related to intracellular requirements of the fungus. Altogether, our data suggests that P. brasiliensis actively responds to the radicals generated endogenously during metabolism and counteracts the oxidative burst of immune cells by inducing the expression of SOD isoforms.

New insights into a complex fungal pathogen: the case of Paracoccidioides spp.

Paracoccidioidomycosis is a systemic mycosis endemic to Latin America, with Paracoccidioides brasiliensis and P. lutzii being the causal agents of this disorder. Several issues have been raised in the 100 years since its discovery and in this article we discuss features of this fascinating fungal pathogen, including its biology, eco-epidemiology and aspects of its pathogenicity. We also consider some of its virulence determinants, the most recent advances in the study of its metabolic pathways and the molecular and genetic research tools developed for this research. We also review the animal models used to study host-fungal interactions and how the host defence mechanisms against this pathogen work.

Macrophage Interaction with Paracoccidioides brasiliensis Yeast Cells Modulates Fungal Metabolism and Generates a Response to Oxidative Stress.

Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.

Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis.

Neutrophils play an important role as effector cells and contribute to the resistance of the host against microbial pathogens. Neutrophils are able to produce extracellular traps (NETs) in response to medically important fungi, including Aspergillus spp., Candida albicans and Cryptococcus gattii. However, NET production in response to Paracoccidioides brasiliensis has yet to be studied. We have demonstrated that human neutrophils produce NETs against both conidia and yeasts of P. brasiliensis. Although the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) did not alter NET production against conidia, it partially suppressed NET formation against P. brasiliensis yeasts. Cytochalasin D or IFN-γ did not affect the production of NETs against the fungus. Additionally, a mutant strain of P. brasiliensis with reduced expression of an alternative oxidase induced significantly higher levels of NETs in comparison with the WT strain. Finally, c.f.u. quantification of P. brasiliensis showed no significant differences when neutrophils were treated with DPI, DNase I or cytochalasin D as compared with untreated cells. These data establish that NET formation by human neutrophils appears to be either dependent or independent of reactive oxygen species production, correlating with the fungal morphotype used for stimulation. However, this mechanism was ineffective in killing the fungus.

Paracoccidioides brasiliensis PbP27 gene: knockdown procedures and functional characterization.

Paracoccidioides brasiliensis PbP27 gene encodes a protein localized in both the fungal cytoplasm and cell wall. The parasitic infectious form produces this protein preferentially with the gene's expression varying between the fungus phylogenetic species. The biological function of the native p27 has yet to be determined during either growth of the yeast or host infection. Therefore, in this study, through the use of antisense RNA technology and Agrobacterium tumefaciens-mediated transformation, we generated mitotically stable PbP27 mutants (PbP27 aRNA) with the goal to evaluate the role of p27 in the biology and virulence of this fungus. PbP27 expression was reduced 60-75% in mutants, as determined by real-time PCR in correlation with a decrease in p27 expression. No alterations in the growth curve or in the ability to shift from mycelia to yeast or from yeast to mycelia were observed in PbP27 aRNA strains; however, we did observe a reduction in cell vitality. Moreover, a decrease in cell viability of PbP27 aRNA yeast cells after interaction with IFN-γ-stimulated macrophages was detected. Based on these results, we propose that p27 plays a role in yeast cell architecture and represents one of the mechanisms employed by this fungus for its interaction with the monocyte/macrophage system.

Inhibition of PbGP43 expression may suggest that gp43 is a virulence factor in Paracoccidioides brasiliensis.

Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available. This is the first study of gp43 using genetically modified P. brasiliensis.

Caracterización del gen mecA de Staphylococcus aureus resistentes a meticilina aislados de tres grupos poblacionales de la ciudad de Medellín / Characterization of mecA gene of methicillin-resistant Staphylococcus aureus Isolated from three population groups in Medellín

Introducción: Staphylococcus aureus resistente a la meticilina (SARM) es responsable de infecciones intrahospitalarias, las que constituyen una importante causa de morbilidad y mortalidad en nuestro medio, por lo cual la rápida identificación y tipificación molecular de la resistencia como el complejo SSCmec es esencial para entender la epidemiología de la infección. Objetivo: Caracterizar fenotípicamente la resistencia a meticilina y genotípicamente el casete cromosomal SSCmec en cepas de S. aureus aislados de individuos de la ciudad de Medellín mediante PCR múltiple. Materiales y métodos: A 41 aislamientos (hospitalarios y de la comunidad) de S. aureus se les estableció la resistencia a cefoxitin mediante la técnica de Kirby-Bauer y la concentración inhibitoria mínima para oxacilina. Mediante PCR convencional se les confirmó la presencia del gen mecA. Para la tipificación del complejo SSCmec se utilizó PCR múltiple para amplificar 6 loci diferentes de este gen. Resultados: A todos los aislamientos se les confirmó resistencia a meticilina y la presencia del gen mecA, de los cuales 17 fueron clasificados como SSC mec I, 1 como SSC mec II, 21 como SSC mecIV; dos aislamientos no fue posible clasificarlos. Conclusiones: Con el uso de esta técnica clasificamos el 95% de los aislamientos del estudio, encontrando una mayor prevalencia de los SSCmec I y IV. La implementación de esta técnica permite una fácil caracterización de los aislamientos SARM y un apropiado manejo de la información de los integrantes de los comités de infecciones hospitalarios, lo cual podría impactar positivamente en el tratamiento a los pacientes y el control de enfermedades infecciosas intrahospitalarias.

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) is involved in nosocomial infections, representing an important cause of morbidity and mortality. The rapid identification and molecular classification of resistance, such as the SSCmec complex, is essential to understanding the epidemiology of infection. Objective: To phenotypically characterize methicillin resistance and to genotype the SSCmec complex in S. aureus isolates collected from a cohort of patients from Medellín, Colombia. Materials and Methods: Cefoxtin resistance was evaluated in 41 S. aureus isolates, using the Kirby-Bauer method and determining the minimal bactericidal concentration of oxacillin. To confirm the presence of the mecA gene, conventional PCR was performed. The classification of the SSCmec complex was carried out by multiple PCR, amplifying 6 different loci in this gene. Results: Methicillin resistance and the presence of the mecA gene were confirmed in all isolates. A total of 17 were classified as SSCmec I, one as SSCmec II, and 21 SSCmec IV (only two isolates were not classified). Conclusions: Using this method, it was possible to classify 95% of the studied isolates, with a higher prevalence of SSCmec I and IV. The implementation of this technique allows the characterization of MRSA isolates and an appropriate management of the information by the members of the Hospital Infection Committee. Altogether, this method may have a positive impact on the treatment of patients with MRSA infections.

Interaction between Paracoccidioides brasiliensis conidia and the coagulation system: involvement of fibrinogen

The infectious process starts with an initial contact between pathogen and host. We have previously demonstrated that Paracoccidioides brasiliensis conidia interact with plasma proteins including fibrinogen, which is considered the major component of the coagulation system. In this study, we evaluated the in vitro capacity of P. brasiliensis conidia to aggregate with plasma proteins and compounds involved in the coagulation system. We assessed the aggregation of P. brasiliensis conidia after incubation with human serum or plasma in the presence or absence of anticoagulants, extracellular matrix (ECM) proteins, metabolic and protein inhibitors, monosaccharides and other compounds. Additionally, prothrombin and partial thromboplastin times were determined after the interaction of P. brasiliensis conidia with human plasma. ECM proteins, monosaccharides and human plasma significantly induced P. brasiliensis conidial aggregation; however, anticoagulants and metabolic and protein inhibitors diminished the aggregation process. The extrinsic coagulation pathway was not affected by the interaction between P. brasiliensis conidia and plasma proteins, while the intrinsic pathway was markedly altered. These results indicate that P. brasiliensis conidia interact with proteins involved in the coagulation system. This interaction may play an important role in the initial inflammatory response, as well as fungal disease progression caused by P. brasiliensis dissemination.

Involvement of the 90 kDa heat shock protein during adaptation of Paracoccidioides brasiliensis to different environmental conditions.

HSP90 is a molecular chaperone that participates in folding, stabilization, activation, and assembly of several proteins, all of which are key regulators in cell signaling. In dimorphic pathogenic fungi such as Paracoccidioides brasiliensis, the adaptation to a higher temperature, acid pH and oxidative stress, is an essential event for fungal survival and also for the establishing of the infectious process. To further understand the role of this protein, we used antisense RNA technology to generate a P. brasiliensis isolate with reduced PbHSP90 gene expression (PbHSP90-aRNA). Reduced expression of HSP90 decreased yeast cell viability during batch culture growth and increased susceptibility to acid pH environments and imposed oxidative stress. Also, PbHSP90-aRNA yeast cells presented reduced viability upon interaction with macrophages. The findings presented here suggest a protective role for HSP90 during adaptation to hostile environments, one that promotes survival of the fungus during host-pathogen interactions.

The hydrolase PbHAD32 participates in the adherence of Paracoccidioides brasiliensis conidia to epithelial lung cells.

Adherence of the dimorphic pathogenic fungus Paracoccidioides brasiliensis to lung epithelial cells is considered an essential event for the establishment of infection. We have previously shown that the PbHAD32 hydrolase is important in this early stage of the host-P. brasiliensis yeast cells interaction. The aim of this study was to further elucidate the role of PbHAD32 in conidial thermodimorphism and their interaction with lung epithelial cells. Analysis of the PbHAD32 gene expression revealed higher mRNA levels during the conidia to mycelia (C-M) germination when compared to the conidia to yeast (C-Y) transition. Moreover, PbHAD32 was consistently expressed at higher levels upon infection of lung epithelial cells, but to a greater extent when conidia germinated to produce mycelia. Interestingly, at this particular transitional stage, more conidia adhered to epithelial cells than when they were transiting to the yeast form. Altogether our data further corroborates the importance of PbHAD32 during initial adherence to host cells and suggest that the 32-KDa hydrolase may also participate at different stages of the C-M and C-Y conversions.

Kinetic analysis of gene expression during mycelium to yeast transition and yeast to mycelium germination in Paracoccidioides brasiliensis / Análisis de la cinética de expresión de genes durante la transición de micelio a levadura y la germinación levadura a micelio en Paracoccidioides brasiliensis

Introduction: Paracoccidioidomycosis is an endemic systemic mycosis caused by Paracoccidioides brasiliensis, a thermally dimorphic fungus that in tissues and cultures at 37°C grows as a yeast while at lower temperatures (less than 24°C) it becomes a mold; however the genes that rule these processes and their expression are poorly understood. Objective: This research focused on the kinetic expression of certain genes in P. brasiliensis throughout the dimorphic process, one that involves the transition from the mycelium to yeast forms and the germination from the yeast to mycelium form. Materials and methods: A real-time quantitative polymerase chain reaction (RT-qPCR) was optimized to measure the expression of ten genes connected with diverse cellular functions including cell synthesis and wall structure, oxidative stress response, heat shock response, metabolism, proteins’ processing, solute transport across the cell membrane and signal transduction pathways at different time points during the mycelia to yeast transition, as well as in the yeast to mycelia germination processes. Results: Genes involved in cell synthesis and wall structure, metabolism and signal transduction were differentially expressed and highly up-regulated during the yeast to mycelia germination process; on the other hand, genes involved in heat shock response, cell synthesis and wall structure were highly up-regulated during the mycelia to yeast transition process. The remaining genes were differentially regulated during both processes. Conclusion: In this work the up-regulation of certain genes involved in the morphological changes occurring in P. brasiliensis yeast and mycelia forms were confirmed, indicating that these biological processes play an important role during the host-pathogen interactions, as well as in the fungus adaptation to environmental conditions.

Introducción. La paracoccidioidomicosis es una micosis sistémica causada por el hongo termodimorfo Paracoccidioides brasiliensis. En tejidos y cultivos a 37°C crece como levadura, mientras que a temperaturas menores de 24°C crece como un moho. Sin embargo, se conoce poco sobre los genes que regulan estos procesos. Objetivo. Se evaluó la cinética de expresión de algunos genes en P. brasiliensis mediante el proceso de dimorfismo incluida la transición del micelio a levadura y de la germinación de levadura a micelio. Materiales y métodos. Se optimizó una PCR cuantitativa en tiempo real (RT-qPCR) para medir la expresión de diez genes relacionados con diversas funciones celulares que incluyeron: síntesis de pared, respuesta al estrés oxidativo, respuesta al choque térmico, metabolismo, procesamiento de proteínas, trasporte de solutos a través de membranas y transducción de señales, todo ello a diferentes tiempos durante la transición de micelio a levadura, así como de la germinación de levadura a micelio. Resultados. Se encontró que los genes relacionados con síntesis de pared, metabolismo y transducción de señales, se expresaban de manera diferencial y con regulación positiva durante la germinaciónlevadura a micelio, mientras que algunos genes relacionados con respuesta a choque térmico y a síntesis de pared estaban sobreexpresados en la transición de micelio a levadura. Los genes restantes se regularon de manera diferencial en ambos procesos. Conclusiones. En este trabajo se confirma la regulación positiva de algunos genes relacionados con los cambios morfológicos de las fases levadura y micelio en P. brasiliensis, procesos biológicos que juegan un papel de importancia durante la interacción huésped-parásito y durante la adaptación del hongo al ambiente, respectivamente.

Colonización por Staphylococcus aureus en una población de pacientes VIH positivos de la ciudad de Medellín: perfil de sensibilidad antimicrobiana y caracterización de la resistencia a la meticilina / Colonization by Staphylococcus aureus in an HIV-positive patient population of the city of Medellín: antimicrobial susceptibility patterns and characterization of resistance to methicillin

El objetivo del estudio fue determinar prevalencia de colonización nasal por S. aureus, así como el perfil de sensibilidad antimicrobiana, resistencia a meticilina en las cepas aisladas y relación entre el estado de portador, estado inmunológico y administración de antimicrobianos en un grupo de pacientes infectados con el virus de inmunodeficiencia humana (VIH) en la ciudad de Medellín. De esta forma, se tomaron muestras nasales de 151 pacientes ambulatorios VIH positivos. A los aislamientos de Staphylococcus aureus se les evaluó el perfil de sensibilidad antimicrobiana utilizando la técnica de Kirby-Bauer. A los aislamientos resistentes a cefoxitin, se les determinó concentración inhibitoria mínima para oxacilina y vancomicina. A las cepas de S. aureus meticilino resistentes (SARM) se les realizó reacción en cadena de la polimerasa (PCR) para confirmar la presencia del gen MecA. Se encontró una prevalencia de colonización nasal por S. aureus en 37.7% de los pacientes; la colonización por SARM fue 1.9% y por S. aureus sensible a meticilina (SASM) de 35.7%. Los aislamientos SARM se observó sensibilidad para eritromicina 66.6%, clindamicina 100%, gentamicina 100%, tetraciclina 66.6% y vancomicina 100%. La PCR mostró el gen MecA en los aislamientos SARM. Se determinó la prevalencia de colonización por S. aureus en la población estudiada, no se observó relación estadísticamente significativa entre el estado de portador, estado inmunológico, administración de antimicrobianos, períodos previos de hospitalización o sexo en la población estudiada. Los hallazgos microbiológicos se correlacionaron con la presencia del gen Mec A. No se detectaron cepas con sensibilidad disminuida a glicopéptidos ni resistentes a vancomicina.

A 32-kilodalton hydrolase plays an important role in Paracoccidioides brasiliensis adherence to host cells and influences pathogenicity.

One of the most crucial events during infection with the dimorphic fungus Paracoccidioides brasiliensis is adhesion to pulmonary epithelial cells, a pivotal step in the establishment of disease. In this study, we have evaluated the relevance of a 32-kDa protein, a putative adhesion member of the haloacid dehalogenase (HAD) superfamily of hydrolases, in the virulence of this fungus. Protein sequence analyses have supported the inclusion of PbHad32p as a hydrolase and have revealed a conserved protein only among fungal dimorphic and filamentous pathogens that are closely phylogenetically related. To evaluate its role during the host-pathogen interaction, we have generated mitotically stable P. brasiliensis HAD32 (PbHAD32) antisense RNA (aRNA) strains with consistently reduced gene expression. Knockdown of PbHAD32 did not alter cell vitality or viability but induced morphological alterations in yeast cells. Moreover, yeast cells with reduced PbHAD32 expression were significantly affected in their capacity to adhere to human epithelial cells and presented decreased virulence in a mouse model of infection. These data support the hypothesis that PbHad32p binds to extracellular matrix (ECM) proteins and modulates the initial immune response for evasion of host defenses. Our findings point to PbHAD32 as a novel virulence factor active during the initial interaction with host cells in P. brasiliensis.

Identificación de algunos genes asociados al proceso de germinación de la conidia al micelio en Paracoccidioides brasiliensis / Identification of genes associated with germination of conidia to form mycelia in the fungus Paracoccidioides brasiliensis

Introducción. Paracoccidioides brasiliensis es un hongo dimórfico térmico, que a temperatura ambiente se presenta como un moho productor de conidias, mientras que en el huésped se comporta como una levadura de gemación múltiple. Los mecanismos moleculares que rigen la germinación de conidia a micelio aún se desconocen. Objetivo. Estudiar en P. brasiliensis la cinética del proceso de germinación de conidia a micelio y determinar los genes expresados durante este proceso mediante la construcción y el análisis de una librería EST (Expressed Sequence Tag). Materiales y métodos. Para el estudio de la cinética de germinación, se produjeron y aislaron conidias de P. brasiliensis. Estas fueron incubadas en cultivos líquidos a 18°C por 24, 48, 72 y 96 horas, y se examinaron por microscopía de luz. A partir de conidias cultivadas por 96 horas, se construyó y caracterizó una librería EST, la cual representaría los genes expresados durante el proceso de germinación. Resultados. Durante el proceso de germinación de conidia a micelio, se observó 11,7±1,2%, 30±0,6%, 43±1,3% y 66±2,4% de germinación a las 24, 48, 72 y 96 horas de incubación, respectivamente. Además, se obtuvo una librería del proceso de germinación consistente en 129 secuencias agrupadas en cuatro secuencias contiguas y siete secuencias únicas, para un total de 11 posibles genes. Ocho secuencias (72,7%) no habían sido descritas anteriormente en otras librerías informadas para este hongo y podrían representar genes específicos de la germinación de conidia a micelio. Conclusiones. Éste es el primer reporte en el que se identifican genes no descritos anteriormente, que son expresados durante la germinación de conidia a micelio, proceso de gran importancia en la biología de P. brasiliensis.

Introduction. Paracoccidioides brasiliensis is a thermo-dimorphic fungus. At room temperature it grows as a mold that produces conidia, whereas in the vertebrate host it grows as a multiple-budding yeast. The molecular mechanisms involved in the germination from the conidia to the mycelia process remain unknown. Objective. The kinetics of conidia to mycelia germination process were studied in the dimorphic fungus P. brasiliensis. Gene expression during this process was evaluated by construction and analysis of an EST library. Materials and methods. For the germination kinetics study, P. brasiliensis conidia were isolated as single cell units. Then, they were cultured at 18° C in BHI (brain-heart infusion) broth for 24, 48, 72 and 96 hr. After each perion, they were examined by light microscopy. From conidia harvested at 96 hr, an EST library was constructed; at this stage the gene expression was presumed to be maximal for the germination process. Results. During the conidia to the mycelia developmental process, the following germination rates were observed: at 24 hr, 11.7±1.2%; at 48 hr, 30±0.6%; at 72 hr, 43±1.3%; and at 96 hr, 66±2.4%. At the 96 hour stage, an EST library was constructed. It consisted of 129 sequences grouped in 4 contigs and 7 singlets for a total of 11 possible genes. Eight of the sequences had not been described previously in other EST libraries of this fungus. Conclusions. New genes were identified that were expressed during the conidia to the mycelia germination process and may represent genes specific to the germination process.

Purification and partial characterization of a Paracoccidioides brasiliensis protein with capacity to bind to extracellular matrix proteins.

Microorganisms adhere to extracellular matrix proteins by means of their own surface molecules. Paracoccidioides brasiliensis conidia have been shown to be capable of interacting with extracellular matrix proteins. We aimed at determining the presence of fungal proteins that could interact with extracellular matrix protein and, if found, attempt their purification and characterization. Various extracts were prepared from P. brasiliensis mycelial and yeast cultures (total homogenates, beta-mercaptoethanol, and sodium dodecyl sulfate [SDS] extracts) and analyzed by ligand affinity assays with fibronectin, fibrinogen and laminin. Two polypeptides were detected in both fungal forms. SDS extracts that interacted with all the extracellular matrix protein were tested; their molecular masses were 19 and 32 kDa. Analysis of the N-terminal amino acid sequence of the purified 32-kDa mycelial protein showed substantial homology with P. brasiliensis, Histoplasma capsulatum, and Neurospora crassa hypothetical proteins. Additionally, a monoclonal antibody (MAb) produced against this protein recognized the 32-kDa protein in the SDS extracts of both fungal forms for immunoblot. Immunofluorescence analysis revealed that this MAb reacted not only with mycelia and yeast cells, but also with conidia, indicating that this protein was shared by the three fungal propagules. By immunoelectron microscopy, this protein was detected in the cell walls and in the cytoplasm. Both the 32-kDa purified protein and MAb inhibited the adherence of conidia to the three extracellular matrix proteins in a dose-dependent manner. These findings demonstrate the presence of two polypeptides capable of interacting with extracellular matrix proteins on the surface of P. brasiliensis propagules, indicating that there may be common receptors for laminin, fibronectin, and fibrinogen. These proteins would be crucial for initial conidial adherence and perhaps also in dissemination of paracoccidioidomycosis.