Genotyping of clinical isolates of Aspergillus flavus and its relationship with environmental isolates center

Antecedentes: Durante un período de 4 meses, y mientras se llevaba a cabo un muestreo ambiental de aire, se diagnosticaron 2 casos de aspergilosis por Aspergillus flavus en un centro oncohematológico de Buenos Aires, Argentina. Objetivos: Conocer la variabilidad y la relación genética entre los aislamientos clínicos y los ambientales obtenidos en el centro oncohematológico. Métodos: Se utilizaron 2 técnicas de genotipificación con diferente poder discriminatorio (RAPD y AFLP). Una matriz de similitud genética fue calculada usando el método de Jaccard y fue la base para la cons- trucción de un dendrograma por el método de UPGMA. Se estimó el nivel de variabilidad genética por medio del porcentaje de loci polimórficos, número de alelos efectivos y heterocigosidad esperada, y el índice de asociación (I A ) . Resultados: El dendrograma mostró que los aislamientos de A. flavus recuperados de los pacientes no se relacionaron genéticamente con los del ambiente nosocomial. Los valores más altos de diversidad gené- tica correspondieron a los aislamientos ambientales. El I A estimado para todos los aislamientos sugiere eventos de recombinación. Conclusiones: Los pacientes 1 y 2 no fueron infectados con los aislamientos obtenidos del ambiente hospitalario. Los aislamientos clínicos y ambientales de A. flavus mostraron alta variabilidad genética entre ellos

AFLP analysis reveals high genetic diversity but low population structure in Coccidioides posadasiiisolates from Mexico and Argentina

BACKGROUND: Coccidioides immitis and C. posadasii cause coccidioidomycosis, a disease that is endemic to North and South America, but for Central America, the incidence of coccidioidomycosis has not been clearly established. Several studies suggest genetic variability in these fungi; however, little definitive information has been discovered about the variability of Coccidioides fungi in Mexico (MX) and Argentina (AR). Thus, the goals for this work were to study 32 Coccidioides spp. isolates from MX and AR, identify the species of these Coccidioides spp. isolates, analyse their phenotypic variability, examine their genetic variability and investigate the Coccidioides reproductive system and its level of genetic differentiation. METHODS: Coccidioides spp. isolates from MX and AR were taxonomically identified by phylogenetic inference analysis using partial sequences of the Ag2/PRA gene and their phenotypic characteristics analysed. The genetic variability, reproductive system and level of differentiation were estimated using AFLP markers. The level of genetic variability was assessed measuring the percentage of polymorphic loci, number of effective allele, expected heterocygosity and Index of Association (I(A)). The degree of genetic differentiation was determined by AMOVA. Genetic similarities among isolates were estimated using Jaccard index. The UPGMA was used to contsruct the corresponding dendrogram. Finally, a network of haplotypes was built to evaluate the genealogical relationships among AFLP haplotypes. RESULTS: All isolates of Coccidioides spp. from MX and AR were identified as C. posadasii. No phenotypic variability was observed among the C. posadasii isolates from MX and AR. Analyses of genetic diversity and population structure were conducted using AFLP markers. Different estimators of genetic variability indicated that the C. posadasii isolates from MX and AR had high genetic variability. Furthermore, AMOVA, dendrogram and haplotype network showed a small genetic differentiation among the C. posadasii populations analysed from MX and AR. Additionally, the I(A) calculated for the isolates suggested that the species has a recombinant reproductive system. CONCLUSIONS: No phenotypic variability was observed among the C. posadasii isolates from MX and AR. The high genetic variability observed in the isolates from MX and AR and the small genetic differentiation observed among the C. posadasii isolates analysed, suggest that this species could be distributed as a single genetic population in Latin America

Disk diffusion method for fluconazole susceptibility testing of Candida spp. isolates

Fil: Rodero, L. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Córdoba, Susana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Vivot, W. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Campo, M. Universidad Católica Argentina. Cátedra de Microbiología; Argentina.Fil: Corfield, P. Universidad Católica Argentina. Cátedra de Microbiología; Argentina.Fil: Olguín, C. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Cuirolo, A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Soria, M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Guelfand, L. Universidad Católica Argentina. Cátedra de Microbiología; Argentina.Fil: Canteros, C. E. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Davel, Graciela Odelsia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Red de Vigilancia de la Resistencia a los Antimicrobianos WHONET; Argentina.Se estudiaron 1193 aislamientos clínicos para estandarizar y evaluar un método de difusión con discos de fluconazol de lectura visual, que permita detectar levaduras sensibles al antifúngico. Las especies analizadas fueron: Candida albicans (n=584), Candida parapsilosis (n=196), Candida tropicalis (n=200), Candida glabrata (n=113), Candida krusei (n=50), Candida spp. y otras levaduras oportunistas (n=50). Los discos fueron manufacturados en el INEIANLIS “Dr. Carlos G. Malbrán”. Se midieron los halos de inhibición del crecimiento producidos por fluconazol y la concentración inhibitoria mínima (CIM) por el método de referencia M27-A2 modificado por EUCAST. Se establecieron los valores de corte del método de difusión en: ³ 16 mm para levaduras sensibles a fluconazol (CIM £ 8 μg/ml), entre 9 y 15 mm para sensibles dependientes de la dosis (CIM = 16-32 mg/ml) y £ 8 mm para resistentes (CIM ³ 64 μg/ml). El método de difusión tuvo 94,7% de concordancia con el de referencia, con 0,2% de errores very major y 0,3% de errores major. La reproducibilidad inter e intralaboratorio fue muy buena. Para detectar aislamientos sensibles a fluconazol, este método resulta confiable y de bajo costo; sin embargo, es conveniente que los aislamientos con halos £ 15 mm sean reevaluados por el método de referencia. (EN) In order to standardize and evaluate a disk diffusion method with visual reading to detect in vitro fluconazole susceptibility of yeast, 1193 clinical isolates were tested. These included 584 Candida albicans, 196 Candida parapsilosis, 200 Candida tropicalis, 113 Candida glabrata, 50 Candida krusei and 50 Candida spp. and other opportunistic yeasts. The disks were manufactured in the INEI-ANLIS “Dr. Carlos G. Malbrán”. The disk diffusion method results were compared to MIC results obtained by the reference CLSI M27-A2 broth microdilution method modified by EUCAST. The interpretative breakpoints for in vitro susceptibility testing of fluconazole were established at: zone diameter ³ 16 mm for MIC £ 8 μg/ml (susceptible isolates), between 9 and 15 mm for MIC = 16-32 mg/ml (susceptible dose-dependent isolates), and £ 8 mm for MIC ³ 64 μg/ml (resistant isolates). Overall agreement between the two methods was 94.7%, with 0.2% very major errors, and 0.3% major errors. Inter - and intralaboratory agreement was good. The disk diffusion method for drug susceptibility testing of Candida spp. isolates is inexpensive, reliable and reproducible. However, when the inhibition zone diameter is £ 15 mm, it is advisable to test the isolate by the reference microdilution method

Disk diffusion method for fluconazole susceptibility testing of Candida spp. isolates

Fil: Rodero, L. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Córdoba, Susana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Vivot, W. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Campo, M. Universidad Católica Argentina. Cátedra de Microbiología; Argentina.Fil: Corfield, P. Universidad Católica Argentina. Cátedra de Microbiología; Argentina.Fil: Olguín, C. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Cuirolo, A. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Soria, M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Guelfand, L. Universidad Católica Argentina. Cátedra de Microbiología; Argentina.Fil: Canteros, C. E. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Davel, Graciela Odelsia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Red de Vigilancia de la Resistencia a los Antimicrobianos WHONET; Argentina.Se estudiaron 1193 aislamientos clínicos para estandarizar y evaluar un método de difusión con discos de fluconazol de lectura visual, que permita detectar levaduras sensibles al antifúngico. Las especies analizadas fueron: Candida albicans (n=584), Candida parapsilosis (n=196), Candida tropicalis (n=200), Candida glabrata (n=113), Candida krusei (n=50), Candida spp. y otras levaduras oportunistas (n=50). Los discos fueron manufacturados en el INEIANLIS “Dr. Carlos G. Malbrán”. Se midieron los halos de inhibición del crecimiento producidos por fluconazol y la concentración inhibitoria mínima (CIM) por el método de referencia M27-A2 modificado por EUCAST. Se establecieron los valores de corte del método de difusión en: ³ 16 mm para levaduras sensibles a fluconazol (CIM £ 8 μg/ml), entre 9 y 15 mm para sensibles dependientes de la dosis (CIM = 16-32 mg/ml) y £ 8 mm para resistentes (CIM ³ 64 μg/ml). El método de difusión tuvo 94,7% de concordancia con el de referencia, con 0,2% de errores very major y 0,3% de errores major. La reproducibilidad inter e intralaboratorio fue muy buena. Para detectar aislamientos sensibles a fluconazol, este método resulta confiable y de bajo costo; sin embargo, es conveniente que los aislamientos con halos £ 15 mm sean reevaluados por el método de referencia. (EN) In order to standardize and evaluate a disk diffusion method with visual reading to detect in vitro fluconazole susceptibility of yeast, 1193 clinical isolates were tested. These included 584 Candida albicans, 196 Candida parapsilosis, 200 Candida tropicalis, 113 Candida glabrata, 50 Candida krusei and 50 Candida spp. and other opportunistic yeasts. The disks were manufactured in the INEI-ANLIS “Dr. Carlos G. Malbrán”. The disk diffusion method results were compared to MIC results obtained by the reference CLSI M27-A2 broth microdilution method modified by EUCAST. The interpretative breakpoints for in vitro susceptibility testing of fluconazole were established at: zone diameter ³ 16 mm for MIC £ 8 μg/ml (susceptible isolates), between 9 and 15 mm for MIC = 16-32 mg/ml (susceptible dose-dependent isolates), and £ 8 mm for MIC ³ 64 μg/ml (resistant isolates). Overall agreement between the two methods was 94.7%, with 0.2% very major errors, and 0.3% major errors. Inter - and intralaboratory agreement was good. The disk diffusion method for drug susceptibility testing of Candida spp. isolates is inexpensive, reliable and reproducible. However, when the inhibition zone diameter is £ 15 mm, it is advisable to test the isolate by the reference microdilution method

HpaC Controls Substrate Specificity of the Xanthomonas Type III Secretion System

The Gram-negative bacterial plant pathogen Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to inject bacterial effector proteins into the host cell cytoplasm. One essential pathogenicity factor is HrpB2, which is secreted by the T3S system. We show that secretion of HrpB2 is suppressed by HpaC, which was previously identified as a T3S control protein. Since HpaC promotes secretion of translocon and effector proteins but inhibits secretion of HrpB2, HpaC presumably acts as a T3S substrate specificity switch protein. Protein–protein interaction studies revealed that HpaC interacts with HrpB2 and the C-terminal domain of HrcU, a conserved inner membrane component of the T3S system. However, no interaction was observed between HpaC and the full-length HrcU protein. Analysis of HpaC deletion derivatives revealed that the binding site for the C-terminal domain of HrcU is essential for HpaC function. This suggests that HpaC binding to the HrcU C terminus is key for the control of T3S. The C terminus of HrcU also provides a binding site for HrpB2; however, no interaction was observed with other T3S substrates including pilus, translocon and effector proteins. This is in contrast to HrcU homologs from animal pathogenic bacteria suggesting evolution of distinct mechanisms in plant and animal pathogenic bacteria for T3S substrate recognition

Aconitase B Is Required for Optimal Growth of Xanthomonas campestris pv. vesicatoria in Pepper Plants

The aerobic plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) colonizes the intercellular spaces of pepper and tomato. One enzyme that might contribute to the successful proliferation of Xcv in the host is the iron-sulfur protein aconitase, which catalyzes the conversion of citrate to isocitrate in the tricarboxylic acid (TCA) cycle and might also sense reactive oxygen species (ROS) and changes in cellular iron levels. Xcv contains three putative aconitases, two of which, acnA and acnB, are encoded by a single chromosomal locus. The focus of this study is aconitase B (AcnB). acnB is co-transcribed with two genes, XCV1925 and XCV1926, encoding putative nucleic acid-binding proteins. In vitro growth of acnB mutants was like wild type, whereas in planta growth and symptom formation in pepper plants were impaired. While acnA, XCV1925 or XCV1926 mutants showed a wild-type phenotype with respect to bacterial growth and in planta symptom formation, proliferation of the acnB mutant in susceptible pepper plants was significantly impaired. Furthermore, the deletion of acnB led to reduced HR induction in resistant pepper plants and an increased susceptibility to the superoxide-generating compound menadione. As AcnB complemented the growth deficiency of an Escherichia coli aconitase mutant, it is likely to be an active aconitase. We therefore propose that optimal growth and survival of Xcv in pepper plants depends on AcnB, which might be required for the utilization of citrate as carbon source and could also help protect the bacterium against oxidative stress

Evolutionary and Experimental Assessment of Novel Markers for Detection of Xanthomonas euvesicatoria in Plant Samples

BACKGROUND: Bacterial spot-causing xanthomonads (BSX) are quarantine phytopathogenic bacteria responsible for heavy losses in tomato and pepper production. Despite the research on improved plant spraying methods and resistant cultivars, the use of healthy plant material is still considered as the most effective bacterial spot control measure. Therefore, rapid and efficient detection methods are crucial for an early detection of these phytopathogens. METHODOLOGY: In this work, we selected and validated novel DNA markers for reliable detection of the BSX Xanthomonas euvesicatoria (Xeu). Xeu-specific DNA regions were selected using two online applications, CUPID and Insignia. Furthermore, to facilitate the selection of putative DNA markers, a customized C program was designed to retrieve the regions outputted by both databases. The in silico validation was further extended in order to provide an insight on the origin of these Xeu-specific regions by assessing chromosomal location, GC content, codon usage and synteny analyses. Primer-pairs were designed for amplification of those regions and the PCR validation assays showed that most primers allowed for positive amplification with different Xeu strains. The obtained amplicons were labeled and used as probes in dot blot assays, which allowed testing the probes against a collection of 12 non-BSX Xanthomonas and 23 other phytopathogenic bacteria. These assays confirmed the specificity of the selected DNA markers. Finally, we designed and tested a duplex PCR assay and an inverted dot blot platform for culture-independent detection of Xeu in infected plants. SIGNIFICANCE: This study details a selection strategy able to provide a large number of Xeu-specific DNA markers. As demonstrated, the selected markers can detect Xeu in infected plants both by PCR and by hybridization-based assays coupled with automatic data analysis. Furthermore, this work is a contribution to implement more efficient DNA-based methods of bacterial diagnostics

Epidemiology of Invasive Fungal Infections in Latin America

The pathogenic role of invasive fungal infections (IFIs) has increased during the past two decades in Latin America and worldwide, and the number of patients at risk has risen dramatically. Working habits and leisure activities have also been a focus of attention by public health officials, as endemic mycoses have provoked a number of outbreaks. An extensive search of medical literature from Latin America suggests that the incidence of IFIs from both endemic and opportunistic fungi has increased. The increase in endemic mycoses is probably related to population changes (migration, tourism, and increased population growth), whereas the increase in opportunistic mycoses may be associated with the greater number of people at risk. In both cases, the early and appropriate use of diagnostic procedures has improved diagnosis and outcome

Immunodiagnosis of endemic mycoses and bronchopulmonary aspergilosis: A multicenter study in Argentina

Fil: Canteros, C. E. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Rivas, M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Soria, M. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Lee, W. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Perrotta, Diego. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Rodero, L. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Davel, Graciela Odelsia. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Micología; Argentina.Fil: Berducci, O. Grupo EMMB; Argentina.Fil: Bonardello, N. Grupo EMMB; Argentina.Fil: Castro, H. Grupo EMMB; Argentina.Fil: Chacón, Y. Grupo EMMB; Argentina.Fil: Cendán Colombo, L. Grupo EMMB; Argentina.Fil: De Vechi, M. Grupo EMMB; Argentina.Fil: Errecalde, G. Grupo EMMB; Argentina.Fil: Fernández, N. Grupo EMMB; Argentina.FIl: Gorostiaga, J. L. Grupo EMMB; Argentina.Fil: López, C. Grupo EMMB; Argentina.Fil: Mackay, P. Grupo EMMB; Argentina.Fil: Gonzalez, R. Grupo EMMB; Argentina.Fil: Cacace, María Luisa. Grupo EMMB; Argentina.Fil: Mestron, S. Grupo EMMB; Argentina.Fil: Mónaco, L. Grupo EMMB; Argentina.Fil: Nardin, M. E. Grupo EMMB; Argentina.Fil: Ramos, L. Grupo EMMB; Argentina.Fil: Pagella, H. Grupo EMMB; Argentina.Fil: Petrussi, N. Grupo EMMB; Argentina.Fil: Pizarro, M. R. Grupo EMMB; Argentina.Fil: Sánchez, R. Grupo EMMB; Argentina.Fil: Saporiti, A. M. Grupo EMMB; Argentina.Fil: Tichellio, A. G. Grupo EMMB; Argentina.Fil: Tiraboschi, N. Grupo EMMB; Argentina.Fil: Tonelli, L. Grupo EMMB; Argentina.Fil: Zanuso, A. Grupo EMMB; Argentina.Se realizó entre 01-04-2000 y 30-03-2001, un estudio de corte transversal, para conocer la frecuencia relativa de las enfermedades por hongos dimorfos y Aspergillus spp. en la República Argentina y evaluar la certeza en el diagnóstico de los laboratorios de diferentes áreas geográficas. Participaron 25 centros de salud provenientes de 12 provincias y de la Ciudad Autónoma de Buenos Aires. Fueron analizados en el laboratorio de origen 965 sueros de pacientes con sospecha clínica de histoplasmosis (HP), paracoccidioidomicosis (PCM), coccidioidomicosis (CM) y aspergilosis. Todos los sueros positivos y el 35% de los negativos fueron reevaluados en el laboratorio de referencia por inmunodifusión doble en agar. La concordancia entre los resultados obtenidos en los centros de origen y el de referencia fue de 98,8%. Se detectaron anticuerpos específicos en 120 sueros correspondientes a 98 pacientes. El 71,4% (70 casos) de los diagnósticos correspondió a micosis endémicas (HP, PCM y CM) y el resto a aspergilosis. PCM fue diagnosticada en 47,9% (47 casos), aspergilosis en 28,6% (28 casos), HP en13,3% (13 casos) y CM en 10,2% (10 casos). La participación en este estudio fue voluntaria y no todos los centros del país estaban representados, sin embargo, las frecuencias de enfermedades fúngicas fueron las esperadas y coincidentes con estudios previos realizados a nivel nacional. (EN) In order to contribute to the knowledge of the relative frequency of chronic fungal diseases and assess the performance of diagnostic laboratories in Argentina, a multicenter study was performed with the participation of 25 medical centers located in 12 different provinces and Buenos Aires City. Between 04-01- 2000 and 03-30-2001, 965 serum specimens from patients clinically suspected of having histoplasmosis (HP), paracoccidioidomycosis (PCM), coccidioidomycosis (CM) or aspergilosis were analyzed. Agar immunodiffusion tests (IDD) were done locally. All positive and 35% of negative sera were retested in the reference center. Results of laboratories of origin showed 98.8% concordance with those of reference center. Antibodies against any of the etiological agents were detected in 120 specimens from 98 patients. Endemic mycoses (HP, PCM and CM) were diagnosed in 70 patients (71.4%) and aspergilosis in 28 (28.6%). The frequencies of the different mycoses in decreasing order w ere PCM 47 patients (47.9%), aspergilosis 28 patients (28.6%), HP 13 patients (13.3%) and CM 10 patients (10.2%). The study was carried out on a voluntary basis and some areas of the country were not represented. However, the frequencies were in range with the expected rates in the population under study