Benznidazole biotransformation and multiple targets in <i>Trypanosoma</i> cruzi revealed by metabolomics

&lt;b&gt;Background&lt;/b&gt;&lt;p&gt;&lt;/p&gt; The first line treatment for Chagas disease, a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, involves administration of benznidazole (Bzn). Bzn is a 2-nitroimidazole pro-drug which requires nitroreduction to become active, although its mode of action is not fully understood. In the present work we used a non-targeted MS-based metabolomics approach to study the metabolic response of T. cruzi to Bzn.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Methodology/Principal findings&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Parasites treated with Bzn were minimally altered compared to untreated trypanosomes, although the redox active thiols trypanothione, homotrypanothione and cysteine were significantly diminished in abundance post-treatment. In addition, multiple Bzn-derived metabolites were detected after treatment. These metabolites included reduction products, fragments and covalent adducts of reduced Bzn linked to each of the major low molecular weight thiols: trypanothione, glutathione, γ-glutamylcysteine, glutathionylspermidine, cysteine and ovothiol A. Bzn products known to be generated in vitro by the unusual trypanosomal nitroreductase, TcNTRI, were found within the parasites, but low molecular weight adducts of glyoxal, a proposed toxic end-product of NTRI Bzn metabolism, were not detected.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Conclusions/significance&lt;/b&gt;&lt;p&gt;&lt;/p&gt; Our data is indicative of a major role of the thiol binding capacity of Bzn reduction products in the mechanism of Bzn toxicity against T. cruzi

Salmonella enterica Subclinical Infection: Bacteriological, Serological, Pulsed-Field Gel Electrophoresis, and Antimicrobial Resistance Profiles-Longitudinal Study in a Three-Site Farrow-to-Finish Farm

Fil: Vigo, German B. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Cappuccio, J. A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Pineyro, Pablo E. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Salve, Angela. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Machuca, Mariana A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Quiroga, Maria A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Moredo, Fabiana. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Giacoboni, Gabriel. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Cancer, Jose L. Private practitioner; Argentina.Fil: Caffer, María Ines. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Pichel, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Perfumo, Carlos J. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.The aim of this surveillance was to study both Salmonella spp. shedding patterns and the time course of serological response in farrow-to-finish reared pigs from a subclinically infected farm. Antimicrobial resistance profile, molecular subtyping, and the relationship among the isolates were determined by pulsed-field gel electrophoresis (PFGE). A farrow-to-finish farm of 6000 sows, with a history of Salmonella Typhimurium septicemia, was selected. A longitudinal bacteriological and serological study was conducted in 25 sows before farrowing (M=S1) and in 50 offspring at 21 (M=S2), 35 (M=S3), 65 (M=S4), 86 (M=S5), 128 (M=S6), and 165 (M=S7) days of age. Serum antibodies were tested using Herdcheck Swine Salmonella antibody test kit (Idexx Laboratories, ME). Bacteria were isolated from pooled fecal samples. Suspected isolates were confirmed by conventional biochemical assays, and those identified as Salmonella spp. were serotyped. A variation between seropositive percentages and positive fecal samples was observed. Serologically positive pigs decreased from S1 to S4, and subsequently increased from S4 to S7. The percentages of fecal positive culture increased from M1 to M3, and then declined in M4, increased in M5, and were negative in M6 and M7. In the study three serovars, Salmonella 3,10:e,h:-, Salmonella Muenster, and Salmonella Bovismorbificans, were identified with low pathogenicity for swine. Three multidrug resistance strains (one belonged to Salmonella 3,10:e,h:- and two belonged to Salmonella Muenster) were found. PFGE results showed three different but closely related patterns among the 13 isolates of Salmonella Bovismorbificans, and two patterns for the three Salmonella Muenster and Salmonella 3,10:e,h:- isolates. This longitudinal study established critical points of Salmonella spp. infection in the farm and the production stages, where appropriate control measures must be taken. PFGE showed clonal relationships in each serovar. Antibiotic resistance profiles should be periodically included due to public health concerns

Salmonella enterica Subclinical Infection: Bacteriological, Serological, Pulsed-Field Gel Electrophoresis, and Antimicrobial Resistance Profiles-Longitudinal Study in a Three-Site Farrow-to-Finish Farm

Fil: Vigo, German B. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Cappuccio, J. A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Pineyro, Pablo E. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Salve, Angela. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Machuca, Mariana A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Quiroga, Maria A. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Moredo, Fabiana. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Giacoboni, Gabriel. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Cancer, Jose L. Private practitioner; Argentina.Fil: Caffer, María Ines. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Pichel, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Perfumo, Carlos J. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.The aim of this surveillance was to study both Salmonella spp. shedding patterns and the time course of serological response in farrow-to-finish reared pigs from a subclinically infected farm. Antimicrobial resistance profile, molecular subtyping, and the relationship among the isolates were determined by pulsed-field gel electrophoresis (PFGE). A farrow-to-finish farm of 6000 sows, with a history of Salmonella Typhimurium septicemia, was selected. A longitudinal bacteriological and serological study was conducted in 25 sows before farrowing (M=S1) and in 50 offspring at 21 (M=S2), 35 (M=S3), 65 (M=S4), 86 (M=S5), 128 (M=S6), and 165 (M=S7) days of age. Serum antibodies were tested using Herdcheck Swine Salmonella antibody test kit (Idexx Laboratories, ME). Bacteria were isolated from pooled fecal samples. Suspected isolates were confirmed by conventional biochemical assays, and those identified as Salmonella spp. were serotyped. A variation between seropositive percentages and positive fecal samples was observed. Serologically positive pigs decreased from S1 to S4, and subsequently increased from S4 to S7. The percentages of fecal positive culture increased from M1 to M3, and then declined in M4, increased in M5, and were negative in M6 and M7. In the study three serovars, Salmonella 3,10:e,h:-, Salmonella Muenster, and Salmonella Bovismorbificans, were identified with low pathogenicity for swine. Three multidrug resistance strains (one belonged to Salmonella 3,10:e,h:- and two belonged to Salmonella Muenster) were found. PFGE results showed three different but closely related patterns among the 13 isolates of Salmonella Bovismorbificans, and two patterns for the three Salmonella Muenster and Salmonella 3,10:e,h:- isolates. This longitudinal study established critical points of Salmonella spp. infection in the farm and the production stages, where appropriate control measures must be taken. PFGE showed clonal relationships in each serovar. Antibiotic resistance profiles should be periodically included due to public health concerns

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

PKA regulatory subunits mediate synergy among conserved G-protein-coupled receptor cascades

G-protein-coupled receptors sense extracellular chemical or physical stimuli and transmit these signals to distinct trimeric G-proteins. Activated Gα-proteins route signals to interconnected effector cascades, thus regulating thresholds, amplitudes and durations of signalling. Gαs- or Gαi-coupled receptor cascades are mechanistically conserved and mediate many sensory processes, including synaptic transmission, cell proliferation and chemotaxis. Here we show that a central, conserved component of Gαs-coupled receptor cascades, the regulatory subunit type-II (RII) of protein kinase A undergoes adenosine 3′-5′-cyclic monophosphate (cAMP)-dependent binding to Gαi. Stimulation of a mammalian Gαi-coupled receptor and concomitant cAMP-RII binding to Gαi, augments the sensitivity, amplitude and duration of Gαi:βγ activity and downstream mitogen-activated protein kinase signalling, independent of protein kinase A kinase activity. The mechanism is conserved in budding yeast, causing nutrient-dependent modulation of a pheromone response. These findings suggest a direct mechanism by which coincident activation of Gαs-coupled receptors controls the precision of adaptive responses of activated Gαi-coupled receptor cascades

Serovars of Salmonella enterica subspecies enterica and its antimicrobial resistance in slaughterhouse pigs

Fil: Ibar, M. P. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Vigo, G. Universidad Nacional de La Plata. Cátedra de Microbiología; Argentina.Fil: Pineyro, P. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Caffer, María Inés. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Servicio de Enterobacterias; Argentina.Fil: Quiroga, P. Universidad de Buenos Aires. Departamento de Microbiología; Argentina.Fil: Perfumo, C. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Centrón, D. Universidad de Buenos Aires. Departamento de Microbiología; Argentina.Fil: Giacoboni, G. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Se realizó un estudio para determinar la prevalencia de Salmonella y sus serovariedades en cerdos de faena, para evaluar sus perfiles de resistencia a los antimicrobianos y para conocer la presencia de integrones de clase 1 como posibles reservorios de resistencia. A partir de un total de 386 muestras de porcinos provenientes de cuatro frigoríficos de las provincias de Buenos Aires y de Santa Fe (Argentina), se identificaron 93 (24,1%) cepas de Salmonella enterica subespecie enterica, 52 (55,9%) de contenido cecal y 41 (44,1%) de nódulo linfático ileocecal. Se hallaron 13 serovariedades de S. enterica, las más prevalentes fueron S. Schwarzengrund, S. Heidelberg, S. subespecie I 6,8:e,h:-, S. Derby y S. Bredeney. Se probaron 15 antimicrobianos por el método de dilución en agar: amikacina, gentamicina, ciprofloxacina, cefalotina, cefotaxima, enrofloxacina, fosfomicina, polimixina-B, tetraciclina, cloranfenicol, estreptomicina, trimetoprima-sulfametoxazol, ampicilina, nitrofurantoína y ácido nalidíxico. Según se estableció mediante la determinación de la CIM, el 73% de las cepas de S. enterica subespecie enterica fueron sensibles a todos los antimicrobianos probados. Se observó resistencia a tetraciclina en 24 (25,8%) de las 93 cepas, a cloranfenicol en 22 (23,7%), a estreptomicina en 22 (23,7%) a trimetoprima-sulfametoxazol en 20 (21,5%), a ampicilina en 18 (19,4%), a nitrofurantoína en 3 (3,2%) y a ácido nalidíxico en 3 (3,2%). Algunos aislamientos de S. Typhimurium, S. Heildelberg, S. Derby y S. Orion presentaron multirresistencia y portaban el gen de la integrasa clase 1. Los mayores porcentajes de resistencia correspondieron a los antimicrobianos habitualmente utilizados en veterinaria y en las explotaciones porcinas

Serovars of Salmonella enterica subspecies enterica and its antimicrobial resistance in slaughterhouse pigs

Fil: Ibar, M. P. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Fil: Vigo, G. Universidad Nacional de La Plata. Cátedra de Microbiología; Argentina.Fil: Pineyro, P. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Caffer, María Inés. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Servicio de Enterobacterias; Argentina.Fil: Quiroga, P. Universidad de Buenos Aires. Departamento de Microbiología; Argentina.Fil: Perfumo, C. Universidad Nacional de La Plata. Cátedra de Patología Especial; Argentina.Fil: Centrón, D. Universidad de Buenos Aires. Departamento de Microbiología; Argentina.Fil: Giacoboni, G. Universidad Nacional de La Plata. Laboratorio de Diagnóstico e Investigaciones Bacteriológicas; Argentina.Se realizó un estudio para determinar la prevalencia de Salmonella y sus serovariedades en cerdos de faena, para evaluar sus perfiles de resistencia a los antimicrobianos y para conocer la presencia de integrones de clase 1 como posibles reservorios de resistencia. A partir de un total de 386 muestras de porcinos provenientes de cuatro frigoríficos de las provincias de Buenos Aires y de Santa Fe (Argentina), se identificaron 93 (24,1%) cepas de Salmonella enterica subespecie enterica, 52 (55,9%) de contenido cecal y 41 (44,1%) de nódulo linfático ileocecal. Se hallaron 13 serovariedades de S. enterica, las más prevalentes fueron S. Schwarzengrund, S. Heidelberg, S. subespecie I 6,8:e,h:-, S. Derby y S. Bredeney. Se probaron 15 antimicrobianos por el método de dilución en agar: amikacina, gentamicina, ciprofloxacina, cefalotina, cefotaxima, enrofloxacina, fosfomicina, polimixina-B, tetraciclina, cloranfenicol, estreptomicina, trimetoprima-sulfametoxazol, ampicilina, nitrofurantoína y ácido nalidíxico. Según se estableció mediante la determinación de la CIM, el 73% de las cepas de S. enterica subespecie enterica fueron sensibles a todos los antimicrobianos probados. Se observó resistencia a tetraciclina en 24 (25,8%) de las 93 cepas, a cloranfenicol en 22 (23,7%), a estreptomicina en 22 (23,7%) a trimetoprima-sulfametoxazol en 20 (21,5%), a ampicilina en 18 (19,4%), a nitrofurantoína en 3 (3,2%) y a ácido nalidíxico en 3 (3,2%). Algunos aislamientos de S. Typhimurium, S. Heildelberg, S. Derby y S. Orion presentaron multirresistencia y portaban el gen de la integrasa clase 1. Los mayores porcentajes de resistencia correspondieron a los antimicrobianos habitualmente utilizados en veterinaria y en las explotaciones porcinas