Generation and selection of anti-flagellin monoclonal antibodies useful for serotyping Salmonella enterica

In developing countries, bacterial acute gastroenteritis continues to be an important cause of morbidity and mortality among young children. Salmonellosis constitutes a major cause of infectious enteritis worldwide, most of them associated to the consumption of contaminated food products. Traditionally, Salmonella has been classified in serovars based on varieties of O and H surface antigens. In the present work we generated and characterized a panel of anti-flagellin monoclonal antibodies (MAbs) in order to select antibodies useful for detecting the H surface antigen. Four different MAbs were obtained by somatic hybridization of splenocytes. We found two MAbs that recognised regions of flagellin conserved among different Salmonella serovars. Other two MAbs recognised structures restricted to Salmonella enterica sv. Typhimurium, being one of them suitable for agglutination tests. Using a diverse panel of S. enterica serovars with different H antigen varieties we confirmed that this MAb agglutinates specifically S. Typhimurium (antigenic formula: 4,12:i:1,2) or other serovars expressing flagellar factor i. In conclusion, we generated a valuable immunochemical tool to be used in simple assays for serotyping of epidemiologically relevant strains. The capacity to characterize specific strains and determine the primary sources of Salmonella contamination generate valuable information of the epidemiology of this microorganism, contributing to the improvement of public health.Fil: Hiriart, Yanina. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigaciones del Sistema Inmune; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Serradell, Maria de Los Angeles. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Martinez, Araci. Universidad de la República; UruguayFil: Sampaolesi, Sofia. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Cátedra de Microbiología General; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Gonzalez Maciel, Maria Dolores. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigaciones del Sistema Inmune; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Chabalgoity, Jose Alejandro. Universidad de la República; UruguayFil: Yim, Lucia. Universidad de la República; UruguayFil: Algorta, Gabriela. Universidad de la República; UruguayFil: Rumbo, Martín. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigaciones del Sistema Inmune; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Lokalno liječenje kroničnog ulcerativnog stomatitisa pasa primjenom mezenhimnih matičnih stanica iz adipoznog tkiva - prikaz slučaja

Canine chronic ulcerative stomatitis (CCUS) is a condition characterized by chronic pain, associated with oral ulceration, inflammation and mucosal necrosis. We report the case of a 5-year-old female Akita Inu canine with sialorrhea, halitosis, oral ulcers, submandibular lymphadenopathy and substantial oral discomfort. After a poor response to conventional scaling and antibiotic treatment, the patient improved when prednisolone was administered at a dose of 1mg / kg daily. Aggressive immunosuppressive treatments and teeth extractions were ruled out, so cellular therapy was started. This study evaluates the effect of allogeneic thawed and expanded canine adipose tissue-derived mesenchymal stem cells (AT-MSCs) therapy in a case of CCUS. Four local injections of AT-MSCs (3.5 to 8×106 cells) were administered every 3–4 weeks under general anesthesia and immediately after dental scaling. A higher dose of AT-MSCs (22×106 cells) was injected 5 months later under the same conditions. After the AT-MSCs treatment, the patient showed an improvement in oral lesions and reduction of oral pain for at least 4 weeks. We concluded that a dose of 1×106 AT-MSCs/kg body weight reduced the symptoms of CCUS for at least one month in this case. To our knowledge, this is the first description of allogeneic AT-MSC therapy on canine chronic ulcerative stomatitis.Kronični ulcerativni stomatitis u pasa (CCUS) obilježava kronični bol, povezan s oralnom ulceracijom, upalom i sluzničnom nekrozom. Ovo je prikaz slučaja petogodišnje ženke pasmine akita inu sa sijalorejom, halitozom, oralnim ulceracijama, submandibularnom limfadenopatijom i supstancijalnim oralnim smetnjama. Nakon slabog odgovora na konvencionalno čišćenje kamenca i antibiotičko liječenje, stanje se poboljšalo s primjenom prednizolona u dozi od 1mg/kg na dan. Odgođena je agresivna imunosupresija i vađenje zubi te je započeta terapija matičnim stanicama. U istraživanju je procijenjen učinak alogenskih odmrznutih i ekspandiranih mezenhimnih matičnih stanica iz adipoznog tkiva (AT-MSCs) u liječenju CCUS-a. Lokalno su primijenjene četiri injekcije AT-MSC-a (3,5 do 8 × 106 stanica) svaka 3 – 4 tjedna pod općom anestezijom i neposredno nakon čišćenja zubnog kamenca. Veća doza AT-MSC-a (22 × 106 stanica) primijenjena je pet mjeseci poslije pod jednakim uvjetima. Nakon liječenja AT-MSC-om došlo je do poboljšanja u cijeljenju oralnih lezija i smanjenju boli tijekom namanje četiri tjedna. Zaključujemo da, u ovomslučaju, doza od 1 × 106 AT-MSC-a po kilogramu tjelesne mase ublažava simptome CCUS-a tijekom najmanje jednog mjeseca. Ovo je prema našim spoznajama prvi opis alogenskog liječenja kroničnog ulcerativnog stomatitisa u pasa AT-MSC-om

Community-associated methicillin-resistant Staphylococcus aureus in children treated in Uruguay

Introduction: Staphylococcus aureus produces a variety of diseases among children, ranging from skin and soft tissue infections to invasive life-threatening diseases. Since 1990, an increasing number of diseases produced by community-associated methicillin-resistant S. aureus (CA-MRSA) isolates have been reported. The aim of this study was to describe the importance and the microbiological characteristics of S. aureus isolates recovered from children treated at the Hospital Pediátrico del Centro Hospitalario ?Pereira Rossell? (HP-CHPR); focusing on invasive diseases caused by CA-MRSA isolates, as well as some clinical aspects of the diseases they have produced. Methodology: One hundred and twenty-five S. aureus isolates recovered from the HP-CHPR between 2003 and 2006 from children with invasive (n=89) and superficial diseases (n=36) were included. Genotypic and phenotypic characteristics of S. aureus isolates and relevant clinical aspects of each child were studied. Results: CA-MRSA isolates accounted for 73% of all S. aureus recovered from invasive (mainly bone and joint) infections, pneumonia and bacteraemia. The most common CA-MRSA strain recovered from invasive (n=65) and superficial (n=36) diseases had the following features: pulsotype A (type USA1100), SCCmec cassette type IV, Panton-Valentine Leukocidin genes positive, susceptibility to trimethoprim-sulfamethoxazole without the inducible macrolide-lincosamide-streptogramin B (iMLSB) resistance phenotype. No association between genotypic characteristics of invasive CA-MRSA isolates and clinical outcomes was found. Conclusions: CA-MRSA isolates produced a wide spectrum of invasive diseases in a public paediatric hospital between 2003 and 2006. Microbiologic characterization suggests the spread of an adapted CA-MRSA clone lacking erm genesFil: Pardo, Lorena. Universidad de la Republica; UruguayFil: Vola, Magdalena. Universidad de la Republica; UruguayFil: Macedo Viñas, Marina. Universidad de la Republica; UruguayFil: Machado, Virginia. Universidad de la Republica; UruguayFil: Cuello, Dianna. Universidad de la Republica; UruguayFil: Mollerach, Marta Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Castro, Marta. Universidad de la Republica; UruguayFil: Pirez, Catalina. Universidad de la Republica; UruguayFil: Varela, Gustavo. Universidad de la Republica; UruguayFil: Algorta, Gabriela. Universidad de la Republica; Urugua

Vigilancia del uso de antibióticos en el Hospital Pediátrico del Centro Hospitalario Pereira Rossell: susceptibilidad antimicrobiana; gasto y consumo de antibióticos

Introducción: La utilización de guías para el tratamiento antibiótico empírico constituye una estrategia fundamental para contener el aumento sostenido en la resistencia bacteriana y en los gastos en antibióticos. En 1998, en el Hospital Pediátrico del Centro Hospitalario Pereira Rossell (HP-CHPR) se adecuaron las guías de antibioticoterapia empírica para las infecciones más frecuentes asistidas en salas generales y en emergencia, en base a la epidemiología local. Desde esa fecha se comenzó la vigilancia del uso de antibióticos. Objetivos: Analizar los resultados de esta vigilancia sobre el gasto y el consumo de antibióticos y sobre la susceptibilidad antimicrobiana. Metodología: Se determinó el gasto generado por los antibióticos recomendados en las guías de atención hospitalaria, en salas generales y en emergencia (penicilina, aminopenicilinas, cefalosporinas, macrólidos). Para estimar el consumo en pacientes hospitalizados se calculó la dosis diaria definida (DDD)/100 camas día para cada uno de los antibióticos. Se compararon los resultados de gasto y consumo 2001-2002. Se comparó la susceptibilidad antimicrobiana de los gérmenes más frecuentes del período 2001-2002 con la previa a la adecuación de las guías terapéuticas. Resultados: Los antibióticos recomendados representaron en el año 2001, 57% del gasto en antibióticos (2.206.652,57 pesos) y en 2002, 54% (1.441.280 pesos). Los antibióticos más utilizados fueron las aminopenicilinas. El consumo de cefuroxime intravenoso disminuyó 60% (de 13.1 a 5.36 DDD/100 camas-día). El consumo de ceftriaxona intravenosa y de amoxicilina oral aumentó 38% y 16%, respectivamente. Para el resto de los antibióticos el consumo se mantuvo constante. Las tasas de susceptibilidad antimicrobiana de los gérmenes más frecuentes (S. pneumoniae, S. aureus, E. coli, Shigella spp) a los antibióticos recomendados en las guías terapéuticas se mantuvieron por encima de 80%. Conclusiones: Estos son los primeros resultados de la vigilancia del uso de antibióticos en este hospital y demuestran la vigencia y la aceptación de las guías de antibioticoterapia empírica propuestas. Es difícil interpretar la disminución del gasto en antibióticos debido a la variación en el precio de estos medicamentos en este período

Generation and selection of anti-flagellin monoclonal antibodies useful for serotyping Salmonella enterica

In developing countries, bacterial acute gastroenteritis continues to be an important cause of morbidity and mortality among young children. Salmonellosis constitutes a major cause of infectious enteritis worldwide, most of them associated to the consumption of contaminated food products. Traditionally, Salmonella has been classified in serovars based on varieties of O and H surface antigens. In the present work we generated and characterized a panel of anti-flagellin monoclonal antibodies (MAbs) in order to select antibodies useful for detecting the H surface antigen. Four different MAbs were obtained by somatic hybridization of splenocytes. We found two MAbs that recognised regions of flagellin conserved among different Salmonella serovars. Other two MAbs recognised structures restricted to Salmonella enterica sv. Typhimurium, being one of them suitable for agglutination tests. Using a diverse panel of S. enterica serovars with different H antigen varieties we confirmed that this MAb agglutinates specifically S. Typhimurium (antigenic formula: 4,12:i:1,2) or other serovars expressing flagellar factor i. In conclusion, we generated a valuable immunochemical tool to be used in simple assays for serotyping of epidemiologically relevant strains. The capacity to characterize specific strains and determine the primary sources of Salmonella contamination generate valuable information of the epidemiology of this microorganism, contributing to the improvement of public health.Laboratorio de Investigaciones del Sistema InmuneFacultad de Ciencias Exacta

Generation and selection of anti-flagellin monoclonal antibodies useful for serotyping Salmonella enterica

In developing countries, bacterial acute gastroenteritis continues to be an important cause of morbidity and mortality among young children. Salmonellosis constitutes a major cause of infectious enteritis worldwide, most of them associated to the consumption of contaminated food products. Traditionally, Salmonella has been classified in serovars based on varieties of O and H surface antigens. In the present work we generated and characterized a panel of anti-flagellin monoclonal antibodies (MAbs) in order to select antibodies useful for detecting the H surface antigen. Four different MAbs were obtained by somatic hybridization of splenocytes. We found two MAbs that recognised regions of flagellin conserved among different Salmonella serovars. Other two MAbs recognised structures restricted to Salmonella enterica sv. Typhimurium, being one of them suitable for agglutination tests. Using a diverse panel of S. enterica serovars with different H antigen varieties we confirmed that this MAb agglutinates specifically S. Typhimurium (antigenic formula: 4,12:i:1,2) or other serovars expressing flagellar factor i. In conclusion, we generated a valuable immunochemical tool to be used in simple assays for serotyping of epidemiologically relevant strains. The capacity to characterize specific strains and determine the primary sources of Salmonella contamination generate valuable information of the epidemiology of this microorganism, contributing to the improvement of public health.Laboratorio de Investigaciones del Sistema InmuneFacultad de Ciencias Exacta

Enteropathogens Associated with Acute Diarrhea in Children from Households with High Socioeconomic Level in Uruguay

Infectious diarrhea, a common disease of children, deserves permanent monitoring in all social groups. To know the etiology and clinical manifestations of acute diarrhea in children up to 5 years of age from high socioeconomic level households, we conducted a descriptive, microbiological, and clinical study. Stools from 59 children with acute community-acquired diarrhea were examined, and their parents were interviewed concerning symptoms and signs. Rotavirus, adenovirus, and norovirus were detected by commercially available qualitative immunochromatographic lateral flow rapid tests. Salmonella, Campylobacter, Yersinia, and Shigella were investigated by standard bacteriological methods and diarrheagenic E. coli by PCR assays. We identified a potential enteric pathogen in 30 children. The most frequent causes of diarrhea were enteropathogenic E. coli (EPEC), viruses, Campylobacter, Salmonella, and Shiga-toxin-producing E. coli (STEC). Only 2 patients showed mixed infections. Our data suggest that children with viral or Campylobacter diarrhea were taken to the hospital earlier than those infected with EPEC. One child infected with STEC O26 developed “complete” HUS. The microbiological results highlight the importance of zoonotic bacteria such as atypical EPEC, Campylobacter, STEC, and Salmonella as pathogens associated with acute diarrhea in these children. The findings also reinforce our previous communications about the regional importance of non-O157 STEC strains in severe infant food-borne diseases

Generation and selection of anti-flagellin monoclonal antibodies useful for serotyping Salmonella enterica

In developing countries, bacterial acute gastroenteritis continues to be an important cause of morbidity and mortality among young children. Salmonellosis constitutes a major cause of infectious enteritis worldwide, most of them associated to the consumption of contaminated food products. Traditionally, Salmonella has been classified in serovars based on varieties of O and H surface antigens. In the present work we generated and characterized a panel of anti-flagellin monoclonal antibodies (MAbs) in order to select antibodies useful for detecting the H surface antigen. Four different MAbs were obtained by somatic hybridization of splenocytes. We found two MAbs that recognised regions of flagellin conserved among different Salmonella serovars. Other two MAbs recognised structures restricted to Salmonella enterica sv. Typhimurium, being one of them suitable for agglutination tests. Using a diverse panel of S. enterica serovars with different H antigen varieties we confirmed that this MAb agglutinates specifically S. Typhimurium (antigenic formula: 4,12:i:1,2) or other serovars expressing flagellar factor i. In conclusion, we generated a valuable immunochemical tool to be used in simple assays for serotyping of epidemiologically relevant strains. The capacity to characterize specific strains and determine the primary sources of Salmonella contamination generate valuable information of the epidemiology of this microorganism, contributing to the improvement of public health.Laboratorio de Investigaciones del Sistema InmuneFacultad de Ciencias Exacta

Genomic and phenotypic variation in epidemic-spanning Salmonella enterica serovar Enteritidis isolates.

BACKGROUND: Salmonella enterica serovar Enteritidis (S. Enteritidis) has caused major epidemics of gastrointestinal infection in many different countries. In this study we investigate genome divergence and pathogenic potential in S. Enteritidis isolated before, during and after an epidemic in Uruguay. RESULTS: 266 S. Enteritidis isolates were genotyped using RAPD-PCR and a selection were subjected to PFGE analysis. From these, 29 isolates spanning different periods, genetic profiles and sources of isolation were assayed for their ability to infect human epithelial cells and subjected to comparative genomic hybridization using a Salmonella pan-array and the sequenced strain S. Enteritidis PT4 P125109 as reference. Six other isolates from distant countries were included as external comparators.Two hundred and thirty three chromosomal genes as well as the virulence plasmid were found as variable among S. Enteritidis isolates. Ten out of the 16 chromosomal regions that varied between different isolates correspond to phage-like regions. The 2 oldest pre-epidemic isolates lack phage SE20 and harbour other phage encoded genes that are absent in the sequenced strain. Besides variation in prophage, we found variation in genes involved in metabolism and bacterial fitness. Five epidemic strains lack the complete Salmonella virulence plasmid. Significantly, strains with indistinguishable genetic patterns still showed major differences in their ability to infect epithelial cells, indicating that the approach used was insufficient to detect the genetic basis of this differential behaviour. CONCLUSION: The recent epidemic of S. Enteritidis infection in Uruguay has been driven by the introduction of closely related strains of phage type 4 lineage. Our results confirm previous reports demonstrating a high degree of genetic homogeneity among S. Enteritidis isolates. However, 10 of the regions of variability described here are for the first time reported as being variable in S. Enteritidis. In particular, the oldest pre-epidemic isolates carry phage-associated genetic regions not previously reported in S. Enteritidis. Overall, our results support the view that phages play a crucial role in the generation of genetic diversity in S. Enteritidis and that phage SE20 may be a key marker for the emergence of particular isolates capable of causing epidemics