Survey of selected pathogens in free-ranging pinnipeds in Uruguay.

Marine mammals, regarded as sentinels of aquatic ecosystem health, are exposed to different pathogens and parasites under natural conditions. We surveyed live South American fur seals Arctocephalus australis and South American sea lions Otaria flavescens in Uruguay for Leptospira spp., canine distemper virus (CDV), Mycobacterium spp., Toxoplasma gondii, and Neospora caninum. Samples were collected from 2007 to 2013. The seroprevalence of Leptospira spp. was 37.6% positive, 50.9% negative, and 11.5% suspect for A. australis (n = 61) while for O. flavescens (n = 12) it was 67% positive, 25% negative, and 8% suspect. CDV RNA was not detected in any of the analyzed samples. Most animals tested seropositive to tuberculosis antigens by WiZo ELISA (A. australis: 29/30; O. flavescens: 20/20); reactivity varied with a novel ELISA test (antigens MPB70, MPB83, ESAT6 and MPB59). Seroprevalence against N. caninum and T. gondii was 6.7 and 13.3% positive for O. flavescens and 0 and 2.2% positive for A. australis respectively. To evaluate possible sources of infection for pinnipeds, wild rats Rattus rattus and semi-feral cats Felis catus were also tested for Leptospira spp. and T. gondii respectively. Water samples tested for Leptospira revealed saprofitic L. bioflexa. Pathogenic Leptospira were detected in the kidneys of 2 rats, and cats tested positive for T. gondii (100%). These results represent a substantial contribution to the study of the health status of wild pinnipeds in Uruguay.

Bacterial pathogens associated with bloody diarrhea in Uruguayan children.

Diarrheal disease continues to be a serious health problem, especially in developing countries. Bloody diarrhea represents approximately 20-30% of all cases and has higher morbidity and mortality. Treatment with antibiotics is beneficial in cases of Shigella, Campylobacter, Yersinia and Salmonella infection, principally in those children with a higher risk of invasive disease. The aims of this study were to detect the bacterial agents associated with bloody diarrhea in children and to determine their antimicrobial susceptibility patterns. Between June 2001 and January 2008, 249 children with bloody diarrhea were studied. Shigella and Shiga toxin-producing Escherichia coli (STEC) were recovered from 48 (19.3%) and 3 (1.2%) of the total of cases, respectively. In 49 out of 249 children, in whom other enteropathogens were investigated, we recovered Campylobacter jejuni from 7 children (14.3%), Salmonella spp. from 2 (4.1%) and Aeromonas spp. from 1 (2%) in addition to Shigella from 7 children (14.3%). Thirty-four (70%) Shigella isolates showed resistance to ampicillin and 13 (27%) to trimethoprim-sulfamethoxazole. All Shigella isolates were susceptible to nalidixic acid, ciprofloxacin and ceftriaxone. Salmonella and STEC isolates were susceptible to all antibiotics assayed. Thus, the use of trimethoprim-sulfamethoxazole or ampicillin would not be appropriate for the empirical treatment of Shigella - associated diarrhea.

Prevalence of Salmonella enterica in poultry and eggs in Uruguay during an epidemic due to Salmonella enterica serovar Enteritidis.

Salmonella enterica serovar Enteritidis (S. Enteritidis) is frequently associated with food-borne disease worldwide. Poultry-derived products are a major source. An epidemic of human infection with S. Enteritidis occurred in Uruguay, and to evaluate the extent of poultry contamination, we conducted a nationwide survey over 2 years that included the analysis of sera from 5,751 birds and 12,400 eggs. Serological evidence of infection with Salmonella group O:9 was found in 24.4% of the birds. All positive sera were retested with a gm flagellum-based enzyme-linked immunosorbent assay, and based on these results, the national prevalence of S. Enteritidis infection was estimated to be 6.3%. Salmonellae were recovered from 58 of 620 pools made up of 20 eggs each, demonstrating a prevalence of at least 1 in every 214 eggs. Surprisingly, the majority of the isolates were not S. Enteritidis. Thirty-nine isolates were typed as S. Derby, 9 as S. Gallinarum, 8 as S. Enteritidis, and 2 as S. Panama. Despite the highest prevalence in eggs, S. Derby was not isolated from humans in the period of analysis, suggesting a low capacity to infect humans. Microarray-based comparative genomic hybridization analysis of S. Derby and S. Enteritidis revealed more than 350 genetic differences. S. Derby lacked pathogenicity islands 13 and 14, the fimbrial lpf operon, and other regions encoding metabolic functions. Several of these regions are present not only in serovar Enteritidis but also in all sequenced strains of S. Typhimurium, suggesting that these regions might be related to the capacity of Salmonella to cause food-borne disease.

[Detection and characterization of Shiga toxin-producing Escherichia coli from clinical cases and food in Uruguay]. / Detección y caracterización de Escherichia coli productor de toxina Shiga a partir de casos clínicos y de alimentos en Uruguay.

We have assessed the frequency of Shiga toxin-producing Escherichia coil (STEC) in clinical and food samples as well as studied the genotypic and phenotypic characteristics of the recovered strains. One hundred ninety eight fecal samples from children with bloody diarrhea (BD), 14 from children with hemolytic uremic syndrome (HUS), 220 ground beef samples and 4 STEC isolates from other beef-derived products were analyzed. The STEC strains were isolated from 3 (1.5%) children with bloody diarrhea, 1 (7%) from a child with HUS and 4 (1.8%) from ground beef samples. All strains were eae and ehxA positive. The serotypes found were: O157:H7 (9 strains), O26:H11 (2), O111: NM (1) and O145:HNT (1). All O157:H7 STEC strains harbored the eae subtype gamma1, O26:H11 and O145:HNT strains, subtype beta1 and O111:NM strain, subtype gamma2/theta. The STEC strains of the same serogroup showed high genetic diversity. In Uruguay, STEC is not frequently isolated from cases of bloody diarrhea in children. However, all the recovered STEC strains carried the genes associated with severe disease and 2 out of 3 children infected with STEC developed HUS. Ground beef and other food products might be important vehicles for O157:H7 strains.

Detección y caracterización de Escherichia coli productor de toxina Shiga a partir de casos clínicos y de alimentos en Uruguay / Detection and characterization of Shiga toxin - producing Escherichia coli from clinical cases and food in Uruguay

Establecimos la frecuencia de aislamiento de Escherichia coli productor de toxina Shiga (STEC) a partir de muestras clínicas y de alimentos, así como las características fenotípicas y genotípicas de las cepas recuperadas. Se analizaron 198 muestras fecales de niños con diarrea sanguinolenta (DS), 14 muestras fecales de niños con síndrome urémico hemolítico (SUH) y 220 muestras de carne picada. También se estudiaron 4 cepas STEC aisladas de alimentos embutidos. Se recuperó STEC de 3 (1,5%) de los niños con DS, de 1 (7%) niño con SUH y de 4 (1,8%) de las muestras de carne picada. Todas las cepas fueron eae y ehxA positivas. Los serotipos detectados fueron: O157:H7 (9 cepas), O26:H11 (2 cepas), O111:NM (1 cepa) y O145:HNT (1 cepa). Todas las cepas O157:H7 portaron el subtipo eae-g1; las cepas O26:H11 y O145:HNT portaron el subtipo eae-b1 y la cepa O111:NM portó el subtipo eae-g2/q. Las cepas STEC del mismo serogrupo mostraron alta diversidad genética. En Uruguay STEC no sería agente frecuente de diarrea con sangre en niños. Sin embargo, las cepas recuperadas presentaron los genes asociados con enfermedad severa y 2 de los 3 niños infectados con STEC evolucionaron a SUH. La carne picada y otros alimentos serían vehículos importantes de O157:H7.

We have assessed the frequency of Shiga toxin-producing Escherichia coli (STEC) in clinical and food samples as well as studied the genotypic and phenotypic characteristics of the recovered strains. One hundred ninety eight fecal samples from children with bloody diarrhea (BD), 14 from children with hemolytic uremic syndrome (HUS), 220 ground beef samples and 4 STEC isolates from other beef-derived products were analyzed. The STEC strains were isolated from 3 (1.5%) children with bloody diarrhea, 1 (7%) from a child with HUS and 4 (1.8%) from ground beef samples. All strains were eae and ehxA positive. The serotypes found were: O157:H7 (9 strains), O26:H11 (2), O111: NM (1) and O145:HNT (1). All O157:H7 STEC strains harbored the eae subtype g1, O26:H11 and O145:HNT strains, subtype b1 and O111:NM strain, subtype g2/q. The STEC strains of the same serogroup showed high genetic diversity. In Uruguay, STEC is not frequently isolated from cases of bloody diarrhea in children. However, all the recovered STEC strains carried the genes associated with severe disease and 2 out of 3 children infected with STEC developed HUS. Ground beef and other food products might be important vehicles for O157:H7 strains.

[Genetic environment of CTX-M-2 in Klebsiella pneumoniae isolates from hospitalized patients in Uruguay]. / Entorno genético de CTX-M-2 en aislamientos de Klebsiella pneumoniae provenientes de pacientes hospitalizados en Uruguay.

We studied two CTX-M-2-producing Klebsiella pneumoniae clinical strains, K96005 and K13, isolated from hospitalized patients in Uruguay, during 1996 and 2003, respectively. The genomic surroundings of bla(CTX-M-2) were characterized by PCR-mapping and DNA sequencing. Our results show that blaCTX-M-2 is included in a complex class-1 integron (InK13), associated with an orf513 in both isolates. The genetic array of the integron, aac(6')-lb, bla(OxA,2), orfD (gene cassette region), associated with an orf513-bla(CTX-M-2), seems to be widely disseminated over the Rio de la Plata region.

Association of broad-spectrum antibiotic use with faecal carriage of oxyiminocephalosporin-resistant enterobacteriaceae in an intensive care unit.

The link between administration of antibiotics and detection of third-generation-cephalosporin-resistant (TGCR) enterobacteriaceae in faeces was studied in patients in a burns intensive care unit (ICU). The presence of extended-spectrum beta-lactamase producers was also determined in these isolates. At least two rectal swab samples were taken from 43 of 72 patients admitted to the ICU from January 1998 to June 1999. Antibiotic resistance tests were performed for all isolated enterobacteriaceae using the methods of the National Committee for Clinical Laboratory Standards. Only 10 out of 30 antibiotic-treated patients showed TGCR enterobacteriaceae in faeces. Fisher's exact test showed a relationship between the administration of oxyiminocephalosporins (third-generation cephalosporins) (P=0.002) or carbapenems (P=0.003) and the isolation of TGCR enterobacteriaceae from faeces. The administration of oxyiminocephalosporins led to the selection of resistant strains in the faecal flora.

Comparative molecular study of Mycobacterium tuberculosis strains, in times of antimicrobial drug resistance.

Strains of Mycobacterium tuberculosis were compared using two DNA fingerprinting techniques: Restriction Fragment Length Polymorphism (RFLP) and Double-Repetitive-Element-PCR (DRE-PCR). Two of these strains: IH1 (susceptible to isoniazid) and IH2 (resistant to isoniazid) were recovered from cases of pulmonary tuberculosis which occurred in two brothers who lived together. The first one was recognized on July 1999, and the second was diagnosed one year later. IH1 and IH2 showed the same pattern of bands with both molecular tests. These results suggest that single drug chemoprophylaxis may occasionally select resistant strains for that drug, which can eventually cause disease and be recognized through these tests. Strains IH3, IH4 and IH5 were obtained from sputum samples of 3 different patients, and intra-laboratory cross-contamination was suspected when it was realized that the 3 positive materials had been consecutively processed the same day by the same worker in the same biological safety cabinet. Again, the 3 strains revealed identical band patterns with RFLP and DRE-PCR, confirming the posed suspicion. The results with DRE-PCR were obtained after only 8 hours of work, without the need for subcultures. This procedure allows quick correction of treatment conducts, avoiding unnecessary exposure of people and bacteria to antimicrobial drugs.

An attenuated Salmonella Enteritidis strain derivative of the main genotype circulating in Uruguay is an effective vaccine for chickens.

We have recently reported that Salmonella enterica serovar Enteritidis (S. Enteritidis) strains circulating in Uruguay, are unevenly distributed among different genetic subtypes, with a predominant genotype that is a common contaminant of poultry-derived food and that accounts for the vast majority of human cases of food-borne disease. Herein, we describe the construction of a genetically-defined aroC derivative (LVR02) of a local strain of S. Enteritidis belonging to the major genetic type. We demonstrated the attenuation and the immunogenicity of that strain in a mouse model, and evaluated it as a vaccine for commercial layer chickens. LVR02 proved to be stable, attenuated, innocuous, immunogenic and to induce protective immunity against a S. Enteritidis challenge when used for oral vaccination. A single oral dose of LVR02 administered to newly hatched chickens induced protection against oral challenge with the parental virulent strain, preventing systemic and persistent intestinal infection and significantly reducing the shedding of the challenge strain in birds' feces. A second vaccine dose at 15 days post-hatching boosted the immunogenicity of the vaccine, and strengthened the protection achieved with a single dose. This strain may represent the basis of a live vaccine to be included in national control programs to reduce circulation of this pathogen in the country.

Comparative molecular study of Mycobacterium tuberculosis strains, in times of antimicrobial drug resistance / Estudio molecular comparativo de cepas de Mycobacterium tuberculosis, en tiempos de resistencia antimicrobiana a los fármacos

Strains of Mycobacterium tuberculosis were compared using two DNA fingerprinting techniques: Restriction Fragment Length Polymorphism (RFLP) and Double-Repetitive-Element-PCR (DRE-PCR). Two of these strains: IH1 (susceptible to isoniazid) and IH2 (resistant to isoniazid) were recovered from cases of pulmonary tuberculosis which occurred in two brothers who lived together. The first one was recognized on July 1999, and the second was diagnosed one year later. IH1 and IH2 showed the same pattern of bands with both molecular tests. These results suggest that single drug chemoprophylaxis may occasionally select resistant strains for that drug, which can eventually cause disease and be recognized through these tests. Strains IH3, IH4 and IH5 were obtained from sputum samples of 3 different patients, and intra-laboratory cross-contamination was suspected when it was realized that the 3 positive materials had been consecutively processed the same day by the same worker in the same biological safety cabinet. Again, the 3 strains revealed identical band patterns with RFLP and DRE-PCR, confirming the posed suspicion. The results with DRE-PCR were obtained after only 8 hours of work, without the need for subcultures. This procedure allows quick correction of treatment conducts, avoiding unnecessary exposure of people and bacteria to antimicrobial drugs.

Se compararon cepas de Mycobacterium tuberculosis utilizando 2 procedimientos de ADN fingerprinting: polimorfismo de los fragmentos de restricción (RFLP) y Double-Repetitive-Element-PCR (DRE-PCR). Dos de las cepas: IH1 (susceptible a isoniazida) e IH2 (resistente a isoniazida) se recuperaron a partir de casos de tuberculosis pulmonar que ocurrieron en dos hermanos convivientes. La primera fue aislada en julio de 1999 y la segunda un año después. IH1 e IH2 mostraron el mismo patrón de bandas por ambos procedimientos. Estos resultados sugieren que la quimioprofilaxis con una sola droga puede ocasionalmente seleccionar mutantes resistentes, las cuales pueden causar enfermedad y ser reconocidas por estos procedimientos. Las cepas IH3, IH4 e IH5 fueron aisladas de 3 pacientes diferentes, y examinadas por probable contaminación cruzada dentro del laboratorio ya que fueron procesadas el mismo día, por el mismo operador y en la misma cabina de seguridad biológica. Nuevamente, las 3 cepas revelaron el mismo patrón de bandas por RFLP y por DRE-PCR, confirmando la sospecha. Los resultados de la DRE-PCR se obtuvieron luego de 8 horas de trabajo, sin necesidad de subcultivos. Esta técnica permitiría la rápida correción de pautas de tratamiento, evitando la exposición innecesaria de personas y bacterias a drogas antimicrobianas.

Serotipos, perfil plasmídico y antibiotipos de cepas de Shigella flexneri aisladas de niños menores de 5 años con diarrea sanguinolenta usuarios de los servicios de Salud Pública / Serotypes, serum profile and anti-biotypes of Shigella flexneri cultures isolated from infants under 5 years with diarrhea sanguinolenta users of Public Health Services

Introducción: en niños menores de 5 años con diarrea sanguinolenta, Shigella flexneri es el agente bacteriano más frecuentemente recuperado en nuestro medio. La shigellosis es una de las enfermedades infecciosas en las cuales el tratamiento con antimicrobianos es efectivo. La elección empírica del antimicrobiano adecuado es problemática debido a la resistencia de Shigella a diversos antibióticos. Esta situación estimuló el interés en el desarrollo de vacunas para el control de esta enfermedad. Debido a que algunas vacunas están orientadas a promover la respuesta inmune serotipo específica, es importante establecer la distribución de los serotipos prevalentes en la población que interesa inmunizar. El objetivo de este estudio fue caracterizar 50 cepas de Shigella flexneri aisladas a partir de niños con diarrea sanguinolenta, recuperadas en 4 encuestas etiológicas de gastroenteritis. Método: a cada una se le realizó: serotipificación, estudio del patrón de lipopolisacárido, perfil plasmídico y estudio de sensibilidad a diferentes antimicrobianos. Resultados: los seroptipos prevalentes fueron 2a, 3c, 4, 6, y 1. Se identificaron 10 antibotipos diferentes. En los cultivos del serotipo 2a se hallaron 3 patrones plasmídicos; el 5 fue el más frecuente, seguido por el 6 y el 7. El análisis de la evolución de los antibiotipos circulantes mostró una tendencia hacia la aparición de tipos con mayor espectro de resistencia. Conclusiones: vista esta evolución, y de acuerdo a los resultados obtenidos, resulta de interés ensayar un inmunógeno que incluya los determinantes "O" específicos de los serotipor 2a, 1, 3c, 4, 6 y el de Shigella sonnei.

Comparative molecular study of Mycobacterium tuberculosis strains, in times of antimicrobial drug resistance.

Strains of Mycobacterium tuberculosis were compared using two DNA fingerprinting techniques: Restriction Fragment Length Polymorphism (RFLP) and Double-Repetitive-Element-PCR (DRE-PCR). Two of these strains: IH1 (susceptible to isoniazid) and IH2 (resistant to isoniazid) were recovered from cases of pulmonary tuberculosis which occurred in two brothers who lived together. The first one was recognized on July 1999, and the second was diagnosed one year later. IH1 and IH2 showed the same pattern of bands with both molecular tests. These results suggest that single drug chemoprophylaxis may occasionally select resistant strains for that drug, which can eventually cause disease and be recognized through these tests. Strains IH3, IH4 and IH5 were obtained from sputum samples of 3 different patients, and intra-laboratory cross-contamination was suspected when it was realized that the 3 positive materials had been consecutively processed the same day by the same worker in the same biological safety cabinet. Again, the 3 strains revealed identical band patterns with RFLP and DRE-PCR, confirming the posed suspicion. The results with DRE-PCR were obtained after only 8 hours of work, without the need for subcultures. This procedure allows quick correction of treatment conducts, avoiding unnecessary exposure of people and bacteria to antimicrobial drugs.

Primer aislamiento en Uruguay de Escherichia Coli, productora de toxina Shiga del serotipo O157: H7 en una niña con síndrome urémico hemolítico / First Uruguayan isolation of Shiga toxin-producing Escherichia coli O157: H7 in female infant with hemolytic syndrome

En mayo de 2002 se aisló por primera vez en Uruguay Escherichia coli O157:H7, productora de toxina shiga a partir del coproductivo de una niña de 16 meses procedente de Melo, con diagnóstico de síndrome urémico hemolítico. La cepa, productora de toxinas shiga tipo 2 y tipo 2 variante humana a, era genéticamente distinta de las cepas circulantes en Argentina.

Efecto del Lactobacillus casei administrado en el suero de rehidratación oral, en el tratamiento de la enfermedad diarreica aguda / Efficacy of Lactobacillus GG in oral rehidratation solution

El probiótico Lactobacillus GG es efectivo en promover una recuperación rápida de la diarrea aguda infantil producida por rotavirus. Hay poca información, sobre el rol de este agente en el efecto sobre la diarrea producida por otros gérmenes, tampoco hay evidencia de su eficacia administrada en la sal de hidratación oral para pscientes con diarrea de diversas causa. Método: Niños de 1 mes a 3 años de edad con enfermedad diarreica aguda fueron enrolados en una investigación doble ciego. Pacientes fueron randomizados y colocados en al grupo A recibiendo sal de hidratación y placebo, y en el grupo recibiendo, el mismo con el agragado de una preparación viva de Lactobacuillus GG. Luego de la hidratación en las 4 ó 6 horas se les ofreció su dieta habitual. Resultados: enrolamos 97 pacientes 52 del grupo A y 45 del grupo B. Duración de la diarrea luego del enrolamiento fue 7 días para el grupo A y 4 días para el grupo B (p<0.005).Para los niños rotavirus positivos la diarrea duró 6 días para el grupo A y 3 días para el grupo B (p<0.005).La diarrea duró más de 10 días, 5 para el grupo A y 1 para el grupo B. Conclusión: Administrar sal de hidratación oral conteniendo Lactobacillus GG a niños con enfermedad diarreica aguda es segura y resulta en duración menor de esta y tiene menos chance de pasar a curso prolongado. (AU)

Analysis of the O-antigen chain length distribution during extracellular and intracellular growth of Shigella flexneri.

This study has established that there were no changes in the general structure of LPS of Shigella flexneri M90T either when the bacteria grew free in the cytoplasm of the eucaryotic host cell or during extracellular growth in liquid LB medium at 37 degrees C. In both cases there was a similar bi-modal O-antigen chain length distribution with the mean modal values between 1 and 2, and between 11 and 14 subunits. This suggests that the intracellular localization is not a significant stimulus perceived by Shigella to regulate the length of its O-side chains. However, when the bacteria grew under extracellular conditions (liquid medium) at 30 degrees C, even though there were no changes in the modal values, the pattern of the O-antigen chain length distribution of LPS was different, with an increase in the amount of the long chains relative to the short chains.

Etiology of children's diarrhea in Montevideo, Uruguay: associated pathogens and unusual isolates.

We studied microorganisms associated with infant diarrhea in a group of 256 children admitted to a public pediatric hospital in Montevideo, Uruguay. Diagnostic procedures were updated to optimize detection of potential pathogens, which were found in 63.8% of cases, and to be able to define their characteristics down to molecular or antigenic type. Coinfection with two or more agents was detected in more than one-third of positive studies. Escherichia coli enteric virotypes, especially enteropathogenic E. coli (EPEC), were shown to be prevalent. Rotavirus, Cryptosporidium, Campylobacter (mainly Campylobacter jejuni), and Shigella flexneri were also often identified. Enterotoxigenic E. coli, Salmonella, and Giardia lamblia were sporadically recognized. Unusual findings included two enteroinvasive E. coli strains, one Shigella dysenteriae 2 isolate, and a non-O:1 Vibrio cholerae culture. EPEC bacteria and S. flexneri (but not Salmonella) showed unusually frequent antimicrobial resistance, especially towards beta-lactam antibiotics, which is the subject of ongoing work.

Alimentos que pueden consumir los ninos.Tiempo optimo de hervido de la leche que se utiliza en los laticinios para lactantes, en nuestro medio. / Food that can be consumed by children.Best time to boil the milk used in milk products for babies in our milieu

El Departamento de Educacion Sanitaria del Ministerio de Salud Publica aconseja hervir la leche con destino al lactante sin determinar tiempo. Morquio ensenaba: "hervir cinco minutos, enfriar los biberones y mantenerlos frios". Proveyendose de leche pasteurizada a 200.000 ninos en la capital y aledanos se procuro conocer la realidad y el mejor consejo posible, concluyendose: es necesario que la distribucion de la leche cumpla las disposiciones que establece el Decreto del 24 de enero de 1954, de la Intendencia Municipal de Montevideo, por las que la temperatura de la leche al llegar al consumidor debe ser no mayor de 10oC. sobre cero. Los cinco minutos aconsejados por Morquio pueden no ser de estricto cumplimiento si la higiene de los utensilios y el hervido del agua de la mezcla, son correctos. La conservacion en frio,durante 24 horas a no mas de 10oC. asegura la potabilidad de la mezcla. Si no se usa heladera los cinco minutos de hervido son de rigor. La educacion sanitaria debe llegar al hogar por multiples medios; al efecto los murales confeccionados por Salud Publica deben contener en uno, todas las secuencias, desde el rechazo de la leche que no llegue fria al consumidor,hasta el final -conservacion en frio- con lo cual la vista del conjunto sera aleccionante