First Release of the Bacterial Biobank of the Urban Environment (BBUE).

Metagenomics is providing a broad overview of bacterial functional diversity; however, culturing and biobanking are still essential for microbiology. Here, we present the Bacterial Biobank of the Urban Environment (BBUE), a sizable culture collection for long-term storage and characterization of the microbiota associated with urban environments relevant for public health.

A Naturally Occurring Deletion in FliE from Salmonella enterica Serovar Dublin Results in an Aflagellate Phenotype and Defective Proinflammatory Properties.

Salmonella enterica serovar Dublin is adapted to cattle but is able to infect humans with high invasiveness. An acute inflammatory response at the intestine helps to prevent Salmonella dissemination to systemic sites. Flagella contribute to this response by providing motility and FliC-mediated signaling through pattern recognition receptors. In a previous work, we reported a high frequency (11 out of 25) of S Dublin isolates lacking flagella in a collection obtained from humans and cattle. The aflagellate strains were impaired in their proinflammatory properties in vitro and in vivo The aim of this work was to elucidate the underlying cause of the absence of flagella in S Dublin isolates. We report here that class 3 flagellar genes are repressed in the human aflagellate isolates, due to impaired secretion of FliA anti-sigma factor FlgM. This phenotype is due to an in-frame 42-nucleotide deletion in the fliE gene, which codes for a protein located in the flagellar basal body. The deletion is predicted to produce a protein lacking amino acids 18 to 31. The aflagellate phenotype was highly stable; revertants were obtained only when fliA was artificially overexpressed combined with several successive passages in motility agar. DNA sequence analysis revealed that motile revertants resulted from duplications of DNA sequences in fliE adjacent to the deleted region. These duplications produced a FliE protein of similar length to the wild type and demonstrate that amino acids 18 to 31 of FliE are not essential. The same deletion was detected in S Dublin isolates obtained from cattle, indicating that this mutation circulates in nature.

[A five-year experience with zoonotic Salmonella at a pediatric reference centre]. / Un quinquenio de experiencia (2005-2010) con infecciones por Salmonella spp en un centro nacional de referencia en pediatría.

BACKGROUND: Salmonella can cause asymptomatic infections, diarrhea, bacteremia and focal infections such as meningitis and osteomyelitis. AIM: To describe clinical and microbiological aspects of infections by Salmonella spp. in children in a pediatric referral hospital: Centro Hospitalario Pereira Rossell, in Montevideo, Uruguay. MATERIALS AND METHODS: Descriptive and retrospective study of 46 patients, from which Salmonella spp was isolated between January 1, 2005 and December 31, 2010. RESULTS: Salmonella spp was isolated in 46 children younger than 15 years old. 18 were below 2 years old and 5 children below three months. 24% of the children had risk factors, such as HIV infection, oncological diseases and malnutrition; low birth weight and pneumonia were associated conditions. No deaths were reported. The serotypes more frequently found were: Typhimurium and Enteritidis. Most of the strains were susceptible to ampicillin and third generation of cephalosporins. DISCUSSION: Diarrhea with blood was the predominant clinical presentation, and there were no outbreaks. Typhimurium and Enteritidis were the most common serotypes. Based on the profiles of susceptibility antimicrobial, we could maintain the same recommendations until the moment suggested. CONCLUSIONS: we must consider the Salmonella infection in febrile children under risk of an invasive bacterial disease, with or without focal infection.

Un quinquenio de experiencia (2005-2010) con infecciones por Salmonella spp en un centro nacional de referencia en pediatría / A five-year experience with zoonotic Salmonella at a pediatric reference centre

Resumen Introducción: Salmonella sp puede causar infecciones asintomáticas, gastroenteritis, bacteriemia e infecciones focales como meningitis y osteomielitis. Objetivo: Describir aspectos microbiológicos y clínicos de las infecciones por Salmonella spp en niños en un hospital de referencia pediátrico Centro Hospitalario Pereira Rossell. Montevideo Uruguay. Material y Métodos: Estudio descriptivo y retrospectivo de pacientes en quienes se aislara Salmonella spp en el período 1 de enero de 2005 al 31 de diciembre de 2010. Resultados: Se aisló Salmonella spp en 46 niños menores de 15 años. Dieciocho eran menores de 2 años y 5 niños menores de tres meses. 24% de los pacientes presentaba factores de riesgo (infección por VIH; enfermedad hemato-oncológica, desnutrición) y co-morbilidades (bajo peso al nacer y neumonía). No hubo fallecidos. Los serotipos más frecuentes fueron: Typhimurium y Enteritidis. La mayoría de las cepas eran sensibles a ampicilina y cefalosporinas de tercera generación. Discusión: La presentación clínica predominante fue diarrea con sangre, no se presentaron brotes. Basados en los perfiles de susceptibilidad antimicrobiana, se pueden mantener las recomendaciones hasta el momento sugeridas. Conclusiones: Se debe tener en cuenta la infección por Salmonella sp en niños febriles con riesgo de enfermedad bacteriana invasora, con o sin focalidad.

Background: Salmonella can cause asymptomatic infections, diarrhea, bacteremia and focal infections such as meningitis and osteomyelitis. Aim: To describe clinical and microbiological aspects of infections by Salmonella spp. in children in a pediatric referral hospital: Centro Hospitalario Pereira Rossell, in Montevideo, Uruguay. Materials and Methods: Descriptive and retrospective study of 46 patients, from which Salmonella spp was isolated between January 1, 2005 and December 31, 2010. Results: Salmonella spp was isolated in 46 children younger than 15 years old. 18 were below 2 years old and 5 children below three months. 24% of the children had risk factors, such as HIV infection, oncological diseases and malnutrition; low birth weight and pneumonia were associated conditions. No deaths were reported. The serotypes more frequently found were: Typhimurium and Enteritidis. Most of the strains were susceptible to ampicillin and third generation of cephalosporins. Discussion: Diarrhea with blood was the predominant clinical presentation, and there were no outbreaks. Typhimurium and Enteritidis were the most common serotypes. Based on the profiles of susceptibility antimicrobial, we could maintain the same recommendations until the moment suggested. Conclusions: we must consider the Salmonella infection in febrile children under risk of an invasive bacterial disease, with or without focal infection.

Repression of flagella is a common trait in field isolates of Salmonella enterica serovar Dublin and is associated with invasive human infections.

The nontyphoidal Salmonella enterica serovar Dublin is adapted to cattle but infrequently infects humans, very often resulting in invasive infections with high levels of morbidity and mortality. A Salmonella-induced intestinal acute inflammatory response is postulated as a mechanism to prevent bacterial dissemination to systemic sites. In S. enterica serovar Typhimurium, flagella contribute to this response by providing motility and FliC-mediated activation of pattern recognition receptors. In this study, we found 4 Salmonella enterica isolates, with the antigenic formula 9,12:-:-, that, based on fliC sequence and multilocus sequence type (MLST) analyses, are aflagellate S. Dublin isolates. Interestingly, all were obtained from human bloodstream infections. Thus, we investigated the potential role of flagella in the unusual invasiveness exhibited by S. Dublin in humans by analyzing flagellation and proinflammatory properties of a collection of 10 S. Dublin human clinical isolates. We found that 4 of 7 blood isolates were aflagellate due to significantly reduced levels of fliC expression, whereas all 3 isolates from other sources were flagellated. Lack of flagella correlated with a reduced ability of triggering interleukin-8 (IL-8) and CCL20 chemokine expression in human intestinal Caco-2 cells and with reduced early inflammation in the ceca of streptomycin-pretreated C57/BL6 mice. These results indicate that flagella contribute to the host intestinal inflammatory response to Salmonella serovar Dublin and suggest that their absence may contribute to its systemic dissemination through dampening of the gut immune response. Analysis of FliC production in a collection of cattle isolates indicated that the aflagellate phenotype is widely distributed in field isolates of S. Dublin.

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.

Genomic Comparison of the Closely Related Salmonella enterica Serovars Enteritidis and Dublin.

The Enteritidis and Dublin serovars of Salmonella enterica are closely related, yet they differ significantly in pathogenicity and epidemiology. S. Enteritidis is a broad host range serovar that commonly causes gastroenteritis and infrequently causes invasive disease in humans. S. Dublin mainly colonizes cattle but upon infecting humans often results in invasive disease.To gain a broader view of the extent of these differences we conducted microarray-based comparative genomics between several field isolates from each serovar. Genome degradation has been correlated with host adaptation in Salmonella, thus we also compared at whole genome scale the available genomic sequences of them to evaluate pseudogene composition within each serovar.Microarray analysis revealed 3771 CDS shared by both serovars while 33 were only present in Enteritidis and 87 were exclusive to Dublin. Pseudogene evaluation showed 177 inactive CDS in S. Dublin which correspond to active genes in S. Enteritidis, nine of which are also inactive in the host adapted S. Gallinarum and S. Choleraesuis serovars. Sequencing of these 9 CDS in several S. Dublin clinical isolates revealed that they are pseudogenes in all of them, indicating that this feature is not peculiar to the sequenced strain. Among these CDS, shdA (Peyer´s patch colonization factor) and mglA (galactoside transport ATP binding protein), appear also to be inactive in the human adapted S. Typhi and S. Paratyphi A, suggesting that functionality of these genes may be relevant for the capacity of certain Salmonella serovars to infect a broad range of hosts.

Naturally occurring motility-defective mutants of Salmonella enterica serovar Enteritidis isolated preferentially from nonhuman rather than human sources.

Salmonellosis represents a worldwide health problem because it is one of the major causes of food-borne disease. Although motility is postulated as an important Salmonella virulence attribute, there is little information about variation in motility in natural isolates. Here we report the identification of a point mutation (T551 → G) in motA, a gene essential for flagellar rotation, in several Salmonella enterica serovar Enteritidis field isolates. This mutation results in bacteria that can biosynthesize structurally normal but paralyzed flagella and are impaired in their capacity to invade human intestinal epithelial cells. Introduction of a wild-type copy of motA into one of these isolates restored both motility and cell invasiveness. The motA mutant triggered higher proinflammatory transcriptional responses than an aflagellate isolate in differentiated Caco-2 cells, suggesting that the paralyzed flagella are able to signal through pattern recognition receptors. A specific PCR was designed to screen for the T551 → G mutation in a collection of 266 S. Enteritidis field isolates from a nationwide epidemic, comprising 194 from humans and 72 from other sources. We found that 72 of the 266 (27%) isolates were nonmotile, including 24.7% (48/194) of human and 33.3% (24/72) of food isolates. Among nonmotile isolates, 15 carried the T551 → G mutation and, significantly, 13 were recovered from food, including 7 from eggs, but only 2 were from human sources. These results suggest that the presence of paralyzed flagella may impair the ability of S. Enteritidis to cause disease in the human host but does not prevent its ability to colonize chickens and infect eggs.

Differential phenotypic diversity among epidemic-spanning Salmonella enterica serovar enteritidis isolates from humans or animals.

Nontyphoidal salmonellae are major causes of food-borne disease worldwide. In Uruguay, Salmonella enterica serovar Enteritidis was the most commonly isolated serovar throughout the last decade, with a marked epidemic period between 1995 and 2004. In a previous study, we conducted comparative genomics of 29 epidemic-spanning S. Enteritidis field isolates, and here we evaluated the pathogenic potential of the same set of isolates using several phenotypic assays. The sample included 15 isolates from human gastroenteritis, 5 from invasive disease, and 9 from nonhuman sources. Contrary to the genetic homogeneity previously observed, we found great phenotypic variability among these isolates. One-third of them were defective in at least one assay, namely, 10 isolates were defective in motility, 8 in invasion of Caco-2 cells, and 10 in survival in egg albumen. Twelve isolates were tested for invasiveness in 3-day-old chickens, and five of these were significantly less invasive than the reference strain. The two oldest preepidemic isolates were reduced in fitness in all assays, providing a plausible explanation for the previous negligible incidence of S. Enteritidis in Uruguay and supporting the view that the introduction or emergence of a more virulent strain was responsible for the marked rise of this serovar. Further, we found differences in fitness among the isolates which depended on the source of isolation. A total of 1 out of 14 isolates from human gastroenteritis, but 6 out of 13 isolates from other sources, was impaired in at least two assays, suggesting enhanced fitness among strains able to cause intestinal disease in humans.

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.

Random amplified polymorphic DNA and phenotyping analysis of Salmonella enterica serovar enteritidis isolates collected from humans and poultry in Uruguay from 1995 to 2002.

Molecular and phenotyping techniques were applied to study Salmonella enterica serovar Enteritidis strains both from human cases of infection and of avian origin isolated in Uruguay from 1995 to 2002. A group of 62 isolates was subjected to random amplified polymorphic DNA (RAPD) assay and analysis of antibiotic resistance patterns. Twenty-one of these strains were further characterized by phage typing and analysis of their protein expression profiles. RAPD fingerprinting with five different primers discriminated 10 different genetic profiles. Of the 62 strains tested, 48 had a single major genetic profile, whereas the other nine profiles were evenly distributed among the other strains. The genetic diversity was greater among strains of animal origin than among isolates of human origin. Comparative examination of the results obtained by RAPD analysis and phenotypic analysis and by strain source provided evidence of the reliable discriminatory power of RAPD analysis in our study. Six avian isolates with antibiotic resistance were detected: two were nalidixic acid resistant and four had a particular beta-lactam resistance pattern. The last four isolates all had the same unusual phage type (phage type 4b); however, RAPD analysis differentiated them into two groups. Two isolates with unique RAPD profiles were recovered from distinct human cases, suggesting that the technique differentiates unrelated strains. Overall, the results show the existence of a predominant genetic type that is present in poultry and that is transmitted to humans. There are also several other genotypes, but only a few of them could be recovered from human sources, suggesting the existence of different pathogenic traits among strains circulating in the country.