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Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objective Contribute to the knowledge of the relationship between genetic and pathogenic characteristics of meningeal cryptococcosis-producing strains from Uruguay. Methods We have included a total of 21 isolates of Cryptococcus spp from human cases of meningoencephalitis. We start from the general strain collection of the mycology laboratory of the department of parasitology and mycology, medical school, University of the Republic. The strain was sectioned taking into account the phenotypic diversity and genetic diversity through a previous analysis by PCR fingerprinting, for study by MLST. MLST was performed according to the protocol available at: http://mlst.mycologylab.org/defaultinfo.aspx?Page=CryptoHome. Infection, intracellular survival, and cell proliferation assays were performed. The murine macrophage cell line J774 (ATCC TIB 67) was used. Results Of the 21 strains studied, in 13 we obtained an ST that had been previously reported (9 C. neoformans complex and 4 C. gattii complex). For the remaining 8 strains, we obtained new allelic combinations to which new STs were assigned, which were deposited in the MLST database for Cryptococcus spp. (http://mlst.mycologylab.org) (Table 1). All strains were phagocytosed and all were able to persist within macrophages at 24 h post-infection, although there was great variability between strains (Table 1). In most of the strains, the control of macrophages prevailed through cell lysis mechanisms over Cryptococcus spp, with one proliferation index (PI) ˂1.0. In the proliferation assay, only 4 strains managed to proliferate within the macrophages, 3 of them correspond to C. gattii complex, genotypes VGI and VGII. The 4th strain that achieved proliferation was corresponding to the VNI genotype. Conclusion We obtained a clear differentiation of in vitro behavior between the strains corresponding to C. neoformans complex and C. gattii complex. The strains belonging to C. gattii complex presented less phagocytosis and greater persistence and intracellular proliferation.

The Enteritidis and Dublin serovars of Salmonella enterica are phylogenetically closely related yet differ significantly in host range and virulence. S. Enteritidis is a broad-host-range serovar that commonly causes self-limited gastroenteritis in humans, whereas S. Dublin is a cattle-adapted serovar that can infect humans, often resulting in invasive extraintestinal disease. The mechanism underlying the higher invasiveness of S. Dublin remains undetermined. In this work, we quantitatively compared the proteomes of clinical isolates of each serovar grown under gut-mimicking conditions. Compared to S. Enteritidis, the S. Dublin proteome was enriched in proteins linked to response to several stress conditions, such as those encountered during host infection, as well as to virulence. The S. Enteritidis proteome contained several proteins related to central anaerobic metabolism pathways that were undetected in S. Dublin. In contrast to what has been observed in other extraintestinal serovars, most of the coding genes for these pathways are not degraded in S. Dublin. Thus, we provide evidence that S. Dublin metabolic functions may be much more affected than previously reported based on genomic studies. Single and double null mutants in stress response proteins Dps, YciF, and YgaU demonstrate their relevance to S. Dublin invasiveness in a murine model of invasive salmonellosis. All in all, this work provides a basis for understanding interserovar differences in invasiveness and niche adaptation, underscoring the relevance of using proteomic approaches to complement genomic studies.

Campylobacter fetus is an important animal pathogen that causes infectious infertility, embryonic mortality and abortions in cattle and sheep flocks. There are two recognized subspecies related with reproductive disorders in livestock: Campylobacter fetus subsp. fetus (Cff) and Campylobacter fetus subsp. venerealis (Cfv). Rapid and reliable detection of this pathogenic species in bulls is of upmost importance for disease control in dairy and beef herds as they are asymptomatic carriers. The aim of the present work was to assess the performance a real-time PCR (qPCR) method for the diagnosis of Campylobacter fetus in samples from bulls, comparing it with culture and isolation methods. 520 preputial samples were both cultured in Skirrow's medium and analyzed by qPCR. The estimated sensitivity of qPCR was 90.9% (95% CI, 69.4%–100%), and the specificity was 99.4% (95% CI, 98.6% - 100%). The proportion of C. fetus positive individuals was 2.1% by isolation and 2.5% by qPCR. Isolates were identified by biochemical tests as Cfv (n = 9) and Cff (n = 2). Our findings support the use of qPCR for fast and accurate detection of C. fetus directly from field samples of preputial smegma of bulls. The qPCR method showed to be suitable for massive screenings because it can be performed in pooled samples without losing accuracy and sensitivity. EEA Balcarce Fil: Delpiazzo, Rafael. Universidad de la República Oriental del Uruguay. Facultad de Veterinaria. Estación Experimental "Dr. Mario A. Cassinoni"; Uruguay. Fil: Barcellos, Maila. Universidad de la República Oriental del Uruguay. Facultad de Ciencias; Uruguay. Fil: Barros, Sofía. Universidad de la República Oriental del Uruguay. Facultad de Ciencias; Uruguay. Fil: Bentacor, Laura. Universidad de la República. Facultad de Medicina; Uruguay. Fil: Fraga, Martín. Instituto Nacional de Investigación Agropecuaria. Estación Experimental La Estanzuela; Uruguay. Fil: Gil, Jorge. Universidad de la República Oriental del Uruguay. Facultad de Veterinaria. Estación Experimental "Dr. Mario A. Cassinoni"; Uruguay. Fil: Iraola, Gregorio. Institut Pasteur de Montevideo. Laboratorio de Genómica Microbiana; Uruguay. Universidad Mayor. Facultad de Ciencias; Chile. Wellcome Genome Campus, Wellcome Sanger Institute; United Kingdom. Fil: Morsella, Claudia Graciela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Fil: Paolicchi, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Fil: Pérez, Ruben. Universidad de la República Oriental del Uruguay. Facultad de Ciencias; Uruguay. Fil: Riet-Correa, Franklin. Instituto Nacional de Investigación Agropecuaria. Estación Experimental La Estanzuela; Uruguay. Fil: Sanguinetti, Margarita. Universidad de la República Oriental del Uruguay. Facultad de Ciencias; Uruguay. Fil: Silva, Alfonso. Universidad de la República Oriental del Uruguay. Facultad de Ciencias; Uruguay. Fil: Silva Silveira, Caroline da. Instituto Nacional de Investigación Agropecuaria. Estación Experimental La Estanzuela; Uruguay. Fil: Caballeros, Lucía. Universidad de la República Oriental del Uruguay. Facultad de Ciencias; Uruguay.

Salmonella enterica serovar Enteritidis is a major cause of foodborne disease in Uruguay since 1995. We used a genomic approach to study a set of isolates from different sources and years. Whole genome phylogeny showed that most of the strains are distributed in two major lineages (E1 and E2), both belonging to MLST sequence type 11 the major ST among serovar Enteritidis. Strikingly, E2 isolates are over-represented in periods of outbreak abundance in Uruguay, while E1 span all epidemic periods. Both lineages circulate in neighbor countries at the same timescale as in Uruguay, and are present in minor numbers in distant countries. We identified allelic variants associated with each lineage. Three genes, ycdX, pduD and hsdM, have distinctive variants in E1 that may result in defective products. Another four genes (ybiO, yiaN, aas, aceA) present variants specific for the E2 lineage. Overall this work shows that S. enterica serovar Enteritidis strains circulating in Uruguay have the same phylogenetic profile than strains circulating in the region, as well as in more distant countries. Based on these results we hypothesize that the E2 lineage, which is more prevalent during epidemics, exhibits a combination of allelic variants that could be associated with its epidemic ability.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In Montevideo (2013-2018), 8 Campylobacter fetus extraintestinal infections were reported. The polyclonal nature of strains revealed by whole-genome sequencing and the apparent lack of epidemiological links was incompatible with a single contamination source, supporting alternative routes of transmission.

Campylobacter fetus is a Gram-negative, microaerophilic bacterium that infects animals and humans. The subspecies Campylobacter fetus subsp. fetus (Cff) affects a broad range of vertebrate hosts and induces abortion in cows and sheep. Campylobacter fetus subsp. venerealis (Cfv) is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus subsp. testudinum (Cft) has been isolated mostly from apparently healthy reptiles belonging to different species but also from ill snakes and humans. Genotypic differentiation of Cff and Cfv is difficult, and epidemiological information is scarce because there are few methods to study the genetic diversity of the strains. We analyze the efficacy of MLST, ribosomal sequences (23S gene and internal spacer region), and CRISPRs to assess the genetic variability of C. fetus in bovine and human isolates. Sequences retrieved from complete genomes were included in the analysis for comparative purposes. MLST and ribosomal sequences had scarce or null variability, while the CRISPR-cas system structure and the sequence of CRISPR1 locus showed remarkable diversity. None of the sequences here analyzed provided evidence of a genetic differentiation of Cff and Cfv in bovine isolates. Comparison of bovine and human isolates with Cft strains showed a striking divergence. Inter-host differences raise the possibility of determining the original host of human infections using CRISPR sequences. CRISPRs are the most variable sequences analyzed in C. fetus so far, and constitute excellent representatives of a dynamic fraction of the genome. CRISPR typing is a promising tool to characterize isolates and to track the source and transmission route of C. fetus infections.

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., Microbiology Resource Announcements, 7 (16), ISSN:2576-098X

Multidrug-resistant Salmonella enterica isolates are an increasing problem worldwide; nevertheless, the mechanisms responsible for such resistance are rarely well defined. Multidrug-resistant S. enterica serovar Typhimurium isolates ST3224 and ST827 were collected from two patients., Multidrug-resistant Salmonella enterica isolates are an increasing problem worldwide; nevertheless, the mechanisms responsible for such resistance are rarely well defined. Multidrug-resistant S. enterica serovar Typhimurium isolates ST3224 and ST827 were collected from two patients. The characteristics of both genomes and antimicrobial resistance genes were determined using next-generation sequencing.

Salmonella enterica serovar Enteritidis is a major agent of foodborne diseases worldwide. In Uruguay, this serovar was almost negligible until the mid 1990s but since then it has become the most prevalent. Previously, we characterized a collection of strains isolated from 1988 to 2005 and found that the two oldest strains were the most genetically divergent. In order to further characterize these strains, we sequenced and annotated eight genomes including those of the two oldest isolates. We report on the identification and characterization of a novel 44 kbp Salmonella prophage found exclusively in these two genomes. Sequence analysis reveals that the prophage is a mosaic, with homologous regions in different Salmonella prophages. It contains 60 coding sequences, including two genes, gogB and sseK3, involved in virulence and modulation of host immune response. Analysis of serovar Enteritidis genomes available in public databases confirmed that this prophage is absent in most of them, with the exception of a group of 154 genomes. All 154 strains carrying this prophage belong to the same sequence type (ST-1974), suggesting that its acquisition occurred in a common ancestor. We tested this by phylogenetic analysis of 203 genomes representative of the intraserovar diversity. The ST-1974 forms a distinctive monophyletic lineage, and the newly described prophage is a phylogenetic signature of this lineage that could be used as a molecular marker. The phylogenetic analysis also shows that the major ST (ST-11) is polyphyletic and might have given rise to almost all other STs, including ST-1974.

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.

We report a 4.99-Mb draft genome sequence of Salmonella enterica subsp. enterica serovar Infantis strain SPE101, isolated from feces of a 5-month-old breast-fed female showing diarrhea associated with severe dehydration and malnutrition. The infection prolonged for 6 months despite antibiotic treatment.

The objectives of this study were to investigate clinical isolates of Salmonella enterica serovar Typhimurium resistant to β-lactam antibiotics, to characterise their mechanisms of antibiotic resistance and to evaluate the possible biological cost of expressing resistance genes. Two oxyimino-cephalosporin-resistant Salmonella isolates obtained from children with diarrhoea were characterised. The occurrence of plasmid-encoded bla CMY-2 genes was confirmed by molecular methods and conjugation assays; transcription levels were determined by quantitative real-time PCR (qRT-PCR). The genomic context of the β-lactamases, replicon type and addiction systems were analysed by PCR. Genomic relatedness of both isolates was studied by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) assays. Growth curves, motility and invasiveness assays in Caco-2 cells were performed to analyse the bacterial fitness of both isolates. Both isolates carried a blaCMY-2-like allele in an IncI plasmid and belonged to the same MLST sequence type (ST19); nevertheless, they showed extensive differences in their PFGE profiles and virulotypes. Isolate STM709 appeared to lack the Salmonella virulence plasmid and displayed less motility and invasiveness in cultured cells than isolate STM910. qRT-PCR showed that isolate STM709 had higher blaCMY-2 mRNA levels compared with STM910. Altogether, the results suggest that a plasmid carrying blaCMY-2 could be disseminating among different clones of S. Typhimurium. Different levels of blaCMY-2 mRNA could have an effect on the fitness of this micro-organism, resulting in lower invasiveness and motility. © 2013 International Society for Chemotherapy of Infection and Cancer., Comisión Sectorial de Investigación Científica (CSIC, Uruguay)

International audience; An epidemiological paradox surrounds Salmonella enterica serovar Enteritidis. In high-income settings, it has been responsible for an epidemic of poultry-associated, self-limiting enterocolitis, whereas in sub-Saharan Africa it is a major cause of invasive nontyphoidal Salmonella disease, associated with high case fatality. By whole-genome sequence analysis of 675 isolates of S. Enteritidis from 45 countries, we show the existence of a global epidemic clade and two new clades of S. Enteritidis that are geographically restricted to distinct regions of Africa. The African isolates display genomic degradation, a novel prophage repertoire, and an expanded multidrug resistance plasmid. S. Enteritidis is a further example of a Salmonella serotype that displays niche plasticity, with distinct clades that enable it to become a prominent cause of gastroenteritis in association with the industrial production of eggs and of multidrug-resistant, bloodstream-invasive infection in Africa.

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.

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.

Background: Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains. Results: We developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 102 and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability. Conclusions: The possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay a good option to establish a new standard in molecular identification and quantification of C. fetus species. EEA Balcarce Fil: Iraola, Gregorio. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Uruguay. Institut Pasteur Montevideo. Unidad de Bioinformática; Uruguay Fil: Pérez, Ruben. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Uruguay Fil: Betancor, Laura. Universidad de la República. Facultad de Medicina. Instituto de Higiene. Departamento de Bacteriología y Virología; Uruguay Fil: Marandino, Ana. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Uruguay Fil: Morsella, Claudia Graciela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: Mendez, Maria Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: Paolicchi, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: Piccirillo, Alessandra. Università degli Studi di Padova. Dipartimento di Biomedicina Comparata e Alimentazione; Italia Fil: Tomás, Gonzalo. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Uruguay Fil: Velilla, Alejandra Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: Calleros, Lucía. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Uruguay

Whole-genome characterisation in clinical microbiology enables to detect trends in infection dynamics and disease transmission. Here, we report a case of bacteraemia due to Campylobacter fetus subsp. fetus in a rural worker under cancer treatment that was diagnosed with cellulitis; the patient was treated with antibiotics and recovered. The routine typing methods were not able to identify the microorganism causing the infection, so it was further analysed by molecular methods and whole-genome sequencing. The multi-locus sequence typing (MLST) revealed the presence of the bovine-associated ST-4 genotype. Whole-genome comparisons with other C. fetus strains revealed an inconsistent phylogenetic position based on the core genome, discordant with previous ST-4 strains. To the best of our knowledge, this is the first C. fetus subsp. fetus carrying the ST-4 isolated from humans and represents a probable case of zoonotic transmission from cattle.

Nonspecific stimulation of lung defenses by repeated oral administration of immunomodulators, such as bacterial extracts, has shown potential for the prevention of respiratory tract infections. Here, we show that intranasal (i.n.) immunization with a bacterial extract formulated as a colloid induces an acute inflammatory response in the lungs characterized by increased production of CCL and CXCL chemokines and a major influx of dendritic cells (DCs) and neutrophils, with a higher proportion of DCs showing an activated phenotype (high CD80/CD86 expression). Cytokine levels measured in bronchoalveolar-lavage samples showed a small increase in the production of tumor necrosis factor alpha and similar levels of the other cytokines measured (interleukin 10 [IL-10], IL-12, and gamma interferon [IFN-γ]) in immunized mice compared with control mice. However, the recall response of primed animals after antigenic challenge induced increased expression of IL-12 and IFN-γ mRNAs in lung homogenates. Overall, all these effects were not due to the lipopolysaccharide content in the bacterial extract. Furthermore, we found that three i.n. doses administered 2 to 3 weeks apart were enough to elicit long-lasting specific serum immunoglobulin G (IgG) and secretory IgA antibody responses. Assessment of IgG subclasses showed a balanced pattern of IgG1-IgG2a responses. The serum total IgE concentrations were also elevated in immunized mice 2 weeks after the third dose, but they significantly decreased soon afterwards. Our results suggest that simple formulations of bacterial extracts administered i.n. are highly immunogenic, eliciting local and systemic immune responses, and may serve as the basis for cost-effective immunotherapies for the prevention and treatment of respiratory infections.

We studied a clinical isolate of Salmonella enterica serotype Enteritidis showing resistance to oxyiminocephalosporins. PCR analysis confirmed the presence of bla CTX-M-14 linked to IS903 in a 95-kb IncI1 conjugative plasmid. Such a plasmid is maintained on account of the presence of a pndAC addiction system. Multilocus sequence typing (MLST) analysis indicated that the strain belongs to ST11. This is the first report of bla CTX-M-14 in Salmonella Enteritidis of human origin in South America. Copyright © 2012, American Society for Microbiology. All Rights Reserved., CSIC (Comisión Sectorial de Investigación Científica, Uruguay); European Community (FP7-HEALTH-2007-223431); Ministerio de Ciencia e Innovación/Programa Bio-Fundamental (BFU2009-09200)

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.

Salmonella enterica serovar Heidelberg ranks among the most prevalent causes of human salmonellosis in the United States and Canada, although it has been infrequently reported in South American and European countries. Most Salmonella infections are self-limiting; however, some invasive infections require antimicrobial therapy. In this work we characterized an oxyimino-cephalosporin resistant S. Heidelberg isolate recovered from an inpatient in a Buenos Aires hospital. CMY-2 was responsible for the ß-lactam resistance profile. S. Heidelberg contained a 97 kb plasmid belonging to the Inc N group harboring blaCMY-2. ISEcp1 was located upstream blaCMY-2 driving its expression and mobilization. The isolate belonged to sequence type 15 and virotyping revealed the presence of sopE gene. In this study we identified the first CMY-2 producing isolate of S. Heidelberg in Argentina and even in South America. Salmonella enterica serovar Heidelberg es uno de los principales agentes causantes de salmonelosis en humanos en Estados Unidos y Canadá, sin embargo, resulta infrecuente en los países de Sudamérica y Europa. En este trabajo se caracterizó un aislamiento de S. Heidelberg resistente a oximino-cefalosporinas recuperado de un paciente internado en un hospital de la Ciudad de Buenos Aires. Se evidenció la presencia de un plásmido de 97 kb perteneciente al grupo de incompatibilidad IncN, portador del gen blaCMY-2. ISEcp1 fue localizado corriente arriba de blaCMY-2, promoviendo su expresión y movilización. El aislamiento de S. Heidelberg correspondió al secuenciotipo 15 y en la virotipificación se detectó el gen sopE. En este trabajo describimos por primera vez la producción de CMY-2 en una cepa de S. Heidelberg en nuestro país y América Latina. Fil: Cejas, Daniela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Vignoli, Rafael. Universidad de la República; Uruguay Fil: Quinteros, Mirta. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas F. J. Muñiz; Argentina Fil: Marino, Ricardo. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas F. J. Muñiz; Argentina Fil: Callejo, Raquel. Gobierno de la Ciudad de Buenos Aires. Hospital de Infecciosas F. J. Muñiz; Argentina Fil: Betancor, Laura. Universidad de la República; Uruguay Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Radice, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The objectives of this study were to investigate clinical isolates of Salmonella enterica serovar Typhimurium resistant to β-lactam antibiotics, to characterise their mechanisms of antibiotic resistance and to evaluate the possible biological cost of expressing resistance genes. Two oxyimino-cephalosporin-resistant Salmonella isolates obtained from children with diarrhoea were characterised. The occurrence of plasmid-encoded bla

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.

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.

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.

A small (8.2-kb) ColE1 plasmid encoding TEM-144 (a new β-lactamase with a ceftazidimase profile) was sequenced by a gene-walking strategy. The blaTEM allele was carried on a Tn2 element, disrupting a Rom protein gene. TEM-144 differs from TEM-1 by two mutations (R164C and E240K) and from the ceftazidime-hydrolyzing TEM-91 by one mutation (T182M). Fil: Vignoli, Rafael. Universidad de la Republica. Facultad de Medicina. Instituto de Higiene.; Uruguay Fil: Cordeiro, Nicolas F.. Universidad de la Republica. Facultad de Medicina. Instituto de Higiene.; Uruguay Fil: García, Virginia. Universidad de la Republica. Facultad de Medicina. Instituto de Higiene.; Uruguay Fil: Mota, María Inés. Universidad de la Republica. Facultad de Medicina. Instituto de Higiene.; Uruguay Fil: Betancor, Laura. Universidad de la Republica. Facultad de Medicina. Instituto de Higiene.; Uruguay Fil: Power, Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Chabalgoity, José A.. Universidad de la Republica. Facultad de Medicina. Instituto de Higiene.; Uruguay Fil: Schelotto, Felipe. Universidad de la Republica. Facultad de Medicina. Instituto de Higiene.; Uruguay Fil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología. Cátedra de Microbiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Ayala, Juan A.. Consejo Superior de Investigaciones Científicas; España. Universidad Autónoma de Madrid; España