Patricio Retamal

Pandemic (H1N1) 2009 in breeding turkeys, Valparaiso, Chile.

Pandemic (H1N1) 2009 virus was detected in breeding turkeys on 2 farms in Valparaiso, Chile. Infection was associated with measurable declines in egg production and shell quality. Although the source of infection is not yet known, the outbreak was controlled, and the virus was eliminated from the birds.

Characterization of MgtC, a virulence factor of Salmonella enterica Serovar Typhi.

The MgtC is a virulence factor in Salmonella Typhimurium that is required for growth at low-Mg2+ concentrations and intramacrophage survival. This gene is codified in a conserved region of the Salmonella pathogenicity island 3 (SPI-3), and is also present in the chromosome of other Salmonella serovars. In this study we characterized the MgtC factor in S. Typhi, a human specific pathogen, by using mgtC and SPI-3 mutant strains. We found that MgtC is the most important factor codified in the SPI-3 of S. Typhi for growth in low-Mg2+ media and survival within human cells. In addition, by using reporter genes we determined that the low-Mg2+ concentration, acidic media and PhoP regulator induce mgtC expression in S. Typhi. We suggest that MgtC is the most important virulence factor codified in the SPI-3 of S. Typhi.

Whole-Genome Sequencing Analysis of Salmonella enterica Serovar Enteritidis Isolates in Chile Provides Insights into Possible Transmission between Gulls, Poultry, and Humans

ABSTRACT Salmonella enterica subsp. enterica serotype Enteritidis is a major cause of human salmonellosis worldwide; however, little is known about the genetic relationships between S. Enteritidis clinical strains and S. Enteritidis strains from other sources in Chile. We compared the whole genomes of 30 S. Enteritidis strains isolated from gulls, domestic chicken eggs, and humans in Chile, to investigate their phylogenetic relationships and to establish their relatedness to international strains. Core genome multilocus sequence typing (cgMLST) analysis showed that only 246/4,065 shared loci differed among these Chilean strains, separating them into two clusters (I and II), with cluster II being further divided into five subclusters. One subcluster (subcluster 2) contained strains from all surveyed sources that differed at 1 to 18 loci (of 4,065 loci) with 1 to 18 single-nucleotide polymorphisms (SNPs), suggesting interspecies transmission of S. Enteritidis in Chile. Moreover, clusters were formed by strains that were distant geographically, which could imply that gulls might be spreading the pathogen throughout the country. Our cgMLST analysis, using other S. Enteritidis genomes available in the National Center for Biotechnology Information (NCBI) database, showed that S. Enteritidis strains from Chile and the United States belonged to different lineages, which suggests that S. Enteritidis regional markers might exist and could be used for trace-back investigations. IMPORTANCE This study highlights the importance of gulls in the spread of Salmonella Enteritidis in Chile. We revealed a close genetic relationship between some human and gull S. Enteritidis strains (with as few as 2 of 4,065 genes being different), and we also found that gull strains were present in clusters formed by strains isolated from other sources or distant locations. Together with previously published evidence, this suggests that gulls might be spreading this pathogen between different regions in Chile and that some of those strains have been transmitted to humans. Moreover, we discovered that Chilean S. Enteritidis strains clustered separately from most of S. Enteritidis strains isolated throughout the world (in the GenBank database) and thus it might be possible to distinguish the geographical origins of strains based on specific genomic features. This could be useful for trace-back investigations of foodborne illnesses throughout the world.

Avian Influenza in wild birds from Chile, 2007–2009

Aquatic and migratory birds, the main reservoir hosts of avian influenza viruses including those with high pathogenic potential, are the wildlife species with the highest risk for viral dissemination across countries and continents. In 2002, the Chilean poultry industry was affected with a highly pathogenic avian influenza strain, which created economic loss and triggered the establishment of a surveillance program in wild birds. This effort consisted of periodic samplings of sick or suspicious animals found along the coast and analyses with standardized techniques for detection of influenza A virus. The aim of this work is to report the detection of three avian influenza strains (H13N2, H5N9, H13N9) in gulls from Chile between 2007-2009, which nucleotide sequences showed highest similitudes to viruses detected in wild birds from North America. These results suggest a dissemination route for influenza viruses along the coasts of Americas. Migratory and synanthropic behaviors of birds included in this study support continued monitoring of avian influenza viruses isolated from wild birds in The Americas and the establishment of biosecurity practices in farms.

Modified Intracellular-Associated Phenotypes in a Recombinant Salmonella Typhi Expressing S. Typhimurium SPI-3 Sequences

A bioinformatics comparison of Salmonella Pathogenicity Island 3 sequences from S. Typhi and S. Typhimurium serovars showed that ten genes are highly conserved. However three of them are pseudogenes in S. Typhi. Our aim was to understand what functions are lost in S. Typhi due to pseudogenes by constructing a S. Typhi genetic hybrid carrying the SPI-3 region of S. Typhimurium instead of its own SPI-3. We observed that under stressful conditions the hybrid strain showed a clear impairment in resistance to hydrogen peroxide and decreased survival within U937 culture monocytes. We hypothesized that the marT-fidL operon, encoded in SPI-3, was responsible for the new phenotypes because marT is a pseudogen in S. Typhi and has a demonstrated role as a transcriptional regulator in S. Typhimurium. Therefore we cloned and transferred the S. Typhimurium marT-fidL operon into S. Typhi and confirmed that invasion of monocytes was dramatically decreased. Finally, our findings suggest that the genomic and functional differences between SPI-3 sequences have implications in the host specificity of Typhi and Typhimurium serovars.

Genetic and phenotypic evidence of the Salmonella enterica serotype Enteritidis human-animal interface in Chile

Salmonella enterica serotype Enteritidis is a worldwide zoonotic agent that has been recognized as a very important food-borne bacterial pathogen, mainly associated with consumption of poultry products. The aim of this work was to determine genotypic and phenotypic evidence of S. Enteritidis transmission among seabirds, poultry and humans in Chile. Genotyping was performed using PCR-based virulotyping, pulse-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Pathogenicity-associated phenotypes were determined with survival to free radicals, acidic pH, starvation, antimicrobial resistance, and survival within human dendritic cells. As result of PCR and PFGE assays, some isolates from the three hosts showed identical genotypic patterns, and through MLST it was determined that all of them belong to sequence type 11. Phenotypic assays show diversity of bacterial responses among isolates. When results were analyzed according to bacterial host, statistical differences were identified in starvation and dendritic cells survival assays. In addition, isolates from seabirds showed the highest rates of resistance to gentamycin, tetracycline, and ampicillin. Overall, the very close genetic and phenotypic traits shown by isolates from humans, poultry, and seabirds suggest the inter-species transmission of S. Enteritidis bacteria between hosts, likely through anthropogenic environmental contamination that determines infection of seabirds with bacteria that are potentially pathogenic for other susceptible organism, including humans.

Salmonella enterica in Pinnipeds, Chile

To the Editor: Several wildlife-associated zoonotic agents have played a major role in the emergence of diseases in humans (1). However, diseases can also emerge in wildlife as a result of human activities, such as contamination of the marine environment and its fauna by the disposal of nontreated human sewage. Salmonella enterica is among the agents identified as causing infection in various marine birds and mammals, including pinnipeds, from different geographic regions (2–4). The objective of our study was to determine whether S. enterica infection occurs in pinnipeds from the Chilean coast. During August–December 2010, we obtained samples from 13 South American sea lions (Otaria flavescens) that the sanitary authority found malnourished and stranded at the northern Chilean beaches of Antofagasta (23°40′S; 70°24′W) and Los Vilos (31°54′S; 71°30′W) (Table). The pinnipeds showed no clinical signs or symptoms of disease; however, rectal swab samples were obtained during their stay for rehabilitation at the Buin Marino facilities (Santiago, Chile). After the animals recovered, they were released to their original habitat. Table Characteristics of South American sea lions (Otaria flavescens) tested for infection with Salmonella spp., Chilean coast, August–December 2010* The swab samples were placed in Cary-Blair transport medium (COPAN, Murrieta, CA, USA) for shipment to the laboratory (Laboratory of Infectious Diseases, University of Chile, Santiago). To isolate bacteria, we placed the swab samples into 5 mL of buffered peptone water (Difco APT broth; Becton Dickinson, Franklin Lakes, NJ, USA), incubated them for 24 h at 42°C with agitation, and then aliquots of the suspension were transferred into the following media: modified semisolid Rappaport-Vassiliadis basal medium (Oxoid, Sao Paulo, Brazil) with novobiocin (20 µg/mL), selenite cysteine broth base (Oxoid), and xylose lysine desoxycholate agar (Difco XLD; Becton Dickinson). After the aliquots were incubated at 37°C for 24–48 h, we identified suspected colonies by using biochemical tests and invA gene detection by PCR (5). Results showed that 2 of the 13 animals were infected with S. enterica strains, which were serotyped as S. enterica serotype Newport and S. enterica serotype Havana (Table), according to the Kauffmann-White scheme (6). Testing showed that the strains were susceptible to the following antimicrobial drugs: ampicillin, chloramphenicol, tetracycline, amoxicillin/clavulanic acid, trimethoprim/sulfamethoxazole, cefotaxime, nalidixic acid, nitrofurantoin, ciprofloxacin, ceftazidime, and cefoxitin (7). Our results confirm S. enterica infection in pinnipeds from Chile and, more broadly, the South American coast and contrast with previous unsuccessful attempts to detect Salmonella spp. in pinnipeds from Valdivia, 2,200 km to the south (8). This finding suggests geographic variability in the epidemiology of infection; however, this possibility must be confirmed in additional studies with more samples and additional regions. S. enterica is an endemic bacterium in Chile that causes infection in humans and domestic animals. The Chilean sanitary authority includes S. enterica infection among the list of notifiable diseases, but surveillance is not conducted for S. enterica in wildlife. However, consideration should be given to changing this situation, given a report suggesting S. enterica as a priority for active surveillance (9). In addition, S. enterica serotypes Newport and Havana have been detected in Chile’s human population (10), strengthening the necessity for official support for initiatives addressing the need to elucidate the epidemiology of Salmonella in aquatic animals.

Serotype-associated polymorphisms in a partial rpoB gene sequence of Salmonella enterica

Salmonella enterica is a zoonotic bacterium with more than 2500 serotypes, which affect a wide range of hosts and produce diverse clinical outcomes. Strain identification usually involves costly and time-demanding procedures. This paper describes the sequencing of a rpoB hypervariable gene segment (847 bp) that allows identification of serotypes in S. enterica strains isolated from several hosts. The nucleotide similarity values among S. enterica serotypes ranged from 98.23% to 99.88%, with potential usefulness for devising a simple one-step sequencing as a first approach for identification of S. enterica strains. In conclusion, the analysis of polymorphisms in the partial rpoB sequence can discriminate S. enterica strains at the subspecies level.

Brucella infection in marine mammals in Antarctica

Infection with Brucella pinnipedialis and Brucella ceti in marine mammals of the northern hemisphere has been extensively reported. In Chile, the National Antarctic Institute (INACH) and the University of Chile have developed a collaborative study establishing the presence of anti- Brucella

Corynebacterium pseudotuberculosis Infection in Patagonian Huemul (Hippocamelus bisulcus)

Abstract Corynebacterium pseudotuberculosis is an intracellular bacteria and the etiologic agent of caseous lymphadenitis in domestic and wildlife species. We report C. pseudotuberculosis infection in Patagonian huemul (Hippocamelus bisulcus) from the Cerro Castillo National Reserve, Region of Aysen, Chile. Subcutaneous abscesses in the abdominal and pectoral regions from two animals were sampled and bacteriologic isolation was performed. In both cases, we isolated a C. pseudotuberculosis strain belonging to the ovine genotype. In addition, one isolate was resistant to ciprofloxacin and streptomycin. We report that H. bisulcus is a susceptible species to this bacterium, which is transmitted by direct or indirect contact with domestic sheep (Ovis aries) and which represents a potential conservation threat to populations of H. bisulcus. Additional research and prevention efforts should be addressed.