Whole-Genome Sequencing of Salmonella enterica Serovar Infantis and Kentucky Isolates Obtained from Layer Poultry Farms in Ecuador

Nontyphoidal Salmonella (NTS) represents one of the leading causes of foodborne gastroenteritis worldwide (1, 2). The global burden of NTS infections is 93.8 million cases, and it is estimated that NTS causes around 155,000 deaths annually (3). Unfortunately, there is a lack of information about surveillance studies of NTS in Latin American countries, and reports on NTS outbreaks are scarce in this region (4). In South American countries, there has been an increase in reports of multidrug-resistant Salmonella enterica subsp. enterica serovar Infantis in clinical, food, and livestock-related samples. That is the case in Peru (5, 6), Brazil (7), and Ecuador, where reports of NTS infections are related to extended-spectrum β-lactamase (ESBL)-producing S. Infantis (8). In Ecuador, this serovar represents the most commonly reported serovar in poultry-related samples (9, 10).

The importance of S. Infantis in the epidemiology of nontyphoidal salmonellosis has been described lately in Europe and the United States (2, 11). In the United States, infections caused by CTX-M-65 S. Infantis have been linked to travelers to South America (Peru and Ecuador) (12), suggesting that this serotype could play an essential role in the epidemiology of human salmonellosis. No reports of Salmonella enterica subsp. enterica serovar Kentucky have been published in Ecuador so far (according to the Scopus and Web of Science databases). In this report, we present the genome sequences of five recent strains of S. Infantis and two strains of S. Kentucky isolated from layer poultry farms in central Ecuador.

From August 2017 to December 2017, an aleatory sampling program in layer farms was carried out as part of routine diagnostics. Briefly, 102 samples from 21 farms in central Ecuador were analyzed by the ISO-6579 method. The samples were preenriched using buffered peptone water (Merck Millipore, Darmstadt, Germany) and incubated at 37°C for 18 to 24 h. Subsequently, the preenrichments were selectively enriched using Rappaport-Vassiliadis broth (Becton, Dickinson, Le Pont de Claix, France) at 41.5°C for 24 h. Next, the selective enrichment was streaked onto xylose lysine deoxycholate (XLD) agar (Merck Millipore) and incubated at 37°C for 24 h. The presumptive colonies were purified in MacConkey agar (Merck Millipore) and incubated at 37°C for 24 h. Biochemical tests were performed to confirm the genus Salmonella (catalase, oxidase, triple sugar iron [TSI], Simmons citrate, Christensen urea agar base, indole reaction). The positive samples were cryopreserved using overnight growth in LB broth (Sigma-Aldrich, St. Louis, USA) supplemented with 30% glycerol (Merck Millipore).

Thirty-one isolates were identified as belonging to the genus Salmonella. Seven isolates were selected according to their sampling origin (strains U822s, U824s, and U842s, Latacunga; U825s, Quero; U845s, Ambato; U827s, Pelileo; and U828s, Cevallos). Isolates were recovered from poultry feeders, environmental swabs, and cloacal swabs (Table 1). Salmonella cultures were grown in LB broth and incubated at 37°C for 24 h. The biomass was used for genomic DNA extraction using the Wizard genomic DNA purification kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions and DNA quantified using the Qubit 3.0 fluorometer (Invitrogen, CA, USA). Genomic libraries were prepared using the Nextera XT library prep kit (Illumina, CA, USA), and whole-genome sequencing (WGS) was performed using 300-bp paired-end read lengths on the MiSeq platform (Illumina). Raw reads were de novo assembled via SKESA v2.2 using the NCBI Prokaryotic Genome Annotation Pipeline (13). The draft genome assembly quality was evaluated using CheckM v1.0.18 (14) and QUAST v4.4 (15).

Draft genomes were analyzed using the tools from the Center for Genomic Epidemiology of the Technical University of Denmark. Using WGS data, the serotypes were determined using SeqSero v1.2 (16). Multilocus sequence types (MLSTs) were determined using MLST v2.0 (17). The presence of plasmids was identified using PlasmidFinder v2.1 (18). The prediction of bacterial pathogenicity was evaluated using PathogenFinder v1.1 (19). Finally, the detection of Salmonella pathogenicity islands was carried out using SPIFinder v1.0 (20). All analyses were performed using default parameters.

The number of contigs ranged from 179 to 322, and their N₅₀ values ranged from 29,447 to 52,705 bp. The draft genomes ranged from 4,744,299 to 5,001,560 bp, with 52.27% average GC content (Table 1). The assemblages showed 100% completeness in all isolates, and the percentage of contamination ranged from 0.08% to 0.39%. The number of annotated genes ranged from 4,788 to 5,100. The SeqSero analysis revealed that the isolates belonged to the S. enterica subsp. enterica serovars Infantis (5) and Kentucky (2). MLST analysis showed that the S. Infantis strains belonged to sequence type 32 (ST32) and the S. Kentucky strains to ST152. The plasmid replicon typing detected the plasmid incompatibility group IncX1 in the S. Kentucky strains only (Table 1).

These draft genomes provide essential information about the circulating clones of Salmonella from layer poultry farms in Ecuador and can serve as references for genome comparison studies.

We thank Gabriela A. Salazar, Elizabeth Sánchez-Salazar, Ricardo Guerrero-López, Alyssa Dickey, and Brian Sauders for their technical assistance.

The sampling part of this study was supported by the Dirección de Investigación y Desarrollo DIDE Universidad Técnica de Ambato (Research and Development Directorate of the Technical University of Ambato, Ecuador; project number 0192-CU-P-2018).