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

Published: August 30, 2024

By: William Calero-Cáceres 1,2; Joyce Villacís 1; Maria Ishida 3; Elton Burnett 4; Christian Vinueza-Burgos 5.

Summary

Author details:

1 UTA-RAM-One Health, Centro de Investigaciones Agropecuarias, Facultad de Ciencias Agropecuarias, Universidad Técnica de Ambato (UTA), Ambato, Ecuador; 2 Faculty of Science and Engineering in Food and Biotechnology, Universidad Técnica de Ambato (UTA), Ambato, Ecuador; 3 New York State Department of Agriculture and Markets, Food Laboratory, Albany, New York, USA; 4 Institute of Parasitology, McGill University, Montreal, Quebec, Canada; 5 Facultad de Medicina Veterinaria y Zootecnia, Universidad Central del Ecuador, Quito, 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 -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).

Whole-Genome Sequencing of Salmonella enterica Serovar Infantis and Kentucky Isolates Obtained from Layer Poultry Farms in Ecuador - Image 1

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 Plasmid Finder 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.

This article was originally published in Microbiology Resource Announcements 9:e00091-20. https://doi.org/ 10.1128/MRA.00091-20. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

References

1. World Health Organization. 2015. WHO estimates of the global burden of foodborne diseases. World Health Organization, Geneva, Switzerland.

2. EFSA, ECDC. 2018. The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2017. EFSA J 16:e05500. https://doi.org/10.2903/j.efsa.2018.5500.

3. Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O’Brien SJ, Jones TF, Fazil A, Hoekstra RM, International Collaboration on Enteric Disease “Burden of Illness” Studies. 2010. The global burden of nontyphoidal Salmonella gastroenteritis. Clin Infect Dis 50:882– 889. https://doi.org/10 .1086/650733.

4. Balasubramanian R, Im J, Lee JS, Jeon HJ, Mogeni OD, Kim JH, Rakotozan drindrainy R, Baker S, Marks F. 2019. The global burden and epidemiology of invasive non-typhoidal Salmonella infections. Hum Vaccin Immunother 15:1421–1426. https://doi.org/10.1080/21645515.2018.1504717.

5. Granda A, Riveros M, Martínez-Puchol S, Ocampo K, Laureano-Adame L, Corujo A, Reyes I, Ruiz J, Ochoa TJ. 2019. Presence of extended-spectrum -lactamase, CTX-M-65 in Salmonella enterica serovar Infantis isolated from children with diarrhea in Lima, Peru. J Pediatr Infect Dis 14: 194 –200. https://doi.org/10.1055/s-0039-1685502.

6. Silva C, Betancor L, García C, Astocondor L, Hinostroza N, Bisio J, Rivera J, Perezgasga L, Pérez Escanda V, Yim L, Jacobs J, García-del Portillo F, SalmoIber CYTED Network, Chabalgoity JA, Puente JL. 2017. Characterization of Salmonella enterica isolates causing bacteremia in Lima, Peru, using multiple typing methods. PLoS One 12:e0189946. https://doi.org/ 10.1371/journal.pone.0189946.

7. Monte DF, Lincopan N, Berman H, Cerdeira L, Keelara S, Thakur S, Fedorka-Cray PJ, Landgraf M. 2019. Genomic features of high-priority Salmonella enterica serovars circulating in the food production chain, Brazil, 2000 –2016. Sci Rep 9:1–12. https://doi.org/10.1038/s41598-019 -45838-0

8. Cartelle Gestal M, Zurita J, Paz y Mino A, Ortega-Paredes D, Alcocer I. 2016. Characterization of a small outbreak of Salmonella enterica serovar Infantis that harbor CTX-M-65 in Ecuador. Braz J Infect Dis 20:406 – 407. https://doi.org/10.1016/j.bjid.2016.03.007.

9. Salazar GA, Guerrero-López R, Lalaleo L, Avilés-Esquivel D, Vinueza Burgos C, Calero-Cáceres W. 2019. Presence and diversity of Salmonella isolated from layer farms in central Ecuador. F1000Res 8:235. https://doi .org/10.12688/f1000research.18233.2.

10. Vinueza-Burgos C, Cevallos M, Ron-Garrido L, Bertrand S, De Zutter L. 2016. Prevalence and diversity of Salmonella serotypes in Ecuadorian broilers at slaughter age. PLoS One 11:e0159567. https://doi.org/10.1371/ journal.pone.0159567.

11. Tate H, Folster JP, Hsu CH, Chen J, Hoffmann M, Li C, Morales C, Tyson GH, Mukherjee S, Brown AC, Green A, Wilson W, Dessai U, Abbott J, Joseph L, Haro J, Ayers S, McDermott PF, Zhao S. 2017. Comparative analysis of extended-spectrum-ß-lactamase CTX-M-65-producing Salmonella enterica serovar Infantis isolates from humans, food animals, and retail chickens in the United States. Antimicrob Agents Chemother 61:e00488-17. https://doi.org/10.1128/AAC.00488-17.

12. Brown AC, Chen JC, Watkins LKF, Campbell D, Folster JP, Tate H, Wasilenko J, Van Tubbergen C, Friedman CR. 2018. CTX-M-65 extendedspectrum -lactamase–producing Salmonella enterica serotype Infantis, United States. Emerg Infect Dis 24:2284 –2291. https://doi.org/10.3201/ eid2412.180500.

13. Souvorov A, Agarwala R, Lipman DJ. 2018. SKESA: strategic k-mer extension for scrupulous assemblies. Genome Biol 19:153. https://doi.org/ 10.1186/s13059-018-1540-z

14. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. 2015. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 25:1043–1055. https://doi.org/10.1101/gr.186072.114.

15. Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075. https:// doi.org/10.1093/bioinformatics/btt086.

16. Zhang S, Yin Y, Jones MB, Zhang Z, Deatherage Kaiser BL, Dinsmore BA, Fitzgerald C, Fields PI, Deng X. 2015. Salmonella serotype determination utilizing high-throughput genome sequencing data. J Clin Microbiol 53:1685–1692. https://doi.org/10.1128/JCM.00323-15.

17. Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, Jelsbak L, Sicheritz-Pontén T, Ussery DW, Aarestrup FM, Lund O. 2012. Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol 50:1355–1361. https://doi.org/10.1128/JCM.06094-11.

18. Carattoli A, Zankari E, Garciá-Fernández A, Larsen MV, Lund O, Villa L, Aarestrup FM, Hasman H. 2014. In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing. Antimicrob Agents Chemother 58:3895–3903. https://doi.org/10.1128/ AAC.02412-14.

19. Cosentino S, Voldby Larsen M, Møller Aarestrup F, Lund O. 2013. PathogenFinder— distinguishing friend from foe using bacterial whole genome sequence data. PLoS One 8:e77302. https://doi.org/10.1371/ journal.pone.0077302.

20. Center for Genomic Epidemiology. 2020. SPIFinder (version 1.0). https:// cge.cbs.dtu.dk/services/SPIFinder/

What is the global burden of Nontyphoidal Salmonella (NTS) infections?

The global burden of NTS infections is 93.8 million cases, and it is estimated that NTS causes around 155,000 deaths annually (3).

What is the significance of multidrug-resistant Salmonella enterica subsp. enterica serovar Infantis in South American countries?

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 -lactamase (ESBL)-producing S. Infantis (8).