# Genomic Characterization of Yersinia enterocolitica Isolates, Costa Rica

Cyril Savin, Gletty Oropeza, Luis Barboza Fallas, Olga Rivas-Solano, Grettel

Chanto, Javier Pizarro-Cerdá

► To cite this version:

Cyril Savin, Gletty Oropeza, Luis Barboza Fallas, Olga Rivas-Solano, Grettel Chanto, et al.. Genomic Characterization of Yersinia enterocolitica Isolates, Costa Rica. Emerging Infectious Diseases, 2025, 31 (4), pp.1529 - 1530. 10.3201/eid3104.240963. pasteur-05015003

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## References

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# Genomic Characterization of *Yersinia enterocolitica* Isolates, Costa Rica

Cyril Savin, Gletty Oropeza, Luis A. Barboza Fallas, Olga Rivas-Solano, Grettel Chanto, Javier Pizarro-Cerdá

Author affiliations: Institut Pasteur, Paris, France (C. Savin, J. Pizarro-Cerdá); Instituto Costarricense de Investigación y Ensenanza en Nutrición y Salud, Cartago, Costa Rica (G. Oropeza, G. Chanto); Instituto Tecnológico de Costa Rica, Cartago (L.A. Barboza Fallas, O. Rivas-Solano)

DOI: https://doi.org/10.3201/eid3104.240963

Data on enteric yersinioses in Central America are limited. Genomic characterization of 78 *Yersinia enterocolitica* isolates from Costa Rica indicated persistent infection-source circulation between animal reservoirs and humans, as well as unusual antimicrobial resistance levels. Our study highlights the importance of genomic surveillance to monitor *Yersinia*-caused infections in Costa Rica.

The *Yersinia* genus encompasses 2 enteropathogenic species, *Y. enterocolitica* and *Y. pseudo-tuberculosis* (1,2). Those bacteria are the cause of foodborne infections that range from mild enteritis, especially in children, to systemic infections in the elderly or patients with underlying disorders (3). Enteric yersiniosis is the third most frequently reported zoonosis in Europe, mainly caused by *Y. enterocolitica* infections (4). Isolates can be classified into nonpathogenic genotypes (1Aa and 1Ab) and 11 other pathogenic genotypes (2).

In France, genomic surveillance based on a *Y. enterocolitica* core genome multilocus sequence typing (cgMLST) scheme was useful in identifying genetically closely related isolates and initiating an epidemiologic investigation with public health authorities to identify a common source of infection (5). Epidemiologic information on enteric yersinioses is scarce in the Americas. We studied *Yersinia* isolates from Costa Rica to evaluate their circulation in the country and to characterize them at the genomic level.

We analyzed 78 isolates collected during 2003–2023; all were of clinical origin except 1 veterinary isolate (Appendix 1, https://wwwnc.cdc.gov/EID/article/31/4/24-0963-App1.pdf). Genomic characterization based on a *Yersinia* 500-gene cgMLST enabled us to identify all isolates as pathogenic *Y. enterocolitica*: 2 isolates belonged to genotype 2/3-5a
<figure><img src="image_2.png" /><figcaption>**Figure.** Phylogenetic tree of 76 *Yersinia enterocolitica* genotype 4 isolates collected in Costa Rica, 2003–2023. The tree was reconstructed with IQ-TREE2 (http://www.iqtree.org) after alignment of the 1,727 genes of the cgMLST *Y. enterocolitica*. Columns show information on province (1), category (2), origin (3), antimicrobial resistance by drug (4–10), and by gene (11–18). Map (inset) shows cases by province. AMC, amoxicillin/clavulanic acid; AMO, amoxicillin; cgMLST, core genome multilocus sequence typing; CXN, cefalexin; FOX, cefoxitin; NAL, nalidixic acid; TET, tetracycline; TIC, ticarcillin.</figcaption></figure>

and the 76 others to genotype 4. All patients (n = 77) experienced enteric infections; we only identified pathogenic isolates. Epidemiologic records of the 77 clinical isolates showed that the sex ratio was 1:1 as expected in other countries (4). Most (76%) of the isolates were collected from patients <20 years of age; 20% were from adults 21–60 years of age. We observed a slight (4%) increase in patients >61 years of age. Most (97%) isolates came from feces, indicating that patients had a digestive form of infection, whereas 3% of the isolates were collected from blood (Appendix 2 Table, https://wwwnc.cdc.gov/EID/article/31/4/24-0963-App2.xlsx). Overall, the epidemiology of *Y. enterocolitica* isolates circulating in Costa Rica was similar to that of other countries (4).

Geographic distribution of the isolates showed that the 78 isolates were distributed in the 7 provinces

of Costa Rica; 60 (77%) isolates were from the Gran Area Metropolitana, a densely populated region around San Jose, the capital city (Figure). Phylogenetic reconstruction of the 76 genotype 4 isolates revealed 4 distinct bacterial subgroups (Figure). No geographic association was found between subgroups or provinces. We conducted a genomic comparison of isolates on the basis of a *Y. enterocolitica* 1,727-gene cgMLST and subsequent clustering of isolates with a 5-mismatch threshold; we found that 51/76 isolates belonged to 9 clusters of >2 isolates (Table; Appendix 1 Figure). We identified 3 clusters with isolates collected over a long period: cluster 6122 for 10 years, cluster 6127 for 13 years, and cluster 6124 for 8 years. Those periods suggest persistence of the source of contamination. We found that cluster 3151 is composed

<table><caption>**Table.** Epidemiologic characteristics of cgMLST clusters of *Yersinia enterocolitica* isolates, Costa Rica*</caption><thead><tr><th rowspan="2">cgMLST cluster</th><th colspan="2">No. isolates from</th><th rowspan="2">Category</th><th rowspan="2">Country</th><th rowspan="2">Isolation dates</th></tr><tr><th>Costa Rica</th><th>No. isolates in cluster</th></tr></thead><tbody><tr><td>6122</td><td>27</td><td>27</td><td>Clinical</td><td>Costa Rica</td><td>2004–2014</td></tr><tr><td>6127</td><td>8</td><td>8</td><td>Clinical</td><td>Costa Rica</td><td>2006–2019</td></tr><tr><td>6180</td><td>3</td><td>3</td><td>Clinical</td><td>Costa Rica</td><td>2014–2016</td></tr><tr><td>3151</td><td>3</td><td>3</td><td>Clinical, veterinary</td><td>Costa Rica</td><td>2018–2022</td></tr><tr><td>6124</td><td>2</td><td>2</td><td>Clinical</td><td>Costa Rica</td><td>2005–2013</td></tr><tr><td>6149</td><td>2</td><td>2</td><td>Clinical</td><td>Costa Rica</td><td>2007–2008</td></tr><tr><td>6164</td><td>2</td><td>2</td><td>Clinical</td><td>Costa Rica</td><td>2008–2011</td></tr><tr><td>6212</td><td>2</td><td>2</td><td>Clinical</td><td>Costa Rica</td><td>2012–2015</td></tr><tr><td>5652</td><td>2</td><td>3</td><td>Clinical</td><td>Costa Rica and France</td><td>2015–2023</td></tr></tbody></table>

<footnote>*Clinical isolates are from human samples. cgMLST, core genome multilocus sequence typing.</footnote>
of mixed clinical and veterinary isolates, suggesting circulation between the animal reservoir and humans (Table; Appendix 2 Table). Of note, cluster 5652 is composed of 2 isolates from Costa Rica and 1 from France. Epidemiologic records of the France isolate showed that the patient was returning from Costa Rica at the time of sampling, suggesting that he was infected during his trip.

Among the 76 genotype 4 isolates, antimicrobial susceptibility testing revealed unusually high levels of resistance to cefoxitin (7.9%), nalidixic acid (23.7%), and tetracycline (9.2%) (Figure; Appendix Table) (6). Nalidixic acid resistance was associated with mutations in the quinolone resistance-determining region of the *gyrA* gene; we identified the previously described S83R mutation (7) in 15 isolates and D87G mutation in 3 isolates. Cefoxitin resistance in 6 isolates was associated with a G751A mutation of the *ampC* gene, identical to the mutation present in the naturally cefoxitin-resistant genotype 2/3-5a. Those 6 isolates are in the upper part of the tree, suggesting the mutation was acquired during emergence of the branch (Figure). In 6/7 tetracycline-resistant isolates, resistance was associated with acquisition of the *tetC* gene located on a 13.7 kb plasmid circulating in other enterobacteria. Those 6 isolates are on the upper branch of the tree, suggesting that plasmid acquisition occurred during emergence of the branch (Figure). The other tetracycline-resistant isolate was associated with the *tetA* gene, together with *aph(3')-Ib* and *aph(6)-Id*, *floR*, and *sul2*, located on an 81.2 kb multidrug-resistant plasmid (Figure). The presence of those genes was phenotypically associated with resistance to tetracycline, streptomycin, chloramphenicol, and sulfonamides.

In summary, this study brings insights into the distribution of enteropathogenic *Yersinia* species circulating in Costa Rica. Diversity is low because most of the *Y. enterocolitica* isolates belonged to biotype 4. Many closely related isolates were identified within a long timeframe, sometimes between human clinical and veterinary isolates, suggesting a persistence of the source of infection and the circulation between the reservoir and humans. Implementation of routine genomic surveillance in Costa Rica could help to monitor the circulation of closely related *Yersinia* isolates and initiate epidemiologic investigation to implement control measures in the event of an outbreak.

Acknowledgments

We thank the national network of bacteriology laboratories of Costa Rica for their participation in the surveillance of enteropathogenic agents.

This project received funding from Institut Pasteur (Paris, France) and the LabEx Integrative Biology of Emerging Infectious Diseases (grant no. ANR LBX-62 IBEID).

About the Author

Dr. Savin is a microbiologist and deputy director of the *Yersinia* National Reference Laboratory, Institut Pasteur, Paris. His research interests include biological diagnosis, genomics, evolution, and epidemiology of *Yersiniae*.

References

1. Le Guern AS, Savin C, Angermeier H, Brémont S, Clermont D, Mühle E, et al. *Yersinia artesiana* sp. nov., *Yersinia proxima* sp. nov., *Yersinia alsatica* sp. nov., *Yersinia vastinensis* sp. nov., *Yersinia thraica* sp. nov. and *Yersinia occitanica* sp. nov., isolated from humans and animals. *Int J Syst Evol Microbiol.* 2020;70:5363-72. https://doi.org/10.1099/ijsem.0.000417

2. Savin C, Criscuolo A, Guglielmini J, Le Guern AS, Carniel E, Pizarro-Cerdá J, et al. Genus-wide *Yersinia* core-genome multilocus sequence typing for species identification and strain characterization. *Microb Genom.* 2019;5:e000301. https://doi.org/10.1099/mgen.0.000301

3. Cover TL, Aber RC. *Yersinia enterocolitica*. N Engl J Med. 1989;321:16-24. https://doi.org/10.1056/NEJM198907063210104

4. European Food Safety Authority; European Centre for Disease Prevention and Control. The European Union One Health 2022 zoonoses report. EFSA J. 2023;21:e8442. https://doi.org/10.2903/j.efsa.2023.8442

5. Le Guern AS, Savin C, Chereau F, Tessier S, Guglielmini J, Brémont S, et al. A novel cgMLST for genomic surveillance of *Yersinia enterocolitica* infections in France allowed the detection and investigation of outbreaks in 2017-2021. *Microbiol Spectr.* 2024;12:e0050424. https://doi.org/10.1128/spectrum.00504-24

6. Abdel-Haq NM, Papadopol R, Asmar BI, Brown WJ. Antibiotic susceptibilities of *Yersinia enterocolitica* recovered from children over a 12-year period. *Int J Antimicrob Agents.* 2006;27:449-52. https://doi.org/10.1016/j.ijantimicag.2005.12.008

7. Capilla S, Ruiz J, Goñi P, Castillo J, Rubio MC, Jiménez de Anta MT, et al. Characterization of the molecular mechanisms of quinolone resistance in *Yersinia enterocolitica* O:3 clinical isolates. *J Antimicrob Chemother.* 2004;53:1068-71. https://doi.org/10.1093/jac/dkh225

Address for correspondence: Cyril Savin and Javier Pizarro-Cerdá, Unité de Recherche Yersinia, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex 15, France; e-mail: cyril.savin@pasteur.fr and javier.pizarro-cerda@pasteur.fr
DOI: https://doi.org/10.3201/eid3104.240963

EID cannot ensure accessibility for supplementary materials supplied by authors. Readers who have difficulty accessing supplementary content should contact the authors for assistance.

# Genomic Characterization of *Yersinia enterocolitica* Isolates, Costa Rica

## Appendix 1

### Materials and Methods

#### Bacterial Isolation

This study includes 78 isolates collected between 2003 and 2023 in the 7 provinces of Costa Rica. The 77 clinical isolates were obtained by the Institute for Research and Teaching in Nutrition and Health (INCIENSA) in the context of public health surveillance of enteric diseases. Direct culturing without enrichment allowed to recover isolates from patients presenting various symptoms, mostly acute diarrhea. A total of 77 isolates from the stock culture collection of the National Reference Center of Bacteriology were taken from storage at -70°C and passaged on blood agar. Identification and susceptibility cards were inoculated according to the directions of the manufacturer and processed in the Vitek2 instrument. In addition, conventional biochemical tests and miniaturized biochemical assays (API 20E) were performed.

The veterinary isolate, collected from a pig tonsil, was obtained by the Technological institute of Costa Rica, San Carlos campus, in the context of a research project related to food borne pathogens. Twenty-five g of pig tonsil was inoculated in 225 mL of peptone sorbitol bile (PSB) broth (Millipore, Burlington, Massachusetts, United States) and incubated at 4°C. Every 7 days, for a month, 1 mL of the culture was subcultured in 100 mL of PSTA Enrichment Broth Base (Himedia, Pennsylvania, United States) and incubated at 28°C for 48 hours. Then, 10 µl were streaked onto semi-solid Cefsulodin-Irgasan-Novobiocin Agar (CIN) (Yersinia Selective Agar Base and Yersinia Selective Supplement, Millipore, Burlington, Massachusetts, United States). As indicated in the ISO 10273:2017 method (1), *Y. enterocolitica* colonies on CIN agar are typically small and smooth, with a red center and a translucent rim, and when examined with obliquely transmitted light, they are non-iridescent and finely granular.
## DNA Extraction and Genome Sequencing

Isolates were grown for 18 hours at 28°C in LB broth under agitation at 150 rpm. DNA extraction was performed with the Maxwell RSC Cultured Cells DNA kit (reference AS1620, Promega, Madison, USA) on a Maxwell CSC 48 Instrument (Promega, Madison, USA) according to the manufacturer's instructions. DNA quantity and purity were assessed using a Qubit fluorometer (Thermo FisherScientific, Waltham, MA, USA). Genomic libraries were prepared using the Nextera XT Sample Kit (Illumina, San Diego, CA, USA), and DNA sequencing was performed on a NextSeq 500 platform (Illumina) using 2× 150-bp paired-end runs. Raw data were assembled using the fq2dna pipeline (https://gitlab.pasteur.fr/GIPhy/fq2dna). To identify plasmids present in isolates IP51152 and IP51348, genomic DNA extraction was performed using a PureLink Genomic DNA mini kit (reference K182002, Invitrogen) following the manufacturer's instructions. Long-read genome sequencing was performed using Oxford Nanopore Technologies (ONT) with the Plasmidsaurus service (www.plasmidsaurus.com). Hybrid genome assembly using both Illumina and ONT sequencing data was performed as previously described (https://github.com/rrwick/Perfect-bacterial-genome-tutorial) (2).

## Genomic Characterization and Molecular Typing

Taxonomic assignment of the isolates was performed using a 500-gene cgMLST Yersinia as previously described (3). Molecular typing of the isolates was performed using a 1,727-genes cgMLST *Y. enterocolitica* followed by clustering of the isolates with a maximum of 5 allelic differences as previously described (4). Phylogenetic reconstruction was carried out on the 1,727 genes of the cgMLST *Y. enterocolitica* scheme after their extraction using the "Export" plugin at https://bigsdb.pasteur.fr/yersinia. For each isolate, the genes were concatenated and then aligned using mafft v7.467 (5). From this alignment, a phylogenetic reconstruction was performed with IQ-TREE2 (6) using a bootstrap value of 1,000 and by first determining the best substitution model thanks to the "ModelFinder" implemented in IQ-TREE2.

## Antimicrobial Susceptibility Testing and Genotypic Prediction of Antimicrobial Resistance

The antibiotic resistance profiles of the isolates were determined by disk diffusion method on Mueller-Hinton II agar according to the European Committee on Antimicrobial Susceptibility Testing (https://www.eucast.org) recommendation with an incubation temperature of 28°C. Antibiotics tested were amoxicillin (AMO – 20 µg), cefoxitin (FOX - 30 µg),
cefotaxime (COX – 5 µg), ceftazidime (CZD – 10 µg), ciprofloxacin (CIP – 5 µg), nalidixic acid (NAL – 30 µg), cefalexin (CXN – 30 µg), cotrimoxazole (Trimethoprim – Sulfamethoxazole, SXT 1.25 + 23.75 µg), amoxicillin + clavulanic acid (AMC, 20 µg + 10 µg), ticarcilline (TIC – 75 µg), tetracycline (TET – 30 µg), ertapenem (ETP – 10 µg), and amikacin (AKN – 30 µg). In addition, sulfonamides (SSS – 300 µg), chloramphenicol (CHL – 30 µg), and streptomycin (SMN – 10 µg) were tested for the multidrug resistant isolate IP51348.

The presence of antimicrobial resistance genes presence in the genomes was identified using the Resfinder Database version 4.3.3 (7).

## Data Visualization

Epidemiologic, phenotypic and genetic data along with the phylogenetic tree were visualized using Microreact www.microreact.org (8). The cgMLST *Y. enterocolitica* profiles of the 76 *Y. enterocolitica* genotype 4 isolates were exported from the BIGSdb database using "Export plugin" at https://bigsdb.pasteur.fr/yersinia and the minimum spanning tree was built using BioNumerics v.7.6 (Applied-Maths, Sint-Martens-Latem, Belgium).

## Data Availability

The cgMLST allelic profiles of the 78 isolates have been made publicly available in the *Yersinia* BIGSdb database (https://bigsdb.pasteur.fr/yersinia). Sequence data was made publicly available in NCBI/EBI/DDJJ databases (BioProject no. PRJNA1128560).

## Supplementary References

1. International Organization for Standardization. ISO 10273:2017. Microbiology of food and animal feeding stuffs—horizontal method for the detection of presumptive pathogenic *Yersinia enterocolitica* [cited 2025 Feb 26]. https://www.iso.org/standard/63329.html

2. Wick RR, Judd LM, Holt KE. Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing. PLOS Comput Biol. 2023;19:e1010905. PubMed https://doi.org/10.1371/journal.pcbi.1010905

3. Savin C, Criscuolo A, Guglielmini J, Le Guern AS, Carniel E, Pizarro-Cerdá J, et al. Genus-wide *Yersinia* core-genome multilocus sequence typing for species identification and strain characterization. Microb Genom. 2019;5:e000301. PubMed https://doi.org/10.1099/mgen.0.000301
4. Le Guern AS, Savin C, Chereau F, Tessier S, Guglielmini J, Brémont S, et al. A novel cgMLST for genomic surveillance of *Yersinia enterocolitica* infections in France allowed the detection and investigation of outbreaks in 2017–2021. *Microbiol Spectr.* 2024;12:e0050424. [PubMed](https://doi.org/10.1128/spectrum.00504-24)

5. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. *Mol Biol Evol.* 2013;30:772–80. [PubMed](https://doi.org/10.1093/molbev/mst010)

6. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. *Mol Biol Evol.* 2020;37:1530–4. [PubMed](https://doi.org/10.1093/molbev/msaa015)

7. Bortolaia V, Kaas RS, Ruppe E, Roberts MC, Schwarz S, Cattoir V, et al. ResFinder 4.0 for predictions of phenotypes from genotypes. *J Antimicrob Chemother.* 2020;75:3491–500. [PubMed](https://doi.org/10.1093/jac/dkaa345)

8. Argimón S, Abudahab K, Goater RJE, Fedosejev A, Bhai J, Glasner C, et al. Microreact: visualizing and sharing data for genomic epidemiology and phylogeography. *Microb Genom.* 2016;2:e000093. [PubMed](https://doi.org/10.1099/mgen.0.000093)
<figure><img src="image_3.png" /><figcaption>**Appendix 1 Figure.** Minimum spanning tree of the 76 *Yersina enterocolitica* genotype 4 isolates collected from 2003 to 2023 in Costa Rica. The tree was built based on the allelic profiles of the 1,727 genes *cgMLST Y. enterocolitica*. Circles represent the different profiles and sizes are proportional to the number of isolates within. All the allelic profiles with a maximum of 5 allelic distance were collapsed in the same circle. Branch lengths, reflecting the allelic differences between the profiles using square root scaling, are indicated on the branches. Cluster numbers are indicated close to each circle. When ≥2 isolates, the number of isolates present in a cluster are indicated within the circles.</figcaption></figure>