ABSTRACT

Objectives.

To determine the proportion of Salmonella enterica in fecal samples of live pigs with suspected salmonellosis analyzed at the diagnostic unit of the University of Antioquia, Colombia between 2019 and 2021, and examine the serotypes and antimicrobial resistance patterns.

Methods.

This was a laboratory-based cross-sectional study of routine data on fecal samples received from pig farms in all nine subregions of Antioquia state, Colombia. Salmonella spp. detection at the university is done using enrichment, selective culture, and polymerase chain reaction. Serotypes were identified using the Kauffmann–White scheme and isolates were tested for antimicrobial susceptibility using broth microdilution.

Results.

Of 653 samples tested, 149 (23%) were positive for S. enterica. Nine serotypes were identified. The most common were Salmonella Typhimurium (56%) and its monophasic variant (35%). Resistance to ampicillin (70%) was most frequently observed, followed by ciprofloxacin (55%), and sulfamethoxazole–trimethoprim (52%). No isolates were resistant to amikacin and gentamicin. Multidrug resistance (resistance to ≥ 3 classes of antibiotics) was observed in 61 (44%) isolates. Multidrug resistance was highest in S. Typhimurium (57%) compared with the other serotypes. Serotype was associated with multidrug resistance (p = 0.01), but age of the pig and sub-region were not.

Conclusions.

The proportion of Salmonella spp. and the associated high levels of multidrug resistance are of concern and may indicate irrational use of antimicrobials and poor management practices in pig production systems in the region. Strengthened surveillance is needed to monitor and improve farm management practices and the use of antimicrobials in farms in Colombia.

Keywords
Salmonella enterica; serogroup; drug resistance, multiple; swine; Colombia

RESUMEN

Objetivos.

Determinar la proporción de Salmonella enterica en muestras fecales de cerdos vivos con presunta salmonelosis analizadas en la unidad de diagnóstico de la Universidad de Antioquia (Colombia) entre el 2019 y el 2021, así como examinar los serotipos y los patrones de resistencia a los antimicrobianos.

Métodos.

Se trata de un estudio transversal de laboratorio sobre datos ordinarios de muestras fecales provenientes de granjas porcinas de las nueve subregiones del departamento de Antioquia (Colombia). La detección de Salmonella spp. en la universidad se realiza mediante el enriquecimiento, el cultivo selectivo y la reacción en cadena de la polimerasa. Se identificaron los serotipos con el esquema de Kauffmann-White y se examinaron las cepas aisladas para determinar la susceptibilidad antimicrobiana mediante microdilución en caldo.

Resultados.

De las 653 muestras analizadas, 149 (23%) dieron un resultado positivo para S. enterica. Se identificaron nueve serotipos. Los más comunes fueron Salmonella typhimurium (56%) y su variante monofásica (35%). La resistencia a la ampicilina fue la observada con mayor frecuencia (70%), seguida de la resistencia al ciprofloxacino (55%) y al sulfametoxazol-trimetoprima (52%). Ninguna cepa aislada fue resistente a la amikacina y la gentamicina. Se observó resistencia a múltiples fármacos (resistencia a tres o más clases de antibióticos) en 61 cepas (44%). La resistencia a múltiples fármacos fue más elevada en el caso de S. typhimurium (57%) en comparación con los otros serotipos. Se asoció el serotipo con la resistencia a múltiples fármacos (p = 0,01), a diferencia de la edad del cerdo y la subregión.

Conclusiones.

La proporción de Salmonella spp. y los elevados niveles asociados de resistencia a múltiples fármacos son preocupantes y pueden ser un indicativo de uso irracional de antimicrobianos y malas prácticas de gestión en los sistemas de producción porcina de la región. Es necesario reforzar la vigilancia para dar seguimiento y mejorar las prácticas de gestión agropecuaria y el uso de antimicrobianos en las granjas en Colombia.

Palabras clave
Salmonella enterica; serogrupo; resistencia a múltiples medicamentos; porcinos; Colombia

RESUMO

Objetivos.

Determinar a proporção de Salmonella enterica em amostras de fezes de suínos vivos com suspeita de salmonelose analisadas na unidade de diagnóstico da Universidade de Antioquia, Colômbia, entre 2019 e 2021, e examinar seus sorotipos e padrões de resistência a antimicrobianos.

Métodos.

Estudo transversal, de base laboratorial, utilizando dados de rotina de amostras de fezes recebidas de suinocultores em todas as nove sub-regiões do estado de Antioquia, Colômbia. A detecção de Salmonella spp. na Universidade é feita por enriquecimento, cultura seletiva e reação em cadeia da polimerase. Os sorotipos foram identificados usando o esquema de Kauffmann-White, e os isolados foram testados quanto à suscetibilidade aos antimicrobianos pelo método de microdiluição em caldo.

Resultados.

Das 653 amostras testadas, 149 (23%) foram positivas para S. enterica. Foram identificados nove sorotipos. Os mais comuns foram Salmonella Typhimurium (56%) e sua variante monofásica (35%). A resistência à ampicilina (70%) foi observada com maior frequência, seguida pela resistência ao ciprofloxacino (55%) e ao sulfametoxazol/trimetoprima (52%). Nenhum isolado apresentou resistência à amicacina ou gentamicina. Multirresistência (resistência a ≥ 3 classes de antibióticos) foi observada em 61 isolados (44%). A multirresistência foi mais comum em S. Typhimurium (57%), em comparação aos outros sorotipos. Foi constatada associação da multirresistência com sorotipos (p = 0,01), mas não com idade do suíno ou sub-região.

Conclusões.

A proporção de Salmonella spp. e os níveis elevados associados de multirresistência a antimicrobianos aqui constatados são preocupantes, e podem indicar uso irracional de antimicrobianos e práticas inadequadas de manejo nos sistemas de suinocultura da região. É preciso fortalecer a vigilância para monitorar e melhorar as práticas de manejo agrícola e o uso de antimicrobianos em fazendas na Colômbia.

Palavras-chave
Salmonella enterica; sorogrupo; resistência a múltiplos medicamentos; suínos; Colômbia

Salmonellosis is a gastrointestinal infection caused by Salmonella enterica, a gram-negative bacterium. In humans, this infection is generally contracted through the consumption of contaminated food of animal origin (primarily eggs, pork meat, and chicken meat), although other foods, such as manure-contaminated vegetables and fruits, have also been linked to transmission (¹1. Martínez-Avilés M, Garrido-Estepa M, Álvarez J, de la Torre A. Salmonella surveillance systems in swine and humans in Spain: a review. Vet Sci. 2019;6(1):20. doi: 10.3390/vetsci6010020
https://doi.org/10.3390/vetsci6010020... , ²2. Salmonella (non typhoid). Geneva: World Health Organization; 2018 [accessed 2021 Jul 22]. Available from: https://www.who.int/es/news-room/fact-sheets/detail/salmonella-(non-typhoidal)
https://www.who.int/es/news-room/fact-sh... ). In the European Union, pork is one of the main sources of human salmonellosis after eggs and egg products (¹1. Martínez-Avilés M, Garrido-Estepa M, Álvarez J, de la Torre A. Salmonella surveillance systems in swine and humans in Spain: a review. Vet Sci. 2019;6(1):20. doi: 10.3390/vetsci6010020
https://doi.org/10.3390/vetsci6010020... , ²2. Salmonella (non typhoid). Geneva: World Health Organization; 2018 [accessed 2021 Jul 22]. Available from: https://www.who.int/es/news-room/fact-sheets/detail/salmonella-(non-typhoidal)
https://www.who.int/es/news-room/fact-sh... ). S. enterica also causes disease in pigs in a variety of clinical forms. Thus, salmonella infection has substantial economic, public health, and animal health consequences (³3. Ostanello F, De De Lucia A. On-farm risk factors associated with Salmonella in pig herds. Large Anim Rev. 2020;26(3):133–40.). Transmission can occur through pig-to-pig contact, because of infected animals arriving from other farms, or feed contamination from the feces of other carrier animals (⁴4. Ferrari RG, Rosario DKA, Cunha-Neto A, Mano SB, Figueiredo EES, Conte-Junior CA. Worldwide epidemiology of Salmonella serovars in animal-based foods: a meta-analysis. Appl Environ Microbiol. 2019;85(14). doi: 10.1128/AEM.00591-19
https://doi.org/10.1128/AEM.00591-19... ).

S. enterica is widely distributed in domestic and wild animals and over 2 600 serotypes have been identified (⁵5. Oliván Iglesia R, Acín Tresaco C, Mainar Jaime RC. El papel del cerdo en la incidencia de la infección por salmonella en la población humana [The role of the pig in the incidence of salmonella infection in the human population]. Zaragoza: University of Zaragoza; 2019.). The S. enterica serotypes that mainly cause clinical disease in pigs are S. Choleraesuis and S. Typhimurium. S. Typhimurium is the most frequent serotype detected in pigs. It is sometimes associated with diarrhea in young pigs and is also a common source of food poisoning in humans (⁶6. Minh DK, Hounmanou YMG, Mai HBT, Olsen JE, Dalsgaard A. Prevalence and genomic characterization of Salmonella Weltevreden in commercial pig feed. Vet Microbiol. 2020;246:108725. doi: 10.1016/j.vetmic.2020.10872
https://doi.org/10.1016/j.vetmic.2020.10... , ⁷7. Griffith RW, Carlson SA, Krull AC. Salmonellosis. In: Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW, Zhang J, editors. Diseases of swine, 11th edition. Hoboken, NJ: John Wiley & Sons, Inc.; 2019. doi: 10.1002/9781119350927
https://doi.org/10.1002/9781119350927... ).

Salmonellosis in pigs is treated using antimicrobials including amoxicillin–clavulanic acid, ampicillin, ceftiofur, ciprofloxacin, chloramphenicol, florfenicol, gentamicin, sulfamethoxazole–trimethoprim, tetracycline, and macrolide antibiotics such as tilmicosin – many of these antimicrobials are important for human therapy (⁸8. Ayala-Romero C, Ballen-Parada C, Rico-Gaitan M, Chamorro-Tobar I, Zambrano-Moreno D, Poutou-Piñales R, et al. Prevalencia de Salmonella spp., En ganglios mesentéricos de porcinos en plantas de beneficio Colombianas [Prevalence of Salmonella spp., in mesenteric pig’s ganglia at Colombian benefit plants]. Rev MVZ Cordoba. 2018;23(1):6474–86. doi: 10.21897/rmvz.1242
https://doi.org/10.21897/rmvz.1242... ). Antimicrobials are used in animals not only for treatment, but also for controlling the spread of infection (metaphylaxis), preventing infection (prophylaxis), improving feed efficiency, and promoting growth (⁹9. Moreno-Switt AI, Pezoa D, Sepúlveda V, González I, Rivera D, Retamal P, et al. Corrigendum: Transduction as a potential dissemination mechanism of a clonal qnrB19-carrying plasmid isolated from Salmonella of multiple serotypes and isolation sources. Front Microbiol. 2019;11:547. doi: 10.3389/fmicb.2020.00547
https://doi.org/10.3389/fmicb.2020.00547... ).

Although strict regulations exist about the use of antimicrobials in many countries, the enforcement is often weak which has resulted in indiscriminate use of antimicrobials. This overuse has contributed to the emergence of multidrug-resistant (MDR) strains of bacteria (¹⁰10. Casana Rico C. El uso de antibióticos en la industria alimentaria y su contribución al desarrollo de resistencias. determinantes de la diseminación de la resistencia a la colistina [The use of antibiotics in the food industry and its contribution to the development of resistance. determinants of the spread of colistin resistance]. Madrid: Universidad Complutense; 2017.–¹²12. Bravo Aguilar MJ. Diagnóstico general de sistemas productivos porcinos en el municipio de Concepción (Antioquia) [General diagnosis of pig production systems in the municipality of Concepción (Antioquia)] [Dissertation]. Caldas: Unilasallista Corporación Universitaria; 2017.). Antimicrobial-resistant Salmonella strains have been found in chicken, pig, and other foods all around the world (¹³13. Ren D, Chen P, Wang Y, Wang J, Liu H, Liu H. Phenotypes and antimicrobial resistance genes in Salmonella isolated from retail chicken and pork in Changchun, China. J Food Saf. 2017;37(2):e12314. doi: 10.1111/jfs.12314
https://doi.org/10.1111/jfs.12314... ). When humans are infected with resistant Salmonella strains, treatment can be difficult, increasing the likelihood of treatment failure, and even death (¹⁴14. Caniça M, Manageiro V, Abriouel H, Moran-Gilad J, Franz CMAP. Antibiotic resistance in foodborne bacteria. Trends Food Sci Technol. 2019;84:41–4. doi:10.1016/J.TIFS.2018.08.001
https://doi.org/10.1016/J.TIFS.2018.08.0... , ¹⁵15. Joint FAO/WHO Expert meeting in collaboration with OIE on foodborne antimicrobial resistance: role of the environment, crops and biocides. Meeting report. Geneva: World Health Organization & Rome: Food and Agriculture Organization; 2019.). Hence, it is vital to understand the antibiotic resistance patterns of Salmonella spp. infections in pigs.

Limited evidence exists about the prevalence and antibiotic resistance patterns of Salmonella spp. in pigs in Colombia (⁴4. Ferrari RG, Rosario DKA, Cunha-Neto A, Mano SB, Figueiredo EES, Conte-Junior CA. Worldwide epidemiology of Salmonella serovars in animal-based foods: a meta-analysis. Appl Environ Microbiol. 2019;85(14). doi: 10.1128/AEM.00591-19
https://doi.org/10.1128/AEM.00591-19... , ⁵5. Oliván Iglesia R, Acín Tresaco C, Mainar Jaime RC. El papel del cerdo en la incidencia de la infección por salmonella en la población humana [The role of the pig in the incidence of salmonella infection in the human population]. Zaragoza: University of Zaragoza; 2019.,¹⁶16. Ujueta Rodríguez S, Araque Marín A. Detección de residuos antimicrobianos en músculo, hígado y riñón de cerdo expendidos en Bogotá, Colombia [Detection of antimicrobial residues in muscle, liver and kidney of pigs sold in Bogotá, Colombia]. Revista U.D.C.A Actualidad & Divulgación Científica, 2016;19(2):371–9. doi: 10.31910/rudca.v19.n2.2016.91
https://doi.org/10.31910/rudca.v19.n2.20... ). Previous studies have provided baseline data on antimicrobial resistance patterns of Salmonella spp. in pigs in the country (⁵5. Oliván Iglesia R, Acín Tresaco C, Mainar Jaime RC. El papel del cerdo en la incidencia de la infección por salmonella en la población humana [The role of the pig in the incidence of salmonella infection in the human population]. Zaragoza: University of Zaragoza; 2019., ⁷7. Griffith RW, Carlson SA, Krull AC. Salmonellosis. In: Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW, Zhang J, editors. Diseases of swine, 11th edition. Hoboken, NJ: John Wiley & Sons, Inc.; 2019. doi: 10.1002/9781119350927
https://doi.org/10.1002/9781119350927... , ⁸8. Ayala-Romero C, Ballen-Parada C, Rico-Gaitan M, Chamorro-Tobar I, Zambrano-Moreno D, Poutou-Piñales R, et al. Prevalencia de Salmonella spp., En ganglios mesentéricos de porcinos en plantas de beneficio Colombianas [Prevalence of Salmonella spp., in mesenteric pig’s ganglia at Colombian benefit plants]. Rev MVZ Cordoba. 2018;23(1):6474–86. doi: 10.21897/rmvz.1242
https://doi.org/10.21897/rmvz.1242... ). However, data from fecal samples in live animals with suspected salmonellosis are limited (⁴4. Ferrari RG, Rosario DKA, Cunha-Neto A, Mano SB, Figueiredo EES, Conte-Junior CA. Worldwide epidemiology of Salmonella serovars in animal-based foods: a meta-analysis. Appl Environ Microbiol. 2019;85(14). doi: 10.1128/AEM.00591-19
https://doi.org/10.1128/AEM.00591-19... ). Furthermore, no studies have investigated the factors associated with MDR. In addition, with the exception of one study in four regions of Colombia (¹⁷17. Pulecio-Santos S, Bermúdez-Duarte P, Suárez-Alfonso MC. Susceptibilidad antimicrobiana de aislamientos de Salmonella entérica obtenidos del pre-beneficio y de porcinos en Colombia [Antimicrobial susceptibility of Salmonella enterica isolated during the pre-harvest period in swine in Colombia]. Rev Salud Pública (Bogata). 2015;17:106–19.), most of the previously published studies (⁸8. Ayala-Romero C, Ballen-Parada C, Rico-Gaitan M, Chamorro-Tobar I, Zambrano-Moreno D, Poutou-Piñales R, et al. Prevalencia de Salmonella spp., En ganglios mesentéricos de porcinos en plantas de beneficio Colombianas [Prevalence of Salmonella spp., in mesenteric pig’s ganglia at Colombian benefit plants]. Rev MVZ Cordoba. 2018;23(1):6474–86. doi: 10.21897/rmvz.1242
https://doi.org/10.21897/rmvz.1242... , ¹⁰10. Casana Rico C. El uso de antibióticos en la industria alimentaria y su contribución al desarrollo de resistencias. determinantes de la diseminación de la resistencia a la colistina [The use of antibiotics in the food industry and its contribution to the development of resistance. determinants of the spread of colistin resistance]. Madrid: Universidad Complutense; 2017.–¹²12. Bravo Aguilar MJ. Diagnóstico general de sistemas productivos porcinos en el municipio de Concepción (Antioquia) [General diagnosis of pig production systems in the municipality of Concepción (Antioquia)] [Dissertation]. Caldas: Unilasallista Corporación Universitaria; 2017.) were conducted in specific municipalities, with small sample sizes and limited generalizability (¹⁸18. Arias Ortiz LJ, Valencia Silva JH. Análisis de los planes de manejo sanitario en granjas porcícolas en el municipio de Donmatías, Antioquia [Analysis of sanitary management plans in pig farms in the municipality of Donmatías, Antioquia]. Fusagasugá: Universidad de Cundinamarca; 2019 [accessed 2021 Jul 22]. Available from: https://repositorio.ucundinamarca.edu.co/handle/20.500.12558/1848
https://repositorio.ucundinamarca.edu.co... ). Therefore, the aim of the present study was to: (i) determine the proportion of S. enterica in fecal samples of pigs with suspected salmonellosis which were analyzed at the veterinary diagnostic laboratory of the University of Antioquia, Colombia, between 2019 and 2021; and (ii) describe the serotypes and antibiotic resistance patterns.

METHODS

Study design, sample and period

This was a laboratory-based cross-sectional study based on the analysis of routine data from fecal samples of pigs with suspected salmonellosis which were received at the diagnostic unit of the Faculty of Agrarian Sciences at the University of Antioquia, Colombia from January 1, 2019 to June 30, 2021. Pigs with gastrointestinal symptoms such as diarrhea and other asymptomatic pigs in contact with them were considered animals with suspected salmonellosis.

Setting

Antioquia is one of the 32 departments (states) in Colombia with a population of 5.8 million; it is divided into nine sub-regions. Antioquia accounts for about one third of the pig population in the country (¹⁹19. Giraldo-Cardona JP, Gualdrón-Ramírez D, Chamorro-Tobar I, Pulido-Villamarín A, Santamaría-Durán N, Castañeda-Salazar R, et al. Salmonella spp. prevalence, antimicrobial resistance and risk factor determination in Colombian swine farms. Pesq Vet Bras. 2019;39(10):816–22. doi: 10.1590/1678-5150-pvb-6156.
https://doi.org/10.1590/1678-5150-pvb-61... , ²⁰20. Evaluaciones Agrícolas por consenso y UMATAS municipales. Medellín: Gobernación de Antioquia, Secretaría de Agricultura y Desarrollo Rural; 2017). This study was conducted in the diagnostic unit of the University of Antioquia which regularly receives fecal samples from all the sub-regions of the state.

Sample collection

Fecal samples of pigs with suspected salmonellosis are brought to the laboratory by the clients (farmers, veterinarians, or relevant associations). These samples could be from an individual animal or a pool of animals. Upon arrival at the laboratory, clients fill out a request form. Samples that have been adequately maintained with cold chain requirements (2–8 °C.) and are of a sufficient quantity (minimum of 25 g of feces) are investigated further following the sample acceptance standards of ISO/IEC 17025, 2017 (²¹21. ISO/IEC 17025. General requirements for the competence of testing and calibration laboratories. Geneva: International Organization for Standardization; 2017.).

Isolation, identification and serotyping

The basic laboratory procedure for the diagnosis of Salmonella spp. has five steps: pre-enrichment; selective enrichment; isolation in selective media; differential biochemical tests; and determination of the serotype (²²22. Microbiology of the food chain – horizontal method for the detection, enumeration and serotyping of Salmonella – Part 1: Detection of Salmonella spp EN ISO 6579-1. Geneva, International Organization for Standardization; 2017.). First, 10–25 g of the sample are transferred to 225 mL of buffered peptone water, shaken for 2 minutes, and incubated at 35 °C for 18–24 hours. For selective enrichment, the previously incubated sample together with 0.1 mL of the buffered peptone water are inoculated into 10 mL of Rappaport-Vassiliadis broth and incubated at 42 °C for 18–24 hours. After this incubation, the broth cultures are plated on Hektoen agar and xylose lysine deoxycholate agar and then incubated at 35 °C for 18–24 hours. Blue or blue-green colonies (with or without a black center) on Hektoen agar and clear to pinkish-red colonies (with or without a black center) on xylose lysine deoxycholate agar are considered presumptive of Salmonella spp. Colonies with typical Salmonella spp. characteristics on triple sugar agar and lysine iron agar are put through polymerase chain reaction (PCR) amplification of the invA gene (²³23. Ferretti R, Mannazzu I, Cocolin L, Comi G, Clementi F. Twelve-hour PCR-based method for detection of Salmonella spp. in food. Appl Environ Microbiol. 2001;67(2):977–8. doi: 10.1128/AEM.67.2.977-978.2001
https://doi.org/10.1128/AEM.67.2.977-978... ) and further biochemical characterization is done using the urease, indole, citrate, and motility tests (²⁴24. Carroll KC, Glanz BD, Borek AP, Burger C, Bhally HS, Henciak S, et al. Evaluation of the BD Phoenix automated microbiology system for identification and antimicrobial susceptibility testing of Enterobacteriaceae. J. Clin. Microbiol. 2006;44:3506–9. doi: 10.1128/JCM.00994-06.
https://doi.org/10.1128/JCM.00994-06... ). Serotypes are identified using the Kauffmann–White scheme for classification of somatic (O) and flagellar (H) antigen type (²²22. Microbiology of the food chain – horizontal method for the detection, enumeration and serotyping of Salmonella – Part 1: Detection of Salmonella spp EN ISO 6579-1. Geneva, International Organization for Standardization; 2017.).

Antimicrobial susceptibility testing

The minimum inhibitory concentrations (MIC) of Salmonella isolates are evaluated using a Phoenix automated microbiological system (BD) (²⁵25. Grimont PAD, Weill F-X. Antigenic formulae of the Salmonella serovars. Paris: WHO Collaborating Centre for Reference and Research on Salmonella; 2007.) using the NMIC/ID-406 panel for gram-negative bacteria. This panel includes the following 16 agents: amikacin, ampicillin, ampicillin–sulbactam, cefazolin, cefepime, cefoxitin, ceftazidime, ceftriaxone, ciprofloxacin, ertapenem, gentamicin, imipenem, meropenem, piperacillin–tazobactam, sulfamethoxazole–trimethoprim, and tigecycline. The MIC results, both of the antibiotics used for the antibiogram and those used for confirmation of extended-spectrum beta-lactamases (ESBL), are interpreted by the Phoenix expert system.

The MIC for the Salmonella isolates tested were interpreted as resistant, intermediate, or susceptible based on the guidelines of Clinical and Laboratory Standards Institute (²⁶26. Performance standards for antimicrobial susceptibility testing, 31st edition. Pittsburgh, PA: Clinical and Laboratory Standards Institute; 2021.). Escherichia coli ATCC 25922 and S. Enteritidis ATCC 13076 were used as quality control bacteria for each assay. Strains with MIC break-point results for ciprofloxacin that included ≤ 0.125 μg/mL were tested by 5 μg ciprofloxacin diffusion disk to confirm susceptibility results. This is needed because the ciprofloxacin breakpoint specific for Salmonella spp. in the guidelines of the Clinical and Laboratory Standards Institute is sensitive at ≤ 0.06 μg/mL and resistant at ≥ 1 μg/mL (²⁶26. Performance standards for antimicrobial susceptibility testing, 31st edition. Pittsburgh, PA: Clinical and Laboratory Standards Institute; 2021.), and the BD Phoenix^TM NMIC/ID 406 panel has a ciprofloxacin dilution range of 0.125–2 μg/mL (²⁵25. Grimont PAD, Weill F-X. Antigenic formulae of the Salmonella serovars. Paris: WHO Collaborating Centre for Reference and Research on Salmonella; 2007.). Because of this, results ≤ 0.125 μg/mL include values from sensitive (≤ 0.06 μg/mL) to intermediate (0.125 μg/mL).

The categorization of the antimicrobials followed the World Health Organization’s (WHO’s) list of critically important antimicrobials for human medicine (²⁷27. Critically important antimicrobials for human medicine, 6th revision 2018. Geneva: World Health Organization; 2019 [accessed 2021 Jul 22]. Available from: https://apps.who.int/iris/handle/10665/312266
https://apps.who.int/iris/handle/10665/3... ). Antimicrobials were classified as critically important in human medicine, highly important, and important.

Data management and statistical analysis

The variables included in the analysis were: age of the pigs; sub-region; serotype; and antibiotic susceptibility results (susceptible, intermediate, and resistant) for selected antibiotics. It was not possible to record if samples came from an individual animal or a pool of animals because this information was not provided by the clients. Data were extracted from the electronic laboratory database at the Faculty of Agrarian Sciences of the University of Antioquia and were analyzed using EpiData analysis software, version 2.2.2.187 (EpiData Association, Odense, Denmark). The proportion of S. enterica in the samples was calculated by dividing the number of samples positive for S. enterica by the total number of fecal samples processed. For the Salmonella-positive isolates, the number and proportion resistant to individual antimicrobials were calculated. Isolates with resistance to three or more classes of antibiotics were classified as MDR, as defined by a joint group of the European Centre for Disease Prevention and Control and the United States Centers for Disease Control and Prevention (²⁸28. Magiorakos A-P, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268–81. doi: 10.1111/j.1469-0691.2011.03570
https://doi.org/10.1111/j.1469-0691.2011... ). Associations between variables (such as age, sub-region, and serotype) and MDR were assessed using the chi-squared test. Prevalence ratios and 95% confidence intervals were calculated. A p-value of < 0.05 was considered statistically significant.

Ethics

Permission to access the laboratory data was obtained from the head of the Diagnostic Unit, University of Antioquia, respecting confidentiality policies of owner and farm data. Ethics approvals were obtained from the Institutional Committee on the Care and Use of Experimental Animals at the University of Antioquia (UdeA. 141/03-08-2021), the Ethics Advisory Group at the International Union against Tuberculosis and Lung Disease, Paris, France (EAG number: 23-21/02-09-2021), and the ethics committee of the Pan American Health Organization (PAHOERC.0383.01/05-08-2021). We obtained informed consent from the caretakers of the animals who submitted the specimens for their use for research purposes.

RESULTS

Proportion of S. enterica

In all, 653 samples were tested during the study period, and of these 149 (22.8%) were positive for S. enterica (Figure 1). The proportion of Salmonella isolates varied from 0.0% (Occidente) to 40.7% (Oriente) (Table 1).

Serotyping

We identified nine S. enterica serotypes: Typhimurium (83 samples), Typhimurium monophasic variant (S.1,4,12:i:–) (52), Enteritidis (7), Virchow (2), Bovismorbificans (1), Edinburg (1), Heidelberg (1), Infantis (1), and Manhattan (1). The most common serotype was S. Typhimurium (56%), followed by its monophasic variant (35%) (Figure 1).

Antimicrobial resistance of S. enterica serotypes

Of the 149 Salmonella isolates, 139 (93.3%) were tested for antimicrobial susceptibility (Figure 1): for the other 10 samples, it was not possible to obtain new growth of the strains from the first culture for antimicrobial susceptibility testing. Resistance to ampicillin (70% of isolates) was most frequently observed, followed by ciprofloxacin (55%), sulfamethoxazole–trimethoprim (52%), cefazolin (39%), ampicillin–sulbactam (35%), ceftazidime (29%), and cefepime and ceftriaxone (28% each). Resistance to piperacillin–tazobactam was also detected in 9% of isolates (Table 2).

A higher proportion of S. Typhimurium and S. Typhimurium monophasic variant showed resistance to antibiotics of all classes than the other serotypes (Table 3). Strains with MIC break-point results for ciprofloxacin that included ≤ 0.125 μg/mL were tested by 5 μg ciprofloxacin diffusion disk to confirm sensitivity. Of the 17 strains evaluated, five were confirmed as sensitive with a zone diameter ≥ 31 mm. Of the 139 isolates, 40 (29%) were positive for the production of ESBLs. Additionally, in one of the isolates, the presence of a positive ESBL test with resistance to meropenem, imipenem, and ertapenem was observed.

No isolates were resistant to amikacin and gentamicin, and only small proportions were resistant to cefoxitin, ertapenem, imipenem, meropenem, and tigecycline (Table 2).

Factors associated with MDR

Overall, MDR was observed in 61 (44%) isolates. The only variable associated with MDR was the serotype (p = 0.01). The serotype with the highest levels of MDR was S. Typhimurium (57%) (Table 4). The most common resistance profile in S. Typhimurium (20, 45%) was ampicillin–ampicillin/sulbactam–ceftriaxone–ciprofloxacin–sulfamethoxazole/trimethoprim. Age of the pig and sub-region were not associated with MDR.

DISCUSSION

This is the first study from Colombia to assess the proportion and resistance patterns in S. enterica isolated from fecal samples of live pigs with suspected salmonellosis. There were three key findings. First, about one in four samples was positive for S. enterica. with variations in proportion across the sub-regions of Antioquia Second, the most common serotypes were S. Typhimurium and its monophasic variant. Third, about 44% of the isolates showed MDR, with S. Typhimurium exhibiting significantly higher levels of MDR than the other serotypes.

The prevalence of S. enterica in our study (23%) was higher than that of a previous study in Colombia which reported 8% prevalence in fecal samples. The difference in the results can be explained by the fact that the previous study used fecal samples of healthy pigs (¹¹11. Giraldo-Cardona JP, Gualdrón-Ramírez D, Chamorro-Tobar I, Pulido-Villamarín A, Santamaría-Durán N, Castañeda-Salazar R, et al. Salmonella spp. prevalence, antimicrobial resistance and risk factor determination in Colombian swine farms. Pesq Vet Bras, 2019;39:816–22.). Other studies in Colombia have reported a prevalence of Salmonella spp. of 3% in pig carcasses, 13% in environmental samples from slaughterhouses, and 28% in samples of pig mesenteric ganglia (⁸8. Ayala-Romero C, Ballen-Parada C, Rico-Gaitan M, Chamorro-Tobar I, Zambrano-Moreno D, Poutou-Piñales R, et al. Prevalencia de Salmonella spp., En ganglios mesentéricos de porcinos en plantas de beneficio Colombianas [Prevalence of Salmonella spp., in mesenteric pig’s ganglia at Colombian benefit plants]. Rev MVZ Cordoba. 2018;23(1):6474–86. doi: 10.21897/rmvz.1242
https://doi.org/10.21897/rmvz.1242... ).

Our estimates are similar to those of the European Food Safety Authority in 2008 for Greece, Luxemburg, Portugal, and Spain (²⁹29. Report of the task force on zoonoses data collection on the analysis of the baseline survey on the prevalence of Salmonella in slaughter pigs, in the EU, 2006–2007. Part A: Salmonella prevalence estimates. EFSA J. 2008;136:1–111), which reported the prevalence to be between 25% and 30%. A lower prevalence was found in other countries such as Austria, Estonia, Finland, Norway, Poland, and Slovakia where the prevalence was about 5%. These variations might be related to stringent biosecurity standards and better farming practices in these countries (²⁹29. Report of the task force on zoonoses data collection on the analysis of the baseline survey on the prevalence of Salmonella in slaughter pigs, in the EU, 2006–2007. Part A: Salmonella prevalence estimates. EFSA J. 2008;136:1–111). Of note, differences in the results of the prevalence of Salmonella spp. could be due to other factors such as the site of sample collection (farms versus slaughterhouses) and the type of samples (fecal versus non-fecal) (³⁰30. Jensen AN, Dalsgaard A, Stockmarr A, Nielsen EM, Baggesen DL. Survival and transmission of Salmonella enterica serovar Typhimurium in an outdoor organic pig farming environment. Appl Environ Microbiol. 2006;72(3):1833–42. doi: 10.1128/AEM.72.3.1833-1842.2006
https://doi.org/10.1128/AEM.72.3.1833-18... ).

The sub-region with the highest prevalence of Salmonella spp. in Antioquia was Oriente (40%). This finding might be related to suboptimal farming practices and environmental problems with the management of solid waste reported in this region (¹²12. Bravo Aguilar MJ. Diagnóstico general de sistemas productivos porcinos en el municipio de Concepción (Antioquia) [General diagnosis of pig production systems in the municipality of Concepción (Antioquia)] [Dissertation]. Caldas: Unilasallista Corporación Universitaria; 2017., ³¹31. Ostanello F, De De Lucia A. On-farm risk factors associated with Salmonella in pig herds. Large Anim Rev. 2020;26(3):133–40.). The variation in the prevalence across sub-regions could be attributed to the production system and management practices of farms. Sub-regions with lower proportions of Salmonella spp., such as Nordeste (13%), Valle de Aburra (21%), and Norte (21%), have intensive pig production systems and better implementation of good farming practices, such as farm sanitation, hygienic feeding practices, herd management, and health management (²⁰20. Evaluaciones Agrícolas por consenso y UMATAS municipales. Medellín: Gobernación de Antioquia, Secretaría de Agricultura y Desarrollo Rural; 2017, ³¹31. Ostanello F, De De Lucia A. On-farm risk factors associated with Salmonella in pig herds. Large Anim Rev. 2020;26(3):133–40.).

S. Typhimurium and its monophasic variant were the predominant serotypes and accounted for about 90% of all isolates. This finding is similar to the findings in other countries of the European Union, Brazil, China, Japan, and the United States (⁴4. Ferrari RG, Rosario DKA, Cunha-Neto A, Mano SB, Figueiredo EES, Conte-Junior CA. Worldwide epidemiology of Salmonella serovars in animal-based foods: a meta-analysis. Appl Environ Microbiol. 2019;85(14). doi: 10.1128/AEM.00591-19
https://doi.org/10.1128/AEM.00591-19... , ³²32. Guerra Filho JB, Yamatogi RS, Possebon FS, Fernandes SA, Tiba-Casas MR, Lara GH., .et al. Frequency, serotyping and antimicrobial resistance pattern of Salmonella from feces and lymph nodes of pigs. Pesq Vet Bras. 2016;36:1165–70., ³³33. Su JH, Zhu YH, Ren TY, Guo L, Yang GY, Jiao LG, Wang JF. Distribution and antimicrobial resistance of Salmonella isolated from pigs with diarrhea in China. Microorganisms. 2018;6(4):117. doi: 10.3390/microorganisms6040117
https://doi.org/10.3390/microorganisms60... ). Although studies from other parts of the world also report Derby as another common serotype (³⁴34. Bearson SM. Salmonella in swine: prevalence, multidrug resistance, and vaccination strategies. Annu Rev Anim Biosci. 2022;10:373–93. doi: 10.1146/annurev-animal-013120-043304
https://doi.org/10.1146/annurev-animal-0... ), we did not find this serotype in our study, nor did we detect S. Choleraesuis.

Our research showed that Salmonella spp. exhibited high levels of resistance to the most commonly prescribed antibiotics for the treatment of swine salmonellosis, such as ampicillin (70%), ciprofloxacin (55%), and sulfamethoxazole–trimethoprim (52%) (⁸8. Ayala-Romero C, Ballen-Parada C, Rico-Gaitan M, Chamorro-Tobar I, Zambrano-Moreno D, Poutou-Piñales R, et al. Prevalencia de Salmonella spp., En ganglios mesentéricos de porcinos en plantas de beneficio Colombianas [Prevalence of Salmonella spp., in mesenteric pig’s ganglia at Colombian benefit plants]. Rev MVZ Cordoba. 2018;23(1):6474–86. doi: 10.21897/rmvz.1242
https://doi.org/10.21897/rmvz.1242... ). Our values were higher than those of a recent Colombian study (¹⁷17. Pulecio-Santos S, Bermúdez-Duarte P, Suárez-Alfonso MC. Susceptibilidad antimicrobiana de aislamientos de Salmonella entérica obtenidos del pre-beneficio y de porcinos en Colombia [Antimicrobial susceptibility of Salmonella enterica isolated during the pre-harvest period in swine in Colombia]. Rev Salud Pública (Bogata). 2015;17:106–19.), but similar those reported in China (³³33. Su JH, Zhu YH, Ren TY, Guo L, Yang GY, Jiao LG, Wang JF. Distribution and antimicrobial resistance of Salmonella isolated from pigs with diarrhea in China. Microorganisms. 2018;6(4):117. doi: 10.3390/microorganisms6040117
https://doi.org/10.3390/microorganisms60... ). Chloramphenicol, ciprofloxacin, sulfamethoxazole–trimethoprim, and ceftriaxone are the first choices for treating salmonellosis in humans and are designated by WHO as critically and highly important for human medicine (²⁷27. Critically important antimicrobials for human medicine, 6th revision 2018. Geneva: World Health Organization; 2019 [accessed 2021 Jul 22]. Available from: https://apps.who.int/iris/handle/10665/312266
https://apps.who.int/iris/handle/10665/3... ). Brazil, Korea, and Spain have observed similar resistance patterns with percentages of 80%, 94%, and 70%, respectively (³²32. Guerra Filho JB, Yamatogi RS, Possebon FS, Fernandes SA, Tiba-Casas MR, Lara GH., .et al. Frequency, serotyping and antimicrobial resistance pattern of Salmonella from feces and lymph nodes of pigs. Pesq Vet Bras. 2016;36:1165–70., ³⁴34. Bearson SM. Salmonella in swine: prevalence, multidrug resistance, and vaccination strategies. Annu Rev Anim Biosci. 2022;10:373–93. doi: 10.1146/annurev-animal-013120-043304
https://doi.org/10.1146/annurev-animal-0... ). Of concern in our study was the high prevalence of MDR (44%) – much higher than a previous study in Colombia which reported MDR of 12% (¹¹11. Giraldo-Cardona JP, Gualdrón-Ramírez D, Chamorro-Tobar I, Pulido-Villamarín A, Santamaría-Durán N, Castañeda-Salazar R, et al. Salmonella spp. prevalence, antimicrobial resistance and risk factor determination in Colombian swine farms. Pesq Vet Bras, 2019;39:816–22.). MDR was significantly higher in S. Typhimurium than the other serotypes, similar to reports from Brazil and China (³²32. Guerra Filho JB, Yamatogi RS, Possebon FS, Fernandes SA, Tiba-Casas MR, Lara GH., .et al. Frequency, serotyping and antimicrobial resistance pattern of Salmonella from feces and lymph nodes of pigs. Pesq Vet Bras. 2016;36:1165–70., ³³33. Su JH, Zhu YH, Ren TY, Guo L, Yang GY, Jiao LG, Wang JF. Distribution and antimicrobial resistance of Salmonella isolated from pigs with diarrhea in China. Microorganisms. 2018;6(4):117. doi: 10.3390/microorganisms6040117
https://doi.org/10.3390/microorganisms60... ). These trends could be a consequence of unregulated antimicrobial use in pig production (¹¹11. Giraldo-Cardona JP, Gualdrón-Ramírez D, Chamorro-Tobar I, Pulido-Villamarín A, Santamaría-Durán N, Castañeda-Salazar R, et al. Salmonella spp. prevalence, antimicrobial resistance and risk factor determination in Colombian swine farms. Pesq Vet Bras, 2019;39:816–22.), but further investigation on the levels of antimicrobial use in pig farming is needed to evaluate this hypothesis. Salmonella spp. with MDR patterns is a risk to public health as these serotypes can be transmitted to people by different routes such as: indirect transmission through waste management; direct animal–human transmission; or indirect human–food transmission (³⁵35. Mendelson M, Matsoso MP. The World Health Organization global action plan for antimicrobial resistance. S Afr Med J. 2015;105(5):325. doi: 10.7196/samj.9644
https://doi.org/10.7196/samj.9644... ). Such MDR patterns thus complicate therapeutic management of Salmonella spp. infection in humans.

The finding that one of the isolates positive for ESBL was resistant to meropenem, imipenem, and ertapenem could mean the presence of an ESBL with porin closure or a carbapenemase. This finding requires further investigation.

A strength of our study was that we used fecal samples from pigs with suspected salmonellosis, rather than healthy pigs as in previous Colombian studies, which adds to the limited evidence on this topic. In addition, all tests were performed in an ISO 17025 and quality-assured laboratory, and we followed the guidelines of the Clinical and Laboratory Standards Institute for antimicrobial susceptibility testing and interpretation. As such, we aimed to ensure that all our laboratory results were valid and reliable. Another strength was that we had a larger sample than previous studies in Colombia (⁸8. Ayala-Romero C, Ballen-Parada C, Rico-Gaitan M, Chamorro-Tobar I, Zambrano-Moreno D, Poutou-Piñales R, et al. Prevalencia de Salmonella spp., En ganglios mesentéricos de porcinos en plantas de beneficio Colombianas [Prevalence of Salmonella spp., in mesenteric pig’s ganglia at Colombian benefit plants]. Rev MVZ Cordoba. 2018;23(1):6474–86. doi: 10.21897/rmvz.1242
https://doi.org/10.21897/rmvz.1242... , ¹⁰10. Casana Rico C. El uso de antibióticos en la industria alimentaria y su contribución al desarrollo de resistencias. determinantes de la diseminación de la resistencia a la colistina [The use of antibiotics in the food industry and its contribution to the development of resistance. determinants of the spread of colistin resistance]. Madrid: Universidad Complutense; 2017., ¹¹11. Giraldo-Cardona JP, Gualdrón-Ramírez D, Chamorro-Tobar I, Pulido-Villamarín A, Santamaría-Durán N, Castañeda-Salazar R, et al. Salmonella spp. prevalence, antimicrobial resistance and risk factor determination in Colombian swine farms. Pesq Vet Bras, 2019;39:816–22.) and we included samples from all nine Antioquia sub-regions. As a result, we believe the findings are representative of Salmonella spp. infection in pigs in Antioquia.

Our study also had some limitations. The laboratory database did not provide information on several important variables including animal symptoms (whether symptomatic or asymptomatic contacts), type of sample (individual or pooled sample), farm management procedures, and antibiotics used. These variables would have allowed for a more comprehensive and in-depth analysis, and might have provided insights into the reasons for the high prevalence of Salmonella spp. and MDR in Antioquia. In addition, we did not assess tetracycline resistance. Tetracycline resistance has been reported to be high in Salmonella spp. isolated from pigs and its absence from our analysis may have underestimated overall MDR levels (³³33. Su JH, Zhu YH, Ren TY, Guo L, Yang GY, Jiao LG, Wang JF. Distribution and antimicrobial resistance of Salmonella isolated from pigs with diarrhea in China. Microorganisms. 2018;6(4):117. doi: 10.3390/microorganisms6040117
https://doi.org/10.3390/microorganisms60... ).

Despite these limitations, our findings have some important implications. First, the recording system in the laboratory of the University of Antioquia needs to be strengthened to capture all the key variables discussed before. Second, barring evidence from studies such as ours, no surveillance system is in place for Salmonella spp. in pigs in Colombia. Given the importance of the growing pig industry, the high consumption of pork by the people of Colombia, and the potential public health implications, we recommend that a surveillance system be instituted with sentinel sites (at farms and slaughterhouses) established across the country. Such a measure will help to capture nationally representative data and monitor the trends in prevalence and resistance levels of Salmonella spp. Until such a surveillance system becomes a reality, we need more nationally representative studies to monitor the prevalence of Salmonella spp., MDR levels and the practices in the pig production chain (farms, slaughterhouses, storage places, distribution points, and retail outlets) in Colombia. Although fragmented, such information would help to identify the sources of Salmonella spp. at each production stage. It would also help to guide interventions to reduce the prevalence of salmonellosis in pigs and the risk to human consumers. Finally, current methods to detect resistance are phenotypic. For the detection of resistance genes, we recommend using molecular tools such as whole genome sequencing and PCR. This information will aid our understanding of the mechanisms underlying antimicrobial resistance in MDR isolates.

The high levels of MDR could be a consequence of indiscriminate use of antibiotics in pig farms. This possibility calls for strategies to understand and regulate antimicrobial use in animal production in Colombia. These measures could include: (i) education of pig farmers and other workers involved in the pig production chain about good husbandry practices, sanitation, disinfection, and the rational use of antibiotics; and (ii) education of veterinarians to be prudent in prescribing antibiotics. According to the guidelines of WHO’s global action plan on antimicrobial resistance, a One Health approach with a coordinated response of all sectors (human, animal, and environment) is required (³⁵35. Mendelson M, Matsoso MP. The World Health Organization global action plan for antimicrobial resistance. S Afr Med J. 2015;105(5):325. doi: 10.7196/samj.9644
https://doi.org/10.7196/samj.9644... ).

In conclusion, we found a high proportion of Salmonella spp. and high levels of MDR in fecal samples of live pigs with suspected salmonellosis in Antioquia, Colombia. These findings deserve urgent attention and action from both the animal health and public health sectors. Our recommendation to develop a national surveillance system to monitor the prevalence of Salmonella spp. and antimicrobial resistance levels would enable identification of the sources of Salmonella spp. at the different stages of pig production and provide evidence to improve the regulation of antimicrobial use in the animal sector.

Disclaimer.

The authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the Revista Panamericana de Salud Pública / Pan American Journal of Public Health and/or those of the Pan American Health Organization.

Acknowledgements.

This research protocol was developed through the Structured Operational Research and Training Initiative (SORT IT), a global partnership coordinated by the WHO Special Programme for Research and Training in Tropical Diseases (TDR), United Nations Children’s Fund, United Nations Development Programme, and the World Bank. The specific SORT IT program that led to this study protocol included an implementation partnership of: TDR and the Pan American Health Organization (PAHO); WHO country offices of Colombia and Ecuador; Ministry of Health and Social Protection, Colombia; Food and Agriculture Organization, Freetown, Sierra Leone; Sustainable Health Systems, Freetown, Sierra Leone; Tuberculosis Research and Prevention Center Non-Governmental Organization, Yerevan, Armenia; International Union Against Tuberculosis and Lung Diseases, Paris, France and South-East Asia offices, India; Institute of Tropical Medicine, Antwerp, Belgium; Damien Foundation, Brussels, Belgium; Indian Council of Medical Research, National Institute of Epidemiology, New Delhi, India; Jawaharlal Institute of Post Graduate Medical Education, Pondicherry, India; GMERS Medical College Gotri, Vadodara, Gujarat, India; India Medical College Baroda, Gujarat, India; Sri Manakula Vinayagar Medical College, Madagadipet, India; Public Health, Ontario, Canada; Quadram Institute Bioscience, Norwich, United Kingdom; Universidade Federal de Ciencias de Saude de Porto Alegre, Porto Alegre, Brazil; Universidade de Brasilia, Brasilia, Brazil; Universidad de Concepcion, Concepcion, Chile, Universidad de los Andes, Bogata, Colombia; Universidad Pontificia Bolivariana, Tunja, Colombia; Universidad Central del Ecuador, Quito, Ecuador; and Universidad Autónoma de Yucatán, Merida, Mexico.

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