ABSTRACT

Objective.

To describe antimicrobial resistance profiles of Escherichia coli and Salmonella spp. isolated from chicken carcasses and the antimicrobials commonly used in animals in Ecuador and provide information on antimicrobial resistance patterns for implementing evidence-based corrective measures.

Methods.

Meat samples were collected from chicken carcasses in 199 slaughterhouses across Ecuador as part of a national pilot study for monitoring antimicrobial resistance in agricultural sources in 2019. Samples were tested for E. coli and Salmonella spp. Sensitivity to 10 critically important and three highly important antimicrobials (from a human health perspective) was assessed. The country report submitted to the World Organization for Animal Health was accessed to extract the quantity of antimicrobials produced or imported for use in animals.

Results.

Of 383 samples, E. coli was isolated from 148 (39%) and Salmonella spp. from 20 (5%) samples. Ninety percent of the isolates were resistant to at least one critically important antimicrobial. Resistance was highest to erythromycin (E. coli 76%; Salmonella spp. 85%) and tetracycline (E. coli 71%; Salmonella spp. 90%). Critically or highly important antimicrobials (colistin, tetracycline, trimethoprim/sulfamethoxazole) formed the bulk (87%) of antimicrobials used in animals as per the World Organization for Animal Health report.

Conclusions.

High prevalence of antimicrobial resistance in poultry in Ecuador calls for the development of guidelines and regulations on the use of antimicrobials and for engagement with livestock producers. The existing surveillance system needs to be strengthened to improve the monitoring of antimicrobial use and evolving resistance patterns.

Keywords
Microbial sensitivity tests; drug resistance, microbial; beta-lactamases; poultry; operations research; Ecuador

RESUMEN

Objetivo.

Describir los perfiles de resistencia antimicrobiana de las bacterias Escherichia coli y Salmonella spp. aisladas en carne de pollo y los antimicrobianos comúnmente empleados en animales en Ecuador, así como proporcionar información sobre los patrones de resistencia a los antimicrobianos para poner en marcha medidas correctivas basadas en la evidencia.

Métodos.

Se recogieron muestras de carne de pollo en 199 mataderos de todo Ecuador en el marco de un estudio piloto nacional para monitorear la resistencia a los antimicrobianos en fuentes agrícolas en el 2019. Se analizaron las muestras en busca de E. coli y Salmonella spp. Se evaluó la sensibilidad a diez antimicrobianos de importancia crítica y tres muy importantes (para la salud humana). Se accedió al informe de país presentado ante la Organización Mundial de Sanidad Animal para obtener la cantidad de antimicrobianos producidos o importados para su uso en animales.

Resultados.

De 383 muestras, se aisló E. coli en 148 (39%) y Salmonella spp. en 20 (5%). En total, 90% de las cepas aisladas fueron resistentes a al menos un antimicrobiano de importancia crítica. Hubo una mayor resistencia a la eritromicina (E. coli: 76%; Salmonella spp.: 85%) y a la tetraciclina (E. coli: 71%; Salmonella spp.: 90%). Los antimicrobianos de importancia crítica o muy importantes (colistina, tetraciclina, trimetoprima/sulfametoxazol) constituyeron la mayor parte (87%) de los antimicrobianos empleados en animales según el informe de la Organización Mundial de Sanidad Animal.

Conclusiones.

Debido a la alta prevalencia de la resistencia a los antimicrobianos en las aves de corral en Ecuador, son imprescindibles la elaboración de directrices y regulaciones sobre el uso de antimicrobianos y el compromiso con los productores pecuarios. Es necesario fortalecer el sistema de vigilancia existente para mejorar el seguimiento del uso de antimicrobianos y de la evolución de los patrones de resistencia.

Palabras clave
Pruebas de sensibilidad microbiana; farmacorresistencia microbiana; beta-lactamasas; aves de corral; investigación operativa; Ecuador

RESUMO

Objetivo.

Descrever perfis de resistência aos antimicrobianos em Escherichia coli e Salmonella spp. isoladas de carcaças de frango e os antimicrobianos comumente usados em animais no Equador e fornecer informações sobre padrões de resistência aos antimicrobianos para implementar medidas corretivas baseadas em evidências.

Métodos.

Foram coletadas amostras de carne de carcaças de frango em 199 abatedouros em todo o Equador como parte de um estudo piloto nacional para monitorar a resistência aos antimicrobianos de origem agrícola em 2019. Foram testadas amostras de E. coli e Salmonella spp. Foi avaliada a sensibilidade a 10 agentes antimicrobianos de importância crítica e três agentes antimicrobianos muito importantes (do ponto de vista da saúde humana). O relatório do país apresentado à Organização Mundial de Saúde Animal foi acessado para extrair a quantidade de antimicrobianos produzidos ou importados para uso em animais.

Resultados.

De 383 amostras, E. coli foi isolada em 148 (39%) e Salmonella spp. em 20 (5%). Noventa por cento dos isolados foram resistentes a pelo menos um antimicrobiano de importância crítica. A resistência foi maior à eritromicina (E. coli, 76%; Salmonella spp., 85%) e à tetraciclina (E. coli, 71%; Salmonella spp., 90%). Antimicrobianos de importância crítica ou muito importantes (colistina, tetraciclina, trimetoprim/sulfametoxazol) responderam pela maior parte (87%) dos antimicrobianos utilizados em animais, conforme o relatório da Organização Mundial de Saúde Animal.

Conclusões.

A alta prevalência de resistência aos antimicrobianos na avicultura no Equador exige o desenvolvimento de diretrizes e regulamentos sobre o uso de antimicrobianos e o envolvimento com os produtores de gado e avícolas. O sistema de vigilância existente precisa ser reforçado para melhorar o monitoramento do uso de antimicrobianos e a evolução dos padrões de resistência.

Palavras-chave
Testes de sensibilidade microbiana; resistência microbiana a medicamentos; beta-lactamases; aves domésticas; pesquisa operacional; Equador

The World Health Organization (WHO) recognizes antimicrobial resistance (AMR) as one of the most important threats to health in the 21st century, citing the misuse and overuse of antimicrobial products in human and veterinary medicine and food production as primary drivers of drug resistance globally (¹1. World Health Organization. WHO integrated global surveillance on ESBL-producing E. coli using a “One Health” approach: implementation and opportunities. Geneva: WHO; 2021. Available from: https://www.who.int/publications/i/item/who-integrated-global-surveillance-on-esbl-producing-e.-coli-using-a-one-health-approach
https://www.who.int/publications/i/item/... ). Estimates of the number of antibiotics used in agriculture worldwide range from 63 000 to 240 000 tons per year; the considerable uncertainty in these estimates is mainly due to poor surveillance and data collection in many countries (²2. Organización de la Naciones Unidas para la Alimentación y la Agricultura. Concienciación y abogacía para la contención de la resistencia a los antimicrobianos. Rome: FAO; 2018.).

Poultry farms are considered major contributors to AMR because of their extensive use of antimicrobial products, which are used as both growth promoters and prophylactic treatment against bacterial infections in the birds as they are raised (³3. Nhung NT, Chansiripornchai N, Carrique-Mas JJ. Antimicrobial resistance in bacterial poultry pathogens: A review. Front Vet Sci. 2017 Aug 10;4(AUG):126.). Indeed, the presence of antibiotic residues and resistant bacteria in animal urine and stool often combine with farms’ suboptimal waste management and the use of fecal matter as fertilizer to amplify the spread of resistant genes and the selection of resistant bacteria in the environment (¹1. World Health Organization. WHO integrated global surveillance on ESBL-producing E. coli using a “One Health” approach: implementation and opportunities. Geneva: WHO; 2021. Available from: https://www.who.int/publications/i/item/who-integrated-global-surveillance-on-esbl-producing-e.-coli-using-a-one-health-approach
https://www.who.int/publications/i/item/... ). WHO recommends testing for Escherichia coli and Salmonella spp. in chicken samples as part of the integrated AMR surveillance in poultry farms initiative (¹1. World Health Organization. WHO integrated global surveillance on ESBL-producing E. coli using a “One Health” approach: implementation and opportunities. Geneva: WHO; 2021. Available from: https://www.who.int/publications/i/item/who-integrated-global-surveillance-on-esbl-producing-e.-coli-using-a-one-health-approach
https://www.who.int/publications/i/item/... ).

In Latin America, there are several reports on AMR among animals for human consumption; however, the interaction between AMR and antimicrobial use in food production has not been well documented (⁴4. Resistancebank.org [Internet]. Zurich: ETH Zürich; 2019 [cited 2021 Aug 28]. Available from: https://resistancebank.org/
Resistancebank.org... ). Nevertheless, a series of small studies looking into various animal production systems in Ecuador have reported colistin- and multidrug-resistant E. coli, Salmonella spp., and Campylobacter (⁵5. Vinueza-Burgos C, Baquero M, Medina J, De Zutter L. Occurrence, genotypes and antimicrobial susceptibility of Salmonella collected from the broiler production chain within an integrated poultry company. Int J Food Microbiol. 2019;299:1–7. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0168160518304689?via%3Dihub
https://www.sciencedirect.com/science/ar... –⁷7. Sánchez-Salazar E, Gudiño ME, Sevillano G, Zurita J, Guerrero-López R, Jaramillo K, et al. Antibiotic resistance of Salmonella strains from layer poultry farms in central Ecuador. J Appl Microbiol. 2020 May 1;128(5):1347–54.); and multidrug-resistant strains of E. coli have also been found in river water in the capital city of Quito, which possibly indicates that epidemiologically important resistance is being transmitted at sites where humans and animals interact with the environment (⁸8. Ortega-Paredes D, Barba P, Mena-López S, Espinel N, Crespo V, Zurita J. High quantities of multidrug-resistant Escherichia coli are present in the Machángara urban river in Quito, Ecuador. J Water Health. 2020 Feb 1;18(1):67–76. https://doi.org/10.2166/wh.2019.195
https://doi.org/10.2166/wh.2019.195... ).

In response to the rising AMR threat, Ecuador has taken significant steps to address the problem: a five-year national plan to prevent and control AMR was established by ministerial decree in 2019. Key objectives of the decree were to design and implement a national surveillance system for AMR in all areas (human, animal, and environment) and to monitor antimicrobial use in animals (⁹9. Ministerio de Salud Pública del Ecuador. Plan nacional para la prevención y control de la resistencia a antimicrobiana [Internet]. Quito: MSP; 2019. Available from: https://www.salud.gob.ec/wp-content/uploads/2019/10/Plan-Nacional-para-la-prevención-y-control-de-la-resistencia-antimicrobiana_2019_compressed.pdf
https://www.salud.gob.ec/wp-content/uplo... ). The Ecuadorian Ministry of Agriculture and Livestock also prohibited the production, importation, trade, and use of products containing colistin in animals in 2019 (¹⁰10. Agencia de Regulacion y Control Fito y Zoosanitario Ecuador. Resolution 0003. Quito: Agrocalidad; 2019.).

In Ecuador, chicken is widely consumed and the population’s average poultry consumption per capita is high (26 kg per year) compared to the world average (16 kg per year) (¹¹11. Corporación Nacional de Avicultores del Ecuador [Internet]. Quito: CONAVE; 2019 [cited 2021 Jul 23]. Información Sector Avícola (Público). ESTADÍSTICAS DEL SECTOR AVÍCOLA. Available from: https://www.conave.org/informacion-sector-avicola-publico/.
https://www.conave.org/informacion-secto... , ¹²12. Helgi Library [Internet]. Prague: Helgi Analytics; 2021 [cited 2021 Jul 23]. Poultry meat consumption per capita. Available from: https://www.helgilibrary.com/indicators/poultry-meat-consumption-per-capita/.
https://www.helgilibrary.com/indicators/... ). The massive scale of the poultry industry in Ecuador makes it a potentially significant source of AMR. Although several studies have shown that poultry-related AMR may be important in Ecuador, the extent of the problem on a national level is unknown (⁶6. Ortega-Paredes D, de Janon S, Villavicencio F, Ruales KJ, De La Torre K, Villacís JE, et al. Broiler Farms and Carcasses Are an Important Reservoir of Multi-Drug Resistant Escherichia coli in Ecuador. Front Vet Sci. 2020 Nov 25;7:547843., ⁷7. Sánchez-Salazar E, Gudiño ME, Sevillano G, Zurita J, Guerrero-López R, Jaramillo K, et al. Antibiotic resistance of Salmonella strains from layer poultry farms in central Ecuador. J Appl Microbiol. 2020 May 1;128(5):1347–54., ¹³13. Mejía L, Medina JL, Bayas R, Salazar CS, Villavicencio F, Zapata S, et al. Genomic Epidemiology of Salmonella Infantis in Ecuador: From Poultry Farms to Human Infections. Front Vet Sci. 2020 Sep 29;7:547891. https://doi.org/10.3389/fvets.2020.547891
https://doi.org/10.3389/fvets.2020.54789... ).

To address this gap, the Ministry of Agriculture and Livestock conducted a study of bacteria isolated from poultry carcasses in a nationwide sample of slaughterhouses in Ecuador in 2019. The aim was to describe AMR profiles of E. coli and Salmonella spp. isolated from chicken carcasses and the antimicrobials commonly used in animals in Ecuador.

This study provides baseline data on antimicrobial use and resistance in animal production systems to inform surveillance activities and animal health policies and programs in Ecuador and the wider Latin American region.

MATERIALS AND METHODS

Study design and setting

This is a cross-sectional study using data collected by Agrocalidad, the regulatory and control agency for animal and plant health in Ecuador.¹1The dataset used in this article can be made available, on request, by the corresponding author.

Ecuador has a population of approximately 17 million and is divided into 24 provinces, which can be grouped under three regions: the Amazon basin, the Andean highlands, and the coastal region. Poultry production is one of Ecuador’s most important industries: in 2019, all farms together produced around 281 million chickens. The industry is concentrated in the provinces of Guayas, Santo Domingo, and Pichincha and supplies nearly the entire national demand (¹¹11. Corporación Nacional de Avicultores del Ecuador [Internet]. Quito: CONAVE; 2019 [cited 2021 Jul 23]. Información Sector Avícola (Público). ESTADÍSTICAS DEL SECTOR AVÍCOLA. Available from: https://www.conave.org/informacion-sector-avicola-publico/.
https://www.conave.org/informacion-secto... ). There are 546 registered public and private slaughterhouses in the country. Slaughterhouses are regulated by Agrocalidad, which is part of the Ministry of Agriculture and Livestock, one of the three ministries involved in the implementation of the National Plan for the Prevention and Control of AMR (⁹9. Ministerio de Salud Pública del Ecuador. Plan nacional para la prevención y control de la resistencia a antimicrobiana [Internet]. Quito: MSP; 2019. Available from: https://www.salud.gob.ec/wp-content/uploads/2019/10/Plan-Nacional-para-la-prevención-y-control-de-la-resistencia-antimicrobiana_2019_compressed.pdf
https://www.salud.gob.ec/wp-content/uplo... ). Since 2019, antimicrobial use in animals has been monitored and reported to the World Organization for Animal Health (WOAH).

Study period

The samples from the slaughterhouses were collected between 14 May and 28 November 2019. Each slaughterhouse was visited once. Laboratory investigations were carried out over the course of 2019 and 2020. Data on antimicrobials produced or imported for use in animals in 2019 were collected in 2020.

Study population and sampling

Raw chicken meat samples were obtained from healthy animals that had just been slaughtered, before the meat was processed. At the time the pilot study was designed, the frequency of E. coli and Salmonella spp. in such samples was unknown at a national level. For the sample size calculation, the anticipated prevalence of meat samples with bacterial isolates was set at 50% and the desired absolute precision at 5%. This yielded a target sample size of 384 poultry carcasses.

Probability proportionate to size was used to select slaughterhouses and samples to be included. First, the provinces of Ecuador were ranked according to the number of chickens slaughtered per month in 2018. The target number of slaughterhouses and samples to be included per province was determined so that it was proportional to the number of chickens slaughtered. Next, provincial food safety technical officers organized the sampling as part of their routine monitoring visits to slaughterhouses until they reached the target number of samples for their province.

E. coli and Salmonella spp. isolation and antimicrobial susceptibility testing

All microbiological analyses for this study were done at one laboratory in Quito. This central laboratory is part of the Ministry of Agriculture and Livestock (accreditation ISO/IEC 17025 and 9001). The samples were accompanied by certificates containing information about origin, storage, and transport conditions according to the recommendations of the Ecuadorian technical standards (¹⁴14. Instituto Ecuatoriano de Normalización. Control microbiológico de los alimentos: Toma, envío y preparación de muestras para el análisis microbiológico. NTE INEN 1529-2 2013 [Internet]. Quito: INEN;2013:7–12. Available from: https://www.normalizacion.gob.ec/buzon/normas/1529-2-1R.pdf
https://www.normalizacion.gob.ec/buzon/n... ). If any irregularities were detected by the technician at the central laboratory, the corresponding sample was excluded.

The included samples were processed and cultured on selective media following standard operating procedures. The colonies obtained were first examined using a Gram stain; next, the presence of E. coli and Salmonella spp. was confirmed through biochemical testing. For antimicrobial susceptibility testing (AST), the Kirby Bauer method, double disk test (for confirmation of extended-spectrum beta-lactamase-producing strains), and the broth microdilution test (for colistin) were used. AST was interpreted in line with the Clinical and Laboratory Standards Institute (CLSI) Guidelines (¹⁵15. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. Berwyn, PA: CLSI; 2022.).

AST was conducted for 13 antibiotics: gentamicin, chloramphenicol, ceftriaxone, erythromycin, ampicillin, amoxicillin, amoxicillin/clavulanic acid, ampicillin/sulbactam, ciprofloxacin, trimethoprim/sulfamethoxazole, meropenem, tetracycline, and colistin. These drugs were chosen because are part of the National Plan for Surveillance and Control of Contaminants in Primary Production (¹⁶16. Minsterio de Agricultura, Ganadería, Acuacultura y Pesca. Plan Nacional de Vigilancia y Control de Contaminantes en la Producción Primaria [National Plan for Surveillance and Control of Contaminants in Primary Production] [Internet]. Quito: MAGAP; 2017. Available from: https://www.agrocalidad.gob.ec/wp-content/uploads/2020/05/plan-nacional-vigilancia-control-contaminantes-produccion-primaria-064.pdf
https://www.agrocalidad.gob.ec/wp-conten... ).

Data on antimicrobial use in animals

Agrocalidad regulates the registration, control, marketing, and use of veterinary products. Via an online platform, manufacturers and distributors declare product name, class, and amount in kg of antimicrobial agents used in animals for human consumption according to type of use: veterinary medical use (including prevention), growth promotion, or both (¹⁷17. Agencia de Regulación y Control Fito y Zoosanitario. MANUAL PARA EL REGISTRO DE EMPRESAS Y PRODUCTOS DE USO VETERINARIO. Quito: Agrocalidad; 2021.). For the purpose of this study, we obtained the number of antimicrobials imported or produced (in kg) in Ecuador during 2019 from the report sent by Agrocalidad to WHOA in 2020.

Statistical analysis

E. coli and Salmonella spp. resistance patterns and amount of antimicrobials imported/produced (in kg) in Ecuador were summarized using descriptive statistics (counts, proportions, and sums) and presented according to the WHO classification of antimicrobial importance for human medicine (¹⁸18. World Health Organization. Antimicrobianos de importancia crítica para la medicina humana, 6ª revisión. Geneva: WHO; 2019. Available from: https://apps.who.int/iris/handle/10665/331531
https://apps.who.int/iris/handle/10665/3... ). To compare proportions across regions (coastal, Andean highlands, Amazon basin), we used chi-square testing with a level of significance set at p ≤ 0.05.

Ethics

Ethics approval was obtained from the Ethics Committee of the Society for the Fight against Cancer (SOLCA) in Ecuador, the Ethics Committee of the Pan American Health Organization (PAHOERC), and The Union Ethics Advisory Group of the International Union against Tuberculosis and Lung Disease, Paris, France.

RESULTS

AMR profiles of E. coli and Salmonella spp. isolated from chicken carcasses

In this study, chicken meat samples were obtained from 199 slaughterhouses across 20 provinces of Ecuador. Among 383 raw chicken meat samples cultured in selective media, E. coli was isolated from 148 (39%) samples, Salmonella spp. from 20 (5%) samples, and 7 (4%) samples were positive for both bacteria (Figure 1).

All the isolates underwent AST for 10 critically important and three highly important antimicrobials (per the WHO classification). Approximately 90% of the E. coli and Salmonella spp. isolates were resistant to at least one of the antimicrobials considered critically important for human use (Figure 1). The 13 antibiotics tested belonged to 10 classes of antibiotics (2 penicillins and 2 beta lactams). Resistance to three or more classes of antibiotics was considered as multidrug resistance. Among 148 E. coli isolates, 100 (68%) had multidrug resistance. Among 20 Salmonella spp. isolates, 15 (75%) had multidrug resistance.

Among isolates with E. coli, resistance to erythromycin was highest 112 (76%) followed by tetracycline 105 (71%). Nearly half of the isolates were resistant to trimethoprim/sulphamethoxazole (84; 57%), penicillins (71; 48%), or ciprofloxacin (57; 39%). All E. coli isolates were susceptible to carbapenems (Table 1). Extended-spectrum beta-lactamases (ESBL) were detected in 32 (22%) of E. coli isolates.

Among isolates of Salmonella spp., resistance to tetracycline was the highest (18; 90%), followed by erythromycin (17; 85%) and ceftriaxone (16; 80%). While 15 (75%) of the isolates were resistant to penicillins without inhibitors, all Salmonella spp. isolates were susceptible to penicillins with inhibitors and 16 (80%) were intermediate to ciprofloxacin. No resistance was observed against colistin and carbapenems (Table 1). ESBL were detected in 8 (40%) of the Salmonella spp. isolates.

Slaughterhouses in the coastal region contributed most of the samples (246 of 383 samples). Resistance rates among E. coli and Salmonella spp. isolates were similar across all regions of the country, except for the Amazon region, where Salmonella spp. were not recovered, as shown in Table 2. Details of resistance rates within different provinces in each region are shown in Table 3 and 4.

Antimicrobial use in animals raised for human consumption

According to data reported to the WOAH, 134 148 kg of antimicrobials were imported or produced for use in animals (growth promotion, medical use, or both) in Ecuador in 2019. Antimicrobials of the critically important (40%) and highly important group (47%) constituted the bulk of these antimicrobials. Tetracycline and colistin were imported in larger quantities compared to the rest of the antimicrobials (Table 5).

Colistin, a critically important antimicrobial, was used mainly for growth promotion (82%). The rest of the critically imported antimicrobials (ciprofloxacin, erythromycin, gentamicin, and ampicillin) and all the highly important antimicrobials (tetracycline, trimethoprim/sulphamethoxazole, and chloramphenicol) were used for veterinary medical purposes primarily (Table 5).

DISCUSSION

This is the first nationwide study reporting on AMR in the poultry production system of Ecuador, conducted as part of Agrocalidad’s integrated national program for monitoring AMR (¹⁹19. Agencia de Regulación y Control Fito y Zoosanitario. Programa Nacional Integrado de Monitoreo de la Resistencia Antimicrobiana en la Cadena Agroalimentaria de Ecuador Quito: Agrocalidad; 2020.). Nine in ten isolates of E. coli and Salmonella spp. cultured from chicken carcasses sampled from slaughterhouses were resistant to an antimicrobial considered critically important for human health. The findings of this study are important, as they could serve as evidence to inform efforts around antimicrobial stewardship in the animal production industry of Ecuador.

The prevalence of E. coli (39%) and Salmonella spp. (5%) was lower compared to other studies undertaken in Ecuador and other Latin American countries (⁵5. Vinueza-Burgos C, Baquero M, Medina J, De Zutter L. Occurrence, genotypes and antimicrobial susceptibility of Salmonella collected from the broiler production chain within an integrated poultry company. Int J Food Microbiol. 2019;299:1–7. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0168160518304689?via%3Dihub
https://www.sciencedirect.com/science/ar... , ²⁰20. Castro-Vargas RE, Herrera-Sánchez MP, Rodríguez-Hernández R, Rondón-Barragán IS. Antibiotic resistance in Salmonella spp. isolated from poultry: A global overview. Vet World. 2020;13(10):2070–84. https://doi.org/10.14202/vetworld.2020.2070-2084
https://doi.org/10.14202/vetworld.2020.2... ). The prevalence of E. coli in chicken meat samples has been reported to be around 60%–73% and that of Salmonella around 20%–30% in other studies from this region (⁵5. Vinueza-Burgos C, Baquero M, Medina J, De Zutter L. Occurrence, genotypes and antimicrobial susceptibility of Salmonella collected from the broiler production chain within an integrated poultry company. Int J Food Microbiol. 2019;299:1–7. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0168160518304689?via%3Dihub
https://www.sciencedirect.com/science/ar... , ²⁰20. Castro-Vargas RE, Herrera-Sánchez MP, Rodríguez-Hernández R, Rondón-Barragán IS. Antibiotic resistance in Salmonella spp. isolated from poultry: A global overview. Vet World. 2020;13(10):2070–84. https://doi.org/10.14202/vetworld.2020.2070-2084
https://doi.org/10.14202/vetworld.2020.2... ). The prevalence of serovars of Salmonella spp. was not analyzed. In Ecuador, the most common serotype reported in poultry production systems is S. infantis (83%) (²¹21. Vinueza-Burgos C, Cevallos M, Ron-Garrido L, Bertrand S, De Zutter L. Prevalence and Diversity of Salmonella Serotypes in Ecuadorian Broilers at Slaughter Age. PLoS One. 2016;11(7):e0159567. https://doi.org/10.1371/journal.pone.0159567
https://doi.org/10.1371/journal.pone.015... ). In this study, samples were taken after disinfecting the raw chicken meat in slaughterhouses with hyperchlorinated water. This could be a possible cause of the low prevalence rates. There is a need to evaluate and standardize the procedures for sampling and culture to assure comparability of the data.

Isolates of both bacteria (E. coli and Salmonella spp.) showed high rates of resistance to critically important antimicrobials. More than 80% of the samples showed resistance to tetracycline and erythromycin. Studies on AMR profiles of poultry meat in Latin America have reported varying resistance patterns among E. coli and Salmonella isolates (⁶6. Ortega-Paredes D, de Janon S, Villavicencio F, Ruales KJ, De La Torre K, Villacís JE, et al. Broiler Farms and Carcasses Are an Important Reservoir of Multi-Drug Resistant Escherichia coli in Ecuador. Front Vet Sci. 2020 Nov 25;7:547843., ¹³13. Mejía L, Medina JL, Bayas R, Salazar CS, Villavicencio F, Zapata S, et al. Genomic Epidemiology of Salmonella Infantis in Ecuador: From Poultry Farms to Human Infections. Front Vet Sci. 2020 Sep 29;7:547891. https://doi.org/10.3389/fvets.2020.547891
https://doi.org/10.3389/fvets.2020.54789... , ²²22. Donado-Godoy P, Castellanos R, León M, Arevalo A, Clavijo V, Bernal J, et al. The Establishment of the Colombian Integrated Program for Antimicrobial Resistance Surveillance (COIPARS): A Pilot Project on Poultry Farms, Slaughterhouses and Retail Market. Zoonoses Public Health. 2015 Apr;62(Suppl 1):58–69. https://doi.org/10.1111/zph.12192
https://doi.org/10.1111/zph.12192... –²⁴24. Koga VL, Rodrigues GR, Scandorieiro S, Vespero EC, Oba A, De Brito BG, et al. Evaluation of the Antibiotic Resistance and Virulence of Escherichia coli Strains Isolated from Chicken Carcasses in 2007 and 2013 from Paraná, Brazil. Foodborne Pathog Dis. 2015 Jun 1;12(6):479–85. https://doi.org/10.1089/fpd.2014.1888
https://doi.org/10.1089/fpd.2014.1888... ). Differences in geographical contexts and methods used for sample processing could be the reason for this variation.

We found extremely high rates of resistance to critically important antimicrobials in the meat samples. These drugs are the sole or one of limited available therapies to treat serious bacterial infections in humans. Although this is a cause of concern, we believe that this merits further investigation. A few pathways of inquiry that future research can pursue to evaluate the true implications of these findings for human health include but are not limited to (i) establishing the provenance of this AMR, (ii) assessing its relationship with humans through molecular studies, and (iii) evaluating whether the resistant bacteria found in unprocessed meat are retained in the processed or cooked forms that are actually consumed by humans.

Some of these antimicrobials, like tetracycline, are also the mainstay for treating common veterinary infections. Tetracycline was the antibiotic imported for veterinary medical use in the largest quantity by Ecuador in 2019, as per the WOAH report. The possibility of misuse of these antimicrobials in the animal production sectors, thus leading to AMR, cannot be ruled out and must be investigated. There is an urgent need to develop standard guidelines for the rational use of antimicrobials in animals and to put in place mechanisms to ensure strict adherence to these guidelines.

Resistance to critically important antimicrobials was uniformly high (approximately 90%) in all regions of Ecuador, including those with low poultry production such as the Amazon basin. As the poultry industry continues to expand in the country, now is the right time to take measures to promote judicious use of antimicrobials in these regions. This can be done by sensitizing livestock producers to the dangers of AMR and engaging them regarding rational use of these drugs.

No resistance was found to ciprofloxacin in Salmonella spp., one of the most commonly used antibiotics in poultry production. This is contrary to findings from other studies conducted on poultry in Ecuador, which have reported resistance rates to ciprofloxacin ranging from 64% in meat samples to as high as 99% in cecal samples (¹³13. Mejía L, Medina JL, Bayas R, Salazar CS, Villavicencio F, Zapata S, et al. Genomic Epidemiology of Salmonella Infantis in Ecuador: From Poultry Farms to Human Infections. Front Vet Sci. 2020 Sep 29;7:547891. https://doi.org/10.3389/fvets.2020.547891
https://doi.org/10.3389/fvets.2020.54789... , ²¹21. Vinueza-Burgos C, Cevallos M, Ron-Garrido L, Bertrand S, De Zutter L. Prevalence and Diversity of Salmonella Serotypes in Ecuadorian Broilers at Slaughter Age. PLoS One. 2016;11(7):e0159567. https://doi.org/10.1371/journal.pone.0159567
https://doi.org/10.1371/journal.pone.015... ). There is a need to review the procedure of the disc diffusion method used to evaluate antimicrobial sensitivity patterns of ciprofloxacin in this study.

We assessed the association between the resistance patterns found in this study and the antimicrobials that are commonly used in animals in Ecuador. While the resistance to tetracycline and erythromycin was higher in the isolates from chicken carcasses, colistin, tetracycline, and trimethoprim/sulfamethoxazole formed the bulk of antimicrobials used in animals as per the WOAH report on antimicrobial use in animals raised for human consumption (2019). It is pertinent to note that while the isolates were all obtained from poultry, the WOAH report includes antimicrobial use in the animal production sectors as a whole. Therefore, it is not possible to attribute AMR found in this study to high levels of use of any particular antimicrobial. As a next step, antimicrobial use in different animal production industries can be monitored in actual field settings and then triangulated with data on local AMR patterns.

The WOAH report also showed that colistin was the antibiotic mostly commonly used for growth promotion. There are several reports in Ecuador and Latin America about dissemination and high levels of colistin resistance in human, animals, and the environment (²⁵25. Calero-Cáceres W, Tadesse D, Jaramillo K, Villavicencio X, Mero E, Lalaleo L, et al. Characterization of the genetic structure of mcr-1 gene among Escherichia coli isolates recovered from surface waters and sediments from Ecuador. Sci Total Environ. 2022 Feb 1;806:150566., ²⁶26. Quiroga C, Nastro M, Di Conza J. Current scenario of plasmid-mediated colistin resistance in Latin America. Rev Argent Microbiol. 2019 Jan 1;51(1):93–100. https://doi.org/10.1016/j.ram.2018.05.001
https://doi.org/10.1016/j.ram.2018.05.00... ). Consequently, in 2019 Agrocalidad issued a ban on the use of this antibiotic in animals in Ecuador (²⁷27. Agencia de Regulación y Control Fito Zoosanitario. Resolución 0003-Se prohibe la fabricación, importación, comercialización, registro y uso de productos que contengan colistina (Polimixina E) para uso o consumo animal en Ecuador [Internet]. Quito: Agrocalidad; 2019. Available from: https://www.agrocalidad.gob.ec/wp-content/uploads/2020/05/dol6.pdf
https://www.agrocalidad.gob.ec/wp-conten... ). Resistance to colistin was found to be low (2.7%) in this study. However, in view of the reports of resistance from other studies and the prevailing ban, it is important to closely regulate the use of this critically important antimicrobial and to monitor the emergence of resistance through continuous surveillance.

To the best of our knowledge, this is the first study from Ecuador that reports on AMR in the poultry industry from a sample of slaughterhouses across the country. The study also has certain limitations. There were limitations in the procedures for sample collection and AST used in this study, which may have influenced the resistance rates reported here. AST was conducted for a panel of 13 antibiotics that were considered important in the Ecuadorian context. Therefore, we are not able to comment on resistance to other antimicrobials that may be of importance from a human health perspective. It is advisable to include a wider range of antibiotics in future studies.

In conclusion, the high rates of AMR in the poultry industry calls for Agrocalidad to strengthen the existing surveillance program in order to improve the monitoring of antimicrobial use in animals and evolving resistance patterns in the country. Policies need to be developed to regulate the use of antimicrobials of critical or high importance in human health. This study also paves the way for future research studies that can help to better understand the implications of the AMR reported in this study for human health.

Disclaimer.

Authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the RPSP/PAJPH or the Pan American Health Organization (PAHO).

Acknowledgments.

This research protocol was developed through the Structured Operational Research and Training Initiative (SORT IT), a global partnership coordinated by TDR, UNICEF, UNDP, World Bank, WHO Special Programme for Research and Training in Tropical Diseases hosted at the World Health Organization. The specific SORT IT program that led to this study protocol included an implementation partnership of TDR and the Pan American Health Organization (PAHO), the PAHO country offices in Colombia and Ecuador; the Ministry of Agriculture and Livestock of Ecuador; World Organization for Animal Health of Ecuador; Agency for Plant and Animal Health Regulation and Control—Agrocalidad, Ecuador; The International Union Against Tuberculosis and Lung Disease headquarters in Paris, France, and Southeast Asia office, India; Institute of Tropical Medicine, Antwerp, Belgium; Damien Foundation, Belgium; Indian Council of Medical Research—National Institute of Epidemiology; Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER); GMERS Medical College Gotri Vadodara Gujarat, India; India Medical College Baroda, Gujarat, India; Sri Manakula Vinayagar Medical College, India; Universidade Federal de Ciências de Saúde de Porto Alegre, Brazil; Universidade de Brasília, Brazil; Autonomous University of Yucatán, Mexico. We are grateful to Janet Ousley from Médecins sans Frontières (MSF) for editorial support; to Pilar Donado, Coordinator of the Global Health Research Unit (GHRU) on Antimicrobial Surveillance from the Colombian Corporation for Agricultural Research—Agrosavia for her technical support; and Andrés Salguero of Agrocalidad for the report on the use of antimicrobials in animals in Ecuador in 2019.

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