Veterinaria (Montevideo), 2026 62(226), https://doi.org/10.29155/VET.62.226.7

Artículo original

Occurrence and antimicrobial resistance profiles of enterobacteria in treated poultry manure and agricultural soils in Uruguay

Resistencia antimicrobiana y ocurrencia de enteropatógenos bacterianos en estiércol de aves de corral y en suelos agrícolas en Uruguay

Resistência antimicrobiana e ocorrência de bactérias enteropatógenas em dejetos de aves de corte e em solos agrícolas no Uruguai

Yamila Figueroa^{1, 2} https://orcid.org/0000-0002-2218-482X

Carla Stoletniy¹ https://orcid.org/0009-0002-7066-7460

Jimena Posada¹ https://orcid.org/0009-0003-0662-6696

Juan Silvera^{1, 2} https://orcid.org/0009-0006-8950-4118

Pablo Zunino^{1, 2} https://orcid.org/0000-0003-2375-1408

Ana Umpiérrez^{1, 2} https://orcid.org/0000-0002-5897-0400

¹Departamento de Microbiología, Instituto de Investigaciones Biológicas Clemente Estable, Montevideo, Uruguay.

²Centro de Investigación en Ciencias Ambientales, Montevideo, Uruguay. Correspondence author: aumpierrez@iibce.edu.uy / anitaump@gmail.com

Cómo citar este artículo:

Figueroa, Y., Stoletniy, C., Posada, J., Silvera, J., Zunino, P., & Umpiérrez, A. (2026). Occurrence and antimicrobial resistance profiles of enterobacteria in treated poultry manure and agricultural soils in Uruguay. Veterinaria (Montevideo), 62(226), e20266222607. https://doi.org/10.29155/VET.62.226.7

Abstract

Land application of poultry production manure requires safe management strategies to evaluate and mitigate the dissemination of antimicrobial-resistant bacteria and opportunistic pathogens into agroecosystems This study evaluated the presence of zoonotic pathogens and their antimicrobial resistance profiles in poultry manure before and after composting, anaerobic digestion, and Quick Wash treatments, as well as in soils fertilized with these amendments. All samples were processed through bacterial isolation, molecular identification, and susceptibility testing against 17 antibiotics.

A total of 106 isolates were collected and screened. Enterobacteria predominated (98.6 %), mainly Klebsiella spp. and E. coli. Among 37 E. coli isolates, virulence genes compatible with EPEC were identified in three isolates, and other virulence factors in four additional isolates. Antibiotic resistance was observed across all isolates from treated manure and soil, particularly against β-lactams, quinolones, aminoglycosides, and fosfomycin. Twelve multidrug-resistant isolates were identified, mainly Klebsiella spp., Enterobacter spp., and E. coli, with resistance associated with AmpC β-lactamases. None of mcr-1, mcr-2, mcr-5 and mcr-9 genes for colistin resistance, were detected.

These findings demonstrate the presence of antibiotic-resistant enterobacteria in soils and poultry manure and also its persistence in poultry manure after physical, chemical and biological treatments. The presence of antibiotic-resistant enterobacteria highlights the need for monitoring and mitigation strategies to control the environmental spread of antimicrobial resistance.

Keywords: Poultry manure, Zoonosis, Antimicrobial resistance, Multidrug resistance, Agricultural soil.

Resumen

La aplicación agrícola de residuos de la producción avícola requiere estrategias de gestión seguras para evaluar y mitigar la diseminación de bacterias resistentes a los antimicrobianos y patógenos oportunistas en los agroecosistemas. Este estudio evaluó la presencia de patógenos zoonóticos y sus perfiles de resistencia antimicrobiana en residuos avícolas antes y después de tratamientos de compostaje, digestión anaerobia y Quick Wash, así como en suelos fertilizados con estas enmiendas. Todas las muestras fueron procesadas mediante aislamiento bacteriano, identificación molecular y pruebas de susceptibilidad frente a 17 antibióticos.

Se recolectaron y analizaron un total de 106 cepas. Predominaron las enterobacterias (98,6 %), principalmente Klebsiella spp. y E. coli. Entre las 37 cepas de E. coli, se identificaron genes de virulencia compatibles con EPEC en tres cepas y otros factores de virulencia en otras cuatro cepas. Se observó resistencia a los antibióticos en todas las cepas procedentes del estiércol tratado y del suelo, en particular contra los β-lactámicos, las quinolonas, los aminoglucósidos y la fosfomicina. Se identificaron doce cepas multirresistentes, sobre todo de Klebsiella spp., Enterobacter spp. y E. coli, con resistencia asociada a las β-lactamasas AmpC. No se detectó ninguno de los genes mcr-1, mcr-2, mcr-5 o mcr-9, responsables de la resistencia a la colistina.

Estos hallazgos demuestran la presencia de enterobacterias resistentes a los antibióticos en el suelo y el estiércol avícola, así como su persistencia tras tratamientos físicos, químicos y biológicos. La presencia de enterobacterias resistentes a los antibióticos resalta la necesidad de estrategias de monitoreo y mitigación para controlar la propagación ambiental de la resistencia a los antimicrobianos.

Palabras clave: Residuos avícolas, Zoonosis, Resistencia antimicrobiana, Multirresistencia, Suelos tratados.

Resumo

A aplicação de resíduos da produção avícola no solo exige estratégias de gestão seguras para avaliar e mitigar a disseminação de bactérias resistentes a antimicrobianos e patógenos oportunistas em agroecossistemas. Este estudo avaliou a presença de patógenos zoonóticos e seus perfis de resistência antimicrobiana em resíduos avícolas antes e após tratamentos de compostagem, digestão anaeróbia e Quick Wash, bem como em solos fertilizados com essas emendas. Todas as amostras foram processadas por meio de isolamento bacteriano, identificação molecular e testes de suscetibilidade a 17 antibióticos.

Um total de 106 isolados foi coletado e analisado. As enterobactérias predominaram (98,6 %), principalmente Klebsiella spp. e. coli. Entre os 37 isolados de E. coli, foram identificados genes de virulência compatíveis com EPEC em três isolados e outros fatores de virulência em mais quatro isolados. Observou-se resistência a antibióticos em todas as cepas provenientes de esterco tratado e solo, particularmente contra β-lactâmicos, quinolonas, aminoglicosídeos e fosfomicina. Foram identificadas doze cepas multirresistentes, principalmente de Klebsiella spp., Enterobacter spp. e. coli, com resistência associada às β-lactamases AmpC. Nenhum dos genes mcr-1, mcr-2, mcr-5 ou mcr-9, responsáveis pela resistência à colistina, foi detectado.

Esses resultados demonstram a presença de enterobactérias resistentes a antibióticos no solo e no esterco de aves, bem como sua persistência após tratamentos físicos, químicos e biológicos. A presença de enterobactérias resistentes a antibióticos destaca a necessidade de estratégias de monitoramento e mitigação para controlar a disseminação ambiental da resistência antimicrobiana.

Palavras-chave: Resíduos avícolas, Zoonoses, Resistência antimicrobiana, Multirresistência, Solos tratados.

Fecha de recibido: 06/05/2026

Fecha de aceptado: 17/09/2026

Introduction

The emergence and spread of antibiotic-resistant pathogenic bacteria represent one of the main threats to global health. For decades now, the widespread misuse of antimicrobials in both human and veterinary medicine, along with the use of antibiotics as animal growth promoters -nowadays banned in many countries- have significantly contributed to the selection and dissemination of antibiotic-resistant bacteria in the environment (Samreen et al., 2021). Multidrug-resistant (MDR) strains complicate the treatment of human and animal infections, prolong clinical conditions and increase the risk of pathogen spread (Leibler et al., 2009). In this sense, the World Health Organization (WHO) periodically updates the list of bacterial priority pathogens of public health importance, where multidrug-resistant (MDR) Gram-negative Enterobacterales -such as Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp.- are designated as critical priority pathogens (World Health Organization, 2024). Identifying and monitoring these MDR Gram-negative bacteria across environmental and agricultural settings is essential from a One Health framework, as the spread of resistance to critical antimicrobials directly compromises last-resort therapeutic options required for managing life-threatening clinical infections in humans, particularly in intensive care units (ICUs) (World Health Organization, 2024).

Intensification and industrialization of animal production have improved yields and reduced costs, but they have also negatively impacted animal welfare, increased environmental contamination and promoted the dissemination of antibiotic-resistant pathogens, elevating the risk of zoonotic diseases (Leibler et al., 2009). Such spillover of pathogens consequently affects workers in poultry establishments, where an increased prevalence of enteric bacterial infections has been reported (Price et al., 2007; van den Bogaard et al., 2001).

The large volumes of manure generated in poultry production create the need for safe management and valorization. Manure is widely recognized for its nutrient content, supporting its use as organic fertilizer, and legislation for its application has been established in several countries. In Uruguay, regulatory frameworks for manure management have been in place since 2018 (Ministerio de Ganadería, Agricultura y Pesca, 2018). Ensuring the safety of this type of waste before reuse is critical, particularly because pathogens from animals can cause over 60 % of zoonotic infections in humans (Chala & Hamde, 2021), and in many cases carry several antimicrobial resistance genes.

Different zoonotic enterobacteria have been documented in Uruguayan production systems. Shiga toxin-producing Escherichia coli (STEC), the etiological agent of bloody diarrhea and hemolytic uremic syndrome, has been isolated from healthy dairy calves, animals with neonatal diarrhea, and calves deceased from enteric disease, sometimes showing multidrug resistance (Coppola et al., 2020; Fernández et al., 2023; Umpiérrez et al., 2022). Salmonella enterica serovar Enteritidis has been recovered from eggs (Betancor et al., 2010), and multidrug-resistant enteric S. enterica has been detected in cattle (Casaux et al., 2019; 2023).

Beyond primary zoonotic pathogens such as E. coli and Salmonella spp., other members of the Enterobacteriaceae family -including K. pneumoniae, Enterobacter spp., Proteus spp., and Serratia spp. as well as other Gram negative species like Vibrio fluvialis-, play critical roles as opportunistic pathogens in both veterinary and human medicine. In poultry, these bacteria are frequently associated with opportunistic infections, including respiratory disorders, omphalitis, and systemic infections, particularly under intensive production conditions (Abreu et al., 2023). In humans, species such as K. pneumoniae and Enterobacter spp. are leading causes of healthcare-associated and community-acquired infections, such as pneumonia, urinary tract infections, and sepsis (Ullah et al., 2024). Importantly, these species frequently harbor intrinsic or mobile antimicrobial resistance determinants (e.g., AmpC β-lactamases and extended-spectrum β-lactamases), acting as major environmental reservoirs and vehicles for horizontal gene transfer between livestock, the environment, and humans (Motlhalamme et al., 2024).

Pathogenic microorganisms like E. coli and Salmonella spp. can survive for months in fresh poultry manure and poorly managed wastewaters (Kyakuwaire et al., 2019). Even so, due to its nutrient composition, poultry manure has potential as a fertilizer, and different strategies have been developed for its valorization. These include: (i) composting, an aerobic biological process transforming organic matter into nutrient-rich fertilizer, where thermophilic temperatures usually eliminate pathogens (Ayilara et al., 2020); (ii) anaerobic digestion, used for biogas production and manure stabilization, in which mesophilic microorganisms play a key role, and the thermal control can limit pathogen survival (Xu et al., 2022); and (iii) Quick Wash, a USDA-developed technology that reduces soluble phosphorus, recovers nutrients, decreases odors, and generates products suitable as soil amendments or bioenergy sources (Szogi et al., 2015). Additionally, one strategy to enhance the composting processes is to use effective microorganisms (EM). This microbial consortiums have been used with the objective to increase enzymatic activities, promote biodegradation of organic matter and accelerate the composting process (Van Fan et al., 2018). While all these strategies offer significant benefits, it is critical to ensure that treatments are effective in eliminating pathogenic microorganisms and reducing the risk of spreading antimicrobial resistance in the environment.

The objective of this study was to evaluate the presence of the zoonotic pathogens Shiga toxin-producing Escherichia coli (STEC) and Salmonella spp., as well as to isolate and characterize the diversity and antimicrobial resistance profiles of Enterobacteriaceae (including opportunistic pathogens such as Klebsiella spp. and Enterobacter spp.) in poultry manure from broiler and breeders farms before and after treatment processes, and in soils treated with these organic amendments. To this end, four sample groups were evaluated: composted poultry manure (CM), bioreactor-treated poultry manure (BR), Quick Wash-treated poultry manure (QW), and agricultural soil amended with these treated poultry manure products (ST).

Materials and methods

Origin of the samples

A total of 88 poultry manure samples were processed: composted poultry manure (CM) (n = 73), bioreactor-treated poultry manure (BR) (n = 12) and Quick Wash (QW) (n = 3), with each group including a subset of untreated poultry manure. Poultry manure samples were processed progressively upon arrival at the laboratory throughout the study duration. Initial sampling and raw manure collection were carried out within the framework of a project supported by the National Institute of Agricultural Research (INIA) (project code: FPTA-374), the full primary results of which are beyond the scope of the present manuscript. Because raw poultry litter was routinely supplied to the processing facilities from commercial farms, specific flock- or batch-level traceability prior to laboratory delivery was outside the operational scope of this study.

In addition, 18 agricultural soil samples were collected from a controlled field trial designed to evaluate the agricultural application of these treated manure products. The soil evaluation comprised untreated soil (ST-US), baseline/initial soil (ST-SI), and soil amended with each treatment (ST-CM, ST-BR, and ST-QW). Throughout this manuscript, CM, BR and QW are referred to as treated poultry manure, whereas ST refers to agricultural soil after amendment application. Additionally, the composting processes included a subset of composted poultry manure treated with MEN® (Cooperativa Entrebichitos) (MEN, spanish acronym for Efficient Native Microorganisms), as these are commonly incorporated to enhance fermentation and pathogen reduction in different substrates such as wastewater (Echeverriborda et al., 2022; Van Fan et al., 2018).

Molecular screening of virulence and marker genes

Total DNA was extracted from the 106 samples to evaluate the presence of STEC genes: Shiga toxin (stx1 and stx2) and intimin (eae), and Salmonella spp. invasin gene (invA) by qPCR, using primers previously designed in our laboratory (González Revello, 2020). DNA extraction was performed using an automated system and PurePrep Pathogen kit (Molgen, Veenendaal, The Netherlands). Samples were diluted 1/10 in sterile PBS prior extraction. The purified nucleic acids were quantified with NanoDrop. Positive control strains were included in each PCR run: reference strain Escherichia coli O26:H11 (ATCC BAA-2196, positive for stx1, stx2 and eae), as well as fully characterized in-house reference strains from our laboratory collection for Escherichia coli O157:H7 (positive for stx1, stx2, and eae) and Salmonella enterica (positive for invA) (González Revello, 2020).

Bacterial isolation

For bacterial isolation, 20 g of each sample was incorporated in a sterile bag containing 200 mL of Tryptose Soy Broth (TSB; Oxoid Ltd., Basingstoke, UK) for 18-24 h at 37 °C. Enriched cultures were replicated on MacConkey (Oxoid Ltd.) and EMB Levine agar plates (BD, Franklin Lakes, NJ, USA) for 18-24 h at 37 °C. Suspected Enterobacteriaceae colonies, such as lactose-fermenting, were selected for further isolation and identification. For Salmonella spp. isolation, an aliquot (1mL) of TSB-enriched primary cultures was seeded in Tetrationate broth (TT; Oxoid Ltd.) for 24 h at 37 °C and replicated on XLD agar plates (Merck KGaA, Darmstadt, Germany) for 18-24 h at 37 °C. Suspected Salmonella spp. colonies were selected. All isolates were stored in TSB-Glycerol at -80 °C.

Phenotypic and molecular characterization of isolates

Lactose-positive colonies were initially identified using conventional biochemical tests (Octavia & Lan, 2014) and further confirmed by 16S rDNA gene sequencing (Macrogen Inc.). Colonies resembling Salmonella spp. on XLD plates (pink-red with black centers) were confirmed by PCR using primers previously designed in our laboratory (González Revello, 2020) and further validated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Screening of virulence genes in E. coli isolates included Shiga toxins genes: stx1, stx2, Intimin adhesin: eae, pili-associated genes: afa8, papC, sfaDE, cytotoxins: cdtIII, cdtIV, and iucD, was performed according to primers and protocols previously described (Umpiérrez et al., 2022). Isolates were classified into virotypes based on these genes’ presence (Kaper et al., 2004). In addition, all isolates confirmed as Klebsiella pneumoniae were screened by PCR for the pilv-I gene, a specific marker for the high-risk ST258 clone (Gomez et al., 2016).

Antimicrobial resistance

The resistance profile to 17 antibiotics was studied by disc-diffusion on Müller-Hinton agar plates (Oxoid Ltd., Basingstoke, UK). A panel of 17 antimicrobial disks (Oxoid Ltd., Basingstoke, UK) was evaluated: Ampicillin (AMP, 10 µg), Cefoxitin (FOX, 30 µg), Cefuroxime (CXM, 30 µg), Ceftriaxone (CRO, 30 µg), Ceftazidime (CAZ, 30 µg), Cefepime (FEP, 30 µg), Amoxicillin-clavulanic acid (AMC, 20/10 µg), Imipenem (IMI, 10 µg), Ciprofloxacin (CIP, 5 µg), Enrofloxacin (ENR, 5 µg), Nalidixic acid (NA, 30 µg), Trimethoprim-sulfamethoxazole (STX, 1.25/23.75 µg), Gentamicin (GM, 10 µg), Amikacin (AK, 30 µg), Tetracycline (TE, 30 µg), Fosfomycin (FOS, 200 µg), and Colistin (COL, 10 µg) was studied using the pre-diffusion protocol (Servicios Antimicrobianos INEI-ANLIS, 2018), and subsequently the variants mcr-1, mcr-2, mcr-5 and mcr-9, for COL transferable resistance were analyzed by PCR in those isolates where a halo of inhibition was detected (Coppola et al., 2022; Papa-Ezdra et al., 2020). CLSI 2025 cut-off values were used for interpretation of the inhibition zone diameters.

Those isolates classified as MDR were further characterized by PCR for the presence of the aac, cmy, fox, shv, pan-CTX-M, qnr, and fosA3 genes, associated with resistance to aminoglycosides, beta-lactams, and quinolones (Bado et al., 2018; García-Fulgueiras et al., 2011).

Results

Zoonotic pathogen screening in treated poultry manure and soil sample

A total of 106 samples were analyzed, including 88 poultry manure amendment samples (CM, BR and QW) and 18 soil samples from the soil experiment (ST). Samples were tested by qPCR for the presence of STEC genes: stx1, stx2, eae or Salmonella spp. invA gene. None of them were positive.

Distribution of obtained isolates

Afterwards, 106 isolates were recovered from the 106 samples. These isolates were identified using selective culture media, with enterobacteria representing 98.6 % (93/106). Genus-level identification was confirmed by 16S rDNA gene sequencing at Macrogen Inc. Although not expected, genera other than Enterobacteriaceae were detected: Staphylococcus spp. (n = 3), Pseudomonas spp. (n = 5) and Vibrio spp (n = 5).

Among enterobacteria, 37.2 % corresponded to Klebsiella spp. (n = 40), 34.5 % to Escherichia sp. (n = 37), 6.5 % to Enterobacter sp. (n = 7), 5.6 % to Proteus spp. (n = 6), 1.9 % to Serratia sp. (n = 2) and 1.3 % to Salmonella sp. (n = 1). Additionally, 16S rDNA sequencing confirmed that all Escherichia spp. isolates were E. coli, and all Vibrio spp. isolates were V. fluvialis.

The only pink-red colony with a black center on XLD agar plates was confirmed by PCR amplification of the invA gene and further identified as Salmonella enterica subsp. enterica by MALDI-TOF.

Virulotype of E. coli isolates and confirmation of Salmonella isolate

Among the 37 E. coli isolates, three virulence genes were detected in seven of them: the iucD gene, involved in iron uptake; the eae gene, which encodes intimin adhesin, which is part of a key secretion system in the virulence of EPEC and some STEC; and the cdtIV gene, which encodes variant IV of a cytotoxin present in Necrotoxigenic E. coli (NTEC) and Extraintestinal E. coli (ExPEC) variants. Of these 7 pathogenic E. coli isolates, 4 were iucD+, 2 were eae+/cdtIV+ and 1 was eae+. The three eae-positive isolates were recovered from CM+MEN, and were classified as EPEC. In contrast, the remaining four isolates (three from CM and one from BR), although carrying virulence genes, were not assigned to any virotype, as the mere presence of these genes is not sufficient for such classification (Kaper et al., 2004).

Additionally, the invA gene was identified in one isolate, which was subsequently confirmed as Salmonella enterica subsp. enterica by MALDI-TOF.

Antibiotic-succeptibility phenotype of the isolates

Among the 106 isolates, 67 (63.2 %) were resistant to one of the tested antibiotics (1 to 8 resistances simultaneously), whereas 39 (36.8 %) were susceptible to all. Resistant isolates were detected in all samples. To compare the resistance profiles isolates were categorized in: (i) isolates sensitive to all antibiotics tested (no resistance), (ii) isolates with resistance to one antibiotic, (iii) isolates with resistance to two antibiotics, and (iv) isolates resistant to three or more antibiotics. In this latter category, isolates were classified as multidrug-resistant (MDR) when they were resistant to at least one agent in three or more different antimicrobial classes (Magiorakos et al., 2012) (Fig. 1).

Among CM, 50 % of the isolates (n = 31) were sensitive to all antibiotics, while 29 % (n = 18) showed resistance to one, 8 % (n = 5) to two and 13 % (n = 8) showed resistance to three or more antibiotics. Within this treatment, 7 isolates were considered MDR, which were mainly associated with Vibrio fluvialis, and displayed elevated resistance to β-lactam antibiotics (Fig. 1). Within BR, 3 % of the isolates (n = 5) were sensitive to all the antibiotics, 37 % (n = 6) showed resistance to one, 12.5 % (n = 2) to two, and 19 % (n = 3) to three or more antibiotics. In this group, where only enterobacteria were isolated, a high prevalence of resistance to β-lactam antibiotics was observed, associated with inducible AmpC enzyme, together with resistance to fluoroquinolones. Also, 3 MDR Enterobacter sp. isolates were detected in this treatment group (Fig. 1).

Only 3 isolates were recovered from the QW group. One was confirmed as Salmonella spp. and two identified as E. coli. One of the E. coli showed resistance to fluoroquinolones and colistin (Fig. 1). Finally, among ST isolates, 12 % (n = 3) did not show resistance to any of the evaluated antibiotics, 32 % were resistant to a single antibiotic (n = 8), 28 % (n = 7) to two, and 28 % (n = 7) showed resistance to three or more antibiotics. Five isolates (four K. pneumoniae and one E. coli) were MDR in this experiment, including 2 isolates resistant to up to eight antibiotics, mostly within the β-lactam class (Fig. 1).

Regarding MDR isolates characterization, none of the tested aminoglycoside, β-lactam, or quinolone resistance genes (aac, cmy, fox, shv, pan-CTX-M, qnr, and fosA3) were detected by PCR.

Figure 1: Antibiotic resistance profiles and multidrug resistance (MDR) among isolates obtained from treated poultry manure and soil samples. CM: composted poultry manure; BR: bioreactor-treated poultry manure; QW: Quick Wash-treated poultry manure; ST: agricultural soil after amendment application. Ampicillin (AMP), Cefoxitin (FOX), Cefuroxime (CXM), Ceftriaxone (CRO), Ceftazidime (CAZ), Cefepime (FEP), Amoxicillin-clavulanic acid (AMC), Imipenem (IMI), Amikacin (AK), Gentamicin (GM), Fosfomycin (FOS), Ciprofloxacin (CIP), Nalidixic Acid (NA), Enrofloxacin (ENR) and Colistin (COL)

Molecular screening of colistin-resistant isolates and K. pneumoniae ST258 clone

A subset of 35 isolates exhibiting phenotypic resistance to colistin according to pre-difusion test was screened for the presence of transferable colistin resistance genes. None of them tested positive for the mcr variants evaluated. Likewise, none of the 38 K. pneumoniae isolates were positive for pilv-I gene, indicating the absence of the ST258 clone in the analyzed isolates.

Discussion

Animal manure is recognized as a significant reservoir of antibiotic residues, resistant bacteria and antibiotic resistance genes (ARGs), representing an important link between agricultural practices and the emergence of antimicrobial resistance worldwide (Zhu et al., 2019).

Several studies have shown that land application of manure without adequate pretreatment causes ecosystem disturbance, significantly increasing the diversity and abundance of ARGs in agricultural soils, favoring their persistence and horizontal transfer in soil and watercourses (Heuer et al., 2011; Hu et al., 2016). In this work, we focused on the isolation of Enterobacteriaceae, which were isolated from both treated poultry manure and soil samples. Our findings are consistent with research documenting that manure-derived bacteria and pathogens can persist in soils for several weeks and even months (Chee-Sanford et al., 2009).

Even though STEC virulence genes were not observed, genes compatible with diarrheagenic EPEC were detected. This pathogen has a high capacity for persistence in the environment, particularly in soil, which underlines the importance of its monitoring and control (Ongeng et al., 2015). The role of external sources, such as irrigation water and certain agricultural practices, in the introduction and dissemination of these pathogens in soil has been documented (Gigliucci et al., 2025). Nevertheless, the available scientific literature reveals a scarcity of studies focusing on the monitoring of EPEC in the context of manure treatment or organic amendments.

In addition, a Salmonella isolate was detected in the bioreactor treatment (BR), consistent with previous reports describing its persistence after biological treatment processes (Tran et al., 2020). Although biological treatment processes may reduce bacterial loads, they may not completely eliminate certain pathogens. However, Salmonella was not detected in the soil samples amended with the BR material. Studies with more samples and isolates are needed to better assess the microbiological safety of this treatment.

Beyond primary zoonotic pathogens such as STEC and Salmonella spp., this study recovered several other Enterobacteriaceae, including Klebsiella spp., Enterobacter spp., Proteus spp., and Serratia spp., from treated poultry manure and agricultural soil samples. Although these bacteria are commonly considered opportunistic rather than primary zoonotic pathogens, their presence in agricultural matrices is relevant from a One Health perspective, particularly because several of these genera include clinically important organisms and may harbor antimicrobial resistance determinants.

Resistance to various antibiotics was detected, primarily to β-lactams, quinolones, aminoglycosides, and fosfomycin. Beta-lactam resistance in Enterobacteriaceae can be mediated by the expression of AmpC β-lactamases, a mechanism that predominantly occurs at the chromosomal level in several species. In our study, phenotypic patterns compatible with AmpC β-lactamase production were observed in isolates of Enterobacter spp., Klebsiella spp., and V. fluvialis. Enterobacter intrinsically harbors an inducible AmpC, which may also be associated with plasmid-borne elements in certain species. The expression of AmpC enables the hydrolysis of first- and second-generation cephalosporins, such as cephalothin, cefazolin, and cefoxitin, as well as most penicillins and β-lactamase inhibitor combinations (Tamma et al., 2019). The detection of isolates showing phenotypic patterns compatible with AmpC production in soil highlights the potential relevance of these environments for the persistence of antibiotic-resistant bacteria. Resistance to broad-spectrum cephalosporins, including CAZ and CRO, was observed in Klebsiella spp. isolates from the soil experiment. This phenotype may be compatible with increased AmpC activity, although the underlying mechanism was not genetically characterized in this study (Tamma et al., 2019, 2021).

Antibiotic-resistant Enterobacteriaceae isolates were detected recovered from all soil samples. Although definitive conclusions cannot be drawn regarding the reduction or persistence of resistant bacteria following the application of different amendments, the presence of antibiotic-resistant Enterobacteriaceae in the baseline soil is noteworthy, highlighting the issue of their occurrence in the natural environment. Several external factors, including irrigation water, proximity to animal and food-production facilities, and wildlife, may contribute to the introduction of antibiotic-resistant bacteria and resistance genes into agricultural soils (Franklin et al., 2024; Gigliucci et al., 2025).

All colistin-resistant isolates belonged to the Enterobacteriaceae family, but none carried mcr-1, mcr-2, mcr-5 or mcr-9 genes, which are widely recognized for their high potential for horizontal dissemination and global epidemiological relevance (Mmatli et al., 2022). The mcr-1 gene is the most broadly distributed worldwide, primarily found in bacteria such as E. coli, Salmonella enterica, and K. pneumoniae. In contrast, the mcr-2 gene, though less prevalent, has also been identified and remains a subject of study due to its potential for horizontal transmission (Hussein et al., 2021). Although none of the colistin-resistant isolates tested positive for these genes, it would be relevant to expand the search to include other variants, which have been described in the scientific literature (Martiny et al., 2022). In Uruguay, mcr-1 has already been documented in E. coli from both human infections and livestock feces, underscoring the importance of integrated One Health surveillance (Coppola et al., 2020; Papa-Ezdra et al., 2020). Furthermore, colistin resistance detected by the disk diffusion method was predominantly observed in Klebsiella spp. strains, consistent with previous studies reporting clinical isolates of this species lacking mcr genes (Niazadeh et al., 2022).

The detection of antibiotic-resistant Enterobacteriaceae across the evaluated treatment groups shows the occurrence of antimicrobial resistance in animal-derived amendments and agricultural soils, despite the application of different management strategies. These results highlight the potential role of poultry manure as a reservoir of resistant bacteria and reinforce the importance of monitoring antimicrobial resistance in agroecosystems relevant to animal production. However, due to the descriptive nature of this study and the reliance on phenotypic profiling, it is not possible to confirm whether specific bacterial strains persisted across treatments or if direct transmission occurred from manure to soil. High-resolution genomic approaches like Whole Genome Sequencing will be necessary in future studies to track specific bacterial lineages and mobile genetic elements across the manure-soil continuum.

Conclusion

In Uruguay, although the National Plan for Antimicrobial Resistance promotes the responsible use of antimicrobials and environmental protection, specific guidelines addressing antibiotic resistance associated with poultry manure are still limited (Ministerio de Salud Pública (MSP) et al., 2025). In this context, the present study provides original local evidence that supports the need to strengthen surveillance and mitigation measures, contributing to a more sustainable and sanitary management of animal manure within a One Health framework.

Acknowledgments

We would like to thank all the technicians and scientists involved in the FPTA-374 project for carrying out the amendments, collecting the samples and conducting the field trial: Amabelia del Pino, Gimena Arrarte, Laura Arló, Virginia Takata, Omar Casanova, Claudia Etchebehere, Natalia Bajsa, Natalia Rodríguez, Guillermo Zinola, Guillermo Espinosa, Gastón Carro, Leticia Rogel, Bruno Romano and Melanie Fagúndez.

We also thank Lic. Agustina Grisaro and Bq. Adriana Sucari from Laboratorio de Ensayos, Stamboulian (Argentina), for their support in the MALDI-TOF identification of the Salmonella enterica subsp. enterica isolate.

Financial support

This work was supported by an INIA project, code: FPTA-374.

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Contribution Note

1.Conceptualization, 2.Data curation, 3.Formal analysis, 4.Funding acquisition, 5.Investigation, 6.Methodology, 7.Visualization, 8.Writing-original draft, 9.Writing-review & editing.

Y. Figueroa contribiuted in: 1,2,3,5,6,8; C. Stoletniy: 2; J. Posada: 2; J. Silvera: 2; P. Zunino: 1,4,6, 7,9. A. Umpiérrez: 1,2,3,4,5,6,7,8,9.

Data Availability

The dataset supporting the results of this study is included within the article.

Nota del editor

El editor José Manuel Verdes aprobó este artículo.