A multicenter genomic epidemiological investigation in Brazil, Chile, and Mexico reveals the diversity and persistence of Salmonella populations in surface waters

Salmonella is a major global health concern, responsible for over 95 million cases and more than 50,000 deaths annually, predominantly in developing countries (1). The pathogen causes disease in a wide range of hosts, survives in various environments for extended periods, and is mainly transmitted to humans through contaminated food and water (2), leading to multiple salmonellosis outbreaks each year (2–4). While traditionally associated with meat and poultry, recent outbreaks increasingly implicate produce, such as onions, alfalfa sprouts, peaches, and papayas (5). One of the primary sources of produce contamination is irrigation water (6–9).

In Latin America, as in other regions, surface waters such as rivers, canals, dams, and lakes are used extensively for crop irrigation. Moreover, studies have reported these water sources are vulnerable to contamination with pathogens and other hazards (9, 10). These water sources are critical in understanding the interconnected health of humans, animals, and the environment, as per the One Health approach. They serve as a reliable indicator of regional health and potential reservoirs of various pathogens, including Salmonella.

Global studies have revealed widespread Salmonella contamination in surface waters (11–15). A meta-analysis examining published reports from several countries estimates the presence of Salmonella in surface water to be 31% (16). Recently, high rates of Salmonella contamination in the surface waters of California (56.4%) and Pennsylvania (49%) have also been reported (17). However, not all Salmonella serovars are epidemiologically relevant, and some seem to pose higher risks to humans (2). Studies have identified both high-risk serovars, such as Newport, Typhimurium, and Enteritidis, and less common types in surface water (11, 17–20).

Research specific to surface waters in Latin America is limited, with existing studies often focusing on small areas or single countries and are of a cross-sectional nature (18, 19, 21, 22). Comprehensive and longitudinal research across the region could reveal the diversity and commonality of Salmonella serovars, their resistome, and their ability to persist and disseminate across different regions in Latin America. This investigation aimed to understand the broader picture of Salmonella’s population in agricultural waters in different regions of Latin America to obtain further insights into its role in food safety and its global diversity.

In this study, we described the presence of a large diversity of Salmonella serovars in surface waters of Latin America, with genomes genetically closely related within each country but more distant or unrelated when comparing among countries.

First, Salmonella isolation rates across different regions varied. This was expected because of the regional differences in ecosystem characteristics and climatic conditions, which could affect Salmonella spread and survival in the environment. For instance, sampled regions in Central Chile have a temperate Mediterranean climate; Central Mexico has arid climates; and Brazil has tropical climates that are usually humid. This variability highlights the complexity of Salmonella’s ecological presence, affected by factors such as temperature and rainfall patterns (38). The research teams from Chile, using data from the first 2 years of the project, already explored environmental and anthropogenic factors that could influence the likelihood of isolating Salmonella locally. They found that the sampling month was the factor with more impact on Salmonella isolation and that the anthropogenic variables evaluated only marginally contributed to this result (21). The other teams in the project are currently exploring local factors influencing the phenomena.

Co-occurrence of multiple Salmonella serovars was observed in almost 40% of samples. Samples from Brazil contained the highest number of serovars per sample (n = 10). This can be explained by the fact that the group in Paraiba State, Brazil, used a triplicate-sampling approach, resulting in the filtering of 30 L of water per sample compared with the 10-L samples used by the other groups, which used uniplicate sampling (10 L). In the remaining groups, we observed up to six isolates per sample. This could also have affected the isolation rate comparability among groups as a larger sample volume resulted in higher detection rates and greater diversity across samples. Other studies have described the presence of more than one Salmonella serovar in a single sample. For instance, a study in the Susquehanna River watershed in Pennsylvania reported the presence of up to 10 different Salmonella serovars per sample, with an average of three (17), which is higher than the average of 1.6 from this study. However, Deaven et al. (17) used CRISPR-SeroSeq, an amplicon-based sequencing tool that allows for all Salmonella present in a sample to be identified. In our study, we picked colonies from plates and sequenced up to 10 isolates per sample. Since one serovar might be more abundant than others by several orders of magnitude, picking colonies representing different populations becomes challenging, and it might underestimate the actual Salmonella diversity in our samples. This has been observed in other matrices, such as chicken carcasses and droppings (39, 40). Consequently, our diversity per sample could be even more substantial than reported.

The frequent isolation of S. Newport, S. Typhimurium, and S. Infantis in our samples is noteworthy, given their high relevance in foodborne salmonellosis outbreaks and the emergence of antimicrobial-resistant strains belonging to these serovars. S. Newport has been frequently implicated in foodborne outbreaks linked to produce in the U.S.: alfalfa sprouts in 2010, cantaloupe in 2012, cucumbers in 2014, papayas in 2017, and onions in 2020 (41–43). S. Newport has also been frequently isolated from tomatoes grown on the East Coast in the U.S. (44). This serovar has been reported among the top five serovars causing human clinical cases in Brazil and Mexico for several years (45, 46). Accordingly, this was the most common serovar found in surface water samples from both countries. Although S. Newport is not among the top five most prevalent human serovars in the last Chilean national report (47), it was among the most frequently identified serovars from Chile. S. Typhimurium has been reported as the first or second most frequent cause of salmonellosis in the three participant countries (45–48), and it has been isolated from several other sources in Latin America, such as domestic animals (49), food of animal origin (46), and water (18, 19). This serovar has also caused foodborne outbreaks in the U.S. linked to tomatoes in 2006, cantaloupes in 2012, and alfalfa sprouts in 2022 (50–52). S. Typhimurium high frequency in surface water indicates that this serovar is widespread in the environments surveyed. Interestingly, S. Typhimurium was detected in only one of the two participating groups in Brazil, probably indicating potential risk factors associated with this serovar. S. Infantis is an emerging serovar worldwide, characterized by the presence of a plasmid carrying multiple AMR genes (53). In Chile, it was the second leading cause of salmonellosis in 2018 (47), and multi-resistant isolates have been isolated from poultry facilities (32), chicken meat (33), pet foods (34), and water (19), among other sources. In Brazil, it has also been isolated from diverse sources, including the poultry industry (32), and it has been among the top 10 clinical human serovars since 2013 (45). In Mexico, the presence of this serovar has been associated with beef (46). Moreover, this serovar was linked to an outbreak involving papayas in the U.S. in 2017 (54). Altogether, the presence of these three serovars among the most frequently isolated across the whole study indicates that high-risk salmonellae contaminate surface waters used for irrigation in Latin America and are widespread in the surveyed areas, potentially becoming a risk to consumers.

Other clinically relevant serovars were found in this study. For instance, serovars that have caused outbreaks linked to produce, such as Agona 4.6% (n = 87), Anatum 2.9% (n = 56), Panama 3.8% (n = 73), Muenchen 2.6% (n = 49), Braenderup 2.5% (n = 42), Adelaide 1.8% (n = 34), Oranienburg 1.4% (n = 26), and Javiana 1.2% (n = 22), were present in all three countries in variable frequencies (Table 2). On the other hand, other relevant serovars were not collected from all three countries. For example, Salmonella Enteritidis has been the top serovar linked to human disease in Chile for several years, and it represents a top disease-causing Salmonella in Brazil and Mexico (45–47). Interestingly, almost all S. Enteritidis isolates were collected in Chile, and none came from Brazil. Salmonella Heidelberg has been consistently reported as a top serovar causing salmonellosis in Brazil (45, 48), and it is linked with several foodborne outbreaks in the U.S. (55–57); this serovar was only isolated in samples from Brazil. These variations were expected and might be due to natural and anthropogenic conditions such as climates, fauna, local policies, and sanitation conditions. For example, in Brazil, vaccination in poultry against S. Enteritidis is widespread (58). Since poultry is the main reservoir for this serovar, this practice could restrict the spread of S. Enteritidis into the environment.

It is estimated that by 2050, AMR will be responsible for the deaths of 10 million people annually (59). Enterobacteriaceae is among the bacterial families most significantly impacted by the rise in AMR rates, and Salmonella is no exception (60). In our study, almost one-third of the Salmonella isolates contained AMR determinants, indicating that AMR genetic elements are widespread in the surveyed areas. Moreover, some isolates carried over 10 AMR determinants and were predicted to be resistant to nine antimicrobial families simultaneously. These findings showed that water environments act as a reservoir for AMR determinants, facilitating their dissemination in the environment and possible transfer to other bacteria. Antimicrobial residues in surface waters might increase this problem, which has been described in at least one of the watersheds sampled in this study (61). These findings encourage us to further investigate the phenotypic antimicrobial resistance traits of Salmonella from this study in the future.

The phylogenetic analysis showed intra-serovar diversity. Isolates from the same serovar but collected in different countries were generally not clonal as shown by the multiple cgMLST analysis, including genomes of single serovars, incrementing the technique’s resolution. This highlights the relevance of performing additional analysis in front of apparently closely related genomes to detect whether the phylogenetic relatedness observed is actual or an artifact of the analysis. Some exceptions include some isolates of worldwide spread serovars Enteritidis, Infantis, and Typhimurium; large PD clusters of these serovars contained not only water isolates from multiple countries in the study but also hundreds of genomes of diverse origins, including clinical, animal, and environmental isolates. Numerous plausible hypotheses can be made about the international transmission of these isolates; however, in the absence of a trace-back investigation, these remain speculative at best. This makes it exceedingly challenging to draw definitive conclusions, as the causes may include international food trade, travel, wildlife migration (e.g., avian migration), biofilms in transoceanic boats, or other factors that remain undefined.

Furthermore, we observed that multiple isolates from the same country and serovar were clonal or closely related, even from samples collected 3 years apart, suggesting the long-term persistence of specific strains in water sources. This persistence could be attributed to various factors, including the ability of Salmonella to form biofilms and survive in diverse hosts, among others (62). This is interesting since surface waters can integrate the environment with both animal production and human activities, serving as a valuable sample in the One Health context.

Interestingly, over 50% of the PD clusters comprised genomes generated in this study did not include genomes from other countries, suggesting that other unique, undiscovered Salmonella may exist in unexplored environments and that diversity is widely more extensive than expected. However, some genomes originated from clinical isolates were phylogenetically close to water genomes of this study with differences as close as 0 SNPs. The closeness between some of the water isolates and isolates from clinical cases, especially in the U.S. and the UK, cannot be dismissed, but the epidemiological link between these genomes has yet to be determined. More importantly, this finding highlights the presence of high-risk Salmonella in surface waters in Latin America.

A significant portion of the study was conducted during the COVID-19 pandemic. The disruptions caused by this worldwide event in this study included changes in sampling schedules and locations, and it could have further impacted the representativeness of our findings. As a result, it is important to note that the Salmonella isolation rates obtained from this research are only applicable to the study design and sampled areas and should not be interpreted as representative of the pathogen’s prevalence in surface waters across Latin America or an individual country.

Foodborne outbreaks associated with produce have been linked to irrigation water and, specifically, to surface waters (9, 63). In Latin America, surface waters are one of the primary water sources for crop irrigation (64); therefore, the presence of high-risk foodborne pathogens in these waters could represent a risk to consumers—the three countries in this study, Brazil, Chile, and Mexico, are prominent food exporters worldwide. Mexico is a leading exporter of produce to its neighbor country, the United States, and Chile exports fruits in counter-season to the northern hemisphere. Brazil mainly exports meats, corn, and soybeans, and it is one of the leading producers of fruit juices worldwide. To date, some vegetable-origin foods from these countries have been linked to foodborne outbreaks in the U.S. For example, Mexican onions contaminated with Salmonella Oranienburg were attributed to causing an outbreak in 2021 (65). Accordingly, our study provides valuable information showing that Salmonella isolates present in surface waters in Latin America are highly genetically related to Salmonella found in different food products and to others that have caused clinical cases of salmonellosis. Therefore, it is crucial to investigate foodborne diseases using the One Health perspective to better understand the dynamics of these types of pathogens in the environment and prevent more cases of foodborne diseases.

Surface waters in agricultural regions of Brazil, Chile, and Mexico were investigated in this study. The genomic information gleaned from our sampling efforts sheds light on the persistence and diversity of Salmonella in these regions. The extensive sampling and sequencing employed in our study distinguish it from previous investigations. For a more comprehensive understanding of the ecological dynamics of Salmonella in agricultural surface waters, we integrated these diverse components. While we observed distinctions and similarities in the distribution of Salmonella serovar prevalence among different countries and time periods, we also examined the resistome and evolutionary connections of the isolates. Even though antibiotic resistance, serovar risks, and epidemics are well-known topics, this study provides current data and context for the agricultural regions surveyed. Furthermore, we highlight the persistence of certain Salmonella strains over time, which has important implications for public health interventions.

This research is supported by the FDA of the U.S. Department of Health and Human Services (HHS) as part of financial assistance award U01FDU001418.

We acknowledge the work of Maria Balkey in data submission, the CFSAN strain curation team and the Salmonella HPOP team for strain management at the FDA. Also, we thank Leonela Díaz, Raul Guevara, and Sebastian Gutierrez from INTA, University of Chile; Francisca Álvarez and Constanza Díaz from Pontificia Universidad Católica de Chile; personnel, social service, undergraduate, and graduate students at the Department of Veterinary Preventive Medicine, Faculty of Veterinary Medicine, Universidad Autónoma de Mexico; Alan Douglas L. Rocha, Laiorayne A. Lima, Gustavo F. C. Sales, Elma L. Leite, Nadyra Jeronimo, and Juliana Alves from LAPOA/CCA/ Federal University of Paraiba; Ana Beatriz S. R. da Silva, Ana Paula S. da Silva, Arthur L.L.de Araújo, Dennys M. Girão, Esther H. R. B. Prado, Francisca E. S. Almeida, Luca O. Valdez, Rossiane M. Souza, and Vinicius C. Moura Medical Microbiology Research Laboratory, Paulo de Góes Institute of Microbiology, Federal University of Rio de Janeiro.