Salmonella enterica subspecies enterica, particularly serovar Newport, remains a leading cause of foodborne illnesses in the United States, implicated in numerous outbreaks associated with a diverse array of food products. This study systematically investigates the virulence of five distinct S. Newport isolates, characterized by varying patterns of pulse-field gel electrophoresis (PFGE) molecular-diagnostic subtyping, using the nematode Caenorhabditis elegans as a host model organism. We conducted viability assays on C. elegans to evaluate how these isolates affect nematode survival. The selected bacterial strains, chosen for their historical significance in foodborne outbreaks yet isolated form environmental sources, were previously sequenced to provide a comprehensive genomic framework. A notable focus of our research was on the nearly genetically identical PFGE types Newport-61 and the Newport-1015 isolates, which differ by a 1.7 Mb genomic inversion. C. elegans survival assays in response to pathogenic-strain infections revealed that one Newport-1015 and the Newport-61 isolates were particularly more virulent compared to other isolates tested. These findings enhance our understanding of the pathogenic potential of environmental S. Newport and highlight the need to understand the regulatory mechanisms that contribute to virulence capacity.

The global dissemination of multidrug-resistant (MDR) Salmonella through the international food trade poses a major One Health concern. We used whole-genome sequencing to characterize Salmonella isolates from poultry meat sold in Chile, including domestic and imported products from Brazil and Argentina. Sixty-one Salmonella isolates were recovered from poultry meat; S. Infantis predominated (59%), followed by S. Heidelberg. Among S. Heidelberg from imported-meat poultry, 92% carried the blaCMY-2 gene, conferring resistance to β-lactams. Given the predominance of S. Infantis in poultry meat, we performed an additional in-depth genomic analysis of 73 S. Infantis isolates obtained from poultry meat (n = 32), surface water (n = 30), and human clinical cases (n = 11). Across sources, phenotypic resistance to ciprofloxacin and third-generation cephalosporins reached 93% and 70%, respectively, and MDR (≥3 antimicrobial classes) occurred in 71% of isolates, largely associated with blaCTX-M-65 and gyrA mutations. The pESI (plasmid of emerging S. Infantis)-like plasmid, harboring antimicrobial resistance and virulence genes, appeared in 94% of isolates. Phylogenetic analyses showed close genetic relationships among food, environmental, and clinical isolates, suggesting potential transmission through contaminated poultry meat or water. These findings emphasize the emergence of MDR S. Infantis in Chile and underscore the need for integrated One Health surveillance and prudent antimicrobial use to mitigate foodborne AMR risks.

[This corrects the article DOI: 10.1371/journal.pone.0343882.].

We assessed the prevalence, persistence, genetic diversity, and AMR dynamics of Salmonella spp. (SAL) in surface waters (SuWa) of Central Mexico. On-site, 953 samples (10 L each) from 49 watersheds were collected in 24 sampling rounds (2019-2023) using the modified Moore swab technique. SAL was isolated and identified using conventional microbiological procedures and sequenced on Illumina platforms. Overall, the prevalence of SAL was 54%, with higher values (χ2=23.9, P<0.0001) observed in Tlaxcala and the State of Mexico (64%) than in the other regions (42-50%). The top 10 serovars (Agona, Anatum, Newport, London, Adelaide, Derby, Senftenberg, Infantis, Typhimurium, and Muenchen) represented nearly half of the population and were systematically isolated across sampling rounds and years. The most frequent antimicrobial resistance (AMR) phenotypes involved older antibiotics (e.g., tetracycline, streptomycin, and chloramphenicol: 36%, 33%, and 24%, respectively). Approximately 12% of the isolates showed resistance to azithromycin, whereas resistance to cephalosporins, carbapenems, amikacin, and fluoroquinolones was rarely observed (~0-5%). One-third of the isolates exhibited multidrug-resistant (MDR) phenotypes and genotypes. The overall pool of AMR determinants (62 genes and four gene point mutations) encoded 15 different resistance mechanisms at the population level. The prevalence of AMR phenotypes to older antibiotics and the abundance of AMR genes against all antimicrobial classes increased in the post-COVID-19 pandemic period (0.1-0.4 and 0.1-0.6 Log10-fold change, respectively). These results underscore the public health risks associated with agricultural SuWa and the need for further research to identify the sources of SAL contamination.

We analyzed the prevalence dynamics, genetic diversity, and AMR dynamics of Salmonella spp. (SAL) across surface waters (SuWa) in Central Mexico. On-site, 953 samples (10 L each) from 49 watersheds were collected in 24 sampling rounds (2019–2024) using the modified Moore swab technique. SAL was isolated and identified using conventional microbiological procedures and sequenced on Illumina platforms. Overall, the prevalence of SAL was 54

Pre-exposure to sub-lethal stress can increase the resistance of foodborne pathogens to inactivation processes, posing potential risks to food safety. This study examined how sub-lethal stress influences the resistance of Salmonella enterica to ultraviolet-C (UV-C) treatments on raw whole almonds (RWAs) and fresh-cut leafy greens (FCLGs), investigated the role of rpoS in stress-induced cross-protection, and evaluated Enterococcus faecium NRRL B-2354 as a surrogate for S. enterica. Additionally, we assessed the survival of sub-lethally stressed cells on FCLGs under cold or temperature abuse condition post-UV-C treatment. A cocktail of three S. enterica strains, along with S. Typhimurium ATCC 14028 and its ΔrpoS mutant (IB43), were exposed to desiccation stress, heat shock, oxidation stress, or acid stress. Afterward, stressed and unstressed cells were inoculated onto RWAs and FCLGs, and treated with UV-C (500 μW/cm2, 60 min). Treated FCLGs were then stored under cold or temperature abuse condition for 7 days. Results showed that acid-stressed S. enterica exhibited greater UV-C resistance on RWAs, while oxidation-stressed cells had increased survival on FCLGs (p < 0.05). Under temperature abuse, unstressed, oxidation-stressed, or acid-stressed S. enterica were inactivated faster, whereas heat-shocked cells persisted until Day 7. Desiccation-stressed cells rebounded temporarily before inactivation by Day 7. IB43 was more susceptible to UV-C (p < 0.05) than the wild-type strain and lacked cross-protection from prior sub-lethal stress exposure, confirming the crucial role of rpoS in UV-C resistance and stress adaptation. NRRL B-2354 demonstrated comparable or greater survival than S. enterica, supporting its use as a suitable surrogate. These findings highlight the influence of sub-lethal stress on UV-C resistance in S. enterica and emphasize the importance of including stress-adapted pathogens in challenge studies to improve food safety.

ABSTRACT Salmonella enterica is a significant foodborne pathogen frequently associated with produce contamination through irrigation with untreated surface water. This study investigated the effects of sampling replication and selective media on S. enterica serovar recovery and diversity in surface waters across Paraíba State, Brazil. Water samples ( n = 200) were collected from 10 reservoirs using modified Moore swabs (MMSs), with three replicates per sampling site, yielding 600 samples. The prevalence of S. enterica was 53.5% in individual samples, increasing to 68.5% when combining results from triplicate samples. Statistical analyses demonstrated that triplicate sampling significantly enhanced both recovery rates and serovar diversity compared with single samples ( P < 0.05). Alpha diversity metrics revealed a 39% increase in serovar richness when using triplicate versus single samples. The parallel use of multiple enrichment broths and selective agars proved essential for maximum serovar detection, as certain serovars were recovered exclusively under specific culture conditions. While triplicate sampling provided optimal detection sensitivity, duplicate sampling emerged as a cost-effective alternative, maintaining sufficient statistical power. These findings demonstrate the effectiveness of MMS combined with replicate sampling for comprehensive S. enterica surveillance in surface waters, with implications for water quality monitoring programs and public health risk assessment. IMPORTANCE Salmonella contamination in irrigation water poses a major threat to food safety, as contaminated produce can cause widespread foodborne illness outbreaks affecting thousands of people. Current water monitoring methods often miss these hazardous bacteria, creating blind spots in our food safety systems. This research addresses a critical gap by demonstrating that taking multiple water samples from the same location, rather than just single samples, improves our ability to detect Salmonella contamination. The study shows that collecting three samples instead of one increases the detection rate from 54% to 69% and reveals nearly 40% more serovars. This enhanced detection capability is crucial for protecting public health, as it provides more accurate information about the occurrence of Salmonella in natural surface waters. Lastly, our findings provide practical guidance for improving surveillance programs worldwide, offering a cost-effective approach that significantly strengthens our defense against Salmonella contamination in the food supply chain.

We report the draft genome sequences of 94 multidrug-resistant Salmonella isolates (S. Infantis, Enteritidis, and Typhimurium) from organic and conventional retail chickens in Maryland, USA (average genome size = 4.97 Mb; guanine-cytosine content = 52.12%). These isolates harbored diverse antimicrobial resistance and virulence genes, Salmonella pathogenicity islands, and plasmids, highlighting potential public health risks.

Considering the increasing reports of Salmonella enterica strains resistant to quinolones, antimicrobials frequently employed as therapeutic agents globally, our goal was to investigate the occurrence of plasmid-mediated quinolone resistance (PMQR) determinants in S. enterica recovered from natural surface waters in Paraíba state, Brazil. Water samples (n = 230) were collected monthly in triplicate using modified Moore swabs from 29 sampling sites belonging to 10 large dams. After conventional microbial isolation, representative isolates (n = 938) were submitted to whole genome sequencing, assembly and annotation. Antimicrobial resistance genes (ARGs) were identified, and core genome multilocus sequence typing (cgMLST) was used to infer phylogenetic relationships. Among recovered S. enterica, 130 (13.9%) isolates harboured PMQR determinants; 124 (95.4%) harboured qnrB19, while 6 (4.6%) harboured qnrS1. Multiple other ARGs associated with resistance to aminoglycosides, β-lactams, sulphonamides, tetracyclines and fosfomycin were identified. The diversity of ARGs and plasmids suggests a highly complex resistance landscape. Phylogenetic analysis revealed clustering by serovar and sequence type but not by resistance profile or geographic origin. The absence of association between phylogeny and ARGs highlights the potential role of horizontal gene transfer in disseminating resistance genes in water. Our findings reinforce the importance of antimicrobial resistance surveillance in surface waters.

This study aimed to detect and characterize Listeria monocytogenes (L. monocytogenes) in irrigation water to help reduce the risk of foodborne illnesses. Over nearly a year, water samples (n = 1050) were collected monthly from 120 locations across four Chilean watersheds to assess the prevalence and genetic diversity of the pathogen. The average detection of L. monocytogenes across all watersheds ranged from 15 % to 25 %, underscoring the variability in L. monocytogenes contamination at various sites. Water type, collection month, watershed, pH and season were the primary determinants of the likelihood of L. monocytogenes in surface waters. The genetic analysis of the isolates revealed that clonal L. monocytogenes isolates were identified across regions and were collected throughout the study period. Genomic characterization revealed that most of the 221 isolates sequenced were of serogroup IVb. Virulence-related genes in LIPI1 were present in almost all isolates, while LIPI3 and LIPI4 were less common. Moreover, although infrequent, we found three isolates carrying antimicrobial resistance genes mefA and msrD. These results highlight the genetic diversity of L. monocytogenes in Chilean freshwater environments. At the same time, the environmental persistence ability of L. monocytogenes strains or the presence of reservoirs across different areas could explain the dissemination and persistence of some strains. Ultimately, we did not identify an association between the presence of Salmonella and L. monocytogenes in these watersheds. Insights from this study are vital for improving monitoring efforts and developing targeted control strategies to mitigate public health risks.

Salmonella enterica remains a major foodborne pathogen globally but little attention has been paid to infrequent serovars in environmental settings. We report the occurrence of 30 rare S. enterica serovars isolated from environmental water sources between 2021 and 2022 in semiarid northeastern Brazil. We conducted two risk-based field campaigns at shoreline access points in 10 reservoirs associated with the three largest river basins in the state. Salmonella enterica was recovered from 175 out of 230 water samples, yielding 2903 isolates. Of these, 938 were selected for whole-genome sequencing (WGS). Genome assembly and downstream analyses identified 65 unique serovars, including 68 isolates belonging to 30 rare serovars. Salmonella Carrau (n = 14), S. Oran (n = 9), S. Gaminara (n = 5), and S. Urbana (n = 4) were the most frequent rare serovars. WGS analysis revealed the presence of antimicrobial resistance genes (ARGs) in all isolates. The highest abundances were associated with ARGs conferrying resistance to aminoglycosides [aac(6')-Iaa (100%)], quinolones (parC:p.T57S [98.1%] and qnrB19 [3.77%]), and fosfomycin (fosA7 [3.77%]). Some isolates carried plasmids (IncX3, IncFII [S], IncFII [Cf], Col [pHAD28], and IncFII [SARC14]) that could facilitate the spread of antimicrobial resistance. Phylogenetic analysis indicated the presence of distinct clades for each serovar. Interestingly, 20 serovars are endemic lineages circulating in Brazil, except S. Kiambu, which belongs to an international lineage. These findings underscore the importance of environmental monitoring and understanding the distribution of Salmonella in water sources to safeguard public health and prevent the spread of antimicrobial resistance.

ABSTRACT Surface waters function as ecological niches where Salmonella enterica can persist and disseminate to fresh produce production systems. We examined the genomic characteristics of S. enterica serotypes Agona (n = 86), Braenderup (n = 47), Muenchen (n = 53), and Panama (n = 69) isolates from surface waters in Chile, Mexico, and Brazil between 2019 and 2022. Mexican isolates consistently displayed a higher occurrence of genotypic antimicrobial resistance (AMR) than Chilean and Brazilian isolates. All S. Agona isolates exhibited the presence of fosA7.2, while qnrB19 emerged as the predominant AMR gene (ARG) among S. Braenderup isolates. S. Muenchen isolates from Chile displayed an absence of any ARGs, while those from Mexico and Brazil predominantly carried qnrB19. Among S. Panama isolates from Chile, aadA1, floR, sat2, and tet(B) were the most prevalent ARGs, whereas those from Mexico and Brazil harbored tet(A), and floR and tet(A) as the leading ARGs, respectively. ARG sharing among isolates and ARG co-occurrence within individual isolates were prevalent across countries and serotypes. All isolates containing integrons exhibited genotypic multidrug resistance. The principal coordinates analysis reveals distinct clustering patterns based on country, serotype, number of ARGs per isolate, and plasmid and integron presence/absence. The whole-genome phylogenetic analysis demonstrates clear clusters, each associated with their respective countries. However, a notable exception was observed with one S. Agona isolate from Brazil closely related to two isolates from Chile, differing by only 18 and 19 single-nucleotide polymorphisms, respectively.IMPORTANCEThis comprehensive study explored the intricate genomic landscapes of S. Agona, Braenderup, Muenchen, and Panama isolates from surface waters across Chile, Mexico, and Brazil. By filling important knowledge gaps related to the genomic characteristics of these serotypes, the research offers a nuanced understanding of these serotypes as potential reservoirs for multidrug resistance. Our findings emphasize the urgency of targeted interventions to mitigate the emergence and dissemination of multidrug-resistant Salmonella enterica. This work underscores the need for informed policies and collaborative efforts to address the risks posed by S. enterica in Latin American surface waters.

Sub-lethal stress can induce cross-protection in pathogens, complicating food processing. We evaluated the combined effects of chlorine dioxide (CD) and ultraviolet-C (UV-C) on sub-lethally stressed Salmonella enterica on raw whole almonds (RWAs) and fresh-cut leafy greens (FCLGs), post-treatment survival on FCLGs, and Enterococcus faecium NRRL B-2354 as a S. enterica surrogate. S. enterica cocktails (RWAs: S. Enteritidis, Newport, Typhimurium; FCLGs: S. Enteritidis Montevideo, Typhimurium) were subjected to desiccation, oxidation, heat shock, or acid stress. Stressed and unstressed S. enterica and NRRL B-2354 were inoculated onto RWAs and FCLGs, and treated with CD (0-0.6 mg/L, 0-30 min) or CD + UV-C [CD (0-0.4 mg/L, 0-15 min) followed by UV-C (254 nm, 0-450 mJ/cm2, 0-15 min)]. Treated FCLGs were stored under cold storage (4 °C) or temperature abuse (35 °C for 2 h, then 4 °C) for seven days. CD + UV-C produced < 5.6- and ≥ 5.6-log reductions on RWAs and FCLGs, respectively, while CD achieved < 2.0- and < 2.5-log reductions, respectively. Desiccation- or oxidation-stressed cells were more resistant (P < 0.05) than unstressed or other stressed cells. Treatment (CD vs. CD + UV-C) and microorganism (S. enterica vs. NRRL B-2354) were dominant factors influencing inactivation. Association rule mining identified CD + UV-C on S. enterica yielding high reductions. Stressed cells declined more rapidly under cold storage, while desiccation-stressed cells persisted under temperature abuse. NRRL B-2354 showed lower reductions than S. enterica (P < 0.05), supporting its use as a surrogate with safety margin. These results support CD + UV-C as an effective hurdle strategy and highlight the importance of stress adaptation in validating interventions.

Multidrug-resistant Salmonella is becoming a public health hazard. This study aimed to investigate the genomic diversity of the selected Salmonella serovars isolated from organic and non-organic chickens using whole-genome sequencing (WGS). A total of 94 Salmonella isolates [Infantis (n = 71), Enteritidis (n = 13), Typhimurium (n = 10)] recovered from organic and non-organic retail chickens were subjected to WGS using Illumina MiSeq. All Salmonella isolates contained at least one antibiotic resistance (AR) gene (ARG). All S. Typhimurium and S. Enteritidis isolates, and 98.6 % of S. Infantis isolates possessed aac(6')-Iaa which predicted resistant to aminoglycosides. Most isolates harbored ARGs for sulfonamides, β-lactamase inhibitors, tetracyclines, and fluoroquinolone/quinolone resistance regardless of chicken types. Fisher's exact test indicated a significantly higher prevalence of ARGs in Salmonella isolated from organic chickens. The virulence genes that stimulate Salmonella pathogenicity island 1 (SPI1) encoding, type three secretion system 1 (TTSS-1) translocated effectors, SPI2 encoding, TTSS-2 translocated effectors, fimbrial adherence determinants, serum resistance, stress adaptation and Mg2+ uptake were observed in every serovar regardless of chicken types. Seven plasmids were detected in Salmonella isolates from non-organic chicken, including IncFIB(pN55391), IncFIB(S), Col(BS512), IncFII(S), IncX1, IncC, and ColpVC, whereas Salmonella isolates from organic chicken carried only three plasmids [IncFIB(pN55391), Col(BS512), and IncC]. Phylogenetic analysis confirmed that a significant portion of the isolates had single nucleotide polymorphism (SNP) distances greater than the threshold (SNP ≤ 20), indicating genetic distance among them. These results suggest that the Salmonella isolates from organic and non-organic chickens possessed various AR and virulence genes and thus have the potential to cause salmonellosis.

Salmonella enterica (S. enterica), a ubiquitous zoonotic foodborne pathogen, remains a worldwide public health hazard and economic burden. In recent years, outbreaks associated with the consumption of plant-based foods probably contaminated by irrigation water highlights the importance of water sources. This study investigated anthropogenic and environmental factors influencing S. enterica occurrence in natural watersheds impacted by agricultural and livestock industries in a 10-month longitudinal study in Paraiba, Brazil. Water samples were obtained from multiple sites within the three major river basins by modified Moore Swabs (MMS) and processed by conventional S. enterica isolation methodologies. Physicochemical parameters, climate, and human activities near the water sources were recorded. A logistic regression model was fitted using Generalized Linear Model (GLM) and further adjusted according to the selected variables using the Least Absolute Shrinkage and Selection Operator (LASSO) method. A non-statistical decision tree model was also fitted using the rpart package in R. Season, rainfall regime, water physicochemical features, and anthropogenic activities were significantly associated with S. enterica contamination. According to the regression tree analysis, rainfall within the sampling month was the strongest predictor of S. enterica recovery, potentially due to leaching from soil or runoff from adjacent human and animal activities. The complexity of multivariate conditions driving S. enterica contamination in surface waters highlights the need for region-specific investigations.

Salmonella enterica serovar Newport is a major contributor to the burden of salmonellosis in the United States. We report the closed genomic sequences of 14 S. Newport isolates collected from various sources in the United States. All consist of one circular chromosome, and eight have at least one circular plasmid.

Surface water ecosystems are intimately intertwined with anthropogenic activities and have significant public health implications as primary sources of irrigation water in agricultural production. Our extensive metagenomic analysis examined 404 surface water samples from four different geological regions in Chile and Brazil, spanning irrigation canals (n = 135), rivers (n = 121), creeks (n = 74), reservoirs (n = 66), and ponds (n = 8). Overall, 50.25 % of the surface water samples contained at least one of the pathogenic or contaminant bacterial genera (Salmonella: 29.21 %; Listeria: 6.19 %; Escherichia: 35.64 %). Furthermore, a total of 1,582 antimicrobial resistance (AMR) gene clusters encoding resistance to 25 antimicrobial classes were identified, with samples from Brazil exhibiting an elevated AMR burden. Samples from stagnant water sources were characterized by dominant Cyanobacteriota populations, resulting in significantly reduced biodiversity and more uniform community compositions. A significant association between taxonomic composition and the resistome was supported by a Procrustes analysis (p < 0.001). Notably, regional signatures were observed regarding the taxonomic and resistome profiles, as samples from the same region clustered together on both ordinates. Additionally, network analysis illuminated the intricate links between taxonomy and AMR at the contig level. Our deep sequencing efforts not only mapped the microbial landscape but also expanded the genomic catalog with newly characterized metagenome-assembled genomes (MAGs), boosting the classification of reads by 12.85 %. In conclusion, this study underscores the value of metagenomic approaches in surveillance of surface waters, enhancing our understanding of microbial and AMR dynamics with far-reaching public health and ecological ramifications.

Paired-end short reads of Illumina HiSeq, MiSeq, and NovaSeq of simulated bacterial communities from fresh spinach and surface water were generated in silico at various sequencing depths. Multidrug-resistant Salmonella enterica serotype Indiana was included in the spinach community, while the water community contained multidrug-resistant Pseudomonas aeruginosa.

Akkermansia muciniphila pretreatment mitigated Listeria monocytogenes infection in mice. A. muciniphila improved gut microbiota disturbed by L. monocytogenes infection and significantly increased the level of intestinal linoleic acid in mice. Linoleic acid strengthened the intestinal epithelial barrier and reduced pathogen translocation partly by regulating NF-κB/MLCK pathway in a GPR40-dependent manner.