Microbiota composition and susceptibility to florfenicol and oxytetracycline of bacterial isolates from mussels (Mytilus spp.) reared on different years and distance from salmon farms.

Chilean aquaculture mainly produces salmonids and molluscs. Salmonid production has been questioned by its excessive use of antimicrobials. This study aimed to investigate the bacterial microbiota composition of Mytilus spp. cultivated near salmonid farms and to determine the minimum inhibitory concentration (MIC) to florfenicol and oxytetracycline of its culturable bacteria. Seven Mytilus farming sites classified according to their proximity to salmon farms as close (CSF) or distant (DSF) were sampled in two years. We analyzed Mytilus microbiota composition through culture-independent methods, and isolated culturable bacteria, and identified those isolates with MIC values ≥ 64 µg mL-1 to florfenicol or oxytetracycline. Results revealed that the alpha diversity was affected by sampling year but not by Mytilus farming site location or its interaction. Nevertheless, in 2018, we observed a significant negative correlation between the alpha diversity of Mytilus microbiota in each farm sites and the tonnes of florfenicol reported for each phytosanitary management area. We detected significant differences in beta diversity and relative abundance of specific bacterial taxa in Mytilus microbiota depending on the proximity to salmon farms and years. A higher proportion of isolates with MIC values ≥ 64 µg mL-1 to both antibiotics was detected in 2019 compared to 2018, but not significant differences were detected according to Mytilus farming site location. However, in 2019, isolates from CSF sites showed higher MIC values for both antibiotics than those from DSF. Bacterial genera corresponding to isolates with MIC values ≥ 64 µg mL-1 represented a low proportion of Mytilus microbiota identified with the culture-independent approach, reflecting the need to implement new methodologies in the study of antimicrobial resistance. These results suggest that the proximity to salmonid farms and sampling year influence the Mytilus microbiota and MIC values of their bacterial isolates; however, other environmental variables should be considered in further studies.

Isolation and characterization of non-O157 Shiga toxin-producing Escherichia coli (STEC) isolated from retail ground beef in Santiago, Chile.

Shiga toxin-producing Escherichia coli (STEC) is one of the main cause of foodborne disease worldwide, but isolation rates or characteristics of this bacteria from ground beef in Chile are unknown. The present study aimed to isolate and characterize non-O157 STEC from ground beef sold at retail in the city of Santiago, Chile. We analyzed 430 ground beef samples for the presence of STEC, and isolated the microorganism in 10% of samples (43/430). We obtained 56 isolates from the 43 positive samples; 55 of these (98.2%) fermented sorbitol. Most isolates (98.2%; 55/56) showed ß-glucoronidase activity, and only six (10.7%; 6/56) were resistant to tellurite. Among the virulence factors studied (stx1, stx2, eae, and hlyA), stx2 was the only virulence factor in 41% of the isolates (23/56), whereas 10.7% (6/56) of isolates carried a combination of three virulence factors (stx1 + stx2 + hlyA). None of the isolates carried the gene eae. Finally, isolates were neither serogroups O157 nor "big six". In conclusion, ground beef sold in Santiago, Chile is contaminated with STEC; however, further studies are required for understanding their virulence potential.

Approaches to empower the implementation of new tools to detect and prevent foodborne pathogens in food processing.

Foodborne pathogens cause an important public health burden, which is estimated in 600 million cases and more than 400,000 deaths, globally every year. The most susceptible populations, such as children under the age of five, the elderly and immunocompromised, account for the majority of the deaths. Food safety incidents, outbreaks, sporadic cases, and recalls have recognized economic impact, estimated at 7 billion every year in the US. Food safety has become a priority, and the implementation of preventive controls and monitoring systems has raised the development of new tools to detect and prevent pathogens in the food chain. Detection tools have evolved quickly, from rapid testing methods to application of genomics and metagenomics. Importantly, to reduce food safety hazards at food processing, the food chain needs to be seen from farm to fork. This review summarized the main findings discussed during the 2016 OECD-sponsored symposium on food safety. These include i) trends in food safety that embrace the need to implement new tools in detection and prevention, ii) the very rapid evolution of technologies to detect foodborne pathogens, iii) holistic approaches to prevent pathogens require a whole chain approach, and iv) key pillars to facilitate global implementations of new tools in food safety.

Synergistic effect of copper and low temperature over Listeria monocytogenes.

The capacity to grow at low temperatures has allowed Listeria monocytogenes to become one of the primary food pathogens to date, representing a major public health problem worldwide. Several works have described the homeostatic response of L. monocytogenes under different copper (Cu) treatments growing at mild temperature (30 °C). The aims of this report were to evaluate if changes in the external concentration of Cu affected viability and Cu homeostasis of L. monocytogenes growing at low temperature. Ours results showed that L. monocytogenes growing at 8 °C had a reduced viability relative to 30 °C when exposed to Cu treatments. This decrease was correlated with an increase in the internal concentration of Cu, probably linked to the transcriptional down-regulation of mechanisms involved in Cu homeostasis. This combined effect of Cu and low temperature showed a synergistic impact over the viability and homeostasis of L. monocytogenes, where low temperature exacerbated the toxic effect of Cu. These results can be useful in terms of the use of Cu as an antibacterial agent.

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

This study examined the diversity and persistence of Salmonella in the surface waters of agricultural regions of Brazil, Chile, and Mexico. Research groups (three in 2019-2020 and five in 2021-2022) conducted a long-term survey of surface water across 5-8 months annually (n = 30 monthly). On-site, each team filtered 10-L water samples with modified Moore Swabs to capture Salmonella, which were then isolated and identified using conventional microbiological techniques. Salmonella isolates were sequenced on Illumina platforms. Salmonella was present in 1,493/3,291 water samples (45.8%), with varying isolation rates across countries and years. Newport, Infantis, and Typhimurium were the most frequent among the 128 different serovars. Notably, 22 serovars were found in all three countries, representing almost half of the 1,911 different isolates collected. The resistome comprised 72 antimicrobial resistance (AMR) genes and six point mutations in three genes. At least one AMR determinant was observed in 33.8% (646/1,911) of the isolates, of which 47.4% (306/646) were potentially multidrug resistant. Phylogeny based on core genome multilocus sequence typing (cgMLST) showed that most isolates clustered according to sequence type and country of origin. Only 14 cgMLST multi-country clusters were detected among the 275 clusters. However, further analysis confirmed that close genetic relatedness occurred mostly among isolates from the same country, with three exceptions. Interestingly, isolates closely related phylogenetically were recovered over multiple years within the same country, indicating the persistence of certain Salmonella in those areas. In conclusion, surface waters in these regions are consistently contaminated with diverse Salmonella, including strains that persist over time.IMPORTANCESalmonella is a leading foodborne pathogen responsible for millions of illnesses, hospitalizations, and deaths annually. Although Salmonella-contaminated water has now been recognized as an important contamination source in the agrifood chain, there is a lack of knowledge on the global occurrence and diversity of Salmonella in surface water. Moreover, there has been insufficient research on Salmonella in surface waters from Latin American countries that are major producers and exporters of agricultural products. Incorporating genetic profiling of Salmonella isolates from underrepresented regions, such as Latin America, enhances our understanding of the pathogen's ecology, evolution, antimicrobial resistance, and pathogenicity. Moreover, leveraging genomic data derived from pathogens isolated from diverse geographical areas is critical for assessing the potential public health risk posed by the pathogen and expediting investigations of foodborne outbreaks. Ultimately, global efforts contribute significantly to reducing the incidence of foodborne infections.

Genomic characterisation of the population structure and antibiotic resistance of Salmonella enterica serovar Infantis in Chile, 2009-2022.

Background: Multidrug-resistant (MDR) Salmonella Infantis has disseminated worldwide, mainly linked to the consumption of poultry products. Evidence shows dissemination of this pathogen in Chile; however, studies are primarily limited to phenotypic data or involve few isolates. As human cases of Salmonella Infantis infections have substantially increased in recent years, this study aimed to characterise the genomic epidemiology and antimicrobial-resistance profiles of isolates obtained from different sources, aiming to inform effective surveillance and control measures. Methods: We sequenced 396 Salmonella Infantis genomes and analysed them with all publicly available genomes of this pathogen from Chile (440 genomes in total), representing isolates from environmental, food, animal, and human sources obtained from 2009 to 2022. Based on bioinformatic and phenotypic methods, we assessed the population structure, dissemination among different niches, and antimicrobial resistance (AMR) profiles of Salmonella Infantis in the country. Findings: The genomic and phylogenetic analyses showed that Salmonella Infantis from Chile comprised several clusters of highly related isolates dominated by sequence type 32. The HC20_343 cluster grouped an important proportion of all isolates. This was the only cluster associated with pESI-like megaplasmids, and up to 12 acquired AMR genes/mutations predicted to result in an MDR phenotype. Accordingly, antimicrobial-susceptibility testing revealed a strong concordance between the AMR genetic determinants and their matching phenotypic expression, indicating that a significant proportion of HC20_343 isolates produce extended-spectrum ß-lactamases and have intermediate fluoroquinolone resistance. HC20_343 Salmonella Infantis were spread among environmental, animal, food, and human niches, showing a close relationship between isolates from different years and sources, and a low intra-source genomic diversity. Interpretation: Our findings show a widespread dissemination of MDR Salmonella Infantis from the HC20_343 cluster in Chile. The high proportion of isolates with resistance to first-line antibiotics and the evidence of active transmission between the environment, animals, food, and humans highlight the urgency of improved surveillance and control measures in the country. As HC20_343 isolates predominate in the Americas, our results suggest a high prevalence of ESBL-producing Salmonella Infantis with intermediate fluoroquinolone resistance in the continent. Funding: Partially supported by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services as part of an award, FDU001818, with 30% percent funded by FDA/HHS; and by Agencia de Investigación y Desarrollo de Chile (ANID) through FONDECYT de Postdoctorado Folio 3230796 and Folio 3210317, FONDECYT Regular Folio 1231082, and ANID-Millennium Science Initiative Program-ICN2021_044.

Unveiling the genomic landscape of Salmonella enterica serotypes Typhimurium, Newport, and Infantis in Latin American surface waters: a comparative analysis.

Surface waters are considered ecological habitats where Salmonella enterica can persist and disseminate to fresh produce production systems. This study aimed to explore the genomic profiles of S. enterica serotypes Typhimurium, Newport, and Infantis from surface waters in Chile, Mexico, and Brazil collected between 2019 and 2022. We analyzed the whole genomes of 106 S. Typhimurium, 161 S. Newport, and 113 S. Infantis isolates. Our phylogenetic analysis exhibited distinct groupings of isolates by their respective countries except for a notable case involving a Chilean S. Newport isolate closely related to two Mexican isolates, showing 4 and 13 single nucleotide polymorphisms of difference, respectively. The patterns of the most frequently detected antimicrobial resistance genes varied across countries and serotypes. A strong correlation existed between integron carriage and genotypic multidrug resistance (MDR) across serotypes in Chile and Mexico (R > 0.90, P < 0.01), while integron(s) were not detected in any of the Brazilian isolates. By contrast, we did not identify any strong correlation between plasmid carriage and genotypic MDR across diverse countries and serotypes.IMPORTANCEUnveiling the genomic landscape of S. enterica in Latin American surface waters is pivotal for ensuring public health. This investigation sheds light on the intricate genomic diversity of S. enterica in surface waters across Chile, Mexico, and Brazil. Our research also addresses critical knowledge gaps, pioneering a comprehensive understanding of surface waters as a reservoir for multidrug-resistant S. enterica. By integrating our understanding of integron carriage as biomarkers into broader MDR control strategies, we can also work toward targeted interventions that mitigate the emergence and dissemination of MDR in S. enterica in surface waters. Given its potential implications for food safety, this study emphasizes the critical need for informed policies and collaborative initiatives to address the risks associated with S. enterica in surface waters.

Experimental evidence of the anti-bacterial activity pathway of copper ion treatment on Mycobacterium avium subsp. paratuberculosis.

Copper causes significant damage to the integrity of many bacteria, mainly at the DNA level, through its redox states, as well as its reactive oxygen species (ROS) generating capacity at the cellular level. But whether these mechanisms also apply to Mycobacterium avium subsp. paratuberculosis (MAP) is unknown. In the present study, we have evaluated whether copper ions produce damage at the DNA level of MAP, either through their redox states or through ROS production. MAP-spiked PBS was first supplemented with different copper chelators (2) and ROS antioxidants (3), followed by treatment with copper ions at 942 ppm. MAP DNA integrity (qPCR, magnetic phage separation) was then evaluated. We found that bathocuproine (BCS), as a chelator, and D-mannitol, as an antioxidant of hydroxyl radicals, had a significant protective effect (P < 0.05) on DNA molecules, and that EDTA, as a chelator, and D-mannitol, as an antioxidant had a significant positive effect (P < 0.05) on the viability of this pathogen in contrast to the control and other chelators and anti-oxidants used. In light of the reported findings, it may be concluded that copper ions within MAP cells are directly related to MAP DNA damage.

Feed Regime Slightly Modifies the Bacterial but Not the Fungal Communities in the Intestinal Mucosal Microbiota of Cobia Fish (Rachycentron canadum).

The bacterial community of the intestinal microbiota influences many host functions, and similar effects have been recently reported for the fungal community (mycobiota). Cobia is a tropical fish that has been studied for its potential in marine aquaculture. However, the study of its bacterial community has been underreported and the mycobiota has not been investigated. We analyzed the gut bacterial and fungal profile present in the intestinal mucosa of reared adult cobias fed two diets (frozen fish pieces (FFPs) and formulated feed (FF)) for 4 months by sequencing the 16S rRNA (V3-V4) and internal transcribed spacer-2 (ITS2) regions using Illumina NovaSeq 6000. No significant differences in the alpha diversity of the bacterial community were observed, which was dominated by the phyla Proteobacteria (~96%) and Firmicutes (~1%). Cobia fed FF showed higher abundance of 10 genera, mainly UCG-002 (Family Oscillospiraceae) and Faecalibacterium, compared to cobia fed FFPs, which showed higher abundance of 7 genera, mainly Methylobacterium-Methylorubrum and Cutibacterium. The inferred bacterial functions were related to metabolism, environmental information processing and cellular processes; and no differences were found between diets. In mycobiota, no differences were observed in the diversity and composition of cobia fed the two diets. The mycobiota was dominated by the phyla Ascomycota (~88%) and Basidiomycota (~11%). This is the first study to describe the gut bacterial and fungal communities in cobia reared under captive conditions and fed on different diets and to identify the genus Ascobulus as a new member of the core fish mycobiota.

Prevalence of Salmonella in Eggs from Conventional and Cage-Free Egg Production Systems and the Role of Consumers in Reducing Household Contamination.

Salmonella is one of the leading causes of foodborne disease worldwide, usually related to contaminated poultry or poultry products, such as eggs. Since egg contamination with Salmonella depends on multiple factors that make it challenging to control, consumers' knowledge about food safety and the proper handling of eggs is crucial. The aims of the study were (1) to determine the prevalence of Salmonella in eggs from conventional and alternative production systems, (2) to characterize the Salmonella isolates according to phenotypic-genotypic and antimicrobial-resistant traits, and (3) to understand how consumers manage the hazards related to egg contamination in the household. A total of 426 egg samples were analyzed (conventional systems = 240; alternative systems = 186). Culture-based and molecular microbiological methods were used to identify Salmonella and bioinformatics analysis of whole genome sequences was used to determine the serotype and antimicrobial-resistant genes. Salmonella enterica serotype Enteritidis was detected only in eggs from alternative systems (1.1%, 2/186). Isolates showed resistance to nalidixic acid (100%, 2/2), and the aac(6')-Iaa gene and a mutation in the gyrA gene were identified in both isolates. Overall, consumers demonstrated knowledge regarding food safety; however, many still engage in practices that pose a risk of acquiring foodborne illnesses.

Transcriptomic response of Enterococcus faecalis to iron excess.

Iron is an essential nutrient for sustaining bacterial growth; however, little is known about the molecular mechanisms that govern gene expression during the homeostatic response to iron availability. In this study we analyzed the global transcriptional response of Enterococcus faecalis to a non-toxic iron excess in order to identify the set of genes that respond to an increment of intracellular iron. Our results showed an up-regulation of transcriptional regulators of the Fur family (PerR and ZurR), the cation efflux family (CzcD) and ferredoxin, while proton-dependent Mn/Fe (MntH) transporters and the universal stress protein (UspA) were down-regulated. This indicated that E. faecalis was able to activate a transcriptional response while growing in the presence of an excess of non-toxic iron, assuring the maintenance of iron homeostasis. Gene expression analysis of E. faecalis treated with H(2)O(2) indicated that a fraction of the transcriptional changes induced by iron appears to be mediated by oxidative stress. A comparison of our transcriptomic data with a recently reported set of differentially expressed genes in E. faecalis grown in blood, revealed an important fraction of common genes. In particular, genes associated to oxidative stress were up-regulated in both conditions, while genes encoding the iron uptake system (feo and ycl operons) were up-regulated when cells were grown in blood. This suggested that blood cultures mimic an iron deficit, and was corroborated by measuring feo and ycl expression in E. faecalis treated with the iron chelating agent 2,2-dipyridil. In summary, our group identified an adaptive transcriptional mechanism in response to metal ion stress in E. faecalis, providing a foundation for future in-depth functional studies of the iron-activated regulatory network.

Genome wide identification of Acidithiobacillus ferrooxidans (ATCC 23270) transcription factors and comparative analysis of ArsR and MerR metal regulators.

Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophilic bacterium that obtains its energy from the oxidation of ferrous iron, elemental sulfur, or reduced sulfur minerals. This capability makes it of great industrial importance due to its applications in biomining. During the industrial processes, A. ferrooxidans survives to stressing circumstances in its environment, such as an extremely acidic pH and high concentration of transition metals. In order to gain insight into the organization of A. ferrooxidans regulatory networks and to provide a framework for further studies in bacterial growth under extreme conditions, we applied a genome-wide annotation procedure to identify 87 A. ferrooxidans transcription factors. We classified them into 19 families that were conserved among diverse prokaryotic phyla. Our annotation procedure revealed that A. ferrooxidans genome contains several members of the ArsR and MerR families, which are involved in metal resistance and detoxification. Analysis of their sequences revealed known and potentially new mechanism to coordinate gene-expression in response to metal availability. A. ferrooxidans inhabit some of the most metal-rich environments known, thus transcription factors identified here seem to be good candidates for functional studies in order to determine their physiological roles and to place them into A. ferrooxidans transcriptional regulatory networks.

Multiple posttranscriptional regulatory mechanisms partner to control ethanolamine utilization in Enterococcus faecalis.

Ethanolamine, a product of the breakdown of phosphatidylethanolamine from cell membranes, is abundant in the human intestinal tract and in processed foods. Effective utilization of ethanolamine as a carbon and nitrogen source may provide a survival advantage to bacteria that inhabit the gastrointestinal tract and may influence the virulence of pathogens. In this work, we describe a unique series of posttranscriptional regulatory strategies that influence expression of ethanolamine utilization genes (eut) in Enterococcus, Clostridium, and Listeria species. One of these mechanisms requires an unusual 2-component regulatory system. Regulation involves specific sensing of ethanolamine by a sensor histidine kinase (EutW), resulting in autophosphorylation and subsequent phosphoryl transfer to a response regulator (EutV) containing a RNA-binding domain. Our data suggests that EutV is likely to affect downstream gene expression by interacting with conserved transcription termination signals located within the eut locus. Breakdown of ethanolamine requires adenosylcobalamin (AdoCbl) as a cofactor, and, intriguingly, we also identify an intercistronic AdoCbl riboswitch that has a predicted structure different from previously established AdoCbl riboswitches. We demonstrate that association of AdoCbl to this riboswitch prevents formation of an intrinsic transcription terminator element located within the intercistronic region. Together, these results suggest an intricate and carefully coordinated interplay of multiple regulatory strategies for control of ethanolamine utilization genes. Gene expression appears to be directed by overlapping posttranscriptional regulatory mechanisms, each responding to a particular metabolic signal, conceptually akin to regulation by multiple DNA-binding transcription factors.

Microbiological Quality and Presence of Foodborne Pathogens in Raw and Extruded Canine Diets and Canine Fecal Samples.

Pet food can be a source of microbiological hazards that might affect companion animals and owners. Even though owners usually rely on conventional pet diets, such as extruded diets, new feeding practices, such as raw meat-based diets (RMBDs), have grown. RMBDs' benefits are still scientifically uncertain, while its risks have been documented. The use of canine RMBDs might increase the exposure to zoonotic pathogens, such as Salmonella spp., Listeria monocytogenes, Campylobacter spp., among others. Identifying pathogen prevalence in canine food and pets is required to contribute to public health measures. The aims of this study were: (1) to compare the microbiological quality of RMBDs and extruded diets (2) to identify and compare the prevalence of Salmonella spp., Campylobacter jejuni, and L. monocytogenes from raw and extruded canine diets and canine fecal samples, and (3) to characterize pet owners according to the diet chosen to be used on their pets, their motivations for using RMBDs, and their knowledge about benefits and risks related to this feeding practice. Conventional and molecular microbiological methods were used to identify pathogen presence from food and fecal samples, while pulsed-field gel electrophoresis (PFGE) was performed to evaluate the clonal relationship between isolates. Aerobic plate counts for RMBDs were higher than those detected for extruded diets. Salmonella spp. and L. monocytogenes were isolated from 35.7% (15/42) RMBDs, while Salmonella spp., C. jejuni, and L. monocytogenes from 33.3% (11/33) fecal samples from RMBD-fed dogs. From the RMBD samples positive to Salmonella spp., chicken was the main meat ingredient composing the diets. PFGE analysis confirmed a genetic association between Salmonella spp. isolates from fecal and raw food samples from the same household. We did not detect pathogens from extruded food samples or feces from extruded-fed dogs. Using a survey, we identified dog owners' unawareness and/or underestimation of risks related to RMBDs. We demonstrated that canine raw pet food might be a source of zoonotic foodborne pathogens that represent a health risk for both humans and pets. While clinical findings caused by the mentioned pathogens vary among pets, the zoonotic potential implies a significant concern.

More Insights about the Efficacy of Copper Ion Treatment on Mycobacterium avium subsp. paratuberculosis (MAP): A Clue for the Observed Tolerance.

BACKGROUND: Scientific evidence is scarce for the antimicrobial effect of copper on bacteria characterized as more resistant. Using Mycobacterium avium subsp. paratuberculosis (MAP), a highly resistant microorganism, as a pathogen model, copper ion treatment has shown a significant bactericidal effect; however, the sustainability of MAP against copper toxicity was also reported in several studies. Accordingly, the present study aimed to evaluate the impacts of copper on MAP. METHODOLOGY: This study considered physicochemical properties and copper concentration in a buffer since it could modulate MAP response during the application of copper treatment. RESULTS: Despite the efficacy of copper ions in significantly reducing the MAP load in Phosphate Buffered Saline, some MAP cells were able to survive. The copper concentration generated by the copper ion treatment device increased significantly with increasing exposure times. MAP bacterial load decreased significantly when treated with copper ions as the exposure times increased. An increase in pH decreased oxygen consumption, and an increase in conductivity was reported after treatment application. CONCLUSIONS: Even with higher concentrations of copper, the efficacy of MAP control was not complete. The concentration of copper must be a key element in achieving control of highly resistant microorganisms.

Evaluation of the Persistence and Characterization of Listeria monocytogenes in Foodservice Operations.

Listeria monocytogenes is a major foodborne pathogen that can contaminate food products and colonize food-producing facilities. Foodservice operations (FSOp) are frequently responsible for foodborne outbreaks due to food safety practices failures. We investigated the presence of and characterized L. monocytogenes from two FSOp (cafeterias) distributing ready-to-eat meals and verified FSOp's compliance with good manufacturing practices (GMP). Two facilities (FSOp-A and FSOp-B) were visited three times each over 5 months. We sampled foods, ingredients, and surfaces for microbiological analysis, and L. monocytogenes isolates were characterized by phylogenetic analyses and phenotypic characteristics. GMP audits were performed in the first and third visits. A ready-to-eat salad (FSOp-A) and a frozen ingredient (FSOp-B) were contaminated with L. monocytogenes, which was also detected on Zone 3 surfaces (floor, drains, and a boot cover). The phylogenetic analysis demonstrated that FSOp-B had persistent L. monocytogenes strains, but environmental isolates were not closely related to food or ingredient isolates. GMP audits showed that both operations worked under "fair" conditions, and "facilities and equipment" was the section with the least compliances. The presence of L. monocytogenes in the environment and GMP failures could promote food contamination with this pathogen, presenting a risk to consumers.

Environmental and anthropogenic factors associated with the likelihood of detecting Salmonella in agricultural watersheds.

Surface water is one of the primary sources of irrigation water for produce production; therefore, its contamination by foodborne pathogens, such as Salmonella, may substantially impact public health. In this study, we determined the presence of Salmonella in surface water and characterized the relationship between Salmonella detection and environmental and anthropogenic factors. From April 2019 to February 2020, 120 samples from 30 sites were collected monthly in four watersheds located in two different central Chile agricultural regions (N = 1080). Water samples from rivers, canals, streams, and ponds linked to each watershed were obtained. Surface water (10 L) was filtrated in situ, and samples were analyzed for the presence of Salmonella. Salmonella was detected every month in all watersheds, with a mean detection percentage of 28% (0%-90%) across sampling sites, regardless of the season. Overall, similar detection percentages were observed for both regions: 29.1% for Metropolitan and 27.0% for Maule. Salmonella was most often detected in summer (39.8% of all summer samples tested positive) and least often in winter (14.4% of winter samples). Random forest analysis showed that season, water source, and month, followed by latitude and river, were the most influential factors associated with Salmonella detection. The influences of water pH and temperature (categorized as environmental factors) and factors associated with human activity (categorized as anthropogenic factors) registered at the sampling site were weakly or not associated with Salmonella detection. In conclusion, Salmonella was detected in surface water potentially used for irrigation, and its presence was linked to season and water source factors. Interventions are necessary to prevent contamination of produce, such as water treatment before irrigation.

CutC is induced late during copper exposure and can modify intracellular copper content in Enterococcus faecalis.

Copper is a micronutrient that is required for proper metabolic functioning of most prokaryotic and eukaryotic organisms. To sustain an adequate supply of copper, a cell requires molecular mechanisms that control the metal content to avoid copper toxicity. This toxicity comes primarily from the reactivity of copper, which can lead to the generation of free radicals. In bacteria, two independent systems are responsible for maintaining the balance of copper within the cells (Cop and Cut family proteins). Previous studies describe CutC as a member of the Cut family that is probably involved in copper homeostasis. However, the role of CutC in copper homeostasis is still unclear. In this work, a homolog of CutC was studied in Enterococcus faecalis, a bacterial model for copper homeostasis. The molecular 3D model of efCutC shows the presence of triose phosphate isomerase (TIM) barrel motifs, previously described in CutC crystals from other organisms, which illustrates the conservation of amino acids with the potential ability to coordinate copper. Through quantitative real-time PCR (qPCR), it was demonstrated that efcutC expression is induced late by copper stimulus, Interestingly this transcriptional response directly correlates with a significant increase in the intracellular copper concentration when the protein is absent in the bacteria, suggesting its participation in mechanisms related to efflux of the metal. Our results describe efCutC as a protein able to respond transcriptionally to copper and to participate in the control of copper homeostasis in E. faecalis. This bacterium is the first reported organism containing a cop operon and an active member of the Cut protein family.

Co-occurrence Interaction Networks of Extremophile Species Living in a Copper Mining Tailing.

Copper mining tailings are characterized by high concentrations of heavy metals and an acidic pH, conditions that require an extreme adaptation for any organism. Currently, several bacterial species have been isolated and characterized from mining environments; however, very little is known about the structure of microbial communities and how their members interact with each other under the extreme conditions where they live. This work generates a co-occurrence network, representing the bacterial soil community from the Cauquenes copper tailing, which is the largest copper waste deposit worldwide. A representative sampling of six zones from the Cauquenes tailing was carried out to determine pH, heavy metal concentration, total DNA extraction, and subsequent assignment of Operational Taxonomic Units (OTUs). According to the elemental concentrations and pH, the six zones could be grouped into two sectors: (1) the "new tailing," characterized by neutral pH and low concentration of elements, and (2) the "old tailing," having extremely low pH (~3.5) and a high concentration of heavy metals (mainly copper). Even though the abundance and diversity of species were low in both sectors, the Pseudomonadaceae and Flavobacteriaceae families were over-represented. Additionally, the OTU identifications allowed us to identify a series of bacterial species with diverse biotechnological potentials, such as copper bioleaching and drought stress alleviation in plants. Using the OTU information as a template, we generated co-occurrence networks for the old and new tailings. The resulting models revealed a rearrangement between the interactions of members living in the old and new tailings, and highlighted conserved bacterial drivers as key nodes, with positive interactions in the network of the old tailings, compared to the new tailings. These results provide insights into the structure of the soil bacterial communities growing under extreme environmental conditions in mines.

Probiotic Yeasts and Vibrio anguillarum Infection Modify the Microbiome of Zebrafish Larvae.

The host microbiome plays an essential role in health and disease. Microbiome modification by pathogens or probiotics has been poorly explored especially in the case of probiotic yeasts. Next-generation sequencing currently provides the best tools for their characterization. Debaryomyces hansenii 97 (D. hansenii 97) and Yarrowia lipolytica 242 (Y. lipolytica 242) are yeasts that protect wildtype zebrafish (Danio rerio) larvae against a Vibrio anguillarum (V. anguillarum) infection, increasing their survival rate. We investigate the effect of these microorganisms on the microbiome and neutrophil response (inflammation) in zebrafish larvae line Tg(Bacmpx:GFP) i114. We postulated that preinoculation of larvae with yeasts would attenuate the intestinal neutrophil response and prevent modification of the larval microbiome induced by the pathogen. Microbiome study was performed by sequencing the V3-V4 region of the 16S rRNA gene and prediction of metabolic pathways by Piphillin in conventionally raised larvae. Survival and the neutrophil response were both evaluated in conventional and germ-free conditions. V. anguillarum infection resulted in higher neutrophil number in the intestinal area compared to non-infected larvae in both conditions. In germ-free conditions, infected larvae pre-inoculated with yeasts showed fewer neutrophil numbers than infected larvae. In both conditions, only D. hansenii 97 increased the survival of infected larvae. Beta diversity of the microbiota was modified by V. anguillarum and both yeasts, compared to non-inoculated larvae. At 3 days post-infection, V. anguillarum modified the relative abundance of 10 genera, and pre-inoculation with D. hansenii 97 and Y. lipolytica 242 prevented the modification of 5 and 6 of these genera, respectively. Both yeasts prevent the increase of Ensifer and Vogesella identified as negative predictors for larval survival (accounting for 40 and 27 of the variance, respectively). In addition, yeast pre-inoculation prevents changes in some metabolic pathways altered by V. anguillarum's infection. These results suggest that both yeasts and V. anguillarum can shape the larval microbiota configuration in the early developmental stage of D. rerio. Moreover, modulation of key taxa or metabolic pathways of the larval microbiome by yeasts can be associated with the survival of infected larvae. This study contributes to the understanding of yeast-pathogen-microbiome interactions, although further studies are needed to elucidate the mechanisms involved.