Integrative genome-centric metagenomics for surface water surveillance: Elucidating microbiomes, antimicrobial resistance, and their associations.

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.

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.

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.

Reducción, reutilización y reciclaje: Una revisión crítica del conocimiento científico sobre las pérdidas y desperdicios de alimentos en Chile

Reducir, reutilizar y reciclar, "regla de las tres R", son estrategias que promueven una actitud responsable hacia el medioambiente y contribuyen al desarrollo sostenible. El objetivo de esta revisión fue analizar las publicaciones científicas generadas en Chile en el área de las pérdidas y desperdicios de alimentos (PDA), con enfoque en su cuantificación y basadas en "la regla de las tres R". Se realizó una búsqueda de literatura en tres bases de datos: Web of Science, Pubmed y Scopus. Los artículos originales seleccionados se clasificaron según tipo de acción, etapa de la cadena de suministro de alimentos, grupo de alimentos, cuantificación de PDA y tipo de estrategia (reducción, reutilización o reciclaje). Se encontraron 5 artículos de revisión y 31 artículos originales. La revalorización de subproductos agroindustriales fue la principal acción involucrada en los estudios de PDA (n= 22). El tipo de subproducto también fue analizado, y se encontró que los compuestos fenólicos provenientes de frutales son los más estudiados. Solo el 32% (12/31) de los artículos reportaron información sobre cuantificación de PDA. La revalorización de subproductos sumada a la recuperación de alimentos para producción de energía (n= 4) y de compostaje (n= 2) hacen que el reciclaje sea la estrategia más investigada. Estos antecedentes evidencian la necesidad de complementar la investigación nacional a nivel de acciones que apunten más hacia la reducción y reutilización de PDA y su cuantificación. Este conocimiento permitirá establecer líneas base y planes de monitoreo que contribuyan al cumplimiento de los Objetivos de Desarrollo Sostenible en Chile.

Reduce, reuse, and recycle "the 3Rs rule" are strategies that promote a responsible attitude towards the environment and contribute to sustainable development. The objective of this review was to analyze the scientific publications generated in Chile in the area of food loss and waste (FLW), with a focus on its quantification and based on "the 3Rs rule". A literature search was carried out in three databases: Web of Science, Pubmed, and Scopus. The selected original articles were classified according to the type of action, stage of the food supply chain, food group, FLW quantification, and type of strategy (reduction, reuse, or recycling). Five review articles and 31 original articles were found. The revalorization of agro-industrial by-products was the main initiative involved in the FLW studies (n= 22). The type of by-product was also analyzed, and it was found that the phenolic compounds from fruits are the most studied. Only 32% (12/31) of the articles reported information on FLW quantification. The revalorization of by-products added to food recovery for energy production (n= 4) and composting (n= 2) make recycling the most researched strategy. These antecedents show the need to complement the national research at the level of actions that point more towards reducing and reusing of FLW and its quantification. This knowledge will allow the establishment of baselines and monitoring plans that contribute to the fulfillment of the Sustainable Development Goals in Chile.

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.

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.

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.

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.

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.

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.

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.

Diversity of Non-O157 Shiga Toxin-Producing Escherichia coli Isolated from Cattle from Central and Southern Chile.

Cattle are the main reservoir of Shiga toxin-producing Escherichia coli (STEC), one of the world's most important foodborne pathogens. The pathogen causes severe human diseases and outbreaks. This study aimed to identify and characterize non-O157 STEC isolated from cattle feces from central and southern Chile. We analyzed 446 cattle fecal samples and isolated non-O157 STEC from 12.6% (56/446); a total of 93 different isolates were recovered. Most isolates displayed ß-glucuronidase activity (96.8%; 90/93) and fermented sorbitol (86.0%; 80/93), whereas only 39.8% (37/93) were resistant to tellurite. A subgroup of 30 representative non-O157 STEC isolates was selected for whole-genome sequencing and bioinformatics analysis. In silico analysis showed that they grouped into 16 different sequence types and 17 serotypes; the serotypes most frequently identified were O116:H21 and O168:H8 (13% each). A single isolate of serotype O26:H11 was recovered. One isolate was resistant to tetracycline and carried resistance genes tet(A) and tet(R); no other isolate displayed antimicrobial resistance or carried antimicrobial resistance genes. The intimin gene (eae) was identified in 13.3% (4/30) of the genomes and 90% (27/30) carried the stx2 gene. A phylogenetic reconstruction demonstrated that the isolates clustered based on serotypes, independent of geographical origin. These results indicate that cattle in Chile carry a wide diversity of STEC potentially pathogenic for humans based on the presence of critical virulence genes.

Cultivable Yeast Microbiota from the Marine Fish Species Genypterus chilensis and Seriolella violacea.

Because of its outstanding biological and industrial importance, many efforts have been made to characterize the mycobiota of new environments and their biochemical and biotechnological potentials. Gut mycobiota can be a source of novel yeasts with the potential to be used as probiotics or have industrial applications. In this work, we characterized two as-yet unexplored yeast communities from the intestinal content of the cultured marine Chilean fishes Genypterus chilensis (G. chilensis) and Seriolella violacea (S. violacea). Yeasts were isolated through culture, identified by sequencing their ITS region, and characterized their enzymatic profile with API®ZYM. Rhodotorula mucilaginosa was identified in both fish species. For the first time, Candida palmioleophila, Candida pseudorugosa, Cystobasidium slooffiae, and a member of the Yamadazyma genus were also identified and described as part of the normal fish gut-microbiota. Furthermore, the diverse enzymatic profile exhibited by some of these isolates suggests that it may be possible to develop novel applications for them, such as new probiotics and other biotechnological applications.

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.

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.

Corrigendum: Transduction as a Potential Dissemination Mechanism of a Clonal qnrB19-Carrying Plasmid Isolated From Salmonella of Multiple Serotypes and Isolation Sources.

[This corrects the article DOI: 10.3389/fmicb.2019.02503.].

Whole-Genome Phylogenetic Analysis Reveals a Wide Diversity of Non-O157 STEC Isolated From Ground Beef and Cattle Feces.

Shiga toxin-producing Escherichia coli (STEC) causes foodborne outbreaks that can lead to complications such as hemolytic uremic syndrome. Their main reservoir is cattle, and ground beef has been frequently associated with disease and outbreaks. In this study, we attempted to understand the genetic relationship among STEC isolated in Chile from different sources, their relationship to STEC from the rest of the world, and to identify molecular markers of Chilean STEC. We sequenced 62 STEC isolated in Chile using MiSeq Illumina. In silico typing was determined using tools of the Center Genomic Epidemiology, Denmark University (CGE/DTU). Genomes of our local STEC collection were compared with 113 STEC isolated worldwide through a core genome MLST (cgMLST) approach, and we also searched for distinct genes to be used as molecular markers of Chilean isolates. Genomes in our local collection were grouped based on serogroup and sequence type, and clusters were formed within local STEC. In the worldwide STEC analysis, Chilean STEC did not cluster with genomes of the rest of the world suggesting that they are not phylogenetically related to previously described STEC. The pangenome of our STEC collection was 11,650 genes, but we did not identify distinct molecular markers of local STEC. Our results showed that there may be local emerging STEC with unique features, nevertheless, no molecular markers were detected. Therefore, there might be elements such as a syntenic organization that might explain differential clustering detected between local and worldwide STEC.

Transduction as a Potential Dissemination Mechanism of a Clonal qnrB19-Carrying Plasmid Isolated From Salmonella of Multiple Serotypes and Isolation Sources.

Antimicrobial resistance is an increasing problem worldwide, and Salmonella spp. resistance to quinolone was classified by WHO in the high priority list. Recent studies in Europe and in the US reported the presence of small plasmids carrying quinolone resistance in Enterobacteriaceae isolated from poultry and poultry products. The aims of this study were to identify and characterize plasmid-mediated quinolone resistance in Salmonella spp. and to investigate transduction as a possible mechanism associated to its dissemination. First, we assessed resistance to nalidixic acid and/or ciprofloxacin in 64 Salmonella spp. and detected resistance in eight of them. Genomic analyses determined that six isolates of different serotypes and sources carried an identical 2.7-kb plasmid containing the gene qnrB19 which confers quinolone resistance. The plasmid detected also has high identity with plasmids reported in the US, Europe, and South America. The presence of similar plasmids was later surveyed by PCR in a local Salmonella collection (n = 113) obtained from diverse sources: food (eggs), wild and domestic animals (pigs, horse, chicken), and human clinical cases. qnrB19-carrying plasmids were found in 8/113 Salmonella tested strains. A bioinformatics analysis including Chilean and previously described plasmids revealed over 95.0% of nucleotide identity among all the sequences obtained in this study. Furthermore, we found that a qnrB19-carrying plasmid can be transferred between Salmonella of different serotypes through a P22-mediated transduction. Altogether our results demonstrate that plasmid-mediated quinolone resistance (PMQR) is widespread in Salmonella enterica of different serotypes isolated from human clinical samples, wild and domestic animals, and food in Chile and suggest that transduction could be a plausible mechanism for its dissemination. The occurrence of these antimicrobial resistance elements in Salmonella in a widespread area is of public health and food safety concern, and it indicates the need for increased surveillance for the presence of these plasmids in Salmonella strains and to assess their actual impact in the rise and spread of quinolone resistance.