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ABSTRACT 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.

ABSTRACTSurface 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.

Summary: 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 β-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.

Abstract Background Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans. This pathogen activates multiple regulatory mechanisms in response to stress, and cobalamin biosynthesis might have a potential role in bacterial protection. Low temperature is a strategy used in the food industry to control bacteria proliferation; however, L. monocytogenes can grow in cold temperatures and overcome different stress conditions. In this study we selected L. monocytogenes List2-2, a strain with high tolerance to the combination of low temperature + copper, to understand whether the cobalamin biosynthesis pathway is part of the tolerance mechanism to this stress condition. For this, we characterized the transcription level of three cobalamin biosynthesis-related genes (cbiP, cbiB, and cysG) and the eutV gene, a transcriptional regulator encoding gene involved in ethanolamine metabolism, in L. monocytogenes strain List2-2 growing simultaneously under two environmental stressors: low temperature (8 °C) + copper (0.5 mM of CuSO4 × 5H2O). In addition, the gene cbiP, which encodes an essential cobyric acid synthase required in the cobalamin pathway, was deleted by homologous recombination to evaluate the impact of this gene in L. monocytogenes tolerance to a low temperature (8 °C) + different copper concentrations. Results By analyzing the KEGG pathway database, twenty-two genes were involved in the cobalamin biosynthesis pathway in L. monocytogenes List2-2. The expression of genes cbiP, cbiB, and cysG, and eutV increased 6 h after the exposure to low temperature + copper. The cobalamin cbiP mutant strain List2-2ΔcbiP showed less tolerance to low temperature + copper (3 mM) than the wild-type L. monocytogenes List2-2. The addition of cyanocobalamin (5 nM) to the medium reverted the phenotype observed in List2-2ΔcbiP. Conclusion These results indicate that cobalamin biosynthesis is necessary for L. monocytogenes growth under stress and that the cbiP gene may play a role in the survival and growth of L. monocytogenes List2-2 at low temperature + copper.

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.

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.

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.

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.

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.

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.

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.

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.

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 β-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.

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.

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.

Listeria monocytogenes is a foodborne pathogen that can cause severe disease in susceptible humans. This microorganism has the ability to adapt to hostile environmental conditions such as the low temperatures used by the food industry for controlling microorganisms. Bacteria are able to adjust their transcriptional response to adapt to stressful conditions in order to maintain cell homeostasis. Understanding the transcriptional response of L. monocytogenes to stressing conditions could be relevant to develop new strategies to control the pathogen. A possible alternative for controlling microorganisms in the food industry could be to use copper as an antimicrobial agent. The present study characterized three L. monocytogenes strains (List2-2, Apa13-2, and Al152-2A) adapted to low temperature and challenged with different copper concentrations. Similar MIC-Cu values were observed among studied strains, but growth kinetic parameters revealed that strain List2-2 was the least affected by the presence of copper at 8°C. This strain was selected for a global transcriptional response study after a 1 h exposition to 0.5 mM of CuSO4 × 5H2O at 8 and 37°C. The results showed that L. monocytogenes apparently decreases its metabolism in response to copper, and this reduction is greater at 8°C than at 37°C. The most affected metabolic pathways were carbohydrates, lipids and nucleotides synthesis. Finally, 15 genes were selected to evaluate the conservation of the transcriptional response in the other two strains. Results indicated that only genes related to copper homeostasis showed a high degree of conservation between the strains studied, suggesting that a low number of genes is implicated in the response to copper stress in L. monocytogenes. These results contribute to the understanding of the molecular mechanisms used by bacteria to overcome a combination of stresses. This study concluded that the application of copper in low concentrations in cold environments may help to control foodborne pathogens as L. monocytogenes in the industry.

In this study we evaluated if zebrafish larvae can be colonized by human gut microorganisms. We tested two strategies: (1) through transplantation of a human fecal microbiota and (2) by successively transplanting aerotolerant anaerobic microorganisms, similar to the colonization in the human intestine during early life. We used conventionally raised zebrafish larvae harboring their own aerobic microbiota to improve the colonization of anaerobic microorganisms. The results showed with the fecal transplant, that some members of the human gut microbiota were transferred to larvae. Bacillus, Roseburia, Prevotella, Oscillospira, one unclassified genus of the family Ruminococcaceae and Enterobacteriaceae were detected in 3 days post fertilization (dpf) larvae; however only Bacillus persisted to 7 dpf. Successive inoculation of Lactobacillus, Bifidobacterium and Clostridioides did not improve their colonization, compared to individual inoculation of each bacterial species. Interestingly, the sporulating bacteria Bacillus clausii and Clostridioides difficile were the most persistent microorganisms. Their endospores persisted at least 5 days after inoculating 3 dpf larvae. However, when 5 dpf larvae were inoculated, the proportion of vegetative cells in larvae increased, revealing proliferation of the inoculated bacteria and better colonization of the host. In conclusion, these results suggest that it is feasible to colonize zebrafish larvae with some human bacteria, such as C. difficile and Bacillus and open an interesting area to study interactions between these microorganisms and the host.

We investigated mechanisms involved in the protection of zebrafish (Danio rerio) larvae by two probiotic candidate yeasts, Debaryomyces hansenii 97 (Dh97) and Yarrowia lypolitica 242 (Yl242), against a Vibrio anguillarum challenge. We determined the effect of different yeast concentrations (104 – 107 UFC/mL) to: i) protect larvae from the challenge, ii) reduce the in vivo pathogen concentration and iii) modulate the innate immune response of the host. To evaluate the role of zebrafish microbiota in protection, the experiments were performed in conventionally raised and germ-free larvae. In vitro co-aggregation assays were performed to determine a direct yeast-pathogen interaction. Results showed that both yeasts significantly increased the survival rate of conventionally raised larvae challenged with V. anguillarum. The concentration of yeasts in larvae tended to increase with yeast inoculum, which was more pronounced for Dh97. Better protection was observed with Dh97 at a concentration of 106 CFU/mL compared to 104 CFU/mL. In germ-free conditions V. anguillarum reached higher concentrations in larvae and provoked significantly more mortality than in conventional conditions, revealing the protective role of the host microbiota. Interestingly, yeasts were equally (Dh97) or more effective (Yl242) in protecting germ-free than conventionally-raised larvae, showing that protection can be exerted only by yeasts and is not necessarily related to modulation of the host microbiota. Although none of the yeasts co-aggregated with V. anguillarum, they were able to reduce its proliferation in conventionally raised larvae, reduce initial pathogen concentration in germ-free larvae and prevent the upregulation of key components of the inflammatory/anti-inflammatory response (il1b, tnfa, c3, mpx and il10, respectively). These results show that protection by yeasts of zebrafish larvae challenged with V. anguillarum relates to an in vivo anti-pathogen effect, the modulation of the innate immune system, and suggests that yeasts avoid the host-pathogen interaction through mechanisms independent of co-aggregation. This study shows, for the first time, the protective role of zebrafish microbiota against V. anguillarum infection, and reveals mechanisms involved in protection by two non-Saccharomyces yeasts against this pathogen.

Listeria monocytogenes has become one of the principal foodborne pathogens worldwide. The capacity of this bacterium to grow at low temperatures has opened an interesting field of study in terms of the identification and classification of new strains of L. monocytogenes with different growth capacities at low temperatures. We determined the growth rate at 8 ºC of 110 strains of L. monocytogenes isolated from different food matrices. We identified a group of slow and fast strains according to their growth rate at 8 °C and performed a global transcriptomic assay in strains previously adapted to low temperature. We then identified shared and specific transcriptional mechanisms, metabolic and cellular processes of both groups; bacterial motility was the principal process capable of differentiating the adaptation capacity of L. monocytogenes strains with different ranges of tolerance to low temperatures. Strains belonging to the fast group were less motile, which may allow these strains to achieve a greater rate of proliferation at low temperature.