Genomic-wide analysis of Salmonella enterica strains isolated from peanuts in Brazil.

Peanut-based products have been associated with Salmonella foodborne outbreaks and/or recalls worldwide. The ability of Salmonella to persist for a long time in a low moisture environment can contribute to this kind of contamination. The objective of this study was to analyse the genome of five S. enterica enterica strains isolated from the peanut supply chain in Brazil, as well as to identify genetic determinants for survival under desiccation and validate these findings by phenotypic test of desiccation stress. The strains were in silico serotyped using the platform SeqSero2 as Miami (M2851), Javiana (M2973), Oranienburg (M2976), Muenster (M624), and Glostrup/Chomedey (M7864); with phylogenomic analysis support. Based on Multilocus Sequence Typing (MLST) the strains were assigned to STs 140, 1674, 321, 174, and 2519. In addition, eight pathogenicity islands were found in all the genomes using the SPIFinder 2.0 (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, SPI-13, SPI-14). The absence of a SPI-4 may indicate a loss of this island in the surveyed genomes. For the pangenomic analysis, 49 S. enterica genomes were input into the Roary pipeline. The majority of the stress related genes were considered as soft-core genes and were located on the chromosome. A desiccation stress phenotypic test was performed in trypticase soy broth (TSB) with four different water activity (aw) values. M2976 and M7864, both isolated from the peanut samples with the lowest aw, showed the highest OD570nm in TSB aw 0.964 and were statistically different (p < 0.05) from the strain isolated from the peanut sample with the highest aw (0.997). In conclusion, genome analyses have revealed signatures of desiccation adaptation in Salmonella strains, but phenotypic analyses suggested the environment influences the adaptive ability of Salmonella to overcome desiccation stress.

Profiling the Bacterial Community of Fermenting Traminette Grapes during Wine Production using Metagenomic Amplicon Sequencing.

Advances in sequencing technology and the relatively easy access to the use of bioinformatics tools to profile microbial community structures have facilitated a better understanding of both culturable and non-culturable microbes in grapes and wine. During industrial fermentation, microbes, known and unknown, are often responsible for product development and off-flavor. Therefore, profiling the bacteria from grape to wine can enable an easy understanding of in situ microbial dynamics. In this study, the bacteria of Traminette grapes must undergoing fermentation, and the final wine were subjected to DNA extraction that yielded 15 ng/µL to 87 ng/µL. The 16S amplicon of the hypervariable region of the V4 region was sequenced, relatively abundant bacteria consisting of phyla Proteobacteria, Actinobacteriota, Firmicutes, Bacteroidota, Fusobacteriota and followed by the Verrucomicrobiota, Halobacterota, Desulfobacterota, Myxococcota, and Acidobacteriota. A Venn diagram analysis of the shared unique operational taxonomic units (OTU) revealed that 15 bacteria phyla were common to both grape must, fermenting stage, and final wine. Phyla that were not previously reported were detected using the 16S amplicon sequencing, as well as genera such as Enterobacteriaceae and Lactobacillaceae. Variation in the organic nutrient use in wine and its impact on bacteria was tested; Traminette R tank containing Fermaid O and Traminette L stimulated with Stimula Sauvignon blanc + Fermaid O. Alpha diversity using the Kruskal-Wallis test determined the degree of evenness. The beta diversity indicated a shift in the bacteria at the fermentation stage for the two treatments, and the final wine bacteria looked similar. The study confirmed that 16S amplicon sequencing can be used to monitor bacteria changes during wine production to support quality and better utilization of grape bacteria during wine production.

Gene expression and cell culture assays reveal cheese isolate Lactococcus lactis MC5 may influence the virulence of Staphylococcus aureus.

Staphylococcus aureus (SA) can thrive in a wide variety of hosts and environments, causing clinical infections and foodborne intoxications. In Brazil, SA is commonly isolated from traditional soft cheeses, especially those prepared from unpasteurized milk. In this research, the isolate S. aureus SABRC1 was evaluated for virulence traits under different conditions, including co-inoculation with Lactococcus lactis MC5 (isolated from "Fresh Minas Cheese"), which produces antibacterial peptides. Results from experiments with Caco-2 culture indicated S. aureus SABRC1 was able to adhere (42.83 ± 1.79%) and to invade (48.57 ± 0.41%) the intestinal cells. On the other hand, L. lactis MC5 presented anti-staphylococcal activity as indicated by agar assays, and it was also able to antagonize intestinal cell invasion by S. aureus. Moreover, Reverse Transcriptase-PCR experiments showed virulence genes of S. aureus SABRC1 (hla, icaA and sea) were differentially expressed under diverse culture conditions, which included Brain Heart Infusion modified or not by the addition of glucose, sodium chloride, milk or cheese. This suggests the virulence of S. aureus SABRC1 is influenced by compounds commonly found in daily diets, and not only by its genetic repertoire, adding a novel level of complexity for controlling infection by this pathogen.

A set of antibiotic-resistance mechanisms and virulence factors in GES-16-producing Klebsiella quasipneumoniae subsp. similipneumoniae from hospital wastewater revealed by whole-genome sequencing.

Klebsiella quasipneumoniae subsp. similipneumoniae has emerged as a human pathogen and sporadic isolates from non-clinical sources were reported. Here, we described the phenotypic- and genomic-characteristics of a multidrug-resistant (MDR) and potentially hypervirulent (MDR-hv) Klebsiella quasipneumoniae subsp. similipneumoniae (KqA1) isolated from hospital wastewater. The antibiotic susceptibility profile of KqA1 was investigated using disk-diffusion method, broth microdilution method, and agar dilution method, and the genetic characteristics of antimicrobial resistance, mobile genetics elements, and virulence were evaluated by genomic DNA sequencing on the Illumina® NovaSeq6000 platform as well as by bioinformatic analysis. Resistome analyses revealed the presence of genes related to resistance to ß-lactams, aminoglycosides, quinolones, tetracyclines, sulfonamides, trimethoprim, chloramphenicol, macrolides, and fosfomycin. New genetic contexts to blaGES-16 (carbapenemase gene) and to fosA (fosfomycin resistance gene) were described. A set of mechanisms that can contribute to antibiotic resistance, commonly detected in Klebsiella spp., was also found including chromosomal mutations, efflux systems, proteins, and regulators. Moreover, KqA1 presented genes related to tolerance to metals (arsenic, copper, nickel, cobalt, magnesium, cadmium, zinc, tellurium, selenium) and to biocides (quaternary-ammonium compounds). The isolate was classified as potentially hypervirulent due to a wide range of virulence factors found associated to regulation, motility, biofilm, effector delivery systems, immune modulation, nutritional/metabolic factors, adherence, invasion, and competitive advantage. The occurrence of MDR-hv KqA1 in hospital wastewater points out how this environment matrix plays a crucial role in the maintenance and selection of critical bacterial pathogens. Regarding One Health perspective, it is evident the need for multidisciplinary implementation of control measures for antibiotic-resistant bacteria, not only in hospital settings but also in a general environmental context to mitigate the dissemination of MDR and hv bacteria.

In silico metatranscriptomic approach for tracking biofilm-related effectors in dairies and its importance for improving food safety.

Sessile microorganisms are usually recalcitrant to antimicrobial treatments, and it is possible that finding biofilm-related effectors in metatranscriptomics datasets helps to understand mechanisms for bacterial persistence in diverse environments, by revealing protein-encoding genes that are expressed in situ. For this research, selected dairy-associated metatranscriptomics bioprojects were downloaded from the public databases JGI GOLD and NCBI (eight milk and 45 cheese samples), to screen for sequences encoding biofilm-related effectors. Based on the literature, the selected genetic determinants were related to adhesins, BAP, flagellum-related, intraspecific QS (AHL, HK, and RR), interspecific QS (LuxS), and QQ (AHL-acylases, AHL-lactonases). To search for the mRNA sequences encoding for those effector proteins, a custom database was built from UniprotKB, yielding 1,154,446 de-replicated sequences that were indexed in DIAMOND for alignment. The results revealed that in all the dairy-associated metatranscriptomic datasets obtained, there were reads assigned to genes involved with flagella, adhesion, and QS/QQ, but BAP-reads were found only for milk. Significant Pearson correlations (p < 0.05) were observed for transcripts encoding for flagella, RR, histidine kinases, adhesins, and LuxS, although no other significant correlations were found. In conclusion, the rationale used in this study was useful to demonstrate the presence of biofilm-associated effectors in metatranscriptomics datasets, pointing out to possible regulatory mechanisms in action in dairy-related biofilms, which could be targeted in the future to improve food safety.

Use of encapsulated lactic acid bacteria as bioprotective cultures in fresh Brazilian cheese.

There is great interest for biopreservation of food products, and encapsulation may be a good strategy to extend the viability of protective cultures. In this study, Lactobacillus paraplantarum FT-259 and Lactococcus lactis QMF 11 were separately encapsulated in casein/pectin (C/P) microparticles, which were tested for antilisterial and anti-staphylococcal activity in fresh Minas cheese (FMC) stored at 8 °C. The encapsulation efficiency for both lactic acid bacteria (LAB) was 82.5%, with viability over 6.2 log CFU/g after storage of C/P microparticles for 90 days under refrigeration. Interestingly, free Lb. paraplantarum and free Lc. lactis grew significantly in refrigerated FMC, both in the presence and absence of pathogens, but only the first significatively grew when encapsulated. Encapsulation increased the antilisterial activity of Lb. paraplantarum in FMC. Moreover, Lc. lactis significantly inhibited listerial growth in FMC in both its free and encapsulated forms, whereas Staphylococcus aureus counts were only significantly reduced in the presence of free Lc. lactis. In conclusion, these results indicate that C/P microparticles are effective carriers of LAB in FMC, which can contribute for the assurance of the safety of this product.

Biofilm growth by Listeria monocytogenes on stainless steel and expression of biofilm-related genes under stressing conditions.

This research was carried out to investigate the differences in adhesion and growth during biofilm formation of L. monocytogenes from different sources and clonal complexes. Biofilm by L. monocytogenes (isolates CLIST 441 and 7: both lineage I, serotype 1/2b, CC3; isolates 19 and 508: both lineage II, serotype 1/2c, CC9) was grown on stainless steel coupons under different stressing conditions (NaCl, curing salts and quaternary ammonium compounds-QAC), to determine the expression of different genes involved in biofilm formation and stress response. CLIST 441, which carries a premature stop codon (PMSC) in agrC, formed high-density biofilms in the presence of QAC (7.5% w/v) or curing salts (10% w/v). Reverse Transcriptase-qPCR results revealed that L. monocytogenes isolates presented differences in transcriptional profile of genes related to biofilm formation and adaptation to environmental conditions. Our results demonstrated how L. monocytogenes can survive, multiply and form biofilm under adverse conditions related to food processing environments. Differences in transcriptional expression were observed, highlighting the role of regulatory gene networks for particular serotypes under different stress responses.

Comparative phylo-pangenomics reveals generalist lifestyles in representative Acinetobacter species and proposes candidate gene markers for species identification.

Acinetobacter species have the potential to invade and colonize immunocompromised patients, therefore being well-known as opportunistic pathogens. Among these bacteria, the species of the Acinetobacter calcoaceticus-Acinetobacter baumannii "complex" (Acb members) emerge as the main often isolated bacteria in clinical specimens. The unequivocal taxonomy is crucial to correctly identify these species and associated with comparative genomic analyses aids to understand their life-styles as well. In this study, all publicly available Acinetobacter species at the date of this study preparation were analyzed. The results revealed that the Acb members are in fact a complex when phenotypic methods are confronted, while for comparative and phylogenomics analyses this term is misleading, since they composed a monophyletic group instead. Nine best gene markers (response regulator, recJ, recG, phosphomannomutase, pepSY, monovalent cation/H + antiporter subunit D, mnmE, glnE, and bamA) were selected for identification of Acinetobacter species. Moreover, representative strains of each species were split according their isolation sources in the categories: environmental, human, insect and non-human vertebrate. Neither niche-specific genome signature nor niche-associated functional and pathogenic potential were associated with their isolation source, meaning it is not the main force acting on Acinetobacter adaptation in a given niche and corroborating that their ubiquitous distribution is a reflex of their generalist life-styles.

Pangenome analyses of LuxS-coding genes and enzymatic repertoires in cocoa-related lactic acid bacteria.

Lactobacillaceae presents potential for interspecific Quorum Sensing (QS) in spontaneous cocoa fermentation, correlated with high abundance of luxS. Three Brazilian isolates from cocoa fermentation were characterized by Whole Genome Sequencing and luxS gene was surveyed in their genomes, in comparison with public databases. They were classified as Lactiplantibacillus plantarum, Limosilactobacillus fermentum and Pediococcus acidilactici. LuxS genes were conserved in core genomes of the novel isolates, but in some non-cocoa related Lactic Acid Bacteria (LAB) it was accessory and plasmid-borne. The conservation and horizontal acquisition of luxS reinforces that QS is determinant for bacterial adaptation in several environments, especially taking into account the luxS has been correlated with modulation of bacteriocin production, stress tolerance and biofilm formation. Therefore, in this paper, new clade and species-specific primers were designed for future application for screening of luxS gene in LAB to evaluate the adaptive potential to diverse food fermentations.

Studies on host-foodborne bacteria in intestinal three-dimensional cell culture model indicate possible mechanisms of interaction.

Spheroids of intestinal cells (Caco-2) were used to evaluate the adhesion/invasion ability of Listeria monocytogenes (pathogen) and Lactobacillus sakei 1 (potential probiotic). Besides, transcriptomic analyses of Caco-2 cells in three dimensional cultures were done, with the aim of revealing possible host-foodborne bacteria interactions. Result of adhesion assay for L. monocytogenes in Caco-2 spheroids was 22.86 ± 0.33%, but it was stimulated in acidic pH (4.5) and by the presence of 2% sucrose (respectively, 32.56 ± 1.35% and 33.25 ± 1.26%). Conversely, the invasion rate of L. monocytogenes was lower at pH 4.5, in comparison with non-stressed controls (18.89 ± 1.05% and 58.65 ± 0.30%, respectively). L. sakei 1 adhered to Caco-2 tridimensional cell culture (27.30 ± 2.64%), with no invasiveness. There were 19 and 21 genes down and upregulated, respectively, in tridimensional Caco-2 cells, upon infection with L. monocytogenes, which involved immunity, apoptosis; cytoprotective responses, cell signalling-regulatory pathways. It was evidenced despite activation or deactivation of several pathways in intestinal cells to counteract infection, the pathogen was able to hijack many host defense mechanisms. On the other hand, the probiotic candidate L. sakei 1 was correlated with decreased transcription of two genes in Caco-2 cells, though it stimulated the expression of 14 others, with diverse roles in immunity, apoptosis, cytoprotective response and cell signalling-regulatory pathways. Our data suggest the use of tridimensional cell culture to mimic the intestinal epithelium is a good model for gathering broad information on the putative mechanisms of interaction between host and bacteria of importance for food safety, which can serve as a basis for further in-depth investigation.

Assessment of Internalin A Gene Sequences and Cell Adhesion and Invasion Capacity of Listeria monocytogenes Strains Isolated from Foods of Animal and Related Origins.

Listeria monocytogenes is a foodborne pathogen of global relevance that causes outbreaks and sporadic cases of listeriosis, acquired through the consumption of contaminated products, including milk or meat products and ready-to-eat meat products subjected to intensive handling. The objective of the present study was to classify L. monocytogenes isolated from various food-related sources in the Federal District of Brazil and surrounding areas to sequence internalin A (inlA) genes from these isolates and assess their adhesion and invasion capacity using Caco-2 cells. In addition, 15 were classified as group I, 3 as group II, and 7 classified as group IV. Premature stop codons (PMSCs) at the nucleotide position 976 (GAA→TAA) of the inlA gene were identified in 5 of the 25 isolates. Adhesion and invasion tests in Caco-2 cells showed that all the isolates were capable of adhesion and cellular invasion, with isolates containing PMSCs exhibiting on average higher invasion capacity than those without PMSCs (p = 0.041) and a median of adhesion very distinctive from those without stop codons. These results are the first report of PMSCs in the inlA gene of L. monocytogenes from the Federal District of Brazil and Brazil.

The Lung Microbiome of Three Young Brazilian Patients With Cystic Fibrosis Colonized by Fungi.

Microbial communities infiltrate the respiratory tract of cystic fibrosis patients, where chronic colonization and infection lead to clinical decline. This report aims to provide an overview of the diversity of bacterial and fungal species from the airway secretion of three young CF patients with severe pulmonary disease. The bacterial and fungal microbiomes were investigated by culture isolation, metataxonomics, and metagenomics shotgun. Virulence factors and antibiotic resistance genes were also explored. A. fumigatus was isolated from cultures and identified in high incidence from patient sputum samples. Candida albicans, Penicillium sp., Hanseniaspora sp., Torulaspora delbrueckii, and Talaromyces amestolkiae were isolated sporadically. Metataxonomics and metagenomics detected fungal reads (Saccharomyces cerevisiae, A. fumigatus, and Schizophyllum sp.) in one sputum sample. The main pathogenic bacteria identified were Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia complex, and Achromobacter xylosoxidans. The canonical core CF microbiome is composed of species from the genera Streptococcus, Neisseria, Rothia, Prevotella, and Haemophilus. Thus, the airways of the three young CF patients presented dominant bacterial genera and interindividual variability in microbial community composition and diversity. Additionally, a wide diversity of virulence factors and antibiotic resistance genes were identified in the CF lung microbiomes, which may be linked to the clinical condition of the CF patients. Understanding the microbial community is crucial to improve therapy because it may have the opposite effect, restructuring the pathogenic microbiota. Future studies focusing on the influence of fungi on bacterial diversity and microbial interactions in CF microbiomes will be welcome to fulfill this huge gap of fungal influence on CF physiopathology.

Metataxonomics contributes to unravel the microbiota of a Brazilian dairy.

For this research communication, 90 samples of a Brazilian dairy were combined into four groups (raw material, final product, food-contact and non-food contact surfaces) and analyzed by metataxonomics based on 16S rRNA gene sequencing. The results showed high alpha-diversity indexes for final product and non-food contact surfaces but, overall, beta-diversity indexes were low. The samples were separated in two main clusters, and the core microbiota was composed by Macrococcus, Alkaliphilus, Vagococcus, Lactobacillus, Marinilactibacillus, Streptococcus, Lysinibacillus, Staphylococcus, Clostridium, Halomonas, Lactococcus, Enterococcus, Bacillus and Psychrobacter. These results highlight that rare taxa occur in dairies, and this may aid the development of strategies for food protection.

Finding a common core microbiota in two Brazilian dairies through culture and DNA metabarcoding studies.

Dairy foods are complex ecosystems composed of microorganisms from different origins that can affect flavor and safety of final products. The objective of this paper is to assess the in-house microbiota of two Brazilian dairies and to discuss the possible implications of the taxa determined for food protection. In total, 27 samples from dairies were cultured in selective (Baird Parker, de Man, Rogosa and Sharpe) and non-selective (Brain Heart Infusion) media, and the isolates were identified by Sanger sequencing. Moreover, metagenomic DNA was directly extracted from samples and the structure of the bacterial community was determined by massive DNA sequencing followed by bioinformatics analyses. The results showed the majority of isolates belonged to the group of lactic acid bacteria, but Enterobacteriaceae, Staphylococcacceae, Bacillaceae, Pseudomonadaceae and Moraxellaceae were also detected. From the reads obtained in metataxonomics analyses, a heatmap was constructed and the top 20 OTUs (operational taxonomic units) were determined. Besides, 12 most prevalent bacterial taxa were assigned to the core microbiota of the dairies evaluated, which included Thiomonas thermosulfata, Alkalibacillus salilacus, Pseudomonas clemancea, Erythrobacter aquimans, Tetragenococcus doogicus, Macrococcus brunensis, Pseudomonas ludensis, Streptococcus dentinousetti, Serratia entomophila, Vagococcus teuberi, Lactococcus fujiensis and Tolumonas auensis. In conclusion, the results reveal the presence of bacteria that may be related to spoilage and also foodborne diseases, in microbial niches that also present rare taxa, highlighting the importance to consider culture-independent results to evaluate and improve food safety.

Characterization of an Environmental Multidrug-Resistant Acinetobacter seifertii and Comparative Genomic Analysis Reveals Co-occurrence of Antimicrobial Resistance and Metal Tolerance Determinants.

Acinetobacter calcoaceticus-Acinetobacter baumannii complex is considered one of the main causes of hospital-acquired infections. Acinetobacter seifertii was recently characterized within this complex and it has been described as an emergent pathogen associated with bacteremia. The emergence of multidrug-resistant (MDR) bacteria, including Acinetobacter sp., is considered a global public health threat and an environmental problem because MDR bacteria have been spreading from several sources. Therefore, this study aimed to characterize an environmental MDR A. seifertii isolate (SAb133) using whole genome sequencing and a comparative genomic analysis was performed with A. seifertii strains recovered from various sources. The SAb133 isolate was obtained from soil of a corn crop field and presented high MICs for antimicrobials and metals. The comparative genomic analyses revealed ANI values higher than 95% of relatedness with other A. seifertii strains than A. calcoaceticus-A. baumannii complex. Resistome and virulome analyses were also performed and showed different antimicrobial resistance determinants and metal tolerance genes as well as virulence genes related to A. baumannii known virulence genes. In addition, genomic islands, IS elements, plasmids and prophage-related sequences were detected. Comparative genomic analysis showed that MDR A. seifertii SAb133 had a high amount of determinants related to antimicrobial resistance and tolerance to metals, besides the presence of virulence genes. To the best of our knowledge, this is the first report of a whole genome sequence of a MDR A. seifertii isolated from soil. Therefore, this study contributed to a better understanding of the genetic relationship among the few known A. seifertii strains worldwide distributed.

Diet-induced remission in chronic enteropathy is associated with altered microbial community structure and synthesis of secondary bile acids.

BACKGROUND: The microbiome has been implicated in the initiation and persistence of inflammatory bowel disease. Despite the fact that diet is one of the most potent modulators of microbiome composition and function and that dietary intervention is the first-line therapy for treating pediatric Crohn's disease, the relationships between diet-induced remission, enteropathy, and microbiome are poorly understood. Here, we leverage a naturally-occurring canine model of chronic inflammatory enteropathy that exhibits robust remission following nutritional therapy, to perform a longitudinal study that integrates clinical monitoring, 16S rRNA gene amplicon sequencing, metagenomic sequencing, metabolomic profiling, and whole genome sequencing to investigate the relationship between therapeutic diet, microbiome, and disease. RESULTS: We show that remission induced by a hydrolyzed protein diet is accompanied by alterations in microbial community structure marked by decreased abundance of pathobionts (e.g., Escherichia coli and Clostridium perfringens), reduced severity of dysbiosis, and increased levels of the secondary bile acids, lithocholic and deoxycholic acid. Physiologic levels of these bile acids inhibited the growth of E. coli and C. perfringens isolates, in vitro. Metagenomic analysis and whole genome sequencing identified the bile acid producer Clostridium hiranonis as elevated after dietary therapy and a likely source of secondary bile acids during remission. When C. hiranonis was administered to mice, levels of deoxycholic acid were preserved and pathology associated with DSS colitis was ameliorated. Finally, a closely related bile acid producer, Clostridium scindens, was associated with diet-induced remission in human pediatric Crohn's disease. CONCLUSIONS: These data highlight that remission induced by a hydrolyzed protein diet is associated with improved microbiota structure, an expansion of bile acid-producing clostridia, and increased levels of secondary bile acids. Our observations from clinical studies of exclusive enteral nutrition in human Crohn's disease, along with our in vitro inhibition assays and in vivo studies in mice, suggest that this may be a conserved response to diet therapy with the potential to ameliorate disease. These findings provide insight into diet-induced remission of gastrointestinal disease and could help guide the rational design of more effective therapeutic diets.

Potential of oxygen and nitrogen reactive intermediates to disperse Listeria monocytogenes from biofilms.

Studying biofilm dispersal is important to prevent Listeria monocytogenes persistence in food processing plants and to avoid finished product contamination. Reactive oxygen and nitrogen intermediates (ROI and RNI, respectively) may trigger cell detachment from many bacterial species biofilms, but their roles in L. monocytogenes biofilms have not been fully investigated. This study reports on ROI and RNI quantification in Listeria monocytogenes biofilms formed on stainless steel and glass surfaces; bacterial culture and microscopy combined with fluorescent staining were employed. Nitric oxide (NO) donor and inhibitor putative effects on L. monocytogenes dispersal from biofilms were evaluated, and transcription of genes (prfA, lmo 0990, lmo 0807, and lmo1485) involved in ROI and RNI stress responses were quantified by real-time PCR (qPCR). Microscopy detected the reactive intermediates NO, peroxynitrite, H2O2, and superoxide in L. monocytogenes biofilms. Neither NO donor nor inhibitors interfered in L. monocytogenes growth and gene expression, except for lmo0990, which was downregulated. In conclusion, ROI and RNI did not exert dispersive effects on L. monocytogenes biofilms, indicating that this pathogen has a tight control for protection against oxidative and nitrosative stresses.

Behavior of Foodborne Pathogens Listeria monocytogenes and Staphylococcus aureus in Mixed-Species Biofilms Exposed to Biocides.

In nature and man-made environments, microorganisms reside in mixed-species biofilms, in which the growth and metabolism of an organism are different from these behaviors in single-species biofilms. Pathogenic microorganisms may be protected against adverse treatments in mixed-species biofilms, leading to health risk for humans. Here, we developed two mixed five-species biofilms that included one or the other of the foodborne pathogens Listeria monocytogenes and Staphylococcus aureus The five species, including the pathogen, were isolated from a single food-processing environmental sample, thus mimicking the environmental community. In mature mixed five-species biofilms on stainless steel, the two pathogens remained at a constant level of ∼105 CFU/cm2 The mixed five-species biofilms as well as the pathogens in monospecies biofilms were exposed to biocides to determine any pathogen-protective effect of the mixed biofilm. Both pathogens and their associate microbial communities were reduced by peracetic acid treatments. S. aureus decreased by 4.6 log cycles in monospecies biofilms, but the pathogen was protected in the five-species biofilm and decreased by only 1.1 log cycles. Sessile cells of L. monocytogenes were affected to the same extent when in a monobiofilm or as a member of the mixed-species biofilm, decreasing by 3 log cycles when exposed to 0.0375% peracetic acid. When the pathogen was exchanged in each associated microbial community, S. aureus was eradicated, while there was no significant effect of the biocide on L. monocytogenes or the mixed community. This indicates that particular members or associations in the community offered the protective effect. Further studies are needed to clarify the mechanisms of biocide protection and to identify the species playing the protective role in microbial communities of biofilms.IMPORTANCE This study demonstrates that foodborne pathogens can be established in mixed-species biofilms and that this can protect them from biocide action. The protection is not due to specific characteristics of the pathogen, here S. aureus and L. monocytogenes, but likely caused by specific members or associations in the mixed-species biofilm. Biocide treatment and resistance are a challenge for many industries, and biocide efficacy should be tested on microorganisms growing in biofilms, preferably mixed systems, mimicking the application environment.

Listeria monocytogenes incidence changes and diversity in some Brazilian dairy industries and retail products.

Listeria monocytogenes can cause listeriosis, a severe foodborne disease. In Brazil, despite very few reported cases of listeriosis, the pathogen has been repeatedly isolated from dairies. This has led the government to implement specific legislation to reduce the hazard. Here, we determined the incidence of L. monocytogenes in five dairies and retail products in the Southeast and Midwest regions of Brazil over eight months. Of 437 samples, three samples (0.7%) from retail and only one sample (0.2%) from the dairies were positive for L. monocytogenes. Thus, the contamination rate was significantly reduced as compared to previous studies. MultiLocus Sequence Typing (MLST) was used to determine if contamination was caused by new or persistent clones leading to the first MLST profile of L. monocytogenes from the Brazilian dairy industry. The processing environment isolate is of concern being a sequence-type (ST) 2, belonging to the lineage I responsible for the majority of listeriosis outbreaks. Also, ST3 and ST8 found in commercialized cheese have previously been reported in outbreaks. Despite the lower incidence, dairy products still pose a potential health risk and the occurrence of L. monocytogenes in dairies and retail products emphasize the need for continuous surveillance of this pathogen in the Brazilian dairy industry.