Genomic and phenotypic characterization of multidrug-resistant Salmonella enterica serovar Reading isolates involved in a turkey-associated foodborne outbreak

Salmonella is a global bacterial foodborne pathogen associated with a variety of contaminated food products. Poultry products are a common source of Salmonella-associated foodborne illness, and an estimated 7% of human illnesses in the United States are attributed to turkey products. From November 2017 to March 2019, the Centers for Disease Control and Prevention reported a turkey-associated outbreak of multidrug-resistant (MDR; resistant to ≥3 antimicrobial classes) Salmonella enterica serovar Reading (S. Reading) linked to 358 human infections in 42 US states and Canada. Since S. Reading was seldom linked to human illness prior to this outbreak, the current study compared genomic sequences of S. Reading isolates prior to the outbreak (pre-outbreak) to isolates identified during the outbreak period, focusing on genes that were different between the two groups but common within a group. Following whole-genome sequence analysis of five pre-outbreak and five outbreak-associated turkey/turkey product isolates of S. Reading, 37 genes located within two distinct chromosomal regions were identified only in the pre-outbreak isolates: (1) an ~5 kb region containing four protein-coding genes including uidA which encodes beta-glucuronidase, pgdA encoding peptidoglycan deacetylase, and two hypothetical proteins and (2) an ~28 kb region comprised of 32 phage-like genes and the xerC gene, which encodes tyrosine recombinase (frequently associated with phage genes). The five outbreak isolates also had a deletional event within the cirA gene, introducing a translational frame shift and premature stop codon. The cirA gene encodes a protein with dual receptor functions: a siderophore receptor for transport of dihydroxybenzoylserine as well as a colicin Ia/b receptor. Significant differences for the identified genetic variations were also detected in 75 S. Reading human isolates. Of the 41 S. Reading isolates collected before or in 2017, 81 and 90% of the isolates contained the uidA and pgdA genes, respectively, but only 24% of the isolates collected after 2017 harbored the uidA and pgdA genes. The truncation event within the cirA gene was also significantly higher in isolates collected after 2017 (74%) compared to before or in 2017 (5%). Phenotypic analysis of the S. Reading isolates for colicin and cefiderocol sensitivities (CirA) and β-methyl-D-glucuronic acid utilization (UidA and accessory proteins) supported the genomic data. Overall, a similar genome reduction pattern was generally observed in both the turkey and human isolates of S. Reading during the outbreak period, and the genetic differences were present in genes that could potentially promote pathogen dissemination due to variation in Salmonella colonization, fitness, and/or virulence

RNA-seq Brings New Insights to the Intra-Macrophage Transcriptome of Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection

Reference Transcriptomes of Porcine Peripheral Immune Cells Created Through Bulk and Single-Cell RNA Sequencing

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B-cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B-cell, conventional CD4 and CD8 αβ T-cells, NK cells, and γδ T-cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T-cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology

Analysis of the Oxidative Stress Regulon Identifies soxS as a Genetic Target for Resistance Reversal in Multidrug-Resistant Klebsiella pneumoniae

In bacteria, the defense system deployed to counter oxidative stress is orchestrated by three transcriptional factors, SoxS, SoxR, and OxyR. Although the regulon that these factors control is known in many bacteria, similar data are not available for Klebsiella pneumoniae. To address this data gap, oxidative stress was artificially induced in K. pneumoniae MGH78578 using paraquat and the corresponding oxidative stress regulon recorded using transcriptome sequencing (RNA-seq). The soxS gene was significantly induced during oxidative stress, and a knockout mutant was constructed to explore its functionality. The wild type and mutant were grown in the presence of paraquat and subjected to RNA-seq to elucidate the soxS regulon in K. pneumoniae MGH78578. Genes that are commonly regulated both in the oxidative stress and soxS regulons were identified and denoted as the oxidative SoxS regulon; these included a group of genes specifically regulated by SoxS. Efflux pump-encoding genes and global regulators were identified as part of this regulon. Consequently, the isogenic soxS mutant was found to exhibit a reduction in the minimum bactericidal concentration against tetracycline compared to that of the wild type. Impaired efflux activity, allowing tetracycline to be accumulated in the cytoplasm to bactericidal levels, was further evaluated using a tetraphenylphosphonium (TPP+) accumulation assay. The soxS mutant was also susceptible to tetracycline in vivo in a zebrafish embryo model. We conclude that the soxS gene could be considered a genetic target against which an inhibitor could be developed and used in combinatorial therapy to combat infections associated with multidrug-resistant K. pneumoniae. IMPORTANCE Antimicrobial resistance is a global health challenge. Few new antibiotics have been developed for use over the years, and preserving the efficacy of existing compounds is an important step to protect public health. This paper describes a study that examines the effects of exogenously induced oxidative stress on K. pneumoniae and uncovers a target that could be useful to harness as a strategy to mitigate resistance

Date palm transcriptome analysis provides new insights on changes in response to high salt stress of colonized roots with the endophytic fungus Piriformospora indica

Salinity is a significant threat that causes considerable yield losses in date palm. The root endophytic fungus Piriformospora indica has proven effective in providing salt stress tolerance to host plants. However, the underlying molecular mechanism facilitating the date palm’s response to P. indica inoculation, and its involvement in the salt stress tolerance, remains unknown. In this study, the colonization of P. indica on date palm seedlings exposed to saline conditions was observed through confocal microscopy, and its impact on gene expressions was evaluated using the transcriptomic analysis. Our findings show that P. indica colonization reinforced the cortical cells, prevented them from plasmolysis and cell death under salinity. The RNAseq analysis produced clean reads ranging from 62,040,451 to 3,652,095 across the treatment groups, successfully assembling into 30,600 annotated genes. Out of them, the number of differentially expressed genes (DEGs) varied across the treatments: i.e., 2523, 2031, and 1936 DEGs were upregulated, while 2323, 959, and 3546 were downregulated in Salt, Fungi, and Fungi+Salt groups, respectively. Furthermore, principal component analysis based on transcriptome profiles revealed discrete clustering of samples from different treatment groups. KEGG and GO pathways enrichment analysis highlighted variation in the number and types of enriched pathways among the treatments. Our study indicated variations in gene expression related to plant hormone biosynthesis and signal transduction (auxin, abscisic acid, gibberellin, and ethylene), ABC transporters, sodium/hydrogen exchanger, cation HKT transporter, transcription factors such as WRKY and MYBs, and the plant immune system (lipoxygenase and jasmonate) of the date palm seedlings. By characterizing the transcriptome of date palm roots under salt stress and with colonization of P. indica, the present findings provide valuable perspectives on the molecular mechanisms responsible for inducing salinity stress tolerance in plants

Silver Nanoparticles Induce a Triclosan-Like Antibacterial Action Mechanism in Multi-Drug Resistant Klebsiella pneumoniae

International audienceInfections associated with antimicrobial-resistant bacteria now represent a significant threat to human health using conventional therapy, necessitating the development of alternate and more effective antibacterial compounds. Silver nanoparticles (Ag NPs) have been proposed as potential antimicrobial agents to combat infections. A complete understanding of their antimicrobial activity is required before these molecules can be used in therapy. Lysozyme coated Ag NPs were synthesized and characterized by TEM-EDS, XRD, UV-vis, FTIR spectroscopy, zeta potential, and oxidative potential assay. Biochemical assays and deep level transcriptional analysis using RNA sequencing were used to decipher how Ag NPs exert their antibacterial action against multi-drug resistant Klebsiella pneumoniae MGH78578. RNAseq data revealed that Ag NPs induced a triclosan-like bactericidal mechanism responsible for the inhibition of the type II fatty acid biosynthesis. Additionally, released Ag+ generated oxidative stress both extra- and intracellularly in K. pneumoniae. The data showed that triclosan-like activity and oxidative stress cumulatively underpinned the antibacterial activity of Ag NPs. This result was confirmed by the analysis of the bactericidal effect of Ag NPs against the isogenic K. pneumoniae MGH78578 ΔsoxS mutant, which exhibits a compromised oxidative stress response compared to the wild type. Silver nanoparticles induce a triclosan-like antibacterial action mechanism in multi-drug resistant K. pneumoniae. This study extends our understanding of anti-Klebsiella mechanisms associated with exposure to Ag NPs. This allowed us to model how bacteria might develop resistance against silver nanoparticles, should the latter be used in therapy

Exposure to sub-inhibitory concentrations of the chemosensitizer 1-(1-naphthylmethyl)-piperazine creates membrane destabilization in multi-drug resistant Klebsiella pneumoniae

Antimicrobial efflux is one of the important mechanisms causing multi-drug resistance (MDR) in bacteria. Chemosensitizers like 1-(1-naphthylmethyl)-piperazine (NMP) can inhibit an efflux pump and therefore can overcome MDR. However, secondary effects of NMP other than efflux pump inhibition are rarely investigated. Here, using phenotypic assays, phenotypic microarray and transcriptomic assays we show that NMP creates membrane destabilization in MDR Klebsiella pneumoniae MGH 78578 strain. The NMP mediated membrane destabilization activity was measured using β-lactamase activity, membrane potential alteration studies, and transmission electron microscopy assays. Results from both β-lactamase and membrane potential alteration studies shows that both outer and inner membranes are destabilized in NMP exposed K. pneumoniae MGH 78578 cells. Phenotypic Microarray and RNA-seq were further used to elucidate the metabolic and transcriptional signals underpinning membrane destabilization. Membrane destabilization happens as early as 15 min post-NMP treatment. Our RNA-seq data shows that many genes involved in envelope stress response were differentially regulated in the NMP treated cells. Up-regulation of genes encoding the envelope stress response and repair systems show the distortion in membrane homeostasis during survival in an environment containing sub-inhibitory concentration of NMP. In addition, the lsr operon encoding the production of autoinducer-2 responsible for biofilm production was down-regulated resulting in reduced biofilm formation in NMP treated cells, a phenotype confirmed by crystal violet-based assays. We postulate that the early membrane disruption leads to destabilization of inner membrane potential, impairing ATP production and consequently resulting in efflux pump inhibition

Reference Transcriptomes of Porcine Peripheral Immune Cells Created Through Bulk and Single-Cell RNA Sequencing

Pigs are a valuable human biomedical model and an important protein source supporting global food security. The transcriptomes of peripheral blood immune cells in pigs were defined at the bulk cell-type and single cell levels. First, eight cell types were isolated in bulk from peripheral blood mononuclear cells (PBMCs) by cell sorting, representing Myeloid, NK cells and specific populations of T and B-cells. Transcriptomes for each bulk population of cells were generated by RNA-seq with 10,974 expressed genes detected. Pairwise comparisons between cell types revealed specific expression, while enrichment analysis identified 1,885 to 3,591 significantly enriched genes across all 8 cell types. Gene Ontology analysis for the top 25% of significantly enriched genes (SEG) showed high enrichment of biological processes related to the nature of each cell type. Comparison of gene expression indicated highly significant correlations between pig cells and corresponding human PBMC bulk RNA-seq data available in Haemopedia. Second, higher resolution of distinct cell populations was obtained by single-cell RNA-sequencing (scRNA-seq) of PBMC. Seven PBMC samples were partitioned and sequenced that produced 28,810 single cell transcriptomes distributed across 36 clusters and classified into 13 general cell types including plasmacytoid dendritic cells (DC), conventional DCs, monocytes, B-cell, conventional CD4 and CD8 αβ T-cells, NK cells, and γδ T-cells. Signature gene sets from the human Haemopedia data were assessed for relative enrichment in genes expressed in pig cells and integration of pig scRNA-seq with a public human scRNA-seq dataset provided further validation for similarity between human and pig data. The sorted porcine bulk RNAseq dataset informed classification of scRNA-seq PBMC populations; specifically, an integration of the datasets showed that the pig bulk RNAseq data helped define the CD4CD8 double-positive T-cell populations in the scRNA-seq data. Overall, the data provides deep and well-validated transcriptomic data from sorted PBMC populations and the first single-cell transcriptomic data for porcine PBMCs. This resource will be invaluable for annotation of pig genes controlling immunogenetic traits as part of the porcine Functional Annotation of Animal Genomes (FAANG) project, as well as further study of, and development of new reagents for, porcine immunology