Project description:A prototype oligonucleotide microarray was designed to detect and identify viable bacterial species with the potential to grow of common beer spoilage microorganisms from the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Probes targeted the intergenic spacer regions (ISR) between 16S and 23S rRNA, which were amplified in a combination of reverse transcriptase (RT) and polymerase chain reaction (PCR) prior to hybridization. This method allows the detection and discrimination of single bacterial species in a complex sample. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non-growing bacteria. The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage microorganisms within mixed population. Keywords: microarray, oligonucleotide, species-specific, detection, beer spoilage bacteria
Project description:Vibrio vulnificus causes severe necrotizing wound infections and life-threatening foodborne infections. While clinical isolates of V. vulnificus are well-established as human pathogens, the pathogenic mechanisms underlying the virulence of food-derived isolates, particularly in the case of wound infections, remain poorly understood. This study aimed to elucidate the pathogenic mechanisms of a highly virulent, seafood-derived V. vulnificus isolate. A molecular survey of 28 V. vulnificus isolates from Shenzhen identified four MARTX toxin types, with the D-type predominating (36%). We characterized a representative shrimp-derived isolate, Vv3, which carries a chromosomal D-type MARTX with an ACD-MCF-ABH-MCF effector architecture. Using a newly established mouse wound infection model, Vv3 induced 100% mortality within 12 hours, with high bacterial loads detected systemically. Pathological analysis revealed severe tissue damage at the infection site, marked by muscle necrosis, and significant distal organ damage. Strikingly, flow cytometry analysis of splenocytes showed a significant depletion of macrophages and lymphocytes, rather than a classic cytokine storm, which was supported by transcriptomic data. To dissect the molecular drivers underlying the pathogenicity of food-derived V. vulnificus, we generated isogenic toxin mutants. In vitro assays demonstrated that the MARTX toxin was the primary mediator of rapid cell death in both macrophages and epithelial cells. Deletion of the GD-rich repeat domain in the MARTX toxin (ΔrtxA-GD) significantly reduced cytotoxicity and allowed cells to maintain their morphology, while deletion of hemolysin (ΔvvhA) had a minor effect. Critically, In vivo mice wound infections indicated that MARTX-deficient mutants with or without deletion of vvhA is unable to cause mortality in mice. These results establish that the D-type MARTX toxin is the dominant virulence determinant in this foodborne isolate, driving mortality through a direct destruction of host cells. This study highlights the severe risk posed by foodborne V. vulnificus in wound exposures and informs that the GD-rich region serves as a potential target for intervention against V. vulnificus infection.
Project description:Background: Microorganisms are the major cause of food spoilage during storage, processing and distribution. Pseudomonas fluorescens is a typical spoilage bacterium that contributes to a large extent to the spoilage process of proteinaceous food. RpoS is considered an important global regulator involved in stress survival and virulence in many pathogens. Our previous work revealed that RpoS contributed to the spoilage activities of P. fluorescens by regulating resistance to different stress conditions, extracellular acylated homoserine lactone (AHL) levels, extracellular protease and total volatile basic nitrogen (TVB-N) production. However, RpoS-dependent genes in P. fluorescens remained undefined. Results: RNA-seq transcriptomics analysis combined with quantitative proteomics analysis basing on multiplexed isobaric tandem mass tag (TMT) labeling was performed for the P. fluorescens wild-type strain UK4 and its derivative carrying a rpoS mutation. A total of 375 differentially expressed genes (DEGs) and 212 differentially expressed proteins (DEPs) were identified in these two backgrounds. The DGEs were further verified by qRT-PCR tests, and the genes directly regulated by RpoS were confirmed by 5’-RACE-PCR sequencing. The combining transcriptome and proteome analysis revealed a role of this regulator in several cellular processes, including polysaccharide metabolism, intracellular secretion and extracellular structures, cell well biogenesis, stress responses, ammonia and biogenic amine production, which may contribute to biofilm formation, stress resistance and spoilage activities of P. fluorescens. Moreover, in this work we indeed observed that RpoS contributed to the production of the macrocolony biofilm’s matrix.