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Transcriptional profiling of biofilms formed on chilled beef by psychrotrophic meat spoilage bacterium, Pseudomonas fragi 1793.

ABSTRACT: Pseudomonas fragi is the predominant bacterial species associated with spoiled aerobically stored chilled meat worldwide. It readily forms biofilms on meat under refrigerated temperature conditions used in the meat industry. Biofilm growth leads to slime development on meat which in turn becomes a major quality defect. To understand the genetic regulation that aids P. fragi to survive under chilled conditions used in the meat industry, as well to obtain an overview of the transcriptomic behavior of this organism when grown as biofilms, RNA sequencing was carried out for the main stages of the P. fragi 1793 biofilm. RNA was extracted at different stages of the biofilm cycle namely initiation, maturation and dispersal. At the same time, the biofilm growth was assessed by fluorescent staining and imaging using confocal laser scanning microscope. The results of RNA sequencing were verified by qRT-PCR using twelve genes that were most significantly up and down regulated at each stage. Differential expression analysis at biofilm maturation revealed 332 significantly upregulated genes and 37 downregulated genes relative to initiation. Differential expression analysis at biofilm dispersal reveled 658 upregulated and 275 downregulated genes relative to initiation. During biofilm maturation and dispersal, genes coding for flp family type IVb pilin, ribosome modulation factor, creatininase were the most upregulated genes while genes encoding for iron uptake systems including TonB-dependent siderophore receptor and taurine transport were significantly down regulated. The results show that protein synthesis and cellular multiplication cease after the biofilm population maximum has reached.

SUBMITTER: Wickramasinghe NN

PROVIDER: S-EPMC7921472 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Transcriptional profiling of biofilms formed on chilled beef by psychrotrophic meat spoilage bacterium, Pseudomonas fragi 1793.

Wickramasinghe Nirmani N NN Ravensdale Joshua J Coorey Ranil R Dykes Gary A GA Chandry Peter S PS

Biofilm 20210217

Pseudomonas fragi is the predominant bacterial species associated with spoiled aerobically stored chilled meat worldwide. It readily forms biofilms on meat under refrigerated temperature conditions used in the meat industry. Biofilm growth leads to slime development on meat which in turn becomes a major quality defect. To understand the genetic regulation that aids P. fragi to survive under chilled conditions used in the meat industry, as well to obtain an overview of the transcrip ...[more]

PMID: 33718862

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Project description:Mesophilic and psychrotrophic populations from refrigerated meat were identified in this study, and the spoilage potential of microbial isolates in packaged beef was evaluated by analyzing the release of volatile organic compounds (VOC) by gas chromatography-mass spectrometry (GC/MS). Fifty mesophilic and twenty-nine psychrotrophic isolates were analyzed by random amplified polymorphic DNA-PCR, and representative strains were identified by 16S rRNA gene sequencing. Carnobacterium maltaromaticum and C. divergens were the species most frequently found in both mesophilic and psychrotrophic populations. Acinetobacter baumannii, Buttiauxella spp. and Serratia spp. were identified among the mesophilic isolates, while Pseudomonas spp. were commonly identified among the psychrotrophs. The isolates were further characterized for their growth at different temperatures and their proteolytic activity in vitro on meat proteins extracts at 7 degrees C. Selected proteolytic strains of Serratia proteamaculans, Pseudomonas fragi, and C. maltaromaticum were used to examine their spoilage potential in situ. Single strains of these species and mixtures of these strains were used to contaminate beef chops that were packed and stored at 7 degrees C. At time intervals up to 1 month, viable counts were determined, and VOC were identified by GC/MS. Generally, the VOC concentrations went to increase during the storage of the contaminated meats, and the profiles of the analyzed meat changed dramatically depending on the contaminating microbial species. About 100 volatiles were identified in the different contaminated samples. Among the detected volatiles, some specific molecules were identified only when the meat was contaminated by a specific microbial species. Compounds such as 2-ethyl-1-hexanol, 2-buten-1-ol, 2-hexyl-1-octanol, 2-nonanone, and 2-ethylhexanal were detectable only for C. maltaromaticum, which also produced the highest number of aldehydes, lactones, and sulfur compounds. The highest number of alcohols and ketons were detected in the headspace of meat samples contaminated by P. fragi, whereas the highest concentrations of some alcohols, such as 1-octen-3-ol, and some esters, such as isoamyl acetate, were produced by S. proteamaculans. In conclusion, different microbial species can contribute to meat spoilage with release of different volatile compounds that concur to the overall quality decrease of spoiling meat.

Project description:Brochothrix thermosphacta is a dominant but poorly studied meat spoilage organism. It is a close relative of the foodborne pathogen Listeria monocytogenes, and Brochothrix constitutes the second genus in the Listeriaceae family. Here, the genomes of 12 B. thermosphacta strains were sequenced, assembled into draft genomes, characterized, and compared with the genomes of Brochothrix campestris and L. monocytogenes Phenotypic properties including biogenic amine production and antibiotic and heavy metal susceptibilities were tested. Comparative genomic analyses revealed a high degree of similarity among the B. thermosphacta strains, with bacteriophage genes constituting a significant proportion of the accessory genome. Genes for the production of the malodorous compounds acetate, acetoin, butanediol, and fatty acids were found, as were stress response regulatory genes, which likely play important roles in the spoilage process. Amino acid decarboxylases were not identified in the genomes, and phenotypic testing confirmed their absence. Orthologs of Listeria virulence proteins involved in virulence regulation, intracellular survival, and surface protein anchoring were found; however, key virulence genes were absent. Analysis of antibiotic susceptibility showed that strains were sensitive to the four tested antibiotics, except for one tetracycline-resistant isolate with plasmid-mediated tetracycline resistance genes. Strains tolerated higher levels of copper and cobalt than of cadmium although not at concentrations high enough to categorize the strains as being resistant. This study provides insight into the Brochothrix genome, links previous phenotypic data and data provided here to the gene inventory, and identifies genes that may contribute to the persistence of this organism in the food chain.IMPORTANCE Despite increasing knowledge and advances in food preservation techniques, microbial spoilage of foods causes substantial losses, with negative social and economic consequences. To better control the contamination and microbial spoilage of foods, fundamental knowledge of the biology of key spoilage bacteria is crucial. As a common meat spoilage organism, B. thermosphacta contributes substantially to spoilage-associated losses. Nonetheless, this organism and particularly its genome remain largely unstudied. This study contributes to improving our knowledge of the Brochothrix genus. Spoilage-relevant pathways and genes that may play a role in the survival of this organism in a food processing environment were identified, linking previous phenotypic data and data provided here to the gene inventory of Brochothrix and establishing parallels to and differences from the closely related foodborne pathogen L. monocytogenes.

Dataset Information

Transcriptional profiling of biofilms formed on chilled beef by psychrotrophic meat spoilage bacterium, Pseudomonas fragi 1793.

Publications

Transcriptional profiling of biofilms formed on chilled beef by psychrotrophic meat spoilage bacterium, <i>Pseudomonas fragi</i> 1793.

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