Quantification of viable Brochothrix thermosphacta in cooked shrimp and salmon by real-time PCR

Mamlouk, Kelthoum; Macé, Sabrina; Guilbaud, Morgan; Jaffrès, Emmanuel; Ferchichi, Mounir; Prévost, Hervé; Pilet, Marie-France; Dousset, Xavier

doi:10.1016/j.fm.2011.09.012

Cited by 37 publications

(23 citation statements)

References 36 publications

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“…Moreover, the use of PMA allowed exclusive quantification of viable cells (21,42) and, consequently, only the P. phosphoreum cells active in spoilage. When real-time PCR was used previously for the enumeration of bacteria in a meat or fish matrix, linear quantifications were reported over a range of at least 5 logs, and quantification limits without enrichment varied from about 2 to 3 log CFU/g (22,24,25,37,43,44), as obtained in the present study.…”

Section: Discussionsupporting

confidence: 79%

“…Finally, to photoactivate the PMA, the tubes were placed in crushed ice with their lids removed and exposed for 5 min to light from a 500-W halogen lamp positioned at a distance of 20 cm. They were shaken occasionally to ensure complete cross-linking of the available DNA (24).…”

Section: Raw Salmon Samplesmentioning

confidence: 99%

“…PMA is an intercalating DNA agent which enters dead cells and binds to DNA, inhibiting subsequent PCR amplification and thereby ensuring quantification of viable bacteria (21). PMA has been used in combination with several real-time PCR methods for quantification of viable bacteria in food, e.g., Campylobacter (22), Listeria monocytogenes (23), Brochothrix thermosphacta (24,25), Vibrio parahaemolyticus (26,27), and Escherichia coli O157:H7 (28).…”

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confidence: 99%

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Development of a Rapid Real-Time PCR Method as a Tool To Quantify Viable Photobacterium phosphoreum Bacteria in Salmon (Salmo salar) Steaks

Macé

Mamlouk

Chipchakova

et al. 2013

Appl Environ Microbiol

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A specific real-time PCR quantification method combined with a propidium monoazide sample treatment step was developed to determine quantitatively the viable population of the Photobacterium phosphoreum species group in raw modified-atmospherepacked salmon. Primers were designed to amplify a 350-bp fragment of the gyrase subunit B gene (gyrB) of P. phosphoreum. The specificity of the two primers was demonstrated by using purified DNA from 81 strains of 52 different bacterial species. When these primers were used for real-time PCR in pure culture, a good correlation (R 2 of 0.99) was obtained between this method and conventional enumeration on marine agar (MA). Quantification was linear over 5 log units as confirmed by using inoculated salmon samples. On naturally contaminated fresh salmon, the new real-time PCR method performed successfully with a quantification limit of 3 log CFU/g. A correlation coefficient (R 2 ) of 0.963 was obtained between the PCR method and classic enumeration on MA, followed by identification of colonies (290 isolates identified by real-time PCR or by 16S rRNA gene sequencing). A good correlation with an R 2 of 0.940 was found between the new PCR method and an available specific conductance method for P. phosphoreum. This study presents a rapid tool for producing reliable quantitative data on viable P. phosphoreum bacteria in fresh salmon in 6 h. This new culture-independent method will be valuable for future fish inspection, the assessment of raw material quality in fish processing plants, and studies on the ecology of this important specific spoilage microorganism. Modified-atmosphere-packed (MAP) fresh fish is increasingly popular in Europe and widely sold in supermarkets as a chilled product. Compared to aerobically stored fresh fish, this kind of packaging, with typical headspace CO 2 concentrations of 20 to 60%, modifies the dominating microbiota mainly by reducing growth of aerobic Gram-negative bacteria like Pseudomonas (1, 2). This results in an extended shelf life which facilitates the chilled distribution and marketing of fresh MAP fish. However, this packaging allows the growth of CO 2 -resistant bacteria, including Gram-positive lactic acid bacteria and the Gram-negative bacterium Photobacterium phosphoreum (2-4). The latter has been identified as the specific spoilage organism (SSO) responsible for trimethylamine production and sensory spoilage of MAP cod (5, 6) and as the main spoilage bacterial species of several chilled marine fish, including cod, garfish, halibut, saithe, salmon, and shrimp (7-15).Multilocus analysis, based on 16S rRNA and gyrB and luxABFE genes, divided strains originally isolated and identified as P. phosphoreum into three distinct clades of P. phosphoreum, Photobacterium iliopiscarium, and Photobacterium kishitanii (16,17). The members of the P. phosphoreum species group have been reported as important for spoilage of raw MAP fish, and they include both luminous and nonluminous variants (5, 18). It is therefore relevant to detect and enumerate this s...

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Section: Discussionsupporting

confidence: 79%

Section: Raw Salmon Samplesmentioning

confidence: 99%

mentioning

confidence: 99%

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Development of a Rapid Real-Time PCR Method as a Tool To Quantify Viable Photobacterium phosphoreum Bacteria in Salmon (Salmo salar) Steaks

Macé

Mamlouk

Chipchakova

et al. 2013

Appl Environ Microbiol

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“…of organoleptically unpleasant compounds (1)(2)(3). This Gram-positive, fermentative bacterium can become the dominant spoilage species of modified atmospherepackaged (MAP) and vacuum-packaged meats when sufficient oxygen is present (3)(4)(5)(6).…”

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confidence: 99%

“…This Gram-positive, fermentative bacterium can become the dominant spoilage species of modified atmospherepackaged (MAP) and vacuum-packaged meats when sufficient oxygen is present (3)(4)(5)(6). Although aerobic spoilage is dominated largely by Gram-negative pseudomonads, B. thermosphacta can also play an important role in shortening the shelf-life of aerobically stored meat, particularly when bacteriostatic agents such as sulfites are used (7)(8)(9)(10)(11).…”

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Insight into the Genome of Brochothrix thermosphacta, a Problematic Meat Spoilage Bacterium

Stanborough

Fegan

Powell

et al. 2017

Appl Environ Microbiol

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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.KEYWORDS Brochothrix, Listeria monocytogenes, meat spoilage, spoilage B rochothrix thermosphacta is one of the most abundant spoilage organisms of fresh and cured meats, fish, and fish products, due to its tolerance to high-salt and low-pH conditions, its ability to grow at refrigeration tempera...

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Microbiological Examination of Seafood

Boziaris

Parlapani

2013

Seafood Processing

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Quantification of viable Brochothrix thermosphacta in cooked shrimp and salmon by real-time PCR

Cited by 37 publications

References 36 publications

Development of a Rapid Real-Time PCR Method as a Tool To Quantify Viable Photobacterium phosphoreum Bacteria in Salmon (Salmo salar) Steaks

Development of a Rapid Real-Time PCR Method as a Tool To Quantify Viable Photobacterium phosphoreum Bacteria in Salmon (Salmo salar) Steaks

Insight into the Genome of Brochothrix thermosphacta, a Problematic Meat Spoilage Bacterium

Microbiological Examination of Seafood

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