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

Abstract

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...

Evaluation of an alternative chromogenic method for the detection and enumeration of enterococci in waters

“…However, a previously published paper about the Enterolert method for the detection of enterococci in recreational waters reported a lower correlation coefficient (r = 068) (Eckner, 1998). Other rapid methods (PCR) that have been evaluated for the quantification of other bacteria in food reported correlation coefficient comparable to results reported in current experiments (Macé et al, 2013).…”

Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimp

“…Value differences were compared using the least significant difference (LSD) method at p=0.05 (Macé et al 2013;Wang et al 2014).…”

“…Although the manufacturer recommends 50 μM of PMA as an optimal concentration for inhibiting PCR amplification of DNA from dead cells, a number of reports in the literature (Zhu et al 2012;Macé et al 2013;Barbau-Piednoir et al 2014;Liu and Mustapha 2014) indicated that it was necessary to optimize the PMA concentration for effectively discriminating and quantifying viable cells. As shown in Table 3, the multiplex real-time PCR amplification signal of viable V. parahaemolyticus and L. monocytogenes cells was not affected by PMA treatment at all (detailed C T values not shown).…”

“…PMA is a DNA intercalating reagent, which only penetrates the cell membranes of dead cells and covalently binds to DNA through photolysis (Nocker and Cheung Camper 2006;Pan and Breidt 2007;Luo et al 2010;Schnetzinger et al 2013;Macé et al 2013;Salam et al 2014). This reaction inhibits subsequent PCR amplification, thereby ensuring quantification of viable bacteria (Nocker and Cheung Camper 2006;Pan and Breidt 2007;Zhu et al 2012;Macé et al 2013;Liu and Mustapha 2014;Salam et al 2014). This reagent has been used in combination with several real-time PCR methods for quantification of viable pathogens in food, such as V. parahaemolyticus (Zhu et al 2012), L. monocytogenes (Pan and Breidt 2007), Campylobacter (Josefsen et al 2010), Salmonella spp.…”

“…For maximum efficiency, multiplex real-time PCR was developed as an automated highthroughput technique for simultaneous and direct detection of more than two pathogens in the same reaction (Kim et al 2012;Garrido et al 2013;Ma et al 2014;Zhang et al 2015). However, one of the limitations of real-time PCR and multiplex real-time PCR methods for the detection of bacteria in food samples is that both viable and dead cells are detected (Kramer et al 2009;Schnetzinger et al 2013;Macé et al 2013;Salam et al 2014). Propidium monoazide (PMA) is used in PCR methods to overcome the problem of detection of dead cells.…”