Quantitative PCR assays as a monitoring tool for bacterial genera in fresh fish fillets

Abstract

Fresh fish fillets are a valuable but highly perishable food, and their rapid microbial deterioration is a drawback for food safety and sustainability of aquaculture, food and retail industries. Quantitative PCR (qPCR) assays based on 16S rRNA gene (16S) sequences were developed for the most abundant bacteria genera detected by metagenomics in fresh or processed fish fillets. The efficiency and specificity of six qPCR assays (for 16S of all bacteria or genera Shewanella, Pseudomonas, Carnobacterium, Janthinobacterium and Massilia) was verified using in silico predictions, cloning, sequencing and phylogenetic analyses of amplicons obtained from refrigerated control or high-pressure processed (HPP) European sea bass (Dicentrarchus labrax) fillets. In HPP sea bass fillets, significant decreases in total bacteria 16S and of Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium 16S compared to control fillets were confirmed by qPCR, after 11 days of refrigerated storage. The qPCR assays were successfully applied to monitor microbial contamination during refrigerated storage of fresh fillets from commercial (retail) sea bass and gilthead sea bream (Sparus aurata). Significant increases in total bacterial and Shewanella, Pseudomonas, Carnobacterium and Janthinobacterium contamination were detected after 7 to 14 days. 16S copy number for total bacteria and the four target genera positively correlated with total viable counts using culture enumeration. 16S of Massilia, that is abundant in fresh fish fillets, did not significantly change during storage. The six validated qPCR assays developed are proposed as specific, sensitive, culture-independent methods for monitoring quality or processing outcomes for fish fillets during cold chain storage.