Comparison of Stomaching versus Rinsing for Recovering Bacterial Communities from Rainbow Trout (Oncorhynchus mykiss) Fillets

Abstract

ABSTRACT The use of high-throughput methods allows a better characterization of food-related bacterial communities. However, such methods require large amounts of high-quality bacterial DNA, which may be a challenge when dealing with a complex matrix that has a low concentration of bacteria, such as fresh fish fillets. Therefore, the choice of method used to recover bacteria from a food matrix in a cost-effective way is critical, yet little information is available on the performance of commonly used methods. We assessed the recovery capacity of two such methods: stomaching and mechanical rinsing. The efficiency of the methods was evaluated through quantitative recovery and compatibility with end-point quantitative PCR (qPCR). Fresh rainbow trout (Oncorhynchus mykiss) fillets were inoculated with a bacterial marker, Brochothrix thermosphacta, at different concentrations (7.52 to 1.52 log CFU/g). The fillets were processed by one of the two methods, and the recovery of the marker in the suspensions was assessed by plate counting and qPCR targeting B. thermosphacta–rpoC. The same analyses were performed on six noninoculated fresh fillets. Stomaching and mechanical rinsing allowed efficient and repeatable recovery of the bacterial communities from the 42 inoculated fillets. No significant differences in recovery ratios were observed between the marker enumerated in the inoculation suspensions and in the corresponding recovery suspensions after rinsing and stomaching. However, the stomaching method allowed too many particles to pass through the filters bag, making necessary a limiting supplementary filtration step. As a consequence, only the rinsing recovery method allowed proper PCR quantification of the inoculated B. thermosphacta. The mean recovered bacterial level of the fillets was approximately 3 log CFU/g. It seems more relevant and cost-effective to recover the endogenous bacterial microbiota of a fish fillet structure using the rinsing method rather than the stomaching method. HIGHLIGHTS