Comparative evaluation of most probable number and direct plating methods for enumeration of Escherichia coli in Ruditapes philippinarum, and effect on classification of production and relaying areas for live bivalve molluscs

A precise and accurate method for enumeration of Escherichia coli is critical to classify production and relaying areas for live bivalve molluscs. Paired comparison of most probable number and direct plating count of E. coli was performed on a total of 918 samples of Manila clams (Ruditapes philippinarum) collected from official monitoring and from a regional shellfish monitoring program conducted from 2011 to 2017 in Emilia Romagna region, Italy. The records of E. coli enumeration resulting from both MPN reference method and alternative TBX count method were compared with McNemar's test using three cut-off values, namely 230, 700 and 4.600 MPN/ 100g, based on the requirements laid down in Regulation (EU) 2019/627. The relative trueness was calculated. Significant differences were observed between the two methods for each investigated cut-off: these incongruities concerned a total of 129 (14%), 131 (14%) and 56 (6%) samples observed as negative with TBX but positive by MPN (respectively using 230, 700 and 4.600 E. coli as cut-off), as well as, quite the opposite, a total of 20 (2,2%), 8 (0,9%) and 5 (0,5%) samples observed negative by MPN but positive with TBX method. A negative bias is reported between the two methods. Applying the outcomes of the two different methods to a real scenario, the Veterinary Competent Authority could classify production and relaying areas in the same area of classification only for 69% of samples. This high degree of disagreement between MPN and direct plating outcomes clearly shows the different and not negligible impact of the two methods on the specific E. coli requirements for the classification of production and relaying areas for R. philippinarum. Analytical methods, either official or validated, if used officially by Veterinary Authority must lead to the same classification of production and relaying areas for live bivalve molluscs.