Enumeration of Enterobacteriaceae in Various Foods with a New Automated Most-Probable-Number Method Compared with Petrifilm and International Organization for Standardization Procedures

An automated most-probable-number (MPN) system (TEMPO, bioMerieux, Marcy l'Etoile, France) for enumeration of Enterobacteriaceae (EB) was compared with methods involving violet red bile glucose agar (VRBG) (International Organization for Standardization ISO method 21528-2) (ISO-VRBG) and Petrifilm (PF-EB). The MPN partitioning (three different volumes with 16 replicates of each) is done automatically in a disposable card. Bacterial growth is indicated by acid production from sugars, lowering the pH of the medium, and quenching the fluorescence of 4-methylumbelliferone. After incubation, the number of nonfluorescent wells is read in a separate device, and the MPN is calculated automatically. A total of 411 naturally contaminated foods were tested, and 190 were in the detection range for all methods. For these results, the mean (+/- standard deviation) counts were 2.540 +/- 1.026, 2.547 +/- 0.995, and 2.456 +/- 1.014 log CFU/g for the ISO-VRBG, PF-EB, and automated MPN methods, respectively. Mean differences were -0.084 +/- 0.460 log units for the automated MPN results compared with the ISO-VRBG and 0.007 +/- 0.450 for the PF-EB results compared with the ISO-VRBG results. The automated MPN method tends to yield lower numbers and the PF-EB method tends to yield higher numbers than does the ISO-VRBG method (difference not significant; Kruskal-Wallis test, P = 0.102). Thus, the average difference was highest between the automated MPN method and the PF-EB method (-0.091 +/- 0.512 log units). Differences between the automated MPN and ISO-VRBG results of >1 log unit were detected in 3.4% of all samples. For 3.9% of the samples, one comparison yielded differences of < 1 log CFU/g and the other yielded > 1 but <2 log CFU/g, which means that the differences are possibly > 1 log CFU/g. For the ISO-VRBG method, confirmation of isolates was necessary to avoid a bias due to the presence of oxidase-positive glucose-fermenting colonies. The automated MPN system yielded results comparable to those of the confirmed Enterobacteriaceae ISO-VRBG method but required only 24 h of analysis time.