An interlaboratory study on the detection method for Escherichia albertii in food using real time PCR assay and selective agars.

Escherichia albertii is an emerging enteropathogen. Although E. albertii-specific detection and isolation methods have been developed, their efficiency on food samples have not yet been systematically studied. To establish a series of effective methods for detecting E. albertii in food, an interlaboratory study was conducted in 11 laboratories using enrichment with modified E. coli broth supplemented with cefixime and tellurite (CT-mEC), real-time PCR assay, and plating on four kinds of selective agars. This study focused on the detection efficiency of an E. albertii-specific real-time PCR assay (EA-rtPCR) and plating on deoxycholate hydrogen sulfide lactose agar (DHL), MacConkey agar (MAC), DHL supplemented with rhamnose and xylose (RX-DHL), and MAC supplemented with rhamnose and xylose (RX-MAC). Chicken and bean sprout samples were inoculated with E. albertii either at 17.7 CFU/25 g (low inoculation level) or 88.5 CFU/25 g (high inoculation level), and uninoculated samples were used as controls. The sensitivity of EA-rtPCR was 1.000 for chicken and bean sprout samples inoculated with E. albertii at low and high inoculation levels. The Ct values of bean sprout samples were higher than those of the chicken samples. Analysis of microbial distribution by 16S rRNA gene amplicon sequencing in enriched cultures of bean sprout samples showed that approximately >96 % of the population comprised unidentified genus of family Enterobacteriaceae and genus Acinetobacter in samples which E. albertii was not isolated. The sensitivity of the plating methods for chicken and bean sprout samples inoculated with a high inoculation level of E. albertii was 1.000 and 0.848-0.970, respectively. The sensitivity of the plating methods for chicken and bean sprout samples inoculated with a low inoculation level of E. albertii was 0.939-1.000 and 0.515-0.727, respectively. The E. albertii-positive rate in all colonies isolated in this study was 89-90 % in RX-DHL and RX-MAC, and 64 and 44 % in DHL and MAC, respectively. Therefore, the sensitivity of RX-supplemented agar was higher than that of the agars without these sugars. Using a combination of enrichment in CT-mEC and E. albertii isolation on selective agars supplemented with RX, E. albertii at an inoculation level of over 17.5 CFU/25 g of food was detected with a sensitivity of 1.000 and 0.667-0.727 in chicken and bean sprouts, respectively. Therefore, screening for E. albertii-specific genes using EA-rtPCR followed by isolation with RX-DHL or RX-MAC is an efficient method for E. albertii detection in food.

Detection of Escherichia albertii in Retail Oysters.

ABSTRACT: Escherichia albertii is an emerging foodborne pathogen. Owing to its distribution in river water, it is important to determine the presence of E. albertii in aquaculture-related foods. In this study, we investigated the distribution of E. albertii in retail oyster samples. A total of 427 raw oyster samples (385 Pacific oysters and 42 Japanese rock oysters) were enriched in modified Escherichia coli broth (mEC) or mEC supplemented with novobiocin (NmEC) at 42°C. The cultures were used for E. albertii-specific nested PCR assay, as well as for E. albertii isolation using deoxycholate hydrogen sulfide lactose agar (DHL), DHL supplemented with rhamnose and xylose, and MacConkey agar supplemented with rhamnose and xylose. The population of E. albertii in nested PCR-positive samples was determined using the most-probable-number (MPN) method. E. albertii isolates were subjected to biochemical and genetic characterization. E. albertii was detected in 5 (1.6%) of 315 Pacific oyster samples (one piece each), 2 (2.9%) of 70 Pacific oyster samples (25 g each), and 2 (4.8%) of 42 Japanese rock oyster samples procured from four geographically distinct regions. A total of 64 E. albertii strains were isolated from eight of the nine nested PCR assay-positive oyster samples, and the MPN value was under the detection limit (<3 MPN/10 g). A specific season or month for detecting E. albertii was not observed in this study, suggesting that the pathogen is present in seawater. All the E. albertii isolates, except one, were positive for the virulence factor eae, indicating that these isolates have the potential to infect humans.