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 (RX-DHL), and MacConkey agar supplemented with rhamnose and xylose (RX-MAC). 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 of 315 (1.6%) Pacific oyster samples (one piece each), 2 of 70 (2.9 %) Pacific oyster samples (25 g each), and 2 of 42 (4.8 %) Japanese rock oyster samples procured from four geographically distant 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.

Microbiology and Epidemiology of Escherichia albertii—An Emerging Elusive Foodborne Pathogen

“…Most E. albertii isolation protocols rely on a single enrichment step using various broths including buffered peptone water (BPW), E. coli broth (EC), modified EC (mEC), novobiocin–mEC (NmEC), tryptic soy broth (TSB), modified TSB (mTSB), or novobiocin–cefixime–tellurite supplemented mTSB (NCT-mTSB) at various temperatures ranging from 20 to 44.5 °C [ 22 , 23 , 37 , 46 , 48 , 49 , 50 ]. The added novobiocin (20 to 25 mg/L) inhibits most Gram-positive bacteria, while in the NCT-mTSM enrichment broth the tellurite (1 mg/L) and cerufuxim (0.05 mg/L) inhibit most Enterobacteria, while growth at temperatures ≥ 42 °C is postulated to inhibit most background microbiota, Shigella spp.…”

“…The added novobiocin (20 to 25 mg/L) inhibits most Gram-positive bacteria, while in the NCT-mTSM enrichment broth the tellurite (1 mg/L) and cerufuxim (0.05 mg/L) inhibit most Enterobacteria, while growth at temperatures ≥ 42 °C is postulated to inhibit most background microbiota, Shigella spp. and E. coli [ 22 , 23 , 37 ]. Enrichment protocols that employ antimicrobial selective pressure though promising, run the risk of selecting only E. albertii strains resistant to these antimicrobial agents especially when they are validated using a few strains from limited sources and or geographical regions.…”

“…Several PCR protocols have been proposed, most of which are based on the lysP , mdh , clpX , yejH , and yejK , EAKF1 _ch4033, and the species-specific cdt gene [ 13 , 19 , 22 , 24 , 33 , 51 ]. The eae gene was also proposed as a gene marker for E. albertii but this was soon discarded as several pathogenic E. coli strains also carry this gene [ 5 , 24 ].…”

“…Post enrichment, cultures are plated on deoxycholate hydrogen sulphide lactose (DHL) or MacConkey (MC) agar and various variations of these media with 1% xylose, 1% rhamnose, and or 1% melibiose supplementation (XR-MC, XR-DHL, XRM-MC, XRM-DHL, and XR-DH) [ 22 , 36 , 56 ]. E. coli utilize these sugars and grow to produce colored colonies.…”

“…For example, in several gastroenteritis outbreaks the causative pathogen was wrongly diagnosed as EPEC instead of eae -positive E. albertii [ 9 , 11 , 12 , 14 , 17 , 18 ]. Because of this, several enrichment broths, isolation, and identification protocols have been developed, however, to date, there is no standard method [ 19 , 20 , 21 , 22 , 23 ]. The specificity and sensitivity for most of these protocols though high [ 24 ], have been evaluated based on a limited number of strains, thus it is not clear if such protocols cover the biodiversity of E. albertii isolates.…”