Evaluating Methods for Detecting Escherichia albertii in Chicken Meat

Abstract

Escherichia albertii is an emerging foodborne pathogen. The source of infection in most foodborne outbreaks is unknown, as it is difficult to isolate E. albertii from suspected foods or water. E. albertii has a broad host range among birds and can be isolated from chicken meat. In this study, PCR assay, enrichment and isolation conditions for detecting E. albertii in chicken meat were evaluated. The growth of 48 E. albertii strains isolated in Japan between 1994 and 2018 was evaluated in modified EC broth (mEC) and mEC supplemented with novobiocin (NmEC) and on different media containing carbohydrates. In addition, the enzyme for nested PCR, the enrichment condition, the most probable number (MPN) method, and agar media were also evaluated for chicken meat. To distinguish E. albertii from presumptive non-E. albertii bacteria, desoxycholate hydrogen sulfide lactose agar (DHL), MacConkey agar (MAC), and these agars supplemented with rhamnose and xylose (RX-DHL and RX-MAC, respectively) were used. All E. albertii strains grew in mEC and NmEC at both 36°C and 42°C and did not utilize rhamnose, sucrose, or xylose. Both the first PCR and nested PCR, using TaKaRa Ex Taq which was 10–100 times superior to the other enzymes, showed positive results in enrichment culture of 25 g of chicken meat inoculated with >20 CFU of E. albertii in mEC and NmEC at 42°C for 22 ± 2 h. Thus, the first PCR was sensitive enough to detect E. albertii in chicken meat. The MPN values in mEC and NmEC were 0.5- and 2.3-fold of inoculated bacterial concentration, respectively. E. albertii in chicken meat was more efficiently isolated with enrichment in NmEC (70.1-100%) and plating onto RX-DHL (85.4%) and RX-MAC (100%) compared to enrichment in mEC (53.5-83.3%) and plating onto DHL (70.1%) and MAC (92.4%), respectively. Thus, optimized conditions for the surveillance of E. albertii contamination in food and investigations of E. albertii outbreaks, including the infectious dose, were clarified.

Proposal of a novel selective enrichment broth, NCT-mTSB, for isolation of Escherichia albertii from poultry samples

“…The Japanese national standard adopts mEC for enrichment of STEC (Hara-Kudo et al, 2016). BPW and mEC have also been used for the isolation of E. albertii (Arai et al, 2021;Maeda et al, 2015). Novobiocin (Merck), potassium tellurite (Thermo Fisher Scientific), and cefixime (Fujifilm Wako Pure Chemical) were used as selective supplements.…”

“…Novobiocin (Merck), potassium tellurite (Thermo Fisher Scientific), and cefixime (Fujifilm Wako Pure Chemical) were used as selective supplements. For isolation of E. albertii, we used XR-DH agar modified by elimination of lactose and sucrose from XR-DHL (Arai et al, 2021;Murakami et al, 2019; Table S2).…”

“…10 CFU of E. albertii were cultivated with nine volumes of enrichment broth. BPW, mEC, and novobiocin supplemented mTSB (N-mTSB); BPW and mEC broths, which have been used for isolation of E. albertii from chicken samples (Arai et al, 2021;Maeda et al, 2015), were used for comparison with NCT-mTSB. N-mTSB was used as a control for NCT-mTSB.…”

“…Existing procedures include those of Maheux et al (2018), who developed an mEA agar for selective isolation of E. albertii from stool samples, and of Hinenoya, et al (2020b), who used XRM-MacConkey agar in which the carbohydrate source lactose was replaced with xylose, rhamnose and melibiose. XR-DHL agar, deoxycholate hydrogen sulphide lactose (DHL) agar supplemented with xylose and rhamnose, also have been used for isolation of E. albertii (Arai et al, 2021;Murakami et al, 2019). However, these agars contain no supplements to impair growth of other competing bacteria, except for bile salts, which mainly inhibit the growth of Gram-positive bacteria.…”

“…Several enrichment broths, including buffered peptone water (BPW) and EC broth, have been used for the isolation of E. albertii (Maeda et al, 2015;Wang et al, 2016). Most recently, Arai et al evaluated the effectiveness of two enrichment broths, modified EC broth (mEC) and mEC supplemented with novobiocin, for the isolation of E. albertii (Arai et al, 2021). The problem is that these broths are modifications of enrichment methods for Enterobacteriaceae, which means that not only E. albertii can grow.…”