Prevalence of Escherichia albertii in Raccoons (Procyon lotor), Japan

Natural reservoirs of Escherichia albertii remain unclear. In this study, we detected E. albertii by PCR in 248 (57.7%) of 430 raccoons from Osaka, Japan, and isolated 143 E. albertii strains from the 62 PCR-positive samples. These data indicate that raccoons could be a natural reservoir of E. albertii in Japan.


Detection of Virulence Genes
We analyzed the presence of virulence genes by colony hybridization assay using 32 Plabeled DNA probes targeting eae, stx1, stx2a, stx2f, Eccdt-IB, and Eccdt-IVB under high stringent conditions, as described previously (3). When stx2 and cdt genes were detected by the colony hybridization assay, subtype-specific PCRs for stx2 (4), Eccdt-I and Eccdt-IV were carried out to determine their subtypes (Appendix Table 1). The entire nucleotide sequence of stx2f genes was determined as described previously (5). PCR amplification was done by Veriti Thermal cycler (Thermo Fisher Scientific, https://www.thermofisher.com) using TaKaRa Taq DNA polymerase (Takara Bio, https://www.takarabio.com). We sequenced the PCR products by cycle sequencing method using BigDye Terminator v1.1 and ABI 3130 Genetic Analyzer (Thermo Fisher Scientific).
To determine each intimin subtype, we determined the entire eae nucleotide sequence, as previously described (5). Predicted amino acid sequences of eae genes were aligned with those of the reference intimin subtypes by the Clustal W program of MEGA6 (https://www.megasoftware.net). The reference intimin subtypes used were from Hinenoya et al. (5). If intimin subtypes of E. albertii raccoon strains were determined to be untypable, the putative amino acid sequences were subjected to BLAST homology search using the tblastn module (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Pulsed-Field Gel Electrophoresis (PFGE)
We performed PFGE as described previously (6). Briefly, fresh bacterial cells were embedded in agarose plug and in situ lysis was carried out to isolate total genomic DNA. The genomic DNA embedded plug was subjected to restriction enzyme digestion with 30 U of XbaI (Takara Bio), and electrophoretic separation of the DNA fragments was done in 1% pulsed-field certified agarose (Bio-Rad Laboratories, https://www.bio-rad.com) on a CHEF Mapper PFGE (Bio-Rad) using 0.5x TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA [pH 8.0]). Run conditions were generated by the autoalgorithm mode of the CHEF Mapper PFGE system for the sizes ranging between 20 and 300 kb, and the running time was 26.93 hours. XbaI-digested genomic DNA of Salmonella Braenderup strain H98121 was used as a molecular size marker.
DNA fingerprints of E. albertii strains were interpreted based on Tenover's criteria (7) and analyzed using Fingerprinting II Software (Bio-Rad) to know their phylogenetic relationships.

Detection of Stx2f Production in stx2f Gene-Positive E. albertii
Production of Stx2f by stx2f gene-positive E. albertii strains was determined by Vero cells cytotoxicity assay, as previously described (3). We prepared crude toxin samples as follows: E. albertii was cultured in 3 mL of lysogenic broth (LB broth, Becton Dickinson) at 37°C for 14 hours. An aliquot of the culture was inoculated into 3 mL of fresh lysogenic broth and cultured until early log phase (≈0.2 optical density at 600 nm). Mitomycin C (Kyowa Hakko Kirin, https://www.kyowakirin.com) was added to the culture at the final concentration of 0.5 µg/mL and further incubated at 37°C for 4 hours aerobically. Culture supernatant was passed through a sterile filter with 0.22-µm pore size (Merck Millipore, https://www.emdmillipore.com), and the filtrate was subjected to cytotoxicity assay.
Neutralization assay of the toxin activity was also carried out using anti-Stx2fA rabbit serum (8), which was preincubated with crude toxin samples at 37°C for 30 min. The mixture was applied to the cytotoxicity assay.