Prevalence data of diarrheagenic Escherichia coli in the fecal pellets of wild rodents using culture methods and PCR assay

Published: 26-07-2020| Version 2 | DOI: 10.17632/9dkfkfnyzs.2
Contributors:
Md Mafizur Rahman,
Sanjin Lim,
Heuk Kim,
Jae Youl Cho,
Yung Park

Description

The Escherichia coli and Shiga toxin-producing E. coli, bacteria in the fecal samples were detected by culture and molecular method. The rodent fecal samples were first cultured in a non-selective buffered peptone water (BPW) and then the enrichment broth containing the samples was streaked with a loop onto the E. coli-selective eosin methylene blue agar (EMB) media and Shiga toxin-producing E. coli-selective cefixime tellurite sorbitol MacConkey agar (CT-SMAC) media and incubated. The plates were examined for colony forming units (CFU) and sub-cultivated was conducted on EMB so that pure colonies could be collected. The E. coli colonies produced metallic sheen color produced on EMB agar media. Sorbitol negative colonies (white or off white color) were detected onto CT-SMAC agar (Fig. S1). We select colorless colony as E. coli O157:H7 positive colonies seem to be STEC-positive on culture media. Finally the STEC E. coli was detected based on morphology, PCR band, and sequence similarity analysis. The E. coli and Shiga toxin-producing E. coli sequences were compared for similarity with bacteria deposited in GenBank using NCBI BLAST, which is available at http://www.ncbi.nlm.nih.gov/. The bacterial colonies on the selective media that had been identified by their morphology were re-identified by PCR using molecular markers. A PCR amplification of hypervariable regions (HVR) the bacterial 16S rRNA gene was performed to confirm whether the colonies on the EMB media belonged to E. coli. We randomly selected a single colony from each of the 26 EMB agar plates and after PCR amplification with the HVR primer set, the target bands for the 16S rRNA gene were found in all 26 single colonies (Fig. S2). Thirteen PCR bands were then sequenced and deposited to the GenBank database. A PCR amplification of the Shiga toxin genes (stx1 and stx2) was performed to confirm whether the colonies on the CT-SMAC media belonged to Shiga toxin-producing E. coli. We randomly selected one or more single colonies from each of the 18 CT-SMAC agar plates. Two single colonies from the CT-SMAC plate for MuRaNo-1, three single colonies from MuApSy-3, and a single colony from the others were selected, which resulted in 21 colonies. The PCR amplification using the Stx1 primer set produced bands for the Shiga toxin gene from all of the 21 single colonies (Fig S3). We then sequenced 13 PCR bands from the 21 colonies. The target PCR bands of 5 E. coli with the stx2 gene (Fig S4) were amplified and sequenced, deposited to the GenBank database. Raw data sequence of 16S gene with their colony ID and GenBank accession No. is provided as fasta text file. In addition, similarly, Shiga toxin gene sequences (stx1 and stx2) of raw data set are described with colony ID and GenBank accession No.

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