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

Wild animals, such as rodents seem to be competent reservoir of bacteria-borne zoonotic diseases which disseminate in human. We investigated the presence of E. coli, Shiga toxin-producing E. coli (STEC), and Salmonella in the feces of six category wild rodent species (Apodemus agrarius, A. peninsulae, A. sylvaticus, Micromys minutus, Myodes regulus, and R. norvegicus) captured from different agricultural regions in South Korea. Among them, A. agrarius, which account for 65% of total (N = 52) individuals, are most widely distributed and abundant in various agroecosystems in South Korea. The bacterial identification was performed by cultural and molecular methods. In cultural method, the fecal cultures from 26 individuals formed colonies on E. coli-selective EMB agar media. Of them, the fecal cultures from 18 individuals also produced colonies on the Shiga toxin-producing E. coli-selective CT-SMAC agar media as well as the EMB agar media. In molecular method, polymerase chain reaction (PCR) was carried out to detect two virulence genes (stx1 and stx2) of isolated E. coli. The amplified dataset of stx1 and stx2 genes of E. coli were sequenced. In this manuscript, E. coli and STEC were detected but there were no Salmonella species. The wild rodents’ data would provide important information on reservoirs of those pathogenic bacteria.


a b s t r a c t
Wild animals, such as rodents seem to be competent reservoir of bacteria-borne zoonotic diseases which disseminate in human. We investigated the presence of E. coli , Shiga toxinproducing E. coli (STEC), and Salmonella in the feces of six category wild rodent species ( Apodemus agrarius, A. peninsulae, A. sylvaticus, Micromys minutus, Myodes regulus, and R. norvegicus ) captured from different agricultural regions in South Korea. Among them, A. agrarius , which account for 65% of total ( N = 52) individuals, are most widely distributed and abundant in various agroecosystems in South Korea. The bacterial identification was performed by cultural and molecular methods. In cultural method, the fecal cultures from 26 individuals formed colonies on E. coli -selective EMB agar media. Of them, the fecal cultures from 18 individuals also produced colonies on the Shiga toxin-producing E. coli -selective CT-SMAC agar media as well as the EMB agar media. In molecular method, polymerase chain reaction (PCR) was carried out to detect two virulence genes ( stx1 and stx2 ) of isolated E. coli . The amplified dataset of stx1 and stx2 genes of E. coli were sequenced. In this manuscript, E. coli and STEC were detected but there were no Salmonella species. The wild rodents' data would provide important information on reservoirs of those pathogenic bacteria. ©

Value of the Data
• The data will provide important information on competent reservoir of several bacteria-borne zoonotic diseases in wild rodent species and their fecal might play an important role in the transmission of pathogens, such as diarrheagenic E. coli bacteria. • The dataset provides information about prevalence of diarrheagenic E. coli and could be used for better management of microbial contamination control. • The data will contribute to understanding the potential factors that lead to increase in foodborne illness of farmers during agricultural production and processing.

Data Description
Wild rodents are competent reservoirs of zoonotic diseases that are responsible for significant economic losses and public health problems [3] . The rodents, such as Apodemus agrarius and A. peninsulae , are very common in South Korea and are widely distributed across agricultural farm areas and mountainous forests [4] . They can disseminate zoonotic microorganisms that are a considerable threat to the health of farmers [ 3 , 4 ]. Therefore, they could be important potential factors that lead to increases in food-borne illness during agricultural production and processing. However, wild rodents of A. agrarius , which account for 34 (65%) of the 52 captured individuals, were most commonly captured in the fields ( Table 1 and 5 ). The E. coli and Shiga toxin-producing E. coli , bacteria in the rodent fecal samples were detected by culture and molecular method. For cultural identification, the E. coli or Shiga toxin-producing E. coli colonies were confirmed based on their colony morphology. The E. coli colonies produced metallic sheen color produced on EMB agar media. Sorbitol negative colonies (colorless) were detected onto CT-SMAC agar media (Supplement Fig. S1). We select colorless colony as E. coli O157:H7 positive colonies seem to be STEC-positive on culture media. Moreover, the bacterial colonies on the selective media that had been identified by their morphology were re-identified by PCR using molecular markers. So, a PCR amplification of 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 positive EMB agar plates ( Table 1 ) and after PCR amplification with the hypervariable region (HVR) of 16S primer set, the target bands for the 16S rRNA gene were found in all 26 single colonies (Supplement Fig. S2). Thirteen PCR bands were then sequenced ( Table 2 ). 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 . The target PCR bands of 21 E. coli with stx1 gene and 5 E. coli with stx2 gene were amplified and sequenced (Supplement Fig. S3 and S4). Of them, 13 Stx1 PCR bands and 5 Stx2 PCR bands were sequenced ( Table 3 and 4 ). 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/ .

Sample collection
We captured wild rodents using Sherman traps in various agroecosystems across South Korea ( Table 1 ). Each captured wild rodent was placed into a disposable vinyl zipper bag and then released after collecting its feces. The fecal samples were brought to the laboratory in ice boxes and processed within three hours. The sample collections were conducted under the permission and guideline of local governments.  'o' = detected, ' × ' = not detected and '-' = not tested.     Table 4 Molecular identification of Shiga toxin-producing E. coli in the rodent fecal samples using Shiga toxin gene (stx2) sequences. Similarity   ' × ' = not detected and '-' = not tested.

Detection of diarrheagenic E. coli bacteria
The E. coli and Shiga toxin-producing E. coli , bacteria in the fecal samples were detected by culturing them using the method described in a previous study [2] . The fecal samples (0.1 g to 1 g) were first cultured in 10 mL non-selective buffered peptone water (BPW) at 37 °C overnight and then a 10 μl enrichment broth of containing the samples was streaked with a loop onto the E. coli -selective eosin methylene blue Agar (EMB) media and Shiga toxin-producing E. coliselective cefixime tellurite sorbitol MacConkey agar (CT-SMAC) media and incubated at 37 °C for 24-48 hrs. 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 or Shiga toxin-producing E. coli colonies were confirmed based on their colony morphology [5] . E. coli (NCPP: 14,034) and E. coli O157:H7 (ATTC-95,150) were used as a positive control. The E. coli colonies produced metallic sheen color produced on EMB agar media. Sorbitol negative colonies (colorless) were detected onto CT-SMAC agar. From each plate, 2 to 3 colonies were picked from CT-SMAC media. We select colorless colony as E. coli O157:H7 positive colonies seem to be STEC-positive on culture media. The STEC colonies have morphological differences in CT-SMAC compared to the general E. coli . Finally, the STEC E. coli was detected based on morphology, PCR band, and sequence analysis.

Extraction of total genomic DNA and PCR amplification
The bacterial colonies on the selective media or differential media had been identified by their morphology and re-identified by PCR using molecular markers. The colonies were streaked onto nutrient agar media and then a single colony was collected using sterilized toothpicks. The colonies were incubated at 35 °C for 18 hrs in 5 ml lactose broth (LB) solution. Genomic DNA was extracted from 1 ml LB culture fluid using a DNeasy Blood and Tissue Kit TM according to the manufacturer's instructions (Valencia, CA, USA).
The PCR amplification was performed using a final 25 μL reaction volume containing 10 mMTris-HCl (pH 8.4), 50 mMKCl, 4 mM MgCl 2 , 200 mM of each dNTP, 50 pmol of each primer, 2 U ExTaq polymerase, and 1 μL of genomic DNA. The E. coli colonies on the EMB were molecularly identified by PCR amplification of the bacterial 16S rRNA gene which was performed using the HVR primer set [6] . The Shiga toxin-producing E. coli colonies on CT-SMAC were molecularly identified by PCR amplification using the Stx1 and Stx2 primer set [ 7 , 8 ].
The PCR reaction was conducted using the following reaction conditions: an initial denaturation for 5 min at 94 °C, followed by 35 cycles of denaturation for 1 min at 94 °C, an annealing temperature of 55 °C (HVR, Stx1, and Stx2 primers) for 60 sec, extension for 1 min at 72 °C, and then a final extension for 10 min at 72 °C. The PCR products were subjected to electrophoresis in 1.0% agarose gel and purified using a DNA gel extraction kit (Qiagen, Valencia. CA, USA). The purified PCR products were sent to Macrogen (South Korea) for sequencing. The obtained sequences were compared with other homologous sequences deposited in GenBank using BLASTN2.2.31 + [1] .

Ethics Statement
The sample collections were conducted under the permission and guideline of local governments.

Declaration of Competing Interest
The authors declare that they have no competing interest or financial relationships which have influenced the work reported in this article.