High-throughput multilocus sequence typing (MLST) using Oxford Nanopore Technologies (ONT) for a large-scale avian Escherichia coli study in Mississippi

Authors: JIA, LINAN - Mississippi State University; ARICK II, MARK - Mississippi State University; HSU, CHUAN-YU - Mississippi State University; PETERSON, DANIEL - Mississippi State University; Evans, Jeffrey - Jeff; Robinson, Kelsy; SUKUMARAN, ANURAJ - Mississippi State University; RAMACHANDRAN, RESHMA - Mississippi State University; ADHIKARI, PRATIMA - Mississippi State University; ZHANG, LI - Mississippi State University

Submitted to: Poultry Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/3/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary: Poultry pathogens remain a constant threat to the U.S. poultry industry. Avian pathogenic Escherichia coli (APEC) is an important poultry pathogen the causes avian colibacillosis and is associated with significant economic losses due to mortality, decreased weight gain, and associated treatment expenditures. APEC disease can widely vary and traditional control relied upon antibiotics. To develop alternative means of control, further characterization of APEC and colibacillosis are needed. Herein, Multilocus sequence typing (MLST) was performed to compare multiple APEC isolates. This research aimed to develop a fast and high-throughput workflow that simultaneously sequences seven alleles of multiple E. coli isolates using the Oxford Nanopore Technology (ONT). Seven housekeeping genes were used for MLST. The sequence type (ST) results were reported and analyzed. With the advantages of the high-throughput and low cost of Nanopore sequencing, this study provides a rapid and cost-effective workflow for E. coli typing.

Technical Abstract: Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis and identifying and accurately distinguishing infectious isolates is critical for controlling APEC transmission. Multilocus sequence typing (MLST) is an accurate and efficient strain identification method for epidemiological surveillance. This research aimed to develop a fast and high-throughput workflow that simultaneously sequences seven alleles of multiple E. coli isolates using the Oxford Nanopore Technology (ONT). Sixty-six E. coli strains were isolated from broiler farms and their genome sequences were obtained using the Illumina sequencing platform. Seven housekeeping genes used for MLST were adenosine kinase (adk), fumarate hydratase (fumC), DNA gyrase subunit B (gyrB), isocitrate dehydrogenase (icd), malate dehydrogenase (mdh), purine-rich element binding protein A (purA), and recA bacterial DNA recombination protein (recA). Seven housekeeping genes were amplified, and amplicons were sequenced on an R9.4 MinION flow cell using the Nanopore GridION sequencer. The sequence type (ST) results were compared with Illumina results to confirm the accuracy. The comparison of reagent and labor costs between Sanger sequencing and ONT was conducted. Moreover, the sequencing protocol was revised by introducing large-scale DNA extraction and multiplex PCR into the protocol and large-scale sequencing of 274 more isolates was conducted using a modified protocol. Finally, the ST results were reported and analyzed. With the advantages of the high-throughput and low cost of Nanopore sequencing, this study provides a rapid and cost-effective workflow for E. coli typing.