Superbug origins: high throughput culture, isolation and sequencing of environmental Acinetobacters

• Main Author: Tan, Ching Hai
• Other Authors: Duan Hongwei
• Format: Final Year Project
• Language: English
• Published: Nanyang Technological University 2023
• Subjects: Engineering::Bioengineering
• Online Access: https://hdl.handle.net/10356/166748
• Institution: Nanyang Technological University

Acinetobacter baumannii (A. baumannii) is an opportunistic nosocomial pathogen [1] responsible for a larger number of deaths globally due to the rise of antibiotic resistance genes. Current methods of bacterial isolation, classification and characterisation are too slow and at times expensive, requiring either large amounts of manpower or expensive machines and algorithms to carry out these processes, making them inaccessible in addition to generating large amounts of plastic waste. To address these issues, new methods and processes were formulated utilising an open-source liquid handler (Opentrons-2), custom grids designed and printed using reusable and sterilisable materials, serial dilution techniques, a custom-written protocol and a custom-developed image processing and segmentation program to maximise the number of environmental samples which could be processed simultaneously. To make the process more user-friendly, a user-interface was developed so they would not have to adjust the source code. Results showed that automating the bacterial culturing process on the OT-2 resulted in higher throughput than the traditional method of streaking as the increased time taken for liquid handler to plate the bacteria could be offset by the user’s ability to run multiple of such machines simultaneously. However, the image processing and segmentation program was unable to quantify the number of colonies in each well due to technical limitations which could be further corrected in future experiments. Given the relative success of these methods and processes, they could be adapted for other microbiological experiments such as antibiotic resistance testing or even other fields of biology such as cell-culturing.