Title of research project,
マレーシア・セランゴール川に沿った薬剤耐性菌のプロファイリング
Antimicrobial resistance profiling along Selangor River, Malaysia
Names of project members (including affiliation),
Associate Professor Dr Lee Choon Weng (Universiti Malaya)
Associate Professor Dr Bong Chui Wei (Universiti Malaya)
Wong Yi You (Universiti Malaya)
Professor Dr Kozo Watanabe (Ehime University)
Professor Dr Tatsuya Kunisue (Ehime University)
Assistant Professor Dr Rumi Tanoue (Ehime University)
Purposes
1. To quantify levels of selected antibiotic residues and antibiotic resistance genes in theSelangor River Basin
2. To determine the impact of anthropogenic and antibiotic pollution on bacterial
assemblage and antibiotic resistance
Methods
Sample collection

A total of 12 water samples were collected from upstream (Site L), middle stream (Site5 / 12

C, G and K) and downstream (Site A) of Selangor River Basin in Peninsular Malaysia.
The map above shows all the stations in the study whereas the table below shows themonth and year of sampling. Some of the selected sites are also shown in the pictures
below.
Sample nameSiteMonthYear

A0120

AJanuary

2020

C0120

CJanuary

2020

A0722

AJuly2022

C0722

CJuly

2022

G0722

G

July

2022

K0722

K

July

2022

A1122

A

November

2022

G0923

G

September

2023

K0923

K

September

2023

A1023

AOctober

2023

C1023

COctober

2023

L1023

LOctober2023

In situ physicochemical parameters [temperature, salinity, pH, and dissolved oxygen(DO)], dissolved inorganic nutrients [nitrate (NO3), nitrite (NO2), ammonium (NH4),phosphate (PO4), and silicate (SiO4)], chemical oxygen demand (COD), biologicaloxygen demand (BOD) and total suspended solid (TSS) were measured.

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Station C

Station G

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Station L

Quantification of selected antibiotic
Water samples collected were kept frozen, and transported to Center for Marine
Environmental Studies (CMES), Ehime University for antibiotics analyses. The
concentration of selected antibiotics from six different classes (Macrolides,fluoroquinolones, tetracyclines, sulfonamides, Trimethoprim, beta lactam) weremeasured using HPLC and LC-Ms/Ms in CMES.
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Bacterial community profile
A volume of 200-500 mL of water samples were filtered on 0.2µm pore size Isopore
membrane, depending on the water turbidity. Bacterial DNA extractions were carried out
using DNEasy PowerWater kit and checked for concentration using Qubit 3.0
fluorometer before sending for sequencing. DNA samples were sent for whole genome
metagenomic analysis via high throughput sequencing (Illumina Novaseq), with library
preparation.
Quantification of antibiotic resistance genes in the metagenomic reads
The processed metagenomic reads were also screened for antibiotic resistance genes
using bioinformatic tools with the Comprehensive Antibiotic Resistance Database(CARD) database. The quantification of antibiotic resistance genes was carried out using
the normalized reads.
Results/Activities
Online meetings via Zoom platform were carried out regularly. Prof Kozo has visited thesampling locations in Malaysia from 5th to 7th of June, 2023. The Collaborative Research
Agreement and a Material Transfer agreement between Ehime University and Universiti
Malaya were signed and completed during the visit.

Picture taken in Universiti Malaya, from left: Assoc. Prof. Dr Lee Choon Weng
(Universiti Malaya), Professor Dr Kozo Watanabe (Ehime University), Associate
Professor Dr Bong Chui Wei (Universiti Malaya), Wong Yi You (Universiti Malaya)
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In February 2024, Wong Yi You went to CMES, Ehime University, Japan to the learn
antibiotic extractions and LC-MS/MS processes to screen for 50 antibiotics, including
Macrolides, fluoroquinolones, tetracyclines, sulfonamides, Trimethoprim, beta lactam,
from the water samples. The extraction and analyses with LC-MS/MS were carried out
with the help of Assistant Professor Dr Rumi Tanoue (Ehime University). A total of eightantibiotics (Trimethoprim, Lincomycin, Clindamycin, Clarithromycin, Ciprofloxacin,
Fluconazole, Metronidazole and Florfenicol) ranging from 0.14 – 5 ng/L were detectedfrom the 12 samples.

Pre-treatment before antibiotic extraction.

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Picture taken in CMES, Japan, on the left is Assistant Professor Dr Rumi Tanoue(Ehime University).
Raw data from the 12 metagenomic sequencings were successfully received. During thestay in Japan, Wong Yi You also learnt data analyses for the metagenome data available.
Currently, analyses using bioinformatic tools for the bacterial community profile andantibiotic resistance genes are ongoing.

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Future challengesThe main challenges will be the subsequent analyses of data using different bioinformatictools. The results from antibiotic detection, and metagenome sequencing will beanalysed along with the environmental factors for temporal and spatial variation.

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