Form 3

Annual Report
LaMer, Ehime University
Date (28, February, 2017)
To Director of LaMer
Principle Investigator : Bong Chui Wei
Affiliation: Institute of Biological
Sciences,

FacultyofScience,UniversityofMalaya, Malaysia
Position: Senior Lecturer

Name in print

Project title
Antibiotic resistance genes in marine aquaculture sites in Peninsular Malaysia

Members of project
NameBong ChuiWei (PI)Contributing
Members
Lee Choon
Weng

Thiang Ee
Lean

Affiliation

PositionContribution part

Institute of BiologicalSciences, Faculty of SeniorScience, University of Lecturer
MalayaProject design, performed theexperiments and analyses ofresults

Institute of Biological Associate
Sciences, Faculty of Professor
Science, University ofMalaya

Project design and analyses ofresults

Institute of Ocean & Postgraduate
Earth Sciences (IOES) Student
University of Malaya

Performed the experiments

LaMer Faculty
member incharge
Satoru Suzuki Leading Academia in Professor
Marine and Environment
Pollution
Research,Centerfor
MarineEnvironmentalStudies,
Ehime, University, Japan

Provided feedback and discussion
on the project design and theoutputs of the results
Contributed
reagents/materials/analysis tools

Aim

To quantify antibiotic resistance genes (tetracycline and sulphonamide) in marineaquaculture.
Procedure
Sampling has been carried out at seven main seafood production districts (Perak, Selangor,Pahang, Kelantan, Penang Island, Malacca and Johor) of Peninsular Malaysia. Water
samples were also collected from upstream to downstream of river estuary in Perakincluding runoff waters from zoo, slaughter house, hospital, aquaculture. Seawater samples
were filtered through 0.2 µm polycarbonate membrane filter (Merk Millipore, Germany) tocollect the total DNA of natural bacterial assemblages. DNA from the membranes wereextracted according to Suzuki et al. (2013). Antimicrobial resistance (AMR) genes: tetM,sul1, sul2 and sul3 were quantified by quantitative PCR (qPCR) using CFX 96 Real-Timesystem (Biorad, Laboratories, Hercules, CA, USA) in CMES which method has been
establised.

Results
We have successfully analyzed the abundance of targeted antibiotic resistance genes (suland tet(M) genes) for all the samples within the schedule. Of the all the resistance genes
analyzed, tet(M), sul1 and sul2 were the most abundant, with detected copy number in total
assemblages in aquaculture farm, ranging from~10-5 – 100/16S, ~10-5 – 10-1/16S, ~10-6 –100/16S, respectively, whereas sul3 gene was at very low abundance or not detected in thetotal assemblage at most of aquaculture farm, with copy number of ~ 10-7 – 10-4/16S. This
indicates that tet(M), sul1 and sul2 are ubiquitous in bacterial communities in marine
aquaculture. Same pattern of sul and tet(M) genes were observed in Perak river estuary.
The copy number of tet(M) and sul (1 & 2) genes were relatively higher compared to theprevious studies in China and the Philippines (Gao et al., 2012; Suzuki et al., 2013; Xiong etal., 2014; Xiong et al., 2015; Niu et al., 2016;). However, the relative abundance for tet(M)and sul (1 & 2) genes are lower compared to wastewater treatment in Norway (Lath et al.,2014).
Among the aquaculture farms, of all the farms 92% of aquaculture farms were detected both
sul1 and sul2, followed by tet(M) (86%) and sul3 (69%). The co-existence of sul genes,
combination of sul1 and sul2 were the most frequent which detected in 30 farms, whereas,
the co-existence of sul1 and sul3 (n=24), sul2 and sul3 (n=23), sul1, sul2 and sul3 (n=23)
were lower. Overall, the frequency of antibiotic resistance genes was tetM>sul2>sul1 >sul3.

Figure 1. The abundance of tet(M), sul1, sul2 and sul3 genes in total assemblages.

Perspectives in Future
Unfortunately, we are unable to analyses the antibiotic residues in marine aquaculture, due
to the time constraints. In future, we hope to have an opportunity to learn the techniques inquantifying antibiotic residues in the water at CMES so we could have more insights into therelationship between antibiotic residues and antibiotic resistance genes (ARGs) in marine
aquaculture. Further study, to explore the abundance and diversity of ARGs in both thecultured and uncultured community in marine aquaculture and their correlations between
antibiotic residues will definitely help us to obtain a better picture of the environmental
reservoirs of antibiotic resistance and their potential impacts on public health.

List of Publication
Two manuscripts are in preparation:a. Sulfonamides residues and sulfonamide resistant bacteria and their genes in Taiping
river estuary, Perakb. Occurrence and distribution of antibiotic resistance gene in marine aquaculture.

Conference Presentation
Lye, YL, Bong, CW, Zhang RJ, Zhang G, Satoru S & Chai, LC (2017). Sulfonamide residues
and resistance genes in Taiping river estuary, Perak. The Third Xiamen Symposium onMarine Environmental Sciences, 9-11 Jan 2017, Xiamen University, China