Resistome and Microbial Profiling of Fish collected from different sources in Duhok, Iraq.

Delveen R. Ibrahim¹and Khalid S. Ibrahim²

¹Department of Biology, Collage of Science, University of Duhok, Duhok, Kurdistan Region, Iraq. (MSc Microbiology, PhD Microbiology and Microbial Genetics). Delveen.ibrahim@uod.ac and https://orcid.org/0009-0003-2914-6003

²Department of Biology, College of Sciences, University of Zakho, Zakho, Kurdistan Region, Iraq. (MSc Fish diseases, PhD Gut Microbiome). Khalid.ibrahim@uoz.edu.krd and https://orcid.org/0000-0002-8585-3429

1. Overview

This is study investigates the resistome and microbial profiling of fish collected from 4 different aquatic environments in Duhok governorate, Kurdistan Region of Iraq: Kharbur river (an early tributary of the Tigris River in Iraq) near Zakho hospital effluent, two artificial fish farms and Mosul dam Lake. The rationale is grounded in the urgent need to understand antimicrobial resistance (AMR) dynamics in aquatic food systems as fish represents a key pathway for resistance microbes to inter human populations.

Despite the growing urgency to address antimicrobial resistance globally, there is currently a lack of funding in Kurdistan region, Iraq to support large scale resistant and microbial profiling studies particularly in food and aquatic environment. This project represents a pioneering efforts and first systematic investigation of microbial communities and resistome in fish from multiple aquatic sources in Duhok filling a critical knowledge gap that has direct implication for food safety, environmental health and public health. Funding will support laboratories analysis (16S rRNA sequencing and metagenomics). Bioinformatics training is not needed as the researchers already have those skills. Local facilities at the central veterinary laboratory in Duhok and collaborating university labs are available. The ethics approval to be sought from the Institutional Review Board if the funding is provided. The project is planned over 10 months with sequential phases from sampling, laboratory work, and bioinformatics and analysis. Reporting data generated will be shared via Open Access repositories in compliance with FAIR principles.

2. Introduction

Antimicrobial resistance (AMR) is pressing global challenge with aquatic systems recognized as major reservoir for resistant microbes and resistance genes (1). Fish are an important component of human's diet providing essential proteins and nutrients. However, fish and aquatic environments can act as a reservoir of antibiotic-resistant bacteria and resistance genes (the resistome). This is particularly concerning in the region where aquaculture, urban wastewater and river system interacts with human activities potentially spreading resistant microbes through the food chain. Fish as both environmental indicators and human food sources can serve as victors for AMR transmission (2). Few previous studies have examined AMR of some cultured bacterial species in Iraqi aquatic systems (3,4), and none have systematically profiled the resistome of fish across different sources in Duhok. This study will investigate the following points:

1. What microbial communities are associated with gut fish from different aquatic environments in the Duhok?

2. What resistance genes are presented in these microbial populations, and how do they vary between sites?

3. How do anthropogenic influences (ex: hospital effluent) shape resistome diversity?

Hypothesis:

H1: Fish from hospital-influenced river will show the highest abundance and diversity of antimicrobial resistance genes.

H2: Fish from artificial fish farms will show distinct resistome profiles due to antibiotic use in aquaculture.

H3: Fish from Mosul dam lake will have the lowest abundance of resistance genes due to reduced human influence.

Impact statement:

Regardless of outcomes, this study will provide baseline data on AMR in aquatic food systems in Iraq, inform sustainable aquaculture practices and contribute to global AMR surveillance efforts. Besides, it will support One Health approach linking environments, food safety and human health. The outcome will provide evidence-based recommendations for safer aquaculture practices, wastewater management and AMR surveillance. While contributing to the achievement of several United Nations sustainable development goals (SDG 3, 6, 9, 12, 14 and 17). Ultimately this study will position Iraq as an emerging contributor to international AMR and microbiome research with benefits standing from local communities to the global scientific community.

3. Methods

Participants/Sample

Target sample: 100 fish (25 per site) from Kharbur river (site near hospitals) two artificial fish farms and Mosul dam. Inclusion: healthy fish of market size. Exclusion: visibly diseased or decomposed fish. Recruitment: local fishermen and aquacultures farms. Sample size of 25% is sufficient to capture community level microbial diversity consistent with the previous microbial ecology studies.

Materials and Procedures

Fish will be collected using sterile techniques transported on ice and dissected to obtain feces from digestive tract (intestine). DNA will be extracted using commercial kits and send for PacBio sequencing Full-Length 16S Amplicon. Microbial profiling will be performed by sequencing the 16S rRNA full-length gene and the resistome analysis will involve metagenomic sequencing. The annotation will use the Comprehensive Antibiotic Resistance Database CARD the comprehensive antibiotic-resistant databases, bioinformatic analysis will be conducted with QIIME2 and PICRUSts to predict antimicrobial resistance genes (ARGs) in the identified bacterial communities. Statistical analysis will include; Alpha and beta diversity indices, multivariate analysis to compare resistome and microbiota between sites, correlation of A prevalence with environmental factor (example proximity to hospital effluent).

Planned Analyses

Processing quality filtering, trimming with Chimera removal. For microbial community profiling, 16S rRNA data will be assessed for alpha diversity (Shannon and Simpson indices) and beta diversity (Bray–Curtis dissimilarity and PCoA). Resistome analysis will be performed by quantifying resistance genes as normalized gene counts. Resistome analysis will be performed by quantifying resistance genes as normalized gene counts. Predictive functional profiling of antimicrobial resistance genes (ARGs) will be conducted using PICRUSts, with annotation against the KEGG and MetaCyc databases. Special focus will be given to pathways associated with polymyxin resistance and peptidoglycan biosynthesis V (β-lactam resistance). Statistical analyses will include PERMANOVA for comparing sites and ANOVA for testing differences in diversity indices and other tests if it is necessary.

Ethics and Data Management

Ethics approval will be obtained from an Institutional Review Board at both the University of Duhok and the University of Zakho, all procedures comply with biosafety standards for handling microbial and genetic material. No human data are collected. Data sharing: raw sequencing data will be deposited in NCBI SRA, and processed datasets will be archived in Open Access in situational repositories in line with Findable, accessible, interprable, reusable (FAIR) data principles.

4. Pilot Data (Optional)

No data available for now.

5. Budget

Item	Quantity	Total
Fish, Sample collection & transport	100 sample	1000$
DNA extraction kits and materials	2 kits	1000$
Metagenomic Sequencing	PacBio Full-Length 16S Amplicon Sequencing for 100	3000$ (an offer)

6. References

1. Bengtsson-Palme, J. and Larsson, D.J., 2015. Antibiotic resistance genes in the environment: prioritizing risks. Nature Reviews Microbiology, 13(6), pp.396-396.

2. EFSA Panel on Biological Hazards (BIOHAZ), Koutsoumanis, K., Allende, A., Álvarez‐Ordóñez, A., Bolton, D., Bover‐Cid, S., Chemaly, M., Davies, R., De Cesare, A., Herman, L. and Hilbert, F., 2021. Role played by the environment in the emergence and spread of antimicrobial resistance (AMR) through the food chain. Efsa Journal, 19(6), p.e06651.

3. Jassim, A.A.R., Abdulhameed, D.B. and Al Shammari, N.R., 2019. Bacterial fish diseases in some semi-close aquaculture systems in Basrah Province, Iraq. Basrah Journal of Agricultural Sciences, 32, pp.75-84.

4. lmashhadany DA, Hassan AA, Rashid RF, Abdulmawjood A, Khan IUH. Assessment and Assay Comparison for Detection of Antimicrobial Residues in Freshwater Aquaculture Fish in Erbil Governorate, Iraq. Antibiotics (Basel). 2024;13(3):225. Published 2024 Feb 28. doi:10.3390/antibiotics13030225

Resistome and Microbial Profiling of Fish collected from different sources in Duhok, Iraq.

Resistome and Microbial Profiling of Fish collected from different sources in Duhok, Iraq.

Funding Progress

Peer Reviews

Related Funding

Request for Proposals - Antimicrobial Resistance (AMR) in Aquaculture

Nonprofit Recipient

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DOI