This document discusses a research project aimed at genomic surveillance of antimicrobial resistance (AMR) in wastewater from hospitals, slaughterhouses, and municipal sources in Owerri, Nigeria, a low-income country. The study seeks to understand the role of wastewater in spreading AMR, the genetic determinants of multidrug-resistant (MDR) bacteria, and to develop molecular methods for AMR surveillance in similar settings. It outlines specific research questions, objectives, and methodologies to assess the risks associated with AMR in these environments.

1. Genomic Surveillance of Antimicrobial
Resistance in Wastewater Sources

2. Background
 The release of untreated or poorly treated wastewater into the environment is being increasingly
recognized as the major factor driving the spread of clinically relevant antimicrobial resistant (AMR)
bacteria and genes (ARB and ARGs) in low- and middle-income countries (LMIC), through the environment
into healthcare facilities and community settings
 Environmental AMR surveillance from the culture-based techniques to also include advanced molecular
methods is paramount to understand and mitigate the spread of ARG in the future
 Using the One Health approach, this proposed research project will employ advanced molecular techniques
to assess the risks associated with AMR in three different wastewater types, namely hospital,
slaughterhouse, and municipal wastewaters in Owerri, southeast Nigeria, with Nigeria serving as a model
LMIC.

3. Statement of Problem
 Wastewaters occupy a critical crossover point for the spread of AMR from the environment to the clinics.
However, this domain remains poorly surveyed in the low and middle income countries (LMICs) unlike in the
high-income settings where state-of-the-art techniques are currently being used for the surveillance of
antimicrobial resistant bacteria and genes in the environment
 Wastewater surveillance (WWS) of ARB and ARGs provides composite samples for an entire population, with
easy access to the mixed community microbiome
 This concept is emerging rapidly, but the clinical utility, sensitivity, and uniformity of WWS of ARB remains
poorly understood especially in LMICs where the burden of AMR is high and advanced molecular methods are
rarely used.
 Therefore, there is a clear need to explore how and if molecular WWS for AMR can be applied in LMICs to
evaluate the risks associated with the release of these wastewaters and if detected AMR loads can be
related to clinical cases and outcomes in the local communities

4. Research questions
 What is the role of wastewaters as drivers of the environmental spread and evolution of AMR in LMICs
using Nigeria as a model LMIC?
 How do the microbiome and resistome structure and the resistance gene mobility from different types of
Nigerian wastewaters differ?
 What are the associated risks for the transfer of these ARGs from environmental isolates to clinical
pathogens?
 To what extent can molecular and non-culture-based methods be used for generating accurate and
reliable data on the epidemiology and burden of AMR from environmental settings in Nigeria?

5. Objectives of the study
The main aim of this project is to perform molecular method-based surveillance of AMR from hospital,
slaughterhouse, and municipal wastewater sources in Owerri, southeast Nigeria as a model LMIC settings
to assess the relative abundance and mobility potential of ARGs from these environmental sources.
 To elucidate the genetic determinants sustaining AMR in MDR isolates and complex microbial communities
from different Nigerian wastewater types and compare them to German wastewaters.
 To determine the virulence of the environmental MDR isolates and their ability to spread ARGs to human
pathogens
 To ascertain the mobility potential of ARGs in the complex microbial communities from the three different
types of wastewater to support risk assessment of possible transmission to human pathogens.
 To identify molecular methods that are transferable to LMIC settings to support local AMR surveillance

6. Research Plan and Methodology
 WP1 – Sampling of wastewaters and isolation and phenotypic characterization of MDR Enterobacterales
 WP 1.1. The Nigerian sampling campaign of three types of wastewaters
 WP 1.2. The German sampling campaign
 WP2 - Molecular analysis of isolate libraries. Whole genome sequence (WGS) analysis
 WP 2.1 Phylogeny/Epidemiology
 WP 2.2 Screening for antibiotic resistance genes
 WP 2.3 Analysis of Mobile genetic elements
 WP 2.4 Determining Virulence factors
 WP3 - Whole-community microbiome, resistome and ARG mobility analysis
 WP3.1 16SrRNA based amplicon sequencing and High-throughput qPCR analysis of ARGs
 WP 3.2 Mobility analysis of selected ARGs based on sequencing results from isolates
 WP4 – Defining indicators for future AMR analysis suitable for application in LMIC