Periodic Reporting for period 1 - ARBUATEM (Antibiotic resistant bacteria and genes, associated with urban agriculture in Low and Middle Income Countries: Ecological and medical perspectives) Reporting period: 2015-10-01 to 2017-09-30 Summary of the context and overall objectives of the project The population growth associated with urbanization and climate change, increase pressures on regional water resources and the need for the (re)use of wastewater for irrigation of agricultural lands worldwide. At the same time, the spread and evolution of antibiotic resistant bacteria in the environment, animals and people is one of the most important threats to global human health. It is estimated that more than 800 million farmers are involved in urban peri-urban agriculture in the world, out of which 200 million rely on raw or diluted wastewater for irrigation. Vegetable crops, because of their higher yield potential, low cost of production, higher nutritional value, and high prices in urban market owing to the increasing population in urban cities, are the most commonly irrigated crops with wastewater in low and middle-income countries. In this project, we used metagenomics to investigate microbial patterns, the presence of pathogenic bacteria, plasmids and antibiotic resistance genes in wastewater used for urban agriculture in two African countries (Burkina-Faso and Cameroon). The aim was to evaluate the epidemiological risks associated with the use of wastewater for urban agriculture in these countries, in terms of spreading bacterial drug resistance.• The first objective was to conduct representative sampling of wastewaters andsoils with samples that represented all the investigated areas. • The second objective was to assess physical-chemical characteristics of collectedsamples, and pollution by selected antibiotics. This was to evaluate the influence of abiotic factors and antibiotic pressure on the dynamics of antibiotic-resistant bacteria and antibiotic-resistance genes.• The third objective was to identify the underlying commonalities / similarities inantibiotic resistant bacteria community structures in wastewaters and soils. This information was to further allow understanding of the influence of wastewater irrigation on the potential establishment of bacterial phyla, which are known to include strains that can act as facultative or opportunistic human pathogen.• The fourth objective was to identify antibiotic resistant genes and evaluate theirabundance in wastewaters and soils. That was to allow a description of antibiotic resistant genes of medical interest, their taxonomic position, and development under continuous pollution.• The fifth objective was to identify plasmid amplicons of clinical relevance. This was to give a broader insight into mobile genetic elements involved in the dissemination of transmissible antibiotic resistance genes. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far 1.2 Explanation of the work carried per WP1.2.1 Work package 1: Sampling, physical-chemical analysis, and antibiotics residues assessment.We conducted two representative samplings (October 2015, and June-July 2016) in the investigated areas (Cameroon (Yaounde and Ngaoundere); Burkina Faso (Ouagadougou)). The physical and chemical analysis of the collected samples were done at the University of Trier (Germany), during the secondment of Bougnom under the supervision of Prof Sören Thiele-Bruhn. The outputs have been delivered. 1.2.2 Work package 2: Direct sequencing, flow cytometry and construction of metagenomic libraries. We successfully extracted DNA from wastewater and soil samples. Extracted DNA was used to conduct sequenced based metagenomics. Shotgun metagenomic sequencing was conducted at ARK-Genomics (University of Edinburgh) and the outputs were delivered. Bougnom used the corresponding bioinformatic pipeline necessary to analyse the data. Flow cytometry was substituted with direct isolation and antibiotic susceptibility testing, using culture based bacterial isolation methods. This was because the samples were affected by the long freezing period. 1.2.3 Work package 3: Identification of ARGs and their genomic contexts, and 1.2.4 Work package 4: Biostatistics and Data Management. We successfully identified ARGs and their genomic contexts from the wastewaters using the appropriate bioinformatic pipeline. We are currently analysing the data from soil DNA sequencing. A detailed Data Management Plan was published on our portal (Section 6 Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Our project aimed to assess the ecological and medical consequences of the use of raw wastewater irrigation in low and middle-income countries and has led to progress beyond the state of the art with regards to the available papers on the topic. The outputs from next generation sequencing data, coupled with ecological parameters are providing essential information on the conditions and factors that lead to the mobilization, selection and movement of antibiotic resistant bacteria from the environment to the clinic. The outputs project revealed that the wastewaters harboured a wide diversity of pathogenic bacteria. Antibiotic resistant genes (ARGs) were detected. These include those encoding resistance to the main antibiotic classes, including extended spectrum β-lactamases and carbapenemases. These are the last available β-lactam antibiotics used to treat bacterial infections in humans and animals. Plasmids (elements responsible for the dissemination of ARGs) were also detected. All the factors involved in antibacterial resistance were found in these wastewaters, thus, revealing them to be reservoirs for the dissemination of antibiotic resistance. The collected information from the project will allow the development of new strategies to be implemented to minimise pollution by antibiotic-resistant bacteria and antibiotic-resistance, genes so preventing infections by such bacteria and onward transmission of antibiotic resistance. These outputs will allow the development of new strategies to be implemented to minimise the spread of bacterial resistance. These strategies are urgently needed, considering the epidemiological risk associated with the used of raw wastewater in urban agriculture (200 million farmers rely on raw or diluted wastewater for irrigation worldwide, covering about 11% of the world food consumption). The completed project is the first one conducted in two LMICs, using the state of art metagenomics. The outputs of the studies, coupled with the public engagement activities, will help to raise public awareness on the problem of antibacterial resistance dissemination from urban agriculture, and the requirement of proper water sanitation in LMICs. Environment provides a reservoir for potential transfer of AR bacteria across international borders. This critical information can then be used to develop targeted antibacterial approaches and influence strategies to reduce the transmission of ARGs. This engagement is a part to address the Global Action Plan on Antimicrobial Resistance (GAP on AMR) by addressing the role of water, sanitation and hygiene in combatting AMR from the environment (including sources, selection and dissemination mechanisms). The fellow has acquired new professional skills in the form of research techniques, scientific oral and written communications, supervision and project management to become an independent researcher. Wastewater sampling Irrigated agricultural field sampling
