Acronym

J1-70027

Department:

Department of Biology

Type of project

ARIS projects

Type of project

Basic research project

Role

Lead

Financing

Slovenian Research And Innovation Agency

Duration

01.03.2026 - 28.02.2029

Total

1,66 FTE

Project manager at BF

Čremožnik Zupančič Jerneja

Abstract

Antimicrobial resistance (AMR) is an important issue today as it has reached dangerously high levels in all parts of the world due to the misuse and overuse of antibiotics. Antibiotic resistance genes (ARGs) have been detected in all environments, including natural, artificial and clinical habitats, over the past decade. Anthropogenic activities, including the clinical use of antibiotics, are widely recognized as the main cause of the spread of ARGs, as numerous genetic mechanisms such as conjugation, transformation and transduction enable microorganisms to evolve, adapt and survive in an environment contaminated with antibiotics and ARGs. The aquatic environment plays a crucial role in the spread of antibiotic resistance genes (ARGs), as it is both the final receiving system for most anthropogenic effluents containing ARGs (municipal wastewater, runoff from fields and livestock facilities, etc.) and the main source of recreational, irrigation and drinking water. The popularity of recreational activities that involve contact with water continues to grow worldwide. However, participation in these activities poses some potential microbiological health risks, as human exposure through skin, eye or ear contact, inhalation and ingestion of ARB and ARG in bathing waters can lead to infections that are difficult to treat.

Most clinically relevant ARGs are not randomly distributed in the genomes of antibiotic-resistant bacteria (ARB), but are bundled in mobile genetic elements (MGEs) such as plasmids or transposons, which allow horizontal transfer of ARGs between bacteria. ARGs/MGEs can be transferred as part of ARB genomes (conjugation), in the capsids of bacteriophages (or simple phages) (transduction) or as free DNA (transfection). It has been shown that the capsids of environmental phages frequently transport ARGs. Since phage genomes rarely encode ARGs, this ARG/MGE content in the capsids consists mainly of more or less randomly encapsulated bacterial DNA. It is therefore of great importance to gain new insights into the role of phages in the environment in the dissemination of ARG/MGE and the compartmentalization of the environment.

Bathing waters are assessed on the basis of compliance with certain standards (total coliform bacteria, E. coli, ... ). In the EU, the quality of bathing water is regulated by the Bathing Water Directive (BWD). Currently, only the number of microorganisms in water is measured in recreational waters to determine quality, but not in sediments, although sediments are heavily contaminated with viruses of fecal origin and indicator phages and the ARG they carry. The current Water Framework Directive(2006/7/EC) monitors water quality at designated bathing waters and focuses on the enumeration of fecal indicator organisms (FIO); E. coli and intestinal enterococci. Phages are completely neglected at this stage in bathing waters, although the Drinking Water Directive (2020/2184) provides for methods to detect somatic and F-specific coliphages during regular monitoring. The Drinking Water Directive does not contain such a paragraph, as the presence and biological risk of phages in bathing waters appears to be non-existent or negligible.

To gain new insights and fill the existing gaps regarding the potential role of phages in the emergence and spread of antibiotic resistance in bathing waters, we will apply different sampling methods for water and sediment, conventional cultivation and cultivation-independent metagenomic analyses. The cultivation-based approach is further required to investigate the actual horizontal transmission, transmission frequency, host and environmental factors influencing the transmission frequency of ARGs via plasmids and/or phages, including their host range, as these data cannot be obtained by bioinformatic analyses alone, and finally, the obtained data will be evaluated with statistical analyses and machine learning-based model predictions.


The proposed project has three main objectives. First, it aims to characterize the phageome of bathing waters at the molecular level using next-generation metagenomic analyses, with a particular focus on antibiotic resistance genes (ARGs) harbored within the phageome. Second, the project seeks to develop a culture-based method for the isolation of bacteriophages from bathing waters, coupled with the simultaneous isolation of bacteria carrying integrated phage DNA (prophages) and the assessment of their antimicrobial resistance (AMR). The third objective is to determine the overall ARG burden in bathing waters and to elucidate the role and frequency of bacteriophage-mediated transmission of these genes in the environment. Despite advances in bioinformatic approaches, culture-based methods remain essential for studying actual horizontal gene transfer events, including transmission frequencies and the host- and environment-related factors influencing ARG dissemination via plasmids and/or bacteriophages. Such approaches are also crucial for determining phage and plasmid host range, information that cannot be reliably obtained through bioinformatic analyses alone.

 

Researchers

 

BF

 

NIB

 

ZRC SAZU

 

UNG

 

The phases of the project and their realization

The project is structured into six thematically and temporally interconnected phases that together enable a comprehensive investigation of the role of bacteriophages in the transmission of antibiotic resistance genes in natural recreational water environments.

Phase 1: Sampling and basic environmental characterization
The first phase involves systematic fieldwork at selected natural bathing sites of Lake Bled. This includes sampling of bathing water and sediments, as well as measurement of basic physicochemical parameters. This phase ensures the collection of representative samples and environmental data that form the essential foundation for all subsequent analyses. The sampling collection will last for 24 months.

Phase 2: Culture-independent molecular analysis of the phageome and ARGs
In the second phase, modern culture-independent molecular approaches will be applied. Following viral particle concentration and viral DNA extraction, metagenomic analysis (shotgun sequencing) will be performed to characterize the phageome and to identify and analyze antibiotic resistance genes (ARGs) present in viral genomes. This phase provides insight into the overall potential of the phageome as a reservoir of ARGs in bathing waters.

Phase 3: Culture-dependent methods for the isolation of bacteria and bacteriophages
The third phase is dedicated to experimental validation of findings obtained through metagenomic analyses. It includes the isolation and identification of selected bacteria (e.g., E. coli and related phylogenetic groups), isolation and purification of bacteriophages from water and sediment samples, and detection of phages carrying ARGs. Particular emphasis will be placed on the isolation of so-called jumbo phages and the analysis of bacterial and phage-associated resistance reservoirs.

Phase 4: Determination of horizontal gene transfer potential
In the fourth phase, the actual potential for horizontal transfer of ARGs in the environment will be assessed. This includes the analysis of phages from selected bacterial isolates, investigation of lysogenic and coliphages in natural bathing waters, and experimental evaluation of ARG transfer via bacteriophages into model bacterial hosts. This phase is crucial for understanding the mechanisms and frequency of resistance gene transmission in natural aquatic ecosystems.

Phase 5: Statistical data processing and advanced analyses
During the fifth phase, all collected data will be comprehensively processed using statistical and bioinformatic approaches, including machine learning methods. The aim is to identify patterns and relationships between environmental factors, phageomes, and ARGs, as well as to perform sustainability assessments and evaluate long-term environmental risks.

Phase 6: Interpretation of results, project management, and dissemination
The final phase covers interpretation of results, risk assessment, project management, and dissemination of findings. It includes partner meetings, identification of sources and dissemination pathways of ARGs, and publication of results in scientific journals and open-access databases, ensuring effective knowledge transfer to the scientific and professional communities in line with the “One Health” approach.