ANTIBIOTIC RESISTANCE SPREAD MEDIATED BY HORIZONTAL GENE TRANSFER IN THE AGRI-FOOD ECOSYSTEM

Antibiotic resistance (AR) is a public problem for human health and food safety. Globalization has contributed to create an intense connection among human and animal health and the environment, allowing bacteria and their genes to move among all these compartments, making a “one-health approach” necessary to counteract this phenomenon. Horizontal gene transfer (HGT), which contributes to AR determinants diffusion, is mediated by three main mechanisms: i) conjugation, ii) transduction, and ii) natural transformation. Several environments linked to the agri-food system are both sources of AR determinants and hot spots of HGT. One of the main routes of AR spread in the agri-food system could be represented by the use of treated wastewater as irrigation source: the reuse of water is indeed a common practice in several countries, including Europe, to fight the water crisis exacerbated by global warming. Wastewater treatment plants (WWTPs) could be one of the main sources of free antibiotic resistance genes (ARGs) which could be released in freshwater bodies. AR determinants present in the treated wastewater would thus enter in the food production through irrigation and could be acquired by pathogenic strains, potentially posing a risk to human health. Even if the presence and the related issues about AR determinants in the environment are well known, there are many aspects which have to be understood e.g. the relative contributions of different sources of AR determinants in the environment, also considering HGT events. Since information about the relationship between environmental HGT and spread of AR determinants is limited, the aim of this PhD thesis was to evaluate the diffusion of ARGs through natural transformation and conjugation in environment or in environmental-like conditions to describe several possible routes of AR spread in the agri-food system. Zooplankton plays a crucial role in waterbodies, being closely linked to bacteria inhabiting aquatic environments in several ecological function, and it establishes a connection with bacterial communities that are inhabitant of the environment in which it lives. Due to the interaction between bacteria and zooplankton, together with the presence of Escherichia coli in waterbodies, derived from human and animal faecal waste, I first evaluated the relationship established between zooplankton, with the model Daphnia obtusa, and E. coli, isolated from it, suggesting that Daphnia could help the bacterium to adapt to the harsh condition that could be found in the freshwater bodies, highlighting the possible role of zooplankton in the diffusion of antibiotic resistant bacteria (ARB) in the agri-food system. The interaction observed between Daphnia and E. coli in the first part of this thesis, together with the knowledge of the presence of ARGs in aquatic environment and the moderate ability to E. coli to acquire DNA through natural transformation, have thus led to the study of natural transformation in zooplankton-associated bacteria, also in terms to unveil the animal influence. Indeed, I studied the natural transformation of the environmental E. coli strain ED1, isolated from D. obtusa, mimicking environmental conditions which could be found in the agri-food system. ED1 ability to acquire exogenous DNA, with a higher frequency than the one of a laboratory strain, together with its ability to thrive in lettuce rhizosphere, underlined the importance to investigate the spread of AR determinants in the agri-food system, especially in the rhizosphere of plants which are usually raw-consumed. Moreover, the possible influence of the zooplankton on natural transformation was investigated through the use of D. obtusa and Acinetobacter baylyi BD413, known to be naturally competent to acquire DNA. A decrease of transformation frequency was observed in presence of Daphnia, due to the degradation of exogenous DNA, highlighting the need of further investigations on zooplankton involvement in ARGs diffusion in aquatic environments. Considering possible routes of diffusion of AR determinants in the agri-food system, i.e. from WWTPs to freshwater bodies and their inhabitant community, to crops and plants, I then devoted my attention on HGT by conjugation in rhizosphere of lettuce, used as model of raw-eaten vegetables. The aim of this work was the construction of a donor strain belonging to the Enterobacteriaceae family and isolated from treated wastewater. Specifically, Klebsiella variicola subsp. variicola was genetically manipulated through the chromosomal tagging with a mCherry gene and a constitutively expressed LacIq gene, and the insertion of the broad host range plasmid pKJK5::gfp::KanR, carrying a green fluorescent protein gene (Gfp) under the control of laclq repressible promoter, and thus resulting in the absence of gfp expression in the donor. The gfp was expressed only in recipient strains, following the mobilization of the plasmid through conjugation. The strain ability to donate the plasmid within the bacterial community of lettuce rhizosphere and its ability to colonize the plant root system were verified, making K. variicola subsp. variicola EEF15::lacIq-pLppmCherry-GmR with plasmid pKJK5::gfp a perfect candidate for the study of conjugation in plants microniches. Finally, I contributed to prepare a critical review on microbial assisted phytodepuration and the use of plant growth promoting bacteria in Constructed Wetland (CW) systems, with a focus on HGT events and the possible spread of AR determinants in the rhizosphere of plants used in phytodepuration. Data collected in this PhD project underline the importance to study the diffusion of AR determinants trough HGT events in the agri-food system, in order to create a rank risk and a risk assessment map to mitigate the diffusion of AR.