Layout 1 SPECIAL SECTION Potentially toxic cyanobacteria blooms in the Southern Alps and the Italian peninsula Antimicrobial resistance in the environment In 2015, the World Health Organization (WHO) declared An- timicrobial Resistance (AMR) to be one of the greatest threats to public health and promoted a Global Action Plan to combat AMR with a One Health approach (WHO, 2015). The OIE (World Or- ganization for Animal Health) and the FAO (Food and Agriculture Organization) subsequently joined the WHO in providing practi- cal and legal tools to set up National Action Plans to reduce the use of antibiotics, promote their surveillance, and carry out further research (e.g., WHO, FAO, OIE, 2016). In 2023, the European Council published a recommendation to member states in an effort to harmonize AMR monitoring in all countries. In order to combat antimicrobial resistance from a One Health perspective, consid- ering the environment as part of the problem, member states have to carry out several actions, including the implementation of i) measures to prevent, monitor, and reduce the spread of AMR in the environment and ii) a surveillance system of AMR also in food, in wastewater, and in the environment (EU, 2023). Based on previously existing programs, a wide variation in the occur- rence of AMR in the different European countries emerged, based on bacterial species, antimicrobial groups, and geographical lo- cation (European Center for Disease Prevention and Control, ECDC, 2022). The highest percentage of resistance to the main classes of antibiotics used in hospitals has been reported in the southern countries. In particular, carbapenem-resistant Gram-neg- ative pathogens represent a serious problem as they cause health- care-associated infections, which are a significant threat to public health (ECDC 2022). The One Health concept recognizes a tight connection be- tween humans, animals and environmental health: diseases are transmitted from animals to humans and vice versa also via the environment. In the last decade, the environmental setting has been acknowledged to influence the diffusion and transmission of Antibiotic-Resistant Bacteria (ARB) and Antibiotic-Resis- tance Genes (ARGs) through various mechanisms (for a recent The complex relationship between cyanobacteria and antibiotics/antimicrobial resistance in the environment: an emerging factor in the One Health vision on antimicrobial resistance Maura Manganelli,1 Emanuela Testai,1 Geoffrey A. Codd2,3 1Environment and Health Department, Istituto Superiore di Sanità (ISS), Rome, Italy; 2University of Stirling, School of Natural Sci- ences, Stirling, UK; 3University of Dundee, School of Life Sciences, Dundee, UK ABSTRACT In 2015, the World Health Organization (WHO) declared Antimicrobial Resistance (AMR) as one of the most critical health issues. It proposed, with the Food and Agriculture Organization (FAO) and OIE (World Organization for Animal Health), to address this by a One Health approach, recognizing the connection between humans, animals, and environmental health. Currently, a hypothesis is de- veloping that cyanobacteria and cyanotoxins may contribute to AMR in water. Recent research appears to suggest: i) an impact of cyan- otoxins on antibiotic-resistance gene transfer between bacteria; ii) a role of cyanobacteria as a reservoir of AMR. Finally, cyanotoxin production appears to be stimulated by cyanobacterial exposure to antibiotics. These findings strengthen the importance of con- sidering the environment in its complexity. Corresponding author: Maura Manganelli, Environment and Health Department, Istituto Superiore di Sanità (ISS), viale Regina Elena 299, 00161 Rome, Italy. E-mail: maura.manganelli@iss.it Key words: toxic cyanobacteria, Antibiotic Resistance, cyanotox- ins. Authors’ contributions: all the authors made a substantive intellec- tual contribution. All the authors have read and approved the final version of the manuscript and agreed to be held accountable for all aspects of the work. Conflict of interest: the authors declare no potential conflict of in- terest. Funding: this short commentary has been developed within the project “Antimicrobial-Resistance and the environmental frame- work in the One Health approach: natural aquatic toxic CyanoBac- teria as target and driver in the evolution and spread of Antibiotic Resistance in Italy” (CyB-AR) funded by the Istituto Superiore di Sanità, grant n. ISS20-6e08998e0c0e. Availability of data and materials: all data generated or analyzed during this study are included in this published article. Received: 26 June 2024. Accepted: 12 October 2024 Publisher’s note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affil- iated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. ©Copyright: the Author(s), 2024 Licensee PAGEPress, Italy Advances in Oceanography and Limnology, 2024; 15:12754 DOI: 10.4081/aiol.2024.12754 This work is licensed under a Creative Commons Attribution- NonCommercial 4.0 International License (CC BY-NC 4.0). Non -co mmerc ial us e o nly M. Manganelli, et al.12 review, see Rzymski et al., 2024). From the environment, both ARB and ARGs can return to humans mainly through exposure to water and food, but studies to exclude transmission via the air and other routes of exposure are still too limited (Stanton et al., 2022). The accumulation of antibiotics and other contami- nants, ARGs, and ARB, in aquatic environments released from domestic, municipal (including hospital), and industrial waste- water, as well as from animal farming and aquaculture plants, can adversely affect environmental health, altering ecosystems. Antibiotics can change not only the composition and size of the recipient bacterial community but can also stimulate the prolif- eration of new ARB through selective pressures (Baquero et al., 2008). This can also happen at concentrations >100-fold lower than the minimal inhibitory concentrations of susceptible species (European Food Safety Authority, EFSA, 2021), that are similar to the low (ng/L) environmental concentrations. The few data available from the field indicate widespread dissemination of certain antibiotics at a global level; for example, surveys carried out in Northern Italy revealed substantial antibiotic concentra- tions downstream of Wastewater Treatment Plant (WWTP) ef- fluents (Castiglioni et al., 2018). Selection in the aquatic environment can, therefore, be a likely event, particularly in hotspots such as wastewater, where multiple selectors are present at the same time at significant concentrations (Karkman et al., 2018). Research is needed to standardize AB re- sistance testing for environmental bacteria, harmonize data for the development of useful databases, and develop AMR risk assess- ment. This should consider the risk of AMR emergence in the en- vironment and should quantify the sub-inhibitory antibiotic concentrations that promote it in different scenarios. It should also evaluate the probability of the spread and transmission of antibi- otic resistance from diffuse sources (Di Cesare et al., 2024) and hotspots, including WWTPs (Hanna et al., 2023), to downstream environments and finally to pathogenic bacteria, in addition to considering traditional eco-toxicological endpoints (Berendonk et al., 2015). Some progress has been made, with respect to min- imal selective antibiotic concentration and the risk of AMR emer- gence in the environment (Murray et al., 2021) and in humans (EFSA, 2021). However, no other potential reservoirs other than bacteria in the environment have been considered until recently. The role of cyanobacteria in antimicrobial resistance Cyanobacteria are photosynthetic prokaryotes, many of which produce potent cyanotoxins, naturally occurring in almost every terrestrial and aquatic habitat (Svircev et al., 2019; Chorus and Welker, 2021), including WWTPs (Romanis et al., 2021). Their presence in water bodies is dramatically increasing due to human demands on water resources, eutrophication, and climate change (Huisman et al., 2018). Risks to human health due to cyanotoxin exposure via drinking water, contaminated food, and recreational activities have been extensively reviewed (Buratti et al., 2017; Chorus and Welker, 2021). Many acute poisonings of wild ani- mals and livestock, after drinking from environmental water sources have been recorded (Svircev et al., 2019). Animals appear to be attracted by cyanobacteria in water even when clean water is accessible and can readily drink sufficient volumes of water containing cyanobacteria blooms, or ingest shoreline scums and mats containing cyanobacteria, which can present a lethal oral dose of cyanotoxins (Wood et al., 2016). Furthermore, some cyanobacteria are used as human and animal food supplements for their high content of vitamins and antioxidant compounds (Chorus and Welker 2021). The complex and diverse relationships between cyanobacteria and antibiotics, ARB and ARG began to be considered only re- cently in a small number of papers (Figure 1). Cyanobacterial pop- ulations are closely associated with a wide range of species-specific bacterial communities, for which they can pro- vide photosynthetically-produced organic matter and oxygen. In- directly, they can thus increase ARB populations present in the environment during the development of cyanobacterial blooms (Zhang et al., 2020). A more direct role of cyanobacteria as an ARG natural reservoir and source has been demonstrated in Lake Taihu, China. Wang et al. (2020) found ARGs in 8 cyanobacterial strains isolated from the lake and then, by laboratory experiment, determined species-specific transfer potentials of mobile genetic elements to 5 cyanobacteria from bacterial donors, as a function of temperature and population density. A survey of over 800 cyanobacterial isolates from habitats, including free-living strains from fresh- and marine waters, terrestrial sources, and symbioses, has found AMR genes to be widely encoded, including in mem- bers of major cyanobacterial orders (Nostocales and Oscillatori- ales), which occur in cyanobacterial blooms and mats (Timms et al., 2023). Cyanobacteria have different inter-specific sensitivities to var- ious classes of antibiotics, up to 5 orders of magnitude (Le Page et al., 2017). The mode of action of antibiotics is assumed to be the same in bacteria and cyanobacteria, and the different suscep- tibility seems to be related, as in bacteria, to diversity in mecha- nisms of uptake (e.g., variability in porin channels for the passage of small molecules, e.g., beta lactams), or in efflux pumps systems to eject AB outside the cell or in the presence of degradative en- zymes (Le Page et al., 2019). Dias et al. (2019) found lower sus- ceptibility in strains isolated from WWTPs than in those from natural aquatic environments. In laboratory experiments, we have also observed intra-specific different susceptibility between Planktothrix strains and between Microcystis aeruginosa strains (manuscript in preparation) within the framework of a small pilot project funded by the Istituto Superiore di Sanità (ISS) in Italy with the aim of investigating aspects of the relationship(s) be- tween cyanobacteria and AMR. Our results indicate that antibi- otics can influence the cyanobacterial community, selecting more resistant strains, whatever the mechanism they use. The development of biofilms in drinking water treatment plants is a further important aspect that has been studied by Xu et al. (2020). They looked for cyanotoxins and ARGs in the complex cyanobacteria-containing biofilms along different steps of a treat- ment system in a Drinking Water Treatment Plant (DWTP). They found both ARGs and cyanotoxins, particularly after water treat- ment with activated carbon, with a weak correlation between the presence of ARGs and the concentration of Microcystins (MCs). Subsequently, they demonstrated in lab experiments that cyan- otoxins enhance the rate of horizontal ARG transfer between bac- teria and speculated that the interactions between ARG and MCs can be increased in DWTP biofilms, favoring the emergence of new antimicrobial-resistant cells (Xu et al. 2020). These authors found an up-regulation of genes involved in conjugative transfer and in genes related to anti-oxidant response and suggested that oxidative stress could increase cell membrane permeability, thus Non -co mmerc ial us e o nly The complex relationship between cyanobacteria and antibiotics/antimicrobial resistance in the environment 13 facilitating the passage of the resistant plasmid. To our knowledge, this is the only study on cyanobacteria, cyanotoxins, and ARGs in biofilms, and on the possibility that microcystins enhance the transfer rate between donor and recipient bacteria in horizontal gene transfer. Further studies are needed to substantiate these re- sults, but if they are confirmed, we hypothesize that toxic benthic cyanobacterial mats, especially in proximity to outflows from WWTPs, could be another important hotspot for the transfer of mobile genetic elements, including ARGs, and recommend that these should be a focus of future studies. A further but not less relevant aspect of the relationships be- tween cyanobacteria and antibiotics, is the production of cyan- otoxins, which can be stimulated after repeated exposures to sub-lethal concentrations of antibiotics (Wu et al. 2020). In our pilot study, we have also observed an increase in cell quota of cyanotoxins during a 14-day exposure to amoxicillin of a mono- clonal strain of Microcystis aeruginosa CCAP 1450/6 in a closed system (Manganelli et al., 2023). Concluding remarks These findings indicate that cyanobacteria have an intricate role in influencing the evolution and environmental dissemina- tion of AMR, potentially acting as conduits between environ- mental AMR and humans and animals via environmental aquatic exposure, water consumption, and food chains, given their ex- tensive diversity. Additionally, our findings indicate that it is cru- cial to investigate whether cyanotoxins, a prominent environmental health concern in drinking water (Buratti et al., 2017; Chorus and Welker, 2021), may exacerbate horizontal gene transfer, or be stimulated by environmental antibiotics or acquired AMR, thereby heightening the risk of cyanotoxin ex- posure via waterborne routes. This need is intensified by the in- creasing evidence that the incidence of infectious microbial disease, and ARB, ARG, and cyanobacterial mass populations are all favored by climate change (Huisman et al., 2018; Rzym- ski et al., 2024). In conclusion, the One Health approach to AMR should con- sider the environment in its complexity and should include cyanobacteria among the environmental drivers of AMR, in an effort to better understand the weight of the various drivers. Mean- while, mitigation measures to reduce toxic cyanobacterial blooms should be implemented with the double aim of reducing both the risk of exposure to cyanotoxins and their impact on the spread of antibiotic resistance. Figure 1. Diagram illustrating the relationships between cyanobacteria, antibiotics, and Antimicrobial Resistance (AMR) from a One Health perspective. Antibiotic-resistant bacteria and antibiotics flow into surface waters (like a lake in the example) from various ter- restrial and aquatic sources. 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