Aeromonas spp. are ubiquitous bacteria primarily recovered from aquatic ecosystems (Janda and Abbott, 2010). They are found in fresh water as well as estuarine and marine waters, and in association with numerous autochthonous aquatic organisms in these environments (Gugliandolo et al., 2008; Janda and Abbott, 2010; Khor et al., 2015; Laviad and Halpern, 2016; Chenia and Duma, 2017). They are also isolated from various habitats such as the gastrointestinal tracts of healthy animals and as transient flora in foods such as vegetables, dairy products, meat, seafood, and drinking water.

Several species are involved in pathologic interaction with numerous animals from the aquatic environment, mainly fishes but also corals, for example (Janda and Abbott, 2010; Hamid et al., 2016; Chenia and Duma, 2017). Some of these species have a major economic impact in aquaculture: the species A. hydrophila, A. caviae, and A. veronii are frequent etiologic agents of fish diseases, e.g., motile Aeromonas septicemia and ulcerative syndrome; A. salmonicida is responsible for fish furunculosis, which can cause death within hours (Menanteau-Ledouble et al., 2016). In addition, the hypervirulent pathotype of A. hydrophila is considered an emerging pathogen responsible for outstanding epidemic outbreaks in farmed warm-water fishes (Rasmussen-Ivey et al., 2016).

Moreover, aeromonads are recognized as emerging pathogens in humans (Janda and Abbott, 2010). The severity of the disease varies from diarrhea to septicemia, depending on individual’s susceptibility, mainly the immune status (Aujoulat et al., 2012; Figueras and Beaz-Hidalgo, 2015; Teunis and Figueras, 2016). The species A. hydrophila, A. veronii, A. caviae, A. dhakensis, and A. media are responsible for more than 85% of human cases of aeromonosis (Figueras and Beaz-Hidalgo, 2015; Teunis and Figueras, 2016). Transmission of pathogenic strains of Aeromonas, including the fecal-oral route, is often due to direct or indirect contact with water (Lamy et al., 2009; Janda and Abbott, 2010; Li et al., 2015; Pal et al., 2016).

To date, widespread of antibiotic-resistant bacteria such as Aeromonas is a major public health issue related to the One Health concept, considering that aeromonads circulate within the major ecosystems: human, animals and water. Occurrence of antibiotic-resistant Aeromonas has been reported in these three ecosystems (Figueira et al., 2011; Piotrowska and Popowska, 2014; Esteve et al., 2015; Li et al., 2015; Patil et al., 2016; Baron et al., 2017). In this context, Aeromonas has been recently proposed as an indicator to assess the spread of antibiotic resistance in the aquatic environment (Berendonk et al., 2015).

Among water environments, estuaries are of particular interest for Aeromonas ecology. They are a transitional zone between a freshwater river and the seawater, characterized by a salinity gradient, where the level of contamination by chemicals and fecal bacteria reflect the land use of the watershed. In this environment, Aeromonas has been isolated in both water and sediments; some isolates exhibited resistance to multiple antibiotics (Henriques et al., 2006; Silva et al., 2014). Thus, in a lagoon estuarine area (Abidjan, Africa), the maximum abundance of Aeromonas was linked to both discharges from urban areas and the river-flow period when the water salinity was low (Marcel et al., 2002). In estuaries, aeromonads were reported to be associated with copepods, the most abundant zooplankton living in the salinity gradient zone and a key component in estuarine food chains. Interestingly, in coastal water in Italy, Gugliandolo et al. (2008) showed that abundance of Aeromonas spp. associated with the copepods is higher than in the water column, suggesting that copepods may function as an aeromonad reservoir. However, today little is known about the putative role of copepods as a vector of pathogenic strains for humans, and the population diversity of Aeromonas associated with copepods remains poorly documented. The topic is complicated by the fact that (i) there are still no simple culturable methods to recover Aeromonas from the environment (Latif-Eugenín et al., 2016) and (ii) the aeromonad population structure is a complex of species, making taxonomic classification difficult. Multilocus phylogenetic analysis (MLPA) and/or whole-genome sequencing are now used for a more precise delineation of the Aeromonas species (Alperi et al., 2010a; Roger et al., 2012; Colston et al., 2014; Talagrand-Reboul et al., 2017), with new species of Aeromonas autochthonous in water environments regularly described (e.g., Marti and Balcázar, 2015; Latif-Eugenín et al., 2016).

The Seine estuary is the largest macrotidal estuary opening into the English Channel, which is characterized by strong anthropic pressure exerted on its watershed (76,650 km²): 30% of the French population is located mainly in its urban areas, with 40% of the country’s economic activity (mostly the chemical industry) and 30% of the national agricultural activity. The estuarine water quality has been extensively studied within the framework of the French multidisciplinary scientific program¹. The microbiological quality of the Seine estuary water is poor, mainly impacted by wastewater treatment plants (WWTPs) treating wastewater from Paris and its suburbs during high flow-periods, while the input of Seine tributaries are predominant during low-flow periods (Garcia-Armisen et al., 2005; Servais et al., 2007; Touron et al., 2007). In addition, high contamination by trace metals, mainly cadmium and lead, and organic compounds such as polycyclic aromatic hydrocarbons, (PAHs), polychlorinated biphenyls (PCBs) and pesticides make the Seine estuary one of the most contaminated in Europe (Carpentier et al., 2002).

This macrotidal estuary is characterized by the presence of a high turbidity zone (HTZ) in the lower estuary (in the mouth of the estuary) where suspended particulate matter (SPM) and the associated contaminants are concentrated. Therefore, in this area the behavior of microorganisms such as fecal bacteria, mainly associated with organomineral particles, is strongly influenced by hydrosedimentary processes (Guézennec et al., 1999; Pachepsky and Shelton, 2011; Malham et al., 2014). During a semidiurnal tidal cycle, bacteria associated with particles suspended in the water column settle during slack high water and then are concentrated in fluid mud at the water–sediment interface. In contrast, at the beginning of a flood, when current velocities increase, bacteria trapped within the fluid mud can be resuspended in the water column (Berthe et al., 2008). Another characteristic of this area is the abundance of Eurytemora affinis, a distinctly dominant copepod species (crustacean, calanoid). In this oligohaline zone, Eurytemora affinis accounts for up to more than 90% of the zooplankton (Mouny and Dauvin, 2002; Devreker et al., 2010). The dynamics of population of Eurytemora affinis was driven by environmental parameters (SPM, salinity and temperature) and consequently – as bacteria dynamic-closely linked to the tidal cycle (Devreker et al., 2010).

Thus, the mouth of Seine estuary is a key environment and provides an outstanding model for studying autochthonous aeromonads in the estuary, and those from human and animal origin, mainly released by treated effluent from WWTPs or watershed run-off via tributaries. The present study aims to compare the abundance and the diversity of Aeromonas populations (i) in the water column, i.e., water and fluid mud, (ii) in association with copepods and (iii) from treated effluent from WWTP released in the same area. For this purpose, a 2-year monitoring campaign was carried out in the Seine estuary, and the diversity of the Aeromonas population was investigated based on a combined culturable and molecular approach, and the phenotypic antibiotic-resistance profile was determined.

Aquatic environments including aquatic organisms are considered Aeromonad’s primary habitat (Janda and Abbott, 2010). Over the last decade, molecular approaches have greatly enhanced the knowledge of Aeromonas diversity in coastal or estuarine waters (Gugliandolo et al., 2008; Silva et al., 2014). Here, we confirm that Aeromonas should be identified using molecular methods for a better understanding of the diversity of Aeromonas in the oligohaline zone of a highly anthropized estuary (Seine). In this environment, only 44.7% of the presumptive Aeromonas, i.e., able to grow on a selective medium, were identified as Aeromonas based on the sequence of radA and/or gyrB genes. Further identification using mass spectrometry (MALDI-TOF) shows that presumptive Aeromonas (radA^-) could be mainly related to Pseudomonas and Serratia. These strains have the same culturable characteristics than Aeromonas on the selective media used in this study (Aeromonas Isolation Agar). However, mass spectrometry is not yet the most accurate method to identify all Aeromonas species or environmental bacteria (Shin et al., 2015). In this environment contaminated by metals, it has been shown that prevalence of Pseudomonas and Enterobacteriaceae as well as Aeromonas is high (Henriques et al., 2006). These results confirmed that housekeeping gene sequencing is the most accurate method to identify Aeromonas strains at the species level for strains recovered from aquatic environments, as described for aeromonads from other sources (Alperi et al., 2010b; Roger et al., 2012; Colston et al., 2014; Talagrand-Reboul et al., 2017).

Here we show that in the mouth of the macrotidal Seine estuary, Aeromonas spp. are detected in water and fluid mud during all hydrological periods. In contrast, in estuary water as well as in the coastal environment, a seasonal variation of Aeromonas abundance was reported. The maximum abundance of Aeromonas was observed in the urban area, during the high river flow period in a eutrophic tropical estuary (Abidjan, Senegal) and in spring in coastal waters (Messina, Italy) (Marcel et al., 2002; Silva et al., 2014). Moreover, in this study, the abundance of Aeromonas in water never exceeded 2 ± 1.7 × 10² CFU.100 mL^-1 and were lower than those reported in Italian and Brazilian anthropized estuaries (10¹ to 10⁶ UFC 100 mL^-1) (Gugliandolo et al., 2008; Silva et al., 2014). These discrepancies could be explained by (i) the methods used for counting Aeromonas, (ii) the impact of hydrosedimentary processes on Aeromonas behavior in a macrotidal estuary, (iii) the land use of the catchment basin that controls inputs of Aeromonas from soils, humans, and animals by surface runoff and waste waters, and (iv) the temperature of the Seine estuary water that is lower than the aeromonad optimal growth temperature of 22–25°C (Janda and Abbott, 2010). We show here that Aeromonas spp. are detected at the sediment–water interface of the fluid mud, which corresponds to the suspended particulate that settles at the bottom during slack water at high tide. Indeed, these results suggest that the behavior of Aeromonas in the mouth of the Seine estuary was strongly influenced by both the hydrology and particle dynamics (Guézennec et al., 1999; Malham et al., 2014). In the mouth of the Seine estuary, occurrence of Aeromonas is mainly related to the upstream input – i.e., treated WWTP effluent or watershed run off – as well as autochthonous Aeromonas able to grow in the oligohaline area of this highly anthropized environment. In addition, a secondary input of Aeromonas was related to the resuspension of surficial sediment, which previously settled on the intertidal mudflats located in this area (Berthe et al., 2008).

In the mouth of Seine estuary, Aeromonas spp. are also associated with Eurytemora affinis whose abundance depends on both their lifecycle and – as bacteria – the hydrosedimentological processes (Mouny and Dauvin, 2002; Devreker et al., 2010). Indeed, here we show that copepods are present when there is a HTZ in the oligohaline area of the Estuary. These results are consistent with a study reported by Devreker et al. (2010) that shows link between both salinity and the dynamic of SPM and those of copepods in the mouth of the Seine estuary. In similar estuarine water (e.g., the Adriatic Sea, Italy), it has also been shown that Aeromonas spp. were associated with copepods (Temora stylifera, Acartia clausii, Centropages typicus, and Paracalanus parvus) (Dumontet et al., 1996; Gugliandolo et al., 2008).

In this study, we show that the diversity of Aeromonas populations from the water column (water and fluid mud) is different from that associated with copepods. In the Seine estuary water column, five species of Aeromonas co-exist: A. salmonicida, A. bestiarum, A. encheleia, A. media, and A. rivipollensis. Indeed, such Aeromonas species could be well adapted to a estuarine environment having a cooler surface water with mean temperature of 13.4°C (min: 5.3°C/ max 21.3°C). Except for A. salmonicida (Henriques et al., 2006), these species, previously observed in farmed fishes (water and fish) (Esteve et al., 1995; Schmidt et al., 2001; Hatha et al., 2005; Behbahani et al., 2014; Bartkova et al., 2016) and in waste water (Figueira et al., 2011; Marti and Balcázar, 2015; Popovic et al., 2015; Vaz-Moreira et al., 2015; Varela et al., 2016), have never been, as far as we know, described in coastal and anthropized estuary waters. Only the species A. hydrophila, A. caviae, A. sobria, and A. veronii have been reported in coastal or estuarine environments (the Adriatic Sea, Italy) (Dumontet et al., 1996; Fiorentini et al., 1998; Gugliandolo et al., 2008). Underestimation of Aeromonas diversity could mainly stem from the identification methodology. However, both the sampling strategy and the water temperature could also explain the differences observed between the diversities of the Aeromonas population in these similar aquatic environments. Consequently, these factors deserve to be precisely reported in further studies. In this study, an input of A. media and A. rivipollensis from the treated influent of the closest WWTP cannot be excluded even if no correlation between abundance of Aeromonas and fecal bacterial indicators was observed. Indeed, in this anthropized estuary, the diversity of Aeromonas populations in the water column probably reflects the coexistence of autochthonous Aeromonas (A. salmonicida, A. bestiarum, A. encheleia), for which the estuary is the primary habitat, and Aeromonas previously released from wastewater. Interestingly, one of the dominant Aeromonas species from WWTP outflow in Monfort and Baleux’s (1990) study was A. caviae, which could possibly be wrongly identified because (i) this species is known to be difficult to phenotypically distinguish from A. media and A. rivipollensis, and (ii) A. rivipollensis was an unrecognized species at time of study.

In the Seine estuary, the Aeromonas population associated with Eurytemora affinis was composed of three species described today as non-human pathogens and was dominated by A. salmonicida. None of these species (A. rivipollensis, A. salmonicida, or A. bestiarum) has been previously described as being associated with copepods. To date, only the species A. hydrophila has been reported in association with copepods, probably due to the lack of resolution of the biochemical methods used in these studies (Dumontet et al., 1996; Gugliandolo et al., 2008). The higher aeromonad density associated with copepods may be due to the known aeromonad ability to produce biofilm and favor copepod colonization, and this should require further study. Interestingly, higher resistance to TCC was observed within the A. salmonicida population associated with copepods. As aeromonads are naturally resistant to Penicillins (Baron et al., 2017), these results suggest the existence of a specific population of A. salmonicida belonging to the microbiota of Eurytemora affinis, although further study is warranted to confirm this assumption.

In the oligohaline zone of a highly anthropized estuary (oceanic climate), Aeromonas spp. (gyrB⁺ radA⁺) are present in the water column (water and fluid mud) and associated with living copepods (Eurytemora affinis). However, the diversity of the Aeromonas populations in the water column (water and fluid mud) is different from those associated with copepods. In the water column, the Aeromonas species autochthonous of the estuary, i.e., A. salmonicida, A. bestiarum, A. encheleia, co-exist with Aeromonas species (A. media, A. rivipollensis) originating from human and animal sources discharged by the WWTP effluent. A. salmonicida are the major species bound to the copepods (Eurytemora affinis), even if A. rivipollensis and A. bestiarum are also detected, while no human pathogenic species were associated with copepods. The proportion of A. salmonicida resistant to the antibiotic (TCC) was higher than in the Aeromonas population in the water column. These results underlined the key role played by this type of anthropized estuary, i.e., as an environment where autochthonous aeromonads and those originating from human and animal sources coexist.

FP, BL, and EJ-B defined the research theme. GC, TB, RL, JF-L, and FP defined sampling strategy and designed methods and experiments. GC carried out the laboratory experiments, strains isolation and antibiotic resistance analysis, FR carried out the phylogeny of aeromonads. GC, FR, TB, and FP analyzed and interpreted the data. FP and GC drafted the manuscript. BL, TB, RL, and JF-L revised the paper critically. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.