Amphibians face anthropogenic pollution threats from different sources, and their associated gut microbiomes are highly sensitive to these pollutants (Jiménez and Sommer 2016). Pesticides are likely to co-occur with antibiotics that are worldwide used in human and veterinary medicine and arrive to aquatic environments from agroecosystems and swine, chicken and cow feedlots without any treatments (Rico et al. 2014). The interactive impact of antibiotics and herbicides on gut microbiota disturbance has rarely been studied (Zhan et al. 2018). The present study reinforced that GBH and CIP, individually and in mixture, altered gut microbiota composition of R. arenarum tadpoles. A decrease in weight mass and stage of development in GBH treatment- was also observed in treatments with microbiota alteration.
It is known that gut microbiota plays key roles in host vital functions as immune-system modulation, digestion, biotransformation and protection against pathogens (Sommer et al. 2013). Therefore, dysbiosis of microbiota, as well as alteration of its functional optimization can lead to severe health problems for hosts (Claus et al. 2016). In this study, decrease on tadpoles’ weight from both individual and mixture pollutants treatments was observed, together with a disruption of normal microbiota community structure. Moreover, in GBH treatment, tadpoles also showed a delay on development. Similar to previous studies that described gut microbiota shifts related to weight and changes on development (Zhang et al. 2020; Chai et al. 2018), these results suggest a relation between microorganism community and the animals’ physiology (Nehra et al. 2016) and enhance the importance of study gut microbiota to estimate impact of environmental chemicals on health and fitness of wildlife, environments and humans (Nguyen et al. 2015).
The taxa that have been found in this study as part of the microbial communities of R. arenarum tadpoles mostly belong to the bacilli Gram (-) Enterobacteriaceae family, also Aeromonas and Pseudomonas species. The genera of bacteria found in the microbial communities of CO tadpoles coincided with those described in pioneering works in the area that share the culture sowing methodology (Hird et al. 1983, Lajmanovich et al. 2001). After two weeks of exposure to GBH, microbial communities from the tadpoles’ gut had significantly changed their structure. The diversity and genus richness indexes of gram-negative bacteria increased considerably, while the dominance of the taxa registered in the control (i.e. E. coli), decreased. GBHs have already been reported to disrupt the gut microbiota of animals likely to live near agricultural sites (i.e., bees, Motta et al. 2018; water fleas Daphnia spp., Suppa et al. 2020). Besides, it was also found that GBH altered the diversity of the soil microbes (Wolmarans 2014) and can favors to certain species which perform less efficiently in the other conditions (Imparato et al. 2016). In general, it is agreed that diversity is given by the variation in environmental conditions and availability of nutrients (Goldfarb et al. 2011). In this sense, the dysbiosis observed here for the microbiota community of GBH-treated R. arenarum tadpoles, given by an increase in diversity and evenness of taxa, could be related to the fact that GLY positively influences the bacterial growth since some Gram (+) and Gram (-) bacteria use it as a source of carbon, nitrogen and phosphorus (Van Eerd et al. 2003). Other alterations in the normal conditions of the intestinal lumen due to xenobiotics as GLY could also contribute to the microbiota shift: it has been suggested that mucous layer of the intestine of mammals is affected by pollutants, and dietary emulsifiers are capable of altering the microbiota (Chassaing et al. 2015; Lozano et al. 2018).
Another explanation for the shift in bacteria community composition in GBH treatment may be related to the ability of some bacteria to transform GLY (Sviridov et al. 2015). Some bacteria transform GLY into aminomethylphosphonic acid (AMPA) by the enzyme glyphosate oxidoreductase, and use this metabolite or directly the GLY molecule to obtain phosphate for their metabolism by C-P bond break down (Imparato et al. 2016). Thus, it can be inferred that GBH application may induce to an artificial selection that stimulates existing bacteria capable of degrading the herbicide (Villarreal-Chiu et al. 2017). In accordance, some of the taxa increased in GBH treatment of our study, such as Enterobacter spp. and Providencia spp., are known to have associative traits to GLY degradation or use as a substrate (Nourouzi et al. 2011, Kryuchkova et al. 2014). From an ecological point of view, the decrease of some bacterial species could release ecological niches that would be occupied by others (Blot et al. 2019). Regarding this, the importance of deepening the study of the relationships between the different taxa that make up the intestinal microbiota here is highlighted, and the imbalance that could be generated between beneficial and pathogenic bacteria for amphibian tadpoles.
Concerning the quantification of CFU, GBH treatment showed a significant increase compared to CO. This effect could be related, among various factors, to host transcriptional changes enriched for lipid and carbon metabolism, as is suggested by Suppa et al. (2020). Moreover, studies on dynamics of bacteria communities of the soil and rhizosphere, associated the increase of fast-growing bacteria abundance with the availability of carbon compounds in GLY presence (Imparato et al. 2016). The increase in the amount of CFU in the GBH treatment probably corresponds to the increase in those taxa capable of degrading GLY. Consequently, the production of AMPA would increase, together with its potential risks to hosts animals and human health (e.g. impairment of DNA reparation and mRNA synthesis, Allemann 2019, de Brito Rodrigues et al. 2019).
On the other hand, most of the studies of the effect of antibiotics on the intestinal microbiota are focused on human health, and warn about the consequences of prolonged treatments and permanent loss of certain fundamental taxa for the maintenance of healthy gut (Jakobsson et al. 2010, Pop et al. 2016). Similarly, it is important to pay attention to the effects that drug residues may have on the bacterial communities of non-target organisms, since these are frequent in water bodies (Peltzer et al. 2017, Hu et al. 2020). In our study, CIP treatment induced dysbiosis on gut microbiota of R. arenarum tadpoles by reduction of taxa diversity and increase dominance of a single genus. Aeromonas spp. represented more than 50% of relative taxa abundance on microbiotal gut community, assuming its resistance to CIP. This result is consistent with other studies that reported multidrug-resistance (including CIP) of Aeromonas spp. from wild animals (Dias et al. 2018).
The presence of dysbiosis on CIP treated tadpole can lead to serious consequences on tadpoles and other wild animals, since Aeromonas spp. identified here (A. veronii and A. hydrophila) had been associated with several diseases in humans and fishes (Rahman et al. 2002, Toranzo et al. 2005, Janda and Abbot, 2010). Skwor et al. (2014) warned about the risk of emerging resistant Aeromonas spp. in the environment and organisms, due to the overuse of antibiotics in both human and veterinary medicine. In addition, Leclercia spp. was another taxon that highlight dysbiosis in CIP treatment, since it did not appear on CO nor GBH treatments. Yehia (2013) reported multiple resistance to antibiotics (including CIP) in Leclercia spp. strains isolated from farm poultry intestinal tracts, and enhanced the health risks remaining on inadequate use of a wide spectrum of antibiotics for different interest.
In the present study, richness and diversity index in the gut microbiota from GBH-CIP treatment were similar to CO, but the taxa composition showed to be different. Some genera from CO as Klebsiella spp. and Pseudomonas spp. were decreased or absent in the mixture treatment. Additionally, some trends observed for individual pollutant treatments were repeated in CHB-CIP: increase of Enterobacter spp. and presence of Proteus spp. (as in GBH), and increase of Aeromonas spp. and presence of Leclercia spp. (similar to CIP treatment). It is more than clear that pressure of both xenobiotics interacts to influence microbiotal community structure. Results observed in GBH-CIP mixture treatment not only confirmed the susceptibility of gut bacterial microbiota in R. arenarum tadpoles to different type of pollutants individually, but also enhance their effects in mixtures, as they are more likely happen in the environment (Ramakrishnan et al. 2019). To the best of our knowledge, this is the first report on disruption of gut microbiota of amphibian tadpoles by a mixture of an antibiotic and herbicide. As it is clearly aimed on a recent review, CECs affect gut bacteria and have great imbalance on host health (Tsiauossis et al. 2019). More studies are need to elucidate how real-life scenarios with complex CECs mixtures can affect tadpole microbiota and ultimately, life aquatic health.
Overall, gut bacterial microbiota demonstrated to be a key endpoint for evaluating the effects of pollutants on non-target animals as amphibians’ tadpoles. Last years, there has been a growing interest and concern about its diversity and structure variation due to changes in environmental conditions and pressures in order to understand its complex symbiotic relations with hosts' life (Evariste et al. 2019), and the bacterial resistance due to exposure to antibiotics such as CIP (Jørgensen et al. 2013).