Contribution of enrofloxacin and Cu²⁺ to the antibiotic resistance of bacterial community in a river biofilm

Highlights

• ENR was a major force in developing the antibiotic resistance of the river biofilm.

• ENR increased the number and abundance of antibiotic resistance genes (ARGs).

• ENR drove the shift of bacterial community in river biofilms.

• The bacterial communities resistant to Cu²⁺ treatment group were resistant to ENR.

• Multidrug efflux gene and MRG coexistence contributed to ENR-resistant bacteria.

Abstract

Pollutants discharged from wastewater are the main cause of the spread of antibiotic resistance in river biofilms. There is controversy regarding the primary contribution of environmental selectors such as antibiotics and heavy metals to the development of antibiotic resistance in bacterial communities. Here, this study compared the effect of environmental safety concentration Cu²⁺ and enrofloxacin (ENR) on the evolution of antibiotic resistance by examining phenotypic characteristics and genotypic profiles of bacterial communities in a river biofilm, and then distinguished the major determinants from a comprehensive perspective. The pollution induced community tolerance in ENR-treated group was significantly higher than that in Cu²⁺-treated group (at concentration levels of 100 and 1000 μg/L). Metagenomic sequencing results showed that ENR significantly increased the number and total abundance of antibiotic resistance genes (ARGs), but there was no significant change in the Cu²⁺- treated group. Compared with Cu²⁺, ENR was the major selective agent in driving the change of taxonomic composition because the taxonomic composition in ENR was the most different from the original biofilm. Comparing and analyzing the prokaryotic composition, the phylum of Proteobacteria was enriched in both ENR and Cu²⁺ treated groups. Among them, Acidovorax and Bosea showed resistance to both pollutants. Linking taxonomic composition to ARGs revealed that the main potential hosts of fluoroquinolone resistance genes were Comamonas, Sphingopyxis, Bradyrhizobium, Afipia, Rhodopseudomonas, Luteimonas and Hoeflea. The co-occurrence of ARGs and metal resistance genes (MRGs) showed that the multidrug efflux pump was the key mechanism connecting MRGs and ARGs. Network analysis also revealed that the reason of Cu²⁺ selected for fluoroquinolones resistant bacterial communities was the coexistence of multidrug efflux gene and MRGs. Our research emphasizes the importance of antibiotics in promoting the development of antibiotic resistant bacterial communities from the perspective of changes in community structure and resistome in river biofilms.

Introduction

The spread of antibiotic resistance in pathogens is a challenge for global public health, and polluted environments have been clearly implicated in the acceleration of the evolution of antibiotic resistance (Allen et al., 2010; Martinez, 2008). Compelling evidence shows that pollutants and pathogens from agriculture, aquaculture (Chen et al., 2018a) and wastewater treatment plants (Jia et al., 2017; Proia et al., 2016) discharge into rivers, causing increased antibiotic resistance in bacterial community and transmission frequency of antibiotic resistance genes (ARGs) (Marti et al., 2014; Tlili et al., 2017). Biofilms dominate the microbial life of rivers, constitute the main habitat of river microorganisms and drive crucial ecological processes (Battin et al., 2016). Due to the high cell density, robust extracellular polymeric substances (EPS) matrix provides sufficient conditions for intercellular communication, which makes river biofilms not only an environmental reservoir of ARGs, but also a hot spot for the dissemination of antibiotic resistance (Abe et al., 2020; Balcazar et al., 2015; Flemming et al., 2016; Guo et al., 2018). Furthermore, following the transportation and trophic transfer of biofilm harboring antibiotic resistance, concerns have been arisen regarding their negative impact on zoobenthos, fish and human health. However, one ongoing difficulty in the development of antibiotic resistance is to disentangle environmental determinants from other stressors in river biofilms.

Previous studies hold contradictory views about the major environmental factors that develop antibiotic resistance of bacterial communities. Some studies believed that the spread of antibiotic resistance genes is caused by antibiotics (Huerta et al., 2013; Zhang et al., 2018). The detection concentration of antibiotics in the aquatic environment ranges from ng/L to μg/L (Graham et al., 2011; Michael et al., 2013), and a higher concentration level (of mg/L) has been found in areas directly exposed to wastewater discharge from pharmaceutical factories (Le and Munekage, 2004). Mobile gene elements such as plasmids (Li et al., 2018; Pinilla-Redondo et al., 2018) and integron-integrase cassettes (Burch et al., 2009, 2014; Gaze et al., 2011; Gillings et al., 2015), have been reported to be involved in the dissemination of antimicrobial resistance. However, some studies have emphasized that the prevalence of ARGs is not significantly related to antibiotics, due to a presence of a natural resistome and heavy metals drive the shift of the resistome (Gao et al., 2012; Mazhar et al., 2021; Wright, 2007; Zou et al., 2021). Two possible reasons are responsible for the contradictions, one of which is the pollution profile of river biofilm. In heavily contaminated sites such as landfills, breeding farms and mining areas, the main environmental selection for bacteria communities comes from high concentrations of heavy metals. Heavy metals shape the structure of bacterial communities, which are the main carriers of antibiotic resistance genes. In clean water areas such as rivers or reservoirs, although the presence of ARGs is affected by wastewater discharge (Czekalski et al., 2014; Huerta et al., 2013), the concentration of heavy metals in wastewater that meets the discharge standards is lower than the mg/L (Chinese National Standards, GB8978-1996), which can be defined as the environmental safety concentration. Whether the environmental safety concentration of heavy metal is still the main factor in promoting the development of antibiotic resistance remains to be explored. Secondly, the assessment of co-selection is not comprehensive. The pollution induced community tolerance (PICT) method (Blanck, 2002) only evaluates the phenotypic characteristics of resistant bacterial communities, and ignores the impact of pollutants on the resistome (Song et al., 2017). Based on metagenomic sequencing, only the correlation analysis between ARGs and environmental factors is performed, and experimental evidences are lacking (Komijani et al., 2021; Wu et al., 2017). Therefore, a comprehensive study is imperatively needed to compare the contribution of antibiotics and heavy metals as selective agents in the development of resistant bacterial community in river biofilm.

We proposed to evaluate contribution of enrofloxacin and environmental safety concentration of Cu²⁺ to the antibiotic resistance of bacterial community in a river biofilm from a comprehensive perspective for the first time. Enrofloxacin, as one of the common fluoroquinolone antibiotic of veterinary use, is harmful to aquatic ecosystems and human health. As a growth-promoting factor in the process of agriculture and aquaculture, Cu²⁺ has an inescapable responsibility in promoting the spread of ARGs (Seiler and Berendonk, 2012). A biofilm culture device was used to develop biofilm with raw river water. The biofilm was exposed to Cu²⁺ and enrofloxacin for 28 days during the cultivation process, covering from the initial bacterial adhesion to the final formation of river biofilm. In order to achieve a comprehensive and reliable comparative study, together with PICT which was used to explore the phenotypic profiles of bacterial communities in biofilm, a metagenomic method was used to analyze the changes of bacterial resistome and community composition under the stress of pollutants. This method provides a new perspective for comparing the role of heavy metals and antibiotics.