River Biofilms Microbiome and Resistome Responses to Wastewater Treatment Plant Effluents Containing Antibiotics

Abstract

Continuous exposure to low concentrations of antibiotics (sub-minimal inhibitory concentration: sub-MIC) is thought to lead to the development of antimicrobial resistance (AMR) in the environmental microbiota. However, the relationship between antibiotic exposure and resistance selection in environmental bacterial communities is still poorly understood and unproven. Therefore, we measured the concentration of twenty antibiotics, resistome quality, and analyzed the taxonomic composition of microorganisms in river biofilms collected upstream (UPS) and downstream (DWS) (at the point of discharge) from the wastewater treatment plant (WWTP) of Poitiers (France). The results of statistical analysis showed that the antibiotic content, resistome, and microbiome composition in biofilms collected UPS were statistically different from that collected DWS. According to Procrustes analysis, microbial community composition and antibiotics content may be determinants of antibiotic resistance genes (ARGs) composition in samples collected DWS. However, network analysis showed that the occurrence and concentration of antibiotics measured in biofilms did not correlate with the occurrence and abundance of antibiotic resistance genes and mobile genetic elements. In addition, network analysis suggested patterns of co-occurrence between several ARGs and three classes of bacteria/algae: Bacteroidetes incertae sedis, Cyanobacteria/Chloroplast, and Nitrospira, in biofilm collected UPS. The absence of a direct effect of antibiotics on the selection of resistance genes in the collected samples suggests that the emergence of antibiotic resistance is probably not only due to the presence of antibiotics but is a more complex process involving the cumulative effect of the interaction between the bacterial communities (biotic) and the abiotic matrix. Nevertheless, this study confirms that WWTP is an important reservoir of various ARGs, and additional efforts and legislation with clearly defined concentration limits for antibiotics and resistance determinants in WWTP effluents are needed to prevent their spread and persistence in the environment.

Life in an unsuspected antibiotics world: River biofilms

“…The relative abundance (mean value) of these two classes of integrons is higher in the biofilms collected from the CR (downstream point) than in the others: CR DWS: Intl1 -0.14, Intl3 -0.02; CR UPS: Intl1 -0.02, Intl3 -0.01; VR: Intl1 -0.06, Intl3 -0. The presence of class 1 and 3 integrons in polluted water and their enrichment in WWTP discharges has already been described in various studies (Gillings et al, 2015;Bengtsson-Palme and Larsson, 2016;Aubertheau et al, 2017;Cacace et al, 2019;Matviichuk et al, 2022). The CR and VR biofilms exhibit a high abundance of Intl1, close to the values observed in urbanized areas and/or exposed to WWTP effluents (see a comparison of results with data from the literature -Figure 3).…”

“…Twenty commonly used antibiotics (6 classes) (Table 1) were measured in all collected samples by the procedure described in Aubertheau et al (2017) and Matviichuk et al (2022). Pressurized liquid extraction (ASE™ 350, Thermo Scientific, Waltham -USA) was used to extract antibiotics from the biofilm samples.…”

“…For instance, Gaze et al (2011) investigated the effect of quaternary ammonium compounds (QACs) (disinfectants, sanitizers, detergents and biocides) in co-selecting ARGs and showed their enrichment due to the predominance of IntI1 as a result of QACs exposure. The phenomenon of co-selection is also closely related to HGT, as well as to the efflux pump mechanism, which is one of the mechanisms responsible for cross-resistance and a frequently used "defence" mechanism in bacterial biofilms (Wales and Davies, 2015;Pal et al, 2017;Cheng et al, 2019;Matviichuk et al, 2022).…”

Metagenomics: An Approach for Unraveling the Community Structure and Functional Potential of Activated Sludge of a Common Effluent Treatment Plant

“…Due to the limitation of the culturability of microbes, the whole spectrum of microbial diversity and their functions cannot be captured; however, using shotgun metagenomics, it is possible to untap the hidden microbial community and their interactions in any given environmental sample (Handelsman, 2004 ; Bodor et al, 2020 ). Using shotgun metagenomics analysis, the influence of wastewater on the genetic composition of sediment microflora was observed in terms of the dissemination of antibiotic resistance gene mobile genetic elements into the receiving aquatic system (Matviichuk et al, 2022 ).…”

Do pharmaceuticals affect microbial communities in aquatic environments? A review

“…The release of residual antimicrobial compounds from WWTPs into the environment is one important cause of this phenomenon. Observations in recent years unambiguously confirm that AMR should be counted among the main threats to the modern world, and this phenomenon, although already causing a significant problem, is likely to intensify(O'Neil, 2014;Matviichuk et al, 2022).…”