A TbPO₄-based capturer for environmental extracellular antibiotic genes by interrogating lanthanide phosphates nanoneedles

Highlights

• A facile approach for extraction of trace amount of eDNA using LnPO₄ nanoneedles was developed.

• Less crystalline LnPO₄ nanoneedles had a higher eDNA adsorption capacity compared to highly crystalline materials.

• The developed approach showed a comparable eDNA extraction efficiency to a commercial extraction method, but less cost.

• Occurrence of ARGs in the eDNA that extracted by TbPO₄ nanomaterial from West River sample were detected.

Abstract

Accurate determination of antibiotic resistance genes (ARGs) in environmental DNA molecules (eDNA) is challenging owing to its low abundance in the aquatic environment. Here we report a facile and cost-efficient approach to extract trace amount of eDNAs in the aquatic environment using LnPO₄ nanomaterials. Among the nanomaterials, less crystalline TbPO₄ nanoneedles was identified as the most prominent candidate for long stranded DNA and short stranded DNA with adsorption efficiency above 97%. The adsorbed DNA was washed off from TbPO₄ nanoneedles by optimized eluant (85% PBS, 15% EtOH, 4 g/L glycine, pH 10.0) with an optimal DNA recovery of 78.83%. Our approach showed a comparable or better eDNA extraction efficiency than a commercial extraction method for different environmental samples, but 89% less cost. The high purity of the extracted eDNA was demonstrated by a high A_260/280 ratio. Using qPCR experiment, the occurrence of six common ARGs in the eDNA were detected with abundance ranging from 4.06 × 10³ to 3.51 × 10⁹ copies/L in river samples. This specific DNA capturer is valuable for the evaluation of spatial and temporal dynamic of ARGs pollution to provide insight into the potential risk with regard to the human health.

Introduction

The widespread use of antibiotics has led to a world-wide epidemic of antibiotic-resistance and propagation of antibiotic resistance genes (ARGs) and antibiotic resistance bacteria (ARB). Hospitals and non-clinical environments including livestock farms and aquaculture systems are major places for overuse of antibiotics, exerting a high selection pressure contributing to the development of ARGs and ARB (Zarei-Baygi and Smith, 2021). Antibiotic residues as well as ARB and ARGs are then released and widely transported in the environment by direct excretion or the irrigation with wastewater or treated effluents, posing great threat to environment and human health. By 2050, antimicrobial resistance infection is expected to become an important factor for global deaths (Sommer et al., 2017).

Transfer of ARGs has become key driving force for the spread of antibiotic resistance among bacteria through the gene transfer process (Jia et al., 2015, Sanderson et al., 2016). Determination of the abundance and spatial dynamics of ARGs is crucial for evaluation of their threat to public health and for development of mitigation strategy to combat antibiotic resistance. ARGs present as both intracellular DNA (iDNA) and extracellular fraction (eDNA) which is secreted from living bacteria or released from dead specimens (Hao et al., 2019). The intracellular ARGs have been found in landfills (Xu et al., 2021), drinking water treatment plants (Ma et al., 2019, Su et al., 2018), wastewater treatment systems (Liu et al., 2018), and recipient environment (Mao et al., 2014; Y. Zhang et al., 2013). Such ARGs have also been detected in tap water samples in countries such as France (Madec et al., 2016), India (Kaur et al., 2020) and China (Zhang et al., 2020) with various abundance. Nevertheless, little is known about the extracellular ARGs (eARGs) pollution owing to their low concentration in the environment, especially in the environmental aquatic systems.

In the past few decades, analysis of target molecule at trace level adopted extraction pretreatment for enrichment purpose. There are several methods for enriching DNA molecules from environmental samples for the eARGs detection and analysis, eg., organic solvent or surfactant assisted precipitation (DeFlaun et al., 1986, Mao et al., 2014, Natarajan et al., 2016), filtration (Liang and Keeley, 2013, Liu et al., 2020), liquid chromatography (Beebee, 1991, Calderón-Franco et al., 2021, Katevatis et al., 2017, Wang et al., 2016), kit extraction (Nukazawa et al., 2018), and combined procedures (Yang et al., 2020). However, highly effective techniques in terms of the cost, time, yield and the impact on environment are still lacking. Recently, DNA adsorbent materials such as montmorillonite clay (Cai et al., 2006), carbon (Kirtane et al., 2020), and Fe₃O₄ (Yuan et al., 2019) have been explored for capturing eDNA through the non-specific van der Waals forces, hydrophobic interactions or cation bridges. For example, Fe₃O₄ has been successfully used to extract eDNA from waste water with a recovery efficiency higher than traditional methods (Yuan et al., 2019). Although promising, their usage is limited by interference from organic species and biological debris.

Lanthanide phosphates (LnPO₄) have recently emerged as appealing materials with applications ranging from sensors to plasma display panels (Li et al., 2015). LnPO₄ possesses a high affinity for molecules with phosphate groups (Zhang et al., 2015, Zhou et al., 2013). The surface lanthanide ions with abundant f-orbitals prefer to bind negatively charged phosphate groups through coordination. Such a selectivity is beneficial for suppressing the nonspecific adsorption of non-phosphorylated environmental pollutants. Moreover, LnPO₄ materials are highly stable and biocompatible (Meenambal et al., 2019), making it suitable for selective eDNA enrichment without causing environmental pollutions. In this work, LnPO₄ materials were used for the first time for eDNA enrichment, and the extraction efficiency of various LnPO₄ (Sm, Eu, Gd, Tb and Dy) nanomaterial toward eDNA in aquatic environment have been systematically explored. Moreover, the influences of crystal structure, DNA types, and eluant composition on DNA adsorption and recovery were analyzed to establish the optimal DNA extraction conditions. This method was also applied to extract eDNA from urban rivers to facilitate identification of six abundant resistance genes in these eDNA samples.