Elsevier

Water Research

Volume 92, 1 April 2016, Pages 188-198
Water Research

A new adsorption-elution technique for the concentration of aquatic extracellular antibiotic resistance genes from large volumes of water

https://doi.org/10.1016/j.watres.2016.01.035Get rights and content

Highlights

  • We present a technique for eARG recovery with 104 times more sensitive than qPCR.

  • With our method, eARGs can be concentrated from large volume of water.

  • We also describe novel nucleic acid adsorption particles with high capacity for eARGs.

  • We determine eARGs concentration in natural water.

Abstract

Extracellular antibiotic resistance genes (eARGs) that help in the transmission and spread of antibiotic-resistant bacteria are emerging environmental contaminants in water, and there is therefore a growing need to assess environmental levels and associated risks of eARGs. However, as they are present in low amounts, it is difficult to detect eARGs in water directly with PCR techniques. Here, we prepared a new type of nucleic acid adsorption particle (NAAP) with high capacity and developed an optimal adsorption-elution method to concentrate eARGs from large volumes of water. With this technique, we were able to achieve an eARG recovery rate of above 95% from 10 L of water samples. Moreover, combining this new method with quantitative real-time PCR (qPCR), the sensitivity of the eARG detection was 104 times that of single qPCR, with the detection limit lowered to 100 gene copies (GCs)/L. Our analyses showed that the eARG load, virus load and certain water characteristics such as pH, chemical oxygen demand (CODMn), and turbidity affected the eARGs recovery rate. However, high eARGs recovery rates always remained within the standard limits for natural surface water quality, while eARG levels in water were lower than the detection limits of single qPCR assays. The recovery rates were not affected by water temperature and heterotrophic plate counts (HPC). The eARGs whatever located in the plasmids or the short-length linear DNAs can be recovered from the water. Furthermore, the recovery rate was high even in the presence of high concentrations of plasmids in different natural water (Haihe river, well water, raw water for drinking water, Jinhe river, Tuanbo lake and the Yunqiao reservoir). By this technology, eARGs concentrations were found ranging from (2.70 ± 0.73) × 102 to (4.58 ± 0.47) × 104 GCs/L for the extracellular ampicillin resistance gene and (5.43 ± 0.41) × 102 to (2.14 ± 0.23) × 104 GCs/L for the extracellular gentamicin resistance gene in natural water for the first time, respectively. All these findings suggest that NAAPs have great potential for the monitoring of eARGs pollution in water.

Introduction

The development of antibiotic resistance is a serious problem globally (Allen, 2010, Cabello, 2006, WHO, 2012). A large number of antibiotic-resistant bacteria (ARB) and even multiple drug-resistant bacteria (MDRB) are found in water (Bessa et al., 2014, Moges et al., 2014, Mohanta and Goel, 2014), and the transfer and spread of antibiotic resistance genes (ARGs) in water is considered to play a key role in the dissemination of ARB (Pruden et al., 2012, Rahube and Yost, 2010, Rizzo et al., 2013).

Two forms of natural ARGs—intracellular ARGs (iARGs) and extracellular ARGs (eARGs)—are present in water(Nielsen et al., 2007, Zhang et al., 2013). iARGs are located in the bacterial compartment and promote ARB dissemination via conjugation and transduction (Mao et al., 2014). Increasing number of researchers focus on iARGs concentration in natural water bodies which is up to 6.7 × 106 GCs/L in natural water (Luo et al., 2010, Wang et al., 2012, Xu et al., 2015). eARGs, unlike iARGs, originate from the lysis of dead ARB or are secreted by live ARB. Thus, with eARGs, dissemination of drug resistance is possible via natural transformation wherein competent non-resistant bacteria acquire antibiotic resistance by taking up eARGs from the aquatic environment under particular (micro) environmental conditions such as biofilm formation and sedimentation(Li, 2001, Molin and Tolker, 2003, Pietramellara et al., 2009). eARGs with genetic mobile platforms such as plasmids, transposons and integrons in the water have been gained increasing attention for their potential contribution to the spread of antibiotic resistance among bacterial communities(Baur et al., 1996, Bennett, 2008, Han et al., 2012, Paget and Simonet, 1994, Stokes, 2011). eARGs are defined as an emerging environmental contaminant that can persist in the aquatic environment for an extended period of time, ranging up to months, with the help of soil colloids and sand particles(Borin et al., 2008, Cai et al., 2006, Pruden et al., 2006, Pietramellara et al., 2009); thus, eARGs in water may pose a serious threat to both human health and ecosystem (Pruden et al., 2006). Investigating the pollution caused by eARGs can not only help in assessing their environmental concentrations and associated risks, but also help to better understand and clarify their contribution to the dissemination of ARGs in water.

According to Zhang et al.(Zhang et al., 2013), the amount of eARGs in extracellular DNA extracts from sludge are usually two to three orders of magnitude lower than the amount of iARGs in the corresponding intracellular DNA extracts(Beebee, 2006, Deflaun, 1986, Zhanbei and Ann, 2013). However, very little is known about eARG pollution in water due to its limited concentration: eARGs are undetectable directly by PCR and qPCR as a result of their low concentration. It is therefore essential to concentrate eARGs from 1 L or more water prior to eARG detection. Nucleic acid precipitation with organic solvents such as ethanol and isopropanol is a widely used technique to purify or concentrate free nucleic acids in aqueous solution. The recovery rate with this procedure is quite high and the extracted nucleic acids have high purity (Deflaun, 1986, Tan, 2009). However, this procedure is rather complicated to perform, requires a lot of time, and causes environmental toxicity because it requires the use of a large amount of organic reagents. In addition, the recovery rate and purity of the nucleic acids obtained are easily affected by the quality of the water. Silica gel membrane adsorptive method, which utilizes the adsorption of spin column (silica gel) to nucleic acid, is another widely applied method in the forms of commercial kits such as E. Z.N.A. gel extraction kit (Omega Bio-Tek) and DNA Isolation kit (Roche). This method is rapid and sensitive, and it is suitable for small volumes of less than 1 mL(Kjeldsen et al., 2010, Metcalf and Weese, 2012). In recent years, filtration environmental DNA capture method has been developed to capture environmental DNA in the water(Jerde et al., 2011). However, only 16% recovery of the spiked extracellular DNA (eDNA) were reported from 200 mL water in the help of the mixed cellulose acetate and cellulose nitrate (MCE) membrane filter(Zhanbei and Ann, 2013). To the best of our knowledge, there are no reported techniques for recovering aquatic eARGs from large volumes of water (1 L or more).

In this study, we described novel nucleic acid adsorption particles [NAAPs, silica gel coated with Al(OH)3] with high capacity for eARGs and established a new adsorption-elution method to concentrate eARGs from large volumes of water with a high efficiency for the first time, which is based on the electrostatic adsorption of eARGs with NAAPs, followed by the interaction disruption of organic eluent which is consisted of 3× broth and 0.05 mol/L glycine (Gly), and then isopropanol precipitation. The effect of the conditions of the procedure, e.g., eluents, flow rate and water quality indicators e.g. pH value, turbidity and chemical oxygen demand (CODMn) on recovery rate were also investigated using antibiotic-resistant plasmids. Moreover, to validate its applicability and efficiency, various natural water samples were explored and eARGs concentrations in natural water were reported for the first time.

Section snippets

Microorganisms

Escherichia coli (ATCC 25922), the bacteriophage MS2 and its host strain E. coli (ATCC 15597) were purchased from the American Type Culture Collection. Pseudomonas aeruginosa PA14, whose gentamicin resistant gene was on the chromosome (Gmr), was provided by Nankai University (Choi and Schweizer, 2006). They were grown in lysogeny broth (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) at 37 °C. The bacteriophage MS2 was prepared and detected by the double-layer plaque assay (

Characteristics of the NAAPs

Granular NAAPs are irregular, with their sizes ranging from 200 to 400 μm (Fig. 1a). However, compared with naked silica gel (Fig. 1b), the surface of NAAPs is more rough, and 4.16%–16.91% of Al elements are found dispersed on the coater. The BET surface area of the NAAPs was determined to be 316.99 m2/g. The isoelectric points (IEPs) of Al(OH)3 were calculated to be between pH 8.5 and 9.3 (Fig. 1c). In addition, the pH of the water had a considerable effect on the adsorption capacity (Fig. 1

Discussion

In this study, we have proposed and described an adsorption-elution technique using particles with high adsorption capacity for nucleic acids that can be used to efficiently concentrate eARGs from large volume of natural water. Combining this new method with qPCR, the detection limit of eARGs in water was improved by 104 times compared to the limit of single qPCR. More important, we quantified the concentrations of some eARGs such as extracellular ampicillin and gentamicin resistance genes in

Conclusions

  • We have established an efficient and simple method to efficiently concentrate eARGs from natural water even when the levels of eARGs are low.

  • This technique can be used to concentrate eARGs from a wide variety of water bodies under different environmental conditions.

  • This new technique would not only help tackle the problem of eARG pollution in water bodies and assess their environmental levels and associated risks, but also help us better understand and clarify their contribution to the

Statement of competing financial interests

The authors declare no competing financial interests related to the publication of this study.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (81372947, 31470234).

We thank Prof. Wei-hui Wu (Nankai University, China) for providing the Pseudomonas aeruginosa PA14.

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