Antibiotic resistance gene abundances associated with antibiotics and heavy metals in animal manures and agricultural soils adjacent to feedlots in Shanghai; China

Abstract

Eight antibiotic resistance genes (ARGs), 7 heavy metals, and 6 antibiotics were quantified in manures and soils collected from multiple feedlots in Shanghai. The samples were analyzed to determine if ARG abundances were associated with heavy metal concentration and independent of antibiotics. The results revealed the presence of chloramphenicol, sulfonamides and tetracyclines at concentration ranges of 3.27–17.85, 5.85–33.37 and 4.54–24.66 mg kg⁻¹, respectively. Typical heavy metals, such as Cu, Zn, and As, were detected at concentration ranges of 32.3–730.1, 75.9–4333.8, and 2.6–617.2 mg kg⁻¹. All ARGs tested were detected in the collected samples except tetB(P), which was absent in animal manures. Overall, sulfonamide ARGs were more abundant than tetracycline ARGs. Except for sulII, only a weak positive correlation was found between ARGs and their corresponding antibiotics. On the contrary, significant positive correlations (p < 0.05) were found between some ARGs and typical heavy metals. For example, sulA and sulIII were strongly correlated with levels of Cu, Zn and Hg. The data demonstrated that the presence of ARGs was relatively independent of their respective antibiotic inducer. In addition to antibiotics, toxic heavy metals, such as Hg, Cu, and Zn, exerted a strong selection pressure and acted as complementary factors for ARG abundance.

Introduction

Antibiotics are commonly used in concentrated animal feeding operations (CAFOs) worldwide to treat animal diseases and promote animal growth. In China, over 8000 t of antibiotics are used as feed additives each year [1]. In Turkey, the usage of veterinary antibiotics in feeding operations has been reported to be 33% of total pharmaceutical consumption[2]. In the USA, 16 million kg of antibiotics are used annually, and 70% is estimated to be used for non-therapeutic purposes [3]. The majority of antibiotics used is excreted in feces and urine and then persist and accumulate in soils after repeated manure application. Recently, higher levels of antibiotic residues were detected in organic vegetable fields compared to traditional vegetable fields [4], [5]. This finding was due to the large amount of manure used in the cultivation of organically grown vegetables.

A major concern from antibiotic contamination of the environment is the rapid and increasing number of antibiotic resistance genes (ARGs) found in bacteria. ARGs are naturally present in the chromosomes of environmental bacteria but are now presenting in plasmids under selection pressure from antibiotics [6], [7]. Under these conditions, they can be transferred between non-pathogens, pathogens, and even distantly related organisms, such as Gram-positive and Gram-negative bacteria, through horizontal gene transfer (HGT) [8], [9]. It was predicted that in the absence of antibiotic selection pressure, antibiotic resistance would disappear. However, the wide dissemination of ARGs and their presence in human pathogens in places without a high antibiotic load indicate that once those elements are present in gene-transfer platforms, the probability of their maintenance in natural ecosystems is high [10], [11]. Therefore, antibiotic resistance genes are now considered pollutants [9].

It is noteworthy that, in addition to emerging non-traditional ARG contaminants, anthropogenic-derived sources of metals represent a major source of contamination in agricultural soils [12]. Mining, processing, and smelting have contaminated soil and water resources throughout the world [13], [14], [15], [16]; the usage of pesticides and mineral elements for animal growth has led to an even wider range of non-point source contamination of heavy metals [17], [18]. Unlike antibiotics, metals are not subject to degradation and can represent a long-term selection pressure.

Recently, an increasing number of reports have suggested that the increasing abundance of ARGs may be due to waste releases of heavy metals; there are known links between heavy metals and antibiotic resistance maintenance and proliferation [19], [20], [21]. Nevertheless, antibiotic residues are still considered the direct and primary selection pressures that accelerate the expansion of the bacterial resistome in the soil. To our knowledge, there is little experimental evidence or quantitative data that has correlated the levels of heavy metals and elevated antibiotic resistance in human impacted environments, such as manure-amended agricultural soils. The extent to which metal levels in the soil affect ARG abundance is still not well known.

In the present study, an area in Shanghai, located at the upper reaches of the Huangpu River and concentrated with large and small scale feedlots, was selected as the research region. Its unique geographic position posed potential antibiotic and ARG contamination risks to the Huangpu River; this river is an extremely important shipping artery and provides approximately 80% of the raw drinking water for Shanghai [22]. Animal manures and manure-amended agricultural soils were sampled from multiple livestock feedlots. Eight ARGs (tetB(P), tetM, tetO, tetW, sulI, sulII, sulIII, and sulA) were quantified as biomarkers for antibiotic resistance potential in manures and soils. These biomarkers were chosen because they are the most common ARGs in the current reports and found in over 90% of identified resistant species [23], [24], [25]. The levels of antibiotics and heavy metals in the collected samples were simultaneously measured. The experimental data were then analyzed to determine the relationship between ARG abundance and distribution and anthropogenic-derived heavy metal content in the presence of antibiotic residues.