Dissipation of the antibiotic sulfamethoxazole in a soil amended with anaerobically digested cattle manure

Highlights

• SMX dissipation in a digestate-amended soil was evaluated in microcosm experiments.

• SMX at a high concentration in soil halved in a few days.

• SMX presence affected the natural microbial community structure and activity.

• The antibiotic promoted the intI1 gene spread among the soil bacteria populations.

• Anaerobically digested cattle manure can act as a source of antibiotics, ARB and ARGs.

Abstract

The application of anaerobically digested cattle manure on agricultural land for both improving its quality and recycling a farm waste is an increasingly frequent practice in line with the circular economy. However, knowledge on the potential risk of spreading antibiotic resistance through this specific practice is quite scarce. The antibiotic sulfamethoxazole (SMX) is one of the most heavily prescribed in veterinary medicine. In this study, SMX dissipation and the possible effects on natural microorganisms were investigated in a soil amended with an anaerobically digested cattle manure produced from a biogas plant inside a livestock farm. Microcosm experiments were performed using amended soil treated with SMX (20 mg/kg soil). During the experimental time (61 days), soil samples were analysed for SMX and N4-acetylsulfamethoxazole, microbial abundance, activity and structure. Furthermore, the prevalence of the intI1 gene was also determined. The overall results showed that, although there was an initial negative effect on microbial abundance, SMX halved in about 7 days in the digestate-amended soil. The intI1 gene found in both the digestate and amended soil suggested that the use of anaerobically digested cattle manure as fertilizer can be a source of antibiotic resistant bacteria (ARBs) and genes (ARGs) in agroecosystems.

Introduction

The extensive use and disposal of antibiotics raise concerns about their occurrence in different environmental matrices [1,2]. In particular, the use of veterinary antibiotics in livestock farming worldwide is expected to increase during the coming years by more than 60% [3]. These compounds are excreted unaltered or as metabolites by the organisms treated and can reach soil and water ecosystems.

Anaerobic digestion of farm waste, including livestock manure, is currently a promising technique for producing biogas, leading to a liquid and solid digestate used in agriculture [4,5] as a good alternative to inorganic fertilizers [6]. As well as energy production, an advantage of anaerobic digestion is the absence (or the significant reduction) of pathogens in the digestate [7] and a complete recycling of the waste. This environmental friendly practice, used in organic farming, does not exclude the likelihood of introducing antibiotics to soil [8]. Consequently, it is very important to assess the potential risk of spreading antibiotic resistance through the application of anaerobically digested cattle manure on agricultural land. Once in the environment, antibiotics can affect natural microbial populations and ecosystem functions [9]. Moreover, depending on their physico-chemical characteristics, antibiotics can be adsorbed to soil [10], transported to surface or groundwaters by leaching and runoff [11,12], up-taken by plants [13,14] or degraded [15,16]. The selection and diffusion of resistant microbial strains and mobile genetic elements can also occur [2,17]. For all these reasons, antibiotics are environmentally considered emerging pseudo-persistent organic pollutants (pseudo-POPs), due to their continual introduction into the environment [18].

Sulfamethoxazole (SMX) is one of the most highly prescribed and consumed antibiotics in human and veterinary medicines and among the most widespread sulfonamides in aquatic and terrestrial ecosystems. It has also been placed on the top ten list of high priority pharmaceuticals identified in an European assessment of pharmaceuticals and personal care products [19]. Once administered, SMX is not completely metabolized; approximately 43% is transformed to N₄-acetyl-sulfamethoxazole (Ac-SMX) and only 15% to other transformation products [20,21]. Consequently, both SMX and its acetylate metabolite (Ac-SMX) can be expected to be found as environmental contaminants. SMX is a polar molecule with a low soil adsorption coefficient [22] and some studies report it as a biodegradable compound with higher degradation rates in soil than in water [23,24]. SMX degradation depends on soil texture, organic carbon content, depth, temperature, presence/absence of light (as it is a photosensitive molecule) and the presence of microbial populations resistant to its effect and able to degrade it [25]. In recent literature, half-lives of SMX have been reported from 4 to 13 days in soil [[26], [27], [28]]. However, some studies suggest that sulfonamides can be more persistent in the environment than in laboratory controlled studies [29]. Some laboratory experiments have found that the application of exogenous organic matter (EOM), such as manure in soil, can stimulate antibiotic biodegradation [27,30], while others have pointed out the inhibition of antibiotic mineralization [31].

The bacterial target of sulfonamides is the enzyme dihydropteroate synthase (DHPS), which is involved in the bacterial folic acid pathway [32]. Sulfonamide resistance is associated with a spontaneous mutation in the dhps (folP) gene or the acquisition of plasmid-borne sul genes (sul1, sul2, sul3) [32,33]. The spread of sul1 genes by horizontal gene transfer occurs rapidly due to their linkage to class 1 integrons (intI1), which are often embedded in DNA mobile elements, such as plasmids and transposons. Class 1 integrons (intI1) have been identified as a primary source of antimicrobial resistance genes and serve as a potential pool of resistance genes in a variety of bacterial species [34]. The intI1 gene has been proposed as a molecular marker for antibiotic resistance in the environment [35].

In this study, SMX dissipation in a soil (containing the natural microbial community) amended with an anaerobically digested cattle manure (digestate) and treated with the SMX antibiotic at a concentration of 20 mg/kg was evaluated. At the same time, the possible antibiotic effects on the microbial community in terms of microbial abundance, community structure (Gram-positive bacteria, Gram-negative bacteria and Fungi) and dehydrogenase activity were investigated. Finally, the prevalence of a molecular marker, the intI1 gene, was determined.