Effects of prescription antibiotics on soil- and root-associated microbiomes and resistomes in an agricultural context

Highlights

• Soil microbiome composition changed due to antibiotics in irrigation water.

• Antibiotics (AB) affected abundance of specific bacterial Orders in the roots.

• Rhizobiales abundance reduced in roots exposed to anbitiotics.

• AB increased intrinsic (mex), acquired (phoP, parC,E) resistance in root endophytes.

• Confirmed the impact of AB in soil and plants' microbiomes and resistomes.

Abstract

The use of treated wastewater for crop irrigation is rapidly increasing to respond to the ever-growing demands for water and food resources. However, this practice may contribute to the spread of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs) in agricultural settings. To evaluate this potential risk, we analyzed microbiomes and resistomes of soil and Lactuca sativa L. (lettuce) root samples from pots irrigated with tap water spiked with 0, 20, or 100 μg L⁻¹ of a mixture of three antibiotics (Trimethoprim, Ofloxacin, Sulfamethoxazole). The presence of antibiotics induced changes in bacterial populations, particularly in soil, as revealed by 16S rDNA sequence analysis. Parallel shotgun sequencing identified a total of 56 different ARGs conferring resistance against 14 antibiotic families. Antibiotic -treated samples showed increased loads of ARGs implicated in mutidrug resistance or in both direct and indirect acquired resistance. These changes correlated with the prevalence of Xantomonadales species in the root microbiomes. We interpret these data as indicating different strategies of soil and root microbiomes to cope with the presence of antibiotics, and as a warning that their presence may increase the loads of ARBs and ARGs in edible plant parts, therefore constituting a potential risk for human consumers.

Introduction

The use and abuse of antibiotics for medical and veterinary uses have induced a significant increase in the presence of antibiotics (ABs), antibiotic resistant bacteria (ARB), and antibiotic resistance genes (ARGs) in the environment (Han et al., 2016; Hu et al., 2016; Koch et al., 2017). ABs are widespread in surface water (Kolpin et al., 2002; Yan et al., 2013), groundwater (Hirsch et al., 1999; Szekeres et al., 2018), and treated and untreated sewage (Gao et al., 2012; Chang et al., 2010; Duong et al., 2008; Osorio et al., 2012). The exposure of bacterial populations to sub-inhibitory AB concentrations may induce AB resistance and contribute to the evolution and spread of ARB, which is already a major health concern at the global scale (Berendonk et al., 2015; WHO, 2014). Some projections identified ARB-related illnesses as a main cause of death worldwide by 2050 (WHO, 2014).

Soil microbiomes are reservoirs of ABs and ARGs due to both natural and anthropogenic processes, although there is a general consensus that the contribution of human-driven inputs have been escalating in recent times. For example, ABs used in healthcare are ultimately discharged into the aquatic environment via wastewater treatment plants (WWTPs). While WWTPs are very efficient in removing the so-called conventional pollutants (ammonia, phosphate, BOD, etc.), they have a very limited capacity to remove various pharmaceuticals and other chemical compounds, including ABs (Michael et al., 2013). In addition, WWTPs have been identified as potential hotspots for the generation and propagation of ARB, as they facilitate the direct contact of bacteria from very different origins with non-controlled concentrations of many bactericidal products, including disinfectants, metals, and ABs (Manaia et al., 2018; Pärnänen et al., 2019). Therefore, WWTPs effluents, the so-called treated wastewaters (TWW), typically present relevant loads of ARB and ARGs (Michael et al., 2013; Le-Minh et al., 2010; Martin et al., 2015).

An increasingly relevant source of ARB and ARGs in agricultural soils comes from the recent tendency of using TWWs for irrigation or fertigation of crops. This practice is meant to address the chronic (and ever increasing) water shortage in many food producing regions in the world, including the Mediterranean (Bouwer, 2000; Pina et al., 2020). While the use of TWW for crop irrigation may appear as an environmentally and economically sound solution to this demand, there is a growing concern that it may lead to the increase of the exposure of humans and livestock to ARG and ARB, presenting potential health risks. Plants can uptake ABs from soil (Ahmed et al., 2015; Boxall et al., 2006; Christou et al., 2017), leading to AB-mediated selective pressure in plant endophytes. The uptake of ABs by the roots and their translocation to leaves and fruits have been observed in both greenhouse and real field experiments (Christou et al., 2019; Goldstein et al., 2014). Leafy (like lettuce) and root vegetables (radish, carrot) show the highest capacity to uptake contaminants of emerging concern, including ABs, followed by fruit vegetables (cucumber, tomato, pepper, beans), and by cereal crops (corn, rice, wheat) (Christou et al., 2019). Similarly, the composition of the endophytic microbiome and of its associated resistome appears to be linked to the corresponding soil microbiome and resistome (Cerqueira et al., 2019a; Cerqueira et al., 2019b; Cerqueira et al., 2019c). The situation is further complicated by the also common practice of using animal manure or sewage sludge as soil amendments, which also constitute rich sources of ABs, ARBs, and ARGs (Martin et al., 2015; Chen et al., 2016; McKinney et al., 2018; Udikovic-Kolic et al., 2014). Whether or not these practices represent for the consumers a significant risk of exposure to ARBs, ARGs, and pathogens is still a matter of discussion (Frankel et al., 2010; van Hoek et al., 2015).

In this study, we investigated changes in microbiomes and resistomes from soil and roots of lettuce plants (Lactuca sativa L.) grown in a controlled mesocosm environment in which plants were irrigated with tap water spiked with different levels of three selected ABs (Trimetoprim, Ofloxacin, Sulfamethoxazole). The hypothesis was that the presence of the ABs will affect the resident microbiome and that this effect will modify both soil and root microbiome and the associated resistomes, therefore increasing the loads of ARGs in the plant tissues. We used non-targeted, high-throughput sequencing methods, as microbiomes are complex and dynamic communities that adapt according the environmental stimuli (Lee et al., 2018). Given this extreme complexity, metagenomic methodologies outcompete culture-dependent methods, which are restricted to species that can be cultured in the lab (Manaia et al., 2018), or targeted quantitative polymerase chain reaction (qPCR) analyses, which are limited to specific sets of genes.

The effects of the presence of ABs in soil and root microbiomes were analyzed using both 16S rDNA amplicon sequencing and whole-genome high-throughput sequencing. Similar approaches have been previously used for analyzing microbiomes from WWTP effluents and sludge (Huang et al., 2020; Yoo et al., 2020; Fang et al., 2018), or from aquaculture settings (Zhao et al., 2018). They are also used to study the effects of adding manure on both soil and root microbiome and resistomes (Guron et al., 2019; Zhang et al., 2020). Our experimental setup differentiates from these studies in that it is specifically designed to test the effect of the addition of antibiotics on the resident microbiome and resistomes, and to analyze their simultaneous effects in soil and root at the metagenomic level. The study is directed to provide information of the impact of the water quality, and particularly, of its content in ABs, in soil microbiomes and in their colonization to crop roots, and how these impacts could influence their associated resistomes. It is also relevant for supporting the limitation of the agricultural use of antibiotics in plant protection strategies, a practice still allows in several countries and, under special circumstances, also in the EU and the USA (Fortunati et al., 2019; McManus et al., 2002).The objective is to contribute to the ongoing discussion on the presence of ABs in water and amendments used to produce eadible agricultural products and their potential impact in public health (Blum et al., 2019).