Reducing residual antibiotic levels in animal feces using intestinal Escherichia coli with surface-displayed erythromycin esterase

Highlights

• Esterase displayed on E. coli cell surface effectively degraded erythromycin.

• Surface-engineered bacteria showed high stability for degrading erythromycin.

• Engineered bacteria colonized mouse gut and reduced erythromycin in feces.

• Elimination of antibiotics from feces prevents its spread to the environment.

Abstract

Antibiotics are widely used in livestock and poultry industries, which results in large quantities of antibiotic residues in manure that influences subsequent treatments. In this study, an Escherichia coli strain was engineered to display erythromycin esterase on its cell surface. The engineered strain (E. coli ereA) efficiently degraded erythromycin by opening the macrocyclic 14-membered lactone ring in solution. Erythromycin (50 mg/L) was completely degraded in a solution by E. coli ereA (1 × 10⁹ CFU/mL) within 24 h. E. coli ereA retained over 86.7 % of the initial enzyme activity after 40 days of storage at 25 °C, and 78.5 % of the initial activity after seven repeated batch reactions in solution at 25 °C. Mice were fed with E. coli ereA and real-time quantitative PCR data showed that E. coli ereA colonized in the mice large intestine. The mice group fed E. coli ereA exhibited 83.13 % decrease in erythromycin levels in their feces compared with the mice group not fed E. coli ereA. E. coli ereA eliminated antibiotics from the source preventing its release into the environment. The surface-engineered strain therefore is an effective alternative agent for treating recalcitrant antibiotics, and has the potential to be applied in livestock and poultry industries.

Introduction

Antibiotics are extensively used in livestock for medical purposes and as growth promoters (Feng et al., 2017). It is estimated that livestock will consume about 105.6 thousand tons of antibiotics by 2030 globally (Lekshmi et al., 2017). However, a large amount of antibiotics is not metabolized or absorbed in animal body; therefore, about 50–90 % of the consumed antibiotics are excreted in the original form or as parent compounds (Tran et al., 2016; Ivanov et al., 2016). As a result, animal manure is one of the main sources of antibiotic contamination, and antibiotics have frequently been found in diverse environments (Kafaei et al., 2018; Michael et al., 2013; Zhang et al., 2013). Antibiotic is one of the emerging organic contaminants with adverse effects on human health and ecosystem (Michael et al., 2013), and is regarded as one of the three biggest dangers by the World Health Organization (Kafaei et al., 2018; Li and Zhang, 2013). Antibiotic ubiquity in the environment promotes selection of antibiotic-resistant bacteria in natural environments, resulting in one of the most serious public health concerns in the 21st century (Kafaei et al., 2018). Moreover, although antibiotics are present at very low concentrations in environment, they are a global ecological problem (Bouki et al., 2013). Residual antibiotics are toxic to aquatic species; for example, they cause a decrease in the population of phytoplankton and zooplankton, and affect the reproductive systems of aquatic species (Kafaei et al., 2018; Fent et al., 2006). In addition, the key components of the aquatic ecosystem may also be affected by the sustained release of antibiotics into the environment (Ivanov et al., 2016; Martínez, 2008). Therefore, it is important to develop an effective method to control antibiotics release from pollution sources.

The antibiotics concentrations range from 1 to 136 mg/kg of dry matter in manure (Ezzariai et al., 2018). Abiotic and biotic treatments have been used for antibiotic residues in animal manure (Ivanov et al., 2016; Michael et al., 2013). For instance, incineration is used as an effective technology to deal with animal manure through ash fertilizers and energy production (Huang et al., 2011). Nevertheless, it may cause air pollution due to the production of large amount of smoke (Skoulou and Zabaniotou, 2007). Compost is a good technology that decreases residual antibiotic levels in animal manure (Guo et al., 2017). However, residual antibiotics (35 mg/kg dry matter) influence the efficiency of composting and reduce the duration of the thermophilic period (Ezzariai et al., 2018). Anaerobic digestion (AD) is widely used to treat animal manure, but the residual antibiotics inhibit the methane production process (Yin et al., 2016). Although these traditional processes have shown good removal efficiencies for other pollutants, more research and investigation is needed regarding antibiotics removal (Barancheshme and Munir, 2018). These conventional technologies are not sufficient to completely remove the antibiotics in manure (Yang et al., 2018). Hence, a new strategy is essential to improve antibiotics removal and reduce the release of antibiotics from manure into the environment (Ou et al., 2015).

Intestinal microbes often play a significant role in the elimination of pollutants, such as adsorption of heavy metals and degradation of organic pollutants (Zhai et al., 2016; Cai et al., 2018). Notably, functional bacterial colonization in animal gut can remediate contaminants (Cai et al., 2018; Sinha et al., 2008). For instance, mice fed Lactobacillus plantarum show accelerated cadmium excretion in their feces (Zhai et al., 2016). Millions of microbial decomposers inhabit the gut of the earthworm and can degrade polycyclic aromatic hydrocarbon pollutants (Sinha et al., 2008). Similarly, antibiotics are consumed and metabolized quickly by fly larvae with the help of intestinal microbes (Cai et al., 2018). This remediation strategy has a low-cost and employs a convenient technology for pollutant removal (Sinha et al., 2008). A large number of bacteria inhabit the large intestine of animals, which can possibly decrease the release of residual antibiotics into the manure. However, intestinal bacteria in animals that eliminate residual antibiotics have rarely been reported. Therefore, antibiotics removal from pollution sources using intestinal bacteria is a possibility.

Some functional enzymes are displayed on the bacterial cell surface, which gives the opportunity to create surface-engineered bacteria to effectively degrade recalcitrant organic contaminants, thereby overcoming limitations such as substrate transport and enzyme purification (Smith et al., 2015; Yang et al., 2015). Phytase display on the Saccharomyces cerevisiae cell surface effectively degrades phytate phosphorus (Chen et al., 2016a). Yeast with surface-displayed laccase can significantly degrade sulfamethoxazole and bisphenol A in wastewater (Margot et al., 2015). Erythromycin esterase originating from E. coli plasmids or Pseudomonas aeruginosa has the potential to degrade erythromycin, which is one of the most widely used macrolide antibiotics, and a recalcitrant pollutant due to its persistence in the environment (de Cazes et al., 2016; Pérez et al., 2017). Additionally, erythromycin is widely used in livestock for growth promoters (Pérez et al., 2017). Erythromycin is metabolized and absorbed in the small intestine and the residual erythromycin is excreted in feces in the original form (Minami et al., 1996). About half of the orally administered erythromycin was excreted into the feces at a dose of 20 mg/kg (Kohno et al., 1989). Therefore, it is hypothesized that erythromycin esterase displayed on E. coli cell surface could be exploited as a new engineered bacteria colonizing the large intestine for the reduction of erythromycin levels in feces. In this study, erythromycin esterase was displayed on E. coli cell surface by ice nucleation protein (Fig. 1). The engineered bacteria were fed to Kunming mice used as an animal model to evaluate erythromycin removal from animal manure.