Persistence of naturally occurring antibiotic resistance genes in the bacteria and bacteriophage fractions of wastewater

Highlights

• ARGs persist to disinfection and inactivation processes.

• ARGs in bacteriophages show longer persistences than in bacteria.

• ARGs persist where Escherichia coli decay.

• The stability of ARGs increases the possibility of the gene being transferred.

• Elimination of ARGs from water would reduce their spread.

Abstract

The emergence and prevalence of antibiotic resistance genes (ARGs) in the environment is a serious global health concern. ARGs from bacteria can be mobilized by mobile genetic elements, and recent studies indicate that phages and phage-derived particles, among others, could play a role in the spread of ARGs through the environment. ARGs are abundant in the bacterial and bacteriophage fractions of water bodies and for successful transfer of the ARGs, their persistence in these environments is crucial. In this study, three ARGs (bla_TEM, bla_CTX-M and sul1) that naturally occur in the bacterial and phage fractions of raw wastewater were used to evaluate the persistence of ARGs at different temperatures (4 °C, 22 °C and 37 °C) and pH values (3, 7 and 9), as well as after various disinfection treatments (thermal treatment, chlorination and UV) and natural inactivation in a mesocosm. Gene copies (GC) were quantified by qPCR; then the logarithmic reduction and significance of the differences between their numbers were evaluated. The ARGs persisted for a long time with minimal reductions after all the treatments. In general, they showed greater persistence in the bacteriophage fraction than in the bacterial fraction. Comparisons showed that the ARGs persisted under conditions that reduced culturable Escherichia coli and infectious coliphages below the limit of detection. The prevalence of ARGs, particularly in the bacteriophage fraction, poses the threat of the spread of ARGs and their incorporation into a new bacterial background that could lead to the emergence of new resistant clones.

Introduction

The development of antibiotic resistance is a common evolutionary process in microorganisms to ensure their survival against other microorganisms. Antibiotic resistance can appear through a process of spontaneous mutation or be acquired via vertical or, more commonly, horizontal gene transfer. The use and misuse of antibiotics over recent decades and the presence of certain concentrations of antibiotics in different environments could have accelerated this phenomenon by exerting a selective pressure. Poor sanitary conditions, inefficient (or the total absence of) sewage treatment, and deficiencies in the control of both human and veterinary infections stimulate the generation and further spread of antibiotic resistant determinants (EFSA-ECDC, 2015, World Health Organization, 2014). The unfortunate consequences are that an estimated 25,000 people in Europe (ECDC/EMEA, 2009) and 23,000 in America die every year because of antibiotic-resistant infections (The White House, 2014).

Antibiotic resistance genes (ARGs) are abundant in water from different bodies of water with different levels of faecal pollution (Li et al., 2015, Rodriguez-Mozaz et al., 2014, Xu et al., 2014). One challenge we face is to evaluate whether the ARGs retain biological activity and therefore remain available to horizontal gene transfer mechanisms, which could lead to the emergence of new resistant clones. Despite their occurrence, detection of ARGs is not included in the analysis of the risk posed by water from different sources. In accordance with European regulations, water recovered from sewage only has to comply with different levels of quality concerning the presence of Escherichia coli, Legionella, Taenia and nematodes as biological parameters (BOE 1620, 2007).

Wastewater treatment processes normally result in significant reductions in the concentration of microorganisms present in sewage, prior to discharge (Lucena et al., 2004, Marín et al., 2015). However, a considerable amount of resistant bacteria are still found in treated sewage (Guardabassi et al., 2002, Huang et al., 2012, LaPara et al., 2011). In addition, under certain inactivating environmental conditions or disinfection treatment, the overall concentration of bacteria decreases, but the percentage of antibiotic resistant bacteria and consequently of ARGs in the total bacterial community could increase during wastewater treatment (Czekalski et al., 2012, Zhang et al., 2009).

Previous studies have evaluated the persistence of ARGs in bacteria following different disinfection processes: chlorination, UV irradiation and ozonation. According to Auerbach et al., 2007, UV irradiation does not affect the number of detectable tet^R gene types. In contrast, other authors using different chlorine concentrations (Yuan et al., 2015) or high UV doses up to 4000 or 124,770 J/m² (Zhuang et al., 2014, McKinney and Pruden, 2012), demonstrate a significant effect on the level of activity of ARGs in wastewater. However, despite ARGs being reduced significantly, a large number were still present after treatment.

Recent studies highlight the role of bacteriophage particles as ARG vehicles in the environment, in accordance with their abundance in human and animal wastewater, surface water and sludge (Calero-Cáceres et al., 2014, Colomer-Lluch et al., 2011, Colomer-Lluch et al., 2014b, Colomer-Lluch et al., 2014a, Marti et al., 2014, Marti et al., 2013). Phage particles containing ARGs enter these biomes from autochthonous bacteria or from the fecal source or pollution (Quirós et al., 2014). There is no information about how disinfection treatments and inactivation processes affect the ARGs present in the bacteriophage fraction, but bacteriophages are known to persist more than bacteria after disinfection procedures (Allué-Guardia et al., 2014, Cantalupo et al., 2011, Wommack et al., 1996). Here, we evaluate the presence of ARGs in the bacteriophage fraction and compare it with their presence in the bacterial fraction when affected by different environmental conditions, and also after different disinfection and natural inactivation processes.