Description

• To identify how the initial soil water content and different C/N-ratios in slurry influences horizontal gene transfer
• To identify dominant fecal bacteria and antibiotic resistant bacteria found in drainage water
• To model the leaching of resistant bacteria and fecal bacteria and determine how slurry C/N-ratio and soil water content influences the bacterial transport. Using this information we will develop a risk assessment tool for the contamination of antibiotic resistance bacteria from agricultural soils as well as suggest a potential alternative to E. coli as the best suited fecal indicator bacteria.

Hypothesis

The slurry soil environment is a "hotspot" for gene transfer.

• Slurry with a high C/N-ratio increase the likelihood of horizontal gene transfer due to an increase in nutrient availability compared to slurry with a low C/N-ratio.
• An increase in soil water content will increase horizontal gene transfer due to increased interaction between nutrients, pollutants and bacteria.

• Slurry with a low C/N-ratio will induce preferential flow because matrix pores are blocked by small solid matter compared to slurry with a high C/N-ratio.
• The effect of increased preferential flow by the slurry with a low C/N-ratio is greater for the soil with a low initial soil water content because of greater infiltration of the slurry as compared to a soil with a higher initial soil water content.

Background

The large production and use of antibiotics and antimicrobials in medicine and agriculture during the last 60 years have benefited both human health and agricultural productivity throughout the world. Unfortunately, there is growing evidence that resistance to antibiotics is increasing in both benign and pathogenic bacteria, and therefore poses as an emerging threat to public and human health in the future. Jones et al. (2008) analyzed approximately 300 outbreaks of human disease associated with new species or variant of an infectious agent from 1940 to 2004 and concluded that 60 % of the events were caused by zoonoses and 20 % of the events were associated with drug-resistance. The importance of antibiotic resistance was recently highlighted in a UK report concluding that this should be added to the UK government's list of threats to national security, alongside pandemic influenza and terrorism (Davis 2013).

Large amounts of antibiotics are used in the animal husbandry and have promoted the development and abundance of antibiotic resistance in the farm environment. Slurry has become a reservoir of antibiotic compounds, resistant bacteria as well as zoonotic pathogens, and its application to arable land poses an environmental risk. Knapp et al. (2010) found that there has been a significant increase of antibiotic genes in agricultural soil from 1940 to 2008. Slurry application is assumed to increase antibiotic resistance genes and selection of resistant bacterial populations in soil (Heuer et al. 2011). Resistance genes are likely located on mobile genetic elements such as plasmids, integrons and/or transposable elements. In a newly published study an important contributor to the spread of antibiotic resistance genes in manure-amended agricultural soils was suggested to be the broad-host range IncP-1 plasmids (Heuer et al. 2012). The horizontal gene transfer from slurry to soil bacteria is an important factor in resistance distribution, because introduced faecal bacteria typically show limited survival and tend to decline below detection limit within months (Agerso et al. 2006; Semenov et al. 2008; Semenov et al.2009). However, horizontal gene transfer from these bacteria to indigenous soil bacteria might increase the persistence of resistance genes in the soil. Slurry has been shown to increase the horizontal transfer of antibiotic resistance genes in soil due to increased levels of nutrients (Heuer & Smalla 2007). There is currently scarce information on slurry properties and how these influence the likelihood of gene transfer. It is known that that the soil microbial changes are influenced by the volume and degradability of the waste product. The presence of easily degradable compounds may result in a rapid increase in biomass and favors the growth of copiotrophic compared to oligotrophic organisms (Fierer et al. 2007). Manure with high dry matter content will generally have a high C/N-ratio whereas slurry with less dry matter will have a low C/N-ratio. Therefore, the use of manure tends to favor oligotrophics and potentially increase gene transfer. The spatial isolation of soil bacteria is assumed to prevent plasmid-mediated gene transfer between populations (Heuer & Smalla 2012). In a soil with higher soil water content a continuous water film or flowing water would increase this probability.

In order to evaluate the risk of contaminating fresh water a combined knowledge of survival and persistence in the soil is needed along with detailed leaching studies. Several studies including the recently terminated Pathos project (https://pathos.geus.net) have shown a high potential for transport of zoonotic and antibiotic resistant bacteria from slurry treated topsoil to subsurface layers or underlying aquifers (Chee-Sanford et al. 2001; Chee-Sanford et al. 2009; Bech et al. 2010; Bech et al. 2011) or has been associated with Danish drinking water (Ethelberg et al. 2005; Gubbels et al.2012). However, despite extensive prior research, many gaps still remain in our knowledge of bacterial fate due to a complicated transport patterns through the vadoze zone processes in which bacteria may migrate. Preferential flow commonly generates high-speed, high volume flow with minimal exchange with the soil matrix (Lin et al. 1997) and as such has a dominant influence on infiltration and contamination of receiving waters (McMurry et al. 1998; Artz et al. 2005). Many previous studies have tested the leaching of fecal indicator bacteria E. coli under unrealistic conditions that may have led to biased results. We saw in a resent experiment that leaching of tetracycline resistant bacteria took place during nonsaturated conditions and that relatively low precipitation intensities was enough to generate bacterial transport. This challenges some of the existing literature, as saturation may not be as an important factor as currently believed. In addition previous studies have focused on coliform bacteria, total bacteria and total bacterial biomass, fecal coliform bacteria and fecal streptococci as major indicator microorganisms. However, recent studies and review have detailed the lack of correlation between indicator microorganisms and the presence of pathogenic bacteria in surface water (Duris et al. 2009; Shelton et al. 2011). However, further research is needed to conclude and potentially suggest an alternative fecal indicator bacterium.