The effects of veterinary antibiotics on soil microbial communities

Abstract

The possible environmental “side effects” of pharmaceuticals to the environment have not yet been investigated extensively. Among the veterinary drugs, the antibiotics are an important group, due to their extensive use and their potential to also affect environmental bacteria. The main entry path for veterinary antibiotics into the environment is the application of manure of treated farm animals onto agricultural land. In this thesis, it was investigated whether antibiotics excreted with animal manure can affect soil bacteria. Antibiotic-related changes in soil bacterial communities were studied in microcosms.

The main technique applied is named pollution-induced community tolerance (PICT). PICT is based on the idea that the exposure to a toxicant leads to an increase in the tolerance of the community as a whole to that toxicant, e.g. by the disappearance of sensitive species. This tolerance increase, in turn, can be used to detect toxic effects of a substance. For PICT analyses with antibiotics based on substrate utilization in multiwell plates, it turned out that soil nutrient amendment (such as manuring) is essential.

Changes in the community composition can also become apparent by changes in the pattern of different organic substances the bacteria can respire (CLPP). In a methodological analysis, it was shown how CLPP tests could be improved by new calculation methods and practical test details. A third technique, based on the composition of the community DNA (16S rDNA DGGE), was applied to study the most numerous community members.

Three different classes of antibiotics were tested with these methods. The PICT method was able to demonstrate effects for all three classes, while both CLPP and DGGE failed to detect the effect of one antibiotic each. Different methods should thus be applied simultaneously in bacterial ecotoxicology. Further, tetracyclines were more toxic than sulfonamides and macrolides: the community doubled its tolerance at a concentration of 1.3 mg oxytetracycline per kg of soil. As tetracyclines have been detected in agricultural soils in significant concentrations, they are more likely to pose a risk than the other classes. It has further been investigated whether the specificity of PICT studies is limited by co-tolerance, thus an increase in tolerance to a toxicant the community was not exposed to. In this study, bacteria were only co-tolerant to very similar substances.

Antibiotic resistance was another research question addressed. It is not known to what extent antibiotic resistant bacteria prevail in the environment, and how that is linked with antibiotic use in humans and farm animals. In PCR analyses, resistance genes detected in pig manure were shown to be transferred to soils. However, this was found in microcosms, but not in field studies. The “background” of resistance at the field sites before manure application was already high, such that no effect of the manure application on the resistance gene diversity could be found. It is concluded that the relevance of antibiotic use and the concomitant resistance development in the environment cannot yet sufficiently be determined, but must be investigated further.