Schlüsselwort: MALDI‐TOF

Comparative epidemiological and molecular biological analysis of methicillin-resistent Staphylococcus aureus (MRSA) from livestock and its environment Within the scope of this present study, selected samples which were isolated in the course of a partial project between the Free University Berlin and the Veterinary University Hannover from 2009 to 2011, have been further investigated. The objective of that previous study was to comprehensively determine the occurrence of MRSA in barn air and housing environment in animal husbandries of different animal species in Germany. To estimate an appropriate transfer risk of airborne MRSA from livestock to other animal populations or local residents, field studies have been performed in conventional pig and poultry farms. Within the following thesis the emission of MRSA from barn air to the vicinity of the farms should be proven by using molecular biological analysis. Furthermore, a differentiated evaluation regarding to the tenacity and pathogenicity of transmitted and sedimented MRSA should be performed. Finally, results of macrorestriction analysis were compared to an approach of using protein spectra obtained by MALDI-TOF-MS for molecular epidemiology. Overall 238 selected MRSA isolates originating from four fattening pig farms and two breeding pig farms as well as two broiler and four turkey fattening farms were further investigated. The spa- typing demonstrated that in pig barns and its environment the spa-types t011, t108 and t1451 were identified most frequently, whereas t011, t5452 and t034 dominated in the inside and outside of the turkey farms respectively t1430 in the broiler farms. The majority of the selected isolates (87%) were assigned to clonal complex 398. All so-called non-CC398 strains (13%) were assigned to sequence type ST9 or ST5. A simultaneous detection of the same MRSA spa types inside the barns and within the whole farm confirmed the hypothesis of the previous study that an emission via the airborne route has been occured. The clonal analysis was performed by Cfr9I pulsed-field gel electrophoresis. Therefore 124 MRSA isolates (60 from pig farms, 34 from broiler farms and 30 from turkey farms) were chosen. The results demonstrated the presence of MRSA clones in the barn air and housing environment of investigated animal husbandrys. In addition, MRSA clones could also be detected in air samples from inside as well as from outside on the downwind site up to a distance to 50m and 150m, respectively. Because air sampling was performed simultaneously we can assume a proven airborne transmission of MRSA from livestock. In order to compare transmitted and sedimented MRSA under changing environmental conditions, pig isolates from summer and winter samplings were investigated. As a result, MRSA clones were detected in samples from winter and the following summer at identical distances. This confirms the hypothesis of the previous study that sedimented MRSA strains show high tenacity in the environment by surviving longer periods on soil surfaces. Additionally, a series of 38 samples were selected for microarray analysis. Investigated MRSA strains displayed only a low presence of virulence associated genes. Resistance genes for beta-lactam antimicrobials were detected in all isolates. Genes mediating resistance to other antimicrobial classes were also frequently detected. Furthermore, the Panton-Valentine Leukozidin gene and the Toxic Shock Syndrome Toxin gene could not be identified in any of the positive MRSA strains. While all investigated MRSA strains might be able to form bacterial biofilms, genes for staphylococcal entertoxins were found in only 31,6 % of the investigated isolates. However, the detection of antibiotic resistance genes or genes encoding for SE does not mean that appropriate genes will be expresses and own a pathogenic potential for humans and animals Finally protein mass spectra obtained by MALDI-TOF-MS and macrorestriction analysis were compared with each other in regard of their usability for molecular epidemiology. Phylogenetics on the basis of PFGE analysis is widely accepted. Cluster analysis of protein spectra differed substantially from the widly PFGE ones. For this reason using MALDI-TOF-MS for typing MRSA for epidemiology purpose is not recommended yet. However, further investigations on this topic seem to be necessary and useful.