Molecular and ecological aspects of MRSA ST398 colonization in pigs

Abstract

The aim of this thesis was to understand the successful colonization of MRSA ST398 in pigs and the factors that may contribute to this colonization. The first part describes the development and application of an ex vivo pig nasal mucosa explants model to study MRSA colonization. The second part studies the ecology of MRSA ST398, especially with respect to the possible relationship between staphylococcal flora on the generation of MRSA ST398 in the farm environment and the influence of organic husbandry on MRSA ST398 prevalence in pig farms. Pig animal models have been used to study colonization in pigs. However, these models have some disadvantages such as unstable colonization, limited bacterial detection as well as infection that may result in death of the animal. In this study the nasal mucosa explants model was investigated as a novel tool to study MRSA ST398 colonization in pigs. The cultivation of the nasal mucosa explants did not show changes in morphology and viability during cultivation of the tissue for 72h. Nasal mucosa explants, inoculated with three MRSA ST398 strains isolated from a pig carrier (S0462) and a human patient (S0385-1 and S0385-2) showed three different colonization patterns. One strains (S0462) showed significant increase of the number of bacteria, strain S0385-1 showed stable colonization whereas strain S0385-2 showed a decline of bacteria. Visualization of the explants during MRSA colonization showed no tissue damage during MRSA colonization. Next, we investigated the global gene changes during ex vivo colonization of MRSA ST398. Microarray data showed that genes involved mainly in metabolic processes were up- or down-regulated during experimental colonization while most genes encoding virulence factors were down-regulated during ex vivo colonization. Two genes were selected to study their potential contribution to maintenance of colonization: vwbp and scpA. Unfortunately, single knockout mutant strains did not show any phenotypic differences in the colonization pattern ex vivo, which indicates that these selected genes, do not play a crucial role in maintenance of colonization ex vivo. Phage therapy as potential intervention strategy was studied in vivo and in the ex vivo model but no reduction of MRSA numbers was observed. In both settings, application of muporicin resulted in an almost complete reduction of MRSA. In conclusion, the ex vivo model is a suitable tool not only to study S. aureus colonization in pigs, but can also be applied for preliminary screening of new decolonization treatments and would help to reduce the number of experimental animals. In the second part of this thesis it was shown that methicillin-resistant coagulase-negative staphylococci on pig farms may act as a source for development of new MRSA variants. On biological pig farms the number of MRSA was lower which could be due to different types of farm management. The global understanding of the ecology of MRSA ST398 is helpful to develop and implement new strategies to limit possible transfer of MRSA from livestock to humans.