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Pawel Tulinski

Thursday 12 September 2013

Molecular and ecological aspects of MRSA ST398 colonization in pigs

Promotor: Prof. dr. Jaap Wagenaar
Defence: 12 September 2013

The aim of this thesis was to extend our knowledge about MRSA ST398 colonization in pigs and to focus on the ecology of MRSA ST398 on pig farms. For these reasons this thesis has been divided into two parts. The first part describes the development and application of an ex vivo model to study MRSA colonization. The second part focuses on unanswered questions about 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.

Ex vivo model

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 bacterial inoculation that may result in death of the animal. A suitable alternative system to gain better understanding of nasal colonization is the use of porcine nasal mucosa explants in which bacterial and host factors can be evaluated under controlled conditions. Chapter 2 describes porcine nasal mucosa explants as a novel tool to study MRSA ST398 colonization in pigs. The cultivation of the nasal mucosa explants resulted in lack of changes in morphology and viability during cultivation of the tissue for 72h. Moreover, nasal mucosa explants were inoculated with three MRSA ST398 strains isolated from a pig carrier (S0462) and a human patient (S0385-1 and S0385-2). MRSA S0385-2 strain was a laboratory variant of S0835-1 with a phage integrated in the beta-hemolysin gene (hlb), which resulted in a lack of lysis of red blood cells. Three different colonization patterns were observed. One strains (S0462) showed significant increase of the number of CFU, isolate S0385-1 showed stable bacterial number and isolate S0385-2 showed decline of bacteria numbers. These observations suggest that different strains utilize distinct mechanisms during their interaction with nasal tissue. Moreover, visualization of the explants during MRSA colonization showed no tissue damage during MRSA colonization. Remarkable is the fact that all MRSA ST398 isolates initially adhered to the epithelium with similar numbers of cells (around 106 CFU/cm2). We hypothesized that initial adherence of bacterial cells to the epithelium is not essential for maintenance of colonization. Next, we investigated the global gene changes during ex vivo colonization of MRSA ST398. Chapter 3 describes the study on the global gene expression pattern of MRSA ST398 during ex vivo colonization. The microarray data showed that genes involved mainly in metabolic processes were up- or down-regulated during experimental colonization while 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. This study showed that mechanisms responsible for successful colonization of MRSA ST398 in pigs are multifactorial. More extensive studies, e.g., based on proteome analysis, are needed to fully understand the molecular mechanism responsible for successful colonization of MRSA ST398 in livestock.

Livestock are a potential MRSA reservoir for humans. Eradication of MRSA ST398 from livestock will reduce the source of transmission of MRSA ST398 transmission from animals to humans. Until now, there is no such method available to limit MRSA colonization in livestock. Phage therapy is a possible alternative treatment, which may be applied to livestock. Chapter 4 illustrates the first example to phage therapy in pigs. Bacteriophages were able to kill bacteria in vitro, nevertheless this effect of the bacteriophages could not be observed in the animal experiment. Similar results were obtained in an ex vivo experiment using the ex vivo nasal explants model. . In both settings, application of muporicin resulted in an almost complete reduction of MRSA. The reason as to why bacteriophages were not able to limit MRSA colonization in animals and ex vivo is unclear. One of the reasons may be differences in S. aureus surface protein expression which may limit the accessibility for bacteriophage to target the bacterial cell surface. Additionally, Chapter 4 shows that the ex vivo model is a powerful tool not only to study S. aureus colonization in pigs, but can also be applied in intervention studies. Moreover, the use of the explants model in the preliminary screening of new decolonization treatments would help to reduce the number of experimental animals required.

In summary of the first part of this thesis, in the ex vivo models the normal cell-cell contacts, the cell-extracellular matrix contacts and the three-dimensional structure of the tissue are preserved. The nasal mucosa explant model can be further applied to various studies in order to investigate the complexity of bacterial colonization in pigs as well as in other animals. Furthermore, the ex vivo model can be used as a platform to screen new treatment strategies both in veterinary and public health settings.

Ecology of MRSA ST398

The generation of novel MRSA strains in the environment is speculative. It is assumed that CNS found in the environment could serve as a source for SCCmec for the development of new MRSA strains. However, this observation was obtained from SCCmec typing in CNS isolated from various animals and it was not compared with MRSA isolated from the same niche. Chapter 5 described the first study of t Staphylococcus species harboring SCCmec on pig farms. A total of 4 SCCmec types (III, IV, V and ND) and 3 subtypes of SCCmec type IV (IVa, IVc and IVvar) were identified in 10 staphylococcal species. This study demonstrated that a reservoir of mecA-positive CNS coincides with S. aureus on pig farms. Moreover, the presence of the same SCCmec types among CNS and S. aureus supports the hypothesis that on pig farms CNS may act as a reservoir for the exchange of SCCmec.

This study revealed the importance of monitoring not only the prevalence of MRSA but also the companion staphylococcal flora as a reservoir of antibiotic resistance genes, which may be transferred between staphylococcal species.

A second unanswered question in the ecology of MRSA ST398 is the possible relation between antibiotic usage and prevalence of MRSA ST398 on pig farms. It has been suggested that antibiotic consumption in livestock is linked with high prevalence of MRSA ST398 in livestock. However, a risk factor analysis investigated on conventional pig farms in The Netherlands did not find a significant association between antibiotic consumption on the farm and MRSA prevalence. The study presented in Chapter 6 describes the prevalence of MRSA on organic pig farms in The Netherlands. In comparison to conventional farms in The Netherlands, the average antibiotic usage on the organic farms is on average 70% lower. Moreover, the prevalence of MRSA on organic pig herds was also lower when compared with conventional herds (21% vs. 71%). However, the correlation between antibiotic consumption on the farms and prevalence of MRSA was also not clear in our study. The data presented in Chapter 6 suggest that different farm management can be responsible for a lower MRSA prevalence on organic farms when compared with conventional farms.

In summary, the second part of this thesis (Chapter 5 and 6) manifests the beginning of our understanding of the ecology of MRSA ST398 on pig farms, the presence of staphylococci on pig farms and the potential influence of different types of farm management in the development of novel MRSA ST398 in livestock. The global understanding of the ecology of MRSA ST398 may be helpful to develop and implement new strategies to limit possible transfer of MRSA from livestock to humans. Identification of the molecular mechanisms behind HGT of SCCmec between staphylococci needs to be unraveled and remains a challenge.