Studies of Pseudomonas aeruginosa and its virulence factors in a mouse model of dermal infection
Geerlings, Peter (2013) Studies of Pseudomonas aeruginosa and its virulence factors in a mouse model of dermal infection. PhD thesis, Murdoch University.
Pseudomonas aeruginosa is an opportunistic pathogen of hosts with impaired immune function or concurrent disease, most notably patients with Cystic Fibrosis and those who have been severely burned. It has an extensive array of virulence factors (VFs) which enable it to colonise and penetrate host tissue. It is responsible for a high percentage of nosocomial infections and is becoming increasingly antibiotic resistant, so there is an immediate need for novel treatments of severe P. aeruginosa infections. Following severe traumatic injury to the skin, the dermis and epidermis may be destroyed, leaving the fascia and endothelium of the vasculature as the final structural barriers protecting the circulation and preventing bacteraemia. Little has been reported on the mechanisms used by P. aeruginosa to penetrate the fascia and enter the circulation, because conventional animal models of dermal injury do not allow for quantitative study of bacterial pathogenesis and host responses. To address this, a novel transdermal chamber was designed, manufactured and implanted into mice allowing for repeated sampling and quantitative analysis of bacterial replication, VFs, and host responses. Despite the bacterial load in the chamber exudate being similar to normal control mice, mice immunosuppressed using cyclophosphamide (CP) had significantly higher levels of Exotoxin A (ETA), P. aeruginosa elastase (PAE) and Phospholipase C (PLC), and developed fatal bacteraemia when compared to immunocompetent mice.
It was hypothesised that raised levels of ETA and PAE contributed to the pathogenesis of P. aeruginosa in CP treated mice by increasing the permeability of the fascia and endothelium allowing bacteria to enter the circulation. To test this, purified ETA and PAE were added to the chambers of immunocompetent mice, followed by fluorescently labelled dextran to assess efflux measured as a change in dextran concentration in the chamber exudate over time. ETA caused sustained local depletion of leukocytes and fibroblasts, reduction in exudate volume and weight loss, but did not change the dextran concentration in the chamber exudate compared to control mice. PAE was proinflammatory, increased exudate volume, damaged fascial tissue, induced local haemorrhage, and resulted in a decrease in dextran concentration in the chamber compared to controls. Combining ETA and PAE in the chamber resulted in increased weight loss, haemoglobinaemia and increased accumulation of dextran in the local draining lymph nodes.
Preliminary trials using ovine anti-sera to P. aeruginosa antigens in infected chambers of CP-treated mice increased their survival. Collectively, these results show that the increased concentration of VFs, not bacterial load, facilitated bacterial translocation across the fascia and endothelium, and uncovers the underlying significance of the superficial fascia as a defensive barrier against infectious agents. Furthermore, this study suggests that neutrophils are crucial to reducing VF concentration, and that topically applied heterologous anti-P. aeruginosa antibodies may be useful in preventing fatal bacteraemia in severe dermal infections of susceptible patients.
|Publication Type:||Thesis (PhD)|
|Murdoch Affiliation:||School of Veterinary and Life Sciences|
|Supervisor:||Stumbles, Phil and Penhale, John|
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