Pathogenesis of Actinobacillus pleuropneumoniae : role of toxins and fimbriae

Actinobacillus pleuropneumoniae causes porcine pleuropneumonia, a disease that occurs world-wide and affects growing pigs of all ages. Infection of pigs with A. pleuropneumoniae can result in high morbidity and mortality. The present work contributes to the understanding of the pathogenesis of A. pleuropneumoniae by providing important novel insights into several major virulence traits of the pathogen. The RTX toxins ApxI, ApxII and ApxIII are essential for the development of clinical symptoms and lesions typical for A. pleuropneumoniae infections. However, most serotypes of A. pleuropneumoniae produce in vivo more than one toxin. To assess the relative contribution of ApxI and ApxII to the infection, the ability of different Apx mutants of A. pleuropneumoniae serotype 1 to induce lesions was investigated. The results indicate that the production of more than one toxin enhances virulence of A. pleuropneumoniae. Adherence to host tissue is regarded as an important first step in the colonisation of the host. In this thesis, we describe an in vitro infection model to study the interaction of A. pleuropneumoniae with primary cultures of porcine lung epithelial cells [LEC]. The efficient binding of A. pleuropneumoniae to LEC indicates that adherence to epithelial cells in the lower respiratory tract may constitute an important step in the pathogenesis. In search for the mechanism of the adherence event, the role of LPS was investigated. On the basis of the results with LEC, LPS seems not to be involved in adherence of A. pleuropneumoniae to cells of the lower respiratory tract. In an attempt to identify factors involved in adherence of A. pleuropneumoniae, we focussed on fimbriae. Fimbriae mediate adherence in a number of species and have been isolated recently from A. pleuropneumoniae. The genome of A. pleuropneumoniae was found to contain at least four genes involved in type IV fimbriae biogenesis. The functionality of the fimbriae genes was demonstrated by expression of these genes from a constitutively active promoter, which resulted in fimbrial subunits and in fimbriae protruding from the bacterial cell surface. The involvement of fimbriae in adherence to LEC could not be demonstrated. Noteworthy is the presence of an alanine in contrast to the consensus glycine at position -1 from the cleavage site of the fimbrial subunit protein ApfA. This appears to be an intrinsic trait of ApfA. The apparent lack of fimbriae expression under routine laboratory growth conditions led us to investigate the conditions necessary for fimbriae promoter activity using a promoter trap vector. The fimbriae promoter appears to be intact but is subject to regulation. Expression of the fimbriae promoter was dependent on the growth phase, was seen only in chemically defined medium and was strongly induced in A. pleuropneumoniae that were adhering to primary cultures of LEC. The latter suggests that contact with epithelial cells may be a trigger for fimbriae production. The importance of a more natural setting for the induction of fimbriae promoter activity was further underlined by our finding that the fimbriae promoter was active in vivo after endobronchial inoculation of pigs

Response to Letter to the Editor: 'Human skin allografts: Storage medium at 4 degrees C and viability'

Trauma Surger

Evaluation of saline, RPMI and DMEM/F12 for storage of split-thickness skin grafts

Radiolog

Decontamination of burn wounds using a cold atmospheric pressure plasma jet

Decontamination of burn wounds using a cold atmospheric pressure plasma jet C.A.J. van Gils, S. Hofmann, B. Boekema and P. Bruggeman Eindhoven University of Technology, Department of Applied Physics, group EPG, P.O. Box 513, 5600 MB Eindhoven In the treatment of burn wounds bacterial infections are a common problem. Next to conventional treatments, atmospheric pressure plasmas might be able to provide additional means to reduce the bacterial concentration in a burn wound. In this work the effects of an argon atmospheric pressure plasma jet on both the gram negative bacterium pseudomonas aeruginosa and human keratinocytes and fibroblasts are investigated. Pseudomonas aeruginosa is a common cause of infection in burn wound patients. Keratinocytes are the predominant cells in the epidermis and fibroblasts are critical for wound healing. The influence of discharge power, duty cycle, treatment time and the addition of air on the survivability of the bacteria and cells is determined for two different electrode geometries. To be able to directly treat tissue the gas temperature has to be near room temperature and the UV production has to be limited. The gas temperature in the plasma as well as outside the plasma is estimated from the rotational temperature of the rotational bands of OH(A-X) and N2(C-B) and thermocouple measurements respectively. Also the relative UV generated by the plasma is measured

Decontamination of burn wounds using a cold atmospheric pressure plasma jet

Decontamination of burn wounds using a cold atmospheric pressure plasma jet C.A.J. van Gils, S. Hofmann, B. Boekema and P. Bruggeman Eindhoven University of Technology, Department of Applied Physics, group EPG, P.O. Box 513, 5600 MB Eindhoven In the treatment of burn wounds bacterial infections are a common problem. Next to conventional treatments, atmospheric pressure plasmas might be able to provide additional means to reduce the bacterial concentration in a burn wound. In this work the effects of an argon atmospheric pressure plasma jet on both the gram negative bacterium pseudomonas aeruginosa and human keratinocytes and fibroblasts are investigated. Pseudomonas aeruginosa is a common cause of infection in burn wound patients. Keratinocytes are the predominant cells in the epidermis and fibroblasts are critical for wound healing. The influence of discharge power, duty cycle, treatment time and the addition of air on the survivability of the bacteria and cells is determined for two different electrode geometries. To be able to directly treat tissue the gas temperature has to be near room temperature and the UV production has to be limited. The gas temperature in the plasma as well as outside the plasma is estimated from the rotational temperature of the rotational bands of OH(A-X) and N2(C-B) and thermocouple measurements respectively. Also the relative UV generated by the plasma is measured

SPS-neutralization in tissue samples for efficacy testing of antimicrobial peptides

Background: Accurate determination of the efficacy of antimicrobial agents requires neutralization of residual antimicrobial activity in the samples before microbiological assessment of the number of surviving bacteria. Sodium polyanethol sulfonate (SPS) is a known neutralizer for the antimicrobial activity of aminoglycosides and polymyxins. In this study, we evaluated the ability of SPS to neutralize residual antimicrobial activity of antimicrobial peptides [SAAP-148 and pexiganan; 1% (wt/v) in PBS], antibiotics [mupirocin (Bactroban) and fusidic acid (Fucidin) in ointments; 2% (wt/wt))] and disinfectants [2% (wt/wt) silver sulfadiazine cream (SSD) and 0.5% (v/v) chlorhexidine in 70% alcohol].Methods: Homogenates of human skin models that had been exposed to various antimicrobial agents for 1 h were pipetted on top of Methicillin-resistant Staphylococcus aureus (MRSA) on agar plates to determine whether the antimicrobial agents display residual activity.To determine the optimal concentration of SPS for neutralization, antimicrobial agents were mixed with PBS or increasing doses of SPS in PBS (0.05-1% wt/v) and then 105 colony forming units (CFU)/mL MRSA were added. After 30min incubation, the number of viable bacteria was assessed. Next, the in vitro efficacy of SAAP-148 against various gram-positive and gram-negative bacteria was determined using PBS or 0.05% (wt/v) SPS immediately after 30 min incubation of the mixture. Additionally, ex vivo excision wound models were inoculated with 105 CFU MRSA for 1 h and exposed to SAAP-148, pexiganan, chlorhexidine or PBS for 1 h. Subsequently, samples were homogenized in PBS or 0.05% (wt/v) SPS and the number of viable bacteria was assessed.Results: All tested antimicrobials displayed residual activity in tissue samples, resulting in a lower recovery of surviving bacteria on agar. SPS concentrations at >= 0.05% (wt/v) were able to neutralize the antimicrobial activity of SAAP-148, pexiganan and chlorhexidine, but not of SSD, Bactroban and Fucidin. Finally, SPS-neutralization in in vitro and ex vivo efficacy tests of SAAP-148, pexiganan and chlorhexidine against gram-positive and gram-negative bacteria resulted in significantly higher numbers of CFU compared to control samples without SPS-neutralization.Conclusions: SPS was successfully used to neutralize residual activity of SAAP-148, pexiganan and chlorhexidine and this prevented an overestimation of their efficacy.Immunogenetics and cellular immunology of bacterial infectious disease