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

Burn-Induced Local and Systemic Immune Response: Systematic Review and Meta-Analysis of Animal Studies

Contains fulltext : 284423.pdf (Publisher’s version ) (Open Access

Considerations in designing and testing plasma devices for medical applications

Cold atmospheric plasma has been shown to have great potential for many applications on or in the human body, such as disinfection, wound healing, and cancer treatment. Several medical plasma devices have been developed and have obtained CE certification. However, the route there and into clinical practice is not straightforward. Already during the design stage, many matters need to be taken into account: (1) application and user requirements, (2) medical device regulations, e.g. on electrical safety and electromagnetic compatibility, and (3) production-related issues. Subsequent research should be conducted using a setup that resembles the clinical situation as closely as possible, since seemingly insignificant factors can have a large influence on the plasma and its effects. To ensure that the CE marked device will actually be adopted in clinical practice requires further actions during the research and development process: the demands and concerns of all parties directly and indirectly involved in its use should be identified and at least the crucial parties should be acquainted with plasma medicine and the specific medical device. Some examples will be given from the R&D process of a new flexible volume Dielectric Barrier Discharge (vDBD)

Considerations in designing and testing plasma devices for medical applications

Cold atmospheric plasma has been shown to have great potential for many applications on or in the human body, such as disinfection, wound healing, and cancer treatment. Several medical plasma devices have been developed and have obtained CE certification. However, the route there and into clinical practice is not straightforward. Already during the design stage, many matters need to be taken into account: (1) application and user requirements, (2) medical device regulations, e.g. on electrical safety and electromagnetic compatibility, and (3) production-related issues. Subsequent research should be conducted using a setup that resembles the clinical situation as closely as possible, since seemingly insignificant factors can have a large influence on the plasma and its effects. To ensure that the CE marked device will actually be adopted in clinical practice requires further actions during the research and development process: the demands and concerns of all parties directly and indirectly involved in its use should be identified and at least the crucial parties should be acquainted with plasma medicine and the specific medical device. Some examples will be given from the R&D process of a new flexible volume Dielectric Barrier Discharge (vDBD)

Review: Lessons Learned From Clinical Trials Using Antimicrobial Peptides (AMPs)

Antimicrobial peptides (AMPs) or host defense peptides protect the host against various pathogens such as yeast, fungi, viruses and bacteria. AMPs also display immunomodulatory properties ranging from the modulation of inflammatory responses to the promotion of wound healing. More interestingly, AMPs cause cell disruption through non-specific interactions with the membrane surface of pathogens. This is most likely responsible for the low or limited emergence of bacterial resistance against many AMPs. Despite the increasing number of antibiotic-resistant bacteria and the potency of novel AMPs to combat such pathogens, only a few AMPs are in clinical use. Therefore, the current review describes (i) the potential of AMPs as alternatives to antibiotics, (ii) the challenges toward clinical implementation of AMPs and (iii) strategies to improve the success rate of AMPs in clinical trials, emphasizing the lessons we could learn from these trials

Antibacterial plasma at safe levels for skin cells

Plasmas produce various reactive species, which are known to be very effective in killing bacteria. Plasma conditions, at which efficient bacterial inactivation is observed, are often not compatible with leaving human cells unharmed. The purpose of this study was to determine plasma settings for inactivation of Pseudomonas aeruginosa, without damaging skin cells in vitro under the same treatment conditions. An RF argon plasma jet excited with either continuous or time modulated (20 kHz, 20% duty cycle) voltages was used. To compare these two operation modes, only the input voltage was adjusted in order to obtain the same average power (1.7 W) for both modes. All other settings, i.e. gas flow, distance plasma tip to liquid surface, were kept constant. Bacteria or skin cells in physiological salt solution were exposed to direct non-contact plasma treatments. Short plasma treatments of up to 2 min resulted in a high reduction of bacterial numbers and did not affect dermal fibroblasts or keratinocytes. Bacterial inactivation has been previously ascribed to peroxynitrite, nitrite and H2O2 while eukaryotic cell viability is proposed to be reduced in the long term by the presence of H2O2 and is less affected by reactive nitrogen species. The remote RF plasma jet treatment was highly effective for bacterial inactivation while skin cell viability was preserved

Safety and bactericidal efficacy of cold atmospheric plasma generated by a flexible surface Dielectric Barrier Discharge device against Pseudomonas aeruginosa in vitro and in vivo

BACKGROUND: Cold atmospheric plasma (CAP), which is ionized gas produced at atmospheric pressure, could be a novel and potent antimicrobial therapy for the treatment of infected wounds. Previously we have shown that CAP generated with a flexible surface Dielectric Barrier Discharge (sDBD) is highly effective against bacteria in vitro and in ex vivo burn wound models. In the current paper, we determined the in vitro and in vivo safety and efficacy of CAP generated by this sDBD device. METHODS: The effect of CAP on DNA mutations of V79 fibroblasts was measured using a hypoxanthine-guanine-phosphoribosyltransferase (HPRT) assay. Furthermore, effects on cell proliferation, apoptosis and DNA damage in ex vivo burn wound models (BWMs) were assessed using immunohistochemistry. Next, 105 colony forming units (CFU) P. aeruginosa strain PAO1 were exposed to CAP in a 3D collagen-elastin matrix environment to determine the number of surviving bacteria in vitro. Finally, rat excision wounds were inoculated with 107 CFU PAO1 for 24 h. The wounds received a single CAP treatment, repeated treatments on 4 consecutive days with CAP, 100 µL of 1% (wt/wt) silver sulfadiazine or no treatment. Wound swabs and punch biopsies were taken to determine the number of surviving bacteria. RESULTS: Exposure of V79 fibroblasts to CAP did not increase the numbers of mutated colonies. Additionally, the number of proliferative, apoptotic and DNA damaged cells in the BWMs was comparable to that of the unexposed control. Exposure of PAO1 to CAP for 2 min resulted in the complete elimination of bacteria in vitro. Contrarily, CAP treatment for 6 min of rat wounds colonized with PAO1 did not effectively reduce the in vivo bacterial count. CONCLUSIONS: CAP treatment was safe but showed limited efficacy against PAO1 in our rat wound infection model