Hannah Trøstrup

Polymorphonuclear Leukocytes Restrict Growth of Pseudomonas aeruginosa in the Lungs of Cystic Fibrosis Patients

ABSTRACT Cystic fibrosis (CF) patients have increased susceptibility to chronic lung infections by Pseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate the in vivo growth physiology of P. aeruginosa within lungs of chronically infected CF patients. A novel, quantitative peptide nucleic acid (PNA) fluorescence in situ hybridization (PNA-FISH)-based method was used to estimate the in vivo growth rates of P. aeruginosa directly in lung tissue samples from CF patients and the growth rates of P. aeruginosa in infected lungs in a mouse model. The growth rate of P. aeruginosa within CF lungs did not correlate with the dimensions of bacterial aggregates but showed an inverse correlation to the concentration of polymorphonuclear leukocytes (PMNs) surrounding the bacteria. A growth-limiting effect on P. aeruginosa by PMNs was also observed in vitro, where this limitation was alleviated in the presence of the alternative electron acceptor nitrate. The finding that P. aeruginosa growth patterns correlate with the number of surrounding PMNs points to a bacteriostatic effect by PMNs via their strong O2 consumption, which slows the growth of P. aeruginosa in infected CF lungs. In support of this, the growth of P. aeruginosa was significantly higher in the respiratory airways than in the conducting airways of mice. These results indicate a complex host-pathogen interaction in chronic P. aeruginosa infection of the CF lung whereby PMNs slow the growth of the bacteria and render them less susceptible to antibiotic treatment while enabling them to persist by anaerobic respiration.

Pseudomonas aeruginosa biofilm aggravates skin inflammatory response in BALB/c mice in a novel chronic wound model

Chronic wounds are presumed to persist in the inflammatory state, preventing healing. Emerging evidence indicates a clinical impact of bacterial biofilms in soft tissues, including Pseudomonas aeruginosa (PA) biofilms. To further investigate this, we developed a chronic PA biofilm wound infection model in C3H/HeN and BALB/c mice. The chronic wound was established by an injection of seaweed alginate‐embedded P. aeruginosa PAO1 beneath a third‐degree thermal lesion providing full thickness skin necrosis, as in human chronic wounds. Cultures revealed growth of PA, and both alginate with or without PAO1 generated a polymorphonuclear‐dominated inflammation early after infection. However, both at days 4 and 7, there were a more acute polymorphonuclear‐dominated and higher degree of inflammation in the PAO1 containing group (p < 0.05). Furthermore, PNA‐FISH and supplemented DAPI staining showed bacteria organized in clusters, resembling biofilms, and inflammation located adjacent to the PA. The chronic wound infection showed a higher number of PAO1 in the BALB/c mice at day 4 after infection as compared to C3H/HeN mice (p < 0.006). In addition, a higher concentration of interleukin‐1beta in the chronic wounds of BALB/c mice was observed at day 7 (p < 0.02), despite a similar number of bacteria in the two mouse strains. The present study succeeded in establishing a chronic PA biofilm infection in mice. The results showed an aggravating impact of local inflammation induced by PA biofilms. In conclusion, our findings indicate that improved infection control of chronic wounds reduces the inflammatory response and may improve healing.

What Is New in the Understanding of Non Healing Wounds Epidemiology, Pathophysiology, and Therapies

Chronic wounds are a growing socioeconomic problem in the western world. Knowledge on recalcitrant wounds relies on in vitro studies or clinical observations, and there is emerging evidence on the clinical impact of bacterial biofilm on skin healing. Chronic wounds are locked in the inflammatory state of wound healing, and there are multiple explanations for this arrest with the theory of exaggerated proteolysis as the most commonly accepted. Previously, there has not been enough focus on the different etiologies of chronic wounds compared to acute, healing wounds. There is an urgent need to group chronic wounds by its cause when searching for possible diagnostic or therapeutic targets. Good wound management should therefore consist of recognition of basic wound etiology, irrigation, and debridement in order to reduce microbial and necrotic load, frequently changed dressings, and appropriate antimicrobial and antibiofilm strategies based on precise diagnosis. Representative sampling is required for diagnosis and antimicrobial treatment of wounds. The present review aims at describing the impact of biofilm infections on wounds in relation to diagnosing, treatment strategies, including experimentally adjuvant approaches and animal models.

Bead-size directed distribution of Pseudomonas aeruginosa results in distinct inflammatory response in a mouse model of chronic lung infection

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by biofilms, tolerant to antibiotics and host responses. Instead, immune responses contribute to the tissue damage. However, this may depend on localization of infection in the upper conductive or in the peripheral respiratory zone. To study this we produced two distinct sizes of small alginate beads (SB) and large beads (LB) containing P. aeruginosa. In total, 175 BALB/c mice were infected with either SB or LB. At day 1 the quantitative bacteriology was higher in the SB group compared to the LB group (P < 0·003). For all time‐points smaller biofilms were identified by Alcian blue staining in the SB group (P < 0·003). Similarly, the area of the airways in which biofilms were identified were smaller (P < 0·0001). A shift from exclusively endobronchial to both parenchymal and endobronchial localization of inflammation from day 1 to days 2/3 (P < 0·05), as well as a faster resolution of inflammation at days 5/6, was observed in the SB group (P < 0·03). Finally, both the polymorphonuclear neutrophil leucocyte (PMN) mobilizer granulocyte colony‐stimulating factor (G‐CSF) and chemoattractant macrophage inflammatory protein‐2 (MIP‐2) were increased at day 1 in the SB group (P < 0·0001). In conclusion, we have established a model enabling studies of host responses in different pulmonary zones. An effective recognition of and a more pronounced host response to infection in the peripheral zones, indicating that increased lung damage was demonstrated. Therefore, treatment of the chronic P. aeruginosa lung infection should be directed primarily at the peripheral lung zone by combined intravenous and inhalation antibiotic treatment.

The phagocytic fitness of Leucopatches may impact the healing of chronic wounds

Chronic non‐healing wounds are significantly bothersome to patients and can result in severe complications. In addition, they are increasing in numbers, and a challenging problem to the health‐care system. Handling of chronic, non‐healing wounds can be discouraging due to lack of improvement, and a recent explanation can be the involvement of biofilm infections in the pathogenesis of non‐healing wounds. Therefore, new treatment alternatives to improve outcome are continuously sought‐after. Autologous leucopatches are such a new, adjunctive treatment option, showing promising clinical effects. However, the beneficial effect of the patches are not understood fully, although a major contribution is believed to be from the release of stimulating growth factors from activated thrombocytes within the leucopatch. Because the leucopatches also contain substantial numbers of leucocytes, the aim of the present study was to investigate the activity of the polymorphonuclear neutrophils (PMNs) within the leucopatch. By means of burst assay, phagocytosis assay, migration assay, biofilm killing assay and fluorescence in‐situ hybridization (FISH) assay we showed significant respiratory burst in PMNs, active phagocytosis and killing of Pseudomonas aeruginosa by the leucopatch. In addition, bacterial‐induced migration of PMNs from the leucopatch was shown, as well as uptake of P. aeruginosa by PMNs within the leucopatch. The present study substantiated that at least part of the beneficial clinical effect in chronic wounds by leucopatches is attributed to the activity of the PMNs in the leucopatch.

Chronic urinary tract infections in patients with spinal cord lesions - biofilm infection with need for long-term antibiotic treatment

Patients suffering from spinal cord injuries resulting in complete or incomplete paraplegia or tetraplegia are highly disposed to frequent, recurrent or even chronic urinary tract infections (UTIs). The reason for the increased risk of acquiring UTIs is multifactorial, including reduced sensation of classical UTI symptoms, incomplete bladder emptying, frequent catheterizations or chronic urinary tract catheters. Biofilms in relation to UTIs have been shown both on catheters, on concrements or as intracellular bacterial communities (IBCs). Due to the increased risk of acquiring recurrent or chronic UTIs and frequent antibiotic treatments, patients experience an increased risk of being infected with antibiotic‐resistant bacteria like extended‐spectrum β‐lactamase–producing Escherichia coli or Klebsiella spp., but also bacteria like Pseudomonas aeruginosa inherently resistant to several antibiotics. Diagnosing the UTI can also be challenging, especially distinguishing harmless colonization from pathogenic infection. Based on a previous study showing activation of humoral immune response toward UTI pathogens in patients with spinal cord lesions (SCL), the present mini review is an evaluation of using antibody response as an indicator of chronic biofilm UTI. In addition, we evaluated the effect of long‐term treatment with antibiotics in patients with SCLs and chronic UTI, defined by culturing of a uropathogen in the urine and elevated specific precipitating antibodies against the same uropathogen in a blood sample. Elimination of chronic UTI, decrease in specific precipitating antibody values and avoiding selection of new multidrug‐resistant (MDR) uropathogens were the primary markers for effect of treatment. The results of this evaluation suggest that the long‐term treatment strategy in SCL patients with chronic UTI may be effective; however, randomized prospective results are needed to confirm this.

Hyperbaric oxygen therapy augments tobramycin efficacy in experimental Staphylococcus aureus endocarditis

Staphylococcus aureus infective endocarditis (IE) is a serious disease with an in-hospital mortality of up to 40%. Improvements in the effects of antibiotics and host responses could potentially benefit outcomes. Hyperbaric oxygen therapy (HBOT) represents an adjunctive therapeutic option. In this study, the efficacy of HBOT in combination with tobramycin in S. aureus IE was evaluated. A rat model of S. aureus IE mimicking the bacterial load in humans was used. Infected rats treated subcutaneously with tobramycin were randomised into two groups: (i) HBOT twice daily (n = 13); or (ii) normobaric air breathing (non-HBOT) (n = 17). Quantitative bacteriology, cytokine expression, valve vegetation size and clinical status were assessed 4 days post-infection. Adjunctive HBOT reduced the bacterial load in the aortic valves, myocardium and spleen compared with the non-HBOT group (P = 0.004, <0.001 and 0.01, respectively) and improved the clinical score (P <0.0001). Photoplanimetric analysis and weight of valve vegetations showed significantly reduced vegetations in the HBOT group (P <0.001). Key pro-inflammatory cytokines [IL-1β, IL-6, keratinocyte-derived chemokine (KC) and vascular endothelial growth factor (VEGF)] were significantly reduced in valves from the HBOT group compared with the non-HBOT group. In conclusion, HBOT augmented tobramycin efficacy as assessed by several parameters. These findings suggest the potential use of adjunctive therapy in severe S. aureus IE.

Animal models of chronic wound care: the application of biofilms in clinical research

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Pseudomonas aeruginosa biofilm hampers murine central wound healing by suppression of vascular epithelial growth factor: Pseudomonas aeruginosa biofilms in wounds

Biofilm‐infected wounds are clinically challenging. Vascular endothelial growth factor and host defence S100A8/A9 are crucial for wound healing but may be suppressed by biofilms. The natural course of Pseudomonas aeruginosa biofilm infection was compared in central and peripheral zones of burn‐wounded, infection‐susceptible BALB/c mice, which display delayed wound closure compared to C3H/HeN mice. Wounds were evaluated histopathologically 4, 7 or 10 days post‐infection. Photoplanimetry evaluated necrotic areas. P. aeruginosa biofilm suppressed vascular endothelial growth factor levels centrally in BALB/c wounds but increased peripheral levels 4–7 days post‐infection. Central zones of the burn wound displayed lower levels of central vascular endothelial growth factor as observed 4 and 7 days post‐infection in BALB/c mice compared to their C3H/HeN counterparts. Biofilm suppressed early, centrally located S100A8/A9 in BALB/c and centrally and peripherally later on in C3H/HeN wounds as compared to uninfected mice. Peripheral polymorphonuclear‐dominated inflammation and larger necrosis were observed in BALB/c wounds. In conclusion, P. aeruginosa biofilm modulates wounds by suppressing central, but inducing peripheral, vascular endothelial growth factor levels and reducing host response in wounds of BALB/c mice. This suppression is detrimental to the resolution of biofilm‐infected necrosis.

Immune Modulating Topical S100A8/A9 Inhibits Growth of Pseudomonas aeruginosa and Mitigates Biofilm Infection in Chronic Wounds

Pseudomonas aeruginosa biofilm maintains and perturbs local host defense, hindering timely wound healing. Previously, we showed that P. aeruginosa suppressed S100A8/A9 of the murine innate host defense. We assessed the potential antimicrobial effect of S100A8/A9 on biofilm-infected wounds in a murine model and P. aeruginosa growth in vitro. Seventy-six mice, inflicted with a full-thickness burn wound were challenged subcutaneously (s.c.) by 106 colony-forming units (CFUs) of P. aeruginosa biofilm. Mice were subsequently randomized into two treatment groups, one group receiving recombinant murine S100A8/A9 and a group of vehicle controls (phosphate-buffered saline, PBS) all treated with s.c. injections daily for up to five days. Wounds were analyzed for quantitative bacteriology and contents of key inflammatory markers. Count of blood polymorphonuclear leukocytes was included. S100A8/A9-treatment ameliorated wound infection, as evaluated by quantitative bacteriology (p ≤ 0.05). In vitro, growth of P. aeruginosa was inhibited dose-dependently by S100A8/A9 in concentrations from 5 to 40 μg/mL, as determined by optical density-measurement (OD-measurement) and quantitative bacteriology. Treatment slightly augmented key inflammatory cytokine Tumor Necrosis Factor-α (TNF-α), but dampened interferon-γ (IFN-γ) levels and blood polymorphonuclear count. In conclusion, topical S100A8/A9 displays remarkable novel immune stimulatory and anti-infective properties in vivo and in vitro. Importantly, treatment by S100A8/A9 provides local infection control. Implications for a role as adjunctive treatment in healing of chronic biofilm-infected wounds are discussed.