Pseudomonas aeruginosa biofilm hampers murine central wound healing by suppression of vascular epithelial growth factor.

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.

Antibiotic penetration and bacterial killing in a Pseudomonas aeruginosa biofilm model.

OBJECTIVES: Treating biofilm infections successfully is a challenge. We hypothesized that biofilms may be considered as independent compartments with particular pharmacokinetics. We therefore studied the pharmacokinetics and pharmacodynamics of tobramycin in a seaweed alginate-embedded biofilm model. METHODS: Seaweed alginate beads containing Pseudomonas aeruginosa were cultured in LB medium, sampled at day 1, 3, 5 or 7 and examined for the effect of treatment with tobramycin for 30 min. Treated beads were homogenized and the number of cfu was determined. The antibiotic concentration in the solution of homogenized beads was measured. Finally, beads were examined for live cells by Syto9 staining and for dead cells by propidium iodide staining using a confocal laser scanning microscope. RESULTS: The antibiotic level in each bead was relatively stable (range 30-42 mg/L; MIC = 1.5 mg/L). There were fewer cfu in the tobramycin-treated beads than the non-treated beads (P < 0.016) and bacterial killing was reduced as the culture period increased from 1 to 7 days. Throughout the study period, increasing size and more superficial positioning of the microcolonies within the beads were demonstrated by confocal laser scanning microscopy. More dead cells (measured by propidium iodide staining) were observed in the treated group of beads, which supports the results obtained by culture. CONCLUSIONS: The present study, simulating the clinical pharmacokinetics of tobramycin, demonstrates fast absorption of tobramycin in an in vitro biofilm model. In addition, this model system enables parallel investigation of pharmacokinetics and pharmacodynamics, providing a model for testing new treatment strategies.

Bacteriophage therapy and infective endocarditis - is it realistic?

Infective endocarditis (IE) is a severe infection of the inner heart. Even with current standard treatment, the mean in-hospital mortality is as high as 15-20%, and 1-year mortality is up to 40% for left-sided IE. Importantly, IE mortality rates have not changed substantially over the past 30 years, and the incidence of IE is rising. The treatment is challenging due to the bacterial biofilm mode of growth inside the heart valve vegetations, resulting in antibiotic tolerance. Achieving sufficient antibiotic anti-biofilm concentrations in the biofilms of the heart valve vegetations is problematic, even with high-dose and long-term antibiotic therapy. The increasing prevalence of IE caused by antibiotic-resistant bacteria adds to the challenge. Therefore, adjunctive antibiotic-potentiating drug candidates and strategies are increasingly being investigated. Bacteriophage therapy is a reemerging antibacterial treatment strategy for difficult-to-treat infections, mainly biofilm-associated and caused by multidrug-resistant bacteria. However, significant knowledge gaps regarding the safety and efficacy of phage therapy impede more widespread implementation in clinical practice. Hopefully, future preclinical and clinical testing will reveal whether it is a viable treatment. The objective of the present review is to assess whether bacteriophage therapy is a realistic treatment for IE.

Bacteriophages for the treatment of pseudomonas-infected vascular prosthesis.

We present a case report detailing therapeutic application of two lytic antipseudomonal bacteriophages to treat a chronic relapsing Pseudomonas aeruginosa infection of a prosthetic aortic graft. As there are currently no Danish laboratories offering phages for clinical therapy, and this case, to our knowledge represents the first applied phage therapy in Denmark, the practical and regulatory aspects of offering this treatment option in Denmark is briefly reviewed along with the clinical case.

Efficacy of a synthetic antimicrobial peptidomimetic versus vancomycin in a Staphylococcus epidermidis device-related murine peritonitis model.

OBJECTIVES: Biofilm-forming Staphylococcus epidermidis is a prevalent cause of peritonitis during peritoneal dialysis. We compared the efficacy of a synthetic antimicrobial peptidomimetic (Ltx21) versus vancomycin in a murine model mimicking a device-related peritonitis. METHODS: Silicone implants, pre-colonized with an S. epidermidis biofilm, were inserted into the peritoneal cavity of BALB/c mice. Three groups (36 mice in each) with pre-colonized implants received intraperitoneal treatment with Ltx21, vancomycin or placebo. Mice were euthanized on day 3 (n = 12), day 6 (n = 12) or day 8 (n = 12) post-implantation. Controls were mice with sterile implants (n = 18) and mice without surgery (n = 6). Bacterial reductions in cfu were analysed from implants and peritoneal fluid (PF). Inflammatory responses in serum and PF were measured. RESULTS: Vancomycin resulted in a stronger reduction in cfu counts, both on pre-colonized implants and in PF, compared with Ltx21 and placebo. Complete bacterial clearance of the implants was not achieved in any of the groups. The implants pre-colonized with S. epidermidis 1457 resulted in a low-grade peritonitis. We observed, only on day 6, a significant increase in the PF leucocyte count in the group with pre-colonized implants compared with the group with sterile implants (P = 0.0364). CONCLUSIONS: Treatment with vancomycin or Ltx21 was not sufficient to achieve complete bacterial clearance of implants, underlining the difficulties of treating such infections. The low-grade infection may attenuate the inflammatory response and contribute to impaired bacterial clearance.

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.

Hyperbaric oxygen therapy counteracts Pseudomonas aeruginosa biofilm micro-compartment phenomenon in murine thermal wounds.

Background: Biofilm antibiotic tolerance is partly explained by the behavior of a biofilm as an independent pharmacokinetic micro-compartment. Hyperbaric oxygen therapy has been shown to potentiate antibiotic effects in biofilms. The present study investigates the effect of hyperbaric oxygen therapy (HBOT) on the biofilm micro-pharmacokinetic/pharmacodynamic behavior of tobramycin in an animal biofilm model. Methods: Full-thickness necroses were created mid-scapular on mice by means of a thermal lesion. After four days, three 16 h seaweed alginate biofilm beads containing Pseudomonas aeruginosa PAO1 were inserted under the necrosis, and three beads were inserted under the adjacent non-affected skin. The mice were randomized to three groups I) HBOT for 1.5 h at 2.8 atm and 0.8 mg tobramycin/mouse subcutaneously; II) Tobramycin as monotherapy, same dose; III) Saline control group. Half the number of mice from group 1 and 2 were sacrificed, and beads were recovered in toto after 3 h and the other half and the placebo mice were sacrificed and beads collected after 4.5 h. Results: Lower CFUs were seen in the burned group receiving HBOT at 3 and 4.5 h compared to beads in the atmospheric environment (p = 0.043 and p = 0.0089). At 3 h, no CFU difference was observed in the non-burned skin (HBOT vs atmospheric). At 4.5 h, CFU in the non-burned skin had lower CFUs in the group receiving HBOT compared to the corresponding atmospheric group (p = 0.02). CFU was higher in the burned skin than in the non-burned skin at 3 h when HBOT was applied (p = 0.04), effect faded out at 4.5 h.At both time points, the tobramycin content in the beads under burned skin were higher in the HBOT group than in the atmospheric groups (p = 0.031 and p = 0.0078). Only at 4.5 h a higher tobramycin content was seen in the beads under the HBOT-treated burned skin than the beads under the corresponding non-burned skin (p = 0.006). Conclusion: HBOT, as an anti-biofilm adjuvant treatment of chronic wounds, counteracts biofilm pharmacokinetic micro-compartmentalization through increased available tobramycin and augmented bacterial killing.

Hyperbaric oxygen therapy augments ciprofloxacin effect against Pseudomonas aeruginosa biofilm infected chronic wounds in a mouse model.

Introduction: Chronic wounds have a compromised microcirculation which leads to restricted gas exchange. The majority of these hypoxic wounds is infested with microorganisms congregating in biofilms which further hinders the antibiotic function. We speculate whether this process can be counteracted by hyperbaric oxygen therapy (HBOT). Methodology: Twenty-eight BALB/c mice with third-degree burns were included in the analyses. Pseudomonas aeruginosa embedded in seaweed alginate beads was injected under the eschar to mimic a biofilm infected wound. Challenged mice were randomized to receive either 4 days with 1 x ciprofloxacin combined with 2 × 90 min HBOT at 2.8 standard atmosphere daily, 1 x ciprofloxacin as monotherapy or saline as placebo. The mice were clinically scored, and wound sizes were estimated by planimetry daily. Euthanasia was performed on day 8. Wounds were surgically removed in toto, homogenized and plated for quantitative bacteriology. Homogenate supernatants were used for cytokine analysis. Results: P. aeruginosa was present in all wounds at euthanasia. A significant lower bacterial load was seen in the HBOT group compared to either the monotherapy ciprofloxacin group (p = 0.0008), or the placebo group (p < 0.0001). IL-1ß level was significantly lower in the HBOT group compared to the placebo group (p = 0.0007). Both treatment groups had higher osteopontin levels than the placebo group (p = 0.002 and p = 0.004). The same pattern was seen in the S100A9 analysis (p = 0.01 and p = 0.008), whereas no differences were detected between the S100A8, the VEGF or the MMP8 levels in the three groups. Conclusion: These findings show that HBOT improves the bactericidal activity of ciprofloxacin against P. aeruginosa wound biofilm in vivo. HBOT in addition to ciprofloxacin also modulates the host response to a less inflammatory phenotype.

Risk factors for methicillin-resistant Staphylococcus aureus colonization in a level-IV neonatal intensive care unit: a retrospective study.

Objective: To identify risk factors associated with methicillin-resistant Staphylococcus aureus (MRSA) colonization in neonatal patients during an MRSA outbreak to minimize future outbreaks. Design: Retrospective case-control study. Setting: Level-IV neonatal intensive care unit (NICU) at Copenhagen University Hospital, Rigshospitalet, Denmark. Patients: Neonates with either MRSA or methicillin-susceptible Staphylococcus aureus (MSSA). Methods: Methicillin-resistant Staphylococcus aureus-positive neonates were matched with those colonized or infected with MSSA in a 1:1 ratio. The control group was selected from clinical samples, whereas MRSA-positive neonates were identified from clinical samples or from screening. A total of 140 characteristics were investigated to identify risk factors associated with MRSA acquisition. The characteristics were categorized into three categories: patient, unit, and microbiological characteristics. Results: Out of 1,102 neonates screened for MRSA, between December 2019 and January 2022, 33 were MRSA positive. They were all colonized with an MRSA outbreak clone (spa type t127) and were included in this study. Four patients (12%) had severe infection. Admission due to respiratory diseases, need for intubation, need for peripheral venous catheters, admission to shared rooms with shared toilets and bath facilities in the aisles, and need for readmission were all correlated with later MRSA colonization (P < 0.05). Conclusion: We identified clinically relevant diseases, procedures, and facilities that predispose patients to potentially life-threatening MRSA infections. A specific MRSA reservoir remains unidentified; however, these findings have contributed to crucial changes in our NICU to reduce the number of MRSA infections and future outbreaks.

Immune Response to Biofilm Growing Pulmonary Pseudomonas aeruginosa Infection.

Biofilm infections are tolerant to the host responses and recalcitrance to antibiotic drugs and disinfectants. The induced host-specific innate and adaptive immune responses by established biofilms are significantly implicated and contributes to the course of the infections. Essentially, the host response may be the single one factor impacting the outcome most, especially in cases where the biofilm is caused by low virulent opportunistic bacterial species. Due to the chronicity of biofilm infections, activation of the adaptive immune response mechanisms is frequently experienced, and instead of clearing the infection, the adaptive response adds to the pathogenesis. To a high degree, this has been reported for chronic Pseudomonas aeruginosa lung infections, where both a pronounced antibody response and a skewed Th1/Th2 balance has been related to a poorer outcome. In addition, detection of an adaptive immune response can be used as a significant indicator of a chronic P. aeruginosa lung infection and is included in the clinical definitions as such. Those issues are presented in the present review, along with a characterization of the airway structure in relation to immune responses towards P. aeruginosa pulmonary infections.

Murine burn lesion model for studying acute and chronic wound infections.

Acute wounds, such as thermal injury, and chronic wounds are challenging for patients and the healthcare system around the world. Thermal injury of considerable size induces immunosuppression, which renders the patient susceptible to wound infections, but also in other foci like the airways and urinary tract. Infected thermal lesions can progress to chronic wounds with biofilm making them more difficult to treat. While animal models have their limitations, murine wound models are still the best tool at the moment to identify strategies to overcome these challenges. Here, we present a murine burn model, which has been developed to study biofilm formation, the significance of wound healing, and for identifying novel treatment candidates. Investigating the effect of a thermal injury in mice, we observed that 48 h after introduction of the injury, the mice showed a reduction in polymorphonuclear neutrophil granulocytes (PMNs) and a reduced capacity for phagocytosis and oxidative burst. Regarding the chronic wound, Pseudomonas aeruginosa biofilm arrested wound healing and kept the wound in an inflammatory state, but suppressing PMN function by means of the PMN factor S100A8/A9, corresponding to observations in human venous leg ulcers. Monotherapy and dual treatment with S100A8/A9 and ciprofloxacin on P. aeruginosa biofilm-infected murine wounds have been investigated. In combination, S100A8/A9 and ciprofloxacin reduced the bacterial quantity, lowered the proinflammatory response, and increased anti-inflammatory cytokines after 4 days of treatment. When the treatment was prolonged, an additional prevention of resistance development was detected in all the dual-treated mice. In the present review, we provide data on using the murine model for research with the aim of better understanding pathophysiology of wounds and for identifying novel treatments for humans suffering from these lesions.

Animal models of chronic and recurrent Pseudomonas aeruginosa lung infection: significance of macrolide treatment.

Animal models of human diseases are invaluable and inevitable elements in identifying and testing novel treatments for serious diseases, including severe infections. Planning and conducting investigator-initiated human trials are generally accepted as being enormously challenging. In contrast, it is often underestimated how much planning, including background and modifying experiments, is needed to establish a relevant infectious disease animal model. However, representative animal infectious models, well designed to test generated hypotheses, are useful to improve our understanding of pathogenesis, virulence factors and host response and to identify novel treatment candidates and therapeutic strategies. Such results can subsequently proceed to clinical testing if suitable. The present review aims at presenting all the pulmonary Pseudomonas aeruginosa infectious models we have knowledge of and the detailed descriptions of established animal models in our laboratory focusing on macrolide therapy are presented.

Analysis of antibiotic use patterns in Danish hospitals 2015-2021 using an adapted version of the who aware classification.

BACKGROUND: AWaRe is a tool to categorise and guide antibiotic use. Antibiotics are classified in three groups: Access, Watch and Reserve. The Access group contains first choice antibiotics for 25 of the most common infections. Antibiotics in the Watch and Reserve groups should be restricted to cases that cannot be effectively treated with drugs from the Access group. OBJECTIVES: The primary aim of this study was to evaluate and adapt the WHO 2019 AWaRe classification for use with antibiotic usage data in Danish hospitals. The secondary aim was to study the usefulness of the abxaware; software package for visualisation and analysis of temporal trends in antibiotic use patterns. METHODS: We obtained data on purchases of antibiotics in Danish hospitals from January 2015 to July 2021. Sixty-seven unique drugs had been purchased. To better correspond with Danish guidelines, we moved two drugs one AWaRe level upwards. To help aggregate antibiotics according to AWaRe and visualise use patterns, we developed an R package, abxaware. RESULTS: After adding two drugs that were not included in the original AWaRe classification nearly all antibiotics (>99%) used in Danish hospitals were covered. The abxaware software package for R is a useful tool to help aggregate, visualise and analyse antibiotic use patterns. CONCLUSIONS: With minor modifications, we adapted the AWaRe classification to cover most antibiotics used in Danish hospitals and to reflect Danish treatment guidelines. The abxaware package is a useful tool to aggregate and plot antibiotic usage data according to the AWaRe classification and to test for non-random variation in the percentage use of Access antibiotics.

Lactoferricin-inspired peptide AMC-109 augments the effect of ciprofloxacin against Pseudomonas aeruginosa biofilm in chronic murine wounds.

OBJECTIVES: Chronic wounds are characterised by prolonged inflammation, low mitogenic activity, high protease/low inhibitor activity, microbiota changes and biofilm formation, combined with the aetiology of the original insult. One strategy to promote healing is to terminate the parasitism-like relationship between the biofilm-growing pathogen and host response. Antimicrobial peptide AMC-109 is a potential treatment with low resistance potential and broad-spectrum coverage with rapid bactericidal effect. We aimed to investigate whether adjunctive AMC-109 could augment the ciprofloxacin effect in a chronic Pseudomonas aeruginosa wound model. METHODS: Third-degree burns were inflicted on 33 BALB/c mice. Pseudomonas aeruginosa embedded in seaweed alginate was injected sub-eschar to mimic biofilm. Mice were randomised to receive AMC-109, combined AMC-109 and ciprofloxacin, ciprofloxacin, or placebo for 5 days followed by sample collection. RESULTS: A lower bacterial load was seen in the double-treated group compared with either monotherapy group (AMC-109, p = 0.0076; ciprofloxacin, p = 0.0266). To evaluate the innate host response, cytokines and growth factors were quantified. The pro-inflammatory response was dampened in the double-treated mice compared with the mono-ciprofloxacin-treated group (p = 0.0009). Lower mobilisation of neutrophils from the bone marrow was indicated by reduced G-CSF in all treatment groups compared with placebo. Improved tissue remodelling was indicated by the highest level of tissue inhibitor of metalloproteases and low metalloprotease level in the double-treated group. CONCLUSION: AMC-109 showed adjunctive antipseudomonal abilities augmenting the antimicrobial effect of ciprofloxacin in this wound model. The study indicates a potential role for AMC-109 in treating chronic wounds with complicating biofilm infections.

Chicken IgY reduces the risk of Pseudomonas aeruginosa urinary tract infections in a murine model.

Introduction: Urinary tract infections (UTIs) with Pseudomonas aeruginosa are a severe problem in disposed patients in modern healthcare. Pseudomonas aeruginosa establishes recalcitrant biofilm infections and can develop antibiotic resistance. Gargling with avian egg yolk anti-Pseudomonas antibodies (IgY) has shown clinical effect in preventing onset of chronic P. aeruginosa lung infections in patients with cystic fibrosis (CF). Therefore, we speculated whether passive intravesically administered IgY immunotherapy could be a novel strategy against P. aeruginosa UTIs. Aim: To evaluate if prophylactic repurposing of anti-Pseudomonas IgY can prevent UTIs with P. aeruginosa in a UTI mouse model. Materials and methods: In vitro, P. aeruginosa (PAO1 and PAO3) was mixed with increasing concentrations of specific anti-Pseudomonas IgY (sIgY) or non-specific control IgY (cIgY) and/or freshly isolated human neutrophils. Bacterial growth was evaluated by the optical density at 600 nm. In vivo, via a temporary transurethral catheter, 10-week-old female Balb/c mice were intravesically infected with 50 ml of a bacterial suspension and sIgY, cIgY, or isotonic NaCl. IgY and NaCl were either co-instilled with the bacteria, or instilled prophylactically, 30 min prior to infection. The animals were euthanized 20 h after infection. Vesical bacteriology was quantified, and cytokine expression in the bladder homogenate was measured by multiplex cytokine assay. Results: In vitro, sIgY concentrations above 2.5% reduced bacterial growth in a dose-dependent manner. In vivo, a UTI lasting for minimum 7 days was established by installing 5 × 106 colony-forming units (CFU) of P. aeruginosa PAO1. sIgY reduced vesical bacterial load if co-installed with P. aeruginosa PAO1. Prophylactic sIgY and cIgY reduced bacterial load when compared to isotonic NaCl. CXCL2 and G-CSF were both increased in infected bladders compared to non-infected controls which had non-detectable levels. Co-installation of sIgY and bacteria nearly completely inhibited the inflammatory response. However, the cytokine levels in the bladder did not change after prophylactic administration of sIgY or cIgY. Conclusion: Prophylactic sIgY significantly reduces the amount of bacteria in the bladder in a mouse model of P. aeruginosa cystitis and may serve as a novel non-antibiotic strategy in preventing P. aeruginosa UTIs.

Extensive microbiological respiratory tract specimen characterization in critically ill COVID-19 patients.

Microbial co-infections may contribute to the pulmonary deterioration in COVID-19 patients needing intensive care treatment. The present study portrays the extent of co-infections in COVID-19 ICU patients. Conventional culture, molecular detections for atypical aetiologies, QiaStat-Dx® respiratory panel V2 detecting 21 respiratory pathogens and ribosomal DNA genes 16S/18S amplicon-based microbiome analyses were performed on respiratory samples from 34 COVID-19 patients admitted to the ICU. Potential pathogens were detected in seven patients (21%) by culturing, in four patients (12%) by microbiome analysis and in one patient (3%) by respiratory panel. Among 20 patients receiving antibiotics prior to ICU admission, fungi (3 Candida albicans, 1 C. tropicalis, 1 C. dubliniensis) were cultured in 5 (15%) endotracheal aspirates. Among 14 patients who were antibiotic-naive at ICU admission, two patients (6%) had bacterial respiratory pathogens (Staphylococcus aureus, Streptococcus pseudopneumoniae) cultured in their endotracheal aspirates. Microbiome analysis recognized four potential respiratory pathogens (3 Haemophilus influenza, 1 Fusobacterium necrophorum) isolated in samples from four other patients (12%). QiaStat-Dx® respiratory panel V2 detected adenovirus in one patient (3%). The prevalence of pulmonary microbial co-infections is modest among COVID-19 patients upon admission to ICU. Microbiome analysis complements conventional microbial diagnostics in characterization of respiratory co-infections.

Adjunctive S100A8/A9 Immunomodulation Hinders Ciprofloxacin Resistance in Pseudomonas aeruginosa in a Murine Biofilm Wound Model.

Objective: Pseudomonas aeruginosa is known to contribute to the pathogenesis of chronic wounds by biofilm-establishment with increased tolerance to host response and antibiotics. The neutrophil-factor S100A8/A9 has a promising adjuvant effect when combined with ciprofloxacin, measured by quantitative bacteriology, and increased anti- and lowered pro-inflammatory proteins. We speculated whether a S100A8/A9 supplement could prevent ciprofloxacin resistance in infected wounds. Method: Full-thickness 2.9cm2-necrosis was inflicted on 32 mice. On day 4, P.aeruginosa in seaweed alginate was injected sub-eschar to mimic a mono-pathogenic biofilm. Mice were randomized to receive ciprofloxacin and S100A8/A9 (n=14), ciprofloxacin (n=12) or saline (n=6). Half of the mice in each group were euthanized day 6 and the remaining day 10 post-infection. Mice were treated until sacrifice. Primary endpoint was the appearance of ciprofloxacin resistant P.aeruginosa. The study was further evaluated by genetic characterization of resistance, means of quantitative bacteriology, wound-size and cytokine-production. Results: Three mice receiving ciprofloxacin monotherapy developed resistance after 14 days. None of the mice receiving combination therapy changed resistance pattern. Sequencing of fluoroquinolone-resistance determining regions in the ciprofloxacin resistant isolates identified two high-resistant strains mutated in gyrA C248T (MIC>32µg/ml) and a gyr B mutation was found in the sample with low level resistance (MIC=3µg/ml). Bacterial densities in wounds were lower in the dual treated group compared to the placebo group on both termination days. Conclusion: This study supports the ciprofloxacin augmenting effect and indicates a protective effect in terms of hindered ciprofloxacin resistance of adjuvant S100A8/A9 in P.aeruginosa biofilm infected chronic wounds.

Immune Responses to Pseudomonas aeruginosa Biofilm Infections.

Pseudomonas aeruginosa is a key pathogen of chronic infections in the lungs of cystic fibrosis patients and in patients suffering from chronic wounds of diverse etiology. In these infections the bacteria congregate in biofilms and cannot be eradicated by standard antibiotic treatment or host immune responses. The persistent biofilms induce a hyper inflammatory state that results in collateral damage of the adjacent host tissue. The host fails to eradicate the biofilm infection, resulting in hindered remodeling and healing. In the present review we describe our current understanding of innate and adaptive immune responses elicited by P. aeruginosa biofilms in cystic fibrosis lung infections and chronic wounds. This includes the mechanisms that are involved in the activation of the immune responses, as well as the effector functions, the antimicrobial components and the associated tissue destruction. The mechanisms by which the biofilms evade immune responses, and potential treatment targets of the immune response are also discussed.

Azithromycin potentiates avian IgY effect against Pseudomonas aeruginosa in a murine pulmonary infection model.

Cystic fibrosis (CF) patients are at risk of acquiring chronic Pseudomonas aeruginosa lung infections. The biofilm mode of growth of P. aeruginosa induces tolerance to antibiotics and the host response; accordingly, treatment failure occurs. Supplemental azithromycin has proven beneficial in CF owing to potential immunomodulatory mechanisms. Clinical studies have demonstrated a reduction in exacerbations in CF patients by avian IgY anti-Pseudomonas immunotherapy. We hypothesise that azithromycin pre-treatment could potentiate the observed anti-Pseudomonas effect of IgY opsonisation in vivo. Evaluation of phagocytic cell capacity was performed using in vitro exposure of azithromycin pre-treated human polymorphonuclear neutrophils to IgY opsonised P. aeruginosa PAO3. A murine lung infection model using nasal planktonic P. aeruginosa inoculation and successive evaluation 24 h post-infection was used to determine lung bacteriology and subsequent pulmonary inflammation. Combined azithromycin treatment and IgY opsonisation significantly increased bacterial killing compared with the two single-treated groups and controls. In vivo, significantly increased bacterial pulmonary elimination was revealed by combining azithromycin and IgY. A reduction in the inflammatory markers mobiliser granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein 2 (MIP-2) and interleukin 1 beta (IL-1ß) paralleled this effect. Combination of azithromycin and anti-Pseudomonas IgY potentiated the killing and pulmonary elimination of P. aeruginosa in vitro and in vivo. The augmented effect of combinatory treatment with azithromycin and IgY constitutes a potential clinical application for improving anti-Pseudomonas strategies.