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.

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.

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.

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.

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.

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.

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.

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.

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.

A case report of polymicrobial bacteremia with Weissella confusa and comparison of previous treatment for successful recovery with a review of the literature.

Weissella confusa is a Gram-positive coccus and a commensal bacterium of the human gastrointestinal tract with a potential to cause invasive infections. We report the presence of W. confusa in the blood of a 25-year-old male patient with Crohn's disease, short bowel syndrome treated with home parenteral nutrition, and a history of recurrent bloodstream infections, admitted to our hospital with fever and malaise. A polymicrobial culture of W. confusa and Aeromonas hydrophila was identified from blood, for which treatment with meropenem and metronidazole was initiated. The literature was searched for previous cases of infection with W. confusa . In total, 14 reports describing infection of 28 patients were found, most cases presenting with bacteremia. The previous reports have described variable susceptibility to antibiotics; however, all were reported to be vancomycin resistant. Because of its similarities to other vancomycin-resistant cocci, isolates of W. confusa might be difficult to identify with traditional methods. Infection may be facilitated by its natural vancomycin resistance, leading to severe infection in hosts with underlying diseases. We describe the treatment of previous cases of infection and suggest treatment methods shown effective in other cases. Vancomycin is often used as treatment of infection with Gram-positive organisms, but this may need to be reevaluated, as several pathogenic bacteria are intrinsically vancomycin resistant. A review on reported treatments of bacteremia by W. confusa suggests the use of daptomycin, amoxicillin-clavulanate or piperacillin/tazobactam as recommendable antibiotic regimens.

Efficacy and safety of azithromycin maintenance therapy in primary ciliary dyskinesia (BESTCILIA): a multicentre, double-blind, randomised, placebo-controlled phase 3 trial.

BACKGROUND: Use of maintenance antibiotic therapy with the macrolide azithromycin is increasing in a number of chronic respiratory disorders including primary ciliary dyskinesia (PCD). However, evidence for its efficacy in PCD is lacking. We aimed to determine the efficacy and safety of azithromycin maintenance therapy for 6 months in patients with PCD. METHODS: The Better Experimental Screening and Treatment for Primary Ciliary Dyskinesia (BESTCILIA) trial was a multicentre, double-blind, parallel group, randomised, placebo-controlled phase 3 trial done at 6 European PCD clinics (tertiary paediatric care centres and university hospitals in Denmark, Germany, Netherlands, Switzerland, and UK). Patients with a confirmed diagnosis of PCD, aged 7-50 years old, and predicted FEV1 greater than 40% were recruited. Participants were randomly assigned (1:1), stratified by age and study site, via a web-based randomisation system to azithromycin 250 mg or 500 mg as tablets according to bodyweight (

In vivo demonstration of Pseudomonas aeruginosa biofilms as independent pharmacological microcompartments.

BACKGROUND: Pseudomonas aeruginosa is difficult to eradicate from the lungs of cystic fibrosis (CF) patients due to biofilm formation. Organs and blood are independent pharmacokinetic (PK) compartments. Previously, we showed in vitro biofilms behave as independent compartments impacting the pharmacodynamics. The present study investigated this phenomenon in vivo. METHODS: Seaweed alginate beads with P. aeruginosa resembling biofilms, either freshly produced (D0) or incubated for 5 days (D5) were installed s.c in BALB/c mice. Mice (n = 64) received tobramycin 40 mg/kg s.c. and were sacrificed at 0.5, 3, 6, 8, 16 or 24 h after treatment. Untreated controls (n = 14) were sacrificed, correspondingly. Tobramycin concentrations were determined in serum, muscle tissue, lung tissue and beads. Quantitative bacteriology was determined. RESULTS: The tobramycin peak concentrations in serum was 58.3 (±9.2) mg/L, in lungs 7.1 mg/L (±2.3), muscle tissue 2.8 mg/L (±0.5) all after 0.5 h and in D0 beads 19.8 mg/L (±3.5) and in D5 beads 24.8 mg/L (±4.1) (both 3 h). A 1-log killing of P. aeruginosa in beads was obtained at 8h, after which the bacterial level remained stable at 16 h and even increased in D0 beads at 24 h. Using the established diffusion retardation model the free tobramycin concentration inside the beads showed a delayed buildup of 3 h but remained lower than the MIC throughout the 24 h. CONCLUSIONS: The present in vivo study based on tobramycin exposure supports that biofilms behave as independent pharmacological microcompartments. The study indicates, reducing the biofilm matrix would increase free tobramycin concentrations and improve therapeutic effects.

Synergistic effect of immunomodulatory S100A8/A9 and ciprofloxacin against Pseudomonas aeruginosa biofilm in a murine chronic wound model.

The majority of chronic wounds are associated with bacterial biofilms recalcitrant to antibiotics and host responses. Immunomodulatory S100A8/A9 is suppressed in Pseudomonas aeruginosa biofilm infected wounds. We aimed at investigating a possible additive effect between S100A8/A9 and ciprofloxacin against biofilms. MATERIALS/METHODS: Thirty-two mice were injected with alginate-embedded P. aeruginosa following a third-degree burn. The mice were randomized into four groups receiving combination ciprofloxacin and S100A8/A9 or monotherapy ciprofloxacin, S100A8/A9 or a placebo and evaluated by host responses and quantitative bacteriology in wounds. In addition, in vitro checkerboard analysis was performed, with P. aeruginosa and ascending S100A8/A9 and ciprofloxacin concentrations. RESULTS: S100A8/A9 augmented the effect of ciprofloxacin in vivo by lowering the bacterial quantity compared to the placebo arm and the two monointervention groups (P < 0.0001). S100A8 and 100A9 were increased in the double-treated group as compared to the monointervention groups (P = 0.032, P = 0.0023). Tissue inhibitor of metalloproteinases-1 and keratinocyte\chemokine chemoattractant-1 were increased in the double-intervention group compared to the S100A8/A9 group (P = 0.050, P = 0.050). No in vitro synergism was detected. CONCLUSION: The observed ciprofloxacin-augmenting effect of S100A8/A9 in vivo was not confirmed by checkerboard analysis, indicating dependence on host cells for the S100A8/A9 effect. S100A8/A9 and ciprofloxacin is a promising therapy for optimizing chronic wound treatment.

Adjunctive dabigatran therapy improves outcome of experimental left-sided Staphylococcus aureus endocarditis.

BACKGROUND: Staphylococcus aureus is the most frequent and fatal cause of left-sided infective endocarditis (IE). New treatment strategies are needed to improve the outcome. S. aureus coagulase promotes clot and fibrin formation. We hypothesized that dabigatran, could reduce valve vegetations and inflammation in S. aureus IE. METHODS: We used a rat model of severe aortic valve S. aureus IE. All infected animals were randomized to receive adjunctive dabigatran (10 mg/kg b.i.d., n = 12) or saline (controls, n = 11) in combination with gentamicin. Valve vegetation size, bacterial load, cytokine, cell integrins expression and peripheral platelets and neutrophils were assessed 3 days post-infection. RESULTS: Adjunctive dabigatran treatment significantly reduced valve vegetation size compared to controls (p< 0.0001). A significant reduction of the bacterial load in aortic valves was seen in dabigatran group compared to controls (p = 0.02), as well as expression of key pro-inflammatory markers keratinocyte-derived chemokine, IL-6, ICAM-1, TIMP-1, L-selectin (p< 0.04). Moreover, the dabigatran group had a 2.5-fold increase of circulating platelets compared to controls and a higher expression of functional and activated platelets (CD62p+) unbound to neutrophils. CONCLUSION: Adjunctive dabigatran reduced the vegetation size, bacterial load, and inflammation in experimental S. aureus IE.