Rifampicin reduces plasma concentration of linezolid in patients with infective endocarditis.

BACKGROUND: Linezolid in combination with rifampicin has been used in treatment of infective endocarditis especially for patients infected with staphylococci. OBJECTIVES: Because rifampicin has been reported to reduce the plasma concentration of linezolid, the present study aimed to characterize the population pharmacokinetics of linezolid for the purpose of quantifying an effect of rifampicin cotreatment. In addition, the possibility of compensation by dosage adjustments was evaluated. PATIENTS AND METHODS: Pharmacokinetic measurements were performed in 62 patients treated with linezolid for left-sided infective endocarditis in the Partial Oral Endocarditis Treatment (POET) trial. Fifteen patients were cotreated with rifampicin. A total of 437 linezolid plasma concentrations were obtained. The pharmacokinetic data were adequately described by a one-compartment model with first-order absorption and first-order elimination. RESULTS: We demonstrated a substantial increase of linezolid clearance by 150% (95% CI: 78%-251%), when combined with rifampicin. The final model was evaluated by goodness-of-fit plots showing an acceptable fit, and a visual predictive check validated the model. Model-based dosing simulations showed that rifampicin cotreatment decreased the PTA of linezolid from 94.3% to 34.9% and from 52.7% to 3.5% for MICs of 2 mg/L and 4 mg/L, respectively. CONCLUSIONS: A substantial interaction between linezolid and rifampicin was detected in patients with infective endocarditis, and the interaction was stronger than previously reported. Model-based simulations showed that increasing the linezolid dose might compensate without increasing the risk of adverse effects to the same degree.

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.

Biofilm formation on endovascular aneurysm repair (EVAR) grafts-a proof of concept in vitro model.

OBJECTIVES: An endovascular aneurysm repair (EVAR) graft is a catheter-implanted vascular prosthesis and is the preferred treatment for patients with aortic aneurysm. If an EVAR graft becomes the focus of infection, the treatment possibilities are limited because it is technically difficult to remove the graft to obtain source control. This study examines whether Pseudomonas aeruginosa and Staphylococcus aureus form biofilm on EVAR prostheses. METHODS: EVAR graft sections were exposed to bacteria at 102 or 108 colony forming units (CFU)/mL in lysogeny broth and Krebs-Ringer at 37°C, bacterial biofilm formation was evaluated by scanning electron microscopy and counting CFU on the graft sections after antibiotic exposure at × 10 minimal inhibitory concentration. Bacteria were tested for tolerance to benzylpenicillin, tobramycin, and ciprofloxacin. RESULTS: Bacterial exposure for 15 minutes established biofilms on all prosthesis fragments (6/6 replicates). After 4 hours, bacteria were firmly attached to the EVAR prostheses and resisted washing. After 18-24 hours, the median CFU/g of EVAR graft reached 5.2 × 108 (1.15 × 108-1.1 × 109) for S. aureus and 9.1 × 107 (3.5 × 107-6.25 × 108) for P. aeruginosa. Scanning electron microscopy showed bacterial attachment to the graft pieces. There was a time-dependent development of tolerance with approximately 20 (tobramycin), 560 (benzylpenicillin), and 600 (ciprofloxacin) times more S. aureus surviving antibiotic exposure in 24- compared with 0-hour-old biofilm. Five (tobramycin) and 170 times (ciprofloxacin) more P. aeruginosa survived antibiotic exposure in 24- compared with 0-hour-old biofilms. DISCUSSION: Our results show that bacteria can rapidly adhere to and subsequently form antibiotic-tolerant biofilms on EVAR graft material in concentrations equivalent to levels seen in transient bacteraemia in vivo. Potentially, the system can be used for identifying optimal treatment combinations for infected EVAR prosthesis.

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.

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.

Dynamics of a Staphylococcus aureus infective endocarditis simulation model.

Infective endocarditis (IE) is a serious infection of the inner surface of heart, resulting from minor lesions in the endocardium. The damage induces a healing reaction, which leads to recruitment of fibrin and immune cells. This sterile healing vegetation can be colonized during temporary bacteremia, inducing IE. We have previously established a novel in vitro IE model using a simulated IE vegetation (IEV) model produced from whole venous blood, on which we achieved stable bacterial colonization after 24 h. The bacteria were organized in biofilm aggregates and displayed increased tolerance toward antibiotics. In this current study, we aimed at further characterizing the time course of biofilm formation and the impact on antibiotic tolerance development. We found that a Staphylococcus aureus reference strain, as well as three clinical IE isolates formed biofilms on the IEV after 6 h. When treatment was initiated immediately after infection, the antibiotic effect was significantly higher than when treatment was started after the biofilm was allowed to mature. We could follow the biofilm development microscopically by visualizing growing bacterial aggregates on the IEV. The findings indicate that mature, antibiotic-tolerant biofilms can be formed in our model already after 6 h, accelerating the screening for optimal treatment strategies for IE.

Potential Advances of Adjunctive Hyperbaric Oxygen Therapy in Infective Endocarditis.

Patients with infective endocarditis (IE) form a heterogeneous group by age, co-morbidities and severity ranging from stable patients to patients with life-threatening complications with need for intensive care. A large proportion need surgical intervention. In-hospital mortality is 15-20%. The concept of using hyperbaric oxygen therapy (HBOT) in other severe bacterial infections has been used for many decades supported by various preclinical and clinical studies. However, the availability and capacity of HBOT may be limited for clinical practice and we still lack well-designed studies documenting clinical efficacy. In the present review we highlight the potential beneficial aspects of adjunctive HBOT in patients with IE. Based on the pathogenesis and pathophysiological conditions of IE, we here summarize some of the important mechanisms and effects by HBOT in relation to infection and inflammation in general. In details, we elaborate on the aspects and impact of HBOT in relation to the host response, tissue hypoxia, biofilm, antibiotics and pathogens. Two preclinical (animal) studies have shown beneficial effect of HBOT in IE, but so far, no clinical study has evaluated the feasibility of HBOT in IE. New therapeutic options in IE are much needed and adjunctive HBOT might be a therapeutic option in certain IE patients to decrease morbidity and mortality and improve the long-term outcome of this severe disease.

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.

Anti-biofilm Approach in Infective Endocarditis Exposes New Treatment Strategies for Improved Outcome.

Infective endocarditis (IE) is a life-threatening infective disease with increasing incidence worldwide. From early on, in the antibiotic era, it was recognized that high-dose and long-term antibiotic therapy was correlated to improved outcome. In addition, for several of the common microbial IE etiologies, the use of combination antibiotic therapy further improves outcome. IE vegetations on affected heart valves from patients and experimental animal models resemble biofilm infections. Besides the recalcitrant nature of IE, the microorganisms often present in an aggregated form, and gradients of bacterial activity in the vegetations can be observed. Even after appropriate antibiotic therapy, such microbial formations can often be identified in surgically removed, infected heart valves. Therefore, persistent or recurrent cases of IE, after apparent initial infection control, can be related to biofilm formation in the heart valve vegetations. On this background, the present review will describe potentially novel non-antibiotic, antimicrobial approaches in IE, with special focus on anti-thrombotic strategies and hyperbaric oxygen therapy targeting the biofilm formation of the infected heart valves caused by Staphylococcus aureus. The format is translational from preclinical models to actual clinical treatment strategies.

Distinct contribution of hyperbaric oxygen therapy to human neutrophil function and antibiotic efficacy against Staphylococcus aureus.

Staphylococcus aureus (SA) causes superficial and severe endovascular infections. The present in vitro study investigates the anti-SA mechanisms of hyperbaric oxygen therapy (HBOT) on direct bacterial killing, antibiotic potentiation, and polymorphonuclear leukocyte (PMN) enhancement. SA was exposed to isolated human PMNs, tobramycin, ciprofloxacin, or benzylpenicillin. HBOT was used as one 90-min session. Bacterial survival was evaluated after 4 h by quantitative bacteriology. PMN functionality as reactive oxygen species (ROS) production was measured by means of dihydrorhodamine 123 analysis. We showed that HBOT exhibits significant direct anti-SA effects. HBOT increased the anti-SA effects of PMNs by 18% after PMA stimulation (p = 0.0004) and by 15% in response to SA (p = 0.36). HBOT showed an additive effect as growth reductions of 26% to sub-MICs of tobramycin (p = 0.0057), 44% to sub-MICs of ciprofloxacin (p = 0.0001), and 26% to sub-MICs of penicillin (p = 0.038). The present in vitro study provides evidence that HBOT has differential mechanisms mediating its anti-SA effects. Our observation supports the clinical possibility for adjunctive HBOT to augment the host immune response and optimize the efficacy of antibiotic treatments.

Novel human in vitro vegetation simulation model for infective endocarditis.

Infective endocarditis (IE) is a heart valve infection with high mortality rates. IE results from epithelial lesions, inducing sterile healing vegetations consisting of platelets, leucocytes, and fibrin that are susceptible for colonization by temporary bacteremia. Clinical testing of new treatments for IE is difficult and fast models sparse. The present study aimed at establishing an in vitro vegetation simulation IE model for fast screening of novel treatment strategies. A healing promoting platelet and leucocyte-rich fibrin patch was used to establish an IE organoid-like model by colonization with IE-associated bacterial isolates Staphylococcus aureus, Streptococcus spp (S. mitis group), and Enterococcus faecalis. The patch was subsequently exposed to tobramycin, ciprofloxacin, or penicillin. Bacterial colonization was evaluated by microscopy and quantitative bacteriology. We achieved stable bacterial colonization on the patch, comparable to clinical IE vegetations. Microscopy revealed uneven, biofilm-like colonization of the patch. The surface-associated bacteria displayed increased tolerance to antibiotics compared to planktonic bacteria. The present study succeeded in establishing an IE simulation model with the relevant pathogens S. aureus, S. mitis group, and E. faecalis. The findings indicate that the IE model mirrors the natural IE process and has the potential for fast screening of treatment candidates.

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.

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.

Computed tomography-guided high-dose-rate brachytherapy in hepatocellular carcinoma: safety, efficacy, and effect on survival.

PURPOSE: To determine the safety and efficacy of computed tomography (CT)-guided brachytherapy in hepatocellular carcinoma (HCC). METHODS AND MATERIALS: A total of 83 patients were recruited, presenting with 140 HCC- lesions. Treatment was performed by CT-guided high-dose-rate (HDR) brachytherapy with an iridium-192 source. The primary endpoint was time to progression; secondary endpoints included local tumor control and overall survival (OS). A matched-pair analysis with patients not receiving brachytherapy was performed. Match criteria included the Cancer of the Liver Italian Program (CLIP) score, alpha-fetoprotein, presence, and extent of multifocal disease. For statistical analysis, Kaplan-Meier and Cox regression were performed. RESULTS: Mean and median cumulative TTP for all patients (n = 75) were 17.7 and 10.4 months. Five local recurrences were observed. The OS after inclusion reached median times of 19.4 months (all patients), 46.3 months (CLIP score, 0), 20.6 months (CLIP score, 1) 12.7 months, (CLIP score, 2), and 8.3 months (CLIP score, >or=3). The 1- and 3-year OS were 94% and 65% (CLIP score, 0), 69% and 12% (CLIP score, 1), and 48% and 19% (CLIP score, 2), respectively. Nine complications requiring intervention were encountered in 124 interventions. Matched-pair analysis revealed a significantly longer OS for patients undergoing CT-guided brachytherapy. CONCLUSION: Based on our results the study treatment could be safely performed. The study treatment had a beneficial effect on OS in patients with advanced HCC, with respect to (and depending on) the CLIP score and compared with OS in a historical control group. A high rate of local control was also observed, regardless of applied dose in a range of 15 to 25 Gy.