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 (n¼ 12), day 6 (n¼ 12) or day 8 (n¼ 12) post-implantation. Controls were mice with sterile implants (n¼ 18) and mice without surgery (n ¼6). 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 ¼ 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. Keywords: biofilms, device-related peritonitis, mouse model Introduction Gram-positive bacteria, in particular coagulase-negative staphylococci, are a prevalent cause of peritoneal dialysis catheter-related peritonitis. 1 This is a serious complication that may lead to catheter removal, peritoneal membrane dysfunction and transfer to haemodialysis. 2 Peritonitis is believed to occur by bacterial entrance to the peritoneal cavity through catheter colonization. 2 Staphylococcal biofilm formation on catheters results in a reduced ability to combat such infections, both by the host immune system and by conventional treatment with antibiotics. Synthetic antimicrobial peptidomimetics (SAMPs) are novel antimicrobial agents derived from cationic antimicrobial peptides that are widespread in nature. 5 Their modes of action are not completely resolved. However, a central mechanism is bacterial membrane disruption, affecting both dormant and dividing bacteria. 7 This study aimed to investigate the efficacy of a SAMP (Ltx21) versus vancomycin in a murine model mimicking devicerelated Staphylococcus epidermidis biofilm-associated peritonitis. We assessed bacterial clearance and the host innate immune response to understand the pathophysiological mechanisms involved. Methods Bacterial isolates and MICs S. epidermidis 1457, used in this experiment, was originally isolated from a central venous catheter infection. S. epidermidis 1457 forms a thick biofilm under the in vitro growth conditions used in this study. 7 All three SAMPs have the same tripeptide sequence, with two arginine moieties providing their cationic properties and a modified tryptophan providing the lipophilic bulk. The SAMPs differ by C-terminal modifications, of which Ltx21 has an additional phenylalanine attached compared with Ltx9 and Ltx5. For Ltx21 the MIC was 6 mg/L (determined by the microbroth dilution method) and the minimal biofilm inhibitory concentration was 60 mg/L (determined by the Alamar blue method), comparable to those values previously reported for Ltx5 and Ltx9. 7 Species confirmation of small colony variants (SCVs) was performed with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Animals and animal ethics One hundred and thirty-two female BALB/c mice (Taconic M&B A/S Ry, Denmark), aged 7 -8 weeks, were used. Information on the experimental treatment of the animals and the duration of experiments (days) is provided in Device-related S. epidermidis biofilm-associated peritonitis model Silicone implants (5 mm×5 mm×2 mm; Ole Dich, Denmark) were inserted into the murine intraperitoneal cavity in order to mimic a device-related peritonitis. Briefly, the implants were prepared by incubation for 120 h in an S. epidermidis 1457 culture. The inoculum was adjusted to an optical density at 600 nm that was equivalent to that of a 2 McFarland standard in 0.9% NaCl and further suspended in tryptic soy broth (TSB) with 1% glucose to induce biofilm formation. Every 24 h the implants were rinsed in PBS and transferred to a sterile flask containing fresh medium (TSB with 1% glucose). Mice were anaethetized by subcutaneous injections (0.15 mL) in the groin area with a mixture of 0.0375 mL of 0.315 mg/mL fentanyl/10 mg/mL fluanisone (VetaPharma Ltd, UK) and 0.0375 mL of 5 mg/L midazolam (Hameln Pharmaceuticals, Germany) in 0.075 mL of sterile water. Insertion of implants and intraperitoneal treatment were performed as previously described. 9,10 Vancomycin (Sandoz, Australia) and Ltx21 (Lytix Biopharma AS, Tromsø, Norway) were both dissolved in 0.9% NaCl to a final concentration of 1 mg/mL and 0.5 mg/mL, respectively. All animals received intraperitoneal injections (400 mL) every 24 h for up to 7 days. Treatment was initiated 2 h post-implantation. The vancomycin dose was 20 mg/kg, based on previous studies. 11,12 The Ltx21 dose was 10 mg/kg, based on previous toxicology studies, a pilot treatment study and in vitro MIC studies. On the days of implant removal, mice were anaethetized by subcutaneous injection of 0.1 mL of pentobarbital (200 mg/mL) (KVL, Denmark). After general anaesthesia, blood was drawn by cardiac puncture and transferred to tubes containing heparin for fluorescence-activated cell sorting (FACS) (n¼6 animals) or 50 mg/L lepirudin (Refludan, Hoechst, Germany) for complement analysis (n¼6 animals). Peritoneal lavage was performed by injecting 5 mL of PBS into the peritoneal cavity, followed by gently massaging the abdomen before withdrawing the peritoneal fluid (PF). Implants were removed from the peritoneal cavity and transferred to tubes containing 1 mL of NaCl and 20 glass beads (Lenz, Laborglasinstrumente, Germany). Mice were euthanized by removal of the heart under general anaesthesia. Bacteriology Implants removed from the animals were vortexed for 30 s followed by 5 min of sonication at 40 kHz in an ultrasound bath (Bransonic 3510, Branso Ultrasonic Corporation, USA). One hundred microlitres of both the implant-derived suspension and PF was serial diluted and plated on blood agar plates (SSI, Denmark) for bacterial enumeration. The cfu counts were determined after incubation at 378C overnight. Prolonged incubation was necessary to detect SCVs. Cytokines, chemokines and complement Quantification of tumour necrosis factor-a (TNF-a), interleukin-1b (IL-1b), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein 2 (MIP-2) and monocyte chemotactic protein-1 (MCP-1/CCL2) was performed on plasma and/or PF using the Fluorokine MAP system (R&D Systems, UK) in combination with a dual-laser, flow-based sorting and detection analyser (Luminex Corporation, USA), according to the manufacturer's description. Complement factors C3a and C5a from plasma and PF were quantified using ELISA kits (USCN Life Science Inc., Wuhan, China) according to the manufacturer's instructions. Haematological parameters and flow cytometry The total leucocyte concentration and the fractions of granulocytes and macrophages were estimated in PF and blood as previously described. 13 Briefly, the fixed samples were analysed using FACS Canto (Becton Dickinson, USA). Light scatter and logarithmically amplified fluorescence parameters from at least 10 000 events were recorded in list mode after gating on forward light scatter to avoid debris, cell aggregates and bacteria. Statistics The data were analysed using GraphPad Prism version 5 (GraphPad Software, Inc., San Diego, CA, USA) or IMB SPSS Statistics 19. We used the two-way analysis of variance (ANOVA) test with Bonferroni corrections for multiple comparisons. The non-parametric Mann-Whitney U-test was Results Clinical observation Independent of treatment groups the mice exhibited signs of illness such as ruffled fur and reduced activity levels during the first 2 days. The mortality rate for mice with infected implants was 6/108 (vancomycin¼2, Ltx21¼2 and placebo¼2). The death of these mice was not anticipated, and compared with surviving mice they did not exhibit signs of illness or distress prior to death. Bacteriology Untreated pre-colonized implants had cfu counts of 3×10 8 cfu/ implant. Treatment in vivo with vancomycin and Ltx21 resulted in moderate reductions in the bacterial counts on the implants Cellular response We found no indication of an elevated systemic cellular response, either in mice with pre-colonized implants or in mice with sterile implants (data not shown). A local cellular response was observed in PF. Higher levels of leucocytes in PF from mice with pre-colonized implants receiving Ltx21, vancomycin or placebo were observed on day 6 (P ¼ 0.0364) when compared with animals with sterile implants Cytokine response Levels of TNF-a, IL-1b, GM-CSF, MIP-2 and MCP-1/CCL2 were measured in plasma and in PF on days 3, 6 and 8 post-implantation (data not shown). No significant differences were observed between the groups on any days regarding levels of IL-1b, MIP-2 and TNF-a. Mice with sterile implants showed significantly higher levels of MCP-1 in PF (P ¼ 0.005) on day 6 compared with mice with pre-colonized implants treated with placebo. GM-CSF was measured in blood only. Significantly increased values of GM-CSF were detected on days 3, 6 and 8 in mice with pre-colonized implants (P ¼0.0028) receiving Ltx21, vancomycin or placebo compared with mice with sterile implants. Significantly increased values of GM-CSF were also found on day 3 (P ¼0.02) and day 6 (P ¼ 0.04) in animals with sterile implants compared with animals without surgery. Complement activation The activation products of the complement system, C3a and C5a, were measured in plasma and PF. No significant activation of complement was observed within the groups with pre-colonized implants compared with the control groups without surgery. Discussion This murine model mimicking a device-related S. epidermidis biofilm-associated peritonitis enabled us to study the effects of two different treatment regimens and the host innate immune response. Our aim was to investigate whether Ltx21 could eradicate pre-formed S. epidermidis biofilms on peritoneal implants. Vancomycin 7 SAMPs have been found to have high levels of serum albumin binding. 14 Although in vitro time -kill kinetic studies have demonstrated rapid killing of bacteria, 6 protein binding might occur instantaneously upon administration of such peptides. 14 This reduces the amount of available peptide and might explain the reduced efficacy of Ltx21 in vivo compared with the good efficacy observed in vitro. 7,14 Vancomycin resulted in better bacterial clearance than Ltx21. However, complete biofilm clearance was not achieved by any of the two study drugs, despite high intraperitoneal dosing. On days 6 and 8 post-implantation we observed SCVs associated with implants in both treatment groups and in the placebo group. SCVs have previously been associated with persistent, subclinical and resistant infections associated with implanted medical devices. The overall low levels of granulocytes and macrophages in both blood and PF on days 3, 6 and 8 indicated a low-grade infection. Induction of macrophage apoptosis and mechanisms interfering with phagocytosis and macrophage activation has been observed for both S. epidermidis and Staphylococcus haemolyticus. 18,19 Schommer et al. 19 demonstrated that biofilm production by S. epidermidis 1457 resulted in reduced phagocytosis and macrophage activation, yielding low activation of the transcription factor NF-kB, leading to a significantly reduced IL-1b synthesis in mouse macrophage-like cells. Furthermore, in a mouse model, an S. aureus biofilm induced macrophage death and a significant reduction in IL-1b, TNF-a and MCP-1 production. 20 These observations are in line with findings from our study. In general, we found no consistent increase in cytokine production in the infected groups compared with the sterile groups. In S. epidermidis biofilm infections, a recent study reported that granulocytes are recruited and activated, but are not capable of engulfing bacteria embedded in the biofilm. No complement activation was observed in this S. epidermidis biofilm peritonitis model. In contrast, a previous study by our group demonstrated that S. epidermidis 1457 biofilm induced a strong complement activation in an ex vivo full blood model. There are limitations with this study. The model used is a suitable peritonitis model allowing simultaneous sampling of several parameters in response to treatment of an implant-associated biofilm infection. However, the current study could have benefitted from inclusion of additional animals, allowing for prolonged observation of persistence. One could argue that the use of pre-colonized implants is clinically irrelevant. However, in order to establish a biofilm infection to study the efficacy of the two different treatment regimens, we found that pre-colonization was necessary in order to obtain an infection in immunocompetent mice. Conclusions Our observations demonstrate failure of the novel SAMP Ltx21 and vancomycin in efficiently eradicating the S. epidermidis implantassociated biofilm infection. The reduced efficacy of the SAMP in vivo compared with previous in vitro results reflects the importance of performing animal studies. The presence of a persistent implant infection, which is not cleared by the innate immune system, is demonstrated. We demonstrated that this model allows for study of the complex interplay between the host immune system and the effects of antimicrobial treatment

Phenotypes of Non-Attached Pseudomonas aeruginosa Aggregates Resemble Surface Attached Biofilm

For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial populations

Wittgenstein's 'On certainty' in the making : studies into its historical and philosophical background

Deze dissertatie beschriift en analyseert de filosofische en tekstuele ontstaansgeschiedenis van Ludwig Wittgenstein's On Certain! (1969), een postuum gepubliceerd werk samengesteld uit opmerkingen die door Wittgenstein zijn opgetekend in de iaren 1949-1951. ... ZIe: Samenvatting

Viral replication kinetics and in vitro cytopathogenicity of parental and reassortant strains of bluetongue virus serotype 1, 6 and 8

Bluetongue virus (BTV), a segmented dsRNA virus, is the causative agent of bluetongue (BT), an economically important viral haemorrhagic disease of ruminants. Bluetongue virus can exchange its genome segments in mammalian or insect cells that have been co-infected with more than one strain of the virus. This process, may potentially give rise to the generation of novel reassortant strains that may differ from parental strains in regards to their phenotypic characteristics. To investigate the potential effects of reassortment on the virus' phenotype, parental as well as reassortant strains of BTV serotype 1, 6, 8, that were derived from attenuated and wild type strains by reverse genetics, were studied in vitro for their virus replication kinetics and cytopathogenicity in mammalian (Vero) cell cultures. The results indicate that genetic reassortment can affect viral replication kinetics, the cytopathogenicity and extent/mechanism of cell death in.infected cell cultures. In particular, some reassortants of non-virulent vaccine (BTV-1 and BTV-6) and virulent field origin (BTV-8) demonstrate more pronounced cytopathic effects compared to their parental strains. Some reassortant strains in addition replicated to high titres in vitro despite being composed of genome segments from slow and fast replicating parental strains. The latter result may have implications for the level of viraemia in the mammalian host and subsequent uptake and transmission of reassortant strains (and their genome segments) by Culicoides vectors. Increased rates of CPE induction could further suggest a higher virulence for reassortant strains in vivo. Overall, these findings raise questions in regards to the use of modified-live virus (MLV) vaccines and risk of reassortment in the field. To further address these questions, additional experimental infection studies using insects and/or animal models should be conducted, to determine whether these results have significant implications in vivo