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UropathogenicEscherichia coli(UPEC) can undergo extensive filamentation in the host during acute urinary tract infections (UTIs). It has been hypothesised that this morphological plasticity allows bacteria to avoid host immune responses such as macrophage engulfment. However, it is still unclear what properties of filaments are important in macrophage-bacteria interactions. The aim of this work was to investigate the contribution of bacterial biophysical parameters, such as cell size and shape, and physiological parameters, such as cell surface and the environment, to macrophage engulfment efficiency. Viable, reversible filaments of known lengths and volumes were produced in the UPEC strain UTI89 using a variety of methods, including exposure to cell-wall targeting antibiotics, genetic manipulation and isolation from anin vitrohuman bladder cell model. Quantification of the engulfment ability of macrophages using gentamicin-protection assays and fluorescence microscopy demonstrated that the ability of filaments to avoid macrophage engulfment is dependent on a combination of size (length and volume), shape, surface and external environmental factors. UTI89 filamentation was also found to occur independently of the SOS-inducible filamentation genes,sulAandymfM, demonstrating the non-essential requirement of these genes for UTI89 filamentation and their ability to avoid macrophage engulfment. With several strains of UPEC now resistant to current antibiotics, our work identifies the importance of bacterial morphology during infection and may provide new ways to prevent or treat these infections via immune modulation or antimicrobials.Author SummaryUrinary tract infections (UTIs) are one of the most common bacterial infections worldwide with 50% of women suffering from a UTI during their lifetime. Escherichia coli is the primary bacteria responsible for UTIs and is usually found in short rod forms. However, during UTIs E. coli can elongate into extremely long thin shapes called ‘filaments’. Filaments are thought to be advantageous during infections because they are too long to be engulfed and killed by immune cells called macrophages. Due to increasing antibiotic resistance in bacteria there is a strong need for the discovery of new ways to treat infections and this is only possible once we thoroughly understand the mechanisms bacteria employ to overcome our immune response. Therefore, we investigated the effect of E. coli filamentation on macrophage engulfment along with other aspects of bacteria reported to influence engulfment. We found that the ability of filaments to avoid macrophage engulfment is dependent on a combination of size (length and volume), shape, surface and external environmental factors. Our research has highlighted the importance of bacterial shape changes during infections and provided a foundational understanding of macrophage engulfment of filaments. Eventually, this knowledge may reveal new targets for treatment of infections.

Experiments in murine models of urinary tract infection (UTI) show that uropathogenic Escherichia coli (UPEC) form bacterial reservoirs in the bladder tissue that can survive beta lactam antibiotics and give rise to reinfection. The observed reinfection cascade suggests intracellular bacterial persistence as a possible explanation for recurrent UTI in humans. To test this hypothesis in an animal model closer to humans, we here investigated whether UPEC infecting the bladders of experimentally inoculated pigs are able to survive standard oral mecillinam treatment. Moreover, we analyzed the infected pig bladders by microscopy for the presence of intracellular UPEC colonies.Seven pigs were experimentally inoculated with the UPEC cystitis strain, UTI89, to induce cystitis. After 5 days of infections, a 3-day oral treatment with the extracellularly active β-lactam, mecillinam, was initiated. The infection was monitored with regular urine and blood samples. When terminated, whole bladders were removed and homogenized to quantify viable intracellular bacteria. In addition, two pigs were inoculated with UTI89pMAN01 constitutively expressing green fluorescent protein and the bladders subsequently analyzed by microscopy for bacterial location and morphology.Experimental inoculation resulted in cystitis in all animals. After 3-day treatment with mecillinam, no viable UPEC were detectable in urine or bladder homogenates. Microscopy analysis of pig bladders at 12 h post infection, revealed no detectable intracellular bacterial colonies and no filamentous UPEC phenotypes were identified.Pigs experimentally infected with UPEC completely clear their infection upon mecillinam treatment, which contrasts earlier findings from similar experiments in mice. Moreover, the hallmarks of induced UTI in mice, i.e. intracellular bacterial communities and bacterial filamentation, could not be identically reproduced in a pig model of acute UTI. This result suggests that significant differences might exist between UTI in mice and larger mammals, and therefore perhaps also between mice and humans. Additional studies are needed to reveal details on the Escherichia coli acute UTI pathogenesis cascade in larger mammals to assess to which extent observations in mice can be transferred to humans.

Uropathogenic Escherichia coli (UPEC) UTI89 is a well-characterized strain, which has mainly been used to study UPEC virulence during urinary tract infection (UTI). However, little is known on UTI89 key fitness-factors during growth in lab media and during UTI. Here, we used a transposon-insertion-sequencing approach (TraDIS) to reveal the UTI89 essential-genes for in vitro growth and fitness-gene-sets for growth in Luria broth (LB) and EZ-MOPS medium without glucose, as well as for human bacteriuria and mouse cystitis. A total of 293 essential genes for growth were identified and the set of fitness-genes was shown to differ depending on the growth media. A modified, previously validated UTI murine model, with administration of glucose prior to infection was applied. Selected fitness-genes for growth in urine and mouse-bladder colonization were validated using deletion-mutants. Novel fitness-genes, such as tusA, corA and rfaG; involved in sulphur-acquisition, magnesium-uptake, and LPS-biosynthesis, were proved to be important during UTI. Moreover, rfaG was confirmed as relevant in both niches, and therefore it may represent a target for novel UTI-treatment/prevention strategies.

Most uropathogenic Escherichia coli (UPEC) express type-1 fimbriae (T1F), a key virulence factor for urinary tract infection (UTI) in mice. Evidence that conclusively associates this pilus with uropathogenesis in humans has, however, been difficult to obtain. We used an experimental porcine model of cystitis to assess the role of T1F in larger mammals more closely related to humans. Thirty-one pigs were infected with UPEC strain UTI89 or its T1F deficient mutant, UTI89ΔfimH, at inoculum titres of 102 to 108 colony forming units per millilitre. Urine and blood samples were collected and analysed 7 and 14 days post-inoculation, and whole bladders were removed at day 14 and analysed for uroepithelium-associated UPEC. All animals were consistently infected and reached high urine titres independent of inoculum titre. UTI89ΔfimH successfully colonized the bladders of 1/6 pigs compared to 6/6 for the wild-type strain. Intracellular UPEC were detectable in low numbers in whole bladder explants. In conclusion, low doses of UPEC are able to establish robust infections in pigs, similar to what is presumed in humans. T1F are critical for UPEC to surpass initial bottlenecks during infection but may be dispensable once infection is established. While supporting the conclusions from mice studies regarding a general importance of T1F in successfully infecting the host, the porcine UTI models’ natural high, more human-like, susceptibility to infection, allowed us to demonstrate a pivotal role of T1F in initial establishment of infection upon a realistic low-inoculum introduction of UPEC in the bladder.

Background: Catheter-associated urinary tract infection (CAUTI) is a frequent community-acquired infection and the most common nosocomial infection. Here, we developed a novel antimicrobial catheter concept that utilizes a silicone-based interpenetrating polymer network (IPN) as balloon material to facilitate a topical slow-release prophylaxis of antibacterial agents across the balloon to the urinary bladder.Methods: The balloon material was achieved by modifying low shore hardness silicone tubes with a hydrogel interpenetrating polymer in supercritical CO2 using the sequential method. Release properties and antibacterial efficacy of the IPN balloon treatment concept was investigated in vitro and in a porcine CAUTI model developed for the study. In the latter, Bactiguard Infection Protection (BIP) Foley catheters were also assessed to enable benchmark with the traditional antimicrobial coating principle.Results: Uropathogenic Escherichia coli was undetectable in urinary bladders and on retrieved catheters in the IPN treatment group as compared to control that revealed significant bacteriuria (>105 colony forming units/ml) as well as catheter-associated biofilm. The BIP catheters failed to prevent E. coli colonization of the bladder but significantly reduced catheter biofilm formation compared to the control.Conclusion: The IPN-catheter concept provides a novel, promising delivery route for local treatment in the urinary tract.

Urinary tract infection (UTI) is the most common bacterial infectious disease with a high frequency of recurrence and the leading cause of septicemia. In vivo experimentation has contributed significantly to the present-day knowledge on UTI pathogenesis. This research has traditionally been based on murine models of UTI. Occasional conflicting results between UTI in mice and humans and increasing skepticism toward small rodent models in general warrant the need of novel large-animal infection models that better resemble the anatomy and physiology of humans, and thus better mimic the course of infection in humans. Here, we report, to our knowledge, the first large-animal model of cystitis. The model is based on pigs, and the protocol supports the establishment of persistent, non-ascending infection in this animal and is established without invasive surgical procedures, pain, and discomfort for the animal. The course of infection is monitored by cystoscopy, microscopy of bladder biopsies, and biochemical analysis of urine and blood samples. At termination, harvested whole bladders from infected pigs are analyzed for microbiological colonization using microscopy, histology, and viable bacterial counts. The model is a useful tool in future studies of UTI pathogenesis and opens up novel possibilities to bridge the current knowledge obtained from small-animal UTI models to UTI pathogenesis in humans.

Approximately half of all nosocomial bloodstream infections are caused by bacterial colonization of vascular catheters. Attempts have been made to improve devices using anti-adhesive or antimicrobial coatings; however, it is often difficult to bind coatings stably to catheter materials, and the low amounts of drug in thin-film coatings limit effective long-term release. Interpenetrating polymer networks (IPNs) are polymer hybrid materials with unique drug release properties. While IPNs have been extensively investigated for use in tablet- or capsule-based drug delivery systems, the potential for use of IPNs in drug release medical devices remains largely unexplored. Here, we investigated the use of silicone-hydrogel IPNs as a catheter material to provide slow anti-bacterial drug-release functionality. IPN catheters were produced by the sequential method, using supercritical CO2 as a solvent to polymerize and crosslink poly(2-hydroxyethyl methacrylate) (PHEMA) in silicone elastomer. The design was tested against Staphylococcus aureus colonization after loading with dicloxacillin (DCX) alone or in combination with thioridazine (TDZ), the latter of which is known to synergistically potentiate the antibacterial effect of DCX against both methicillin-sensitive and methicillin-resistant S. aureus. The hydrophilic PHEMA component allowed for drug loading in the catheters by passive diffusion and provided controlled release properties. The drug-loaded IPN material inhibited bacterial growth on agar plates for up to two weeks and in blood cultures for up to five days, and it withstood 24 h of seeding with resilient biofilm aggregates. The combined loading of DCX + TDZ enhanced the antibacterial efficiency in static in vitro experiments, although release analyses revealed that this effect was due to an enhanced loading capacity of DCX when co-loaded with TDZ. Lastly, the IPN catheters were tested in a novel porcine model of central venous catheter-related infection, in which drug-loaded IPN catheters were found to significantly decrease the frequency of infection.

Most bacterial infections initiate at the mucosal epithelium lining the gastrointestinal, respiratory, and urogenital tracts. At these sites, bacterial pathogens must adhere and increase in numbers to effectively breach the outer barrier and invade the host. If the bacterium succeeds in reaching the bloodstream, effective dissemination again requires that bacteria in the blood, reestablish contact to distant endothelium sites and form secondary site foci. The infectious potential of bacteria is therefore closely linked to their ability to adhere to, colonize, and invade epithelial and endothelial surfaces. Measurement of bacterial adhesion to epithelial cells is therefore standard procedure in studies of bacterial virulence. Traditionally, such measurements have been conducted with microtiter plate cell cultures to which bacteria are added, followed by washing procedures and final quantification of retained bacteria by agar plating. This approach is fast and straightforward, but yields only a rough estimate of the adhesive properties of the bacteria upon contact, and little information on the ability of the bacterium to colonize these surfaces under relevant physiological conditions. Here, we present a method in which epithelia/endothelia are simulated by flow chamber-grown human cell layers, and infection is induced by seeding of pathogenic bacteria on these surfaces under conditions that simulate the physiological microenvironment. Quantification of bacterial adhesion and colonization of the cell layers is then performed by in situ time-lapse fluorescence microscopy and automatic detection of bacterial surface coverage. The method is demonstrated in three different infection models, simulating Staphylococcus aureus endothelial infection and Escherichia coli intestinal- and uroepithelial infection. The approach yields valuable information on the fitness of the bacterium to successfully adhere to and colonize epithelial surfaces and can be used to evaluate the influence of specific virulence genes, growth conditions, and antimicrobial treatment on this process.

Staphylococcus aureus is a major human pathogen in catheter-related infections. Modifying catheter material with interpenetrating polymer networks is a novel material technology that allows for impregnation with drugs and subsequent controlled release. Here, we evaluated the potential for combining this system with plectasin derivate NZ2114 in an attempt to design an S. aureus biofilm-resistant catheter. The material demonstrated promising antibiofilm properties, including properties against methicillin-resistant S. aureus , thus suggesting a novel application of this antimicrobial peptide.

IntroductionConservative treatment solutions against aortic prosthetic vascular graft infection (APVGI) for inoperable patients are limited. The combination of antibiotics with antibacterial helper compounds, such as the neuroleptic drug thioridazine (TDZ), should be explored.AimTo investigate the efficacy of conservative systemic treatment with dicloxacillin (DCX) in combination with TDZ (DCX+TDZ), compared to DCX alone, against early APVGI caused by methicillin-sensitive Staphylococcus aureus (MSSA) in a porcine model.MethodsThe synergism of DCX+TDZ against MSSA was initially assessed in vitro by viability assay. Thereafter, thirty-two pigs had polyester grafts implanted in the infrarenal aorta, followed by inoculation with 10(6) CFU of MSSA, and were randomly administered oral systemic treatment with either 1) DCX or 2) DCX+TDZ. Treatment was initiated one week postoperatively and continued for a further 21 days. Weight, temperature, and blood samples were collected at predefined intervals. By termination, bacterial quantities from the graft surface, graft material, and perigraft tissue were obtained.ResultsDespite in vitro synergism, the porcine experiment revealed no statistical differences for bacteriological endpoints between the two treatment groups, and none of the treatments eradicated the APVGI. Accordingly, the mixed model analyses of weight, temperature, and blood samples revealed no statistical differences.ConclusionConservative systemic treatment with DCX+TDZ did not reproduce in vitro results against APVGI caused by MSSA in this porcine model. However, unexpected severe adverse effects related to the planned dose of TDZ required a considerable reduction to the administered dose of TDZ, which may have compromised the results.

Staphylococcus aureus is able to disseminate from vascular device biofilms to the blood and organs, resulting in life-threatening infections such as endocarditis. The mechanisms behind spreading are largely unknown, especially how the bacterium escapes immune effectors and antibiotics in the process. Using an in vitro catheter infection model, we studied S. aureus biofilm growth, late-stage dispersal, and reattachment to downstream endothelial cell layers. The ability of the released biofilm material to resist host response and disseminate in vivo was furthermore studied in whole blood and phagocyte survival assays and in a short-term murine infection model. We found that S. aureus biofilms formed in flow of human plasma release biofilm thromboemboli with embedded bacteria and bacteria-secreted polysaccharides. The emboli disseminate as antibiotic and immune resistant vehicles that hold the ability to adhere to and initiate colonisation of endothelial cell layers under flow. In vivo experiments showed that the released biofilm material reached the heart similarly as ordinary broth-grown bacteria but also that clumps to some extend were trapped in the lungs. The clumping dispersal of S. aureus from in vivo-like vascular biofilms and their specific properties demonstrated here help explain the pathophysiology associated with S. aureus bloodstream infections.

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