No abstract

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.

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.

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.

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.

The worldwide increase of antimicrobial resistance (AMR) is a serious threat to human health. To avert the spread of AMR, fast reliable diagnostics tools that facilitate optimal antibiotic stewardship are an unmet need. In this regard, Raman spectroscopy promises rapid label- and culture-free identification and antimicrobial susceptibility testing (AST) in a single step. However, even though many Raman-based bacteria-identification and AST studies have demonstrated impressive results, some shortcomings must be addressed. To bridge the gap between proof-of-concept studies and clinical application, we have developed machine learning techniques in combination with a novel data-augmentation algorithm, for fast identification of minimally prepared bacteria phenotypes and the distinctions of methicillin-resistant (MR) from methicillin-susceptible (MS) bacteria. For this we have implemented a spectral transformer model for hyper-spectral Raman images of bacteria. We show that our model outperforms the standard convolutional neural network models on a multitude of classification problems, both in terms of accuracy and in terms of training time. We attain more than 96% classification accuracy on a dataset consisting of 15 different classes and 95.6% classification accuracy for six MR–MS bacteria species. More importantly, our results are obtained using only fast and easy-to-produce training and test data.

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.