Establishing the efficacy of phage therapy for multi-drug resistant Klebsiella pneumoniae urinary tract infections

Rationale:
Antimicrobial resistance (AMR) is a major health care and societal concern and was the cause of up to 4.5 million deaths, worldwide, in 2019. Klebsiella pneumoniae contributes significantly to the acquisition and transmission of antibiotic resistance and is the 3rd cause of death associated with AMR in the world [1]. In France, K. pneumoniae is the 3rd leading cause of urinary tract infection (UTI), with prevalence increasing every year [2].
Among healthy individuals, 60% of women and 5-14% of men will experience UTI in their lifetime, with up to 44% recurring within six months [3]. In people with bladder dysfunction due to peripheral or central neurological lesions (neurobladder), UTI account for almost half of healthcare-associated infections with a major negative impact on morbidity and mortality [4]. In this population, K. pneumoniae is one of the two most prevalent multi-drug resistant (MDR) uropathogens [5]. Critically, MDR neurobladder UTI are associated with an increased risk of treatment failure and kidney failure, prolonged hospital stays, and higher morbidity and mortality [5-7]. Thus, uropathogen drug resistance is a major clinical challenge that will only become more difficult to manage as antibiotic resistance spreads. One way to address this challenge is by the development of non-antibiotic-based therapies to manage recurrent UTI in these vulnerable patients. The use of therapeutic bacteriophages represents one such promising alternative. However, data from in vivo UTI models and use in humans is limited.

Objectives:
The global objective of this study is to demonstrate the efficacy of bacteriophages targeting clinical MDR K. pneumoniae strains from neurobladder UTI in a preclinical model of UTI, as a crucial step to validate their use in patients. Specifically, the PhD student will (1) select, sequence, analyze the in vitro interactions of phages with Klebsiella pneumoniae strains isolated from neurobladder patients with recurrent UTI, to define the best phage cocktail that kills Klebsiella pneumoniae, (2) optimize a preclinical mouse MDR Klebsiella pneumoniae UTI model to assess the efficacy of the phage cocktail, and (3) assess immune responses to phage therapy in vivo.

Methodology:
In Aim 1, isolated bacteriophages targeting MDR Klebsiella pneumoniae clinical strains will be characterized by sequence, burst size, efficiency of plating, pH, and temperature stability. Mutant bacteria resistant to the bacteriophages will be isolated and their genomes sequenced to identify the receptors of these bacteriophages. Together these analyses will define cocktails of active bacteriophages targeting different bacterial receptors. Cocktail efficacy will be increased using in vitro targeted evolution by the Appelmans protocol [8]. The stability and efficiency of the optimized bacteriophages will be tested in urine and in an in vivo Galleria mellonella larvae infection model. This work will be performed in the CIMI laboratory (Dr Eckert) and is estimated to take 12-18 months.
In Aim 2, to establish an MDR Klebsiella pneumoniae UTI model, strains from neurobladder patients will be intravesically instilled via catheter into female and male mouse bladders, using a protocol routinely used in the Pasteur lab [9]. Infection efficacy will be assessed by determining bladder bacterial load at specific timepoints post-infection (e.g., 1, 2, 7, 14, 28 days post-infection). Resolution kinetics will be determined by urine sampling. Up to 5 MDR Klebsiella pneumoniae strains will be tested to identify strains that achieve at least 105 CFU per bladder at 24 hours to enable assessment of bacterial burden reduction after phage therapy. Phage activity will be assessed by treating mice at different time points (e.g., 1, 2, 7 days post-infection). Phages will be given by intraperitoneal or intravesical injection, alone or with antibiotics and bladders will be collected 24 hours post-treatment to determine bacterial burden and number of phages in comparison to untreated groups.
For Aim 3, bladder homogenates will be collected for analysis of cytokines by multi-analyte assays (e.g. Luminex). Flow cytometry will be used to determine whether phage therapy alters the cellular immune response. Aims 2 and 3 will be performed in the Institut Pasteur lab (Dr Calin) and is estimated to take 18-24 months.

Expected results and perspectives:
While Aim 3 depends upon Aim 1 and 2, we are confident this project is feasible as Dr Calin already established that at least three Klebsiella pneumoniae strains isolated from neurobladder patients infect mice to levels that will allow assessment of whether phage therapy is efficacious. Additionally, at least 40 phages that target Klebsiella pneumoniae strains have been identified and will be tested against patient MDR Klebsiella pneumoniae strains (CIMI lab, Dr Eckert). We expect that this work will identify and validate a phage cocktail with efficacy against MDR Klebsiella pneumoniae in a robust preclinical model, and establish whether this approach synergizes with host immune responses, providing a foundation for the development of a clinical trial to treat neurobladder patients with phages. Both the CIMI team and the Institut Pasteur team investigate new therapeutic approaches to fight antibiotic-resistant bacterial infections. Dr Eckert is a clinical microbiology specialist with extensive expertise in antimicrobial resistance and Klebsiella pneumoniae phage therapy. Dr Calin is an infectious diseases physician with extensive clinical expertise in the management of relapsing UTI in complicated clinical settings, such as neurobladder. Dr Calin is currently working on mouse UTI models in Dr Ingersoll’s team “Mucosal Inflammation and Immunity” at Institute Pasteur and developed the MDR Klebsiella pneumoniae UTI mouse model. The PhD candidate will learn diverse skills and techniques in the CIMI and Pasteur environment, working with BSL2 pathogens and performing imaging with spinning disk microscopy, flow cytometry, mass spectrometry, and bioinformatics in the two labs and in technical core facilities.

[1] C. J. L. Murray et al., Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis, The Lancet, 2022, link
[2] SPF, Enquête nationale de prévalence des infections nosocomiales et des traitements anti-infectieux en établissements de santé, mai-juin 2017. link
[3] Y.-C. Chen et al., Emerging Non-Antibiotic Options Targeting Uropathogenic Mechanisms for Recurrent Uncomplicated Urinary Tract Infection, Int. J. Mol. Sci., 2023, link
[4] M. E. García Leoni et al., Management of urinary tract infection in patients with spinal cord injuries, Clin. Microbiol. Infect., 2003, link
[5] V. Šámal et al., The prevalence of antibiotic-resistant and multidrug-resistant bacteria in urine cultures from inpatients with spinal cord injuries and disorders: an 8-year, single-center study, BMC Infect. Dis., 2022, link
[6] M. H. Rabadi et al., Predictors of Mortality in Veterans with Multiple Sclerosis in an Outpatient Clinic Setting, Int. J. MS Care, 2017, link
[7] C.-Y. Hsiao et al., Risk Factors for Development of Septic Shock in Patients with Urinary Tract Infection, BioMed Res. Int., 2015, link
[8] B. H. Burrowes et al., Directed in Vitro Evolution of Therapeutic Bacteriophages: The Appelmans Protocol, Viruses 2019 link
[9] A. Zychlinsky Scharff et al., Sex differences in IL-17 contribute to chronicity in male versus female urinary tract infection, JCI Insight, 2019, link

The Centre for Immunology and Microbial Infections (Cimi) is a mixed research unit created in 2014. It is affiliated to Sorbonne University, Inserm and CNRS. The Cimi is located on the campus of Sorbonne University’s Faculty of Medicine at the Pitié-Salpêtrière site in Paris.

The research conducted at the Cimi explores  how the immune system functions and host-pathogen interactions, but also aims to develop immune-based therapeutic strategies and new ways to fight infectious diseases.

Our team (EDIRA) mainly aims to elucidate the mechanisms of emergence and diffusion of multiple antimicrobial resistance (AMR) focusing on the Multi Drug Resistance (MDR) Mycobacteria, including Mycobacterium tuberculosis (Mtb) and MDR Enterobacteriaceae, especially Klebsiella pneumoniae species.

The team also uses several specific infection models to evaluate the in vivo activity of new antibiotic- or non-antibiotic (such as bacteriophages) -based strategies able to treat mycobacteria infections, or prevent / suppress colonisation by AMR Enterobacteriaceae.

The IP team : https://institutcochin.fr/en/equipes/mucosal-inflammation-and-immunity

How does inflammation impact mucosal immunity in the bladder? A balanced inflammatory response is crucial to fight disease while preventing collateral damage. Inflammation drives pathogen clearance, regulates responses to non-infectious disease, and establishes immune memory, whereas inappropriate inflammation induces disease susceptibility or tissue damage. This is especially true in mucosal tissues, which form a critical barrier against the external environment.
The bladder mucosa is highly vulnerable to urinary tract infection (UTI), cancer, and chronic inflammatory diseases of unknown etiology, which all display sex differences in their prevalence. Notably, multidrug resistant uropathogens make up 3 of the top 5 bacteria on the WHO priority list of organisms needing new antibiotics. Thus, in the absence of new non-antibiotic therapies, UTI may go from difficult-to-treat to impossible-to-treat. Bladder cancer is the 5th most common cancer in men and the 11th in women, and is one of the most expensive cancers to treat due to its recurrent nature.
Host-directed immunotherapies may offer new treatment options for bladder diseases. Our work examines innate and adaptive immune responses to UTI and bladder cancer to identify targets for immunomodulation to treat multi-drug resistant UTI and to understand the response to immunotherapies for bladder cancer through fundamental and translational research. We find that a balance of three factors drives “ideal inflammation” in the bladder: the impact of innate immune responses on memory, sex-mediated immune differences, and the bladder microenvironment. Our research projects focus on (1) how innate immune responses shape adaptive immunity, (2) the influence of sex differences on immunity, and (3) (immuno)modulation of the microenvironment to optimize bladder health. Commonalities with other mucosa suggest that our discoveries may extend more broadly to infection or cancer in other mucosal tissues.

M2 degree with skills in bacteriology, knowledge with phages will be a plus.
Experience with work in L2/A2 environment. Experience with animal experimentation is a plus. WIllingness to work with mice is necessary.
English read, written and spoken essential, level B2 minimum,
Capacity to work autonomously and independently in a team environment
Enthusiasm, flexibility, capacity to interact with academic and industrial partners