Abstract :
[en] Klebsiella pneumoniae, a Gram-negative bacterium, is divided into two distinct pathotypes: the classical (cKp), associated with nosocomial infections, and the hyper-virulent (hvKp), targeting healthy individuals, both exhibiting mechanisms of antibiotic resistance (Gu et al., 2018; Russo et al., 2018; Russo & Marr, 2019; Snitkin et al., 2012). In this context, this thesis focuses on exploring phage therapy, a treatment modality based on the use of phages, as a potential response to the urgency of finding new therapeutic approaches.
The first objective of this thesis was to isolate new phages, that could potentially be used to fight cKp K. pneumoniae infections. Specifically, two phages were isolated against a strain responsible for cystitis, with K3 capsular type and belonging to ST13: vB_KpnP_K3-ULINTkp1 and vB_KpnP_K3-ULINTkp2. They underwent thorough characterization to assess their potential in phage therapy. The results revealed that both phages replicated in vivo and improved the survival of infected and treated Galleria mellonella larvae, with a better efficiency for ULINTkp2. Moreover, ULINTkp2 had the ability to lyse different Klebsiella strains, while ULINTkp1 seemed specific to K3 strains. They also exhibited relatively short attachment and replication times. In silico, no lysogeny or virulence genes were identified. Additionally, these phages demonstrated good stability in an environment simulating urine. These results pave the way for further in-depth research on these phages, exploring their potential for treating K. pneumoniae infections, especially cystitis, which are becoming increasingly resistant to conventional treatments.
The second objective of this thesis was to study the impact of the phage vB_KpnP_K1-ULIP33 on the intestinal microbiota in the context of hvKp K. pneumoniae with K1 capsular type. This is crucial, especially for oral treatments. The in vitro model, called the simulator of the human intestinal microbial ecosystem (SHIME®), which simulates the digestive tract from the stomach to the descending colon, was used (Molly et al., 1994). The results firstly revealed that the phage dissipated more quickly than predicted by mathematical simulations, assuming the absence of interaction between the phage and its host. Regarding intestinal health, the production of short-chain fatty acids and the quantification of various health intestinal biomarkers remained stable, indicating a healthy simulated intestinal microbiota. Despite taxonomic differences between the repetitions of the experiment, the study is as a proof of concept for phage therapy in a long-term SHIME® model, and demonstrated the low impact of the phage ULIP33 on the intestinal microbiota. This opens the door to future research on the application of ULIP33 in phage therapy.
The third objective of this thesis was to explore the potential of the phage ULIP33 as a preventive intestinal decolonization tool for its hvKp K1 ST23 host bacterium in the SHIME® model. In the hospital setting, a significant percentage of patients are colonized by antibiotic-resistant enterobacteria, such as K. pneumoniae. Regarding this, K. pneumoniae K1 was injected into the system to simulate a microbiota hosting such bacteria. Then, ULIP33 was inoculated to assess its potential as a decolonizer. The results revealed different intestinal microbiota implantation in each repetition. In repetition 2, the phage disappeared more rapidly than expected by the mathematical model, suggesting, as a first hypothesis, an absence of interaction with its host and a possible poor implantation of the bacterium. As a second hypothesis, it is conceivable that the phage replicated rapidly, resulting in the eradication of the bacterium, followed by the phage's elimination from the system. In repetitions 1 and 3, the phage persisted and replicated, with bacterial concentration decreasing at certain time points. However, an increase in bacterial concentration was observed at the end of the experiment. Moreover, in repetition 3, this increase in bacterial concentration was more pronounced than that observed when the bacterium was injected alone (experiment conducted in parallel as a control). Several hypotheses could explain these phenomena, including the emergence of phage-resistant bacteria or biofilm production. Additionally, in repetition 1, the bacterium implanted better in the experiment with the phage than in the control experiment. A plausible explanation is the impact of ULIP33 on non-host bacteria in the microbiota, creating a favorable ecological niche for the implantation of exogenous K. pneumoniae. In conclusion, the potential emergence of phage-resistant bacteria and biofilms in the SHIME® system following the addition of ULIP33, in a simulated microbiota hosting its host bacterium, does not favor its role as a therapeutic agent in the field of phage therapy.
This thesis has deepened the understanding of phages and their potential in fighting K. pneumoniae infections, whether of the cKp or hvKp pathotype. The objectives were covered with a methodical approach, ranging from the isolation of new phages to their thorough characterization, studying their impact on the intestinal microbiota, and their potential as a decolonization tool. All the results obtained highlight the importance of continuing research to better understand the interaction mechanisms between phages and their hosts, as well as the potential implications of phage therapy in the context of the intestinal microbiota.
