Innovative Antibiotic Combinations for Hypervirulent Klebsiella pneumoniae - PROJECT SUMMARY/ABSTRACT Hypervirulent Klebsiella pneumoniae (hvKP) can cause life-threatening infections in otherwise healthy people that are associated with high mortality rates of up to 40%. In contrast to hvKP, infections caused by classical K. pneumoniae (cKP) are often restricted to immunocompromised individuals. Increased production of capsular polysaccharide (CPS) is a primary driver of hypervirulence in KP, which enables the bacteria to evade the host immune response, including the complement system. Although hvKP have historically remained susceptible to most antibiotics, multidrug resistance and hypervirulence have recently converged in K. pneumoniae (MDR- hvKP). Increased CPS production by MDR-hvKP may also directly compromise antibiotics by prohibiting them from binding to the bacterial outer membrane or by reducing target site penetration. The traditional approach to select antibiotics for cKP infections is often applied to the treatment of hvKP, focusing primarily on in vitro susceptibilities. However, this approach fails to consider other unique characteristics of the bacteria, such as CPS levels, and has never been validated for MDR-hvKP. Our central hypothesis is that excess CPS expression in MDR-hvKP reduces the activity of certain antibiotics, which can be overcome with targeted therapy that maximizes bacterial killing and capsule inhibition. In preliminary studies, we generated isogenic strains that displayed different levels of CPS from their parent MDR-hvKP clinical isolate. Then we evaluated the response of these strains to a standard-of-care antibiotic (ceftazidime/avibactam) in the hollow fiber infection model and showed that increased CPS led to diminished antibiotic activity, despite no changes in MIC. We also found that pre-treatment with subinhibitory concentrations of the polymyxins significantly decreased CPS, which then sensitized KP to killing by the complement system and ceftazidime/avibactam. Thus, treatment approaches for MDR-hvKP that combine antibiotics with a CPS-inhibitor adjuvant are highly promising. Leveraging an innovative approach, this proposal will assess the interaction between antibiotics and CPS to facilitate rational development of combinations for MDR-hvKP. In Aim 1, we will elucidate the interplay between capsule and antibiotics. Antibiotics that retain maximal activity in the presence of high CPS production and adjuvants that decrease CPS will be identified. In Aim 2, we will rationally develop and validate a combination treatment approach for MDR-hvKP that maximizes synergy between a CPS-inhibitor adjuvant and antibiotic. CRISPR interference (CRISPRi) will be used to verify that inhibition of the CPS synthesis pathway is a viable target and that its inhibition can sensitize bacteria to killing by antibiotics and complement. Leading combinations will be evaluated in a pre-clinical mouse model. This project will generate significant and novel insights into the interplay between CPS and antibiotic exposure in MDR-hvKP to facilitate rational development of antibiotic therapies.
