Bacteriophage cocktail to target diverse lineages of multidrug-resistant …

Description (excerpt)

Attorney Docket No.15969-039PC0 Bacteriophage cocktail to target diverse lineages of multidrug-resistant Klebsiella pneumoniae FEDERAL FUNDING NOTICE The invention was made with United States Government support from Congressionally Directed Medical Research Programs (CDMRP) Peer Reviewed Medical Research Program (PRMRP) Investigator Award No. PR182667. The U.S. government has certain rights in the invention. REFERENCE TO ELECTRONIC SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on October 15, 2024, is named “15969039PC0_seq_listing.xml” and is 740,376 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND 1. Field of the Invention The present invention is directed to the field of antibacterial phages for the treatment of multidrug resistant Klebsiella pneumoniae infection, and in particular to antibacterial phage cocktails comprising newly isolated K. pneumoniae phage Ekq1. 2. Background About 1.27 million people died from drug-resistant bacterial infections in 2019, such as multidrug-resistant (MDR), extensively drug-resistant (XDR), or pan-drug resistant (PDR) ESKAPEE infections (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli), which cause a major threat to public health, especially for people who are immunocompromised or critically ill. Attorney Docket No.15969-039PC0 Klebsiella pneumoniae is a ubiquitous gram-negative bacterium and frequently causes hospital-acquired bacteremia, pneumonia, urinary tract, and other infections, with a high rate of drug resistance. The death rates of K. pneumoniae infection are 50% from pneumoniae and 20- 40% from bloodstream infection (septicemia), and WHO listed carbapenem-resistant and 3rd generation cephalosporin-resistant K. pneumoniae as a critical priority pathogen in 2024. Because of the fast global spread of multidrug-resistant (MDR) and hypervirulent K. pneumoniae, there is a growing need for alternative or adjunctive antibacterial treatments. Bacteriophages (phages) are natural enemies of bacteria and generally safe, and phage therapy has been identified as a valid approach to treat K. pneumoniae infection (Herridge et al., 2020, Eskenazi et al., 2022, and Doub et al., 2022). However, K. pneumoniae is a highly diverse pathogen, and K. pneumoniae phages usually have limited host ranges (Kęsik-Szeloch et al, 2013). Most of K. pneumoniae phages are active against 1-5% of strains, and such narrow specificity of phages and high diversity of bacterial isolates complicate the development of durable phage therapeutics. SUMMARY The purpose of the study presented in this disclosure is to design and preclinically test a phage cocktail containing a few phages targeting recent MDR K. pneumoniae isolates from military hospitals in the U.S. and other countries. It is intended here to provide a fixed and modularized cocktail of bacteriophages for the treatment of combat trauma-associated infections caused by MDR K. pneumoniae. There are problems when developing anti-bacterial phages. First, broadly phage- susceptible strains cause very limited morbidity and do not cause lethality, which is shown in Examples using a mouse lung model without carbapenems (CP) treatment (phage-neutrophil synergy). Second, hypervirulent K. pneumoniae strains are phage-resistant. However, it has been suggested that phages with broader activity can be isolated on near- neighbor species (Jensen et al., 1998 and Wu et al., 2007). Therefore, in this disclosure, a K. pneumoniae phage, EKq1 was isolated using a near-neighbor species of K. pneumoniae, i.e., Klebsiella quasipneumoniae, and the phage EKq1 was proved capable of lysing some MDR K. pneumoniae clinical isolates. Attorney Docket No.15969-039PC0 Herein, phage cocktails comprising EKq1 are introduced. Those phage cocktails comprise other 4 or 5 compatible Klebsiella phages in addition to EKq1. In one aspect, a phage cocktail comprising five Klebsiella phages (KPM1) is provided, which comprises AFR4, KEN39, KEN42, Ekq2, and Ekq1. In another aspect, a phage cocktail comprising six Klebsiella phages (KPM1-1) is provided, which comprises EKp148 in addition to the core 5 phages of KPM1. In another aspect, another six Klebsiella phage cocktail (KPM2) is introduced, which comprises phage KEN18-2-1 instead of EKp148 in addition to the core 5 phages of KPM1 In another aspect, a seven Klebsiella phage cocktail (KPM3) is introduced, which comprises a recombinant phage 15882-3 generated by in vitro phage evolution procedure using a phage mixture comprising KEN22, KEN25, KEN37, and KEN39 K. pneumoniae phages as well as phage KEN18-2-1 in addition to the core 5 phages of KPM1 The cocktails can be used as main ingredients of pharmaceutical compositions, and as standard- or adjunctive treatment against MDR, XDR, and PDR K. pneumoniae infection either alone or in combination with antibiotics. Also, methods of using the same for the treatment of respiratory infections caused by K. pneumoniae is presented. For antibacterial therapeutic purposes, other phage variants comprising a nucleic acid sequence of at least 97% sequence identity to the sequence of those phages aforementioned, or a fragment thereof can also be contemplated for being included in a phage cocktail. In a certain aspect, a phage cocktail comprising a smaller number of phages can be contemplated. That is, a 2-phage cocktail, 3-phage cocktail, or 4-phage cocktail may be considered if they infect the same range of host bacterial strains, which will reduce production cost. Further, each phage can also be used individually as an adjunctive means to ant