Methods of Identifying Bacteriophages that can Infect and Kill Host-Adapted Pathogenic Bacteria

Abstract

The subject matter of the instant invention relates to methods of enhancing harvesting of phages against a targeted host bacteria, as well as methods of identifying phages likely to have an enhanced propensity to infect and kill an infectious pathogenic bacteria in vivo, from samples comprising phages. The invention also relates to phage libraries, pharmaceutical compositions, methods of treatment, and phage-based diagnostic methods and methods of detecting bacteria related thereto.

What is claimed is: 
     
         1 . A phage library comprising phages harvested against targeted host bacteria from a sample comprising said phages, said harvesting method comprising culturing aliquots of said sample in a plurality of in vitro cultures comprising said targeted host bacteria in various concentrations of homogenates of mammalian organ, muscle, and bone. 
     
     
         2 . The phage library of  claim 1 , wherein said culturing step produces one or more changes in the targeted host bacteria that occurs in vivo during host-adaptation. 
     
     
         3 . A phage library comprising phages with enhanced propensity to infect and kill an infectious pathogenic bacteria in vivo, wherein said phages are identified according to a method comprising:
 a. culturing the infectious pathogenic bacteria in a plurality of in vitro cultures comprising various concentrations of homogenates of mammalian organ, muscle, and bone;   b. culturing a sample comprising phages in said plurality of in vitro cultures from step a; and   c. assaying said plurality of in vitro cultures to identify phages that can infect and kill the infectious pathogenic bacteria in vitro in said various concentrations of homogenates of mammalian organ, muscle and bone.   
     
     
         4 . The phage library of  claim 2  or  claim 3 , wherein said culturing produces changes in expression of one or more genes encoding bacterial surface features used as phage receptors. 
     
     
         5 . The phage library of  claim 1  or  claim 3 , wherein said phage library is against a MDR bacterial pathogen selected from the group consisting of  Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa , and  Enterobacter  spp. 
     
     
         6 . The phage library of  claim 1  or  claim 3 , wherein the sample comprising phages comprises one or more phages found in nature. 
     
     
         7 . The phage library of  claim 1  or  claim 3 , wherein the sample comprising phages is collected from one or more natural and/or man-made sources. 
     
     
         8 . The phage library of  claim 7 , wherein said one or more natural and/or man-made sources is selected from the group consisting of soil, water treatment plants, raw sewage, sea water, lakes, rivers, streams, cesspools, animal intestines, human intestines, manure or other fecal matter, organic substrates, biofilms, and medical/hospital sources. 
     
     
         9 . The phage library of  claim 1  or  claim 3 , wherein the culturing is under further various conditions selected from the group consisting of temperature, time, osmotic pressure, pH, CO2 percentage, O2 percentage, nutrient concentration(s), carbon source(s), carbon source concentration(s), growth factor concentration(s), hormone concentration(s), in vitro culture surface characteristics, and concentration of inducer(s) of bacterial virulence factors. 
     
     
         10 . The phage library of  claim 9 , wherein the nutrients are selected from the group consisting of amino acids, carbohydrates, vitamins, and minerals. 
     
     
         11 . The phage library of  claim 10 , wherein the minerals are selected from the group consisting of iron and magnesium. 
     
     
         12 . The phage library of  claim 1  or  claim 3 , wherein the mammalian organ, muscle, and bone are from a mouse. 
     
     
         13 . The phage library of  claim 1  or  claim 3 , wherein the various concentrations of homogenates of mammalian organ, muscle, and bone are 5-25% by weight of said plurality of in vitro cultures. 
     
     
         14 . The phage library of  claim 1  or  claim 3 , wherein the organ is selected from the group consisting of liver, brain, heart, spleen, and kidney. 
     
     
         15 . The phage library of  claim 1  or  claim 3 , wherein the plurality of in vitro cultures further comprise one or more additional culture additives selected from the group consisting of whole or fractionated mammalian serum, whole or fractionated mammalian plasma, and whole mammalian blood. 
     
     
         16 . The phage library of  claim 15 , wherein said whole or fractionated mammalian serum is selected from the group consisting of human serum, animal serum, and a combination thereof. 
     
     
         17 . The phage library of  claim 15 , wherein said whole or fractionated mammalian serum is added to said plurality of in vitro cultures at a concentration of 0-15%. 
     
     
         18 . The phage library of  claim 17 , wherein the concentration is 7.5%. 
     
     
         19 . The phage library of  claim 16 , wherein the animal serum is fetal bovine serum (FBS). 
     
     
         20 . The phage library of  claim 15 , wherein said whole or fractionated mammalian plasma is selected from the group consisting of human plasma, animal plasma, and a combination thereof. 
     
     
         21 . The phage library of  claim 15 , wherein said whole or fractionated mammalian plasma is added to said plurality of in vitro cultures at a concentration of 0-15%. 
     
     
         22 . The phage library of  claim 21 , wherein the concentration is 7.5%. 
     
     
         23 . The phage library of  claim 15 , wherein said whole mammalian blood is selected from the group consisting of human blood, animal blood, and a combination thereof. 
     
     
         24 . The phage library of  claim 15 , wherein said whole mammalian blood is added to said plurality of in vitro cultures at a concentration of 0-15%. 
     
     
         25 . The phage library of  claim 24 , wherein the concentration is 5%. 
     
     
         26 . The phage library of  claim 23 , wherein the animal blood is sheep blood. 
     
     
         27 . The phage library of  claim 15 , wherein said fractionated mammalian serum and said fractionated mammalian plasma may be fractionated by heat, centrifugation, or biochemically using column chromatography prior to addition to the plurality of in vitro cultures. 
     
     
         28 . The phage library of  claim 15 , wherein said whole or fractionated mammalian serum and said whole or fractionated mammalian plasma may or may not be heat inactivated. 
     
     
         29 . A composition comprising one or more phages of the phage library of  claim 1  or  claim 3 . 
     
     
         30 . A method of treating a bacterial infection in a subject in need thereof comprising administering to the subject an effective amount of the composition of  claim 29 . 
     
     
         31 . A method of diagnosing or detecting bacteria in a biotic or abiotic sample comprising using one or more phages of the phage library of  claim 1  or  claim 3 .