Patent Application
20230074892
Widespread and inappropriate uses of chemical antibiotics have selected for multi-drug resistant (MDR) bacterial pathogens, presenting more frequently in human infections and contributing significantly to morbidity. Some bacteria even show evolved resistance to ‘drugs of last resort’, resulting in emergent strains that are pan-drug-resistant (PDR). One example is the Gram-negative bacterium, Pseudomonas aeruginosa, a prevalent opportunistic MDR pathogen that is poised to become a common PDR disease problem. Humans readily encounter P. aeruginosa, which thrives in both natural and artificial environments, varying from lakes and estuaries to hospitals and household sink drains. P. aeruginosa causes biofilm-mediated infections, including catheter associated urinary tract infections, ventilator associated pneumonia, and infections related to mechanical heart valves, stents, grafts and sutures (Cole, S. J., et al., Infection and Immunity 82, 2048-2058 (2014)). Individuals with cystic fibrosis, severe burns, surgical wounds and/or compromised immunity are particularly at risk for P. aeruginosa infections, especially acquired in hospitals.
P. aeruginosa is a ubiquitous Gram-negative, rod-shaped bacterium prevalent in natural and artificial environments (Remold, S. K., et al. Microb Ecol. 62(3), 505-17 (2011)). Adaptation to different habitats has allowed P. aeruginosa to persist in many human-associated environments, most notably in hospitals, where it is increasingly associated with nosocomial infections (Emori, T. G., et al., Clin Microbiol Rev. 6(4), 428-42 (1993)). These infections are difficult to manage, in part due to intrinsic antibiotic resistance resulting from decreased membrane permeability, active antibiotic efflux, and other chromosomally encoded enzymes. Further complicating the problem of P. aeruginosa infections are their ability to form biofilms, herein referred to as “P. aeruginosa biofilms” or “Pseudomonas aeruginosa biofilms”. Biofilm-mediated infections are notoriously difficult to manage, having seemingly much higher resistance to chemical antimicrobials (Stewart, P. S., et al., Lancet 358(9276), 135-08 (2001)) and often form following sub-lethal concentrations of antibiotics (Hoffman, L. R., et al., Nature 436(7054), 1171-5 (2005)). This elevated resistance may be due to exopolymeric substances in the biofilm matrix that slow diffusion of antibiotics and reduce effective concentrations. Furthermore, slow-growing cells present in the biofilm (e.g., persister cells) may have sufficiently reduced metabolisms to withstand bacteriostatic antibiotics that target metabolically active bacteria (Lewis, K. Biochemistry (Mosc). 70(2), 267-74 (2005)). As a result, biofilms may also act as a reservoir for the dissemination of infections throughout the body which could greatly prolong infection duration and severity. Prosthetic vascular graft infections are of significant concern due to the elevated mortality and morbidity rates. A common culprit, P. aeruginosa, presents a serious challenge due to its intrinsic antibiotic resistance and ability to form biofilms on prosthetic material.
Prosthetic vascular graft infections are catastrophic events which present serious challenges to surgeons and place heavy economic burdens on patients and the healthcare system. The reported incidence can vary from 0.6% to 9.5% depending on the site of the vascular graft (Kieffer, E., et al., J Vasc Surg. 33(4), 671-8 (2001); Schild, A. F., et al., J Vasc Access 9(4), 231-5 (2008)). There are currently no clear algorithms for the management of prosthetic vascular graft infections. The basic principles, however, involve systemic antibiotics, debridement of infected tissue, partial or complete graft excision, and secondary revascularization (Bunt, T. J. Cardiovasc Surg. 9(3), 225-33 (2001)). However, many patients presenting with vascular graft infections have significant comorbidities and are often critically ill, making surgical management even more difficult and in certain cases ill-advised. Despite best management, mortality and morbidity rates remain high with conservative estimates for both over 20% (O'Connor, et al., S. J Vasc Surg. 44(1), 38-45 (2006); Perera, G. B., et al., Vasc Endovascular Surg. 40(1), 1-10 (2006)). Reinfection rates are also significant after initial treatment, highlighting the inadequacy of current treatment modalities at eradicating the infecting organism. As the number of procedures involving vascular grafts continues to rise with an aging population and increasing prevalence of atherosclerosis and diabetes, new strategies are sorely needed.
P. aeruginosa infections are notoriously difficult to manage due to low antibiotic permeability of the outer membrane and mechanisms of antibiotic resistance that allow cross resistance to multiple classes and types of antibiotics. Clinically significant levels of antibiotic resistance are mostly caused by interplay between the efficient outer membrane (OM) permeability barrier, ubiquitous periplasmic β-lactamases, and multi-drug resistance (MDR) efflux pumps. These pumps have broad substrate specificity and may act synergistically with the permeability barrier to result in significant intrinsic resistance to many antimicrobials. These pumps expel the antimicrobial from the cell into the surrounding space, and the antimicrobials then have to pass through the OM permeability barrier to regain entry to the cell. Thus, the MDR pumps can effect significant resistance even when their transporter activity is quite low, as long as the OM functions as an effective barrier.
Synergy between efflux and the permeability barrier is necessary for effective drug resistance. Efflux pumps are transport proteins that are found in both Gram-positive and -negative bacteria, as well as in eukaryotic organisms. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multi-drug resistance (MDR). Efflux pumps can also impact iron uptake, bile tolerance, quorum sensing, and other host colonization factors.
In the bacteria domain, there are five major families of efflux transporter: MF (major facilitator), MATE (multidrug and toxic efflux), RND (resistance-nodulation-division), SMR (small multidrug resistance) and ABC (ATP binding cassette). All these systems utilize the proton motive force as an energy source, apart from the ABC family, which utilizes ATP hydrolysis to drive the export of substrates. Transporters that efflux multiple substrates, including antibiotics, did not arise in response to the stresses of the antibiotic era. All bacterial genomes studied contain several different efflux pumps; this indicates their ancestral origins. It has been estimated that ˜5-10% of all bacterial genes are involved in transport and a large proportion of these encode efflux pumps.
The interaction between selection from antibiotics and phages, along with its role in driving bacterial evolution, remain unclear, in part because these interactions depend on the environment, specific phage species, and the bacterial hosts involved in these interactions. Potential evolutionary interactions between drug resistance and phage resistance mechanisms in bacteria have been previously identified, including both positive and negative interactions that are highly genotype-dependent. For example, Pseudomonas aeruginosa bacteria that evolve resistance to phage 14/1 simultaneously become more resistant to antibiotics, whereas P. aeruginosa that evolve resistance to phage tivp become less resistant to antibiotics.
Escherichia coli, bacteria that evolve resistance to phage TLS also lose antibiotic resistance. Such interactions demonstrate that multiple selection pressures sometimes cause bacteria to evolve mutations with trade-up potential (the ability to increase fitness on two traits simultaneously), whereby phages contribute to the problems of increased antibiotic resistance and virulence; in other cases, the mutations have trade-off potential, whereby phages reduce the problem of antibiotic resistance. In both cases of trade-offs and trade-ups, the mutation selected for one function has a pleiotropic effect on another function. This type of pleiotropy, sometimes called molecular gene pleiotropy occurs when a single gene affects multiple traits. For example, the evolution of bacterial resistance to phage can occur via mutations that also decrease cellular resource acquisition (a form of antagonistic pleiotropy), and mutations that improve bacterial growth on one carbon source may improve fitness during growth on another (a form of synergistic pleiotropy). In these ways, pleiotropy can have significant effects on evolution within bacterial populations, shifting phenotypes that are not directly selected.
Pleiotropy in the evolution of phage resistance and antibiotic resistance can result in either decreased (antagonistic pleiotropy) or increased (synergistic pleiotropy) sensitivity to antibiotics. Bacteria-phage interactions can be highly dependent on cell membrane proteins and other surface structures, such as outer membrane proteins and lipopolysaccharides, and in some cases, those structures also contribute to antibiotic resistance. In particular, multi-drug efflux pumps are protein complexes spanning the inner and outer membranes of some bacteria, such as the homologous TolC-AcrAB system in E. coli and OprM-MexAB system in P. aeruginosa. These efflux systems confer resistance to multiple antibiotics, acting as generalized transporters for multiple antibiotic classes as well as detergents, dyes, and bile acids. The outer membrane protein (OMP) components (TolC or OprM) are membrane-spanning beta barrels, with peptide loops that extend outside of the cell. The extracellular loops of OMPs are frequently exploited by phages as the specific binding sites for initiating phage infection. When phages use these OMPs as receptors, bacteria face selection for reduced or modified OMPs. Phages that use OMPs involved in antibiotic resistance, like the previously-characterized TolC-targeting phage TLS, might impose selection on bacterial populations to evolve phage resistance while pleiotropically losing antibiotic resistance.
A need exists in the art to develop alternative methods for the management of antibiotic efflux of MDR in bacteria, like P. aeruginosa and E. coli. The present invention addresses this need.
As described herein, the present invention relates to compositions comprising bacteriophage OMKO1, LPS-5, TIVP-H6, LPS-TLTL, TIVP-27, SFA1-1, SF60B, SFNHSI, SF37B, PG-I1, and U136B, and methods of use thereof.
In one aspect, the invention provides a method of increasing antibiotic sensitivity and/or decreasing virulence in a pathogenic bacteria. The method comprises contacting the bacteria with a lytic bacteriophage selected from the group consisting of OMKO1, LPS-5, TIVP-H6, LPS-TLTL, TIVP-27, SFA1-1, SF60B, SFNHSI, SF37B, PG-I1, and U136B.
In certain embodiments, the bacteria are contacted with bacteriophage at a multiplicity of infection of bacteriophage to bacteria in the range of about 0.05 to about 50.
In certain embodiments, the bacteriophage binds at least one molecule selected from the group consisting of an O-antigen, efflux pump, Type IV pilus, LPS (core), OmpA, OmpC, TolC, and peptidoglycan in the bacteria. In certain embodiments, the bacteriophage binds TolC and LPS.
In certain embodiments, the bacteriophage binds a protein of a Mex efflux pump. In certain embodiments, the Mex efflux pump is a surface exposed protein. In certain embodiments, the Mex protein is selected from the group consisting of OprM, MexA, MexB, MexX, and MexY.
In certain embodiments, the method further comprises contacting the pathogenic bacteria with an antibiotic.
In certain embodiments, the pathogenic bacteria is drug resistant. In certain embodiments, the pathogenic bacteria is a multi-drug resistant (MDR) bacteria.
In certain embodiments, the bacteriophage comprises a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-13.
In certain embodiments, the bacteriophage is administered to a subject in need thereof.
In another aspect, the invention provides a pharmaceutical composition comprising one or more lytic bacteriophages selected from the group consisting of OMKO1, LPS-5, TIVP-H6, LPS-TLTL, TIVP-27, SFA1-1, SF60B, SFNHSI, SF37B, PG-I1, and U136B.
In certain embodiments, the bacteriophage binds at least one molecule selected from the group consisting of an O-antigen, Type IV pilus, LPS (core), OmpA, OmpC, TolC, and peptidoglycan on multi-drug resistant (MDR) bacteria.
In certain embodiments, the one or more bacteriophages comprise a nucleotide sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-13.
In certain embodiments, the pharmaceutical composition further comprises an antibiotic.
In another aspect, the invention provides a method of treating a bacterial infection in a subject in need thereof. The method comprises administering any of the pharmaceutical compositions disclosed herein to the subject with the bacterial infection.
In certain embodiments, the pharmaceutical composition is administered directly to a site of the bacterial infection.
In certain embodiments, the method further comprises administering an antibiotic to the subject. In certain embodiments, the antibiotic is administered before or after or co-administered with the pharmaceutical composition.
In certain embodiments, the bacterial infection is drug resistant. In certain embodiments, the bacterial infection is multi-drug resistant.
In certain embodiments, the pathogenic bacteria is associated with a biofilm. In certain embodiments, the pathogenic bacteria is in a biofilm.
In certain embodiments, the pathogenic bacteria is Pseudomonas aeruginosa, a Shigella species, Staphylococcus aureus, or Escherichia coli.
In another aspect, the invention provides a method of disrupting a pathogenic bacteria associated with a biofilm. The method comprises contacting the bacteria with a lytic bacteriophage selected from the group consisting of OMKO1, LPS-5, TIVP-H6, LPS-TLTL, TIVP-27, SFA1-1, SF60B, SFNHSI, SF37B, PG-I1, and U136B.
In another aspect, the invention provides a method of preventing formation of a biofilm on a surface. The method comprises contacting the surface with a lytic bacteriophage selected from the group consisting of OMKO1, LPS-5, TIVP-H6, LPS-TLTL, TIVP-27, SFA1-1, SF60B, SFNHSI, SF37B, PG-I1, and U136B.
In certain embodiments, the bacteriophage binds a molecule selected from the group consisting of an O-antigen, efflux pump, Type IV pilus, LPS (core), OmpA, OmpC, and TolC in the bacteria and the bacteria either genetically resists bacteriophage infection or becomes infected and lysed by the bacteriophage, wherein the genetically resistant bacteria have impaired efflux pumps and increased sensitivity to one or more antibiotics.
In certain embodiments, the method further comprises contacting the bacteria or surface with the one or more antibiotics.
In another aspect, the invention provides a composition comprising an LPS-5 bacteriophage targeting Pseudomonas aeruginosa, the bacteriophage having a genome comprising a nucleic acid sequence comprising SEQ ID NO: 1.
In another aspect, the invention provides a composition comprising an OMKO1 bacteriophage targeting Pseudomonas aeruginosa, the bacteriophage having a genome comprising a nucleic acid sequence comprising SEQ ID NO: 2.
In another aspect, the invention provides a composition comprising a TIVP-H6 bacteriophage targeting Pseudomonas aeruginosa, the bacteriophage having a genome comprising a nucleic acid sequence comprising SEQ ID NO: 3.
In another aspect, the invention provides a composition comprising an LPS-TLTL bacteriophage targeting Pseudomonas aeruginosa, the bacteriophage having a genome comprising a nucleic acid sequence comprising SEQ ID NO: 4.
In another aspect, the invention provides a composition comprising an TIVP-27 bacteriophage targeting Pseudomonas aeruginosa, the bacteriophage having a genome comprising a nucleic acid sequence comprising SEQ ID NO: 5.
In another aspect, the invention provides a composition comprising an SFA1-1 bacteriophage targeting a Shigella spp. bacteria, the bacteriophage having a genome comprising a nucleic acid sequence comprising SEQ ID NO: 6.
In another aspect, the invention provides a composition comprising an SF60B bacteriophage targeting a Shigella spp. bacteria, the bacteriophage having a genome comprising a nucleic acid sequence comprising SEQ ID NO: 7.
In another aspect, the invention provides a composition comprising an SFNHSI bacteriophage targeting a Shigella spp. bacteria, the bacteriophage having a genome comprising a nucleic acid sequence comprising SEQ ID NO: 8.
In another aspect, the invention provides a composition comprising an SF37B bacteriophage targeting a Shigella spp. bacteria, the bacteriophage having a genome comprising a nucleic acid sequence comprising SEQ ID NO: 9.
In another aspect, the invention provides a composition comprising a PG-I1 bacteriophage targeting Staphylococcus aureus, the bacteriophage having a genome comprising at least one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10-12.
In another aspect, the invention provides a composition comprising a U136B bacteriophage targeting E. coli, the bacteriophage having a genome comprising a nucleic acid sequence comprising SEQ ID NO: 13.
In another aspect, the invention provides a method of treating a bacterial infection in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of any of the compositions disclosed herein.
In certain embodiments, the bacterial infection is drug-resistant or multi-drug resistant.
The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings exemplary embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
As more alternative therapies are considered to combat the rise of antibiotic resistant biofilm-associated infections, the use of bacteriophages, as described herein, presents a novel strategy to manage these difficult to manage infections.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.
It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
As used herein, the articles “a” and “an” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.
As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of 20% or within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
As used herein, the terms “antibacterial activity” and “antimicrobial activity” with reference to a bacteriophage, isolated bacteriophage protein (or variant, derivative or fragment thereof), or bacteriophage product, are used interchangeably to refer to the ability to kill and/or inhibit the growth or reproduction of a microorganism, in particular, the bacteria of the species or strain that the bacteriophage infects. In certain embodiments, antibacterial or antimicrobial activity is assessed by culturing bacteria: gram-positive bacteria (e.g., S. aureus), gram-negative bacteria (e.g., K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa) or bacteria not classified as either gram-positive or gram-negative, according to standard techniques (e.g., in liquid culture, on agar plates), contacting the culture with a bacteriophage or bacteriophage product and monitoring cell growth after the contact. For example, in a liquid culture, the bacteria may be grown to an optical density (“OD”) representative of a mid-point in exponential growth of the culture; the culture is exposed to one or more concentrations of one or more bacteriophage or bacteriophage product, and the OD is monitored relative to a control culture. Decreased OD relative to a control culture is representative of a bacteriophage or bacteriophage product exhibiting antibacterial activity (e.g., exhibits lytic killing activity). Similarly, bacterial colonies can be allowed to form on an agar plate, the plate exposed to a bacteriophage or bacteriophage product, and subsequent growth of the colonies evaluated related to control plates. Decreased size of colonies, or decreased total numbers of colonies, indicate a bacteriophage product.
By “attenuated” is meant the bacterium has a decreased virulence with respect to a wild-type bacterium. In particular, a bacterium has an attenuated virulence of about 10, 20, 30, 40, 50, 60, 70, 80% or more decrease in virulence as compared to a wild-type bacterium.
As used herein the terms “bacteriophage” or “phage” refer to a virus that infects bacteria. A “lytic bacteriophage” is an actively infecting bacteriophage, i.e. one that is in the lytic (virulent) cycle (as opposed to the (dormant) lysogenic cycle). In the lytic cycle, the phage takes over the host cell's machinery for its own replication, ultimately killing (lysing) the host cell to produce its own progeny. The term “bacteriophage products” refers to polynucleotides, polypeptides, or fragments, variants, or derivatives thereof, isolated from a bacteriophage of the invention, which polynucleotide, polypeptide, or fragment, variant, or derivative thereof, exhibits a biological function or activity associated with the bacteriophage from which it was isolated or derived (e.g., antimicrobial or antibacterial activity (e.g., lytic cell killing)).
By “effective amount” is meant the amount required to reduce or improve at least one symptom of a respiratory disorder, condition or disease relative to an untreated patient. The effective amount of airway epithelial cells used for therapeutic treatment of the respiratory disorder, condition or disease varies depending upon the manner of the specific disorder, condition or disease, extent of the disorder, condition or disease, and administration of the cells, as well as the age, body weight, and general health of the subject.
The term “efflux pump” refers to an active, protein transporter localized in the cell membrane that exports substrate(s). In bacteria, five classes of efflux pumps exist: MF (major facilitator), MATE (multidrug and toxic efflux), RND (resistance-nodulation-division), SMR (small multidrug resistance) and ABC (ATP binding cassette).
The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.
“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
A “vector” is a composition of matter that comprises a gene and that may be used to deliver the gene to the interior of a cell. Vector refers to any plasmid containing the gene that is capable of moving foreign sequences into the genomes of a target organism or cell.
As used herein, the term “fragment” as applied to a nucleic acid, is less than the whole.
By “host” or “host cell” is meant a cell, such as a mammalian cell, that harbors a pathogen, such as a bacterium. The pathogen can infect the host cell.
By “immune response” is meant the actions taken by a host to defend itself from pathogens or abnormalities. The immune response includes innate (natural) immune responses and adaptive (acquired) immune responses. Innate responses are antigen non-specific. Adaptive immune responses are antigen specific. An immune response in an organism provides protection to the organism against bacterial infections when compared with an otherwise identical subject to which the composition or cells were not administered or to the human prior to such administration.
By “infection” is meant a colonization of the host. Infection of a host can occur by entry through a membrane of the host, such as a phage passing through the cell membrane of a bacterium.
The term “bacterial infection” means the invasion of the host organism, animal or plant, by pathogenic bacteria. This includes the excessive growth of bacteria which are normally present in or on the body of the organism, but more generally, a bacterial infection is any situation in which the presence of a bacterial population(s) is damaging to a host organism. Thus, for example, an organism suffers from a bacterial infection when excessive numbers of a bacterial population are present in or on the organism's body, or when the effects of the presence of a bacterial population(s) is damaging to the cells, tissue, or organs of the organism.
By “infectious disease” is meant a disease or condition in a subject caused by a pathogen that is capable of being transmitted or communicated to a non-infected subject. Non-limiting examples of infectious diseases include bacterial infections, viral infections, fungal infections, and the like.
The term “isolated” refers to a material or an organism, such as bacteria, that is free to varying degrees from components or other organisms that normally accompany it as found in its native state. Isolated denotes a degree of separation from an original source or surroundings. An isolated bacterium is sufficiently free of other bacteria such that any contaminants do not materially affect growth, pathogencity, infection, etc. or cause other adverse consequences. That is, bacteria are isolated if they are substantially free of bacteria or materials. Purity and homogeneity are typically determined using analytical techniques, for example, single cell culturing. The term “purified” can denote that a cell gives rise to essentially one population.
By “multi-drug resistant,” “multi-drug resistance” or “MDR” is meant antimicrobial resistance to the effects of antibiotics or other antimicrobial drugs.
By “non-pathogenic” is meant an inability to cause disease.
By “pathogen” is meant an infectious agent, such as bacteria, capable of causing infection, producing toxins, and/or causing disease in a host.
By “disrupt” is meant to kill bacteria and/or to inhibit, slow, stop, or prevent bacterial replication and/or growth.
By “associated with a biofilm” is meant that the pathogen is present in and/or on a biofilm or forms a biofilm.
A “portion” of a polynucleotide means at least about twenty sequential nucleotide residues of the polynucleotide. It is understood that a portion of a polynucleotide may include every nucleotide residue of the polynucleotide.
“Proliferation” is used herein to refer to the reproduction or multiplication of similar forms, especially of bacteria. That is, proliferation encompasses production of a greater number of bacteria, and can be measured by, among other things, simply counting the numbers of bacteria, measuring incorporation of 3H-thymidine into the bacteria, and the like.
As used herein, “sample” or “biological sample” refers to anything, which may contain the cells of interest (e.g., cancer or tumor cells thereof) for which the screening method or treatment is desired. The sample may be a biological sample, such as a biological fluid or a biological tissue. In one embodiment, a biological sample is a tissue sample including pulmonary arterial endothelial cells. Such a sample may include diverse cells, proteins, and genetic material. Examples of biological tissues also include organs, tumors, lymph nodes, arteries and individual cell(s). Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid or the like.
The term “strain” means bacteria or bacteriophage having a particular genetic content. The genetic content includes genomic content as well as recombinant vectors. Thus, for example, two otherwise identical bacterial cells would represent different strains if each contained a vector, e.g., a plasmid, with different phage open reading frame inserts.
A “subject” as used herein, may be a human or non-human organism. Non-human organisms include, but are not limited to, livestock, pets, aquaculture organisms, cultivated plants and crops. Preferably, the subject is human.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or improving an infectious disease or condition and/or one or more symptoms associated therewith. It will be appreciated that, although not precluded, treating an infectious disease or condition and/or one or more symptoms associated therewith does not require that the disorder, condition, disease or symptoms associated therewith be completely ameliorated or eliminated.
In the context of treating a bacterial infection a “therapeutically effective amount” or “pharmaceutically effective amount” indicates an amount of a composition comprising bacteriophage which has a therapeutic effect. This generally refers to the lysis of bacterial cells or, to some extent, of the acquisition of resistance (genetic evolution) of bacterial cells to bacteriophage infection.
By “virulence” is meant a degree of pathogenicity of a given pathogen or the ability of an organism to cause disease in another organism. Virulence refers to an ability to invade a host organism, cause disease, evade an immune response, and produce toxins.
By “bacterial virulence” is meant a degree of pathogenicity of bacteria. Bacterial virulence includes causing infection or disease in a host, producing agents that cause or enhance disease in a host, producing agents that cause or enhance disease spread to another host, and causing infection or disease in another host.
By “virulent” or “pathogenic” is meant a capability of a bacterium to cause a severe disease.
By “wildtype” is meant a non-mutated version of a gene, allele, genotype, polypeptide, or phenotype, or a fragment of any of these. It may occur in nature or produced recombinantly.
In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
“Phage therapy”, the application of lytic bacteriophages (or “phages”; viruses of bacteria) for the bio-control of bacteria, is one method for treating multi-drug-resistant (MDR) bacterial infections: the use of lytic (virulent) bacteriophages (bacteria-specific viruses) as self-amplifying ‘drugs’ that specifically target and kill bacteria. Lytic phages bind to one or more specific proteins on the surfaces of particular bacterial hosts, an intimacy that led to development of phage therapy as a biocontrol strategy which predated use of broad-spectrum chemical antibiotics. Due to the recent precipitous rise in antibiotic resistance, phage therapy has seen revitalized interest among Western physicians, buoyed by successful clinical trials demonstrating safety and efficacy.
However, one limitation of phage therapy is the abundant evidence that bacteria readily evolve resistance to phage infection. While multiple mechanisms of phage resistance exist, phage attachment to a receptor binding-site exerts selection pressure for bacteria to alter or down-regulate expression of the receptor, thereby escaping phage infection. Given the certainty of evolved phage-resistance, modern approaches to phage therapy must acknowledge and capitalize on this inevitability. Genetic trade-offs are often observed in biology, where organisms evolve one trait that improves fitness (a relative advantage in reproduction or survival), while simultaneously suffering reduced performance in another trait.
Described herein is an evolutionary-based strategy that forces a genetic trade-off: utilize phages that drive MDR bacterial pathogens to evolve increased phage resistance thereby increasing sensitivity to chemical antibiotics. Thus, this approach to phage therapy should be doubly effective; success is achieved when phage lyse the target bacterium, and success is also achieved when bacteria evolve phage resistance because they suffer increased sensitivity to antibiotics.
Many strains of bacteria have become antibiotic resistant, and some have become resistant to multiple antibiotics and chemotherapeutic agents, the phenomenon of multi-drug resistance. Some strains have become resistant to practically all of the commonly available agents. For example, methicillin-resistant Staphylococcus aureus (MRSA) is resistant to not only methicillin (which was developed to fight against penicillinase-producing S. aureus) but also aminoglycosides, macrolides, tetracycline, chloramphenicol, and lincosamides. Such strains are also resistant to disinfectants, and MRSA can act as a major source of hospital-acquired infections. An old antibiotic, vancomycin, was resurrected for treatment of MRSA infections. However, transferable resistance to vancomycin is now quite common in Enterococcus and found its way finally to MRSA.
The emergence of “pan-resistant” gram-negative strains, notably those belonging to P. aeruginosa and A. baumanii, occurred more recently, after most major pharmaceutical companies stopped the development of new antibacterial agents. Hence, there are almost no agents that could be used against these strains, in which an outer membrane barrier of low permeability and an array of efficient efflux pumps are combined with multitudes of specific resistance mechanisms.
Efflux pumps belonging to the resistance-nodulation-division (RND) family of transporters are the major multi-drug efflux (Mex) mechanism in both E. coli and P. aeruginosa. The pumps in this family consist of three components that function via active transport to move numerous molecules, including antibiotics, out of the cell: an antiporter that functions as a transporter (e.g., MexB, Mex D, MexF, MexY), an outer membrane protein that forms a surface-exposed channel (e.g., OprC, OprB, OprG, OprD, OprI, OprH, OprP, OprO, OprM, OprJ, OprN), and a periplasmic membrane fusion protein that links the two proteins (e.g., MexA, MexC, MexE, MexH, MexX). This system is the major efflux pump associated with intrinsic resistance among 17 possible RND efflux pumps in P. aeruginosa. P. aeruginosa is more resistant than E. coli due to a highly impermeable OM and the presence of multiple efflux systems. Inactivation of the Mex efflux pump renders P. aeruginosa more vulnerable to antibiotics than the average E. coli strain.
In one aspect, the invention includes a composition comprising a lytic bacteriophage selected from the group consisting of LPS-5, OMKO1, TIVP-H6, LPS-TLTL, TIVP-27, SFA1-1, SF60B, SFNHSI, SF37B, PG-I1, and U136B.
In certain embodiments, the bacteriophage binds a molecule of an efflux pump on pathogenic bacteria, drug resistant bacteria, multi-drug resistant (MDR) bacteria, and/or pan-drug resistant (PDR) bacteria.
In one embodiment, the bacteriophage binds a protein, such as a surface exposed protein, of a Mex efflux pump. In another embodiment, the Mex protein is selected from the group consisting of OprM, MexA, MexB, MexX, and MexY. In various embodiments the lytic bacteriophage is selected from the group consisting of OMKO1, LPS-5, TIVP-H6, LPS-TLTL, TIVP-27, SFA1-1, SF60B, SFNHSI, SF37B and PG-I1. In various embodiments the bacteriophage binds a molecule selected from the group consisting of O-antigen, efflux pump, Type IV pilus, LPS (core), OmpA, OmpC and peptidoglycan.
In certain embodiments, the bacteriophage utilizes the antibiotic efflux pump protein TolC and LPS.
In yet another embodiment, the composition further comprises an antibiotic. The antibiotic includes any commonly available agent, such as an antibiotic selected from, but not limited to, amoxicillin, erythromycin, penicillin, ciprofloxacin, azithromycin, ceftolozane/taxobactam, ceftazidime/acibactiam, tetracycline, imipenem/carbapenem, and any combination thereof.
The present invention also includes a pharmaceutical composition comprising the bacteriophage described herein. Pharmaceutical compositions comprise the bacteriophage in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); Magnetic Resonance and Computerized Tomography contrast agents; and preservatives. Compositions of the present invention are preferably formulated for intravenous administration.
Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
In another aspect, the invention includes a composition or a pharmaceutical composition comprising the bacteriophage described herein, wherein the bacteriophage disrupts the multi-drug resistant (MDR) bacteria, Pseudomonas aeruginosa.
Details regarding various bacteriophage of the invention are presented in Table 1, below.
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
Pseudomonas
aeruginosa
Shigella spp.
Shigella spp.
Shigella spp.
Shigella spp.
Staphylococcus
aureus
E. coli
In another aspect, the invention provides an LPS-5 bacteriophage targeting Pseudomonas aeruginosa. In certain embodiments, LPS-5 binds a molecule of O-antigen (of a lipopolysaccharide). In certain embodiments, the LPS-5 bacteriophage has a genome comprising a nucleic acid sequence comprising SEQ ID NO: 1.
In another aspect, the invention provides an OMKO1 bacteriophage targeting Pseudomonas aeruginosa. In certain embodiments OMKO1 binds a molecule of an efflux pump and/or Type IV pilus. In certain embodiments, the OMKO1 bacteriophage has a genome comprising a nucleic acid sequence comprising SEQ ID NO: 2.
In another aspect, the invention provides an TIVP-H6 bacteriophage targeting Pseudomonas aeruginosa. In certain embodiments, TIVP-H6 binds a molecule of type IV pilus. In certain embodiments, the TIVP-H6 bacteriophage has a genome comprising a nucleic acid sequence comprising SEQ ID NO: 3.
In another aspect, the invention provides an LPS-TLTL bacteriophage targeting Pseudomonas aeruginosa. In certain embodiments, LPS-TLTL binds a molecule of LPS (core). In certain embodiments, the LPS-TLTL bacteriophage has a genome comprising a nucleic acid sequence comprising SEQ ID NO: 4.
In another aspect, the invention provides an TIVP-27 bacteriophage targeting Pseudomonas aeruginosa. In certain embodiments, TIVP-27 binds a molecule of type IV pilus. In certain embodiments, the TIVP-27 bacteriophage has a genome comprising a nucleic acid sequence comprising SEQ ID NO: 5.
In another aspect, the invention provides an SFA1-1 bacteriophage targeting a Shigella spp. Bacteria. In certain embodiments, SFA1-1 binds a molecule of OmpA. In certain embodiments, the SFA1-1 bacteriophage has a genome comprising a nucleic acid sequence comprising SEQ ID NO: 6.
In another aspect, the invention provides an SF60B bacteriophage targeting a Shigella spp. bacteria. In certain embodiments, SF60B binds a molecule of OmpC. In certain embodiments, the SF60B bacteriophage has a genome comprising a nucleic acid sequence comprising SEQ ID NO: 7.
In another aspect, the invention provides an SFNHSI bacteriophage targeting a Shigella spp. bacteria. In certain embodiments, SFNHSI binds a molecule of OmpC. In certain embodiments, the SFNHSI bacteriophage has a genome comprising a nucleic acid sequence comprising SEQ ID NO: 8.
In another aspect, the invention provides an SF37B bacteriophage targeting a Shigella spp. bacteria. In certain embodiments, SF37B binds a molecule of OmpC. In certain embodiments, the SF37B bacteriophage has a genome comprising a nucleic acid sequence comprising SEQ ID NO: 9.
In another aspect, the invention provides a PG-I1 bacteriophage targeting Staphylococcus aureus. In certain embodiments, PG-I1 binds a molecule of peptidoglycan. In certain embodiments, the PG-I1 bacteriophage has a genome comprising at least one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10-12:
In another aspect, the invention provides a U136B bacteriophage targeting an E. coli bacteria. In certain embodiments, U136B binds a molecule of TolC. In certain embodiments, U136B binds a molecule of LPS. In certain embodiments, the U136B bacteriophage has a genome comprising a nucleic acid sequence comprising SEQ ID NO: 13.
Tolerable variations of the bacteriophage sequences will be known to those of skill in the art. For example, in some embodiments the bacteriophage comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in any one SEQ ID NOs: 1-13.
In another aspect, the invention includes a method of increasing antibiotic sensitivity and/or decreasing virulence in pathogenic bacteria. In some embodiments, the pathogenic bacteria are drug resistant. In some embodiments, the pathogenic bacteria are multi-drug resistant (MDR) bacteria. In some embodiments, the pathogenic bacteria are pan-drug resistant (PDR) bacteria.
The method comprises contacting the pathogenic bacteria with a lytic bacteriophage selected from the group consisting of OMKO1, LPS-5, TIVP-H6, LPS-TLTL, SFA1-1, SF60B, SFNHSI, SF37B, and U136B. In certain embodiments, the bacteriophage binds a molecule of an efflux pump, OmpA, OmpC, Type IV pilus, LPS or peptidoglycan in the bacteria and the bacteria either genetically resists bacteriophage infection or becomes infected and lysed by the bacteriophage. The genetically resistant bacteria have impaired efflux pumps and increased sensitivity to antibiotics, or have impaired virulence factors, such as reduced production of pyocyanin or elastase, that are responsible for decreased virulence.
In one embodiment, the bacteria are contacted with bacteriophage at a multiplicity of infection (MOI) of bacteriophage to bacteria in the range of about 0.0001 to about 1010. The MOI may range from about 0.0002 to about 109, from about 0.0003 to about 108, from about 0.0004 to about 107, from about 0.0005 to about 106, from about 0.0006 to about 105, from about 0.0007 to about 10,000, from about 0.0008 to about 5,000, from about 0.0009 to about 2,500, from about 0.001 to about 1,000, from about 0.005 to about 500, from about 0.01 to about 100, from about 0.05 to about 50, from about 0.1 to about 10, or any range therebetween.
In another embodiment, the method further comprises contacting the genetically resistant bacteria with an antibiotic. The antibiotic includes any of the antibiotics described herein, any commonly known agent, and any combination thereof.
In another embodiment, the method comprises or further comprises contacting the genetically resistant bacteria with one or more antibiotics, e.g., 1-100 antibiotics or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more antibiotics.
In yet another aspect, the invention includes a method of treating a bacterial infection in a subject in need thereof. In certain embodiments, the bacterial infection is drug resistant or multi-drug resistant (e.g. caused by a drug resistant or multi-drug resistant bacteria). The method comprises administering the pharmaceutical composition as described herein to the subject with the bacterial infection. In one embodiment, the composition is administered directly to a site of the bacterial infection. In another embodiment, the method further comprises administering an antibiotic as described herein to the subject. In one such embodiment, the antibiotic is co-administered with the pharmaceutical composition. In another such embodiment, the antibiotic is administered before or after the pharmaceutical composition is administered.
In some embodiments, the antibiotic can be administered minutes, hours, days, or weeks, before or after the pharmaceutical composition is administered, e.g.: 1, 5, 10, 15, 20, 30, or 45 minutes; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or 22 hours; 1, 2, 3, 4, 5, or 6 days; or 1 or 2 weeks, or any amount of time there between.
In another aspect, the invention includes a method of disrupting pathogenic bacteria associated with a biofilm and compositions for use thereof. In another aspect, the invention includes a method of preventing formation of a biofilm.
In some embodiments, the biofilm is on Dacron and/or any other prosthetic material.
In some embodiments, the pathogenic bacteria are associated with a biofilm. In some embodiments, the pathogenic bacterium is Pseudomonas aeruginosa. In some embodiments, the Pseudomonas aeruginosa is a Pseudomonas aeruginosa biofilm.
In various embodiments, the bacteriophage are selected from among the bacteriophage listed in Table 1.
In certain embodiments, the bacteriophage comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-13. In certain embodiments, the bacteriophage comprises a nucleic acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in any one SEQ ID NOs: 1-13.
In the clinical settings, compositions of the invention can be administered to a subject by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical formulation of the composition can be administered by inhalation, topically, locally or systemically, e.g., by intravenous injection, intramuscular injection, intraperitoneal injection, retro- or peribulbar injection.
The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the manifestation of symptoms associated with the disease or condition. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
Administration of the composition of the present invention to a subject, such as a mammal, for example a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or condition in the subject. An effective amount of the composition necessary to achieve a therapeutic effect may vary according to factors such as the extent of implantation; the time of administration; the duration of administration; other drugs, compounds or materials used in combination with the composition; the state of the disease or disorder; age, sex, weight, condition, general health and prior medical history of the subject being treated; and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the composition without undue experimentation.
Actual dosage levels of the cells in the pharmaceutical formulations of this invention may be varied so as to obtain an amount of the composition that are effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
Routes of administration of the compositions of the invention include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, topical, intra-orbital, intra-aural, intra-articular, and topical administration.
Suitable formulation of the compositions and dosages include, for example, dispersions, suspensions, solutions, beads, pellets, magmas, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal, ocular or oral administration, aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like.
It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations set forth in the examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, differentiation methods, engineered tissues, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.
The materials and methods employed in these experiments are now described.
Bacterial growth conditions and media: Bacteria were grown in LB broth with 10 g tryptone, 5 g yeast extract, and 10 g/L NaCl. LB agar included 15 g/L agar and LB top agar included 7.5 g/L agar unless otherwise noted. Overnight culture incubation was performed at 200 RPM shaking at 37° C.
Phage library screens: To isolate phage U136B, thirty-three phage isolates collected previously from various sources were screened. Each isolate was screened using the plaque spot test on host lawns of wild-type and knockout E. coli from the Keio collection obtained from the Yale Coli Genetic Stock Center (
Phage TIVP-H6 (Myovirus) was discovered by screening for phages that infect Pseudomonas aeruginosa strains which overproduce the virulence factor pyocyanin, and determining that evolution of phage resistance led to reduced pyocyanin production in bacterial mutants.
Phages LPS-5 (Podovirus) and LPS-TLTL were discovered by screening for phages that interact with LPS of P. aeruginosa, indicated by classic ‘rough-colony-forming’ phenotypes when phage-resistant mutants grow on agar (Luzar and Montie (1985) Infection and Immunity 50:572-576).
Phages SF37B, SF60B, SFNHSI, and SFA1-1 were discovered by screening phage isolates from various sources on Shigella strains PES77 and/or BW25113.
Phage PG-I1 was discovered by screening phage isolates from various sources on Staphylococcus aureus bacteria.
Mutant selection procedure: To select for phage-resistant bacteria, wild-type bacteria and phages were mixed, then plated onto LB agar and incubated overnight at 37° C. To ensure the isolation of independent mutations, each replicate was grown from a single isolated colony. To confirm the multiplicity of infection (MOI, the ratio of phage particles to bacterial cells) on the agar plate, bacteria and phage were plated in triplicate and counted cfu/mL and pfu/mL, respectively. From each mutant-selection plate, a random colony was picked, plus any colonies of notable morphology, and streaked onto LB agar plate and incubated at 37° C. overnight. The colonies were re-streaked to obtain double-purified isolates and each grown in 10 mL LB at 37° C. with shaking overnight. Freezer stocks of each mutant were archived in 20% glycerol, stored at −80° C.
Assessment of phage resistance: 10 μL of a high titer stock of phage (˜109 pfu/mL) was streaked along ˜10 cm lines on LB agar plates. Phage streaks were allowed to dry, and 1 μL samples of overnight cultures of each bacterium were streaked perpendicularly across the phage lines in duplicate. Plates were allowed to dry and incubated overnight at 37° C. Bacterial streaks were scored for signs of phage lysis. Isolates with signs of lysis were scored as susceptible to phage while isolates without lysis were scored as phage resistant.
Bacterial growth curves for phage resistance assays: Cultures of wild type and other bacteria (e.g., phage-resistant and gene-knockout strains) were grown to exponential phase as measured by optical density at 600 nm wavelength (OD600) of 0.25-0.38. The exponential-phase cultures were diluted 1:5 into wells with LB to a total volume of 200 μL with phage to reach the target MOI. Cultures were incubated at 37° C. with shaking at 288 RPM for 18 h and OD600 read every 2 minutes using an automated spectrophotometer (TECAN microplate reader).
Bacterial growth curves for comparison of differing mutants: Bacterial cultures were preconditioned in LB medium overnight from individual colonies, then transferred to fresh LB in 200 μl total volumes. Cultures were incubated at 37° C. with shaking and optical density was monitored at 5-min intervals for 24 h by a TECAN microplate reader.
Single step growth curves: Overnight cultures of bacteria were diluted 1:100 into 10 mL pre-warmed LB broth and subjected to shaking at 200 RPM at 37° C. until exponential phase. These cultures were then inoculated into pre-warmed LB at an initial concentration of ˜2*107 cfu/mL to begin the assay. Phage were added to an initial concentration of 2*108 pfu/mL, for an initial MOI of 10. The assay flasks were incubated with shaking at 200 RPM at 37° C. Every 10 min, 50-μL samples were removed from each flask, transferred to a Spin-X column (CLS8160 Sigma), and spun at 14,000 rpm for 1 min. At 2 min and 12 min, bacterial samples were obtained from flasks with and without phage, and were diluted and plated in duplicate as above, in order to preliminarily assess infection rates. Within a week, each filtered phage sample was diluted in a 1:10 dilution series in sterile LB and 100 μL diluted samples were plated in soft agar overlay with 4 mL 7.5 g/L LB soft agar and 100 μL overnight bacteria (e.g.,
Transmission electron microscopy: Images of high-titer phage lysates were collected at the Yale Electron Microscopy facility in the Center for Cellular and Molecular Imaging. Samples were negatively stained with 2% uranyl acetate and imaged with FEG 200 kV transmission EM.
Broth microdilution method for antibiotic MICs: To examine colistin MIC, an original colistin solution (Alfa Aesar) with a concentration of 50 mg/mL was diluted to the following test concentrations in LB medium: 12.5, 25, 50, 100, 150, 200, 250, 300, 350, and 400 ng/mL. Bacterial cultures were grown from single colonies overnight in 10 mL of LB incubated at 37° C. with shaking. Each culture was diluted to ˜2*105 cfu/mL in LB. MICs for colistin were measured by dispensing 100 μL of a given colistin concentration into each column and 100 μl of diluted bacteria into each row of a 96 well plate and incubating at 37° C. with shaking for 18 hr. Bacterial growth was scored visually as a binary variable (no growth vs. any visible growth). The lowest colistin concentration at which the well was completely clear was the value recorded as the MIC. For cases where replicates gave different answers, we report the mode.
Statistical analysis: All analyses were performed in R Studio (R Development Core Team (2010) R: A language and environment for statistical computing. (R Foundation for Statistical Computing, Vienna, Austria) using custom scripts.
Genome sequence analysis: Genome sequences were collected at the University of Pittsburgh Microbial Genome Sequencing (MiGS) center. Genomic DNA libraries were prepared using a modified Illumina Nextera library preparation (Baym M, et al. (2015) PLoS One 10(5):e0128036). Paired-end reads of 151 bp length were collected to a final coverage of ˜100-fold across the reference genome. Sequences were analyzed using breseq with default sequence end-trimming mode (Baym M, et al. (2015) PLoS One 10(5):e0128036; Deatherage D E & Barrick J E (2014) Methods Mol Biol 1151:165-188).
Complementation assays for gene knockouts: Electrocompetent bacterial cells were made by inoculating 10 mL LB broth with 100 μL of overnight bacterial cultures grown in LB broth. These cells were then incubated and shaken at 37° C. for 2-3 hours until they reached an OD600 of approximately 0.6. Next, the cells were centrifuged at 4,000 rpm for 10 min. The supernatant was poured off, then 10 mL of chilled 10% glycerol was added to the pellet. After resuspending the pellet by vortexing, the cells were centrifuged for 5 min at 4,000 rpm, then the supernatant was poured off. This glycerol wash cycle was performed 4 times. Finally, the cells were resuspended in 150 μL of 10% glycerol and were frozen at −80° C. or immediately electroporated. Plasmid DNA from ASKA clones (Kitagawa M, et al. (2005) DNA Res 12(5):291-299) (
Bacteriophage enumeration and efficiency of plating for complementation assays: Soft agar overlay tubes were prepared by mixing 4 mL 7.5 g/L soft agar, 4 μL of 30 p,g/mL chloramphenicol (for transformed strains only), 20 μL of 100 mM IPTG, and 100 μL of a bacterial overnight culture (grown in LB broth). These soft agar overlays were poured over LB plates with added chloramphenicol. For non-transformed strains, no chloramphenicol was added. A 1:10 dilution series was performed for 4 phage stocks from 10−1 through 10−7, and 2 μL of each dilution from each phage stock was spotted onto every bacterial overlay. After these spots were completely dry, the plates were incubated overnight at 37° C. Finally, pfu/mL was calculated for each plate, and the EOP was calculated by dividing the pfu/mL of a test strain by that of the wild type.
Assessment of tetracycline resistance: Overnight cultures were diluted in 1:10 dilution series in sterile LB, and 2 μL spots of each dilution were immediately plated on LB agar plates containing varying concentrations of antibiotic: 0, 0.12, 0.25, 0.5, 1.0, 2.0, and 4.0 pg/mL tetracycline. Plates were allowed to dry and incubated for two days at 37° C. The MIC of tetracycline was recorded for each isolate as the concentration at which colonies no longer formed at the 10−5 spotted dilution. For cases where replicates gave different answers, the mode was reported.
Preparation of phage stocks for the evolution experiment: 20 μL of cryogenically frozen U136B phage stock was diluted in sterile LB broth in a 1:10 dilution series. 100 μL of each dilution was plated with 100 μL of an overnight culture of DH5a in 4 mL 7.5 g/L LB soft agar overlay on standard LB agar and incubated overnight at 37° C. Ten isolated plaques were randomly chosen from the 10−7 dilution plate, suspended in 750 μL sterile LB, and filtered through a 0.22 p,m Spin-X column (CLS8160 Sigma) at 14,000 rpm for 1 min. The ten phage stocks were enumerated by top agar overlay plating and subsequently diluted in sterile LB to a target concentration of 1.0*107 pfu/mL. 200 μL samples of each replicate phage stock were combined in duplicate with 200 μL sterile 40% glycerol solution and frozen at −80° C.
Preparation of bacterial cultures for the evolution experiment: Cryogenically-frozen E. coli strain BW25113 was streaked onto LB agar and incubated overnight at 37° C. Twenty separate isolated colonies were randomly chosen and suspended in 10 mL sterile LB in separate flasks. Cultures were incubated overnight at 37° C., with 200 rpm shaking. Samples of these founding cultures were archived by combining 900 μL of each replicate bacterial culture with 900 μL sterile 40% glycerol solution in duplicate, and frozen at −80° C. The experimental populations were established by adding 9.8 mL sterile LB to 50 mL glass Erlenmeyer flasks and then inoculating with 100 μL of one of the BW25113 bacterial cultures described above (each flask receiving a distinct starting culture) to an estimated concentration of 2*107 cfu/mL (assuming final E. coli overnight densities in LB are 2*109 cfu/mL). For the +phage populations, 100 μL of one of the diluted U136B phage stocks prepared above was then added to 10 of the flasks (each flask receiving a distinct starting culture) to a concentration of 1*105 pfu/mL. For the 10 control populations, 100 μL sterile LB broth were added in place of phage. Flasks were incubated overnight at 37° C., with 200 rpm shaking.
Serial passaging in the evolution experiment: Each day (24 h±2 h), the appearance of each flask was recorded and 100 μL from each experimental flask were transferred into 9.9 mL sterile LB broth in fresh flasks. To reduce risk of contamination, the transfer micropipette was wiped down with 70% ethanol on a between each transfer, and flasks with phage and without phage were alternated to enable detection of cross contamination. To archive the evolved samples each day, 900 μL samples of each replicate experimental volume were combined in duplicate with 900 μL sterile 40% glycerol solution and frozen at −80° C.
Daily enumeration of bacteria during evolution experiment: Each day, 1:10 serial dilutions of each experimental volume were prepared in sterile 0.85% NaCl up to 10−8, and 2 μL spots of each dilution were plated on both standard LB agar plates and LB agar plates prepared with tetracycline. Plates were allowed to dry and then incubated overnight at 37° C. The following day, the most dilute spot with colonies present was identified and colonies were counted in order to determine the total bacterial population density and the TetR bacterial population density for each replicate flask.
Daily enumeration of phage during evolution experiment: The dilution series prepared above was also used to enumerate phage daily. For the 10 flasks with phage, 2 μL spots of each dilution were plated with 100 μL of an overnight culture of DH5a in 4 mL 7.5 g/L LB soft agar overlay on standard LB round plates (when square plates were used, 125 μL DH5a was used in 5 mL soft agar) and incubated overnight at 37° C. For the flasks without phage, only the least dilute concentration was plated as a check for phage contamination. The following day, the most dilute spot with plaques present was identified and plaques were counted in order to determine the phage titer.
Isolation of bacteria from evolution experiment: Two individual isolates were obtained from each population at day 5 and day 10. Isolated colonies were selected in the most dilute spots that contained colonies on the bacterial enumeration plates described above. One colony was picked from the total bacterial population (on the standard LB plates) while one colony was picked from the LB plates containing 0.5 p,g/mL tetracycline. The colonies were streaked onto sterile LB plates and incubated overnight at 37° C. For each isolate, the colony most distant from its neighbors was streaked onto a new sterile LB plate and incubated overnight at 37° C. For each re-streaked isolate, the colony most distant from its neighbors was suspended in sterile LB broth and incubated overnight at 37° C., with 200 rpm shaking. To archive the isolated bacteria, 900 μL samples of each bacterial culture were combined in duplicate with 900 μL sterile 40% glycerol solution and frozen at −80° C.
Kirby Bauer colistin disk diffusion assay: Mueller Hinton plates were prepared by pipetting 25 mL of Mueller Hinton agar into each plate, then allowed to dry for two days. Plates were then used either immediately or refrigerated until use. Overnight bacterial cultures of each strain were prepared by incubating a single bacterial colony in 10 mL LB broth at 37° C. for 18-20 hours. After incubation, 1:10 dilutions of each bacterial strain were prepared by combining 900 μL 0.85% NaCl solution with 100 μL bacterial inoculation. To prepare the disk diffusion plates, 100 μL of each bacterial dilution were pipetted onto a Mueller Hinton plate then spread evenly across the plate with a sterile plastic L spreader. Plates were then allowed to dry and cardboard colistin disks (ThermoFisher, #CT0017B) containing 10 μg colistin were placed in the center of the plates using sterile forceps. Plates were incubated for 20-24 hours at 37° C. Finally, to measure the diameters of the zones of inhibition (ZOI), three straight lines, each at a different angle, were drawn through the center of the ZOI using a ruler. The diameter of the ZOI was measured along the drawn lines.
INSeq transposon mutant library preparation. The insertion-sequence (1NSeq) transposon mutant library was made via conjugation. A donor bacterial strain containing a plasmid was conjugated with a wild type recipient strain on agar plates, at a ratio of 1:1 donor to recipient. After 3 hours incubation at 37° C., conjugation mixtures were plated for single colonies on plates with antibiotic to isolate transconjugants that were resistant to both markers (i.e., marker on plasmid and on recipient chromosome). Following 24 hours incubation at 37° C., colonies were scraped into LB medium using a sterile spatula and stored in 20% glycerol at −80° C.
INSeq screen. Aliquots of the library were thawed on ice, washed once in LB and resuspended to a concentration of approximately 107 CFU/mL in LB and incubated at 37° C. for 1 hour, with shaking. A sample of test phage was added, in triplicate, to a final concentration of approximately 105 PFU/mL, for MOI≈0.01. Following overnight incubation, cultures were harvested and genomic DNA was extracted and prepared for sequencing. DNA libraries were pooled and sequenced at the Yale Center for Genome Analysis via the Illumina HiSeq2500 system. Sequences were analyzed using scripts modified from Goodman et al (Cell Host Microbe 6, 279-289 (2009)). Briefly, using Python scripts adapted from analysis packages previously described (Goodman et al. (2011) Nat Protoc 6, 1969-1980), sequencing reads were indexed by barcode, and transposon sequence was trimmed leaving 16 bp of adjacent genomic DNA. These 16 bp sequences were aligned to a reference genome using Bowtie2, by counting the number of reads for each insertion site, normalizing to counts per million reads and binning by gene (Langmead and Salzberg (2012) Nat Methods 9, 357-359). Transposon insertions mapping to the distal 5% ends of any coding region as well as transposon insertions mapping to intergenic regions were filtered out during analysis. A Z-test was performed using the log ratio of normalized output count to normalized input count, and Q-values from false discovery rate (FDR) correction of <0.05 were considered significant for further analysis.
Efficiency of plating, and adsorption assays. Efficiency of plating (EOP) was measured as the ratio of the test phage titer on the experimental strain to its titer on the WT strain. Phage binding to cells (adsorption) was measured over a 20 minute adsorption assay (Kropinski. Bacteriophages. (Humana Press, 2009), pp. 151-155), which estimates the rate of ‘disappearance’ of phage particles (i.e., reduction in titer) in liquid LB medium over time when viruses are challenged to attach to bacterial cells.
The results of the experiments are now described.
Bacteria frequently encounter selection by both antibiotics and lytic bacteriophages. However, the evolutionary interactions between antibiotics and phages remain unclear, in particular whether and when phages can drive evolutionary trade-offs with antibiotic resistance. Herein, Escherichia coli phage U136B is described. Its reliance on two host factors involved in different antibiotic resistance mechanisms: 1) the efflux pump protein TolC, and 2) the structural barrier molecule lipopolysaccharide (LPS), was demonstrated. Since TolC and LPS contribute to antibiotic resistance, phage U136B should select for their loss or modification, thereby driving a trade-off between phage resistance and either of the antibiotic resistance mechanisms. To test this hypothesis, fluctuation experiments and experimental evolution were used to obtain phage resistant mutants. Using these mutants, the accessibility of specific mutations (revealed in the fluctuation experiments) were compared to their actual success during ecological competition and coevolution (revealed in the evolution experiments). Both tolC and LPS-related mutants arose readily during fluctuation assays, with tolC mutations becoming more common during the evolution experiments. In support of the trade-off hypothesis, phage resistance via tolC mutations occurred with a corresponding reduction in antibiotic resistance in many cases. However, contrary to the hypothesis, some phage resistance mutations pleiotropically conferred increased antibiotic resistance. The molecular mechanisms underlying this surprising pleiotropic result, consideration for practical aspects of phage application, and the importance of ecology in evolution of phage resistance are discussed herein. It is envisioned that phages are useful for the reversal of antibiotic resistance, but that such applications will need to account for both unexpected pleiotropy and evolutionary context.
To identify phages that use the antibiotic efflux pump protein TolC for infection, a collection of E. coli phages were screened for the inability to form plaques on a tolC knockout. This screen identified phage U136B, a curly-tailed phage with siphophage morphology (
In addition to outer membrane protein receptors, many phages also require LPS receptors in their initial contact with the host cell. To assess whether U136B relies on LPS, a screen was conducted on a panel of genes involved in LPS synthesis (the “rfa” genes, named originally for the rough (′rf) colony phenotype of some strains with altered LPS structures). All of the rfa gene knockouts available in the Keio collection were used (Baba T, et al. (2006)Mol Syst Biol 2:2006 0008) (
Phage host range depends largely on receptor availability and receptor structure, so host specificity can be constrained within species. U136B's host range was tested on a panel of divergent E. coli strains and more distant Gram-negative bacteria relatives (
E. coli phage resistance mutations were examined to determine potential evolutionary paths bacterial populations may take when evolving in the presence of phage. To do this, modified fluctuation assays were used, which reveal pre-existing phage resistance mutations that have had minimal time to compete with one another. Twenty independent cultures of E. coli BW25113 (K strain, U136BS, TetracyclineR, ColistinR) were plated with phage U136B, yielding a mutant frequency of 4.0×10−6. One phage-resistant mutant colony was randomly picked from each culture. After streaking for double-isolation, the genomes of each mutant were sequenced. Either tolC mutations or LPS-related mutations were observed in 100% of these mutants (
To determine if the phage resistant mutants had altered resistance to antibiotics, in particular decreased antibiotic resistance via antagonistic pleiotropy, their minimum inhibitory concentrations (MICs) for tetracycline and colistin were determined and compared to their wild-type, phage-sensitive parental strains. Tetracycline is an antibiotic that binds to the bacterial ribosome and prevents peptide chain elongation. Resistance to tetracycline can occur by efflux through the TolC-AcrAB efflux pump, and it was predicted that phage resistance mutations in tolC would result in decreased resistance to effluxed antibiotics. Colistin is a polypeptide antibiotic in the polymyxin class and used as a drug of last resort. Previously, a large screen of E. coli knockouts suggested that changes to several of the rfa LPS synthesis genes may result in increased colistin resistance (summarized in
Supporting the antagonistic pleiotropy hypothesis, all six tolC mutants had reduced resistance to tetracycline compared to the parental strain and a similar phenotype to the tolC knockout control (
Fluctuation experiments revealed bacterial mutations in tolC and LPS-synthesis genes, so it was tested if these same types of mutations would be selected in evolving communities of bacteria and phage. Compared to fluctuation experiments where phage-resistant bacteria have not competed with one another, evolution experiments expose the fitness costs of mutations to selection, altering mutation frequencies over time. The phage resistance mutations may vary in their general fitness costs, in their degree of phage resistance, or the potential for phages to counteract the various resistance mechanisms via coevolution. It was expected that the tolC mutations may arise more quickly and outcompete the LPS-synthesis mutants, as growth curve data demonstrated that tolC mutants are generally more fit than LPS-synthesis mutants (
To test these ideas, a 10-day serial-passaging evolution experiment was conducted. Twenty populations with E. coli BW25113 were founded: 10 populations with phage U136B (“+phage”) and 10 control populations without phage (“−phage”), each passaged serially for 10 days. Each day, total bacterial density (
Again consistent with antagonistic pleiotropy, the populations evolved with phage U136B show a decreased frequency in the tetracycline-resistant phenotype relative to total population size. This effect was present both before phage extinctions occurred on Day 5 (
To investigate the genetic basis of adaptation and patterns of pleiotropy in the evolved populations, one random bacterial clone from each of the control (−phage) and treatment (+phage) populations was isolated on Day 10. Consistent with the hypothesis that +phage populations would face selection against TolC, after 10 days of evolution, the isolates from the +phage populations showed a predominance of tolC mutations and no LPS-related mutations (
The interactions between bacteriophages, antibiotic-resistant bacteria, and antibiotic-sensitive bacteria are complex. One reason detailed knowledge about their ecological and evolutionary interactions is lacking is that few phages have been tested for interactions with antibiotic resistance genes. To gain insight into such relationships, a collection of 33 environmental and commercial E. coli phages were screened for their reliance on the antibiotic efflux pump gene tolC. The screen identified phage U136B (
The role of competition and community in pleiotropic evolution: Comparing the mutations and phenotypes that arise during fluctuation experiments to those from evolution experiments provides an opportunity to compare the accessibility of specific mutations (in this case, tolC and LPS-related mutations) to their actual success during ecological competition and coevolution. Fluctuation experiments revealed that both tetracycline-sensitive (tolC) and colistin-sensitive (LPS-related) mutants arise readily, with LPS-related mutants dominating numerically (
The outcomes of competition over evolutionary time will also be influenced by how long phage persist in the treatment environment. While all 10 of the +phage populations started under identical conditions and contained tolC mutants, these populations otherwise widely varied in terms of phage persistence (
Obtaining a better understanding of community evolution and ecological variability is important in predicting dynamics of therapeutic phages in patients, especially since these complex environments contain other community members that can impact evolutionary outcomes. Such interdisciplinary work may include incorporating evolutionary factors and environmental heterogeneity into computational models of phage infection, tracking phage population sizes in infection models and clinical trials, and including both pharmacokinetic/pharmacodynamic (PK/PD) and phage dosing (rather than relying on a single initial inoculum). Together, these approaches may help to better control phage infection and limit persistence to therapeutic windows.
The effects of specific phage-resistance mutations on pleiotropic traits: The specific tolC mutations observed varied in their effects on the gene as well as on the resulting tetracycline phenotype. Two types of tolC phage-resistance mutations were observed: one type that lost the efflux function of TolC, and another type that retained the efflux function (
Data presented herein reveal that phage resistant mutants with LPS-synthesis gene mutations have decreased resistance to the antibiotic colistin. These data are consistent with the results of a high-throughput screen that identified some rfa genes involved in colistin resistance (
LPS-related changes may also have unintended, negative consequences in pathogenic bacterial populations. The mutation screen uncovered phage-resistant mutants that had increased resistance to tetracycline through changes to LPS (
The evolution of pleiotropic traits beyond antibiotic resistance: LPS is a crucial determinant of host range and medically relevant phenotypes like antigenicity and virulence. Many phages currently being developed for therapeutic use are reliant on LPS, so understanding the evolutionary implications of phage-LPS interactions is important in predicting if and when LPS phage selection may change bacterial virulence and resistance phenotypes. In particular, these interactions are important to predicting how phages will interact with their hosts, including potential collateral damage to commensal bacteria and the evolution of host-range expansion during treatment. Additionally, if LPS-related phage resistance also results in reduced LPS-associated virulence, then this resistance mechanism might be a useful means for reducing bacterial load while also evolutionarily reducing infection severity.
Overall, these results highlight the importance of considering community-level effects, such as competition, when measuring pleiotropic effects of phage resistance. Phage U136B and others like it are useful for the direct treatment of pathogenic infections.
Bacteriophages OMKO1, TIVP-H6, SFA1-1, SF60B, and SF37B were tested to determine if the phage could grow on the target bacterial species and that the phage could not grow on the target species when at least one of multiple putative molecules to which the bacteriophage is intended to bind was knocked out. In each case, the bacteriophage formed plaques on the target bacterium but did not form plaques when the molecule to which it was intended to bind was knocked out (
Bacteriophages LPS-5 and LPS-TLTL in Table 1 were not tested for binding to putative LPS receptors. Rather, it was observed that spontaneous mutants of the target Gram-negative bacteria (Pseudomonas aeruginosa) with evolved resistance to each of these phages showed classic “rough” colony phenotypes, associated with loss of the O-polysaccharide, the most external LPS moiety (Hancock et al. (1983) Infect. Immun. 42:170-177). Consistency of these observed rough-colony phenotypes led to the conclusion that phages LPS-5 and LPS-TLTL putatively bind to LPS of P. aeruginosa.
The binding target of phage PG-I1 in Table 1 is putatively determined to be peptidoglycan, because of the genetic relatedness of the phage sequence to other phages in the Kayvirus genus that infect S. aureus by using wall techoic acid or other portions of peptidoglycan as the binding receptor.
The binding receptor(s) for bacteriophages SFNHSI and TIVP-27 in Table 1 have not been putatitvely determined at this time using laboratory experiments.
Evolution of resistance in Shigella bacteria to phages SFA1-1, SF37B and SF60B can occur through mutations that alter or delete OmpA and/or OmpC. Shigella bacteria are facultative intracellular pathogens of humans, which replicate in epithelial cells of the colon to cause dysentery disease. Mutational changes in ompA and ompC genes of these bacteria are known to be associated with reduced virulence or loss of virulence in Shigella species, because these proteins abolish intercellular spread of the bacteria. Therefore, selection exerted by phages SFA1-1, SF37B and SF60B can cause Shigella bacteria to evolve a trade-off: evolution of phage resistance causes reduced virulence or avirulence in target bacteria.
Deletion of type-IV pili is shown to attenuate virulence in P. aeruginosa, by impairing pyocyanin production, twitching motility, and biofilm formation (Persat et al. (2015) Proc. Natl. Acad. Sci. 112:7563-7568). Phage TIVP-H6 selects for phage resistance in P. aeruginosa via bacterial alteration or loss of type-IV pili. Data showed that phage TIVP-H6 caused statistically significantly (P<0.001) reduced pyocyanin production in 48-hour cultures of PA14, similar to controls using sub-inhibitory concentration of erythromycin (Kanoh and Rubin (2010) Clinical Microbiology Reviews 23:590-615) (
Deletion of O antigen can reduce ability for CF-associated P. aeruginosa strains to produce extracellular elastase (Leone et al. (2008) European Journal of Clinical Microbiology and Infectious Disease 27:1093-1099), and can cause re-sensitivity to hydrophobic antibiotics and impair iron uptake by bacteria (Delcour (2009) Biochimica and Biophysica Acta 1794:808-816). This strongly suggests a mechanism for phage LPS-5 to select for phage resistance in P. aeruginosa via LPS mutations that decrease elastase production and drive antibiotic re-sensitivity. These data used classic Congo-elastase assays (Sachar et al. 1955) Proceedings of the Society for Experimental Biology and Medicine 90:323-326) to confirm that elastase was below the limit of detection in 48-hour-old cultures of phage resistant mutants of PA01 (
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.
The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62/844,515 filed May 7, 2019 and U.S. Provisional Patent Application No. 63/017,369, filed Apr. 29, 2020, each of which are hereby incorporated by reference in their entireties herein.
This invention was made with government support under AI144345 awarded by National Institutes of Health and under 0939454 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US20/31855 | 5/7/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63017369 | Apr 2020 | US | |
| 62844515 | May 2019 | US |
