# Patent

## Title
Engineered enzymatically active bacteriophage and methods for dispersing biofilms

## Abstract

The present invention is directed to engineered enzymatically active bacteriophages that are both capable of killing the bacteria by lysis and dispersing the bacterial biofilm because they have been also engineered to express biofilm-degrading enzymes, particularly dispersin B (DspB), an enzyme that hydrolyzes β-1,6-N-acetyl-D-glucosamine, a crucial adhesion molecule needed for biofilm formation and integrity in Staphylococcus and E. coli, including E. coli K-12, as well as clinical isolates.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of the a U.S. provisional patent application No. 61/014,518 filed Dec. 18, 2008, the content of which is incorporated herein by its entirety.


GOVERNMENT SUPPORT
This invention was made with Government Support under Contract No. EF-0425719 awarded by the National Science Foundation. The Government has certain rights in the invention.


BACKGROUND OF THE INVENTION
Bacterial biofilms are sources of contamination that are difficult to eliminate in a variety of industrial, environmental and clinical settings.
Biofilms are polymer structures secreted by bacteria to protect bacteria from various environmental attacks, and thus result also in protection of the bacteria from disinfectants and antibiotics. Biofilms may be found on any environmental surface where sufficient moisture and nutrients are present. Bacterial biofilms are associated with many human and animal health and environmental problems. For instance, bacteria form biofilms on implanted medical devices, e.g., catheters, heart valves, joint replacements, and damaged tissue, such as the lungs of cystic fibrosis patients. Biofilms also contaminate surfaces such as water pipes and the like, and render also other industrial surfaces hard to disinfect.
For example, catheters, in particular central venous catheters (CVCs), are one of the most frequently used tools for the treatment of patients with chronic or critical illnesses and are inserted in more than 20 million hospital patients in the USA each year. Their use is often severely compromised as a result of bacterial biofilm infection which is associated with significant mortality and increased costs. Catheters are associated with infection by many biofilm forming organisms such as Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis and Candida albicans which frequently results in generalized blood stream infection. Approximately 250,000 cases of CVC-associated bloodstream infections occur in the US each year with an associated mortality of 12%-25% and an estimated cost of treatment per episode of approximately $25,000. Treatment of CVC-associated infections with conventional antimicrobial agents alone is frequently unsuccessful due to the extremely high tolerance of biofilms to these agents. Once CVCs become infected the most effective treatment still involves removal of the catheter, where possible, and the treatment of any surrounding tissue or systemic infection using antimicrobial agents. This is a costly and risky procedure and re-infection can quickly occur upon replacement of the catheter.
Bacteria in biofilms are highly resistant to antibiotics and host defenses and consequently are persistent sources of infection.
Antibiotic resistance in biofilms poses a significant hurdle to eliminating biofilms with conventional antimicrobial drugs, new anti-biofilm strategies should be explored.
Accordingly, there is a need for improved phages to degrade biofilm
SUMMARY OF THE INVENTION
The present invention is directed to engineered enzymatically active bacteriophages that are both capable of killing the bacteria by lysis and dispersing the bacterial biofilm because they have been also engineered to express biofilm-degrading enzymes, particularly dispersin B (DspB), an enzyme that hydrolyzes β-1,6-N-acetyl-D-glucosamine, a crucial adhesion molecule needed for biofilm formation and integrity in Staphylococcus and E. coli, including E. coli K-12, as well as clinical isolates (Itoh, Y, Wang, X, Hinnebusch, B J, Preston, J F & Romeo, T (2005) J Bacteriol 187: 382-387).
The invention is further directed to the uses of such engineered enzymatically active bacteriophages for removing bacterial biofilms and killing bacteria within such biofilms. In particular, the invention is directed for destroying biofilms associated with Staphylococcus and E. coli, including E. coli K-12, as well as clinical isolates as described in Itoh, et al., (Id.).
Bacteriophages (often known simply as “phages”) are viruses that grow within bacteria. The name translates as “eaters of bacteria” and reflects the fact that as they grow, the majority of bacteriophages kill the bacterial host in order to release the next generation of bacteriophages. Naturally occurring bacteriophages are incapable of infecting anything other than specific strains of the target bacteria, underlying their potential for use as control agents.
Bacteriophages and their therapeutic uses have been the subject of much interest since they were first recognized early in the 20th century. Lytic bacteriophages are viruses that infect bacteria exclusively, replicate, disrupt bacterial metabolism and destroy the cell upon release of phage progeny in a process known as lysis. These bacteriophages have very effective antibacterial activity and in theory have several advantages over antibiotics. Most notably they replicate at the site of infection and are therefore available in abundance where they are most required; no serious or irreversible side effects of phage therapy have yet been described and selecting alternative phages against resistant bacteria is a relatively rapid process that can be carried out in days or weeks.
Bacteriophage have been used in the past for treatment of plant diseases, such as fireblight as described in U.S. Pat. No. 4,678,750. Also, Bacteriophages have been used to destroy biofilms (e.g., U.S. Pat. No. 6,699,701). In addition, systems using natural bacteriophages that encode biofilm destroying enzymes in general have been described. Art also provides a number of examples of lytic enzymes encoded by bacteriophages that have been used as enzyme dispersion to destroy bacteria (U.S. Pat. No. 6,335,012 and U.S. Patent Application Publication No. 2005/0004030). The Eastern European research and clinical trials, particularly in treating human diseases, such as intestinal infections, has apparently concentrated on use of naturally occurring phages and their combined uses (Lorch, A. (1999), “Bacteriophages: An alternative to antibiotics?” Biotechnology and Development Monitor, No. 39, p. 14-17).
For example, PCT Publication No. WO 2004/062677 provides a method of treating bacterial biofilm, wherein the method comprises use of a first bacteriophage that is capable of infecting a bacterium within said biofilm, and a first polysaccharide lyase enzyme that is capable of degrading a polysaccharide within said biofilm. However, other studies have showed that addition of alginate lyase to established P. aeruginosa biofilm caused no observable detachment of biofilm and thu use of lyases would not be optimal for biofilm treatment (Christensen et al., 2001).
WO/2006/137847 describes a bacteriophage that expresses a biofilm degarading enzyme attached to its surface.
We provide a novel modular design strategy in which phage that kill bacteria in a species-specific manner are engineered to express at least one of the most effective EPS-degrading enzymes specific to the target biofilm, particularly, for example, dispersin B.
This strategy permits the development of a diverse library of biofilm-dispersing phage rather than trying to isolate such phage from the environment. By multiplying within the biofilm and hijacking the bacterial machinery, engineered enzymatically-active phage achieves high local concentrations of both enzyme and lytic phage to target multiple biofilm components, even with small initial phage inoculations.
We have discovered that our invention provides rapid phage replication with subsequent bacterial lysis and expression of biofilm-degrading enzymes which renders this two-pronged attack strategy a surprisingly efficient, autocatalytic method for removing bacterial biofilms in environmental, industrial, and clinical settings. FIG. 1 shows a schematic representation of the invention.
As discussed in detail below, we have discovered that this approach is at least about two, or more, such as three or four orders of magnitude more efficient than use of a lytic phage alone. This is a significant improvement over any of the lytic phage therapies described before the present invention.
Also, a significant advantage compared to methods where enzymes are administered as dispersions or an added component to compositions containing lytic phages is that the use of the engineered enzymatically active bacteriophages of the present invention allows one to reduce or eliminate multiple applications of the composition during the treatment of a surface having a bacterial biofilm.
Moreover, unlike the controversial reports regarding use of lyase enzyme we have discovered that the phages expressing one or more biofilm degrading enzymes, for example dispersin B, are consistently effective for destroying mature biofilms.
Also, unlike the large quantities of phage required in the methods previously described, such as 108 PFU of lytic phage by Hanlon et al., the present method is efficient using as little as about 102 PFU of initial engineered phage without the need for reapplication of the phage composition due to its ability to multiply (Hanlon, G. W., Denyer, S. P., Olliff, C. J., and Ibrahim L. J., (2001). Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms. App. Env. Micro. 67, 2746-2753.).
Our design also removes the need to express, purify, and deliver large doses of enzyme to specific sites of infection that may be difficult to access, and should improve the efficacy of phage therapy at removing biofilms. Increasingly cost-effective genome sequencing and synthetic biology technologies, which include the refactoring of phage genomes and large-scale DNA synthesis, further enable the production of engineered enzymatic phage and significantly extend the repertoire of biofilm-degrading phage that have been isolated from the environment (Andrianantoandro, E, Basu, S, Karig, D K & Weiss, R (2006) Mol Syst Biol 2: 2006.0028; Chan, L Y, Kosuri, S & Endy, D (2005) Mol Syst Biol 1: 2005.0018; Itaya, M, Tsuge, K, Koizumi, M & Fujita, K (2005) Proc Natl Acad Sci USA 102: 15971-15976).
In one embodiment, and all the other embodiments of the invention, the invention provides an engineered enzymatically active T7 bacteriophage that expresses dispersin B enzyme (DspB) or a fragment thereof having the enzymatically active site of the dispersin B enzyme operably linked to a strong promoter, such as T7 φ10 promoter, wherein a nucleic acid sequence encoding a gene expanding or enhancing the infectivity and/or replication capacity of the T7 bacteriophage, such as T3 gene 1.2 is operably linked into a unique BclI site in the T7 phage and wherein a φ10-dspB construct is operably linked after the T7 phage capsid gene 10B.
In one embodiment the enzymatically active fragment of DspB has at least about 50% of the activity of the wild-type DspB, in one embodiment the activity is at least about 50-60% including all the integers in between, 60%, 70-100%, 70%, 80%, 90% or more or even more than 100% of the activity compared to the wild-type DspB enzyme.
In one embodiment and all the other embodiments of the invention, the invention provides an engineered enzymatically active T7 bacteriophage that expresses dispersin B enzyme (DspB) operably linked to a strong promoter, such as T7 φ10, wherein a nucleic acid sequence encoding a gene expanding or enhancing the infectivity and/or replication capacity of the T7 bacteriophage, such as T3 gene 1.2, is operably linked to a unique BclI site in the T7 phage and wherein the φ10-dspB construct is operably linked after the capsid gene 10B. In one embodiment, the T7 bacteriophage is T7SELECT415-1.
In one embodiment and all the other embodiments of the invention, the invention provides a method of dispersing bacterial biofilm comprising β-1,6-N-acetyl-D-glucosamine, the method comprising contacting the bacterial biofilm with a composition comprising an enzymatically active T7 bacteriophage that encodes and expresses dispersin B (DspB) operably linked to a strong promoter, such as T7 φ10, wherein a nucleic acid sequence encoding a gene expanding or enhancing the infectivity and/or expression range of the T7 bacteriophage, such as T3 gene 1.2 is operably linked into a unique BclI site in the T7 phage and wherein the φ10-dspB construct is operably linked after capsid gene 10B. In one embodiment, the biofilm is a mature biofilm. In one embodiment, the method comprises one time administration of about 102, 103, 104, or 105 PFU of the engineered enzymatially active bacteriophage.
In one embodiment, the invention provides an engineered lytic T7 bacteriophage comprising a nucleic acid encoding dispersin B operably linked to a strong promoter, such as T7 φ10 promoter and further comprising a nucleic acid encoding T3 1.2 gene, in one embodiment, the phage comprises SEQ ID NO: 9.
In one embodiment, and all the other embodiments of the invention, the administration is performed in vivo, into an animal, such as a human or livestock, wherein the engineered enzymatially active bacteriophage is administered alone or in a pharmaceutically acceptable carrier.
In one embodiment and all the other embodiments of the invention, one first diagnoses the bacterial infection in the animal, such as human or livestock, and then, based on the type of the bacterial infection, one selects a particular phage that is effective against the bacteria and the biofilm that the specific infecting bacteria produces. Bacterial cultures for diagnostic purposes are well known to one skilled in the art. Alternatively, one engineers a phage so that it effectively infects the bacterial strain that has infected the subject animal.
Similarly, a “diagnosis” step is also performed in certain embodiments, wherein other than living surfaces are treated with the methods of the invention. Accordingly, in one embodiment, one diagnoses the bacterium or bacteria infecting a surface, and then engineers a specific phage capable of infecting one or more of the bacteria present on said surface to encode an enzyme capable of digesting the specific biofilm produced by said bacterium or bacteria.
In one embodiment, animal diseases that are typically treated with antibiotics are treated with the methods of the present invention using an engineered phage. Particularly livestock, such as cows, pigs, chicken, sheep and horses are suitable target animals for the treatments of the present invention. Other animals can also be treated using the methods of the invention.
In one embodiment and all the other embodiments of the invention, the administration is performed into or onto non-living objects, such as water pipes, catheters, and other surfaces affected by bacteria and bacteria biofilm.
The administration of the enzymatically active bacteriophage can be performed before, after or concurrently with administration of other antibacterial agents, such as antibiotics and microbicides or agents capable of assisting in biofilm dispersion, such as chelating agents.
In one embodiment, the invention provides an engineered lytic T7 bacteriophage comprising a nucleic acid encoding dispersin B operably linked to T7 φ10 promoter and further comprising a nucleic acid encoding T3 1.2 gene. In one embodiment the nucleic acid encoding the phage comprises SEQ ID NO: 9. In one embodiment, the nucleic acid encoding the phage consists essentially of SEQ ID NO: 9. In one embodiment, the nucleic acid encoding the phage consists of SEQ ID NO: 9.
In another embodiment, the invention provides a method of dispersing bacterial biofilm comprising administering to a surface infected with biofilm an engineered lytic T7 bacteriophage comprising a nucleic acid encoding dispersin B operably linked to T7 φ10 promoter and further comprising a nucleic acid encoding T3 1.2 gene or another gene enhancing or expanding the phage infectivity range.
In one embodiment, the biofilm is a mature biofilm.
In one embodiment, the biofilm comprises β-1,6-N-acetyl-D-glucosamine.
In one embodiment, the method of dispersing bacterial biofilms further comprising a step of prior to administering the bacteriophage, determining if the biofilm comprises β-1,6-N-acetyl-D-glucosamine, and if it does, then administering the engineered lytic bacteriophage.
In one embodiment, the biofilm is formed by Staphylococcus and E. coli, including E. coli K-12, as well as clinical isolates of E. coli. In one embodiment, one first determines, by culturing bacterial sample from the subject or surface, if the subject carries Staphylococcus and E. coli, including E. coli K-12, as well as clinical isolates of E. coli. If the subject is determined to carry at least one of these bacterial strains, the phage of the invention is administered to the subject or surface.
In one embodiment, wherein the administering is performed once.
In one embodiment, the administering is performed before, after or concurrently with an antibiotic or antimicrobial agent.
In one embodiment, the administering is performed before, after or concurrently with a biofilm degrading chemical.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows a two-pronged attack strategy for biofilm removal with enzymatically-active DspB-expressing T7DspB phage. Initial infection of E. coli biofilm results in rapid multiplication of phage and expression of DspB. Both phage and DspB are released upon lysis, leading to subsequent infection as well as degradation of the crucial biofilm EPS component, β-1,6-N-acetyl-D-glucosamine (22).
FIGS. 2A-2C show genomes of engineered phage used for biofilm treatment. FIG. 2A shows the genome of T7SELECT415-1 shows a unique BclI site and capsid gene 10B. FIG. 2B shows a DspB-expressing phage T7DspB was created by cloning T3 gene 1.2 into the unique BclI site and cloning the φ10-dspB construct after capsid gene 10B. FIG. 2C shows a non-DspB-expressing control phage T7control was created by cloning T3 gene 1.2 into the unique BclI site and cloning the control S•Tag insert (included in the T7SELECT415-1 kit) as a fusion with the capsid gene 10B.
FIGS. 3A-3C show assays for E. coli TG1 biofilm levels and phage counts after 24 h with no treatment or with treatment with wild-type phage T7wt, wild-type phage T3wt, non-DspB-expressing control phage T7control, or DspB-expressing phage T7DspB. Error bars indicate s.e.m. FIG. 3A shows a mean absorbance (600 nm) for n=16 biofilm pegs stained with 1% CV, solubilized in 33% acetic acid, and diluted 1:3 in 1×PBS (50). FIG. 3B shows mean cell densities (log10(CFU/peg)) for n=12 biofilm pegs. Pegs treated with T7DspB resulted in a 3.65 log10(CFU/peg) reduction in viable cells recovered from E. coli biofilm compared to untreated biofilm. FIG. 3C shows mean phage counts (log10(PFU/peg)) recovered from media in n=3 microtiter plate wells (wells) or sonication of n=3 biofilm pegs (biofilm), as indicated, after 24 h of treatment with initial inoculations of 103 PFU/well. Both T7control and T7DspB showed evidence of replication with phage counts obtained from the microtiter plate wells or with phage counts recovered from the biofilms after sonication.
FIGS. 4A-4F show time-course curves, dosage response curves, and SEM images for engineered phage treatment targeting E. coli TG1 biofilm. Scale bars are 10 μm. Each data point in parts (A) and (E) represents the mean log 10-transformed cell density of n=12 biofilm pegs. Each data point in parts (D) and (F) represents the mean log 10-transformed phage counts obtained from n=3 microtiter plate wells. Error bars indicate s.e.m. FIG. 4A shows a time course (up to 48 h) of viable cell counts for no treatment (red squares), treatment with T7control (black circles), or treatment with T7DspB (blue crosses) demonstrates that T7DspB significantly reduced biofilm levels compared with T7control. FIG. 4B shows an SEM image of T7DspB-treated biofilm after 20 h shows significant disruption of the bacterial biofilm. FIG. 4C shows an SEM image of untreated biofilm after 20 h shows a dense biofilm. FIG. 4D shows a time course of phage counts obtained after initial inoculation of E. coli TG1 biofilm with 103 PFU/well of T7control (circles) or T7DspB (crosses). Both T7control and T7DspB began to replicate rapidly after initial inoculation. FIG. 4E shows dose response curves of mean cell densities (measured after 24 h of treatment) for T7control (circles) and T7DspB (crosses). For all initial phage inoculations, T7DspB-treated biofilm had significantly lower mean cell densities compared to T7control-treated biofilm. FIG. 4F shows dose response curves of mean phage counts (measured after 24 h of treatment) for T7control (circles) and T7DspB (crosses). For all initial phage inoculations, both T7control and T7DspB multiplied significantly.



DETAILED DESCRIPTION OF THE INVENTION
The present invention provides engineered enzymatically active bacteriophages and their use for efficiently destroying bacteria and bacterial biofilms, particularly bacterial biofilms that comprise β-1,6-N-acetyl-D-glucosamine. In one preferred embodiment methods of destroying or eradicating a mature biofilm and bacteria therein are provided. The methods of the present invention provide at least two orders of magnitude greater efficiency in destruction of bacterial biofilms than any previously known phage-based method that we are aware of.
We engineered bacteriophage with biofilm-degrading enzymatic activity to create a synthetic biology platform for eradicating bacterial biofilms.
The bacteriophage can be any phage that has the capacity to infect a biofilm producing bacterium, such as E. coli, P. aeriginosa, S. aureus, E. fecalis and the like. Such phages are well known to one skilled in the art, and include, but are not limited to, lambda phages, T7, T3, and T-even and T-even like phages, such as T2, and T4, and RB69; also phages such as Pf1, Pf4, Bacteroides fragilis phage B40-8 and coliphage MS-2 can be used. For example, lambda phage attacks E. coli by attaching itself to the outside of the bacteria and injecting its DNA into the bacteria. Once injected into its new host, the phage uses E. coli's genetic machinery to transcribe its genes. Any of the known phages can be engineered to express a biofilm degrading enzyme on its surface, as described herein. Preferably, the bacteriophage is T7, more preferably T7SELECT415-1.
The bacteriophages of the present invention are engineered using the traditional methods of genetic engineering that are well known to one skilled in the art. Based on the description in this specification and sequences provided herein and any other sequences known to one skilled in the art, one can readily prepare and produce the phages of the invention. We used T7SELECT415-1 phage to provide an illustration of engineering the phages of the invention. However, the same principles can be used to create any other phage known to one skilled in the art, such as lambda phages, T3, and other T-odd and T-even like phages, such as T2, T4 and RB69; and Pf1, Pf4, Bacteroides fragilis phage B40-8 and coliphage MS-2 using the principles described throughout the specification.
In one embodiment, one prepares an engineered T7 phage by using the T7SELECT415-1 phage display system (NOVAGEN). The T7select phage is engineered to express DspB intracellularly during infection. The DspB gene can be cloned, for example, from Actinobacillus actinomycetemcomitans genomic DNA (ATCC #700685D) into, for example, pET-9a plasmid (NOVAGEN) under the control of the strong promoter, such as T7 φ10 promoter. In one embodiment, the DspB gene is cloned between the NdeI and BamHI sites, for example, using the forward primer 5′ atataatc catatg aat tgt tgc gta aaa ggc aat tc 3′ (SEQ ID NO: 1) and reverse primer 5′ atatac ggatcc tca ctc atc ccc att cgt ct 3′ (SEQ Id NO: 2). In one embodiment, a stop codon is placed in all three reading frames downstream of the T7SELECT415-1 10B capsid gene followed by the φ10-dspB construct, to allow strong expression of DspB by T7 RNA polymerase during infection (FIG. 2B). The φ10-dspB construct can be isolated, for example, by PCR with the primers 5′ gTA AcT AA cgaaattaat acgactcact atagg 3′ (SEQ ID NO: 3) and 5′ atataa cggccg c aagctt tca ctc atc ccc att cgt ct 3′ (SEQ ID NO: 4)(stop codons in uppercase letters). The product can be used in a subsequent PCR reaction with the primers 5′ tactc gaattc t TAA gTA AcT AA cgaaattaat acgactc 3′(SEQ ID NO: 5) and 5′ atataa cggccg c aagctt tca ctc atc ccc att cgt ct 3′ (SEQ ID NO: 6) to create a construct beginning with stop codons in each reading frame followed by the φ10-dspB construct. Both the product of this PCR reaction and the T7SELECT415-1 DNA can be digested with EcoRI and EagI, purified, ligated together using T4 DNA ligase, and packaged into T7 phage particles with T7select packaging extracts to create phage T7DspB-precursor.
Since wild-type T7 is unable to replicate normally in F-plasmid-containing E. coli, in one embodiment, one can clone a gene that expands its host infectivity and/or replication capacity, such as gene 1.2 from phage T3 into T7DspB-precursor and T7control-precursor to create T7DspB and T7control, respectively, which are able to escape exclusion by the F plasmid (FIGS. 2B and 2C) (33). Genomic DNA from T7DspB-precursor and T7control-precursor is isolated, and T3 gene 1.2 can be cloned from the T3 genome, for example, using primers 5′ cgta tgatca aacg agcagggcga acagtg 3′ (SEQ ID NO: 7) and 5′ cgta tgatca ccactc gttaaagtga ccttaaggat tc 3′(SEQ ID NO: 8) and inserted into the unique BclI site in both the T7DspB-precursor and T7control-precursor, which are then packaged with T7select packaging extracts. The resulting phage are amplified on E. coli BL21 and then plated on E. coli TG1(lacI::kan) to isolate T7DspB (FIG. 2B) and T7control (FIG. 2C).
Bacteria frequently live in biofilms, which are surface-associated communities encased in a hydrated EPS matrix, that is composed of polysaccharides, proteins, nucleic acids, and lipids and helps maintain a complex heterogeneous structure (8, 9). Biofilms constitute an essential and protective lifestyle for bacteria in many different natural and man-made environments, including dental plaques, water pipes, medical devices, and industrial systems (10).
Bacterial biofilms have been implicated as a source of persistent infection and contamination in medical, industrial, and food processing settings due to inherent resistance to antimicrobial agents and host defenses (8, 11-13). Thus, there exists a growing need for novel and effective treatments targeted at biofilms, particularly in light of the continually-worsening problem of antibiotic resistance and the discovery that antibiotic use can even induce biofilm formation (14, 15).
Accordingly, in one embodiment, the present invention provides a method for eradicating bacteria and bacterial biofilm comprising administering to a surface affected with bacterial biofilm an enzymatically active lytic bacteriophage that has been engineered to express an enzyme capable to degrading at least β-1,6-N-acetyl-D-glucosamine.
In one embodiment, the bacteriophage further expresses at least one enzyme selected from the group consisting of enzymes listed in Table A (Xavier et al. Microbiology 151 (2005), 3817-3832).











TABLE A





Agent
Origin
Substrate
Notes/action
Reference















Enzymes











Polysaccharide
Bacteriophage
Enterobacter
Phage glycanases



depolymerase

agglomerans GFP
are very specific.




in monospecies
Action of enzyme




biofilms and in
was observed when




dual-species
added to the




biofilms with
phage-susceptible




Klebsiella
monospecies biofilm,




pneumoniae G1
leading to substantial





biofilm degradation





(Hughes et al., 1998)





A 60 min treatment with





a polysaccharase caused





a 20% reduction in





dual-species biofilm





adhesion (Skillman





et al., 1999)


Alginate lyase,
Pseudomonas
Pseudomonas
Strains of
Boyd &


See, e.g.,
aeruginosa
aeruginosa
P. aeruginosa
Chakrabarty (1994),


sequences with

alginate
overproducing alginate
Appl Environ


the following


lyase detached at a
Microbiol


database entries


higher rate than
60, 2355-2359.


O50660, ALGL_AZOCH;


wild-type


O52195, ALGL_AZOVI;


Q9ZNB7, ALGL_HALMR;


A6V1P7, ALGL_PSEA7;


Q02R18, ALGL_PSEAB;


Q06749, ALGL_PSEAE;


Q1I563, ALGL_PSEE4;


Q4KHY5, ALGL_PSEF5;


P59786, ALGL_PSEFL;


Q3KHR0, ALGL_PSEPF;


B0KGQ9, ALGL_PSEPG;


Q88ND1, ALGL_PSEPK;


Q887Q5, ALGL_PSESM;


Q9L7P2, ALGL_PSESY;


Q4ZXL0, ALGL_PSEU2;


P39049, ALXM_PHOS4;


Q59478, ALYA_KLEPN;


Q59639, ALYA_PSEAL;


Q06365, ALYP_PSESO;





However, other studies





showed that addition





of alginate lyase to





established





P. aeruginosa





biofilm caused no





observable detachment





(Christensen et al.,





2001)


Disaggregatase
Methanosarcina
Methanosarcina
Conditions that are
Xun et al. (1990),


enzyme, see, e.g.,
mazei
mazei
generally unfavourable
Appl Environ


sequences of dag of

heteropolysaccharide
for growth are
Microbiol


three M. mazei

capsule mediating
associated with
56, 3693-3698


strains that are

cell aggregation
disaggregatase


available from the


activity


DDBJ database. The


Accession nos.


are AB036793 (S-6T),


AB052161 (TMA) and


AB052162 (LYC)


Esterases with
Wide range of
Acyl residues
Acetyl residues from
Sutherland


wide specificity
bacteria
from bacterial
intracellular
(2001),




polymers as well
carboxylesterase
Microbiology 147,




as other esters
(EC 3.1.1.1) isolated
3-9





from





Arthrobacter viscosus





removed acetyl residues





from xanthan, alginate,





glucose pentaacetate,





cellobiose octaacetate,





exopolysaccharide





produced by A. viscosus,





deacetylated p-





nitrophenyl





propionate, naphthyl





acetate, isopropenyl





acetate and triacetin





(Cui et al., 1999)





Esterases could alter





the physical





properties of a





biofilm structure


Dispersin B (or
Actinobacillus
Poly-β-1,6-
Causes detachment of


DspB), see, e.g.,
actinomycetemcomitans
GlcNAc implicated
cells from


SEQ ID NO: 11

as an adhesion
A. actinomycetemcomitans




factor for biofilms
biofilms and




of several bacterial
disaggregation of




species
clumps of





A. actinomycetemcomitans





in solution (Kaplan et





al., 2003)





Treatment of





S. epidermidis





biofilms with dispersin





B causes dissolution of





the EPS matrix and





detachment of biofilm





cells from the surface





(Kaplan et al., 2004)





Disrupts biofilm





formation by E. coli,





S. epidermidis,





Yersina pestis and





Pseudomonas fluorescens





(Itoh et al., 2005)


DNase I, see,
Commercial
Extracellular
DNase affects the
Whitchurch


e.g., P00639,
(Sigma-Aldrich)
DNA in
capability of P.
et al.


DNAS1_BOVIN; Q767J3,

Pseudomonas
aeruginosa to form
(2002),


DNAS1_CANFA; Q9YGI5,

aeruginosa
biofilms when
Science


DNAS1_CHICK; Q4AEE3,

biofilms
present in the initial
295, 1487


DNAS1_HORSE; P24855,


development stages.


DNAS1_HUMAN; P49183,


Established biofilms


DNAS1_MOUSE; O42446,


were only affected to


DNAS1_OREMO; P11936,


a minor degree by


DNAS1_PIG; O18998,


the presence of


DNAS1_RABIT; P21704,


DNase


DNAS1_RAT; P11937,


DNAS1_SHEEP; P26295,


DRN1_STREQ; P57487,


END1_BUCAI; Q89AD7,


END1_BUCBP; P25736,


END1_ECOLI; P07059,


END2_BPT4;


Mixtures of
Commercial
S. aureus, S.
Pectinex UltraSP
Johansen et


enzymes

epidermidis, P.
(Novo Nordisk A/S,
al. (1997),




fluorescens and P.
a multicomponent enzyme
Appl Environ




aeruginosa
preparation) reduced
Microbiol




biofilms on steel
the number of bacterial
63, 3724-




and polypropylene
cells in biofilms on
3728




substrata
stainless steel without





any significant





bactericidal activity





(the activity of





Pectinex Ultra is





mainly a degradation





of extracellular





polysaccharides)




S. mutans,
Mutanase and dextranase




Actinomyces viscosus
were shown to remove




and Fusobacterium
oral plaque from




nucleatum
hydroxyapatite, but




biofilms on saliva-
were not bactericidal




coated hydroxyapatite
(Novo Nordisk A/S)









Example of a dispersin B gene can be found, for example, with a database accession number ACCESSION AY228551; VERSION: AY228551.1, GI:30420959, see Sequence ID NO: 11 below:








   1 aattgttgcg taaaaggcaa ttccatatat ccgcaaaaaa caagtaccaa gcagaccgga



  61 ttaatgctgg acatcgcccg acatttttat tcacccgagg tgattaaatc ctttattgat


 121 accatcagec tttccggcgg taattttctg cacctgcatt tttccgacca tgaaaactat


 181 gcgatagaaa gccatttact taatcaacgt gcggaaaatg ccgtgcaggg caaagacggt


 241 atttatatta atccttatac cggaaagcca ttcttgagtt atcggcaact tgacgatatc


 301 aaagcctatg ctaaggcaaa aggcattgag ttgattcccg aacttgacag cccgaatcac


 361 atgacggcga tctttaaact ggtgcaaaaa gacagagggg tcaagtacct tcaaggatta


 421 aaatcacgcc aggtagatga tgaaattgat attactaatg ctgacagtat tacttttatg


 481 caatctttaa tgagtgaggt tattgatatt tttggcgaca cgagtcagca ttttcatatt


 541 ggtggcgatg aatttggtta ttctgtggaa agtaatcatg agtttattac gtatgccaat


 601 aaactatcct actttttaga gaaaaaaggg ttgaaaaccc gaatgtggaa tgacggatta


 661 attaaaaata cttttgagca aatcaacccg aatattgaaa ttacttattg gagctatgat


 721 ggcgatacgc aggacaaaaa tgaagctgcc gagcgccgtg atatgcgggt cagtttgccg


 781 gagttgctgg cgaaaggctt tactgtcctg aactataatt cctattatct ttacattgtt


 841 ccgaaagctt caccaacctt ctcgcaagat gccgcctttg ccgccaaaga tgttataaaa


 901 aattgggatc ttggtgtttg ggatggacga aacaccaaaa accgcgtaca aaatactcat


 961 gaaatagccg gcgcagcatt atcgatctgg ggagaagatg caaaagcgct gaaagacgaa


1021 acaattcaga aaaacacgaa aagtttattg gaagcggtga ttcataagac gaatggggat


1081 gagtga






Bacteriophage treatment has been proposed as one method for controlling bacterial biofilms (16). Phage have been used since the early 20th century to treat bacterial infections, especially in Eastern Europe, and have been shown to decrease biofilm formation (16, 17). For example, phage T4 can infect and replicate within E. coli biofilms and disrupt biofilm morphology by killing bacterial cells (18-20). Phage have also been modified to extend their natural host range. E. coli which produce the K1 polysaccharide capsule are normally resistant to infection by T7, but are susceptible to T7 that have been designed to express K1-5 endosialidase (21). Enzymatic degradation of EPS components is another useful strategy for disrupting biofilms, though bacterial cells are not killed (8, 22, 23). For instance, enzymatic degradation of a cell-bound EPS polysaccharide adhesin known as polymeric β-1,6-N-acetyl-D-glucosamine (PGA) by exogenously-applied dispersin B (DspB) has been demonstrated to reduce biofilms of several different species of bacteria (22).
DspB, an enzyme which is produced by Actinobacillus actinomycetemcomitans, hydrolyzes PGA, a crucial adhesin needed for biofilm formation and integrity in Staphylococcus and E. coli, including E. coli K-12 as well as clinical isolates (22). Reports of natural lytic phage with phage-borne polysaccharide depolymerases have shown that phage-induced lysis and EPS degradation are used in combination in natural systems to reduce bacterial biofilms (24, 25). These depolymerases appear to be carried on the surfaces of phage and degrade bacterial capsular polysaccharides to allow access to bacterial cell surfaces (24). However, the chance that one can isolate a natural phage that is both specific for the bacteria to be targeted and expresses a relevant EPS-degrading enzyme is likely to be low (26).
We engineered T7, an E. coli-specific phage (29, 30), to express DspB intracellularly during infection so DspB would be released into the extracellular environment upon cell lysis (FIG. 1).
We employed a modified T7 strain (NOVAGEN T7SELECT415-1) with several deletions of nonessential genes (FIG. 2A). We cloned the gene coding for DspB (dspB) under the control of the strong T7 φ10 promoter so dspB would be strongly transcribed by T7 RNA polymerase during infection (FIG. 2B). As a control, we cloned an S•Tag insert into the T7 genome so that no DspB would be produced (FIG. 2C).
Accordingly, in one embodiment, the invention provides an engineered T7 E. coli-specific phage, for example T7SELECT415-1 phage, that comprises a nucleic acid encoding dispersin B (DspB) that is expressed intracellularly during phage infection of E. coli, wherein the nucleic acid encoding DspB is operably linked to a strong promoter, such as a T7 φ10 promoter, and wherein the T7 phage is engineered to further encode a gene that enhances and/or expands its infectivity and/or replication capacity, such as T3 gene 1.2. The sequences for the genes are well known to one skilled in the art and readily available from the publicly available databases.
To test the effectiveness of our engineered phage against pre-grown, mature biofilm, we cultivated E. coli TG1(lacI::kan) biofilms in LB media on plastic pegs using the standardized MBEC biofilm cultivation system. We used E. coli TG1 as the target biofilm strain since TG1 forms a thick, mature biofilm and contains the F plasmid (31). The F plasmid enhances biofilm maturation along with other biofilm-promoting factors in E. coli, including PGA, flagellum, cellulose, curli, antigen 43, and other conjugative pili and cell surface adhesins (31, 32). Because T7 is unable to replicate efficiently in F-plasmid-containing E. coli, gene 1.2 from T3 phage was also cloned into the unique BclI site in our engineered T7 phage and control T7 phage to circumvent F-plasmid-mediated exclusion and extend the phage host range (FIGS. 2B and 2C) (33). The control phage and engineered phage were named T7control and T7DspB, respectively (FIGS. 2B and 2C).
To determine whether the T7DspB phage was more effective than the T7control phage, we first employed a crystal violet (CV) assay to assess the amount of biofilm on the pegs after phage treatment. Pre-grown TG1(lacI::kan) biofilm was inoculated with only LB media or infected with 103 plaque forming units per peg (PFU/peg) of T7control or T7DspB phage (FIG. 3A). To assess whether our engineered enzymatic phage was more efficacious than wild-type phage at attacking biofilm despite being made with a modified T7 phage, we also treated biofilm with wild-type T7 (T7wt) or wild-type T3 (T3wt) (FIG. 3A).
After 24 hours of treatment, CV staining of untreated biofilm had a 600 nm absorbance (A600) approximately equal to that for T7wt-treated biofilm (FIG. 3A). Both T3wt-treated biofilm and T7control-treated biofilm were reduced compared with the untreated biofilm: the former had an A600 that was lower than that of untreated biofilm by a factor of 10.3, while the latter had an A600 that was lower than that of untreated biofilm by a factor of 5.6 (FIG. 3A).
However, the amount of biofilm left on the T7DspB-treated pegs was significantly less than that with the non-enzymatic phage treatment types, with an A600 which was less by a factor of 14.5 than that of untreated biofilm and less by a factor of 2.6 than that of T7control-treated biofilm (P=5.4*10−8).
These findings demonstrate that DspB expression in T7DspB is crucial to elevating its biofilm-removing efficacy over that of wild-type phage and non-enzymatic T7control phage (FIG. 3A).
To confirm that the decrease in CV staining corresponded with killing of biofilm cells, we used sonication to obtain viable cell counts (CFU/peg) for bacteria surviving in the biofilms after phage treatment. Pre-grown TG1(lacI::kan) biofilm (prior to treatment) reached a mean cell density of 6.4 log10(CFU/peg) after 24 h of growth (FIG. 3B). After 24 h of additional growth in new LB media with no phage treatment, the untreated biofilm had a mean cell density of 6.9 log10(CFU/peg) (FIG. 3B). T3wt-treated biofilm had a mean cell density that was less than that of T7control-treated biofilm by a factor of 5.9 and greater than that of T7DspB-treated biofilm by a factor of 12 (FIG. 3B). T7control-treated biofilm had a mean cell density of 5.1 log10(CFU/peg) while the mean cell density for T7DspB-treated biofilm was 3.2 log10(CFU/peg), the lowest of all the treatment types (FIG. 3B). The difference in viable cells recovered from T7control-treated biofilm and T7DspB-treated biofilm was statistically significant (P=1.2*10−5). These results are consistent with our CV staining data and demonstrate that DspB-expressing T7DspB phage are substantially more effective at killing E. coli TG1 biofilm compared with wild-type T3wt, wild-type T7wt, and non-DspB-expressing control T7control phage.
Our two-pronged method of biofilm eradication involves expression of DspB and rapid phage replication (FIG. 1). To confirm that our phage multiplied, we obtained PFU counts from media in the microtiter plate wells. By 24 h of treatment, wild-type T7 had not replicated but wild-type T3 had multiplied significantly within the biofilm (FIG. 3C). To compare the amount of phage in the microtiter plate wells with phage residing in the biofilms, we also obtained PFU counts by sonicating the biofilms. After 24 h of treatment, PFU counts for T7control and T7DspB recovered from the microtiter plate wells were several orders of magnitude greater than PFU counts recovered by sonication of the biofilms (FIG. 3C). Overall, PFU counts obtained from the wells and the biofilms were all orders of magnitude greater than the initial inoculation of 103 PFU, confirming that phage multiplication indeed took place (FIG. 3C).
Accordingly, we determined that T7DspB had greater biofilm-removing capability than T7control after 24 h of infection. We also determined the time course of biofilm destruction. As shown in FIG. 4A, by 5 h post-infection, T7DspB-treated biofilm had a mean cell density that was 0.82 log10(CFU/peg) less than T7control-treated biofilm (P=2.0*10−4). At 10 h post-infection, T7DspB-treated biofilm began to settle at a steady-state mean cell density between 3 to 4 log10(CFU/peg), while T7control-treated biofilm flattened out at approximately 5 log10(CFU/peg) by 20 h post-infection (FIG. 4A).
T7DspB-treated biofilms had mean cell densities that were approximately two orders of magnitude lower than T7control-treated biofilms, up to 48 h of total treatment (FIG. 4A).
In addition, T7DspB treatment reduced biofilm levels by about 99.997% (4.5 log10(CFU/peg)) compared with untreated biofilm.
Further, we found no evidence of phage resistance developing over the long time course of treatment (FIG. 4A).
We also used scanning electron microscopy (SEM) to image the biofilm pegs over the time course of phage treatment in order to directly visualize biofilm dispersal by our enzymatically-active phage (FIG. 4B, FIG. 4C). After 20 h of treatment, T7DspB-treated biofilm (FIG. 4B) was significantly disrupted compared with the untreated biofilm (FIG. 4C).
These results confirm that T7DspB indeed causes both biofilm reduction and bacterial cell killing.
We studied scanning electron microscopy images for untreated, T7control-treated, and T7DspB-treated biofilms. Consistent with time-course data (FIG. 4A), T7DspB-treated biofilm and T7control-treated biofilm were indistinguishable from untreated biofilm at 2 h 25 min post-infection. However, by 4 h post-infection, T7DspB-treated biofilm began to lyse and disperse significantly, while T7control-treated biofilm was still largely undisturbed. By 10 h post-infection, significant amounts of cell debris were seen in both T7control-treated and T7DspB-treated biofilms. At 20 h post-infection, T7control-treated and T7DspB-treated biofilms had been disrupted by phage treatment, but T7DspB-treated biofilm was composed largely of cell debris and had fewer intact cells than T7control-treated biofilm.
To verify that phage replication was occurring over time, we obtained PFU counts in the microtiter wells. As seen in FIG. 4D, both T7control and T7DspB began to replicate within the bacterial biofilm as early as 50 minutes post-infection. By about 190 minutes, T7control and T7DspB PFU/peg approached steady-state levels of approximately 8 to 9 log10(PFU/peg), indicating that phage replication had occurred (FIG. 4D). T7DspB PFU/peg were generally higher than T7control PFU/peg but not by orders of magnitude as was the case for CFU counts per peg. This is because the T7 burst size ( ̃250 PFU per infective center) (34) multiplied by the number of the extra cells killed by T7DspB, compared with T7control, equals extra PFU/peg that are insignificant compared with the PFU levels already reached by T7control. We did not note any significant differences in burst sizes and growth rates between T7DspB and T7control.
Considering that the above experiments were carried out with initial inoculations of 103 PFU/peg, which translates to a multiplicity of infection (MOI) of about 1:103,4 (FIG. 4A), we next determined the effect of changing the initial MOI on biofilm removal. With low phage doses, repeated rounds of phage multiplication and DspB expression should promote biofilm dispersal and allow more bacterial cells to be accessible for subsequent phage infection. With high phage doses, initial DspB production post-infection should also be very disruptive to biofilm integrity.
As shown in FIG. 4E, T7DspB was much more effective than T7control at removing biofilm at all inoculation levels tested, ranging from 101 PFU/peg to 105 PFU/peg. A dose-dependent effect of phage inoculation on biofilm destruction was observed, with larger inoculations leading to lower mean cell densities, particularly for T7DspB (FIG. 4E).
At inoculation levels greater than or equal to 102 PFU/peg, mean cell densities (CFU/peg) for T7DspB-treated biofilm were significantly lower than those for T7control-treated biofilm by a factor of 49-232 (FIG. 4E).
Thus, at low and high initial inoculations, DspB-expressing T7 is more efficacious at disrupting E. coli TG1 biofilm compared with non-DspB-expressing control T7. All phage dosages tested exhibited phage multiplication within the biofilm (FIG. 4F).
Without wishing to be bound by a theory, these results together show that DspB-expressing phage has significantly improved efficacy in real-world situations where the ability to deliver high levels of phage to biofilms may be limited or where sustained phage replication is less likely, e.g., in the gastrointestinal tract of cholera patients (35, 36).
Accordingly, we demonstrated that our novel engineered phage which express biofilm-degrading enzymes are more efficacious at removing bacterial biofilms than non-enzymatic phage alone. Therefore, we have described and taught a phage design that can be adapted to work in other phage and with other biofilm-degrading enzymes to target a wide range of biofilms.
Thus, engineered bacteriophage treatment provides a novel addition to the therapies or treatment methods available for use against bacterial biofilms in medical, industrial, and biotechnological settings (17).
In one embodiment, the bacteria to be targeted using the phage used in the methods of the invention include E. coli, S. epidermidis, Yersina pestis and Pseudomonas fluorescens. 
The described phage system can also be designed to include directed evolution for optimal enzyme activity, delaying cell lysis or using multiple phage promoters to allow for increased enzyme production, targeting multiple biofilm EPS components with different proteins. One can also target multi-species biofilm with a cocktail of different species-specific engineered enzymatically-active phage, and use a combination therapy using the engineered phage of the invention and antibiotics or combinations thereof that are well known to one skilled in the art to improve the efficacy of both types of treatment.
The phages of the invention can also be used together with other antibacterial or bacteriofilm degrading agents or chemicals such as EGTA, a calcium-specific chelating agent, effected the immediate and substantial detachment of a P. aeruginosa biofilm without affecting microbial activity, NaCl, CaCl2 or MgCl2, surfactans and urea.
Phage therapy has begun to be accepted in industrial and biotechnological settings. For example, the FDA recently approved the use of phage targeted at Listeria monocytogenes as a food additive (37). Despite the fact that phage therapy has several challenges that must be overcome before it will be accepted in Western medicine for treating humans (17), phage therapies have been used successfully in Eastern Europe for over 60 years. It has been shown, for example, that combination therapy with antibiotics and phage may alleviate the development of phage resistance (26, 36, 38). Long-circulating phage has been isolated that also avoids RES clearance to increase in vivo efficacy (35). Accordingly, the methods of the present invention are applicable to human and other animal treatment although clinical trials may be needed to establish their specific tolerance. However, our experiments have already shown that these methods are effective in dispersing biofilms, including biofilms present in human organs, such as colon or lungs.
The specificity of the phage for host bacteria is both an advantage and a disadvantage for phage therapy. Specificity allows human cells as well as innocuous bacteria to be spared, potentially avoiding serious issues such as drug toxicity or Clostridium difficile overgrowth that can arise with antibiotic use. C. difficile infection is characterized by diarrhea and colitis, and has increased in severity in recent years (42). Antibiotic therapy is believed to alter the microbial flora in the colon due to lack of target specificity, thus allowing C. difficile to proliferate and cause disease (43). Furthermore, the ability of our engineered phage to utilize the local bacterial synthetic machinery to produce biofilm-degrading enzymes means that exogenously-applied enzymes, which could have unintended effects on off-target biofilms, are not needed.
However, host specificity means that a well-characterized library of phage must be maintained so that an appropriate therapy can be designed for each individual infection (26). The diversity of bacterial infections implies that it may be difficult for any particular engineered phage to be a therapeutic solution for a wide range of biofilms. Accordingly, in one embodiment, the invention provides use of engineered enzymatically active phage cocktails that comprise at least two, three, four, five, 6, 7, 8, 9, 10 or even more different phages that have different hosts to cover wider a range of target bacteria. In one embodiment, at least one of the phages in the cocktail is an engineered lytic T7 bacteriophage comprising a nucleic acid encoding dispersin B enzyme operably linked to a T7 φ10 promoter and further comprising a nucleic acid encoding T3 1.2 gene. In one embodiment, at least one of the phages comprises a nucleic acid with SEQ ID NO: 9.
One skilled in the art can make a collection of enzymatically-active engineered phage by cost-effective, large-scale DNA sequencing and DNA synthesis technologies described and well known to one skilled in the art (see, e.g., 2, 4, 44). Sequencing technologies allows the characterization of collections of natural phage that have been used in phage typing and phage therapy for many years (45, 46). Accordingly, a skilled artisan can use synthetic technologies as described herein to add biofilm-degrading enzymes to produce new, modified phage.
Furthermore, rational engineering methods with new synthesis technologies can be employed to broaden phage host range. For example, T7 can be modified to express K1-5 endosialidase, allowing it to effectively replicate in E. coli that produce the K1 polysaccharide capsule (21). We took advantage of gene 1.2 from phage T3 to extend our phage host range to include E. coli that contain the F plasmid, thus demonstrating that multiple modifications of a phage genome can be done without significant impairment of the phage's ability to replicate (33). Bordetella bacteriophage use a reverse-transcriptase-mediated mechanism to produce diversity in host tropism which can also be used according to the methods of the present invention to create a phage that encodes a biofilm degrading enzyme, such as dispersin B, and is lytic to the target bacterium or bacteria (47, 48). In addition, utilizing enzymes, such as DspB, that target important adhesins which are common to a broad range of bacterial species, including clinical strains, allow enzymatically-active phage to be applicable to treatment of a greater number of infections (22). The many biofilm-promoting factors required by E. coli K-12 to produce a mature biofilm are likely to be shared among different biofilm-forming bacterial strains and are thus also targets for engineered enzymatic bacteriophage (32).
The enzymatically active bacteriophage of the invention can be formulated in combination with one or more pharmaceutically-acceptable anti-microbial agents. In this regard, combinations of different antimicrobial agents may be tailored to target different (or the same) microorganisms that contribute towards morbidity and mortality. The pharmaceutically acceptable anti-microbial agents of the present invention are suitable for internal administration to an animal, for example human. However, if the phage of the invention is to be used in industrial sterilizing, sterilizing chemicals such as detergents, disinfectants, and ammonium-based chemicals (e.g. quaternary ammonium compounds such as QUATAL) can be used in combination with, or prior to or after the treatment with the phage. Such sterilizing chemicals are typically used in the art for sterilizing industrial work surfaces (e.g. in food processing, or hospital environments), and are not suitable for administration to an animal.
Strong promoter useful according to the present invention are well known to a skilled artisan. Similarly, various genes that can enhance or expand the infectivity and/or replication range of a phage are well known to a skilled artisan.
The present invention also provides pharmaceutical compositions comprising an engineered lytic T7 bacteriophage comprising a nucleic acid encoding dispersin B operably linked to T7 φ10 promoter, e.g., a phage comprising a nucleic acid encoding SEQ ID NO: 9, and further comprising a nucleic acid encoding T3 1.2 gene, and a pharmaceutically acceptable excipient. Suitable carriers for the enzymatically active lytic phages of the invention, for instance, and their formulations, are described in Remington' Pharmaceutical Sciences, 16th ed., 1980, Mack Publishing Co., edited by Oslo et al. Typically an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic. Examples of the carrier include buffers such as saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7.4 to about 7.8. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g. liposomes, films or microparticles. It will be apparent to those of skill in the art that certain carriers may be more preferable depending upon for instance the route of administration and concentration of the a enzymatically active bacteriophage being administered.
Administration to human may be accomplished by means determined by the underlying condition. For example, if the phage is to be delivered into lungs of an individual, inhalers can be used. If the composition is to be delivered into any part of the gut or colon, coated tablets, suppositories or orally administered liquids, tablets, caplets and so forth may be used. A skilled artisan will be able to determine the appropriate way of administering the phages of the invention in view of the general knowledge and skill in the art.
Practice of the present invention will employ, unless indicated otherwise, conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, protein chemistry, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning: A Laboratory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, eds.), Cold Spring Harbor Laboratory Press, 1989; DNA Cloning, Volumes I and II (D. N. Glover, ed), 1985; Oligonucleotide Synthesis, (M. J. Gait, ed.), 1984; U.S. Pat. No. 4,683,195 (Mullis et al.,); Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins, eds.), 1984; Transcription and Translation (B. D. Hames and S. J. Higgins, eds.), 1984; Culture of Animal Cells (R. I. Freshney, ed). Alan R. Liss, Inc., 1987; Immobilized Cells and Enzymes, IRL Press, 1986; A Practical Guide to Molecular Cloning (B. Perbal), 1984; Methods in Enzymology, Volumes 154 and 155 (Wu et al., eds), Academic Press, New York; Gene Transfer Vectors for Mammalian Cells (J. H. Miller and M. P. Calos, eds.), 1987, Cold Spring Harbor Laboratory; Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds.), Academic Press, London, 1987; Handbook of Experiment Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds.), 1986; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, 1986.
The following Examples are provided to illustrate the present invention, and should not be construed as limiting thereof.
Some embodiments of the invention are provided in the following paragraphs:
1. An engineered lytic T7 bacteriophage comprising a nucleic acid encoding dispersin B enzyme or an β-1,6-N-acetyl-D-glucosamine degrading enzymatically active fragment thereof operably linked to a strong promoter and further comprising a nucleic acid encoding a gene to enhance or expand infectivity and or replication capacity of the lytic T7 bacteriophage.
2. The engineered lytic T7 bacteriophage of paragraph 1, wherein the nucleic acid encoding a gene to enhance or expand infectivity and or replication capacity of the lytic T7 bacteriophage encodes T3 1.2 gene.
3. The engineered lytic T7 bacteriophage of paragraphs 1 or 2, wherein the strong promoter is T7 φ10.
4. A method of dispersing bacterial biofilm comprising administering to a surface infected with biofilm the method comprising administering to the surface an engineered lytic T7 bacteriophage comprising a nucleic acid encoding dispersin B enzyme or an β-1,6-N-acetyl-D-glucosamine degrading enzymatically active fragment thereof operably linked to a strong promoter and further comprising a nucleic acid encoding a gene to enhance or expand infectivity and or replication capacity of the lytic T7 bacteriophage.
5. The method of paragraph 4, wherein the nucleic acid encoding a gene to enhance or expand infectivity and or replication capacity of the lytic T7 bacteriophage encodes T3 1.2 gene.
6. The method of paragraphs 4 or 5, wherein the strong promoter is T7 φ10.
7. The method of paragraph 4, wherein the biofilm is a mature biofilm.
8. The method of paragraph 4, wherein the biofilm comprises β-1,6-N-acetyl-D-glucosamine.
9. The method of paragraph 8 further comprising a step of prior to administering the bacteriophage, determining if the biofilm comprises β-1,6-N-acetyl-D-glucosamine, and if it does, then administering the engineered lytic bacteriophage.
10. The method of paragraph 4, wherein the biofilm is formed by bacteria selected from the group consisting of Staphylococcus and E. Coli, including E. Coli K-12 strain, and clinical isolates of E. coli. 
11. The method of paragraph 4, wherein the administering is performed once.
12. The method of paragraph 4, wherein the administering is performed before, after or concurrently with an antibiotic or antimicrobial agent.
13. The method of paragraph 4, wherein the administering is performed before, after or concurrently with a biofilm degrading chemical.
EXAMPLES
Bacterial strains, bacteriophage, and chemicals. E. coli TG1 (F′traD36 lacIqΔ(lacZ) M15 proA+B+/supE Δ(hsdM-mcrB)5 (rk- mk- McrB-) thi Δ(lac-proAB)) was obtained from Zymo Research (Orange, Calif.). The strain TG1 (lacI::kan) used to grow biofilm was created by one-step inactivation of the lacI gene by a kanamycin-resistance cassette (49). E. coli BL21 was obtained from NOVAGEN Inc. (San Diego, Calif.). Wild-type T7 (ATCC #BAA-1025-B2) and T3 (ATCC #11303-B3) were purchased from ATCC (Manassas, Va.). Standard chemicals were obtained from sources as described in Supporting Methods.
Construction and purification of engineered phage. Our engineered T7 phage was created using the T7SELECT415-1 phage display system (NOVAGEN) with standard molecular biology techniques. Instead of cloning DspB onto the phage surface, we designed the T7select phage to express DspB intracellularly during infection. The dspB gene was cloned from Actinobacillus actinomycetemcomitans genomic DNA (ATCC #700685D) under the control of the strong T7 φ10 promoter downstream of the T7SELECT415-1 10B capsid gene and stop codons in all three reading frames to create T7DspB-precursor (FIG. 2B). Packaging of the modified genome was done with the T7select packaging extracts. The control phage, T7control-precursor, was constructed by cloning the T7select control S•Tag insert into the T7SELECT415-1 genome (FIG. 2C). Since wild-type T7 cannot replicate normally in F-plasmid-containing E. coli, we cloned gene 1.2 from phage T3 into the unique BclI site in T7DspB-precursor and T7control-precursor to create T7DspB and T7control, respectively, which are able to escape exclusion by the F plasmid (FIGS. 2B and 2C) (33). The resulting phage were amplified on E. coli BL21 and plated on E. coli TG1 (lacI::kan) to isolate T7DspB (FIG. 2B) and T7control (FIG. 2C), which were confirmed by PCR to have T3 gene 1.2.
Prior to biofilm treatment, T7DspB and T7control were amplified on E. coli BL21 and purified. 12 mL of BL21 overnight cultures were diluted with 12 mL of LB in 125 mL flasks, inoculated with 30 μL of high-titer phage stock, and allowed to lyse at 37° C. and 300 rpm for 3 h. Lysed cultures were clarified by centrifuging for 10 minutes at 10,000 g and filtering the supernatants through NALGENE #190-2520 0.2 μm filters (Nalge Nunc International, Rochester, N.Y.). The purified solutions were centrifuged in a Beckman SW.41T rotor for 1 h at 29,600 rpm to concentrate the phage. The supernatants were removed and pellets were resuspended in 0.2 M NaCl, 2 mM Tris-HCl pH 8.0, and 0.2 mM EDTA. Phage suspensions were reclarified in tabletop microcentrifuges at maximum speed ( ̃13,200 rpm) for 10 minutes. The purified supernatants were finally diluted in 0.2 M NaCl, 2 mM Tris-HCl pH 8.0, and 0.2 mM EDTA for treatment. Appropriate amounts of phage were added to LB+kanamycin (30 μg/mL) for treatment, as described below. Phage purified by this protocol were no more effective at reducing bacterial biofilm levels compared with phage purified by centrifugation with CsCl step gradients.
All phage PFU counts were determined by combining phage with 300 μL of overnight E. coli BL21 culture and 4-5 mL of 50° C. LB top agar (0.7% w/v agar). This solution was mixed thoroughly, poured onto LB agar plates, inverted after hardening, and incubated for 4-6 h at 37° C. until plaques were clearly visible.
Biofilm growth and treatment. All experiments were performed in LB media+kanamycin (30 μg/mL). E. coli biofilms were grown with the MBEC Physiology & Genetics Assay (MBEC BioProducts Inc., Edmonton, Canada), which consists of a 96-peg lid that fits into a standard 96-well microtiter plate. Each well was inoculated with 150 μL of media containing 1:200 dilutions of overnight cultures which had been grown at 37° C. and 300 rpm. Control wells with only media but no bacteria were included. MBEC lids were placed in the microtiter plates, inserted into plastic bags to prevent evaporation, and placed in a shaker (HT Infors MINITRON) for 24 h at 35° C. and 150 rpm to form biofilm on the pegs.
For all treatments except for the dose response experiment, 103 PFU of phage were combined with 200 μL LB+kanamycin (30 μg/mL) in each well in new microtiter plates (COSTAR #3370). For the dose response experiment, 101, 102, 103, 104, or 105 PFU were combined with 200 μL LB+kanamycin (30 μg/mL) in each well. Wells with only media but no phage were included as untreated biofilm controls. MBEC lids with 24 h pre-grown E. coli biofilm were removed from their old 96-well microtiter plates, and placed into the new microtiter plates and back into plastic bags in a shaker at 35° C. and 150 rpm for treatment. After specified amounts of time for the time-course experiment or 24 h for all other experiments, MBEC lids were removed and the amounts of biofilm remaining were assayed by CV staining or viable cell counting, as described below.
Crystal violet (CV) staining assay. Crystal violet staining of MBEC pegs was carried out, after rinsing three times with 1× phosphate-buffered saline (PBS), using a standard, previously reported protocol as described in Supporting Methods (50).
Viable cell count assay. We obtained viable cell counts by disrupting biofilms on the pegs in a sonicating water bath. MBEC pegs were first rinsed three times with 200 μL of 1×PBS and placed into fresh microtiter plates (NUNC #262162) containing 145 μL of 1×PBS in each well, which completely covered the biofilms growing on the pegs. To prevent further infection of bacteria by phage, 20 ng of T7 Tail Fiber Monoclonal Antibody (NOVAGEN) was added to each well. MBEC lids and plates were placed in a Branson Ultrasonics #5510 sonic water bath (Danbury, Conn.) and sonicated for 30 minutes at 40 kHz to dislodge bacteria in biofilms into the wells. Serial dilutions were performed and plated on LB agar+kanamycin (30 μg/mL) plates. Colony-forming units were counted after overnight incubation at 37° C.
Scanning electron microscopy. SEM was performed according to MBEC recommendations (51).
Phage counts. At indicated time points (FIG. 4D) or after 24 h of treatment (FIG. 3C and FIG. 4F), media from n=3 microtiter wells for each treatment type were serially diluted to obtain PFU counts for phage in the liquid phase. To obtain PFU counts for phage residing in biofilms at 24 h post-infection (FIG. 3C), MBEC pegs were rinsed three times with 200 μL of 1× phosphate-buffered saline (PBS) and placed into fresh microtiter plates (NUNC #262162) containing 145 μL of 1×PBS in each well, which completely covered the biofilm on the pegs. No T7 Tail Fiber Monoclonal Antibody was added. The MBEC lids and plates were placed in a Branson Ultrasonics #5510 sonic water bath (Danbury, Conn.) and sonicated for 30 minutes at 40 kHz to dislodge bacteria and phage residing in biofilms into wells. Serial dilutions were performed to obtain PFU counts for phage in biofilms.
Statistical analysis. Student's unpaired two-sided t-test was used to test for statistical significance as described in Supporting Methods. For the CV staining assays, the dataset size for each treatment type was n=16; for the CFU assays, n=12 pegs per treatment type were used.
Standard chemicals. T4 DNA ligase and all restriction enzymes were obtained from New England Biolabs, Inc. (Ipswich, Mass.). PCR reactions were carried out using PCR SuperMix High Fidelity from INVITROGEN (Carlsbad, Calif.). Restriction digests of T7 genomic DNA were purified with the QIAEX II kit, while purification of all other PCR reactions and restriction digests was carried out with the QIAQUICK Gel Extraction or PCR Purification kits (QIAGEN, Valencia, Calif.). All other chemicals were purchased from Fisher Scientific, Inc. (Hampton, N.H.).
Construction and purification of engineered phage. Our engineered T7 phage was created using the T7SELECT415-1 phage display system (NOVAGEN). Instead of cloning DspB onto the phage surface, we designed the T7select phage to express DspB intracellularly during infection. The dspB gene was cloned from Actinobacillus actinomycetemcomitans genomic DNA (ATCC #700685D) into the pET-9a plasmid (Novagen) under the control of the strong T7 φ10 promoter in between the NdeI and BamHI sites using the forward primer 5′ atataatc catatg aat tgt tgc gta aaa ggc aat tc 3′ (SEQ ID NO: 1) and reverse primer 5′ atatac ggatcc tca ctc atc ccc att cgt ct 3′ (SEQ Id NO: 2) (restriction sites underlined). We placed a stop codon in all three reading frames downstream of the T7SELECT415-1 10B capsid gene followed by the φ10-dspB construct, so dspB would be strongly transcribed by T7 RNA polymerase during infection (FIG. 2B). The φ10-dspB construct was isolated by PCR with the primers 5′ gTA AcT AA cgaaattaat acgactcact atagg 3′ (SEQ ID NO: 3) and 5′ atataa cggccg c aagctt tca ctc atc ccc att cgt ct 3′ (SEQ ID NO: 4) (stop codons in uppercase letters); the product was used in a subsequent PCR reaction with the primers 5′ tactc gaattc t TAA gTA AcT AA cgaaattaat acgactc 3′(SEQ ID NO: 5) and 5′ atataa cggccg c aagctt tca ctc atc ccc att cgt ct 3′ (SEQ ID NO: 6) to create a construct beginning with stop codons in each reading frame followed by the φ10-dspB construct. Both the product of this PCR reaction and the T7SELECT415-1 DNA were digested with EcoRI and EagI, purified, ligated together using T4 DNA ligase, and packaged into T7 phage particles with T7select packaging extracts to create phage T7DspB-precursor. The control phage, T7control-precursor, was constructed by cloning the T7select control S•Tag insert into the T7SELECT415-1 phage genome and packaging the genome using T7select packaging extracts (FIG. 2C). Phage T7DspB-precursor and T7control-precursor were routinely amplified on E. coli BL21 and verified by DNA sequencing.
Since wild-type T7 is unable to replicate normally in F-plasmid-containing E. coli, we cloned gene 1.2 from phage T3 into T7DspB-precursor and T7control-precursor to create T7DspB and T7control, respectively, which are able to escape exclusion by the F plasmid (FIGS. 2B and 2C) (33). Genomic DNA from T7DspB-precursor and T7control-precursor was isolated using the QIAGEN Lambda Midi Kit. T3 gene 1.2 was cloned from the T3 genome using primers 5′ cgta tgatca aacg agcagggcga acagtg 3′ (SEQ ID NO: 7) and 5′ cgta tgatca ccactc gttaaagtga ccttaaggat tc 3′ (SEq ID NO: 8) and inserted into the unique BclI site in both the T7DspB-precursor and T7control-precursor, which were then packaged with T7select packaging extracts. The resulting phage were amplified on E. coli BL21 and then plated on E. coli TG1(lacI::kan) to isolate T7DspB (FIG. 2B) and T7control (FIG. 2C), which were confirmed by PCR to have gene 1.2 from T3.
Crystal violet staining assay. MBEC pegs were rinsed three times with 200 μL of 1× phosphate-buffered saline (PBS) and placed into fresh microtiter plates with wells containing 200 μL of 1% CV. After 15 minutes of incubation at room temperature, the stained MBEC pegs were washed three times with 200 μL of 1×PBS and placed into fresh microtiter plates containing 200 μL of 33% acetic acid to solubilize the dye for 15 minutes (50). To avoid oversaturating the absorbance detector, 66.7 μL of the solubilized dye was added to 133.3 μL of 1×PBS; the absorbance at 600 nm of this mixture was assayed in a TECAN SPECTRAFLUOR Plus plate reader (Zurich, Switzerland). The mean A600 nm of wells corresponding to pegs with no biofilm growth was used as a blank measurement to correct all other A600 nm absorbances.
Scanning electron microscopy. Scanning electron microscopy was carried out with a Carl Zeiss Supra 40 VP SEM using Carl Zeiss SMARTSEM V05.01.08 software. Biofilm pegs were broken off at indicated time points and washed three times in 1×PBS. The pegs were then fixed in 2.5% glutaraldehyde in 0.1 M cacodylic acid (pH 7.2) for 2 to 3 h at room temperature. Subsequently, the pegs were air dried for at least 120 h, and mounted and examined by SEM in VPSE mode with EHT=7.5 kV. Each peg was examined at several locations prior to imaging to ensure that characteristic images were acquired. Images were frame- or line-integrated using the SMARTSEM software to achieve better resolution.
Statistical analysis. Data for time-course CFU counts were collected from three independent experiments; all other CFU data were obtained from single experiments in time. Absorbance from crystal violet staining assays or CFU counts from viable cell count assays were evaluated for statistically significant differences using Student's unpaired two-sided t-test (assuming unknown and unequal variances) with an alpha level of 0.05 implemented in MATLAB version 7.0.01 (MATHWORKS, Natick, Mass.). All CFU data were log 10-transformed prior to analysis. All absorbance data and log 10-transformed CFU data were verified to be approximately normally distributed using the qqplot( ) function in MATLAB version 7.0.1 to meet the assumptions of the t-test. Error bars in figures indicate standard error of the mean (s.e.m).
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## Claims

1. A method of dispersing a bacterial biofilm on a surface comprising administering to said surface a genetically engineered lytic T7 bacteriophage wherein the bacteriophage comprises a nucleic acid encoding dispersin B enzyme operably linked to a strong promoter and a nucleic acid encoding a protein to enhance or expand infectivity and or replication capacity of the lytic T7 bacteriophage, wherein the nucleic acid encoding the protein to enhance or expand infectivity and or replication capacity of the lytic T7 bacteriophage encodes T3 1.2 protein.

2. The method of claim 1, wherein the strong promoter is T7 φ10.

3. The method of claim 1, wherein the biofilm is a mature biofilm.

4. The method of claim 1, wherein the biofilm comprises β-1,6-N-acetyl-D-glucosamine.

5. The method of claim 4 further comprising a step of prior to administering the bacteriophage, determining if the biofilm comprises β-1,6-N-acetyl-D-glucosamine, and if it does, then administering the engineered lytic bacteriophage.

6. The method of claim 1, wherein the biofilm is formed by—Staphylococcus and E. coli, including E. coli K-12 strain, and clinical isolates of E. coli.

7. The method of claim 1, wherein the administering is performed once.

8. The method of claim 1, wherein the administering is performed before, after or concurrently with an antibiotic or antimicrobial agent.

9. The method of claim 1, wherein the administering is performed before, after or concurrently with a biofilm degrading chemical.

10. The method of claim 1, wherein the phage further comprises a terminator sequence.