Multiple host range bacteriophage with hybrid tail fibres

Abstract

Modified bacteriophage, uses thereof, and compositions containing the modified bacteriophage are described. The compositions are useful for human treatment and may treat various conditions, including bacterial infections.

Claims

1. A modified bacteriophage, capable of infecting a plurality of different target bacteria, which bacteriophage includes an α/β small acid-soluble spore protein (SASP) gene encoding a SASP which is toxic to the target bacteria; wherein the bacteriophage is non-lytic; and wherein the bacteriophage expresses a hybrid host range determinant protein which comprises an amino acid sequence from a plurality of different bacteriophages; wherein said bacteriophage comprises an inactivated lysis gene, and wherein the hybrid host range determinant protein comprises a tail fibre protein comprising a receptor binding region for binding to the target bacteria and a region linking the receptor binding region to the body of the bacteriophage, wherein the receptor binding region is a C-terminal receptor binding region and the region linking the C-terminal receptor binding region to the body of the bacteriophage is an N-terminal region, and wherein each of the C-terminal and N-terminal regions are from a different bacteriophage than each other; and further wherein the N-terminal region comprises amino acids 1 to 628 of the tail fibre protein and the C-terminal region comprises amino acids 629 to 964 of the tail fibre protein, based on the amino acid sequence of bacteriophage Phi33, and/or wherein the C-terminal region has no more than 96% amino acid sequence identity with the C-terminal region of bacteriophage Phi33.

2. A modified bacteriophage according to claim 1, wherein the C-terminal region is from any one of bacteriophage Phi33, LBL3, SPM-1, F8, PB1, KPP12, LMA2, SN, 14-1, JG024, NH4, PTP47, PTP92, C36 and PTP93.

3. A modified bacteriophage according to claim 2, wherein the C-terminal region amino sequence identity is less than 80%, or less than 70%, or less than 60%.

4. A modified bacteriophage capable of infecting a plurality of different Pseudomonas target bacteria, which bacteriophage includes an α/β small acid-soluble spore protein (SASP) gene encoding a SASP which is toxic to the target bacteria; wherein the bacteriophage is non-lytic; and wherein the bacteriophage expresses a hybrid host range determinant protein which comprises an amino acid sequence from a plurality of different bacteriophages, wherein the hybrid host range determinant comprises a hybrid tail fibre protein, or a tail fibre protein which comprises a receptor binding region for binding to the target bacteria and a region linking the receptor binding region to the body of the bacteriophage, wherein the receptor binding region is a C-terminal receptor binding region and the region linking the C-terminal receptor binding region to the body of the bacteriophage is an N-terminal region, wherein each of the C-terminal and N-terminal regions are from a different bacteriophage, wherein the N-terminal region comprises amino acids 1 to 628 of the tail fibre protein and the C-terminal region comprises amino acids 629 to 964 of the tail fibre protein, based on the amino acid sequence of bacteriophage Phi33, and wherein the C-terminal region has no more than 96% amino acid sequence identity with the C-terminal region of bacteriophage Phi33, and wherein the N-terminal region has at least 95% amino acid sequence identity with the N-terminal region of bacteriophage Phi33.

5. A modified bacteriophage capable of infecting a plurality of different target bacteria, which bacteriophage includes an α/β small acid-soluble spore protein (SASP) gene encoding a SASP which is toxic to the target bacteria; wherein the bacteriophage expresses a hybrid host range determinant protein which comprises an N-terminal region and a C-terminal region from different bacteriophages, wherein the N-terminal region comprises amino acids of a first bacteriophage having at least 95% identity to amino acids 1 to 628 of the tail fibre protein of bacteriophage Phi33 and the C-terminal region comprises amino acids of a second bacteriophage corresponding to amino acids 629 to 964 of the tail fibre protein of bacteriophage Phi33, and wherein the C-terminal region has no more than 96% amino acid sequence identity with the C-terminal region of amino acids 629 to 964 of bacteriophage Phi33.

6. A modified bacteriophage according to claim 5, wherein at least one of the target bacteria is Pseudomonas.

7. A modified bacteriophage according to claim 5, wherein the bacteriophage is non-lytic.

8. A modified bacteriophage according to claim 7, wherein said inactivated lysis gene is inactivated by insertion of a SASP gene.

9. A modified bacteriophage according to claim 8, wherein said inserted SASP gene is SASP-C.

10. A modified bacteriophage according to claim 8, wherein said inserted SASP gene is SASP-C from Bacillus megaterium.

11. A modified bacteriophage according to claim 5, wherein the expression of the SASP gene is under the control of a constitutive promoter which provides for the expression of toxic levels of SASP when the modified bacteriophage is present in multiple copies in the target bacterium.

12. A modified bacteriophage according to claim 11, wherein said promoter is selected from pdhA, rpsB, pgi, fda, and lasB.

13. A modified bacteriophage according to claim 5, wherein: (i) the hybrid tail fibre protein comprises the C-terminal receptor binding region of bacteriophage PTP47 and the N-terminal region of bacteriophage Phi33 or (ii) the C-terminal receptor binding region of bacteriophage PTP92 and the N-terminal region of bacteriophage Phi33.

14. A composition comprising a modified bacteriophage according to claim 5 and a pharmaceutically acceptable carrier.

15. The composition of claim 14, further comprising at least one other modified bacteriophage which is capable of infecting target bacteria, which includes a SASP gene encoding a SASP which is toxic to the target bacteria and which is non-lytic.

16. A method of treatment of bacterial infection in a subject in need thereof, which comprises administering to the subject an effective amount of a modified bacteriophage capable of infecting a plurality of different target bacteria according to claim 5.

17. A method according to claim 16, wherein the bacterial infection comprises a localised organ infection or a multi-organ infection, or a topical infection, oral infection, respiratory infection, eye infection or blood stream infection.

18. The method of claim 16, which is for human therapy.

19. A method of bacterial decontamination, which comprises treating surface bacterial contamination, land remediation or water treatment, with at least one modified bacteriophage according to claim 5.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) This invention will now be described in more detail, by way of example only, and with reference to the accompanying drawings, in which:

(2) FIGS. 1A-1C is a schematic diagram showing construction of plasmids containing lacZΔM15 and Phi33 endolysin;

(3) FIGS. 2A-2D is a schematic diagram showing construction of plasmids with replaced tail fibre sections;

(4) FIGS. 3A-3D is a schematic diagram showing construction of phage with hybrid tail fibre genes, which may be subsequently modified to have endolysin replaced by SASP-C according to the invention;

(5) FIGS. 4A-4D is a schematic diagram showing construction of phage with further hybrid tail fibre genes, which may be subsequently modified to have endolysin replaced by SASP-C according to the invention;

(6) FIGS. 5A-5B is a schematic diagram showing construction of bacteriophage with hybrid tail fibre genes, in which the lacZα marker has been removed;

(7) FIGS. 6A-6D is a schematic diagram showing construction of plasmids in which the endolysin gene is replaced by SASP-C;

(8) FIGS. 7A-7B is a schematic diagram showing production of further bacteriophage according to the invention;

(9) FIGS. 8A-8D is a schematic diagram showing construction of plasmids in which the endolysin gene is replaced by SASP-C codon optimised for expression in P. aeruginosa;

(10) FIG. 9 shows the sequence of the SASP-C gene from Bacillus megaterium strain KM (ATCC 13632), which has been codon optimised for expression in P. aeruginosa;

(11) FIGS. 10A-10D is a schematic diagram showing production of bacteriophage in which the endolysin gene is replaced by SASP-C which has been codon optimised for expression in P. aeruginosa;

(12) FIGS. 11A-11D is a schematic diagram showing production of further bacteriophage according to the invention;

(13) FIGS. 12A-12B is a schematic diagram showing production of further bacteriophage according to the invention;

(14) FIGS. 13A-13E are a CLUSTAL 2.1 multiple sequence alignment of the tail fibre genes from Phage SPM-1, F8, PB1, C36, LBL3, Phi33, LMA2, KPP12, JG024, PTP92, NH-4, 14-1, PTP47, SN. Sequence divergent C-terminal region shaded in grey, sequence conserved N-terminal region unshaded; and

(15) FIG. 14 shows a bar graph of kill of P. aeruginosa by Phi33, PTP92 and a mixture of PTP110 and PTP114 (labelled “Mix”). Bacteria and phage were incubated together at 37° C. and samples removed at 0 and 3 hours for viable cell counting.

(16) FIGS. 15A-15B. 24 hour time-kill curve showing the in vitro efficacy of PT3.8 against P. aeruginosa strains 3503 (FIG. 15A) and ATCC 27853 (FIG. 15B). Cultures were grown in Luria bertani (LB) broth supplemented with 5 mM calcium chloride, 5 mM magnesium sulphate and 0.1% glucose, at 37° C.

(17) FIG. 16. 60 minute time-kill curve showing the in vitro efficacy of PT3.8 against P. aeruginosa strain 3503. Cultures were grown in Luria bertani (LB) broth supplemented with 5 mM calcium chloride, 5 mM magnesium sulphate and 0.1% glucose, at 37° C.

(18) FIG. 17. In vivo efficacy of PT3.8 in a murine lung model of infection. Line labelled “0 hour” shows viable cell numbers in the lung tissue at the time of treatment with either vehicle (tris buffered saline containing 1 mM magnesium sulphate, 10 mM calcium chloride and 10% v/v glycerol) or PT3.8 (2 hours post infection with P. aeruginosa). The viable cell counts in lung tissue at 2 and 22 hours post treatment are shown for each animal in each group (group size=6), the geomean for each data set is represented by a horizontal line. For the 22 hour vehicle group, accurate values could be ascertained for only 3 of 6 animals, the viable cell counts for the other 3 animals were >10{circumflex over ( )} 11 cfu/ml.

GENERIC PRODUCT COVERING A SINGLE TAIL FIBRE WITHIN AN INDIVIDUAL PHAGE, OR A MIX OF PHAGES WHERE EACH TYPE OF PHAGE HAS A SINGLE, DIFFERENT TAIL FIBRE

(19) Summary of the genetic modification of a lytic bacteriophage to render it non-lytic, and such that it carries one of a number of possible tail fibre variants, in addition to SASP-C under the control of a promoter that usually controls expression of the 30S ribosomal subunit protein S2 gene (rpsB).

(20) Genes can be removed and added to the phage genome using homologous recombination. There are several ways in which phages carrying foreign genes and promoters can be constructed and the following is an example of such methods.

(21) For the construction of a Phi33 derivative it is shown how, using an E. coli/P. aeruginosa broad host range vector, as an example only, the existing tail fibre, or a section of the tail fibre, in the bacteriophage genome may be replaced by an alternative tail fibre or tail fibre section from a different bacteriophage, via homologous recombination. It is also shown as an example only, how the resulting phage may be rendered non-lytic, and how additional DNA sequences, such as the SASP-C gene from B. megaterium under the control of a P. aeruginosa rpsB promoter, or the SASP-C gene from B. megaterium, codon optimised for expression in P. aeruginosa, under the control of a P. aeruginosa fda promoter may be added to the bacteriophage genome via homologous recombination.

(22) A tail fibre gene, or section of a tail fibre gene, from an alternative phage may be cloned between two regions of Phi33 DNA that flank the native tail fibre, or section thereof, along with a lacZα genetic marker, in a broad host range E. coli/P. aeruginosa vector. This plasmid may be introduced into P. aeruginosa, and the resulting strain infected with Phi33. Following harvesting of progeny phage, double recombinants in which the native Phi33 tail fibre or tail fibre section, has been replaced by the new tail fibre or tail fibre section and lacZα, may be isolated by plaquing on a suitable P. aeruginosa (lacZΔM15.sup.+) host strain using medium containing a chromogenic substrate that detects the action of β-galactosidase.

(23) In a subsequent step, the lacZα marker may be removed from the bacteriophage genomes by making versions of the previously described tail fibre region recombination plasmids that do not contain the lacZα marker, introducing the new plasmids into suitable P. aeruginosa host strains and infecting with the previously modified bacteriophage derivatives of Phi33 carrying the corresponding alternative tail fibre gene, or section thereof, along with the lacZα marker. Recombinants that retain the new tail fibre or tail fibre section, but from which lacZα has been removed, may be isolated by plaquing on a suitable P. aeruginosa (lacZΔM15.sup.+) host strain using medium containing a chromogenic substrate that detects the action of β-galactosidase.

(24) In a subsequent step, a similar homologous recombination may be used to replace the endolysin gene of Phi33, or of any of the previously described Phi33 derivatives, or similar bacteriophage or similar derivatives, with the gene for SASP-C, under the control of a P. aeruginosa rpsB promoter, while simultaneously adding an E. coli lacZα reporter gene for the identification of recombinant phage. A region consisting of SASP-C controlled by the rpsB promoter, and the E. coli lacZα allele, may be cloned between two regions of Phi33 that flank the endolysin gene, in a broad host range E. coli/P. aeruginosa vector. This plasmid may be transferred to a suitable P. aeruginosa (endolysin.sup.+ lacZΔM15.sup.+) strain, and the resulting strain infected by Phi33 or the previously constructed Phi33 derivative (from which the initial lacZα marker has been removed). Progeny phage may be harvested and double recombinants identified by plaquing on P. aeruginosa (endolysin.sup.+ lacZΔM15.sup.+), looking for acquisition of the new lacZα reporter on medium containing a chromogenic substrate that detects the action of β-galactosidase.

(25) In an alternative subsequent step, a similar homologous recombination may be used to replace the endolysin gene of Phi33, or of any of the previously described Phi33 derivatives, or similar bacteriophage or similar derivatives, with the gene for SASP-C that has been codon optimised for expression in P. aeruginosa, under the control of a P. aeruginosa fda promoter, while simultaneously adding an E. coli lacZα reporter gene for the identification of recombinant phage. A region consisting of codon optimised SASP-C controlled by the fda promoter, and the E. coli lacZα allele, may be cloned between two regions of Phi33 that flank the endolysin gene, in a broad host range E. coli/P. aeruginosa vector. This plasmid may be transferred to a suitable P. aeruginosa (endolysin.sup.+ lacZΔM15.sup.+) strain, and the resulting strain infected by Phi33 or the previously constructed Phi33 derivative (from which the initial lacZα marker has been removed). Progeny phage may be harvested and double recombinants identified by plaquing on P. aeruginosa (endolysin.sup.+ lacZΔM15.sup.+), looking for acquisition of the new lacZα reporter on medium containing a chromogenic substrate that detects the action of β-galactosidase.

(26) Since these bacteriophage to be modified are lytic (rather than temperate), another requirement for these described steps of bacteriophage construction is the construction of a suitable host P. aeruginosa strain that carries either the E. coli lacZΔM15 gene at a suitable location in the bacterial genome, or both the Phi33 endolysin gene and the E. coli lacZΔM15 at a suitable location in the bacterial genome, to complement the lacZα or Δendolysin, lacZαphenotypes of the desired recombinant bacteriophage. As an example, the construction of these P. aeruginosa strains may be achieved via homologous recombination using an E. coli vector that is unable to replicate in P. aeruginosa. The genomic location for insertion of the endolysin and lacZΔM15 transgenes should be chosen such that no essential genes are affected and no unwanted phenotypes are generated through polar effects on the expression of adjacent genes. As an example, one such location may be immediately downstream of the P. aeruginosa strain PAO1 phoA homologue.

(27) The E. coli lacZΔM15 allele may be cloned into an E. coli vector that is unable to replicate in P. aeruginosa, between two regions of P. aeruginosa strain PAO1 genomic DNA that flank the 3′ end of phoA. This plasmid may be introduced into P. aeruginosa and isolates having undergone a single homologous recombination to integrate the whole plasmid into the genome selected according to the acquisition of tetracycline (50 μg/ml) resistance. Isolates (lacZΔM15.sup.+) which have undergone a second homologous recombination event may then be isolated on medium containing 10% sucrose (utilising the sacB counter-selectable marker present on the plasmid backbone).

(28) The Phi33 endolysin gene and the E. coli lacZΔM15 allele may be cloned into an E. coli vector that is unable to replicate in P. aeruginosa, between two regions of P. aeruginosa strain PAO1 genomic DNA that flank the 3′ end of phoA. This plasmid may be introduced into P. aeruginosa and isolates having undergone a single homologous recombination to integrate the whole plasmid into the genome selected according to the acquisition of tetracycline (50 μg/ml) resistance. Isolates (endolysin.sup.+, lacZΔM15.sup.+) which have undergone a second homologous recombination event may then be isolated on medium containing 10% sucrose (utilising the sacB counter-selectable marker present on the plasmid backbone).

(29) In a subsequent step, a similar homologous recombination may be used to remove the lacZα marker from the previously described, (lacZα.sup.+) Phi33 derivatives that have been modified to replace the endolysin gene with the gene for SASP-C, under the control of a P. aeruginosa rpsB promoter. A region consisting of SASP-C controlled by the rpsB promoter, may be cloned between two regions of Phi33 that flank the endolysin gene, in a broad host range E. coli/P. aeruginosa vector. This plasmid may be transferred to a suitable P. aeruginosa (endolysin.sup.+ lacZΔM15.sup.k) strain, and the resulting strain infected by the previously described (lacZα.sup.+) Phi33 derivatives that have been modified to replace the endolysin gene with the gene for SASP-C, under the control of a P. aeruginosa rpsB promoter. Progeny phage may be harvested and double recombinants identified by plaquing on P. aeruginosa (endolysin.sup.+ lacZΔM15.sup.+), looking for loss of the lacZα reporter on medium containing a chromogenic substrate that detects the action of β-galactosidase.

(30) In an alternative subsequent step, a similar homologous recombination may be used to remove the lacZα marker from the previously described, (lacZα.sup.+) Phi33 derivatives that have been modified to replace the endolysin gene with the gene for SASP-C, codon optimised for expression in P. aeruginosa, under the control of a P. aeruginosa fda promoter. A region consisting of SASP-C, codon optimised for expression in P. aeruginosa, controlled by the fda promoter, may be cloned between two regions of Phi33 that flank the endolysin gene, in a broad host range E. coli/P. aeruginosa vector. This plasmid may be transferred to a suitable P. aeruginosa (endolysin.sup.+ lacZΔM15.sup.+) strain, and the resulting strain infected by the previously described (lacZα.sup.+) Phi33 derivatives that have been modified to replace the endolysin gene with the gene for SASP-C, codon optimised for expression in P. aeruginosa, under the control of a P. aeruginosa fda promoter. Progeny phage may be harvested and double recombinants identified by plaquing on P. aeruginosa (endolysin.sup.+ lacZΔM15.sup.+), looking for loss of the lacZα reporter on medium containing a chromogenic substrate that detects the action of β-galactosidase.

(31) Experimental Procedures

(32) PCR reactions to generate DNA for cloning purposes may be carried out using Herculase II Fusion DNA polymerase (Agilent Technologies), depending upon the melting temperatures (T.sub.m) of the primers, according to manufacturers instructions. PCR reactions for screening purposes may be carried out using Taq DNA polymerase (NEB), depending upon the T.sub.m of the primers, according to manufacturers instructions. Unless otherwise stated, general molecular biology techniques, such as restriction enzyme digestion, agarose gel electrophoresis, T4 DNA ligase-dependent ligations, competent cell preparation and transformation may be based upon methods described in Sambrook et al., (1989). Enzymes may be purchased from New England Biolabs or Thermo Scientific. DNA may be purified from enzyme reactions and prepared from cells using Qiagen DNA purification kits. Plasmids may be transferred from E. coli strains to P. aeruginosa strains by conjugation, mediated by the conjugation helper strain E. coli HB101 (pRK2013). A chromogenic substrate for β-galactosidase, S-gal, that upon digestion by β-galactosidase forms a black precipitate when chelated with ferric iron, may be purchased from Sigma (S9811).

(33) Primers may be obtained from Sigma Life Science. Where primers include recognition sequences for restriction enzymes, additional 2-6 nucleotides may be added at the 5′ end to ensure digestion of the PCR-amplified DNA.

(34) All clonings, unless otherwise stated, may be achieved by ligating DNAs overnight with T4 DNA ligase and then transforming them into E. coli cloning strains, such as DH5α or TOP10, with isolation on selective medium, as described elsewhere (Sambrook et al., 1989).

(35) An E. coli/P. aeruginosa broad host range vector, such as pSM1080, may be used to transfer genes between E. coli and P. aeruginosa. pSM1080 was previously produced by combining the broad host-range origin of replication from a P. aeruginosa plasmid, oriT from pRK2, the tetAR selectable marker for use in both E. coli and P. aeruginosa, from plasmid pRK415, and the high-copy-number, E. coli origin of replication, oriV, from plasmid pUC19.

(36) An E. coli vector that is unable to replicate in P. aeruginosa, pSM1104, may be used to generate P. aeruginosa mutants by allelic exchange. pSM1104 was previously produced by combining oriT from pRK2, the tetAR selectable marker for use in both E. coli and P. aeruginosa, from plasmid pRK415, the high-copy-number, E. coli origin of replication, oriV, from plasmid pUC19, and the sacB gene from Bacillus subtilis strain 168, under the control of a strong promoter, for use as a counter-selectable marker.

(37) Detection of Phi33-Like Phage (PB1-Like Phage Family) Conserved N-Terminal Tail Fibre Regions by PCR

(38) 1. Primers for the detection of Phi33-like phage-like tail fibre genes in experimental phage samples may be designed as follows:

(39) The DNA sequences of the tail fibre genes from all sequenced Phi33-like phage (including Phi33, PB1, NH-4, 14-1, LMA2, KPP12, JG024, F8, SPM-1, LBL3, PTP47, C36, PTP92 and SN) may be aligned using Clustal Omega, which is available on the EBI website, and the approximately 2 kb-long highly conserved region mapping to the gene's 5′ sequence may be thus identified (positions 31680-33557 in the PB1 genome sequence, Acc. EU716414). Sections of 100% identity among the 11 tail fibre gene sequences may be identified by visual inspection. Three pairs of PCR primers targeting selected absolutely conserved regions, and amplifying PCR products no longer than 1 kb may be chosen as follows: pair B4500 and B4501, defining a 194 bp-long region; pair B4502 and B4503, defining a 774 bp-long region; and pair B4504 and B4505, defining a 365 bp-long region.

(40) Primer B4500 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 31680 to 31697. Primer B4501 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 31851 to 31872. Primer B4502 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 31785 to 31804. Primer B4503 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 32541 to 32558. Primer B4504 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 32868 to 32888. Primer B4505 consists of sequence of PB1 phage genome (Acc. EU716414) ranging from position 33213 to 33232.

(41) TABLE-US-00001 B4500 (SEQ ID NO: 1) 5′-GTGATCACACCCGAACTG-3′ B4501 (SEQ ID NO: 2) 5′-CGATGAAGAAGAGTTGGTTTTG-3′ B4502 (SEQ ID NO: 3) 5′-ACGCCGGACTACGAAATCAG-3′ B4503 (SEQ ID NO: 4) 5′-TCCGGAGACGTTGATGGT-3′ B4504 (SEQ ID NO: 5) 5′-CCTTTCATCGATTTCCACTTC-3′ B4505 (SEQ ID NO: 6) 5′-TTCGTGGACGCCCAGTCCCA-3′
2. Phi33-like tail fibre genes may be detected in experimental phage samples as follows:

(42) Plaques of isolated phage of environmental origin may be picked from agar plates and added to water and incubated for 30 minutes, making plaque soak outs. The plaque soak outs may be diluted and a portion added to PCR reactions containing one or all of the above primer pairs, and PCR may be performed according to a standard protocol. PCR products may be visualised on a 1.5% agarose gel with ethidium bromide staining, and evaluated for their size. PCR products of the correct size for the primer pair used may be gel-extracted and submitted to an external facility for sequencing. Sequencing results may be compared with the available tail fibre gene sequences in order to confirm the identity of the PCR product.

(43) Construction of Plasmids to Generate Pseudomonas aeruginosa Strains Carrying Either the Escherichia coli lacZΔM15 Gene, or Both the Phi33 Endolysin Gene and the Escherichia coli lacZΔM15 Gene, Immediately Downstream of the phoA Locus of the Bacterial Genome
1. Plasmid pSMX200 (FIG. 1), comprising pSM1104 carrying DNA flanking the 3′ end of the P. aeruginosa PAO1 phoA homologue, may be constructed as follows.

(44) A region comprising the terminal approximately 1 kb of the phoA gene from P. aeruginosa may be amplified by PCR using primers B4200 and B4201 (FIG. 1). The PCR product may then be cleaned and digested with SpeI and BglII. A second region comprising approximately 1 kb downstream of the phoA gene from P. aeruginosa, including the 3′ end of the PA3297 open reading frame, may be amplified by PCR using primers B4202 and B4203 (FIG. 1). This second PCR product may then be cleaned and digested with BglII and XhoI. The two digests may be cleaned again and ligated to pSM1104 that has been digested with SpeI and XhoI, in a 3-way ligation, to yield plasmid pSMX200 (FIG. 1).

(45) Primer B4200 consists of a 5′ SpeI restriction site (underlined), followed by sequence located approximately 1 kb upstream of the stop codon of phoA from P. aeruginosa strain PAO1 (FIG. 1). Primer B4201 consists of 5′ BglII and AflII restriction sites (underlined), followed by sequence complementary to the end of the phoA gene from P. aeruginosa strain PAO1 (the stop codon is in lower case; FIG. 1). Primer B4202 consists of 5′ BglII and NheI restriction sites (underlined), followed by sequence immediately downstream of the stop codon of the phoA gene from P. aeruginosa strain PAO1 (FIG. 1). Primer B4203 consists of a 5′ XhoI restriction site (underlined), followed by sequence within the PA3297 open reading frame, approximately 1 kb downstream of the phoA gene from P. aeruginosa strain PAO1 (FIG. 1).

(46) Primer B4200

(47) 5′-GATAACTAGTCCTGGTCCACCGGGGTCAAG-3′ (SEQ ID NO: 7)

(48) Primer B4201

(49) 5′-GCTCAGATCTTCCTTAAGtcaGTCGCGCAGGTTCAG-3′ (SEQ ID NO: 8)

(50) Primer B4202

(51) 5′-AGGAAGATCTGAGCTAGCTCGGACCAGAACGAAAAAG-3′ (SEQ ID NO: 9)

(52) Primer B4203

(53) 5′-GATACTCGAGGCGGATGAACATTGAGGTG-3′ (SEQ ID NO: 10)

(54) 2. Plasmid pSMX201 (FIG. 1), comprising pSMX200 carrying the Phi33 endolysin gene under the control of an endolysin promoter, may be constructed as follows.

(55) The endolysin promoter may be amplified by PCR from Phi33 using primers B4204 and B4205 (FIG. 1). The endolysin gene itself may be amplified by PCR from Phi33 using primers B4206 and B4207 (FIG. 1). The two PCR products may then be joined together by Splicing by Overlap Extension (SOEing) PCR, using the two outer primers, B4204 and B4207. The resulting PCR product may then be digested with AflII and BglII, and ligated to pSMX200 that has also been digested with AflII and BglII, to yield plasmid pSMX201 (FIG. 1).

(56) Primer B4204 consists of a 5′ AflII restriction site (underlined), followed by a bi-directional transcriptional terminator (soxR terminator, 60-96 bases of Genbank accession number DQ058714), and sequence of the beginning of the Phi33 endolysin promoter region (underlined, in bold) (FIG. 1). Primer B4205 consists of a 5′ region of sequence that is complementary to the region overlapping the start codon of the endolysin gene from Phi33, followed by sequence that is complementary to the end of the endolysin promoter region (underlined, in bold; FIG. 1). Primer B4206 is the reverse complement of primer B4205 (see also FIG. 1). Primer B4207 consists of a 5′ BglII restriction site (underlined), followed by sequence complementary to the end of the Phi33 endolysin gene (FIG. 1).

(57) TABLE-US-00002 Primer B4204 (SEQ ID NO: 11) 5′-GATACTTAAGAAAACAAACTAAAGCGCCCTTGTGGCGCTTTAGTTTT ATACTACTGAGAAAAATCTGGATTC-3′ Primer B4205 (SEQ ID NO: 12) 5′-GATTTTCATCAATACTCCTGGATCCCGTTAATTCGAAGAGTCG-3′  Primer B4206 (SEQ ID NO: 13) 5′-CGACTCTTCGAATTAACGGGATCCAGGAGTATTGATGAAAATC-3′ Primer B4207 (SEQ ID NO: 14) 5′-GATAAGATCTTCAGGAGCCTTGATTGATC-3′
3. Plasmid pSMX202 (FIG. 1), comprising pSMX201 carrying lacZΔM15 under the control of a lac promoter, may be constructed as follows.

(58) The lacZΔM15 gene under the control of a lac promoter may be amplified by PCR from Escherichia coli strain DH10B using primers B4208 and B4209 (FIG. 1). The resulting PCR product may then be digested with BglII and NheI, and ligated to pSMX201 that has also been digested with BglII and NheI, to yield plasmid pSMX202 (FIG. 1).

(59) Primer B4208 consists of a 5′ BglII restriction site (underlined), followed by sequence of the lac promoter (FIG. 1). Primer B4209 consists of a 5′ NheI restriction site (underlined), followed by a bi-directional transcriptional terminator and sequence complementary to the 3′ end of lacZΔM15 (underlined, in bold; FIG. 1).

(60) TABLE-US-00003 Primer B4208 (SEQ ID NO: 15) 5′-GATAAGATCTGAGCGCAACGCAATTAATGTG-3′ Primer B4209 (SEQ ID NO: 16) 5′-GATAGCTAGCAGTCAAAAGCCTCCGGTCGGAGGCTTTTGACTTTATT TTTGACACCAGACCAAC-3′
4. Plasmid pSMX203 (FIG. 1), comprising pSMX200 carrying lacZΔM15 under the control of a lac promoter, may be constructed as follows.

(61) The lacZΔM15 gene under the control of a lac promoter may be amplified by PCR from Escherichia coli strain DH10B using primers B4208 and B4209 (FIG. 1). The resulting PCR product may then be digested with BglII and NheI, and ligated to pSMX200 that has also been digested with BglII and NheI, to yield plasmid pSMX203 (FIG. 1).

(62) Primer B4208 consists of a 5′ BglII restriction site (underlined), followed by sequence of the lac promoter (FIG. 1). Primer B4209 consists of a 5′ NheI restriction site (underlined), followed by a bi-directional transcriptional terminator and sequence complementary to the 3′ end of lacZΔM15 (underlined, in bold; FIG. 1).

(63) TABLE-US-00004 Primer B4208 (SEQ ID NO: 15) 5′-GATAAGATCTGAGCGCAACGCAATTAATGTG-3′ Primer B4209 (SEQ ID NO: 16) 5′-GATAGCTAGCAGTCAAAAGCCTCCGGTCGGAGGCTTTTGACTTTATT TTTGACACCAGACCAAC-3′
Genetic Modification of Pseudomonas aeruginosa to Introduce the Phi33 Endolysin Gene and Escherichia coli lacZΔM15 Immediately Downstream of the phoA Locus of the Bacterial Genome
1. Plasmid pSMX202 (FIG. 1) may be transferred to P. aeruginosa by conjugation, selecting for primary recombinants by acquisition of resistance to tetracycline (50 μg/ml).
2. Double recombinants may then be selected via sacB-mediated counterselection, by plating onto medium containing 10% sucrose.
3. Isolates growing on 10% sucrose may then be screened by PCR to confirm that the endolysin gene and lacZΔM15 have been introduced downstream of the P. aeruginosa phoA gene.
4. Following verification of an isolate (PAX20), this strain may then be used as a host for further modification of Phi33-related bacteriophage, where complementation of both an endolysin mutation and a lacZα reporter are required.
Genetic Modification of Pseudomonas aeruginosa to Introduce the Escherichia coli lacZΔM15 Gene Immediately Downstream of the phoA Locus of the Bacterial Genome
1. Plasmid pSMX203 (FIG. 1) may be transferred to P. aeruginosa by conjugation, selecting for primary recombinants by acquisition of resistance to tetracycline (50 μg/ml).
2. Double recombinants may then be selected via sacB-mediated counterselection, by plating onto medium containing 10% sucrose.
3. Isolates growing on 10% sucrose may then be screened by PCR to confirm that lacZΔM15 has been introduced downstream of the P. aeruginosa phoA gene.
4. Following verification of an isolate (PAX21), this strain may then be used as a host for further modification of bacteriophage, where complementation of a lacZα reporter is required.
Construction of a Plasmid to Replace the 3′ Section of the Phi33 Tail Fibre with that of PTP92, Utilising a lacZα Screening Process
1. pSMX204 (FIG. 2), comprising pSM1080 carrying the region immediately downstream of the Phi33 tail fibre gene, may be constructed as follows.

(64) A 1 kb region of Phi33 sequence covering the terminal 20 bases of the Phi33 tail fibre, and the adjacent downstream region, may be amplified by PCR using primers B4222 and B4249 (FIG. 2). The resulting PCR product may then be cleaned and digested with NheI, and ligated to pSM1080 that has also been digested with NheI and then treated with alkaline phosphatase prior to ligation, yielding plasmid pSMX204 (FIG. 2).

(65) Primer B4222 consists of a 5′ NheI restriction site (underlined), followed by sequence from Phi33, approximately 1 kb downstream of the end of the Phi33 tail fibre gene (FIG. 2). B4249 consists of 5′ NheI-KpnI-AvrII restriction sites (underlined), followed by sequence complementary to the 3′ end of the Phi33 tail fibre and sequence immediately downstream of the tail fibre open reading frame (FIG. 2).

(66) TABLE-US-00005 B4222 (SEQ ID NO: 17) 5′-GATAGCTAGCATGGTTTTCACGACCATG-3′ B4249 (SEQ ID NO: 18) 5′-GATAGCTAGCGAGGTACCGACCTAGGTTTTCCAGCGAGTGACGTAA AATG-3′ 
2. pSMX205 (FIG. 2), comprising pSMX204 carrying lacZα, a 3′ section of the PTP92 tail fibre gene sequence, and a region of Phi33 sequence comprising the 5′ end of the tail fibre gene and sequence located immediately upstream of the Phi33 tail fibre gene, may be constructed as follows.

(67) The lacZα open reading frame may be amplified by PCR from pUC19 using primers B4250 and B4252 (FIG. 2). The PTP92 tail fibre 3′ section may be amplified by PCR from PTP92 using primers B4251 and B4254 (FIG. 2). The lacZα open reading frame may then be joined to the PTP92 tail fibre gene 3′ section by SOEing PCR using the outer primers, B4250 and B4254. A region comprising sequence of the 5′ end of the Phi33 tail fibre gene, and sequence located immediately upstream of the Phi33 tail fibre gene, may be amplified by PCR using primers B4253 and B4229 (FIG. 2). This PCR product may then be joined to the PCR product comprising lacZα and the PTP92 tail fibre gene 3′ section, by SOEing PCR using the outer primers B4250 and B4229. The resulting PCR product may then be cleaned and digested with AvrII and KpnI, and ligated to pSMX204 that has also been digested with AvrII and KpnI, yielding plasmid pSMX205 (FIG. 2).

(68) Primer B4250 consists of a 5′ AvrII restriction site, followed by sequence complementary to the 3′ end of the lacZα open reading frame (FIG. 2). Primer B4252 consists of a 5′ section of sequence that overlaps the 3′ end of the PTP92 tail fibre gene (underlined), followed by sequence of the 5′ end of the lacZα open reading frame. Primer B4251 is the reverse complement of primer B4252 (FIG. 2). Primer B4254 consists of 5′ sequence from within the Phi33 tail fibre gene (underlined), followed by sequence within the 3′ end of the PTP92 tail fibre gene (FIG. 2). Primer B4253 is the reverse complement of Primer B4254. Primer B4229 consists of a 5′ KpnI restriction site (underlined), followed by sequence that is complementary to a region approximately 1 kb upstream of the tail fibre gene in Phi33 (FIG. 2).

(69) TABLE-US-00006 Primer B4250 (SEQ ID NO: 19) 5′-GATACCTAGGTTAGCGCCATTCGCCATTC-3′ Primer B4252 (SEQ ID NO: 20) 5′-CTATTCCAGCGGGTAACGTAAAATGACCATGATTACGGATTC-3′  Primer B4251 (SEQ ID NO: 21) 5′-GAATCCGTAATCATGGTCATTTTACGTTACCCGCTGGAATAG-3′ Primer B4254 (SEQ ID NO: 22) 5′-CAAGCGGGCCGGCTGGTCTCTCGGCAATAACTCCTATGTGATC-3′  Primer B4253 (SEQ ID NO: 23) 5′-GATCACATAGGAGTTATTGCCGAGAGACCAGCCGGCCCGCTTG-3′ Primer B4229 (SEQ ID NO: 24) 5′-GATAGGTACCGCGACCGGTCTGTACTTC-3′
Genetic Modification of Phi33 to Replace the 3′ Section of the Tail Fibre Gene with that of PTP92
1. Plasmid pSMX205 (FIG. 2; FIG. 3) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 μg/ml), yielding strain PTA20.
2. Strain PTA20 may be infected with phage Phi33, and the progeny phage harvested.
3. Recombinant phage in which the 3′ end of the Phi33 tail fibre gene has been replaced by that of PTP92, and to which lacZα has been added, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for black plaques, which are indicative of β-galactosidase activity.
4. PCR may be carried out to check that the tail fibre gene has been replaced, and that lacZα is present.
5. Following identification of a verified isolate (PTPX21; FIG. 3), this isolate may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.
Construction of a Plasmid to Replace the 3′ Section of the Phi33 Tail Fibre with that of PTP47, Utilising a lacZα Screening Process
1. pSMX206 (FIG. 2), comprising pSMX204 carrying lacZα, a 3′ section of the PTP47 tail fibre gene sequence, and a region of Phi33 sequence comprising the 5′ end of the tail fibre gene and sequence located immediately upstream of the Phi33 tail fibre gene, may be constructed as follows.

(70) The lacZα open reading frame may be amplified by PCR from pUC19 using primers B4250 and B4258 (FIG. 2). The PTP47 tail fibre 3′ section may be amplified by PCR from PTP47 using primers B4259 and B4260 (FIG. 2). The lacZα open reading frame may then be joined to the PTP47 tail fibre gene 3′ section by SOEing PCR using the outer primers, B4250 and B4260. A region comprising sequence of the 5′ end of the Phi33 tail fibre gene, and sequence located immediately upstream of the Phi33 tail fibre gene, may be amplified by PCR using primers B4261 and B4229 (FIG. 2). This PCR product may then be joined to the PCR product comprising lacZα and the PTP47 tail fibre gene 3′ section, by SOEing PCR using the outer primers B4250 and B4229. The resulting PCR product may then be cleaned and digested with AvrII and KpnI, and ligated to pSMX204 that has also been digested with AvrII and KpnI, yielding plasmid pSMX206 (FIG. 2).

(71) Primer B4250 consists of a 5′ AvrII restriction site, followed by sequence complementary to the 3′ end of the lacZα open reading frame (FIG. 2). Primer B4258 consists of a 5′ section of sequence that overlaps the 3′ end of the PTP47 tail fibre gene (underlined), followed by sequence of the 5′ end of the lacZα open reading frame. Primer B4259 is the reverse complement of primer B4258 (FIG. 2). Primer B4260 consists of 5′ sequence from within the Phi33 tail fibre gene (underlined), followed by sequence within the 3′ end of the PTP47 tail fibre gene (FIG. 2). Primer B4261 is the reverse complement of Primer B4260. Primer B4229 consists of a 5′ KpnI restriction site (underlined), followed by sequence that is complementary to a region approximately 1 kb upstream of the tail fibre gene in Phi33 (FIG. 2).

(72) TABLE-US-00007 Primer B4250 (SEQ ID NO: 19) 5′-GATACCTAGGTTAGCGCCATTCGCCATTC-3′ Primer B4258 (SEQ ID NO: 25) 5′-CTTTTCCAGCGGGTAACGTAAAATGACCATGATTACGGATTC-3′  Primer B4259 (SEQ ID NO: 26) 5′-GAATCCGTAATCATGGTCATTTTACGTTACCCGCTGGAATAG-3′ Primer B4260 (SEQ ID NO: 27) 5′-CAAGCGGGCCGGCTGGTCTCTCGGCAATAACTCCTATGTGATC-3′  Primer B4261 (SEQ ID NO: 28) 5′-GATCACATAGGAGTTATTGCCGAGAGACCAGCCGGCCCGCTTG-3′ Primer B4229 (SEQ ID NO: 24) 5′-GATAGGTACCGCGACCGGTCTGTACTTC-3′
Genetic Modification of Phi33 to Replace the 3′ Section of the Tail Fibre Gene with that of PTP47
1. Plasmid pSMX206 (FIG. 2; FIG. 4) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 μg/ml), yielding strain PTA21.
2. Strain PTA21 may be infected with phage Phi33, and the progeny phage harvested.
3. Recombinant phage in which the 3′ end of the Phi33 tail fibre gene has been replaced by that of PTP47, and to which lacZα has been added, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for black plaques, which are indicative of β-galactosidase activity.
4. PCR may be carried out to check that the tail fibre gene has been replaced, and that lacZα is present.
5. Following identification of a verified isolate (PTPX22; FIG. 4), this isolate may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.
Construction of a Plasmid to Remove the lacZα Marker from PTPX21
1. pSMX207 (FIG. 5), comprising pSM1080 carrying a 3′ section of the PTP92 tail fibre gene, and a region of Phi33 sequence located immediately downstream of the Phi33 tail fibre gene, may be constructed as follows.

(73) The region of Phi33 sequence located immediately downstream of the Phi33 tail fibre may be amplified by PCR using primers B4222 and B4255 (FIG. 5). The 3′ end of the PTP92 tail fibre gene may be amplified by PCR using primers B4256 and B4257 (FIG. 5). These two PCR products may then be joined by SOEing PCR, using the two outer primers B4222 and B4257. The resulting PCR product may then be cleaned, digested with NheI, cleaned again, and ligated to pSM1080 that has also been digested with NheI and then treated with alkaline phosphatase prior to ligation, to yield plasmid pSMX207 (FIG. 5).

(74) Primer B4255 consists of a 5′ section of the end of the PTP92 tail fibre gene (underlined), followed by sequence immediately downstream of the Phi33 tail fibre gene (FIG. 5). Primer B4256 is the reverse complement of primer B4255 (FIG. 5). Primer B4257 consists of a 5′ NheI restriction site (underlined), followed by sequence of the terminal 1 kb of the PTP92 tail fibre gene (FIG. 5).

(75) TABLE-US-00008 Primer B4255 (SEQ ID NO: 29) 5′-CTATTCCAGCGGGTAACGTAAAATGAAATGGACGCGGATCAG-3′  Primer B4256 (SEQ ID NO: 30) 5′-CTGATCCGCGTCCATTTCATTTTACGTTACCCGCTGGAATAG-3′  Primers B4257 (SEQ ID NO: 31) 5′-GATAGCTAGCGGCAATAACTCCTATGTGATC-3′
Genetic Modification of PTPX21 to Remove the lacZα Marker
1. Plasmid pSMX207 (FIG. 5; FIG. 3) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 μg/ml), yielding strain PTA22.
2. Strain PTA22 may be infected with phage PTPX21, and the progeny phage harvested.
3. Recombinant phage in which the lacZα marker has been removed may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for white plaques, which are indicative of loss of β-galactosidase activity.
4. PCR may be carried out to check that the tail fibre gene has been retained, and that lacZα has been removed.
5. Following identification of a verified isolate (PTPX23; FIG. 3), this isolate may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.
Construction of a Plasmid to Remove the lacZα Marker from PTPX22
1. pSMX208 (FIG. 5), comprising pSM1080 carrying a 3′ section of the PTP47 tail fibre gene, and a region of Phi33 sequence located immediately downstream of the Phi33 tail fibre gene, may be constructed as follows.

(76) The region of Phi33 sequence located immediately downstream of the Phi33 tail fibre may be amplified by PCR using primers B4222 and B4262 (FIG. 5). The 3′ end of the PTP47 tail fibre gene may be amplified by PCR using primers B4263 and B4264 (FIG. 5). These two PCR products may then be joined by SOEing PCR, using the two outer primers B4222 and B4264. The resulting PCR product may then be cleaned, digested with NheI, cleaned again, and ligated to pSM1080 that has also been digested with NheI and then treated with alkaline phosphatase prior to ligation, to yield plasmid pSMX208 (FIG. 5).

(77) Primer B4262 consists of a 5′ section of the end of the PTP47 tail fibre gene (underlined), followed by sequence immediately downstream of the Phi33 tail fibre gene (FIG. 5). Primer B4263 is the reverse complement of primer B4262 (FIG. 5). Primer B4264 consists of a 5′ NheI restriction site (underlined), followed by sequence of the terminal 1 kb of the PTP47 tail fibre gene (FIG. 5).

(78) TABLE-US-00009 Primer B4262 (SEQ ID NO: 32) 5′-CTTTTCCAGCGAGTGACGTAAAATGAAATGGACGCGGATCAG-3′  Primer B4263 (SEQ ID NO: 33) 5′-CTGATCCGCGTCCATTTCATTTTACGTCACTCGCTGGAAAAG-3′  Primers B4264 (SEQ ID NO: 34) 5′-GATAGCTAGCGGCAATAACTCCTATGTGATC-3′
Genetic Modification of PTPX22 to Remove the lacZα Marker
1. Plasmid pSMX208 (FIG. 5; FIG. 4) may be introduced into P. aeruginosa strain PAX21 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 μg/ml), yielding strain PTA23.
2. Strain PTA23 may be infected with phage PTPX22, and the progeny phage harvested.
3. Recombinant phage in which the lacZα marker has been removed may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX21, onto medium containing S-gal, looking for white plaques, which are indicative of loss of β-galactosidase activity.
4. PCR may be carried out to check that the tail fibre gene has been retained, and that lacZα has been removed.
5. Following identification of a verified isolate (PTPX24; FIG. 4), this isolate may be plaque purified twice more on P. aeruginosa strain PAX21, prior to further use.
Construction of a Plasmid to Replace the Endolysin Gene of Phi33, PTPX23, PTPX24, and Similar Phage, by rpsB-SASP-C and lacZα
1. Plasmid pSMX209 (FIG. 6), comprising pSM1080 containing regions of Phi33 flanking the endolysin gene, may be constructed as follows.

(79) The region of Phi33 sequence immediately downstream of the endolysin gene may be amplified by PCR using primers B4265 and B4266 (FIG. 6). This PCR product may then be cleaned and digested with NdeI and NheI. The region of Phi33 sequence immediately upstream of the endolysin gene may be amplified by PCR using primers B4267 and B4268 (FIG. 6). This second PCR product may then be cleaned and digested with NdeI and NheI. The two PCR product digests may then be cleaned again and ligated to pSM1080 that has been digested with NheI and treated with alkaline phosphatase prior to ligation. Clones carrying one insert of each of the two PCR products may be identified by PCR using primers B4265 and B4268, and NdeI restriction digest analysis of the purified putative clones, to identify plasmid pSMX209 (FIG. 6).

(80) Primer B4265 consists of a 5′ NheI restriction site (underlined), followed by Phi33 sequence located approximately 340 bp downstream of the Phi33 endolysin gene (FIG. 6). Primer B4266 consists of 5′ NdeI and KpnI restriction sites (underlined), followed by sequence of Phi33 that is located immediately downstream of the endolysin gene (FIG. 6). Primer B4267 consists of a 5′ NdeI restriction site (underlined), followed by sequence that is complementary to sequence located immediately upstream of the Phi33 endolysin gene (FIG. 6). Primer B4268 consists of a 5′ NheI site (underlined), followed by Phi33 sequence that is located approximately 340 bp upstream of the endolysin gene (FIG. 6).

(81) TABLE-US-00010 Primer B4265 (SEQ ID NO: 35) 5′-GATAGCTAGCTTGGCCAGAAAGAAGGCG-3′ Primer B4266 (SEQ ID NO: 36) 5′-GATACATATGTCGGTACCTATTCGCCCAAAAGAAAAG-3′ Primer B4267 (SEQ ID NO: 37) 5′-GATACATATGTCAATACTCCTGATTTTTG-3′ Primer B4268 (SEQ ID NO: 38) 5′-GATAGCTAGCAATGAAATGGACGCGGATC-3′
2. Plasmid pSMX210 (FIG. 6), comprising pSMX209 containing SASP-C under the control of an rpsB promoter, may be constructed as follows.

(82) The SASP-C gene from Bacillus megaterium strain KM (ATCC 13632) may be amplified by PCR using primers B4269 and B4270 (FIG. 6). The resulting PCR product may then be digested with KpnI and NcoI. The rpsB promoter may be amplified by PCR from P. aeruginosa using primers B4271 and B4272 (FIG. 6). The resulting PCR product may then be digested with NcoI and NdeI. The two digested PCR products may then be cleaned and ligated to pSMX209 that has been digested with KpnI and NdeI, yielding plasmid pSMX210 (FIG. 6).

(83) Primer B4269 comprises a 5′ KpnI restriction site, followed by 5 bases, and then a bi-directional transcriptional terminator, and then sequence complementary to the 3′ end of the SASP-C gene from B. megaterium strain KM (ATCC 13632) (underlined, in bold; FIG. 6). Primer B4270 comprises a 5′ NcoI restriction site (underlined), followed by sequence of the 5′ end of the SASP-C gene from B. megaterium strain KM (ATCC 13632) (FIG. 6). Primer B4271 comprises a 5′ NcoI restriction site (underlined), followed by sequence complementary to the end of the rpsB promoter from P. aeruginosa PAO1 (FIG. 6). Primer B4272 comprises a 5′ NdeI restriction site (underlined), followed by sequence of the beginning of the rpsB promoter from P. aeruginosa PAO1 (FIG. 6).

(84) TABLE-US-00011 Primer B4269 (SEQ ID NO: 39) 5′-GATAGGTACCGATCTAGTCAAAAGCCTCCGACCGGAGGCTTTTGACT TTAGTACTTGCCGCCTAG-3′ Primer B4270 (SEQ ID NO: 40) 5′-GATACCATGGCAAATTATCAAAACGCATC-3′ Primer B4271 (SEQ ID NO: 41) 5′-GATACCATGGTAGTTCCTCGATAAGTCG-3′ Primer B4272 (SEQ ID NO: 42) 5′-GATACATATGCCTAGGGATCTGACCGACCGATCTACTCC-3′
3. pSMX211 (FIG. 6), comprising pSMX210 containing lacZα, may be constructed as follows.

(85) lacZα may be PCR amplified using primers B4273 and B4274 (FIG. 6). The resulting PCR product may then be digested with KpnI and ligated to pSMX210 that has also been digested with KpnI and treated with alkaline phosphatase prior to ligation, to yield pSMX211 (FIG. 6).

(86) Primer B4273 consists of a 5′ KpnI restriction site (underlined), followed by sequence complementary to the 3′ end of lacZα (FIG. 6). Primer B4274 consists of a 5′ KpnI restriction site (underlined), followed by sequence of the lac promoter driving expression of lacZα (FIG. 6).

(87) TABLE-US-00012 Primer B4273 (SEQ ID NO: 43) 5′-GATAGGTACCTTAGCGCCATTCGCCATTC-3′ Primer B4274 (SEQ ID NO: 44) 5′-GATAGGTACCGCGCAACGCAATTAATGTG-3′
Genetic Modification of Phi33, PTPX23, PTPX24, and Similar Phage, to Replace the Endolysin Gene with rpsB-SASP-C and lacZα
1. Plasmid pSMX211 (FIG. 6; FIG. 3; FIG. 4; FIG. 7) may be introduced into P. aeruginosa strain PAX20 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 μg/ml), yielding strain PTA24.
2. Strain PTA24 may be infected in individual experiments with phage Phi33, or PTPX23, or PTPX24, or other similar phage, and the progeny phage harvested.
3. Recombinant phage, in which the endolysin gene has been replaced by rpsB-SASP-C and lacZα, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX20, onto medium containing S-gal, looking for black plaques, which are indicative of β-galactosidase activity.
4. PCR may be carried out to check that the endolysin gene has been replaced, and that rpsB-SASP-C and lacZα are present.
5. Following identification of verified isolates (for example, PTPX25 (FIG. 7), PTPX26 (FIG. 3), PTPX27 (FIG. 4)), the isolates may be plaque purified twice more on P. aeruginosa strain PAX20, prior to further use.
Genetic Modification to Remove the lacZα Marker from PTPX25, PTPX26, PTPX27, and Similar Derivatives of Phi33
1. Plasmid pSMX210 (FIG. 6; FIG. 3; FIG. 4; FIG. 7) may be introduced into P. aeruginosa strain PAX20 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 μg/ml), yielding strain PTA25.
2. Strain PTA25 may be infected in individual experiments with phage PTPX25, or PTPX26, or PTPX27, or other similar phage, and the progeny phage harvested.
3. Recombinant phage, in which lacZα marker has been removed, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX20, onto medium containing S-gal, looking for white plaques, which are indicative of loss of β-galactosidase activity.
4. PCR may be carried out to confirm removal of the lacZα marker, while ensuring that rpsB-SASP-C is still present.
5. Following identification of verified isolates (for example, PTP114 (FIG. 7), PTP110 (FIG. 3), PTPX28 (FIG. 4)), the isolates may be plaque purified twice more on P. aeruginosa strain PAX20, prior to further use.
Construction of a Plasmid to Replace the Endolysin Gene of Phi33, PTPX23, PTPX24, and Similar Phage, by Fda-SASP-C (Codon Optimised) and lacZα
1. Plasmid pSMX212 (FIG. 8), comprising pSM1080 containing regions of Phi33 flanking the endolysin gene, may be constructed as follows.

(88) The region of Phi33 sequence immediately downstream of the endolysin gene may be amplified by PCR using primers B4265 and B4310 (FIG. 8). This PCR product may then be cleaned and digested with NheI and XhoI. The region of Phi33 sequence immediately upstream of the endolysin gene may be amplified by PCR using primers B4311 and B4268 (FIG. 8). This second PCR product may then be cleaned and digested with XhoI and NheI. The two PCR product digests may then be cleaned again and ligated to pSM1080 that has been digested with NheI and treated with alkaline phosphatase prior to ligation. Clones carrying one insert of each of the two PCR products may be identified by PCR using primers B4265 and B4268, and XhoI restriction digest analysis of the purified putative clones, to identify plasmid pSMX212 (FIG. 8).

(89) Primer B4265 consists of a 5′ NheI restriction site (underlined), followed by Phi33 sequence located approximately 340 bp downstream of the Phi33 endolysin gene (FIG. 8). Primer B4310 consists of 5′ AvrII and XhoI restriction sites (underlined), followed by sequence of Phi33 that is located immediately downstream of the endolysin gene (FIG. 8). Primer B4311 consists of 5′ XhoI and AvrII restriction sites (underlined), followed by sequence that is complementary to sequence located immediately upstream of the Phi33 endolysin gene (FIG. 8). Primer B4268 consists of a 5′ NheI site (underlined), followed by Phi33 sequence that is located approximately 340 bp upstream of the endolysin gene (FIG. 8).

(90) TABLE-US-00013 Primer B4265 (SEQ ID NO: 35) 5′-GATAGCTAGCTTGGCCAGAAAGAAGGCG-3′ Primer B4310 (SEQ ID NO: 45) 5′-GATACCTAGGTCCTCGAGTATTCGCCCAAAAGAAAAG-3′  Primer B4311 (SEQ ID NO: 46) 5′-GATACTCGAGGACCTAGGTCAATACTCCTGATTTTTG-3′ Primer B4268 (SEQ ID NO: 38) 5′-GATAGCTAGCAATGAAATGGACGCGGATC-3′
2. Plasmid pSMX213 (FIG. 8), comprising pSMX212 containing SASP-C codon optimised for expression in P. aeruginosa, under the control of an fda promoter, may be constructed as follows.

(91) The SASP-C gene from Bacillus megaterium strain KM (ATCC 13632) may be codon optimised for expression in P. aeruginosa (FIG. 9) and synthesised in vitro. The codon optimised SASP-C gene may then be amplified by PCR using primers B4312 and B4313 (FIG. 8). The fda promoter may be amplified by PCR from P. aeruginosa using primers B4314 and B4315 (FIG. 8). The resulting two PCR products may then be joined by splicing by overlap extension (SOEing) PCR, using the outer primers B4312 and B4314 (FIG. 8). The resulting fda-codon optimised SASP-C-terminator PCR product may then be digested with XhoI and AvrII, cleaned, and ligated to pSMX212 that has been digested with XhoI and AvrII, yielding plasmid pSMX213 (FIG. 8).

(92) Primer B4312 comprises a 5′ XhoI restriction site, followed by a bi-directional transcriptional terminator, and then sequence complementary to the 3′ end of the SASP-C gene from B. megaterium strain KM (ATCC 13632) that has been codon optimised for expression in P. aeruginosa (underlined, in bold; FIG. 8). Primer B4313 comprises sequence of the 3′ end of the fda promoter from P. aeruginosa PAO1 (in bold) followed by sequence of the 5′ end of the codon optimised SASP-C gene. Primer B4314 comprises sequence complementary to the 5′ end of the codon optimised SASP-C gene followed by sequence complementary to the 3′ end of the fda promoter from P. aeruginosa PAO1 (FIG. 8). Primer B4315 comprises a 5′ AvrII restriction site (underlined), followed by sequence of the beginning of the fda promoter from P. aeruginosa PAO1 (FIG. 8).

(93) TABLE-US-00014 Primer B4312 (SEQ ID NO: 47) 5′-GATACTCGAGAGTCAAAAGCCTCCGACCGGAGGCTTTTGACTTCAGT ACTTGCCGCCCAG-3′ Primer B4313 (SEQ ID NO: 48) 5′-GATTGGGAGATACGAGAACCATGGCCAACTACCAGAACGC-3′  Primer B4314 (SEQ ID NO: 49) 5′-GCGTTCTGGTAGTTGGCCATGGTTCTCGTATCTCCCAATC-3′ Primer B4315 (SEQ ID NO: 50) 5′-GATACCTAGGAACGACGAAGGCCTGGTG-3′
3. pSMX214 (FIG. 8), comprising pSMX213 containing lacZα, may be constructed as follows.

(94) lacZα may be PCR amplified using primers B4316 and B4317 (FIG. 8). The resulting PCR product may then be digested with XhoI and ligated to pSMX213 that has also been digested with XhoI and treated with alkaline phosphatase prior to ligation, to yield pSMX214 (FIG. 8).

(95) Primer B4316 consists of a 5′ XhoI restriction site (underlined), followed by sequence complementary to the 3′ end of lacZα (FIG. 8). Primer B4317 consists of a 5′ XhoI restriction site (underlined), followed by sequence of the lac promoter driving expression of lacZα (FIG. 8).

(96) TABLE-US-00015 Primer B4316 (SEQ ID NO: 51) 5′-GATACTCGAGTTAGCGCCATTCGCCATTC-3′ Primer B4317 (SEQ ID NO: 52) 5′-GATACTCGAGGCGCAACGCAATTAATGTG-3′
Genetic Modification of Phi33, PTPX23, PTPX24, and Similar Phage, to Replace the Endolysin Gene with Fda-Codon Optimised SASP-C and lacZα
1. Plasmid pSMX214 (FIG. 8; FIG. 10; FIG. 11; FIG. 12) may be introduced into P. aeruginosa strain PAX20 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 μg/ml), yielding strain PTA26.
2. Strain PTA26 may be infected in individual experiments with phage Phi33, or PTPX23, or PTPX24, or other similar phage, and the progeny phage harvested.
3. Recombinant phage, in which the endolysin gene has been replaced by fda-codon optimised SASP-C and lacZα, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX20, onto medium containing S-gal, looking for black plaques, which are indicative of β-galactosidase activity.
4. PCR may be carried out to check that the endolysin gene has been replaced, and that fda-codon optimised SASP-C and lacZα are present.
5. Following identification of verified isolates (for example, PTPX29 (FIG. 12), PTPX30 (FIG. 10), PTPX34 (FIG. 11)), the isolates may be plaque purified twice more on P. aeruginosa strain PAX20, prior to further use.
Genetic Modification to Remove the lacZα Marker from PTPX29, PTPX30, PTPX34, and Similar Derivatives of Phi33
1. Plasmid pSMX213 (FIG. 8; FIG. 10; FIG. 11; FIG. 12) may be introduced into P. aeruginosa strain PAX20 by conjugation, selecting transconjugants on the basis of tetracycline resistance (50 μg/ml), yielding strain PTA27.
2. Strain PTA27 may be infected in individual experiments with phage PTPX29, or PTPX30, or PTPX34, or other similar phage, and the progeny phage harvested.
3. Recombinant phage, in which lacZα marker has been removed, may be identified by plaquing the lysate from step (2) on P. aeruginosa strain PAX20, onto medium containing S-gal, looking for white plaques, which are indicative of loss of β-galactosidase activity.
4. PCR may be carried out to confirm removal of the lacZα marker, while ensuring that fda-codon optimised SASP-C is still present.
5. Following identification of verified isolates (for example, PTP284 (FIG. 12), PTP384 (FIG. 10), PTP385 (FIG. 11)), the isolates may be plaque purified twice more on P. aeruginosa strain PAX20, prior to further use.

REFERENCES

(97) Abedon S T. (2008). Bacteriophage Ecology: Population Growth, Evolution, an Impact of Bacterial Viruses. Cambridge. Cambridge University Press. Chapter 1. Boucher, H. W., Talbot, G. H., Bradley, J. S., Edwards, J. E., Gilbert, D., Rice, L. B., & Bartlett, J. (2009). Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America. Clinical Infectious Diseases, 48: 1-12. Burrowes, B., & Harper, D. R. (2012). Phage Therapy of Non-wound Infections. Bacteriophages in Health and Disease: Bacteriophages in Health and Disease, Chapter 14: 203-216. Carlton, R. M. (1999). Phage therapy: past history and future prospects. Archivum Immunologiae et Therapiae Experimentalis-English Edition 47:267-274. Ceyssens P, Miroshnikov K, Mattheus W, Krylov V, Robben J, Noben J, Vanderschraeghe S, Sykilinda N, Kropinski A M, Volckaert G, Mesyanzhinov V, Lavigne R. (2009). Comparative analysis of the widespread and conserved PB1-like viruses infecting Pseudomonas aeruginosa. Env. Microbiol. 11:2874-2883. Francesconi, S. C., MacAlister, T. J., Setlow, B., & Setlow, P. (1988). Immunoelectron microscopic localization of small, acid-soluble spore proteins in sporulating cells of Bacillus subtilis. J Bacteriol., 170: 5963-5967. Frenkiel-Krispin, D., Sack, R., Englander, J., Shimoni, E., Eisenstein, M., Bullitt, E. & Wolf, S. G. (2004). Structure of the DNA-SspC complex: implications for DNA packaging, protection, and repair in bacterial spores. J. Bacteriol. 186:3525-3530. Gill J J, Hyman P. (2010). Phage Choice, Isolation and Preparation for Phage therapy. Current Pharmaceutical Biotechnology. 11:2-14. Kutateladze, M., & Adamia, R. (2010). Bacteriophages as potential new therapeutics to replace or supplement antibiotics. Trends Biotechnol. 28:591-595. Lee, K. S., Bumbaca, D., Kosman, J., Setlow, P., & Jedrzejas, M. J. (2008). Structure of a protein—DNA complex essential for DNA protection in spores of Bacillus species. Proc. Natl. Acad. Sci. 105:2806-2811. Nicholson W L, Setlow B, Setlow P. (1990). Binding of DNA in vitro by a small, acid-soluble spore protein from Bacillus subtilis and the effect of this binding on DNA topology. J Bacteriol. 172:6900-6906. Rakhuba D V, Kolomiets E I, Szwajcer Dey E, Novik E I. (2010). Bacteriophage Receptors, Mechanisms of Phage Adsorption and Penetration into Host Cell. Polish J. Microbiol. 59:145-155. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning (Vol. 2, pp. 14-9). New York: Cold Spring Harbor Laboratory Press. Scholl, D., Rogers, S., Adhya, S., & Merril, C. R. (2001). Bacteriophage K1-5 encodes two different tail fiber proteins, allowing it to infect and replicate on both K1 and K5 strains of Escherichia coli. J. Virol. 75:2509-2515. Veesler D, Cambillau C. (2011). A Common Evolutionary Origin for Tailed-Bacteriophage Functional Modules and Bacterial Machineries. Microbiol Mol Biol Rev. 75:423-433. Walker, B., Barrett, S., Polasky, S., Galaz, V., Folke, C., Engstrom, G., & de Zeeuw, A. (2009). Looming global-scale failures and missing institutions. Science, 325:1345-1346. WHO (2014) Antimicrobial resistance: global report on surveillance 2014.

(98) TABLE-US-00016 TABLE 1 Host range of Phi33, PTP92, C36 and PTP47 against 44 European clinical isolates of Pseudomonas aeruginosa. Strains were tested for sensitivity to each phage by dropping 10 μl of crude phage lysate onto a soft agar overlay plate inoculated with bacteria. Plates were grown overnight at 32° C. and the strains were scored for sensitivity to each phage by assessing clearance zones at the point of inoculation. Where phage inhibited growth, as seen by clearance of the bacterial lawn, the strain was marked as sensitive (+), and where no inhibition of growth was seen, the strain was marked as not-sensitive (−) Bacterial Strain no. Phi33 PTP47 PTP92 C36 2019 + + − + 2020 + + − + 2021 + + + + 2029 + + − + 2031 + + + + 2039 + + + + 2040 + + − + 2041 + + + + 2042 + + + + 2045 − − + − 2046 + + + + 2047 + + + + 2048 + + + + 2049 + + + + 2050 + + + + 2051 + + − − 2052 − − − − 2053 + + − + 2054 − + − + 2055 + + − + 2056 + + + + 2057 + + + + 2058 + + + + 2483 − − + − 2484 + + − + 2705 + + − + 2706 + + − + 2707 + + + + 2708 + + + + 2709 + + + + 2710 − + + − 2711 + + + + 2712 + + − + 2713 − + + + 2714 + + + + 2715 + + + + 2716 + + − − 2717 − + + + 2718 − + + + 2719 + + − + 2720 + + + + 2721 + + + + 2722 + + + + 2723 + + − +

(99) TABLE-US-00017 TABLE 2 Host range of Phi33, PTP92 and PTP93 against 35 European clinical isolates of Pseudomonas aeruginosa. Strains were tested for sensitivity to each phage by dropping 10 μl of crude phage lysate onto a soft agar overlay plate inoculated with bacteria. Plates were grown overnight at 32° C. and the strains were scored for sensitivity to each phage by assessing clearance zones at the point of inoculation. Where phage inhibited growth, as seen by clearance of the bacterial lawn, the strain was marked as sensitive (+), and where no inhibition of growth was seen, the strain was marked as not-sensitive (−) Isolate Phi33 PTP93 PTP92 2019 + + − 2020 + + − 2029 + + − 2040 + + − 2045 − + + 2053 + + − 2483 − + + 2484 + + − 2705 + − − 2710 − + + 2711 + + + 2712 + + − 2713 − + + 2716 + + − 2717 − + + 2718 − + + 2720 + + + 2721 + + + 2722 + + + 2723 + − − 2728 − + + 2733 + + − 2734 + + + 2740 − + + 2741 + + + 2742 + + + 2743 + + + 2747 + + + 2748 + + + 2749 + + − 2750 + + + 2752 + + + 2753 − + + 2754 + + + 2756 + + +