Patent Application
20070054357
This invention is directed to a method of identifying classes of bacteriophage useful for the control of, for example, Listeria monocytogenes and Salmonella species in environmental, food, medical, veterinary, agricultural, and other settings. More specifically, groups of nucleotide sequences are provided that are present in and identify a class of bacteriophages useful in the control of, for example, Listeria monocytogenes. Likewise, groups of short nucleotide sequences are provided that identify a class of bacteriophages useful in the control of, for example, Salmonella species. The field of the invention is restricted to bacteriophage genomes; the claimed sequences or group of sequences may occur in the genomes of organisms other than bacteriophages without prejudice to the present invention.
There are six major families of bacteriophages including Myoviridae (T-even bacteriophages), Styloviridae (Lambda bacteriophage groups), Podoviridae (T-7 and related bacteriophage), Microviridae (X174 group), Leviviridae (for example, E coli bacteriophage MS2) and Inoviridae as well as coliphages, in general. Other bacteriophage families include members of the Cystoviridae, Microviridae, and Siphoviridae families.
Bacteriophage has been used therapeutically since the early part of the last century. Bacteriophage, which derive their name from the Greek word “phago” meaning “to eat” or “bacteria eaters”, were independently discovered by Twort as well as by D'Herelle in the first part of the twentieth century. Early enthusiasm led to the use of bacteriophage as both prophylaxis and therapy for diseases caused by bacteria. However, the results from early studies to evaluate bacteriophage as antimicrobial agents were variable due to the uncontrolled study design and the inability to standardize reagents. Later, in better designed and controlled studies, it was concluded that bacteriophage were not useful as antimicrobial agents (Pyle, N. J., J. Bacteriol, 12:245-61 (1936); Colvin, M. G., J. Infect. Dis., 51:17-29 (1932); and Boyd et al., Trans R. Soc. Trop. Med. Hyg., 37:243-62 (1944)).
This initial failure of phage as antibacterial agents may have been due to the failure to select for phage that demonstrated high in vitro lytic activity prior to in vivo use. For example, the phage employed may have had little or no activity against the target pathogen, or they may have been used against bacteria that were resistant due to lysogenization or the phage itself may have been lysogenic for the target bacterium (Barrow et al., Trends in Microbiology, 5:268-71 (1997)). However, with better understanding of the phage-bacterium interaction and of bacterial virulence factors, it has been possible to conduct studies which demonstrated the in vivo anti-bacterial activity of the bacteriophage (Asheshov et al., Lancet, 1:319-20 (1937); Ward, W. E., J. Infect. Dis., 72:172-6 (1943); and Lowbury et al., J. Gen. Microbiol., 9:524-35 (1953)). In the U.S. during the 1940's, Eli Lilly Co. commercially manufactured six phage products for human use, including preparations targeted towards Staphylococci, Streptococci and other respiratory pathogens.
With the advent of antibiotics, the therapeutic use of phage gradually fell out of favor in the U.S. and Western Europe, and little subsequent research was conducted. However, in the 1970's and 1980's bacteriophage therapy continued to be utilized in Eastern Europe, most notably in Poland and the former Soviet Union. Alisky et al. conducted a review of all Medline citations where bacteriophage was employed therapeutically from 1966 to 1996 (Alisky et al., J. Infect., 36:5-15 (1998)).
There are also several British studies describing controlled trials of bacteriophage raised against specific pathogens in experimentally infected animal models such as mice and guinea pigs (see, e.g., Smith, H. W. & M. B. Huggins, J. Gen. Microbiol. 128:307-318 (1982); Smith, H. W. & M. B. Huggins, J. Gen. Microbiol, 129:2659-2675 (1983); Smith, H. W. & R. B. Huggins, J. Gen. Microbiol., 133:1111-1126 (1987); and Smith et al., J. Gen. Microbiol., 133:1127-1135 (1987)). These trials measured objective criteria such as survival rates. Efficacy against Staphylococcus, Pseudomonas and Acinetobacter infections were observed. These studies are described in more detail below.
One such study concentrated on improving bioavailability of phage in live animals by modifying the bacteriophage (Merril et al., Proc. Natl. Acad. Sci. USA, 93:3188-3192 (1996)). Reports from the U.S. relating to bacteriophage administration for diagnostic purposes have indicated phage have been safely administered to humans to monitor humoral immune response in adenosine deaminase deficient patients (Ochs et al., Blood, 80:1163-71 (1992)) and for analyzing the importance of cell-associated molecules in modulating the immune response in humans (Ochs et al., Clin. Immunol. Immunopathol., 67:S33-40 (1993)).
Additionally, Polish, Georgian and Russian papers describe experiments where phage was administered systemically, topically or orally to treat a wide variety of antimicrobial resistant pathogens (see, e.g., Shabalova et al., Abstr. 443. In Proceedings of IX International Cystic Fibrosis Congress, Dublin, Ireland; Slopek et al., Archivum. Immunol. Therapiae Experimental, 31:267-291 (1983); and Slopek et al., Archivum Immunol. Therapiae Experimental, 35:569-83 (1987)).
Infections treated with bacteriophage included osteomyelitis, sepsis, empyema, gastroenteritis, suppurative wound infection, pneumonia and dermatitis. Pathogens treated with the bacteriophage include Staphylococci, Streptococci, Klebsiella, Shigella, Salmonella, Pseudomonas, Proteus and Escherichia. Articles have reported a range of success rates for phage therapy between 80-95% with only rare reversible allergic or gastrointestinal side effects. These results indicate that bacteriophage may be a useful adjunct in the fight against bacterial diseases.
Despite the use of bacteriophage for the treatment of diseases in humans, there remains in the art a need for the discovery of novel bacteriophage and methods for using these bacteriophage in several critical areas. One significant need concerns the treatment of processed or unprocessed food products to treat or prevent colonization with undesirable pathogens such as Listeria monocytogenes or Salmonella which are responsible for food-borne illness. A second critical area of need concerns the removal of undesirable bacteria from industrial environments such as food processing facilities to prevent colonization thereof. A third critical area of need concerns the removal of undesirable pathogens such as L. monocytogenes and Salmonella from environments where they may be passed to susceptible humans and animals, such as supermarkets, hospitals, nursing homes, veterinary facilities, and other such environments. Finally, new bacteriophage and methods of using the same are needed for the treatment of human or animal bacterial disease.
The present invention provides nucleic acid sequences that uniquely define useful classes of bacteriophage. These nucleic acid sequences are termed oligonucleotide motifs. The scientific literature does not directly address notion that specific amino acid or nucleic acid motifs identify groups of bacteriophage specific for specific commercially or medically important bacterial pathogens. Blaisdell (Blaisdell et al. Similarities and dissimilarities of phage genomes, Proceedings of the National Academy of Sciences of the United States of America, 93, 5854 (1996)) recognized that the genomes of bacteriophages contain short oligonucleotide signatures that can be used to construct a taxonomy of bacteriophages and show their relatedness to one another. Blaisdell and colleagues focused upon di- and tetra-nucleotides that were not related to host range in any systematic fashion.
In other studies, some degree of homology has been noted at a crude level, however not at the level of sequences of amino acids or nucleotides that would provide guidance to the skilled practitioner. Salgado (1) studied the homology between two individual bacteriophages, Salmonella enterica serovar Typhimurium phage P22 and Salmonella enterica serovar Anatum var. 15+ phage ε34. Using DNA restriction digest patterns, reaction of both phages with antibodies raised to the P22 phage, and the common reactivity of the tailspike proteins with a monoclonal antibody as evidence, the authors concluded that there significant homology between these phages. Since the tailspike proteins are thought to react with the lipopolysaccharide (LPS) of the two Salmonella serovars, the authors concluded that further studies would be required to establish a role for their findings in determining the specificity of the phage. Although the common reaction with a monoclonal antibody provided evidence of an epitope shared between the tailspike proteins, the studies did not demonstrate the absence of this degree of homology, nor the absence of the monoclonal reactivity in phages that failed to react with Salmonella species.
The highly variable protein sequence near the tip of the long tail fiber proteins in T-even phages encodes the adhesins that determine the ability of the phage to bind to specific bacterial hosts according to the studies of Tetart (2). These studies compared the sequences of the adhesins in the distal tail fibers of T-even phage, finding that recombination in this restricted area led to a change in specificity of the adhesins, and, hence, a change in host range.
Certain hosts require that bacteriophage utilize hyaluronidases in order to effect entry. Marciel (3) compared sequences of hyl (hyaluronidase) genes from 13 bacteriophage specific for Streptococcus pyogenes. These investigators noted allelic variation, where the hyl gene from some strains included a motif of a collagen-like domain, and others did not. The studies did not draw any conclusions regarding the effects of allelic variation on host range.
Loessner et al. (4) carried out important studies of murein hydrolases of phage specific for Listeria monocytogenes. Murein hydrolases are enzymes involved in the lysis of the host bacterial cell after phage replication has occurred. Using sequences derived from two phage specific for Listeria monocytogenes, Loessner and colleagues proposed a modular organization of motifs within these enzymes that would, in fact, facilitate a broad host range through ready utilization of pre-existing catalytic and cell wall binding domains in response to changing conditions. The Loessner studies only addressed the lytic phase of bacteriophage infection and did not, except as noted, address issues of host range, since host range is critically determined by the initial attachment of a bacteriophage to a bacterium, and not by lysis.
Chua (5) published a detailed study of the tailspike protein of the lysogenic Shigella flexneri bacteriophage Sf6. The studies focused upon correlating specific motifs with catalytic activity, and relating the specific catalytic activities to the ability to cleave or otherwise modify specific bacterial antigens. The study did not, however, attempt to relate the presence or absence of specific motifs to host range.
Chipman (6) used X-ray diffraction to study similarities of the capsid proteins of the Spirolasma melliferum phage SpV4 to the Chlamydia phage, Chp1, and the coliphages alpha 3, phi K, G4 and phi X174. These studies identified a hydrophobic cavity that they speculated might serve as a common receptor recognition site during host infection. The study did not develop any information concerning motifs that might govern host range.
Some studies address the attachment of bacteriophage integrases to the host genome. Integration is a feature limited to so-called temperate, or lysogenic, bacteriophages. Although motifs found in integrases could conceivably correlate with host range, they are not relevant to this application since lysogenic bacteriophages are expressly excluded from the subject matter of this invention. Examples of studies using sequence motifs to focus on the mechanism of integration of lysogenic bacteriophages include Dorgai (7), Kaneko (8) and Salmi (9).
Crutz-Le Coq and colleagues (10) obtained the complete sequence of the 31754 bp genome of bIL170, a virulent bacteriophage of Lactococcus lactis belonging to the 936 group. Analysis of this sequence identified a 110 to 150 amino acid hypervariable region flanked by conserved 20 amino acid sequences. The authors hypothesized that this sequence could encode molecules involved in host range determination, however no specific attempt was made to distinguish common motifs among phage lytic for a given bacterial strain or species. Meanwhile, Gottlieb (11, 12) sequenced the genome of phi12, a phage related to phi6, solely for purposes of speciation without addressing host range or specificity, save to note a similarity of the phi12 attachment proteins to those of phi13. While Crutz-Le Coq extensively discusses the potential role of sequence variations in specific genes as determinants of host range, there is no anticipation of sequence motifs that would define useful groups of bacteriophage on the basis of specific, defining sequence motifs. Similar approaches include those of Tu (13), who sequenced the mycoplasma P1 genome and assigned provisional functions on the basis of sequence motifs. Weisberg (14) sequenced the lysogenic filamentous phage HK022, and used the sequence information in an evolutionary context to compare strategies developed by phage to deal with similar problems. Likewise, Pfister (15) sequenced psiM2, focusing upon structure-function assignments and sequence comparisons aimed at establishing the evolutionary hierarchy.
In examining bacteriophage phiKZ as a candidate therapeutic phage for Pseudomonas aeruginosa, Mesyanzhinov et al. (16) obtained its complete DNA sequence. Analysis of this phage included identification of the individual genes and identification of motifs common to many phages. The results bolstered the understanding that many phage are derived from common ancestors, but did not identify sequences or motifs potentially involved in determination of host range.
Altieri (17) described a NusB contains a 10 residue Arg-rich RNA-binding motif (ARM) at the N-terminus but is not sequentially homologous to any other proteins. This motif was used to show that this particular lambda protein, NusB, involved in transcriptional control is, through its structure, a member of a class of alpha-helical RNA-binding proteins.
Verheust (18) noted that in tectiviruses, an unusual phage group whose double stranded DNA lies within a lipid vesicle inside a protein coat, those organisms infecting gram-negative bacteria are closely related. Focusing upon tectiviruses infecting gram positive bacteria, these authors found that mutations in a particular motif in GIL01 and GIL16 phages correlate with a switch to a lytic cycle from a temperate cycle. Both bacterial viruses displayed narrow, yet slightly different, host spectrums.
Akulenko (19) focused upon motifs to define by analogy evolutionarily conserved sequences in the catalytic sites of enzymes. Cannistraro examined protein-level structure-function relationships in bacteriophage T7 DNA polymerase (20). Likewise, Lebars (21) explored structural motifs as part of determining the mechanism of action of the T4 RegB endonuclease, building upon the prior functional studies of Sanson (22). Meanwhile Lee (23) determined motifs comprised of critical lysine residues required for the function of the RNA polymerase domain of bacteriophage T7 helicase-primase. Other work, e.g. that of Benevides (24) has focused upon the common structural features of phage capsid proteins that permit their self assembly. Other studies of motifs common among capsid proteins include those of Pederson (25). Kim (26) looked at a motif in the Nun protein of prophage HK022 that was responsible for the exclusion of superinfection by other phage such as lambda. Many papers deal with common structural motifs in encoded proteins that contribute to their function. Among these are the studies of Kumaraswami (27) examining features of the Mor/C family of transcriptional activators in bacteriophage Mu, and Li (28) examining the transcriptional activation domain of the T4 protein MotA.
Some motifs identify sequences encoding catalytically active nucleic acids. An example is that of Lindqvist (29) of the T4 nrdB group I intron likely encoding a ribozyme.
Certain motifs appear in proteins of temperate, or lysogenic phages, that are involved in the mechanism of lysogeny. Motifs occurring in these proteins have been studied with the goal of clarifying the mechanism of lysogeny. These studies are not directed toward defining useful class of bacteriophage. Examples of such studies include Rutkai (30).
Other studies of motifs are directed toward gaining a better understanding of how bacteriophages function. Studies using motifs to understand so-called immunity, whereby phage-infected bacteria are resistant to superinfection by another bacteriophage, include those of Defenbaugh (31) and Stuart (32). Mitchell (33) examined how bacteriophage DNA is loaded into the empty capsids during replication; he found that the enzymes that accomplish this, bacteriophage terminases, contain Walker A motifs that are signatures for ATPase catalytic sites. Other studies identifying motifs playing a role in packaging of bacteriophage nucleic acids are those of Benevides (34), Brunel (35), Mitchell (36), Rao (37), Rodriguez-Casado (38), Kuebler (39), Parker (40), Tuma (41) and Tao (42).
Other investigators sought to identify motifs signifying enzymes involved in replication of phage DNA or DNA repair, such as DNA polymerases and polynucleotide kinases, as well as enzymes involved in excision of temperate phages and DNA methylation. Others examined phosphatases, such as the studies of White (43). Studies of motifs involved in replication include those of Eisenbrandt (44), Galburt (45), Imburgio (46), Karpel (47), Lee (48), Petrov (49), Rezende (50), Sam (51), Wojciak (52), Yeo (53), Bravo (54), Moyer (55), Radlinska (56), Schneider (57), Valentine (58), de Vega (59), Hoogstraten (60), Makeyev (61), Moscoso (62), Tseng (63) and Illana (64).
Although usually thought to be a feature of eukaryotic genomes, some bacteriophage genes may contain introns. For example, the Brussow laboratory (65-67) found that half of Streptococcus thermophilus phage examined contained a group IA2 intron in a lysin gene; this intron was associated with splicing of phage mRNA. A 14 base pair motif in the coding sequence was positively associated with the presence of an intron. Such motifs are useful in predicting whether a given gene will possess an intron, but are not useful in predicting the host range or other biologic properties of a bacteriophage.
Similar work has been carried out examining common motifs required for the function of proteins involved in translation. Examples include the studies of Sengupta (68).
Looking at phage gene transcription, Nechaev (69) found 8 to 10 base pair motifs that are involved in initiating transcription from T4 late promoters. In related studies, Orsini (70) examined the interaction of this same motif with a transcriptional inhibitory factor. Transcription is also regulated in T4 phage by a somewhat divergent family of RegB endonucleases (71) that cleave and process phage RNA's through specific tetranucleotide motifs. Vieu (72) applied similar approaches to identify bacteriophage genetic elements controlling termination of transcription in lambda phage. Additional papers addressing transcriptional control of bacteriophage genes through identification of motifs include Christie (73), Cilley (74), Fromknecht (75), Kim (76), Marshall-Batty (77), Mukhopadhyay (78), Paul (79), Ho (80), Li (81), Pande (82), Urbauer (83), Faber (84), Scharpf (85) and Watnick (86).
Protein motifs are also important in bacteriophage assembly. Bernal (87), for example, specifically focused upon the role of protein folding motifs in the self assembly of bacteriophage alpha3. Other studies dealing specifically with the role of protein motifs in phage assembly include Rentas (88).
Bleuit (89) looked for a conserved motif in the UvsY protein in T4 bacteriophage that correlated with its DNA binding activity as a recombination mediator protein. These investigators studied how modification of the motif structure influenced its function. Melnyk (90) used motifs in the M13 major coat protein to study how specific protein sequences facilitate low affinity dimer formation. In other structural work, Papanikolopoulou (91, 92) looked at protein folding motifs in bacteriophage adhesins. The studies of Qu and colleagues (93) are similar in that they examined the role of coiled-coil motifs in bacteriophage tail fiber assembly. Van Raaij (94) determined the crystal structure of the T4 proteins tail fiber required for adhesion to its E. coli host. This study correlated structure with function, but did not identify motifs that would correlate with host range or specificity. Likewise, Sam (95) used X-ray diffraction to identify a winged helix motif important for the function of the Xis excisionase in bacteriophage lambda, and Knowlton (96) studied NusG structure, since it is a highly conserved protein linked to termination in many prokaryotic species.
A number of studies are purely genetic in nature, focused upon elaboration of structure-function relationships through comparison of sequences of different bacteriophage. For instance, Sau (97) compared sequences of repressor genes of temperate mycobacteriophage L1 with a mutant gene and other mycobacteriophage repressor proteins; these studies were directed solely toward a better understanding of repressor protein function. Other studies of repressors include those of Shearwin (98). In related studies, Lanthaler (99) localized the gene encoding an endonuclease to a phage introns, then further identified sequence motifs shared with other endonucleases. Other substantially genetic studies include those of Lee (100).
Some studies focused upon phage proteins with specific enzymatic motifs, and used variations among such proteins to gain a better understanding of the function of that enzyme. Examples include the studies of Reiter (101), Zhu (102), Chan (103), Goetzinger (104), Lin (105), Logan (106), Rodriguez (107), Rogov (108), Wang (109) and Yin (110).
Other studies, such as that of Romero (111), identified close homologues of bacterial LytA amidases in two lysogenic phage of Streptococcus mitis; structural and functional comparison of the phage enzymes with the bacterial version permitted assignment of specific functions to specific amino acid residues. In related work, Calin-Jageman identified structural RNA motifs that served to inhibit certain bacterial polymerases (112).
For purely technical purposes, Chan (113) used repeated 7- and 8-base nucleic acid motifs within lambda DNA to validate a novel DNA mapping technology. This work did not test for the presence or functional significance of these motifs across different phage.
Interestingly, Dabrowska (114) examined interactions between phages and various eukaryotic cells, observing binding of phages to the membranes of cancer and normal blood cells. Wild-type phage T4 (wtT4) and its substrain HAP1 with enhanced affinity for melanoma cells inhibit markedly and significantly experimental lung metastasis of murine B16 melanoma cells by 47% and 80%, respectively. A possible molecular mechanism of these effects, namely a specific interaction between the Lys-Gly-Asp motif of the phage protein 24 and beta3-integrin receptors on target cells is proposed. It was also shown that anti-beta3 antibodies and synthetic peptides mimicking natural beta3 ligands inhibit the phage binding to cancer cells. This is in line with the well-described beta3 integrin-dependent mechanism of tumor metastasis. It is concluded that the blocking of beta3 integrins by phage preparations results in a significant decrease in tumor invasiveness.
Doulatov (115) examined the ability of Bordetella phage to generate diversity in a gene specifying host tropism—the gene is a reverse transcriptase. Using the Bordetella phage cassette as a signature, they identified numerous related elements in diverse bacteria. These elements constitute a new family of retroelements with the potential to confer selective advantages to their host genomes.
Some papers deal with both motifs and heterogeneity in the gene cassette encoding the holins and lysins required for bacterial lysis. In this regard, Labrie (116) examined heterogeneity in the lysis cassette of Lactococcus lactis phages, with specific reference to the bacteriophage u136 holin. These studies focused specifically upon the presence of motifs encoding specific enzymatic activities, such as amidases or muramidases, but did not address the issue of which activities might confer specificity. Vukov (117, 118) studied control and structure-function relationships in the Listeria monocytogenes bacteriophage A118 holin, but did not address how this contributed to the specificity of this phage for Listeria. Barenboim noted that some holin genes possess motifs that encode two distinct translational start sites, which in turn yield two distinct holins from the same gene (119).
Some work in the literature is directed toward the introduction of non-native motifs into phage to create improved vectors for gene therapy. Examples of this include the work of Piersanti (120).
Oligonucleotides common to a selected group of bacteriophage can be used to screen new bacteriophage to determine whether the new bacteriophage shares in the specificity as the selected group of bacteriophage.
The invention relates to a method using said oligonucleotides to identify bacteriophage of interest.
The invention relates to an isolated bacteriophage comprising at least two of said oligonucleotides.
Additional features and advantages are described herein, and will be apparent from, the following Detailed Description.
The present invention relates to the use of nucleotide sequences to identify classes of bacteriophages useful in controlling or eliminating bacterial pathogens from environmental, food, medical, veterinary, agricultural, and other settings.
At least three type strains of a particular bacterium which is a lytic target of a bacteriophage are selected to comprise a bacterium test data set. A candidate phage then is tested for lytic activity in all of the strains of the bacterium test data set. As controls, bacteria of a related strain, another species or another genus can be used. Preferably, the bacterium test data set comprises at least 4, 5, 6, 7, 8, 9, 10 or more strains of a bacterium of interest.
As the target of the phage taught herein, any bacterium can be used, including, for example, bacteria of the genus Pseudomonas, Clostridium, Enterobacter, Propionibacter, Vibrio, Xanthomonas, Mycoplasma, Acinetobacter, Chlamydia, Acetobacter, Aeromonas, Agrobacterium, Alcaligenes, Anabena, Archaebacteria, Azotobacter, Bacillus, Borrelia, Campylobacter, Citrobacter, Corynebacterium, Cyanobacteria, Desulfovibrio, Enterococcus, Erwinia, Escherichia, Flavobacterium, Hemophilus, Klebsiella, Lactobacillus, Listeria, Mycobacterium, Mycococcus, Pasteurella, Proteus, Rhodobacter, Salmonella, Shigella, Serratia, Staphylococcus, Streptococcus, Streptomyces, Thermus, Yersinia, Actinomyces, Brucella, Lactococcus, Brevibacterium, Clavibacter, Halobacterium, Helicobacter and so on. Type strains can be obtained from the ATCC or can be obtained practicing methods known in the art.
One embodiment comprises a composition of a bacteriophage whose genome contains two or more sequences drawn from the list comprising Table 1. The phage of interest may contain at least 3, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 of the oligonucleotides of interest. This composition of bacteriophages comprises a group of lytic bacteriophages whose host range is specific for Listeria monocytogenes. The bacteriophage in the composition may contain one copy of sequence drawn from the list in Table 1, or it can contain two or more copies of such sequences. A preferred embodiment comprises a composition of two or more genetically distinct bacteriophages, each of whose genomes contains two or more sequences drawn from the list comprising Table 1. In this embodiment, the genome of each bacteriophage in the composition may contain the same sequences drawn from the list in Table 1 as any other bacteriophage in the composition, or it may differ in one, more than one, or all sequences drawn from the list in Table 1. The genomes of bacteriophages in this composition may contain the same number of sequences drawn from the list in Table 1, or they may contain different numbers of such sequences. The genome of each bacteriophage in the composition may contain one copy of sequence drawn from the list in Table 1, or it can contain two or more copies of such sequences.
As used herein, “complement” is meant to indicate a second oligonucleotide that hybridizes to a first oligonucleotide. Thus, if a first oligonucleotide has a sequence ATGC, the complement is TACG. As used herein, “reverse complement” is a second oligonucleotide that hybridizes to a first oligonucleotide taking into account the polarity of the strand, the first oligonucleotide, ATGC presented in the 5′ to 3′ direction, and the reverse complement also presented in the 5′ to 3′ direction and thus would be GCAT.
Lytic phage are expanded clonally as known in the art. Specificity for a bacterium of interest is ascertained practicing methods known in the art. The genome of the phage of interest is obtained practicing methods known in the art.
For each group of genomic bacteriophage sequences, a set of oligonucleotides was computed such that: [1] each oligonucleotide was 3 nucleotides or longer; [2] each oligonucleotide was as long as possible; [3] an oligonucleotide could hybridize to either strand of the bacteriophage genomic sequence; [4] every oligonucleotide was present in every member of the defining group; and [5] no oligonucleotide was present in any member of the other group. As used herein, an oligonucleotide that satisfies at least conditions [1], [3] and [4], and preferably [1], [3]. [4] and [5], is also known as a “motif.” A “motif set” comprises at least two motifs, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more motifs.
Operationally, the first three criteria were met in the following manner:
Step 1. For each phage genome sequence, the position x was set such that x=1 on the plus strand.
Step 2. The oligonucleotide of length L=3 commencing at position x was stored.
Step 3. x was sequentially incremented by 1 nucleotide from x=1 to x=n, where n equals the length of the phage genome.
Step 4. Step 2 was repeated at each position from x=2 to x=n.
Step 5. The oligonucleotide length L was then incremented by 1 and Steps 1 through 4 were repeated.
Step 6. Step 5 was repeated for each length L from L=2 through L=n.
Step 7. Step 1 through Step 6 were repeated except that Step 1 was modified to set the position x to x=1 on the minus strand.
Step 8. The resultant set of oligonucleotides varying in length from L=3 to L=n were then compared to each member of the defining group (e.g. specific for Listeria monocytogenes or Salmonella sp.). The presence of the exact oligonucleotide sequence and the number of occurrences on either the plus or minus strand were recorded.
Step 9. Oligonucleotides occurring in the sequence of each member of the defining group were retained, and those oligonucleotides failing to occur in every member of the defining group were discarded.
Step 10. The set of oligonucleotide motifs remaining after Step 9 were each individually tested for exact occurrence in a set of 407 phage genomes representing the majority of currently known phage genomes as described.
Step 11. Oligonucleotide motifs occurring in any phage sequence other than that of a bacteriophage belonging to the defining group (e.g. specific for Listeria monocytogenes or Salmonella sp.) were discarded. Only oligonucleotide motifs occurring only in the defining group were retained.
The procedures outlined in Step 1 through Step 11 can be accomplished by computational means well-known to those skilled in the art. The methods can range from simple manual string searches to use of more sophisticated homology search algorithms such as BLAST, provided that the search parameters are adjusted to retain short exact matches as significant.
An oligonucleotide of interest is one that is found at least once in the genome of each of the at least three species-specific, lytic phage of interest that comprise the phage test data set. The phage test data set can comprise at least four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more species-specific, lytic phage strains.
A new candidate bacteriophage then is tested for presence of the two or more oligonucleotides by means of detecting including, but are not limited to: [1] isolation of the bacteriophage genome in its entirety or in any sub-portion followed by DNA sequencing by any means including but not limited to dideoxy sequencing, chemical sequencing according to Gilbert and Maxam, and sequencing by mass spectrometry; [2] polymerase chain reaction (PCR) whereby a pair of sequences flanking or framing the target sequence to be amplified are chosen to serve as primer sequences, requiring only that the sequences lie on opposite strands of the bacteriophage DNA and that the 3′ ends of each sequence lie within 10 kb or less of one another; [3] Southern hybridization wherein an intact bacteriophage genome or fragments produced by restriction digestion are transferred to a membrane following electrophoresis and hybridized with one or more DNA probes consisting of labeled single or double-stranded DNA oligonucleotides with sequences corresponding to an oligonucleotide of interest, followed by stringency washes; and [4] dot blotting wherein an intact bacteriophage genome is directly applied to a membrane following and hybridized with one or more DNA probes consisting of labeled single or double-stranded DNA oligonucleotides with sequences corresponding to an oligonucleotide of interest, followed by stringency washes.
Thus, for example, by hybridization means, candidate phage genomic DNA, which can be digested with a restriction endonuclease, is exposed to one or more oligonucleotides of interest, labeled with a reporter molecule. Suitable controls are included to enable quantification of signal so that it can be determined whether the phage genome contains all of the oligonucleotides of interest, or complements thereof, if the oligonucleotides of interest or mixtures thereof are combined in the probe solution.
Another embodiment comprises a composition of a bacteriophage whose genome contains two or more sequences drawn from the list comprising Table 2. This composition of bacteriophages comprises a group of lytic bacteriophages whose host range is specific for Salmonella species. The bacteriophage in the composition may contain one copy of sequence drawn from the list in Table 2, or it can contain two or more copies of such sequences. In other embodiments, the phage contains any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, . . . 283, 284, 285, 286, 287, 288 or 289 oligonucleotides of interest. A preferred embodiment comprises a composition of two or more genetically distinct bacteriophages, each of whose genomes contains two or more sequences drawn from the list comprising Table 2. In this embodiment, the genome of each bacteriophage in the composition contain the same sequences drawn from the list in Table 2 as any other bacteriophage in the composition, or it may differ in one, more than one, or all sequences drawn from the list in Table 2. The genomes of bacteriophages in this composition may contain the same number of sequences drawn from the list in Table 2, or they may contain different numbers of such sequences. The genome of each bacteriophage in the composition may contain one copy of sequence drawn from the list in Table 2, or it can contain two or more copies of such sequences.
The invention does not anticipate that all bacteriophage lytic for Listeria monocytogenes fall within the scope of the compositions where the bacterial genomes contain two or more sequences drawn from the list contained in Table 1. Neither does this invention anticipate that the genomes of all bacteriophage lytic for Salmonella species will contain two or more sequences drawn from the list contained in Table 2.
The invention now will be exemplified by the following non-limiting examples.
The oligonucleotide motifs listed in Table 1 and Table 2 were identified using computational methods available to those skilled in the art. The set of Listeria-specific nucleotide motifs in bacteriophage specific for Listeria monocytogenes shown in Table 1 was obtained through analysis of the sequences of Listeria-specific bacteriophages List-1, List-2, List-3, List-4, List-36, List-38, LMA-34, LMA-57, LMA-94, and LMA-148. The detection and isolation of bacteriophages specific for Listeria monocytogenes is well known in the literature, and obtaining the genomic sequence thereof is likewise obvious to all workers skilled in the art. The set of Salmonella-specific nucleotide motifs in bacteriophage specific for Salmonella species shown in Table 2 was obtained through analysis of the sequences of Salmonella-specific bacteriophages SBA-1781, SDT-15, SHM-125, SHM-135, and SPT-1. The detection and isolation of bacteriophages specific for Salmonella is well known in the literature, and obtaining the genomic sequence thereof is likewise obvious to all workers skilled in the art. For each group of genomic bacteriophage sequences, a set of oligonucleotides was computed such that: [1] each oligonucleotide was 3 nucleotides or longer; [2] each oligonucleotide was as long as possible; [3] any oligonucleotide could hybridize to either strand of the bacteriophage genomic sequence; [4] every oligonucleotide was present in every member of the defining group; [5] no oligonucleotide was present in any member of the other group. Hence, for the Salmonella phage group, the initial set of oligonucleotides was determined that were at least 3 nucleotides long, and would discriminate the Salmonella phage from Listeria phage. The initial analysis identified 2,120 oligonucleotides that would hybridize to the Listeria phage specifically based upon their genomic sequences, but not to the Salmonella phage. In the case of Salmonella phage, a total of 7,878 oligonucleotides were identified that would hybridize to the Salmonella phage specifically based upon the genomic sequences, but not to the Listeria phage.
To refine the analysis further, the initial set of oligonucleotides was compared to a set of 407 phage genomes representing the majority of currently known phage genomes. The phage test data set included free phage genomes that had been identified and sequenced; these sequences were extracted from the NCBI database. In addition, approximately 250 phage genomes were prophage genomes extracted from the sequences of the genomes of their bacterial hosts. These prophage were identified by manual curation of the ends of the prophage based on several criteria including DNA sequence repeats, integrase gene homologies, and insertion sites. The overall data set did not include the sequences of List-1, List-2, List-3, List-4, List-36, List-38, LMA-34, LMA-57, LMA-94, LMA-148, SBA-1781, SDT-15, SHM-125, SHM-135, or SPT-1. The assembled bacteriophage database thus was able to serve as an appropriate control in the analysis of the aforementioned bacteriophage. To perform the analysis, the number of matches of each of the candidate oligonucleotides to each of the phage genomes was recorded. Oligonucleotides from Listeria bacteriophage were accepted only if there were no matches to any other bacteriophage other than bacteriophage specific for Listeria monocytogenes. Similarly, oligonucleotides from Salmonella bacteriophage were accepted only if there were no matches to any other bacteriophage other than bacteriophage specific for Salmonella species.
The foregoing analysis resulted in 44 oligonucleotide motifs specific for bacteriophage that infect Listeria that do not occur in other known phage genomes. Likewise, the analysis resulted in 289 oligonucleotides specific for bacteriophage that infect Salmonella that do not occur in other known phage genomes.
The phage test data set is defined as the genomic sequences of the Listeria-specific bacteriophages List-1, List-2, List-3, List-4, List-36, List-38, LMA-34, LMA-57, LMA-94, and LMA-148, see WO2005059161. The oligonucleotide motifs may occur more than once in any one bacteriophage genome.
The phage test data set is defined as the genomic sequences of the Salmonella-specific bacteriophages SBA-1781, SDT-15, SHM-125, SHM-135, and SPT-1, see WO2005027829. The oligonucleotide motifs may occur more than once in any bacteriophage genomic sequence.
It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.
All references cited herein are herein incorporated by reference in entirety.
E., Sjoberg, B. M., and Fontecave, M., A metal-binding site in the catalytic subunit of anaerobic ribonucleotide reductase, Proceedings of the National Academy of Sciences of the United States of America, 100, 3826 (2003).
