WO2003103578A2

WO2003103578A2 - Bacteriophage and enzymes lytic to salmonellae

Info

Publication number: WO2003103578A2
Application number: PCT/US2003/017469
Authority: WO
Inventors: Delbert L. Harris; Nakhyung Lee
Original assignee: Iowa State University Research Foundation, Inc.
Priority date: 2002-06-05
Filing date: 2003-06-04
Publication date: 2003-12-18
Also published as: WO2003103578A3; AU2003240505A1; AU2003240505A8

Abstract

Isolated phage HL18 having a broad Salmonella host range and compositions comprising HL18 phage, methods of administering the phage and compositions to livestock animals and products to reduce or prevent dissemination of Salmonella. The invention also provides HL18 phage lysins and lyases, enzymatic compositions, and methods of producing and using lysins and lyases from HL18 phage.

Description

BACTERIOPHAGE AND ENZYMES LYTIC TO SALMONELLAE

[0001] This patent application claims the benefit of U.S. provisional patent application serial number 60/385,570, filed June 5, 2002, entitled BACTERIOPHAGE STRAIN AND BACTERIOPHAGE ENZYMES LYTIC TO A BROAD HOST RANGE OF SALMONELLAE. U.S. provisional patent application serial number 60/385,570 is hereby incorporated by reference in its entirety.

REFERENCES

[0002] Several publications are referenced herein. Full citations for these publications are provided below. The disclosures of these publications are incorporated herein by reference in their entirety, unless otherwise noted.

BACKGROUND OF THE INVENTION

[0003] Bacteriophages ("phages") are viruses able to kill susceptible host bacteria through enzymatic degradation of bacterial cell walls. On the basis of this property, the use of phages to cure bacterial diseases, termed "phage therapy," was investigated in the early 20th century as a potential weapon against infections. These early approaches to phage therapy failed to produce a practical and effective phage preparation for treatment or amelioration of bacterial infection. Phage therapy was almost abandoned and replaced by the introduction of antibiotics. The continued use and overuse of antibiotics, however, has resulted in the emergence of virulent bacterial strains that are resistant to presently available antibiotics.

[0004] Bacteriophage resemble human and animal viruses in a number of ways. For example, viruses infect specific cell types and can cause extensive damage to the infected cells. In animals, this can lead to the production of diseases. Likewise, phages have specific bacterial targets and can cause extensive damage to the bacteria. Lytic bacteriophage initially contact their hosts through specific receptors in their tail followed by injection of their DNA into the host bacterial cell. Like a "Trojan Horse," once inside the cell they direct the production of quantities of progeny phage that are released when the bacterium is lysed and killed by the phage. Lytic phages continue to prohferate in an animal as long as bacteria are present to be infected by the phage.

[0005] Enteric bacteria such as Salmonella and Esώerichia coli can cause food-borne illness in humans due to ingestion of contaminated food products. The economic and health consequences of contaminated livestock increase the importance of rinding an inexpensive yet effective method of reducing or eliminating food- borne illness. Concern regarding the overuse of antibiotics has lead to a search for alternative mechanisms to combat livestock- related infection. New classes of antibiotics have not been discovered in the past 30 years. Furthermore, antimicrobial usage in animals is being restricted in order to protect the pubhc health. Concurrently, changes in social awareness and concerns regarding food safety have increased the need for reduced levels of Salmonella in pigs and other animals.

[0006] Salmonella have a variety of serotypes, approximately 2,400, which are identified by the carbohydrate composition of the outer membrane. More than 20 different serotypes of Salmonella cause clinical problems in the field. In addition, Salmonella is an emerging multi-drug resistant bacteria, which cannot be completely controlled with commercial antibiotics. Generally speaking, commercial methods such as antibiotics or vaccination are unlikely to be effective in controlling Salmonella contamination. Multi-drug resistant (MDR) Salmonella typhimurium is an important pathogen which emerged worldwide during the last two decades and now causes great concern to pubhc health. Since eradication of MDR Salmonellae in livestock is very unlikely, problems derived from these organisms might be resolved only by lowering the level of MDR Salmonellae in the food production chain, including livestock. [0007] Previous studies reported that the culture positive rate of

Salmonella in market weight pigs gradually increases from farm to lariage, and ultimately to slaughterhouse. The sources of Salmonella contamination in pigs include environments (transportation vehicles, holding areas, etc.), active Salmonella shedding pigs, and recurrent Salmonella shedding among carriers because of stress during transport or delivery. Healthy pigs become Salmonella culture positive in tissue samples within as few as three hours after infection resulting from exposure to Salmonella infected pigs. No matter what the sources of Salmonella infection into market weight pigs, the rapid dissemination of Salmonella in pigs prior to slaughter is an important risk factor in pork product contamination.

[0008] Early attempts at phage therapy failed primarily due to the hmited host range of most phages. Bacteria are classified according to the carbohydrates moieties located on their outer membrane. These carbohydrates serve as receptors for particular phages. Phage specificity is derived from a phage's ability to adsorb onto these carbohydrate moieties as a first step in the bacterial killing process. A typical phage is specific for very few bacterial strains even among the same bacterial species. Thus, a typical phage has a small host range and is only capable of killing a small subset of bacteria within a single genus of bacteria.

[0009] Previous approaches to phage therapy have focused on increasing the host range of phage compositions by using phage cocktails. These cocktails are typically undefined mixtures of phages that have specificity against many different bacterial strains. See, e.g., U.S. Patent Number 6,121,036. Experimentally, phage cocktails have been shown to reduce diarrhea and the numbers of Escherichia coli in the intestine of piglets. Smith, H.W. and Huggins, M.B., Effectiveness in Treating Experimental Escherichia coli Diarrhea in Calves, Piglets and Lambs, Journal of General Microbiology 129:2659-75 (1983). Phages lytic for Salmonella typhimurium were found to reduce the levels of Salmonella in both the digestive tract and liver of day-old chicks. Berchieri et al., The Activity In The Chicken Alimentary Tract Of Bacteriophages Lytic For Salmonella typhimurium, Res. Microbiol., 142:541-549 (1991).

[0010] However, the use of phage cocktails is associated with the selection of resistant bacteria through long-term use of bacteriophage cocktail treatment. Resistant bacteria may be a more significant health threat than the initial disease, if antibiotics or other bacteriophage are not available to treat the resistant organisms. The reduction of Salmonella in hvestock prior to slaughter can reduce the prevalence of food-borne human salmonellosis acquired through consumption of contaminated meat products. What is needed is a broad host range bacteriophage and methods of using the bacteriophage to effectively control Salmonella in animals without increasing the risk of selecting for resistant pathogenic bacteria.

BRIEF SUMMARY OF THE INVENTION

[0011] This invention provides compositions containing purified

HL18 bacteriophage and methods of using HL18 phage to reduce the amount of Salmonella in animals (e.g., hvestock animals) and meat products. The present inventors have found that the HL18 phage significantly reduces Salmonella in animals. One embodiment of the invention provides a composition comprising isolated HL18. Another embodiment of the invention provides compositions for reducing the amount of Salmonella in a livestock animal comprising at least 10° PFU of HL18 phage. Administration of the compositions of the invention to an animal can reduce the amount of Salmonella in the animal by, for example, at least 10 fold.

[0012] The invention also provides methods for reducing the amount of Salmonella by administering a composition containing HL18 phage in an acceptable carrier to an animal. The invention further provides methods of reducing dissemination of Salmonella in hvestock animals by administering a composition having a Salmonella reducing effective amount of HL18 to a Hvestock animal prior to harvest. Another embodiment of the invention provides methods of harvesting a livestock animal by administering HL18 phage to a livestock animal less than 24 hours prior to harvest in an amount effective to reduce the amount Salmonella; and harvesting the animal. The Salmonella reducing effective amount of HL18 can reduce dissemination of Salmonella infection by, for example, about 10 to about 10,000 fold. Additional embodiments of the invention include methods of reducing the amount of Salmonella in meat products by applying a composition comprising HL18 phage to a meat product (e.g., pork).

[0013] Other embodiments of the invention are directed to methods of producing HL18 bacteriophage enzymes. The invention provides methods of producing lysin by infecting Salmonella cells with HL18 phage, growing the infected cells in a suitable medium for a time sufficient to produce lysin, lysing the cells, and obtaining the lysin. Another embodiment of the invention is directed to methods of producing lyase comprising infecting Salmonella cells with HL18 phage, growing the infected cells in a suitable medium for a time sufficient to produce the lyase, lysing the cells, and obtaining the lyase.

[0014] Additional embodiments of the present invention are set forth in part in the description that follows, and in part will be obvious from the description, or may be learned through the practice of the invention. The objects and advantages of the invention will be attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is an electron micrograph of phage HL18 negatively stained with 2% unranyl acetate. [0016] FIG. 2 is a bar graph showing the reduction of Salmonella levels in pig tonsil, cecum, and colon by administering HL18 and FOl phage.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Salmonella can disseminate into the body organs of hvestock within a few hours after infection. Livestock susceptible to Salmonella infection are at greatest risk when confined with other animals as they are moved from farm to lariage, and ultimately to the slaughterhouse. Thus, Salmonella-ncgaήve Hvestock may be infected through transport and holding in the lariage just prior to slaughter. Rapid dissemination of the Salmonella results in a greatly increased risk of contaminated meat products. An intervention strategy that uses HL18 phage, or enzymes derived from HL18 phage, to reduce the rapid dissemination of Salmonella and, concomitantly, the potential for selection of resistant Salmonella strains in livestock, reduces the prevalence of food-borne salmonellosis in humans via the ingestion of Salmonella contaminated products.

[0018] Multi-drug resistant Salmonella are prevalent in swine and have been linked to human illness and death. Currently, the prevalence on a herd basis as determined by culture of feces ranges from 43.3%, 68% (pooled pen fecal samples) to 83%. The predominant serotypes in the U.S. are S. enterica (subspecies enterica), serovar Typhimurium, Typhimurium var. Copenhagen, Heidelberg, Derby, Worthington, Infantis, Bredeney, and Agona . 87.7% of the Salmonella typhimurium found in clinically normal swine are penta-resistant and 100% of S. Heidelberg isolates are multi-drug resistant. The emergence of these highly pathogenic, multi-resistant strains is a serious threat to the food supply.

[0019] One embodiment of the invention provides a composition comprising an isolated HL18 phage (ATCC # ). The term "isolated

HL18 phage" means a phage preparation substantially free of non-HL18 phage, preferably 90-95% free of non-HL18 phage, more preferably 99% free of non- HL18 phage. Another embodiment of the invention provides HL18 phage in an acceptable carrier (e.g., physiological saline, water, animal feed, Luria broth ("LB")) for administering the phage to hvestock.

[0020] The present inventors isolated HL18 from sewage by an enrichment method using S. typhimurium x4232 as a host strain, propagated in GCA medium and further purified according to methods known in the art. See, e.g.. Nelson et al., PNAS 98, pp. 4107-4112 (2001). Morphologically, HL18 is a distinct member of the Myoviridae family, with an isometric head of about 46nm in diameter and a long tail about lOOnm in length containing tail fibers. See FIG. 1. In contrast, FOl phage has a head of about 55 nm in diameter and a tail of about 50 nm in length. Phage in the Myoviridae family have a dsDNA genome similar to other enteric phages. HL18 phage effectively lysed all tested MDR Salmonellae which have the prototype of antiobigram of ACSSuT of MDR Salmonellae (Table 1). In addition, HL18 phage were able to lyse a broad range of Salmonellae, including, but not limited to, serogroup B, C_b C₂, D,, and E, (Table lc). HL18 phage effectively inhibited the growth of MDR Salmonellae as shown in Tables la, lb, lc, determined by counting the viable numbers of Salmonellae in the samples taken at designated periods of time after phage inoculation.

Table la. In vitro lytic activity of phage HL18 against strains of MDR

Salmonellae

A: Ampicillin, C: Chloramphenicol, S: Streptomycin, Su: Sulfonanaides, T: Tetracycline; ^■"complete confluent lysis [0021] Phages HL03, HL04, and HL18 were found to lyse multiple drug resistant S. enterica Typhimurium, and 5. enterica Heidelberg by spot test. Lawns of the following multi-drug resistant (MDR) serovars of Salmonella were grown for use with phage spot tests (see table lb below). Briefly, 20 ml of phage lysate at a concentration of 108 pfu/ml were dropped on each bacterial lawn. Plates were incubated overnight before being read. The + symbol indicates clear lysis where the phage were spotted on the bacterial lawn.

Table lb. Lytic activity of phages against drug resistant Salmonella enterica serovar Typhimurium and Heidelberg by in vitro assay

Ac: Amoxicillin/Clavulanic acid, A: Ampicillin, C: Chloramphenicol, K: Kanamycin, Ne: Neomycin, Pi: Piperacillin, P: Poperacillin/Tazobactam, Sp: Spectinomycin, S: Streptomycin, Su: Sulfisoxazole, T: Tetracycline, Tc: Ticarcillin/Caluvulanic acid

Table lc. In vitro lytic activity of phage HL18 to various serotypes of Salmonella

[0022] FIG. 2 shows the reduction of Salmonella levels in various swine tissues foUowing administration of HL18 phage or FOl phage. Two week old pigs were assigned to four groups: (1) Salmonella positive control, (2) FOl phage, (3) HL18 phage, and (4) untreated negative control. All pigs in the first three groups were challenged with 7.05 X 10° CFU of S. enterica Typhimurium X4232 Nal^R. The phage particle concentration in the phage lysate was 5.3 X 10° PFU/ml (FOl phage) and 7.5 X 10⁹PFU/ml (HL18 phage) in GCA medium. Pigs in the phage treatment groups received FOl or HL18 phage lysate via oral (20 ml) and intramuscular (6 ml) routes of administration 1 hour after Salmonella chaUenge. All pigs in the FOl and HL18 phage treatment groups subsequently received 15ml of FOl or HL18 phage lysate via oral route 3, 5, and 7 hours post Salmonella chaUenge. Pigs in the Salmonella positive control group received Salmonella culture lysate of S. enterica Typhimurium χ4232 Nal^R that had been sonicated and filtered with a 0.45um filter. Twelve hours post Salmonella challenge, all pigs were sacrificed and the tissues were coUected. The levels of Salmonella in tonsil, cecum content, and colon content were quantitatively determined on XLD agar plates containing 50μg of nalidixic acid/ml. HL18 phage reduced the Salmonella level by approximately 100 fold in tonsil, 1000 fold in cecum, and over 100 fold in colon. The statistical significance of FOl and HL18 phage treatment compared to the Salmonella control group is indicated by asterisks; * (P<0.05), ** (P<0.01). Data are expressed as means +/- SEM. See FIG. 2 and Table 2.

Table 2

* No. of Salmonella culture positive / No. of submitted samples

[0023] The broad host range of HL18 phage permits inactivation of a broad range of Salmonella serotypes and reduces or eHminates the emergence of drug resistant Salmonella strains. HL18 is capable of inactivating ah or a majority of the clinicaUy relevant Salmonella which cause disease in both humans and animals. Derivatives or by-products of the HL18 phage, such as lytic enzymes purified from the phage, can also be used to inactivate Salmonella. In addition, HL18 phage and its derivatives or by-products can be appHed directly to food products to inactivate Salmonella.

[0024] Compositions containing HL18 phage can be provided in a variety of dosage forms (e.g., oral, injectable, aerosol, rectal). Oral dosage forms include HL18 phage formulated in, for example, animal feed, water, bacterial media (e.g., LB), and saline. Injectable dosage forms include HL18 phage formulated in, for example, saline and water. Aerosol dosage forms include HL18 phage formulated in water or saline and combined under pressure with a suitable propellant. Rectal dosage forms include HL18 phage combined in a suppository formulation. Other dosage forms include tablets and capsules for oral administration and creams, lotions, gels, and transdermal patches for topical administration.

[0025] HL18 phage has a broad host range against Salmonella including Salmonella serotypes most frequently isolated from swine with clinical signs of infection (e.g., S. derby, S. choleraesuis kunzendorf, S. typhimurium, S. heidelherβ, S. choleraesuis, S. anatum, S. mbandaka, and S. schwarzengrund). The broad host range of the HL18 phage makes it especially weU suited for use against various serotypes of Salmonella, including Salmonella typhimurium, in a pre-harvest intervention strategy. Furthermore, the use of a phage having a broad host range to Salmonella as a short term, pre-harvest intervention strategy minimizes the risk of developing Salmonella resistant bacteria.

[0026] HL18 phage can be propagated using a host strain of

Salmonella (e.g., Salmonella typhimurium). For example, S. typhimurium χ4232, a nalidixic acid resistant strain, can be used to infect swine and to propagate phages. SalmoneUa inoculum can be prepared by any suitable method, such as growing the bacteria in LB broth. Phage stock or lysate can also be prepared by any suitable method, such as using GCA (glycerol-Casamino acids) medium supplemented with calcium chloride, and filtered prior to use using, for example, a 0.45 micrometer filter. Phage particles can be further purified, if desired, by techniques including polyethylene glycol precipitation, centrifugation, and dialysis.

[0027] An exemplary Salmonella reducing effective amount of HL18 phage is about 10⁶ to about 10¹² plaque forming units (PFU) of HL18 phage. In another embodiment of the invention, at least about 10° to 10^u total PFU of purified HL18 phage are provided, optionally in a carrier. In yet another embodiment of the invention, about 10¹⁰ PFU of purified HL18 phage are provided, optionaUy in an acceptable carrier. Alternatively, HL18 phage can be lyophilized and dissolved in an acceptable carrier just prior to administration. Administration of HL18 phage in a Salmonella reducing amount to an animal wiU typicaUy reduce the amount of Salmonella in the infected animal by at least about 10 to about 100,000 fold.

[0028] HL18 phage can be administered to an animal by a variety of routes, including, for example, oral, inhalation, nasal, intramuscular, intraperitoneal, intrathecal, vaginal, rectal, topical, and combinations thereof. HL18 phage can be administered oraUy to an animal, for example, in a volume of about 1 to about 100 ml of a carrier. In one embodiment of the invention, phage lysate can be used to replace water avadable for consumption by animals prior to leaving the farm, during transport, or during holding. HL18 phage lysate can be provided, for example, in amounts up to about six gaUons in a day depending on the size and number of animals. The concentration of phage in the carrier can be adjusted to any desired level. Alternatively, HL18 phage can be administered by injection in a volume of about 5 to about 50 ml of a carrier. In another embodiment of the invention, HL18 phage is combined with animal feed or water and administered orally to an animal. The HL18 phage animal feed formulation can be provided to animals at selected time intervals (as discussed below) and then replaced with regular animal feed as desired. Alternatively, HL18 phage in an acceptable carrier, such as water or sahne, may be instilled into the nose of the animal with, for example, a syringe with a nasal spray tip in a suitable volume (e.g., about 1 ml). HL18 phage can also be administered in an acceptable carrier via a stomach tube in any suitable volume (e.g., about 50 ml). In yet another alternative, HL18 phage can be formulated in an aerosol by combining the phage in a carrier, such as water or saline, or in a propeUant (e.g., trichloromonofluoromethane, dichlorodifluoromethane, and oleic acid). Aerosol formulations of the HL18 phage can administered in the nasal passage or oral cavity of the animal.

[0029] In one embodiment of the invention, intramuscular injection of

HL18 phage selectively reduces the level and dissemination of Salmonella in body organs and tissue. In another embodiment of the invention, oral administration of HL18 phage selectively reduces the level and dissemination of Salmonella in the gastrointestinal tract of an animal. Alternatively, HL18 phage is administered by both oral and intramuscular injection routes of administration to reduce the level and dissemination of Salmonella in organ systems and within the gastrointestinal tract of an animal.

[0030] Healthy Hvestock animals (e.g., swine, cattle) are exposed and susceptible to Salmonella infection when housed or transported with infected animals. Thus, intervention with HL18 phage treatment following exposure to SalmoneUa and prior to harvest is effective in reducing the amount of Salmonella in an animal. Alternatively, treatment of Hvestock prior to harvest wiU Hmit the risk of contamination of healthy animals in the event the animals were exposed to Salmonella when housed with other animals or transported to the slaughterhouse.

[0031] Compositions according to the invention containing HL18 phage in an acceptable carrier also can be administered to livestock as a short- term treatment. "Short-term" refers to less than about 24 hours to about 12 hours prior to harvest, alternatively less than about 12 to about 6 hours prior to harvest or less than about 3 hours prior to harvest. In contrast to prior use of phage therapy, short term pre-harvest treatment minimizes the risk of developing resistant Salmonella strains.

[0032] HL18 phage can be administered to animals in single or multiple doses. In one embodiment of the invention, HL18 is administered to an animal in a single dose prior to harvest. Alternatively, HL18 phage can be administered in 2 to 5 doses prior to harvest and after exposure of the animal to Salmonella. Each dose may contain, for example, about 10° to about 10¹² PFU of HL18 phage. In another embodiment, a dose contains about 10¹⁰ PFU of HL18 phage. When administered in multiple doses, several routes of administration may be used or combined. For example, phage can be administered in a first dose via injection followed by subsequent oral administration of the phage. Alternatively, HL18 phage can be orally administered in a first dose foUowed by a second dose administered by injection. Any desired combination of routes of administration may be used to optimize administration of the phage and may be determined by one of ordinary skill given the teachings herein.

[0033] As discussed above, exposure and potential exposure to

Salmonella can occur at several stages of an animal's Hfe. For example, Hvestock animals are exposed to Salmonella when they are housed with infected animals, ingest contaminated food or water, during transport with infected animals, or by intentional or accidental infection with Salmonella. The terms "exposed to Salmonella'^'' and "exposure to Salmonella'^'' also refer to animals that may have been exposed to Salmonella whether or not they actuaUy have been exposed to Salmonella. For example, co-mingling animals potentially exposes an animal to Salmonella since animals in the co-mingled group may be infected by or carry Salmonella.

[0034] In yet another embodiment of the invention, phage are administered to an animal in a single dose at about 3 hours foUowing exposure to Salmonella. In another embodiment of the invention, HL18 phage are administered and at about hourly intervals for up to about nine hours foUowing exposure to Salmonella. Alternatively, HL18 phage are administered to an animal at about 3, 5, 7, and 9 hours foUowing exposure to Salmonella. HL18 phage can also be administered to an animal about an hour after exposure to Salmonella and subsequently administered at about 2 and 3 hours after exposure to Salmonella.

[0035] Methods of harvesting Hvestock animals are also provided. The term "harvest" refers to slaughtering a Hvestock animal by methods accepted in the food processing industry. HL18 phage can be administered to animals prior to harvest in an amount effective to reduce the amount of Salmonella in Hvestock animals foUowed by harvest of the animal. Alternatively, HL18 phage in an acceptable carrier is administered to animals at least three hours prior to harvest.

[0036] In another embodiment of the invention, HL18 phage in an acceptable carrier is appHed to animal meat post-harvest in order to reduce the level of Salmonella in a meat product. The term "meat product" refers to any food product made from an animal (e.g., beef, poultry, lamb, pork). Acceptable carriers for the HL18 phage include water, saline, and LB broth. HL18 phage can be applied to the surface of the meat product to, for example, prevent contamination or to reduce the number of bacteria on the surface. Phage can be appHed by, for example, spraying the surface of or soaking the meat product in a solution containing at least about 10⁶ PFU of HL18 phage. HL18 phage can be appHed to meat products at any time foUowing harvest (e.g., processing, curing, cutting, shipping, and packaging). HL18 phage solution can also be provided in a consumer product to treat surfaces and food products to reduce or prevent contamination by Salmonella. HL18 phage solution can be provided in a pump or aerosol spray bottle to facilitate use of the product.

[0037] The present invention also provides new antimicrobial alternatives for multiple drug resistant Salmonella composed of HL18 phage - derived enzymes with lytic activity against Salmonella including bacteriophage - free lysin and lyase enzymes for reduction of Salmonella.

[0038] Enzymes encoded by lytic phage are typically expressed late in a viral infection and favor the release of virions by the lysis of the host ceUs, or may serve to faciHtate viral infection of host caUs. Phage-derived lysozymes (lysins) digest glycoside Hnkages next to peptide-substituted N-acetylmuramic acid residues, leading to peptidoglycan hydrolysis. When lysins are produced endogenously within a host ceU, their action is dependent upon transport across the cytoplasmic membrane. Holins are hydrophobic membrane proteins that form nonspecific pores on the ceU membrane to promote access of lysin to peptidoglycan substrates. However, when lysins are added exogenously, they can degrade the cell waUs of bacteria, causing "lysis from without." This activity of lysins can be utilized as an effective means of eHminating contaminant bacteria. In addition, recombinant lysins can be produced as recombinant fusion proteins without losing their native specific activity.

[0039] Lyases are enzymes normaUy found bound to small spikes attached to the base-plate of phage particles. Gram-negative bacteria are surrounded by a polysaccharide layer, which viruses need to penetrate with their tail spikes in order to access their receptor sites and infect a bacterial ceU. Since the polysaccharide can act as a barrier to infection, most virulent phages possess a polysaccharide-degrading enzyme (lyase). Their role is to cleave specific Hnkages in polysaccharides, yielding oHgosaccharide fragments. About 10¹³ PFU of phage are able to exogenously degrade about 1 g of polysaccharide.

[0040] The polysaccharide layer of Gram-negative bacteria is responsible for the relative impermeability of their outer membrane to noxious materials, such as, antibiotics, lysozymes, bUe salts. If the LPS layer is damaged, the bacteria are susceptible to these noxious agents. Lyase is able to degrade the 0-antigen unit of LPS. Because phage-derived lyases are able to damage the LPS layer of many Gram-negative bacteria, lysin and lyase can act synergisticaUy to kiU Salmonella. In addition, lyase alone degrades the LPS layer of Salmonella and causes the damaged Salmonella to be susceptible to animal immune defense systems. [0041] Lysin and lyase of HL18 phage can be purified according to any suitable method. See, e.g., Nerma, M.; Siddiqui, J.Z. Biochemistry International 1986, 12267-277; Fastrez, J. Phage lysozymes, in Lysozymes Model Enzymes in Biochemistry and Biology, JoUes, P., editor; Birkhauser Verlag Basel: 1996; pp. 35-64; Black, L. W.; Hogness, D. S. Journal of Biological Chemistry 1969, 244 1968-1975; Tsugita, A.; Inouye, M. Journal of Biological Chemistry 1968, 243 391-397; Alderton, G.; Ward, W. H; Fevold, H. L. Journal of Biological Chemistry 1945, 75743-58; Sophanopoulos, A. J.; Van Holde, K. E. Journal of Biological Chemistry 1964, 2302516-2524795. One embodiment of the invention, provides a method of producing lysin by:

[0042] (1) infecting Salmonella cells with HL18 phage;

[0043] (2) growing the infected cells in a suitable medium for a time sufficient to produce lysin;

[0044] ( 3 ) lysing the ceUs; and

[0045] (4) obtaining the lysin.

[0046] In one embodiment of the invention, Salmonella are infected by HL18 phage by preparing a culture of S. Typhimutium LT2 in minimal medium supplemented with 0.2% glucose. Salmonella can be cultured at 37° C with shaking until the OD⁶⁰⁰ reaches about 0.25. Then, phage can be added at about 5-10 multiplication of infection (MOl) into the Salmonella culture. After allowing 15 minutes of static culture for adsorption, shaking can be continued until lysis of the ceUs occurs at about 6 - 7 hours. The culture can be treated with 20 ml of chloroform and shaken vigorously. Phage lysate can then be obtained by centrifugation. Phage particles can be harvested by polyethylene glycol and sodium chloride precipitation. The phage particles can be resuspended and used to prepare phage enzymes (e.g., lyase or lysin). [0047] HL18 lysin and lyase can be prepared as described previously for other phage enzymes. See, e.g., Verma, M.; Siddiqui, J.Z. Biochemistry International 1986, 12 267-277; Rao, G. R; Burma, D. P. Journal of Biological Chemistry 1971, 24664:74-6479. For example, crude phage lysate can be prepared and treated with protamine to precipitate nucleic acids. See, e.g., Doughty, C. C; Hayashi, J. A. Journal of Bacteriology 1962, 83 1058-1068. FoUowing centrifugation, the supematent can be removed and treated with ammonium sulfate to precipitate proteins. The proteins, containing phage enzymes, can be obtained by loading the protein solution on to an AmberHte IRC-50 column, washing the column with 4 to 5 column volumes of washing buffer, and eluting fractions by changing the salt concentration. See, e.g., Tsugita, A.; Inouye, M. Journal of Biological Chemistry 1968, 243 391-397. During the elution step, each fraction can be collected and assayed for lytic activity against Salmonella in spot tests. AU fractions containing lytic activity can be pooled and passed through Sephadex G-50 column. After washing the column several times, the adsorbed protein (e.g., lysin and/or lyase) can be eluted. The purity of the protein preparations can be determined using SDS- PAGE and silver staining. If necessary, the semi-purified lysin can be passed on AmberHte IRO-50 column again and tested for purity. The active fractions can be pooled and dialyzed in Tris buffer. The lytic activity of the purified lysin or lyase can be titrated. Purified lysins can also be used in screening methods to determine the relative the lytic activity of phage-derived lysins and/or lyases.

[0048] Another embodiment of the invention, provides a method of producing lyase by:

[0049] (1) infecting Salmonella ceUs with HL18 phage;

[0050] (2) growing the infected ceUs in a suitable medium for a time sufficient to produce lyase;

[0051] (3) lysing the ceUs; and [0052] (4) obtaining the lyase.

[0053] Lyase derived from HL18 phage is obtained according to the methods described above with regard to lysin.

[0054] It is to be understood that the appHcation of the teachings of the present invention to a specific problem or environment wiU be within the capabiHties of one having ordinary skUl in the art in Hght of the teachings contained herein. Examples of the products of the present invention and processes for their use appear in the foUowing examples.

EXAMPLE 1 Evaluation Of HL18 Phage Or Phage Derived Enzymes In Swine

[0055] An acute infection model for both young pigs and slaughter weight swine is used to evaluate HL18 phage and HL18 phage-derived enzymes (e.g., lysin and/or lyase) based on natural exposure to Salmonella contaminated feces. To create an experimental pen that is contaminated with Salmonella, seeder pigs are inoculated with Salmonella. Two to three days after inoculation, the seeder pigs are removed and the experimental pigs are placed in the pen for six hours. The experimental pigs are removed and placed in treated (various groups separated by route of treatment administration) and untreated pens. HL18 phage (See, e.g.. Table 2) or phage-derived enzyme (e.g., HL18-derived lyase or lysin) is administered by oral and/or intramuscular (IM) routes for efficacy studies. At 1-3 hours post phage/enzybiotic treatment, pigs are killed, and necropsies and Salmonella levels are quantified in various body organ and tissues. See, e.g., Table 2 for HL18 phage treatment results in mandibular lymph node, ileocecal lymph node, Hver, and spleen. Control pigs are pigs that have not been inoculated with Salmonella and have been treated with sonicated, phage-free Salmonella (NalR). [0056] FoUowing the murine typhoid infection model, see, e.g.,

Pfeiffer, CG.; Marcus, S. L.; Steele-Mortimer, O.; Knodler, L. A.; Finlay, B. B. Infection and Immunity 99 A.D., 675690-5698, Salmonella is grown to exponential phase by growing at 37° C in LB broth overnight, cHluted 1:100 into fresh LB, and incubated, with shaking, for four hours. The bacteria are washed in PBS and resuspended in PBS with 2% glucose. Control mice are given PBS with glucose only (infection negative control). Female C57B1/6 mice, aged 6 to 10 weeks, are inoculated orally with approximately half an LD₅₀ of Salmonella ssp after being deprived of water for 4 hours. A second, systemic challenge model is used, with mice receiving half an LD₅₀ per mouse via lateral taU vein injection. Phage lysate of HL18-derived lysin and/or lyase phage proteins (enzybiotics) is administered either IM, IP or orally at the same time as the Salmonella chaUenge and at various times after Salmonella chaUenge (1, 2 and 4 hours post chaUenge). Sonciated Salmonella (Nal-R) lysate can be administered to the phage negative control group. Six hours after phage treatment, the mice are harvested and the level of Salmonella in spleens, Hvers and intestines is determined.

EXAMPLE 2 Isolation Of HL18 Phage Lysine And Lyase Encoding Nucleic Acids

[0057] Phage genomic DNA is purified from HL18 phage lysates using standard methods appHed to phages such as P22. See, e.g., Osborne, M. J.; Rosen, S. M.; Rothfied, L.; Zeleznick, L. D.; Horecker, B. L. Science 1964, 145 783-789; Vander Byl, C; Kropinski, A. M. Journal of ^'Bacteriology 2000, 182 6472-6481. PCR primers with restriction enzyme sites to enable directional cloning are designed. The PCR products are digested with enzymes and purified using a gene cleaning kit. Fragments of lyase or lysin gene are Hgated into pET expression vector (Novagen). The recombinant plasmid pET is transformed into a bacterial host cell such as E.coli LB21 (De3) to produce recombinant lysin or lyase. The PCR product is digested and Hgated into a pET plasmid (Novagen). The recombinant plasmid containing HL18 lyase or lysin gene is transformed into a bacterial host ceU such as E.coli BL 21 (DE3) and expressed.

[0058] Lysin and lyase encoding nucleic acids derived from HL18 phage are identified by various methods. See, e.g., Loessner, M.I.; Wendlinger,G.; Scherer, S. Molecular Microbiology 1995 (6) 1231-1241; Loessner, M. J.; Gaeng, S.; Scherer, S. Journal of Bacteriology 1999, 181 4452- 4460; Loessner, M. J.; Schneider, A.; Scherer, S. Applied and Environmental Microbiology 1996, 62 3057-3060. Genomic phage DNA is fragmented by digestion with multiple restriction enzymes. These genomic DNA fragments are then electroporesed on an agarose gel. DNA fragments of the appropriate size to encode lysin (2kb-3kb) are cut from the gel and repaired with Mung Bean Nuclease (NEB). The repaired DNA fragments are Hgated into plasmid pSP72 under the control of an inducible T7 promoter (Promega). Recombinant plasmids are transformed into E.coli JM109 (Promega), and spread out on LB agar plates with antibiotics. After overnight incubation, a repHca of original plates is made on new agar plates supplemented with IPTG as a T7 promoter inducer, and incubated for 6 hours. The repHca are exposed to chloroform vapor and overlaid with 0.4% semisolid agar containing Salmonella as a substrate for the in vitro lysin assay. After 1 hour of incubation at 37° C, colonies showing lytic phenotype (clear area) on the replica plate are picked from the original plate. Plasmid DNA are isolated from the colonies with lytic phenotypes and sequenced through the ISU sequencing faciHty.

[0059] Genomic DNA from HL18 phage are analyzed by Southern blotting, using homologous probes produced by PCR (DIG probe Synthesis Kit (Roche)). After detection of lyase and/or lysin genes, a second agarose gel is run and the equivalent area of the gel is cut out to recover the DNA. The recovered DNA is repaired with Mung Bean Nuclease (NEB) and the fragments are Hgated into the pSP72 plasmid. The recombinant plasmid is transformed into JM109 bacteria which are plated out and screened with homologous probes. Positive clones are identified and sequenced and the lyase and/or lysin genes from candidate phage are then subcloned into an expression vector.

[0060] Once a gene (either lysin or lyase) is cloned and sequenced, primers are constructed to add directional restriction sites that will allow PCR products to be cloned into an expression vector. With these primers [5' primer with Ncol (NNNCCAGGNNN), 3' primer of Xho (NNNACTCGANNN)], the genes are amphfied from template DNA, digested with Ncol, and Xhol, and Hgated into pET plasmid. After E. coli are transformed by the recombinant plasmid, the E.coli are grown in LB media until the OD₆₀₀ reaches -0.6 . Next, IPTG is added to induce expression of the gene in culture. During this induction period, smaU ahquots of culture media are removed and run on SDS- PAGE to determine protein yield by densitometric screening of the stained gel and by Western blot using an anti-HisTag antibody. See, e.g., Loessner, M.I.; Wendlinger.G.; Scherer, S. Molecular Microbiology 1995 (6) 1231-1241; Loessner, M. J.; Schneider, A.; Scherer, S. Applied and Environmental Microbiology 1996, 62 3057-3060; Hoffman, B. J.; Broadwater, J. A.; Johnson, P.; Harper, J.; Fox, B. G.; Kenealy, W. R Protein Expression and Publication 1995, 6 646-654. Once the culture conditions are optimized, large quantities of lysins and lyases derived from different phages are produced. After culture, ceUs containing CBD-His-Tag fusion proteins are harvested and passed through a French Press foUowed by treatment with protamine, and cetrifugation to produce a clarified extract. CBD-His-Tag fusion proteins are purified from the supernatant with CbinD 100 Resin (Novagen) as foUows: Dry CbinD 100 resin is added directly to the supernatant and incubated. The resulting mixture is centrifuged and washed. Next, the fusion protein is eluted by washing the final peUet in elution buffer and Ekapture agarose foUowed by centrifugation. This recombinant His-Tag fusion protein can be used for in vivo and in vitro testing. If necessary, the fusion protein can be treated with enterokinase to truncate CBD-binding domain from the fusion protein. EXAMPLE 3 Biological Activity of HL18 Lyase and Lysin

[0061] To determine the activity of both lyase and lysin, substrates include Salmonella with different LPS structures, various Salmonella serotyopes with different phage receptor sites, and Micrococcus lysodeikticus as a lysozyme sensitive substrate. See, e.g., Verma, M.; Siddiqui, J.Z. Biochemistry International 1986, 12 267-277. The bacteria are used as either chloroform treated (kiUed) or Hve. In addition, purified Salmonella-d ήvcd LPS is used to assess lyase activity. Lysin activity is determined by its abUity to lyase sensitive ceUs. Target bacteria are incubated with varying amounts of lysin at 37° C. The decrease of turbidity is monitored by spectrophotometry every 30 seconds for 5 minutes. The level of activity is calculated based on the relationship between the changes of absorbance versus time. In addition, killing activity of recombinant lysin is determined by treating various Salmonella strains and completing growth curves to show any differences in lysin activity against various wUd-type and mutant Salmonella strains. Phage-derived lyases are able to degrade the polysaccharide of susceptible bacteria. For the in vitro activity assay of lyase, purified LPS of Salmonella, chloroform treated whole Salmonella, and intact Hve Salmonella are used as substrates. Lyase activity is evaluated by at least two ways, decreasing of turbidity of the substrates after treatment of appropriate amounts of lyase, and determining degrading pattern of LPS after running SDS-PAGE. Various Salmonella serovars that have different carbohydrate structures on their outer membrane are used to determine host specificity of each lyase.

EXAMPLE 4 Enzobiotics

[0062] Enzybiotics are developed based on in vitro lytic assay results using combinations of lyase and lysin. Enzybiotic preparations are tested in a mouse model as a preHminary study in order to determine critical data, such as, minimal effective dose, effective administration route, and timing of enzybiotic administration. SimUarly, the murin typhoid model for salmonellosis is used in prehminary enzybiotic screening studies. On the basis of these results, enzybiotics are administered into pigs as an intervention strategy against Salmonella infection.

[0063] The above description and examples are only illustrative of embodiments which achieve the objects, features, and advantages of the present invention, and it is not intended that the present invention be Hmited thereto. Any modifications of the present invention which come within the spirit and scope of the foUowing claims is considered part of the present invention.

Claims

CLAIMSWe claim:

1. A composition comprising isolated HL18 phage deposited under

ATCC Accession Number .

2. The composition of claim 1, further comprising an acceptable carrier.

3. The composition of claim 1, wherein said phage is present in an

amount of at least 10⁶ PFU.

4. The composition of claim 1, wherein said phage is present in an

amount less than about 1 X 10¹² PFU.

5. The composition of claim 1, wherein said phage is present in an

amount at least about 1 X 10¹⁰ PFU.

6. A composition comprising HL18 phage deposited under ATCC

Accession Number and a carrier material selected from the group

consisting of saline, water, feed, and Luria broth.

7. The composition of claim 6, wherein said phage is present in an

amount of at least 10⁶ PFU.

8. The composition of claim 6, wherein said phage is present in an

amount less than about 1 X 10¹² PFU.

9. The composition of claim 6, wherein said phage is present in an

amount at least about 1 X 10¹⁰ PFU.

10. The composition of claim 6, wherein said composition is an oral

dosage form.

11. The composition of claim 6, wherein said composition is an injectable

dosage form.

12. The composition of claim 6, wherein said composition is an aerosol

dosage form.

13. The composition of claim 6, wherein said composition is a suppository

dosage form.

14. A method of reducing Salmonella in an animal comprising

administering a composition containing HL18 phage to said animal.

15. The method of claim 14, wherein said phage is administered to said

animal from about 24 hours to less than about 3 hours prior to harvest of said

animal.

16. The method of claim 14, wherein said composition further comprises

a material selected from the group consisting of saHne, water, feed, and Luria

broth.

17. The method of claim 14, wherein said composition is administered

from about 12 hours to about 6 hours prior to harvest of said animal.

18. The method of claim 14, wherein said composition is administered

from about 6 hours to about 3 hours prior to harvest of said animal.

19. The method of claim 14, wherein said the composition is administered

less than about 3 hours prior to harvest of said animal.

20. The method of claim 14, wherein said composition is administered in

a single dose.

21. The method of claim 14, wherein said composition is administered in

multiple doses.

22. The method of claim 14, wherein said composition is administered in

from 2 to 5 doses.

23. The method of claim 14, wherein Salmonella in said animal is reduced

by about 10 to about 100,000 fold.

24. The method of claim 21, wherein said composition is administered at

least 1 hour foUowing initial administration of said composition.

25. The method of claim 21, wherein said composition is administered to

at least two times prior to harvest.

26. The method of claim 14, wherein said composition is administered

oraUy.

27. The method of claim 14, wherein said composition is administered

intramuscularly.

28. The method of claim 14, wherein said composition is administered by

injection.

29. The method of claim 14, wherein said composition is admmistered by

instillation.

30. The method of claim 14, wherein said composition is administered as

a first dose and a second dose.

31. The method of claim 30, wherein said first dose is administered

intramuscularly and said second dose is administered orally.

32. The method of claim 30, wherein said first dose is administered at

least one hour after said animal is exposed to Salmonella.

33. The method of claim 31, wherein said second dose is administered at

least one hour after said first dose.

34. A method of harvesting a livestock animal comprising administering a

composition comprising HL18 phage to a livestock animal less than 24 hours

prior to harvest of said animal in an amount effective to reduce the amount

Salmonella; and harvesting said animal.

35. The method of claim 34, wherein said livestock animal is harvested at

least 1 hour after administering said composition to said animal.

36. A method of reducing dissemination of Salmonella in a Hvestock

animal comprising administering a composition comprising a Salmonella

reducing effective amount of phage HL18 to a Hvestock animal prior to harvest

wherein dissemination of Salmonella infection is reduced by about 10 to about

10,000 fold.

37. The method of claim 36, wherein said Salmonella reducing effective

amount is about 10⁶ to about 10¹² PFU.

38. A method of reducing the amount of Salmonella in a meat product

comprising applying a Salmonella reducing effective amount of HL18 phage to a

meat product.

39. The method of claim 38, wherein said Salmonella reducing effective

amount is about 10⁶ PFU.

40. The method of claim 38, wherein said meat product is selected from

the group consisting of beef, poultry, lamb, and pork.

41. The method of claim 38, wherein said phage is appHed by spraying.

42. The method of claim 38, wherein said phage is appHed by soaking.

43. A method of producing lysin comprising infecting Salmonella ceUs

with HL18 phage, growing said infected cells in a suitable medium for a time

sufficient to produce said lysin, lysing said cells, and obtaining said lysin.

44. The lysin produced by the method of claim 43.

45. A method of producing lyase comprising infecting Salmonella ceUs

with HL18 phage, growing said infected ceUs in a suitable medium for a time

sufficient to produce said lyase, lysing said ceUs, and obtaining said lyase.

46. The lyase produced by the method of claim 45.

47. A method of producing lysin comprising isolating a nucleic acid

encoding lysin derived from HL18 phage, transforming a bacterial host ceU with

said nucleic acid, and inducing said bacterial host ceU to express said nucleic acid

to produce said lysin.

48. The lysin produced by the method of claim 47.

49. A method of producing lyase comprising isolating a nucleic acid

encoding lyase derived from HL18 phage, transforming a bacterial host cell with

to produce said lyase.

50. The lyase produced by the method of claim 49.

51. A method of reducing Salmonella in an animal comprising

administering to the animal a composition containing a material selected from

the group consisting of HL18 phage, lyase derived from HL18 phage, or lysin

derived from HL18 phage.