WO1995032716A1

WO1995032716A1 - Methods and products for the control of pathogenic bacteria

Info

Publication number: WO1995032716A1
Application number: PCT/US1995/006670
Authority: WO
Inventors: David P. Franklin
Original assignee: Haar, Jonathan
Priority date: 1994-05-31
Filing date: 1995-05-31
Publication date: 1995-12-07
Also published as: AU3333695A

Abstract

Anti-infective compositions consisting of phospholipids with substituted fatty acid chains and methods of using same are provided. Also provided are immunogenic, e.g., vaccine, compositions comprised of a live strain of bacteria treated with the anti-infective compositions.

Description

METHODSANDPRODUCTSFORTHECONTROLOF PATHOGENICBACTERIA

FIELD OF THE INVENTION This invention is in the field of antimicrobial compositions and therapeutics and pertains to antibacterial phospholipid compositions, antibacterial agricultural wash solutions, immunogenic compositions, and methods of use thereof to prevent infection and contamination by bacterial pathogens.

BACKGROUND OF THE INVENTION A wide variety of microbial pathogens infect the gastrointestinal and respiratory tracts of hosts ranging from fowl to mammals of all kinds, including humans. Bacteria of the genuses Pseudomonas. Escherichia. and Salmonella, for example, are among the most common microbial pathogens and are major causes of gastrointestinal disease throughout the world. Other important genuses of pathogenic bacteria include Klebsiella. Streptococci. Staphylococci. Hemophilus. Legionella. Clostridia. Shigella. Campylobacter and Listeria. Disease states associated with these organisms vary as a function of the pathogen and host species involved, and range greatly in their severity. In humans, for example, the disease states associated with Salmonella range from simple sub-clinical gastroenteritis to typhoid fever.

These pathogens most commonly infect hosts upon ingestion of bacteria-laden foods. For example, pathogenic microorganisms are common contaminants in the processing of meat and poultry for human consumption. Thus, the improper processing of meat and poultry may result in the introduction of these bacteria into human hosts. Similarly, these bacteria exist in the feed of livestock and poultry, thereby potentially infecting these animals.

Recently, the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USD A) proposed new rules for the reduction of contamination by pathogenic microorganisms of meat and poultry produced and sold in the United States ("Pathogenic Reduction: Hazard Analysis Critical Control Point (HACCP) Systems," USDA/FSIS Docket No. 73-016P, February 3, 1995). Noting that pathogenic microorganisms "are widely recognized by scientists to be the most significant causes of food borne illness," the FSIS has proposed that "all slaughterhouse establishments incorporate at least one effective antimicrobial treatment to reduce the levels of microorganisms on carcasses." Amongst the approved antimicrobial treatments, the FSIS lists washes with hot water, chlorinated water, lactic acid, acetic acid, citric acid, and tri- sodium phosphate (TSP). Such washes have varying degrees of effectiveness, may cause discoloration to the meat or poultry, may pose other hazards to consumers (e.g., associations between chlorinated compounds and cancer), may pose other hazards to the environment (e.g., discharges of phosphate-rich effluents into lakes and streams), or may interfere with export of meat and poultry products (e.g., limitations on the use of chlorinated washes in some countries). The FSIS also notes that irradiation is a highly effective pathogen control mechanism but that it cannot easily be integrated into a slaughter operation and is a largely unavailable, capital- intensive process.

The need remains, therefore, for antimicrobial compositions for use in the meat and poultry processing industries for controlling pathogenic bacteria in a simple, safe, and cost- effective manner.

In addition to infection through ingestion of contaminated foods, pathogenic bacteria may be airborne and may infect the respiratory system after inhalation. For example, pathogenic bacteria are commonly found in air conditioning and ventilation systems used in residential and commercial buildings. These systems effectively spread the pathogenic microorganisms throughout the building, thereby exposing anyone inside to virulent bacteria. Thus, there is a need for a simple, safe, and cost-effective manner of reducing or eliminating the presence of pathogenic bacteria in ventilation systems.

Even the beneficial uses which have been developed for some pathogenic bacteria have potentially deleterious side effects. Certain Pseudomonas strains, for example, have been used in bioremediation and decontamination processes. In particular, Pseudomonas aeruginosa has been used in the bioremediation and decontamination of oil spills. This particular strain is able to degrade petroleum and polychlorinated biphenyls ("PCBs") into non-toxic compounds. Introduction of the bacteria into the environment, however, may have a negative impact by, for example, causing disease in animals present at the decontamination site. Similarly, certain Pseudomonas strains have been employed in the prevention of crop damage by facilitating ice crystal nucleation on the surface of fruit in citrus groves. Numerous sub-strains have been utilized, but most are of Pseudomonas aeruginosa origin. Again, however, the introduction of virulent bacteria into the environment may have a devastating effect on mammals low in the food chain, particularly mice. Thus, in these situations in which pathogenic bacteria are intentionally introduced into the environment, there remains a need for a simple, safe and cost-effective manner of controlling their spread and/or virulence.

Finally, the use of agricultural antibiotics has been suggested as playing a potential role in an observed increase in Salmonella infections over the last three decades. This is because of an observed parallel increase in the rate of infections and the number of bacterial isolates that are resistant to one or more microbial agents (Cohen, M.L. and Tauxe, R.V., Science. 234:964-969 (1986)). It is, therefore, desirable to develop new compositions which may be used to combat bacterial infections or contamination but to which the bacteria do not rapidly develop resistance. That is, there is a need for safe and cost-effective antibacterial compositions which can replace and/or supplement current antibiotic treatments.

Until recently, phospholipids have been seen as relatively inert components of the plasma membrane. Phospholipids consist of one or two fatty acyl chains esterified to a polar "head" group. Because of their amphipathic characteristics, phospholipids can form small vesicles or liposomes when dispersed in aqueous solution. Indeed, much current interest in phospholipids has focused on their use in creating liposomes as storage and in vivo delivery vehicles for drugs. Although the nature of the fatty acyl chains of phospholipids, particularly the degree of unsaturation, is known to influence the fluidity of both natural and artificial lipid membranes, specificity within the class of phospholipids is largely considered to be determined by the polar head groups. This is reflected in the nomenclature of the phospholipids: The common phospholipids, such as the phosphatidylcholines, phosphatidylserines, phosphatidylethanol- amines, phosphatidylinositols, phosphatidylglycerols, sphingomyelins, gangliosides, ceramides, and cardiolipins, are all named according to their head groups (see, e.g., Lehninger, A.L., (1982). Principles of Biochemistry. Worth Publishers, Inc., New York). Similarly, "purified" phospholipids are generally obtained from biological sources as crude lipid extracts which are separated by head group but which may contain a mixture of many different phospholipids which differ in their acyl chains (e.g., soy lecithin).

Bovine phosphatidylserines obtained from brain and phosphatidylserine analogs have been described for use in the treatment of auto-immune dysfunctions involving tumor necrosis factor ("TNF") and to treat bacterial and protozoal infections (European Patent Application, 0505817A1, by Delia Valle et al.). The exact nature of the fatty acyl chains of the phosphatidylserines used in these experiments was not disclosed but, presumably, the preparation contained a variety of different phosphatidylserines. Furthermore, although data showing decreased titer of TNF was presented, no theory regarding the mechanism of action of these compounds was provided.

A variety of phosphatidylethanolamine compositions, optionally including glycolipids, have been described for use to inhibit microbial colonization and infection (PCT International Application WO 92/11015, by Krivan et al.). This disclosure also fails to describe the exact nature of the fatty acyl chains of the phosphatidylethanolamines which are claimed to be useful. Rather, the disclosure advances the theory that certain phospholipids in the body act as "receptors" to which bacteria must bind in order to colonize. Therefore, Krivan et al. suggest the use of host phospholipid "receptors" to "provide a means for preventing colonization by ... 'fooling' a microorganism into binding them" and, thereby, preventing the "binding of microorganisms to native receptors on host cells." Thus, these host "receptors" were suggested as competitive inhibitors of bacterial binding and, by extension, colonization. For the bacterial species Chlamydia trachomatis. Krivan et al. isolated a fraction of the total lipid extract of HeLa cells which contained the putative receptor. This fraction was shown to contain at least seven different phosphatidylethanolamines which varied in the lengths of their fatty acyl chains and in the number and placement of double bonds and hydroxyl groups within them. It is unclear from the disclosure whether one or all of these phosphatidylethanolamines acts as the putative Chlamydia receptor.

Krivan et al. also isolated a fraction of the total lipid extract of human red blood cells which contained a putative receptor for binding and colonization of Helicobacter pylori. This fraction contained at least eighteen different "phosphatidylethanolamine-like" fatty acid methyl esters and dimethyl acetals. Again, these lipids differed in acyl chain lengths and in the number and placement of double bonds and it was unclear which may have been the putative Helicobacter "receptor."

Phosphoglycerides in the presence of omega-6-fatty acids have been suggested as therapeutic and prophylactic agents for bacterial, protozoal, and yeast infections. (See United States Patent No. 5,135,922, to Vitale). Based on studies in rats, Vitale teaches that "PC [phosphatidylcholine] and possibly other phosphoglyceride components of biomembranes are believed to protect against the lethal action of bacteria by promoting the clearance of these disease-causing organisms by the reticuloendothelial system (RES) by altering the fluidity, and consequently the function, of the phagocytic cells." Vitale further notes that "because PC has no inhibitory effect on GBS [Group B Streptococcus] in vitro and actually sustains GBS growth in culture, it is reasonable to speculate that PC enhances the immune response of the newborn animal to GBS." The preferred phosphatidylcholine in Vitale's disclosure is one in which linoleic acid is the primary fatty acid and, in his experiments, Vitale employed a 95% pure preparation of soy lecithin in which linoleic acid comprised 65.9% of the total fatty acids.

SUMMARY OF THE INVENTION The present invention provides a new class of safe and cost-effective antibacterial compositions in the form of non-metabolizable phospholipids which inhibit the growth and/or reduce the virulence of pathogenic bacteria. The phospholipids are characterized by the presence of fatty acid chains which render them non-metabolizable by the pathogens in question and, thereby, inhibit their growth and/or render them substantially avirulent. The compositions are effective against a broad spectrum of pathogenic bacteria including those from the genuses Pseudomonas. Escherichia. Salmonella. Klebsiella. Streptococci. Staphylococci. Hemophilus. Legionella. Clostridia. Shigella. Campylobacter and Listeria.

In each of the embodiments described above and below, it is preferred that the non- metabolizable phospholipids are present at a concentration of at least lOμg/ml and, more preferably, that they are present at concentrations of at least lOOμg/ml or 500μg/ml. In each embodiment it is also preferred that, of the total weight of phospholipids present, that metabolizable phospholipids comprise less than 50%, less than 25%, less than 15% and/or less than 5%. In most preferred embodiments, metabolizable phospholipids are absent from the compositions. In each of the embodiments described above and below, preferred non- metabolizable phospholipids are di-palmitoyl phosphatidylserine and l-oleoyl-2-palmitoyl phosphatidylserine. When the bacterial pathogen is Pseudomonas aeruginosa. most preferred non-metabolizable phospholipids are di-palmitoyl phosphatidylserine and l-oleoyl-2-palmitoyl phosphatidylserine. In each embodiment, when the bacterial pathogen is Salmonella typhimurium. the most preferred non-metabolizable phospholipids are di-palmitoyl phosphatidylserine, l-oleoyl-2-palmitoyl phosphatidylserine, di-palmitoyl phosphatidylinositol, l-oleoyl-2-palmitoyl phosphatidylinositol, di-palmitoyl phosphatidylethanolamine, l-oleoyl-2- palmitoyl phosphatidylethanolamine, di-palmitoyl phosphatidylglycerol, l-oleoyl-2-palmitoyl phosphatidylglycerol, di-palmitoyl phosphatidylcholine, l-oleoyl-2-palmitoyl phosphatidylcholine, and l-linoleoyl-2-palmitoyl phosphatidylcholine. In each embodiment, when the bacterial pathogen is Escherichia coli. the most preferred non-metabolizable phospholipid is di-palmitoyl phosphatidylserine. In a particularly preferred embodiment, the antibacterial phospholipids of the present invention are formulated into an agricultural wash solution which can be used in the meat and poultry processing industries both for the disinfection or decontamination of the meat and poultry itself and for the disinfection or decontamination of the workers, machinery, utensils and surfaces involved in meat and poultry processing. In this embodiment, it is particularly preferred that the antibacterial phospholipids of the present invention are phospholipids which are non- metabolizable by Salmonella species and/or by Escherichia coli. In addition, the present invention provides methods of reducing contamination by pathogenic bacteria in meat and poultry processing which comprise contacting the meat and poultry, workers, machinery, utensils and surfaces with the agricultural wash solutions of the present invention. In each of these embodiments, the agricultural wash solution is preferably heated to at least 37°C.

In other embodiments, a method for reducing infections by bacterial pathogens in livestock and poultry is provided in which the feed for these animals is first treated by contacting it with the non-metabolizable phospholipids of the invention. Preferably, the feed is contacted with at least lOg/kg and, more preferably, at least lOOg/kg of the non-metabolizable phospholipids. Preferred phospholipids are as described above.

In yet another aspect, the invention provides a method for decreasing the presence and/or reducing the virulence of pathogenic bacteria in air conditioning and ventilation systems which involves introducing an effective amount of the agricultural wash solution of the present invention into air conditioning and ventilation systems. In these embodiments, a bacterial pathogen of particular interest is Legionella.

Yet a further aspect of the invention includes a method for reducing virulence in bacterial bioremediation and decontamination efforts which involves treating the bacteria with an antibacterial phospholipid composition of the invention.

In still another aspect, the invention provides antibacterial topical compositions containing a non-metabolizable phospholipid in a suitable pharmaceutical carrier. The topical compositions of the invention may optionally also include other common ingredients used in the production of topical compositions and antibacterials. Similarly, the invention provides methods of preventing or treating topical infections by pathogenic bacteria which comprise the application of the antibacterial topical compositions of the invention to human or other animal skin.

In another aspect, the present invention provides immunogenic compositions comprising live, avirulent bacteria, wherein the bacteria are derived from a virulent, pathogenic bacterial strain but have been rendered avirulent by treatment with a non-metabolizable phospholipid. Preferably, this treatment comprises growing the bacterial pathogens in media containing non- metabolizable phospholipids at the concentrations described above. Similarly, the invention provides methods for protecting animals or humans against bacterial infection which comprises administering an effective amount of the immunogenic compositions of the invention to animals, including humans.

All of the above-described methods and compositions of the invention are particularly well suited to applications with bacteria selected from Pseudomonas. Escherichia. Salmonella. Klebsiella. Streptococci. Staphylococci. Hemophilus. Legionella. Clostridia. Shigella. Campylobacter and Listeria.

DETAILED DESCRIPTION OF THE INVENTION The present invention derives, in part, from the discovery that contacting normally virulent bacteria with a phospholipid which cannot be metabolized by the bacteria has the effect of substantially inhibiting their growth and, thereby, rendering them substantially avirulent. This highly surprising result, that non-metabolizable phospholipids can reduce or eliminate the virulence of otherwise pathogenic bacteria, provides new, safe, effective and economical means for preventing and/or treating bacterial infection and/or contamination. As is well known in the art, phospholipids are a ubiquitous component of biological membranes comprising two fatty acid chains esterified to the first and second hydroxyl groups of glycerol. The third hydroxyl group forms an ester linkage with a phosphate group which is also esterified to a polar alcoholic "head group." Although the fatty acid chains may vary widely, phospholipids are generally grouped into classes according to their head groups. Thus, the phosphatidylserines are phospholipids in which the phosphate group of a phosphatidic acid (i.e., a diacylglycerol phosphate) is esterified to the hydroxyl group of serine. Similarly, in the phosphatidylethanolamines, it is the hydroxyl group of ethanolamine that is esterified to the phosphate.

Although most, if not all, organisms include phospholipids in their structure, organisms vary widely both in their ability metabolize (i.e., catabolize) different phospholipids. These differences derive largely from the differing abilities of these organisms to metabolize phospholipids with particular fatty acid chains. Of particular importance, in this respect, is the number, positions, and conformations of double bonds in the fatty acid chains. Thus, although a particular phospholipid may serve as a food source for some organisms, it is well known that the same phospholipid may be non-metabolizable by some strains of bacteria. (See, e.g., Shaw, N. (1974) Advances in Applied Microbiology. 17:63-108; Ratledge, C. Cl 970) Chemistry and Industry. 26:834-854; Finnerty, W.R., and Makula, R.A. (1975) CRC Critical Reviews in Microbiology. 4(1): 1-40).

The present invention, as noted above, derives in part from the surprising discovery that phospholipids which are non-metabolizable by strains of pathogenic bacteria substantially inhibit their growth and render them avirulent. That is, non-metabolizable phospholipids not only fail to serve as a food source for these bacteria but, in addition, inhibit their growth and reduce their virulence even when alternative metabolizable food sources are present in abundance. This is surprising because, should one nutrient become limiting or unavailable, one would expect that the bacteria would "switch" to the utilization of another. This switching mechanism appears to be inhibited if the bacteria encounter one of the non-metabolizable phospholipids of the present invention.

As a result of this discovery, the present invention provides new uses for such non- metabolizable phospholipids, compositions containing such phospholipids for use in the prevention and treatment of bacterial infections and contamination, and new immunogenic compositions including normally virulent bacteria which have been rendered substantially avirulent by such non-metabolizable phospholipid compositions. I. Definitions

As used herein, the term "antibacterial phospholipid composition" refers to a composition containing a phospholipid which, solely by virtue of its constituent fatty acid chains, is non- metabolizable by a pathogenic bacterium. Such a composition is useful in treating virulent pathogens to reduce their virulence, among other uses described in more detail below. In particular, such compositions may be useful in treating infections or preventing contamination by pathogenic bacteria of the genuses Pseudomonas. Escherichia. Salmonella. Klebsiella. Streptococci. Staphylococci. Hemophilus. Legionella. Clostridia. Shigella. Campylobacter and Listeria. It must be emphasized that, even when a fatty acid is metabolizable in its free form, a phospholipid containing that fatty acid may be non-metabolizable (see Examples below). Therefore, a "non-metabolizable phospholipid" may include fatty acyl chains which, when present as free acids or in other moieties, are readily metabolized.

An antibacterial phospholipid composition is one which, when present as the sole carbon source in a minimal essential medium (e.g., phospholipid in HEPES-Hanks buffer) at a concentration of lmg/ml, will not support significant growth of a pathogenic bacterial strain inoculated into the medium. That is, an antibacterial phospholipid composition is one which, when used in culture as described above, will result in a decrease in the titer of the pathogenic bacteria or an increase in titer of less than one order of magnitude over an extended period (e.g., 18 hours) when maintained at optimal growth temperatures for the pathogen in question.

An "agricultural wash solution" is a solution containing at least 0.0001%, and preferably at least 0.001% or 0.01%, by weight of an antibacterial phospholipid composition of the invention. Alternatively, an "agricultural wash solution" is a solution containing at least lOμg/ml, and preferably, at least lOOμg/ml or 500μg/ml, of an antibacterial phospholipid composition of the present invention. The antibacterial phospholipid composition may be dissolved, suspended, or dispersed in any pharmaceutically acceptable carrier. The solution may also optionally contain detergents and other antimicrobial compositions. Such solutions may be used in disinfecting or decontaminating meat and poultry; the hands or clothing of workers in meat and poultry processing operations; the machinery, utensils and surfaces in meat and poultry processing; feed for livestock including meat and poultry; and air-conditioning or ventilation systems.

An "immunogenic composition" is a composition containing at least one pathogen which has been rendered substantially avirulent following treatment with an antibacterial phospholipid composition of the invention and which composition is capable of eliciting an immune response against the pathogen contained therein. If the immune response is protective, this composition may be useful as a vaccine. If the immune response is less than protective, the composition may be useful as an adjuvant for other vaccines, or to enhance therapeutic treatment of such bacterial infections.

II. Antibacterial Phospholipid Compositions

The present invention provides antimicrobial compositions including non-metabolizable phospholipids. These phospholipids are non-metabolizable because of the presence of one or more fatty acid chains which render them non-metabolizable or, more precisely, non- catabolizable, by one or more strains of pathogenic bacteria. The phospholipid compositions of the invention may include a single non-metabolizable phospholipid or, optionally, may contain a mixture of these phospholipids so as to be more effective against a single pathogenic bacterial strain or so as to be effective against multiple strains of pathogenic bacteria. The phospholipids are preferably chosen from the classes of phosphatidylserines, phosphatidylcholines, phosphatidylethanolamines, phosphatidylinositols and phosphatidylglycerols. Other lipids may also be employed, including those chosen from the sphingomyelins, gangliosides, and ceramides. One of skill in the art can readily select or synthesize suitable fatty acid chains for use in the antibacterial phospholipids of the present invention. Particular attention is directed to the number, placement and conformation of double bonds in these fatty acid chains. The fatty acid chains may contain from 12 to 36 but, more preferably, from 14 to 18 carbon atoms with molecular weights ranging from 200-550 but, more preferably, 200-300. As noted above, unsaturated fatty acids and unsaturated bonds are particularly useful in that the number, placement and conformation of such bonds play a critical role in the ability of organisms to metabolize fatty acids and phospholipids containing them. Generally, phospholipids with substituted fatty acid chains may be synthesized using standard organic synthesis techniques. See, for example, N. E. Bednarcyk and W. L. Erikson, Fatty Acid Synthesis and Applications. Chemical Technology Series No. 9. Noyes Corporation, Park Ridge, NJ (1973), incorporated by reference herein. The phospholipids may be diluted in a physiological buffer such as, but not limited to, phosphate buffered saline or HEPES-Hanks buffer, and treated, e.g., by heat or sonication, to obtain solubility. Synthetic compounds are constructed to remain soluble at physiologic pH and temperature, in the range of pH 5 to 8.5 and 32 °C to 40 °C. The non-metabolizable phospholipids of the present invention may also be isolated and purified from biological sources. As noted above, for example, phospholipids are major components of biological membranes. When obtaining a preparation of a non-metabolizable phospholipid from such a source, it should be noted that the source tissue is likely to include a variety of phospholipids and that some of these may be metabolizable. To the extent that these other phospholipids may be metabolized by the pathogen of interest, they will support and promote the growth of the pathogen and, therefore, diminish the efficacy of the phospholipid preparation.

In preferred embodiments, the non-metabolizable phospholipids comprise at least 50% by weight of the total phospholipids in the antibacterial compositions of the invention. Added purification of the non-metabolizable phospholipids may, of course, entail increasing cost and, therefore, a practical trade-off may be made between increased cost and increased efficacy. Thus, in more preferred embodiments, the non-metabolizable phospholipids may comprise at least 75%, 85% or 95% by weight of the total phospholipids in the composition. In the absence of any countervailing concerns, of course, it is preferred that the antibacterial phospholipids of the present invention be essentially free of metabolizable phospholipids.

As described in the Examples below, non-metabolizable phospholipids for use in the present invention may be easily identified by one of ordinary skill in the art for any particular strain of pathogenic bacteria which is sought to be controlled.

A panel of phospholipids can be assembled in, for example, a 96 well microtiter plate. The phospholipids may be diluted to lOOμg/ml and sonicated into a standard medium such as Davis broth. This media is then dispensed into the microtiter plate and inoculated with a fixed titer (e.g., 1 X 10⁴ CFU/ml of the experimental bacteria. The microtiter plate is then incubated at an appropriate temperature for a fixed period. After the incubation period, the growth of the bacteria may be assayed using, for example, a spectrophotometer or viable cell counts. Thus, a large number of phospholipids can be rapidly and simultaneously tested for antibacterial utility. Those phospholipids which are found to inhibit bacterial growth may then be subjected to additional tests, including minimal inhibitory concentration assays and the in vivo virulence and challenge studies described below (see Examples 2 and 3).

As shown in Table 1 , P. aeruginosa and S. typhimurium are capable of growing in buffered phospholipid solutions containing a variety of phospholipids, using these phospholipids as a food source. Some phospholipids, however, failed to support or only minimally supported bacterial growth. These non-metabolizable phospholipids, it should be noted, cannot be described only in terms of their head groups or only in terms of their fatty acyl chains. Rather, particular combinations of the head and tail groups must be specified. Thus, for example, a bacterium may grow well in some phosphatidylserines but not in others. Similarly, a bacterium may grow well in some di-palmitoyl phospholipids but not in others. Finally, as Table 1 shows, a phospholipid which is an effective antibacterial composition against one bacterial strain may be ineffective against another.

For example, the growth of Pseudomonas aeruginosa may be substantially inhibited and its virulence substantially abated by growing the bacteria in, or contacting them with, media or solutions containing the phospholipids l-oleoyl-2-palmitoyl-phosphatidylserine and/or di- palmitoyl-phosphatidylserine. In contrast, other phosphatidylserines, such as di-oleoyl and di- myristoyl-phosphatidylserine not only failed to inhibit these bacteria but, rather, supported their growth nearly as well as Luria broth. Similarly, other l-oleoyl-2-palmitoyl or di-palmitoyl phospholipids, such as the corresponding phosphatidylinositols, were ineffective in inhibiting growth. (See Example 1, Table 1.) For the Salmonella species, the same phospholipids, l-oleoyl-2-palmitoyl and di- palmitoyl-phosphatidylserine, were found to be highly effective antibacterial phospholipid compositions. In addition, the l-oleoyl-2-palmitoyl and di-palmitoyl varieties of phosphatidylinositol, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol were also found to be highly effective. The N-palmitoyl varieties of sphingomyelin and ceramide were also found to be effective, as was l-linoleoyl-2-palmitoyl-phosphatidylcholine. (See Example 1 , Table 1.) For Escherichia coli. the phospholipid di-palmitoyl phosphatidylserine was again found to be highly effective as an antibacterial phospholipid composition. (See Example 4, Table 2.)

Again, it should be emphasized that no particular class of phospholipids designated solely by "head" group was found to be effective. Thus, in contrast to the teachings of the prior art cited above, one cannot say that phosphatidylserines, phosphatidylethanolamines or phosphatidylcholines have general utility. Further, one must be wary lest a mixture of phospholipids is used in which a majority or large fraction of the phospholipids is fully metabolizable and actually serves to support and promote the growth of the pathogens. III. Agricultural Wash Solutions The agricultural wash solutions of the present invention may be prepared by admixing an antibacterial phospholipid composition of the invention in a suitable carrier. The antibacterial phospholipids are chosen on the basis of their ability to reduce the virulence or inhibit the growth of one or more pathogenic bacteria of interest. Thus, for example, in the poultry industry, an agricultural wash solution targeted to S. tvphimurium may be of most interest whereas in beef processing the targeted pathogen may be E. coli. The agricultural wash solutions are prepared by mixing one or more of the non- metabolizable phospholipids with a diluent, such as a saline based buffer, to a final concentration of 10-500 μg/ml of the antibacterial phospholipids. Other types of diluents may be readily selected by one of skill in the art. In addition, other common agricultural wash components may be optionally added, including detergents, dyes or preservatives. The agricultural wash solutions of the present invention may be used to decontaminate meat or fowl during processing. This may be accomplished by washing the meat to be processed in a volume of the agricultural wash solution sufficient to submerse the meat or fowl. Alternatively, the meat or poultry may be rinsed or sprayed with the agricultural wash solution. Desirably, the amount of the non-metabolizable phospholipid in the wash solution is about 10 to 500 μg/ml. The meat or fowl are desirably sprayed with or rinsed or immersed in the wash solution for a sufficient length of time to completely wet the tissue. The wash may be used at an elevated temperature, preferably at least 37°, including temperatures up to 100°C. Lower temperatures, including temperatures as low as 4°C, may also be employed. The amounts, times, and temperatures may be varied by one of skill in the art in the use of such agricultural solutions.

The agricultural wash solutions are also used to disinfect the hands and clothing of workers as well as the machinery, utensils and surfaces used in processing meat or fowl. This is accomplished by rinsing with a sufficient amount of the wash solutions to wet the hands, clothing, machinery, etc. Again, temperatures of at least 37 °C are preferred, although temperatures as high as 100° C and as low as 4°C may also be employed. In a preferred embodiment, the wash solutions of the invention are applied by aerosol or pressurized sprays. Finally, the agricultural wash solutions of the present invention may be used to treat or rinse feed for livestock and poultry. Because pathogenic bacteria may infect these animals through ingestion of contaminated feed, the agricultural wash solutions may be used to reduce contamination and infection by soaking or rinsing the feed (e.g. grain) prior to feeding. This is particularly beneficial in that it may reduce the need for antibiotic treatments of livestock which, in addition to being costly, increase the selective pressures for the evolution of resistant strains. Furthermore, as infections by pathogenic bacteria in these animals are reduced, the potential for transmission of these pathogens to human consumers of the processed meat and poultry is reduced.

The food additives of the invention are made by the same process as the agricultural washes, absent the addition of a detergent. The non-metabolizable phospholipid compositions are added directly to foodstuffs at concentrations ranging from 10-lOOg/kg feed. This concentration range is anticipated to inhibit a wide range of pathogenic bacteria. IV. Reduction of Virulent Bacteria in Ventilation Systems

The antibacterial phospholipid compositions of the present invention may also be used to reduce the levels of contamination and/or virulence of pathogenic bacteria in air conditioning and ventilation system. Of particular interest are antibacterial phospholipid compositions targeted at the bacteria most commonly found in these systems, including Legionella. The bacteria present in these systems may be rendered avirulent upon introduction of an effective amount of the antibacterial phospholipid compositions described above. This may be accomplished by adding an effective amount of the compositions into these systems where bacteria exist. Spraying the non-metabolizable phospholipid compositions in the form of an aerosol is a particularly effective mode of introducing the compositions into air ducts or recessed portions of these systems. Alternatively, in systems with fluid reservoirs, the antibacterial phospholipids may be added directly to the reservoirs in the form of a solution or dissolvable solid. In a preferred embodiment, the antibacterial phospholipid compositions are contained in a suitable diluent, such as a saline-based buffer. Desirably, between 10 and 500 μg/ml of reagent is added to the air conditioning and ventilation systems. Such treatment is desirably repeated periodically. V. Reduction of Virulence of Bacteria Used in Bioremediation and Agriculture

The antibacterial phospholipid compositions of the invention may also be employed to reduce virulence in bioremediation and decontamination procedures in which bacteria, such as Pseudomonas species, are used. In addition, the antibacterial phospholipid compositions of the invention may be applied in agriculture, for example, to reduce virulence in the bacteria used to combat frost damage in citrus groves.

In bioremediation and decontamination efforts, species such as Pseudomonas aeruginosa have been used to degrade petroleum and polychlorinated biphenyls ("PCBs") upon their introduction into an environment polluted by these contaminants. In one aspect of the present invention, prior to their application, bacteria used in bioremediation or decontamination are treated with a non-metabolizable phospholipid composition. This treatment consists simply of exposing the bacteria to, or contacting them with, one of the antibacterial phospholipid compositions. This may be achieved by growing the bacteria in a culture containing one of these phospholipids as, for example, in the same manner described for the treated bacteria in Examples 2 and 3. Following treatment, the bacteria are applied in the same manner as are conventional bacteria for bioremediation and decontamination purposes. Alternatively, untreated bacteria may be initially introduced into a site, such as an oil spill, and the non-metabolizable phospholipids of the invention may subsequently be added to the same site to reduce the virulence or inhibit the spread of these bacteria. The bacteria may, for example, be introduced in untreated form, allowed to substantially remediate or decontaminate the site, and then be treated with the compositions of the invention to reduce their virulence and/or inhibit their further growth. The treatment of these bacteria does not effect their bioremediation or decontamination properties, yet it does render the bacteria avirulent, thus preventing deleterious effects observed in the animal populations upon introduction of virulent bioremedial bacteria into the contaminated environment. Bacteria have also been used as an ice crystal nucleation catalyst to protect citrus trees and other plants from damage associated with frost. These bacteria can be similarly treated with the compositions of the present invention to render them substantially avirulent and/or inhibit their growth. Again, the method of treatment simply entails growing the bacteria in, or otherwise contacting them with, a solution containing at least one of the non-metabolizable phospholipids of the present invention at the concentrations described above. Thus, as an example only, the bacteria might be treated according to the protocol set forth for the treatment group of Example 2. Following treatment, the bacteria are applied in the same manner as are conventional bacteria for frost protection. Alternatively, untreated bacteria may be applied as in the prior art, and then they may be contacted with the antibacterial phospholipids of the present invention by, for example, using a spray of one of the agricultural wash solutions (without detergent) described above. One of skill in this art can readily select the amount, doses and methods of such application.

V. Topical Compositions

The present invention also encompasses topical compositions or medicaments which may be prepared by combining an antibacterial phospholipid composition of the invention with a suitable carrier. The antibacterial phospholipid composition may be admixed with pharmaceutically acceptable carrier components, such as ointments, creams, salves, jellies, pastes and lotions, to a final concentration of 10-500 μg/ml of phospholipid. Suitable carriers are well known to those of skill in the art and include water, petroleum, saline, mineral oils, alcohols, and the like. Other types of carriers may be readily selected by one of skill in the art. Other useful components of such a topical composition may include skin penetration agents, perfumes, thickening agents, stabilizers, surfactants, coloring agents, and the like. One of skill in the art can readily select desired components from those conventionally used. Optionally, other common topically active ingredients may be added, including, for example, commonly used antibiotics and anesthetics.

The topical medicaments of the present invention may be administered as needed to animals, including humans, to prevent or treat bacterial infection. These topical medicaments may be directly applied to the desired areas of the skin surface. A preferred use of the topical medicament is to apply the composition directly to an area of the skin suffering from burn trauma.

VT. Immunogenic Compositions An immunogenic composition according to this invention is prepared by treating a live strain of normally virulent, pathogenic bacteria with an antibacterial phospholipid composition of the invention. In preferred embodiments, the bacterial strain used in such immunogenic compositions is chosen from the genuses Pseudomonas. Escherichia. Salmonella. Klebsiella. Streptococci. Staphylococci. Hemophilus. Legionella. Clostridia. Shigella. Campylobacter and Listeria. However, one of skill in the art may select other appropriate bacteria for the production of the immunogenic compositions of this invention. The bacterial strains useful in preparing the immunogenic composition of the present invention can be obtained from commercial or academic collections, or depositories such as the American Type Culture Collection in Rockville, Maryland, U.S.A., or isolated using known techniques.

An immunogenic composition according to this invention may be prepared by inoculating an appropriate growth medium with the selected bacteria. A selected culture medium may include any media known to those of skill in the art to propagate the particular bacterial strain used in the vaccine composition. For example, a particularly desirable medium formulation for culturing Salmonella is Luria broth.

The medium is inoculated with a suspension of the selected bacteria strain, at preferably a 5-20% inoculation. The culture is thereafter incubated at a temperature between 30°C and 40°C. A more preferred temperature range is 35 °C to 38 °C, with 37°C being particularly preferred. The incubation typically involves aerobic shaking of the culture.

After inoculation of the medium with the particular bacteria strain to be used in the immunogenic composition, the selected bacteria are treated or contacted with an antibacterial phospholipid composition of the invention. In experiments, such "treated" bacteria are compared to controls which are not treated prior to infection of a host or patient.

For example, as described in Example 2, di-palmitoyl-phosphatidylserine in PBS may be added to Pseudomonas aeruginosa medium, and the bacteria may then be incubated at about 4°C for about one hour. Bacteria are then isolated by centrifugation and resuspended in the phospholipid in the late growth or early stationary phase. Additional non-metabolizable phospholipid may then be added to the medium to a final concentration ranging from 10-500 μg/ml.

An immunogenic amount of these treated, avirulent bacteria is formulated in a pharmaceutically acceptable carrier for introduction into an animal. An immunogenic amount of these treated bacteria is desirably between about 0.1 μg to about 1000 mg bacteria per ml. Alternatively, the dose units can be determined in colony forming units (CFU). When so measured, the immunogenic compositions of the invention desirably contain between about 1 X 10⁴ to about 1 X 10⁸ colony forming units (CFU) of a desired, treated bacteria. These doses preferably provide a sufficient amount of avirulent bacteria to compete for required nutrients with virulent bacteria which have already infected the host, thereby acting to prevent or ameliorate infection by virulent pathogens. Hence, the immunogenic compositions of the invention aid in the prevention or retardation of infection by competing with virulent bacteria. These compositions further function to elicit an immunogenic response to the bacteria. Thus, such compositions may be employed to prevent infection, i.e., as vaccines, or to treat infections by retarding the growth and activity of virulent bacteria in an infected animal.

Generally, a suitable dosage unit is between about 1 ml to about 5 ml, depending upon the size, weight, physical condition and species of the animal to which the vaccine is to be administered. Examples of suitable dosage forms include tablets, capsules, syrups, liquids, and aerosols. Other suitable dosage forms can be readily selected by one of skill in the art. Similarly, one of skill in the art can readily determine the number of doses which an animal requires, e.g. a single administration followed by an annual or biannual booster.

The immunogenic or therapeutic compositions of the invention may be formulated to contain other optional components, e.g. carriers, adjuvants, excipients, and the like, currently, the preferred carrier is phosphate buffered saline. However, other such components are well known to those of skill in the art and can be readily selected.

Similarly, if so desired, the compositions of the invention may be formulated to contain other active ingredients, including, e.g. other vaccine or therapeutic agents suitable for prevention or treatment, respectively, of the selected bacterial infection.

The immunogenic compositions of the invention may be administered in a variety of ways which will elicit a therapeutic or immune response, including but not limited to intranasal and oral inoculation. The immunogenic compositions are preferably administered intranasally by diluting the compositions with phosphate buffered saline. Other modes of administration may be employed, where desired, such as subcutaneous, intradermal, intraperitoneal, intramuscular, or intubational. Alternatively, the immunogenic compositions can be admixed for oral administration, e.g. as a food premix for animal feed.

Particularly, because many bacteria have a common mechanism of pathogenesis, e.g., respiratory and gastrointestinal tract infection, it is expected that treatment of the animal with the compositions of this invention will aide in the prevention or treatment of infection by several bacteria including but not limited to Pseudomonas. Escherichia. Salmonella. Klebsiella. Streptococci. Staphylococci. Hemophilus. Legionella. Clostridia. Shigella. Campylobacter and Listeria.

The foregoing description of the invention includes many specific examples which are intended to be illustrative but not limiting of the scope of the invention enabled by the description provided herein. In addition, the following data from experiments conducted according to the present invention, are presented as illustrative of the utility of the invention and are not intended to limit its scope.

EXAMPLE 1 In vitro Assays for Antibacterial Phospholipid Compositions Synthetic phospholipids were obtained from Avanti Polar Lipids, Alabaster, AL. Naturally occurring phospholipids were obtained from Sigma Chemical Co., St. Louis, MO. All phospholipids were stored in chloroform at -20 °C until used. Upon use, the phospholipid samples were diluted 1 : 1 with methanol and an aliquot was removed from the storage tube with a glass pipette or syringe to limit the oxidation from exposure to air. The chloroform/methanol was blown off with a stream of nitrogen and the phospholipids were sonicated into HEPES-Hanks buffer.

A panel of phospholipids was assembled for testing against the bacterial pathogens Pseudomonas aeruginosa and Salmonella typhimurium. Each of the phospholipids was sonicated into HEPES/Hanks buffer at a concentration of 1 mg/ml. Free fatty acids in HEPES/Hanks buffer and plain Luria broth were used as controls. The phospholipid solutions were then dispensed into a 96 well microtiter plate and inoculated with either 2 X 10⁴ of S. typhimurium or 1 X 10⁴ P. aeruginosa. The plates were incubated for 18 hours at 37°C and then bacterial growth was determined. The lipids employed and results are presented in Table 1.

EXAMPLE 2 In vivo Virulence Studies with Pseudomonas aeruginosa The virulence of P. aeruginosa treated according to the invention was compared to the virulence of wild-type P. aeruginosa. Pseudomonas aeruginosa strain AC869 as well as the other Pseudomonas strains used were obtained from Health Effects Research Laboratory, U. S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711.

In the positive control group, BALB/c and CD-I mice (Charles River Laboratories) were administered wild-type Pseudomonas aeruginosa AC869 at doses ranging from 1 X 10⁴ to 5 X 10⁸ CFU intranasally in phosphate buffered saline (PBS). Within seven days, an LD₅₀ of 1 X 10⁵ was calculated by the method of Reed and Muench, American J. Hygiene. 27:493-497 (1938). In the first experimental group, Pseudomonas aeruginosa AC869 was treated with 500 μg/ml di-palmitoyl-phosphatidylserine in PBS for one hour at 4°C according to the invention. Balb/c and CD-I mice were then administered doses similar to those administered the control group. The LD₅₀ of this experimental group was elevated to levels greater than 10⁹ colony forming units (CFU).

In a second experimental group, Pseudomonas aeruginosa AC869 were treated with 500 μg/ml l-oleoyl-2-palmitoyl-phosphatidylserine in PBS for about one hour at 4°C. The Balb/c and CD-I mice were then exposed to the treated organism as for the above groups. Results similar to the other experimental group were obtained - i.e. LD₅₀ was elevated to levels greater than 10⁹ CFU. Autopsy of the mice in the positive control group revealed gross inflammation and fluid accumulation associated with Pseudomonas pneumonia, while the two experimental groups displayed normal morphology with no signs of pneumonia.

EXAMPLE 3 In vivo Challenge Studies with Pseudomonas aeruginosa Mice which were initially infected with treated 1 X 10⁸ CFU Pseudomonas aeruginosa. as described above, were intranasally challenged with 1 X 10⁷ CFU untreated (wild-type) fully virulent AC869 or a clinical isolate from a human cystic fibrosis patient 15-60 days prior to initial infection. The isolate from the cystic fibrosis patient was chosen because Pseudomonas infection in children with cystic fibrosis is a common and severe complication of that disease. The challenge doses were administered to the mice 10 to 60 days after initial infection.

The mortality was considered for three weeks post infection and in control experiments was 100% as compared to 10% in both of the two experimental groups which had been infected with the treated bacteria prior to challenge by the virulent strain. The mice in the control group were dead within 15 days post infection. As an additional control, P. aeruginosa were treated with phospholipids which were shown to be metabolizable using the simple assay of Example 1 (See Table 1). For example, the bacteria were treated with 500 μg/ml ceramides or sphingomyelins, for about 1 hour at 4°C, according to the invention, and mice were then infected with these treated bacteria. Upon later infection with the wild-type, virulent strain, only 25% survived. These experiments reveal that di-palmitoyl-phosphatidylserine and l-oleoyl-2-palmitoyl- phosphatidylserine treated Pseudomonas aeruginosa AC869 demonstrated greatly reduced respiratory tract virulence. The surviving mice had a greatly increased resistance to re-infection with both the same and other strains of Pseudomonas.

EXAMPLE 4 In vitro Inhibition of Salmonella. Escherichia and Pseudomonas In another series of experiments, the ability of Salmonella typhimurium LT2, Escherichia coli 0157:H7, and Pseudomonas aeruginosa AC869 to grow in vitro on murine intestinal mucosa was investigated in the presence or absence of an antibacterial phospholipid of the invention.

Samples were prepared of lmg/ml CD1 murine intestinal mucosa either with or without the addition of lOOμg/ml of sonicated di-palmitoyl phosphatidylserine. The samples were inoculated with 2.3 X 10⁴ CFU S. typhimurium LT2, 1.7 X 10⁴ CFU E. coli 0157:H7, or 2.1 X 10⁴ CFU P. aeruginosa AC869. The samples were incubated for 18 hours at 37°C and then viable counts were conducted on Luria agar.

The results of these experiments, presented in Table 2, indicate that the phospholipid di- palmitoyl-phosphatidylserine is capable of significantly inhibiting the growth of each of these pathogens.

TABLE 1

Pseudomonas Salmonella aeruginosa typhimurium

Lipid growth growth

Luria broth 2.5 X 10⁹ 1.6 X10⁹

Cardiolipin-bovine brain 3.7 X10⁷ 7.3 X 10"

Sphingomyelin N-stearoyl 3.8 X10⁷ 4.6 X10⁴ N-oleoyl 3.2 X10⁷ 2.6 X10⁵ N-palmitoyl 7.4 X10⁷ 5.2 X10³

Gangliosides monosialoganglioside 2.2 X 10⁷ 2.6 X10⁵ disialoganglioside 5.3 X 10⁷ 1.8 X10⁵ asialoganglioside 2.9 X10⁷ 5.5 X10⁴

Ceramide N-palmitoyl 3.3 X 10⁶ 2.5 X 10³ N-oleoyl 6.4 X10⁷ 2.8 X 10⁵ N-nervonoyl 7.2 X10⁷ 3.5 X10⁵

Cerebroside N-palmitoyl 9.9 X10⁶ 4.3 X 10⁴ N-oleoyl 3.4 X10⁷ 1.8 X10⁶ N-stearoyl-dihydrogalacto 7.1 X 10⁷ 8.6 X10⁵

Phosphatidylinositol di-palmitoyl 2.8 X 10⁷ 4.5 X 10³ di-oleoyl 2.5 X 10⁷ 3.6 X10⁶

1-oleoyl 2-palmitoyl 4.7 X10⁷ 8.1 X 10³ di-myristoyl 7.3 X 10⁷ 2.6 X10⁷

Phosphatidylethanolamine di-palmitoyl 2.2 X 10⁶ 2.3 X 10³ di-oleoyl 3.6 X10⁸ 2.8 X10⁶

1-oleoyl 2-palmitoyl 1.0 X10⁷ 2.9 X10³ di-myristoyl 2.8 X10⁸ 6.6 X10⁶ TABLE 1 Continued

Pseudomonas Salmonella aeruginosa typhimurium

Lipid growth growth

Phosphatidylserine di-palmitoyl 5.8 X10² 2.2 X10² di-oleoyl 4.2 X10⁸ 4.8 X10⁷

1-oleoyl 2-palmitoyl 2.3 X 10³ 5.2 X10² di-myristoyl 1.8 X10⁸ 8.6 X10⁷

Phosphatidylcholine di-palmitoyl 8.9 X10⁵ 2.5 X 10³ di-oleoyl 9.2 X10⁷ 5.6 X10⁶

1-oleoyl 2-palmitoyl 7.2 X 10" 3.4 X10³ di-myristoyl 1.3 X10⁷ 4.5 X 10⁶ di-stearoyl 6.1 X 10⁶ 5.6 X10⁵ 1-linoleoyl 2-palmitoyl 2.1 X10⁵ 2.3 X 10³

Phosphatidylglycerol di-palmitoyl 5.8 X10⁶ 2.7 X10³ di-oleoyl 2.0 X 10⁷ 5.6 X10⁵

1-oleoyl 2-palmitoyl 1.9 X10⁶ 6.5 X 10³ di-myristoyl 3.1 X10⁸ 4.6 X10⁶

Myristic Acid 6.7 X10⁷ 7.4 X 10⁶ Palmitic Acid 3.4 X 10⁷ 6.3 X 10⁶ Oleic Acid 6.9 X10⁷ 6.9 X10⁶ Linoleic Acid 2.7 X10⁷ 3.9 X10⁶ Linolenic Acid 5.2 X10⁷ 1.4 X10⁵ Arachidonic Acid 7.8 X 10⁷ 4.7 X10⁶

TABLE 2 log₁₀ increase CFU log₁₀ increase CFU

Bacterial Strain 1 mg/ml CD1 Mucus 1 mg/ml CD1 Mucus with 100 μg/ml PS

S. typhimurium LT2 3.78 0.58

E. coli 0157:H7 3.14 0.57

P. aeruginosa AC869 4.57 0.89

Claims

1. An agricultural wash solution for reducing the virulence or inhibiting the growth of a bacterial pathogen comprising an antibacterial phospholipid composition including at least one phospholipid which is non-metabolizable by said bacterial pathogen; and a carrier solution.

2. An agricultural wash solution as in claim 1 wherein said non-metabolizable phospholipid is present in said wash at a concentration of at least lOμg/ml.

3. An agricultural wash solution as in claim 1 wherein said non-metabolizable phospholipid is present in said wash at a concentration of at least lOOμg/ml.

4. An agricultural wash solution as in claim 1 wherein said non-metabolizable phospholipid is present in said wash at a concentration of at least 500μg/ml.

5. An agricultural wash solution as in any one of claims 1-4 wherein said wash includes phospholipids which are metabolizable by said bacterial pathogen; said metabolizable phospholipids and said non-metabolizable phospholipids have a total weight; and said metabolizable phospholipids comprise less than 50% of said total weight.

6. An agricultural wash solution as in any one of claims 1-4 wherein said wash includes phospholipids which are metabolizable by said bacterial pathogen; said metabolizable phospholipids and said non-metabolizable phospholipids have a total weight; and said metabolizable phospholipids comprise less than 25% of said total weight.

7. An agricultural wash solution as in any one of claims 1-4 wherein said wash includes phospholipids which are metabolizable by said bacterial pathogen; said metabolizable phospholipids and said non-metabolizable phospholipids have a total weight; and said metabolizable phospholipids comprise less than 15% of said total weight.

8. An agricultural wash solution as in any one of claims 1-4 wherein said wash includes phospholipids which are metabolizable by said bacterial pathogen; said metabolizable phospholipids and said non-metabolizable phospholipids have a total weight; and said metabolizable phospholipids comprise less than 5% of said total weight.

9. An agricultural wash solution as in any one of claims 1-4 wherein said wash is essentially free of phospholipids which are metabolizable by said bacterial pathogen.

10. An agricultural wash solution as in any one of claims 1-9 wherein said bacterial pathogen is of a genus selected from the group consisting of Pseudomonas. Escherichia. Salmonella. Klebsiella. Streptococci. Staphylococci. Hemophilus. Legionella. Clostridia. Shigella. Campylobacter and Listeria.

11. An agricultural wash solution wherein as in any one of claims 1 -9 wherein said non-metabolizable phospholipid is selected from the group consisting of di- palmitoyl phosphatidylserine and l-oleoyl-2-palmitoyl phosphatidylserine.

12. An agricultural wash solution as in any one of claims 1-9 wherein said bacterial pathogen is a Pseudomonas aeruginosa and said non-metabolizable phospholipid is selected from the group consisting of di-palmitoyl phosphatidylserine and 1 -oleoyl-2-palmitoyl phosphatidylserine.

13. An agricultural wash solution as in any one of claims 1-9 wherein said bacterial pathogen is a Salmonella typhimurium and said non-metabolizable phospholipid is selected from the group consisting of di-palmitoyl phosphatidylserine, 1- oleoyl-2-palmitoyl phosphatidylserine, di-palmitoyl phosphatidylinositol, l-oleoyl-2- palmitoyl phosphatidylinositol, di-palmitoyl phosphatidylethanolamine, l-oleoyl-2- palmitoyl phosphatidylethanolamine, di-palmitoyl phosphatidylglycerol, 1 -oleoyl-2- palmitoyl phosphatidylglycerol, di-palmitoyl phosphatidylcholine, l-oleoyl-2-palmitoyl phosphatidylcholine, and l-linoleoyl-2-palmitoyl phosphatidylcholine.

14. An agricultural wash solution as in any one of claims 1-9 wherein said bacterial pathogen is an Escherichia coli and said non-metabolizable phospholipid is di-palmitoyl phosphatidylserine.

15. A method of disinfecting or decontaminating meat or poultry, or workers, machinery, utensils and surfaces employed in the processing of meat or poultry, comprising contacting said meat or poultry, or workers, machinery, utensils and surfaces employed in the processing of said meat or poultry, with an agricultural wash solution of any one of claims 1-14.

16. A method as in claim 15 wherein said agricultural wash solution is heated to a temperature of at least 37°C.

17. A method of reducing infections in livestock and poultry, wherein said livestock and poultry are provided with a feed, comprising contacting said feed with an agricultural wash solution of any one of claims 1-14.

18. A method as in claim 17 wherein said feed is contacted with an amount of said non-metabolizable phospholipid equivalent to at least lOg/kg of said feed.

19. A method as in claim 17 wherein said feed is contacted with an amount of said non-metabolizable phospholipid equivalent to at least lOOg/kg of said feed.

20. A method or reducing the virulence or inhibiting the growth of bacterial pathogens in air conditioning or ventilation systems comprising, introducing within said air conditioning or ventilation systems an agricultural wash solution of any one of claims 1-14.

21. A method as in claim 20 wherein said bacterial pathogen is a Legionella species.

22. A method of reducing the virulence or inhibiting the growth of a bacterial pathogen used in bioremediation, decontamination or agriculture, wherein said bacterial pathogen is released into the environment, comprising contacting said bacterial pathogen with an agricultural wash solution of any one of claims 1-14.

23. A method as in claim 22 wherein said contact comprises culturing said bacterial pathogen is said solution.

24. A topical composition for reducing the virulence and or inhibiting the growth of a bacterial pathogen comprising an agricultural wash solution of any one of claims 1-14 and a pharmaceutically acceptable carrier for topical application.

25. A method of treating infections of the skin by bacterial pathogens comprising topically applying an effective amount of the topical composition of claim 24.

26. An immunogenic composition for immunizing against a bacterial pathogen comprising a live strain of said bacterial pathogen which has been contacted with an antibacterial phospholipid composition, said antibacterial phospholipid composition comprising at least one phospholipid which is non-metabolizable by said bacterial pathogen and a pharmaceutically acceptable carrier.

27. A method of making an immunogenic composition for immunizing against a bacterial pathogen comprising the step of contacting a live strain of said bacterial pathogen with an antibacterial composition comprising at least one phospholipid which is non-metabolizable by said bacterial pathogen and combining said contacted bacterial pathogens with a pharmaceutically acceptable carrier.