CA2559662A1 - Novel antibiotic alternatives - Google Patents

Abstract

The present invention relates to two recombinant colicin expression systems, one utilizing a yeast expression system that produces a protein that is inexpensive to purify, and the other utilizing a plasmid expression system to be used as a probiotic culture. The recombinant colicins provide effective alternatives to conventional antibiotics and may be used to improve the efficiency of pork production, and the safety of its products.

Description

TITLE: NOVEL ANTIBIOTIC ALTERNATIVES
BACKGROUND OF THE INVENTION
It is estimated that over 50% of all economic losses in weaned pigs are due to Esclzez°ichia coli infections, causing either diarrhea or edema disease. In addition to the E.
coli strains responsible for disease in pigs, other E. coli, as well as Salmonella, strains also colonize the intestinal tract of pigs. Many of these strains are of major concern for human food safety. The U.S. Centers for Disease Control and Prevention (CDC) estimates that in to the year 2000 over 1.4 million people suffered and more than 600 died, in this country, from foodborne disease caused by Salmozzella and E. coli 0157:H7. The costs attributed to these diseases were approximately $3.1 billion.
The bacterial strains considered primarily responsible for E. coli infections in pigs, F4 (K88) and F18, are not well controlled by traditional prophylactic antibiotic treatments.
With worldwide concerns over the use of prophylactic antibiotics in animal agriculture, the development of alternatives to conventional antibiotics is urgently needed to protect swine from these E. coli infections.
Probiotics have been explored as one alternative to the use of conventional antibiotics. A "probiotic" strategy is one that employs the use of microflora to reduce 2o pathogenic bacteria (including food-bourne pathogens) in the gut. Probiotic techniques involve the introduction of a healthy microbial population to the gastrointestinal (GI) tract, or providing a limiting nutrient, sometimes termed a ''prebiotic", that allows an existing commensal microbial population to expand its role in the gastrointestinal tract. The addition of a non-pathogenic microbial culture to the intestinal tract of food animals in order to reduce colonization or decrease populations of pathogenic bacteria in the gastrointestinal tract is referred to as "competitive exclusion".
Competitive exclusion cultures may be composed of a single strain, several strains, or even several species of microorganisms. Depending on the stage of production, or more specifically, the maturity of the gut, the goal of this culture can be the exclusion of 3o pathogens from the naive gut of a neonatal animal, or the displacement of an already established pathogenic bacterial population.

Some bacteria produce antimicrobial protein compounds (traditional antibiotics, as well as bacteriocins, or colicins) in order to eliminate competitive bacteria, and have therefore shown additional promise for their use in competitive exclusion products. Protein antibiotics are attractive alternatives to conventional antibiotics used in animal feed, since they are not absorbed intact by the animal and, therefore, leave no antibiotic residues in the meat. Additionally, bacteriocins have the potential for very favorable regulatory status by the U.S. Food and Drug Administration. Nisin, a bacteriocin, is generally regarded as safe for use as a food additive for its antimicrobial properties. The possibility of an effective antibiotic alternative being regulated as a food additive, rather than as a new animal drug, is to further incentive for bacteriocin research among the animal health/feed industries.
Colicins are classified as either pore-forming or nuclease colicins based on their mode of bacteriocidal activity, and are further categorized based on their mode of membrane integration in sensitive bacteria. Members of both classes have been shown effective against gram-negative bacteria of concern for animal health and human food safety, such as E. coli and Salmonella strains, and therefore hold promise for use as alternatives to conventional antibiotics in animal diets.
Colicins are a class of bacteriocins produced by, and effective against E.
coli and closely related members of the family Efzte>"obactef~iaceae. Pore-forming colicins are between 387 and 626 amino acids in length, and provide their antibacterial effect by 2o crossing the outer membrane, spanning the periplasm, and inserting into the bacterial inner cell membrane to form voltage-dependent ion channels. The ion leakage caused by these channels uncouples energy expenditures from growth, causing death in cellular targeted bacteria. Nuclease colicins kill sensitive cells by non-specific degradation of DNA or specific cleavage of rRNA.
Shiga toxin producing E. coli strains, such as 0157:H7, which present serious human food safety concerns, have also been shown to be sensitive to colicins.
Doyle et al., U.S. Pat. No. 5,965,128, discloses the use of c~licin producing E. coli as probiotics in cattle to reduce E. coli 0157:H7 shedding. Further, Lyon et al., U.S. Pat. No.
5,549,895, discloses the use of naturally produced colicins for inhibiting E. coli 0157:H7 and other 3o Eschef~ichia species, as well as Slzigella species in food products, on carcasses, and on hard surfaces as a sanitizer.

Although colicins have shown potential as alternatives to conventional antibiotics in animal feed, it would not be cost effective to purify this protein from naturally occurring colicin producing E. coli strains, nor to include the levels of these bacteria necessary to obtain an antimicrobial effect in the feed.
It is therefore a primary obj ective of the present invention to produce alternatives to conventional antibiotics for use in the animal feed industry.
It is a further objective of the present invention to produce recombinant colicins in a yeast expression system.
It is a further objective of the present invention to produce recombinant colicins in a to plasmid expression system.
It is still a further objective of the present invention to produce recombinant colicins that are effective against pathogenic bacteria.
It is yet a further objective of the present invention to produce recombinant colicins that may be used as a probiotic culture.
15 It is yet a further objective of the present invention to produce recombinant colicins that are effective against E. coli and SalT~aoyaella strains of importance to human food safety.
It is a further objective of the present invention to produce recombinant colicins that are effective against pathogenic bacteria that is cost effective.
2o The method and means of accomplishing each of the above objectives as well as others will become apparent from the detailed description of the invention which follows hereafter.
SUMMARY OF THE INVENTION
The present invention provides a method for the production of colicins from a 25 recombinant organism whereby a suitable host organism is transformed with a transformation cassette comprising a gene encoding a pore-forming or nuclease colicin.
The transformed host organism is then cultured under suitable conditions, and the colicins recovered and purified. The invention further provides transformed hosts comprising expression cassettes capable of expressing colicins.
3o The suitable host organism used in the method is bacteria or yeast. The suitable host organism is more particularly selected from the group of genera consisting of Citrobactey~, Entef~obacter, Klebsiella, Ae~obacter, Lactobacillus, Aspe~gillus, Sacclaaromyces, Schizosaccharomyces, Picltia, Candida, HafZSenula, MethylobacteY, Escherichia, Salmonella, Bacillus, St~eptomyces and Pseudomonas.
The invention is also embodied in a transformed host cell comprising a gene encoding for a colicin, and a host cell transformed with the gene, whereby the transformed host produces colicin, whereby the host cell is preferably P. pastoris or E.
coli. The colicins of this invention have bactericidal activity against various strains of organisms that are of concern in animal health and food safety, including strains of E. coli and Salmonella.
DETAILED DESCRIPTION OF THE DRAWINGS
1o FIG. 1 illustrates the effect of Colicin E1 on the growth of EsclZef°iclaia coli F4 (K88).
FIG. 2 illustrates the effect of Colicin E1 on the growth of Escherichia coli F18.
FIG. 3 illustrates the effect of Colicin N on the growth of Escherichia coli (K88).
15 FIG. 4 illustrates the effect of Colicin N on the growth of Escherichia coli F18.
FIG. 5 illustrates the effect of colicins A, N, and E1 (4.1 ~,g/ml for each individual colicin) on increase in optical density (600 nm) of E. coli 057:H7 strain 933.
O, control; ~, colicin A-treated; D, colicin E1-treated; and 1, colicin N-treated cultures.
FIG. 6 illustrates the effect of colicins A, N, and E1 (4.1 ~.g/ml for each individual 2o colicin) on increase in optical density (600 nm) of E. coli 057:H7 strain 86-24. O, control;
~, colicin A-treated; D, colicin E1-treated; and 1, colicin N-treated cultures.
FIG. 7 illustrates the effect of colicins A, N, and E1 on the maximal specific growth rates (h-1) of E. coli 057:H7 strain 933. D, colicin A-treated; O, colicin E1-treated;
and ~, colicin N-treated cultures.
25 FIG. 8 illustrates the effect of colicins A, N, and E1 on the maximal specific growth rates (h-1) of E. coli 057:H7 strain 86-24. D, colicin A-treated; O, colicin E1-treated; and ~, colicin N-treated cultures.
FIG. 9 illustrates the effect of colicins A, N, and El on bacterial populations (CFU/ml) of E. coli 057:H7 strain 933 after 6 h of incubation. D, colicin A-treated; O, 3o colicin El-treated; and ~, colicin N-treated cultures.

FIG. 10 illustrates the effect of colicins A, N, and El on bacterial populations (CFU/ml) of E. coli 057:H7 strain 86-24 after 6 h of incubation. D, colicin A-treated; O, colicin El-treated; and ~, colicin N-treated cultures.
FIG. 11 illustrates the lowest colicin E1 concentrations (~g/ml) on the maximal specific growth rate (h'1) of E. coli 0157:H7 strains. Error bars indicate standard deviations.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention provides a method for biological production of colicins in a recombinant organism. The method incorporates a microorganism containing a gene 1o coding for a pore-forming colicin, such as colicins, A, B, E1, Ia, and N
(Morton et al., 1983; Mankovich et al., 1986; Pugsley, 1987), or a nuclease colicin, such as E2, E8, E9, E7, E5, E4, DF13, E6, or E3. Such colicins are easily isolated from nonpathogenic strains of E, coli using methods well known in the art. In comparison to conventional antibiotic prophylactic therapy, the present method provides a relatively inexpensive and environmentally responsible means of protecting swine and other animals from E. coli infections. Two pore-forming colicins in particular, namely E1 and N, have been demonstrated to be especially effective against the F18 and F4 strains of E.
coli, respectively. The colicins are produced as described above and purified using conventional methods, such as affinity chromatography.
The following definitions are to be used to interpret the claims and specification.
The terms "host cell" or "host organism" refer to a microorganism capable of receiving foreign or heterologous genes and of expressing those genes to produce an active gene product. The terms "organism(s)" and "microorganism(s)" shall be used interchangeably and will refer to prokaryotic and eulcaryotic organisms that exist in nature as single cells, where each cell is capable of sustaining life independently of other cells of the same type.
The terms "foreign gene", "foreign DNA", "heterologous gene" and "heterologous DNA" refer to genetic material native to one organism that has been placed within a host organism by various means.
3o The terms "recombinant organism" and "transformed host" refer to any organism having been transformed with heterologous or foreign genes. The recombinant organisms of the present invention express foreign genes encoding pore-forming or nuclease colicins.
"Gene" refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding) and following (3' non-coding) the coding region. The terms "native" and "wild-type" refer to a gene as found in nature with its own regulatory sequences.
The terms "encoding" and "coding" refer to the process by which a gene, through the mechanisms of transcription and translation, produces an amino acid sequence. It is understood that the process of encoding a specific amino acid sequence includes DNA
sequences that may involve base changes that do not cause a change in the encoded amino to acid, or which involve base changes which may alter one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence.
It is therefore understood that the invention encompasses more than the specific exemplary sequences.
Modifications to the sequence, such as deletions, insertions, or substitutions in the sequence which produce silent changes that do not substantially affect the functional 15 properties of the resulting protein molecule are also contemplated. For example, alteration in the gene sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated.
Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic 20 residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a biologically equivalent product. In some cases, it may in fact be desirable to make mutants of the sequence in order to study the effect of alteration on 25 the biological activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity in the encoded pro ducts.
The term "expression" refers to the transcription and translation to gene product from a gene coding for the sequence of the gene product.
3o The terms "plasmid", "vector", and "cassette" refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA
sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitate transformation of a particular host cell. "Expression cassette" refers to a specific vector containing a foreign gene and l0 having elements in addition to the foreign gene that allow for enhanced expression of that gene in a foreign host.
The terms "transformation" and "transfection" refer to the acquisition of new genes in a cell after the incorporation of nucleic acid. The acquired genes may be integrated into chromosomal DNA or introduced as extrachromosomal replicating sequences. The term "transformant" refers to the product of a transformation.
The term "genetically altered" refers to the process of changing hereditary material by transformation or mutation.
Recombinant organisms containing the necessary genes that will encode colicins in accordance with this invention may be constructed using techniques well known in the art.
2o In the present invention, genes encoding colicins A, B, E1, Ia, and N were isolated in the laboratory from nonpathogenic strains of E. coli obtained from the National Collection of Type Cultures (NCTC, London, UK) and used to transform host strains, such as E. coli DHSa,, ECL707, AA200, JM109, or W1485; Saccha~o~~yces eerevisiae;
Lactobacillus; P.
pastor~is or the Klebsiella pneumoniae strains ATCC 25955 or ECL 2106.
Methods of obtaining desired genes from a bacterial genome are common and well l~nown in the art of molecular biology. For example, if the sequence of the gene is known, suitable genomic libraries may be created by restriction endonuclease digestion and may be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA may be amplified using standard primer directed amplification methods 3o such as polymerase chain reaction (PCR) (U.S. Pat. No. 4,683,202) to obtain amounts of DNA suitable for transformation using appropriate vectors.

Genes encoding colicins are well known in the art, as are their sources. See e.g.
Pugsley and Oudega, 1987 and Giilor et al., 2004. It is contemplated that any gene encoding a pore-forming or nuclease colicin or having pore-forming or nuclease colicin-like activity is suitable for the purposes of the present invention, wherein that activity is capable of forming ion chamlels in the plasma membrane of bacteria, resulting in membrane depolarization, or capable of non-specific degradation of DNA or specific cleavage of rRNA in sensitive cells.
Suitable host cells for the recombinant production of colicin may be either prokaryotic or eukaryotic (yeast). Preferred hosts include Gitrobacter, Efate~°obacte~, to Klebsiella, Ae~obacte~, Lactobacillus, Aspe~gillus, Saccha~omyces, Schizosaccha~omyces, Pichia, Candida, Hansenula, Methylobacte~, Esche~~ichia, Salmonella, Bacillus, StYepto~rayces and Pseudonaohas. Most preferred in the present invention are Pichia and Escherichia species, with P. pastoris and E. coli being most preferred for cost reasons.
The present invention provides a variety of vectors and transformation and expression cassettes suitable for the cloning, transformation and expression of colicins into a suitable host cell. Suitable vectors can be derived, for example, from a bacteria or a yeast. Protocols for obtaining and using such vectors are known to those in the art.
(Sambrook et al., Molecular Cloning: A Laboratory Manual--volumes 1,2,3 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)).
2o Suitable bacterial vectors for use in the invention are those that can be replicated in the host cells listed above. For example, in a preferred embodiment of the invention, constitutive expression of an E. coli colA gene is accomplished in Lactobacillus by placing this gene downstream of a strong Lactobacilli promoter sequence (Djordjevic et al., 1997).
This plasmid and promoter sequence combination has been used successfully to express an E. coli gusA gene in L. gasseri (Russell and I~laenhammer, 2001).
In a preferred embodiment using a yeast system, expression of an E. coli colA
gene is placed in correct reading frame behind the alpha-factor signal peptide of Saccla~omyees cey~evisiae. This signal peptide has been shown to direct efficient secretion of recombinant E. coli proteins in yeast systems (Brake et al., 1984; Rodriguez et al., 1999;
Stahl, 2001).
3o In this embodiment, expression is preferably controlled by the glyceraldehyde-3-phosphate dehydrogenase promoter or alcohol oxidase I promoter, from a methylotropic yeast, Pichia pastoris. These promoters direct constitutive or inducible expression, respectively, of the gene of interest.
Typically, the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA
fragment which controls transcriptional termination. It is most preferred when both control regions are derived from genes homologous to the transformed host cell although it is to be understood that such control regions need not be derived from the genes native to the specific species to chosen as a production host. Examples of suitable vectors for use in the invention include Pgemt-Easy (E. coli T vector for subcloning PCR products); pGAPz (an integrative P.
pastoris expression vector, also an E. coli shuttle vector; pGAPza (an integrative P.
pastor-is expression vector, also an E. coli shuttle vector); pPICZa (an integrative P.
pastoris expression vector, also an E. coli shuttle vector); pPICZ (an integrative P, pasto~is expression vector, also an E. coli shuttle vector), and pTRK664 (Lactobacillus expression vector, and an E. coli shuttle vector).
Initiation control regions or promoters, which are useful to drive expression of the colicin encoding genes in the desired host cell, are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the present 2o invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGI~, PHOS, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccha~ofnyces); Lactococcus lactis lacA and Lactobacillus acidophilus ATCC

(useful for expression in Lactobacilli ); slpA AOX1 (useful for expression in Pichia); and lac, trp, ?~. PL, ~.PR, T7, tac, and trc (useful for expression in E. coli).
Termination control regions may also be derived from various genes native to the preferred hosts.
Once suitable cassettes are constructed they are used to transform appropriate host cells. Introduction of the cassette containing the genes encoding the colicins, either separately or together into the host cell may be accomplished by known procedures such as by transformation (e.g., using calcium-permeabilized cells, electroporation) or by 3o transfection using a recombinant phage virus. (Sambrook et al., supra.) Fermentation media in the present invention must contain suitable carbon substrates. Suitable substrates may include, but are not limited to, monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose, or mixtures thereof, and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Additionally, the carbon substrate may also be one-carbon substrates such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated.
W addition to utilization of one and two carbon substrates, methylotrophic to organisms are also known to utilize a number of other carbon-containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeast are known to utilize the carbon from methynamine to form trehalose or glycerol (Bellion et al., Microb. Growth C1 Compd., [Int. Symp.], 7th (1993), 415-32. Editor(s): Murrell, J. Collin; Kelly, Don P. Publisher: Intercept, Andover, UK).
Similarly, various species of Cahdida will metabolize alanine or oleic acid (Sinter et al., Arch. Microbiol., 153(5), 485-9 (1990)). Hence, the source of carbon utilized in the present invention may encompass a wide variety of carbon-containing substrates and will only be limited by the requirements of the host organism.
Although it is contemplated that and of the above mentioned carbon substrates and 2o mixtures thereof are suitable in the present invention, preferred carbon substrates are monosaccharides, onigosaccharides, polysaccharides, and one-carbon substrates.
More preferred are sugars such as glucose, fructose, glycerol, sucrose and single carbon substrates such as methanol and carbon dioxide.
In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for glycerol production.
Typically, cells are grown at 28-40°C in appropriate media. Preferred growth media in the present invention are common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, MS broth, Yeast Peptone Dextrose, BMMY, GMMY, or Yeast Malt Extract (YM) broth. ~ther defined or synthetic growth l0 media may also be used and the appropriate medium for growth of the particular microorgaiusm will be known by someone skilled in the art of microbiology or fermentation science.
Suitable pH ranges for the fermentation are between pH 4.0 to pH 9Ø
Reactions may be performed under aerobic or anaerobic conditions. The present invention may be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable.
Methods for the purification of proteins and polypeptides from fermentation media are known in the art. For example, polypeptides can be obtained from cell media by l0 subj ecting the reaction mixture to extraction with an organic solvent, distillation, ultrafiltration, and ion exchange chromatography, and column chromatography.
The recombinant colicins of this invention may be identified directly by submitting the media to functional assay or high pressure liquid chromatography (HPLC) analysis. The levels of expression of the colicin proteins are measured by bacterial inhibition assays and other 15 methods well known in the art.
The following examples are offered to illustrate but not limit the invention.
Thus, they are presented with the understanding that various modifications may be made and still be within the spirit of the invention.

Colicin Production, Purification, and Efficacy Colicin Production and Purification Colicin producing E. coli strains, obtained from the National Collection of Type 25 Cultures (Public Health Laboratory Service, London, England), were inoculated into Luria Broth (LB) to a starting OD6oo ~ 0.1, and incubated with shaking at 37°C. When the cultures reached an OD6oo = 0.9 colicin production was induced by the addition of 0.2 U
Mitomycin C (Sigma)/mL culture. The cell free supernatant was obtained by centrifugation 5.5 h later, and concentrated by ultrafiltration in a stir-cell apparatus (Amicon, Millipore, 30 Bedford, MA) across a regenerated cellulose membrane with a 30 lcDa cut-off (Millipore).
The concentrated sample was then desalted against 10 mM Tris-Cl, pH 8 and purified by ion exchange chromatography. The desalted samples were applied to a column containing Q Sepharose (Amersham Biosciences, Piscataway NJ), equilibrated with 10 mM
Tris-Cl, pH 8.0 (equilibration buffer), and exhaustively washed with the equilibration buffer. The bound protein was eluted with a continuous NaCl gradient using an AKTAprime chromatography system (Amersham Bioscience). The fractions containing the highest concentrations of colicin, determined by SDS-PAGE followed by Coomasie Blue staining, .
were pooled and concentrated by ultrafiltration. The protein concentrations of these samples were determined in each pooled sample (Lowry et al., 1951), and the percentage colicin was determined by densitometry using a 16 bit mexapixel CCD camera, FluorChem 8800, and FluorChem IS800 software (Alpha Innotech, San Leandro, CA).
to Inhibition of Growth Assays Pure cultures of E. coli F4 (K88) and F18 were obtained from the culture collection at the USDA-ARS Federal Food Safety Research Unit (College Station, TX). These cultures were grown overnight in LB at 37°C with shaking, and then used to inoculate a flaslc of LB to an 0D6oo ~ 0.05. The freshly inoculated LB was then aliquoted (SmL) into culture tubes containing various colicin doses. The volume of the colicin doses was made constant by the addition of 10 mM Tris, pH 8. The total volume of each dose, including the 0 ~,g/mL colicin control, was 175 ~,L. These tubes were then incubated with shaking at 37°C, and their 0D6oo determined hourly for six hours. Quantitative determination of the colony forming units (CFU) of the E. coli strains were obtained by serial dilutions and direct plating on LB, initially and 3h post-inoculation. There experiments were repeated in triplicate, and the values presented are means.
RESULTS
Production and Purification of Colicins Yields of 1.1 mg of purified Colicin N/L of culture and 7.6 mg of purified Colicin El/L of culture were obtained with the aforementioned production and purification strategy. The purity of the Colicin N and E1 isolates were 30% and 85%, respectively, as determined by densitometry.
Efficacy of Colicin E1 against Esehericlaia coli F4 (K88) and F18 Colicin E1 significantly reduced the growth rate of E. coli F4 (K88) with 10 ~g/mL
of culture (FIG. 1). A dose of 50 ~.g/mL was needed to inhibit all growth of F4 (K88) for 6 hours. A significant reduction in the growth of F18 was seen with as little as .25 ~,g Colicin E1/mL of culture, and a complete inhibition of growth for 6 hours was seen with 1 ~,g/mL of culture (FIG. 2). Colicin E 1 showed bactericidal activity against both E. coli F4 (K88) and F18. After 3 hours of incubation with 50 ~.g of Colicin E1/mL of media, there was approximately one log less F4 (K88) CFU/mL than in the initial inoculum (Table 1).
This dose caused an approximately three log difference in F4 (K88) CFUImL
between the treated and untreated cultures. These same trends were also seen when F18 was incubated with only 1 ~.g of Colicin E1/mL of media. A dose of 100 p,g of Colicin E1/mL
caused a greater decline in E. coli F18 CFU/mL., but did not eliminate viable cells after three hours of incubation.
to Effect of Colicins on the Viability of Esche~ichia coli F4 (K88) and F18 After 3 Hours Incubation hzitial CFU/mL were 6 x 10' and 1 x 10~ for E. coli F4 (K88) and F18, respectively.
Colicin E1 Colicin N
E. coli Dose, p,g/mL CFU/mL Dose, ~,g/mL CFU/mL
F4 (K88) 0 3 x 109 0 3 x 109 50 5x106 10 1.1x10' 200 4 x 106 50 6 x 105 F 18 0 2 x 109 0 2 x 109 1 1.2x106 50 1x106 100 5 x 104 100 4 x 105 Efficacy of Colicin N Against E. coli F4 (K88) and F18 Colicin N was effective in inhibiting the growth of E. coli F4 (K88) and F18 at doses of 1 and 10 p.g/mL of culture, respectively. (FIG. 3 and 4). To completely inhibit the growth of these strains for 6 hours required 25 p,g of Colicin N/mL of F4 (K88) and 50 ~,g/mL of F18 (FIG. 3 and 4). A 10 p,g of Colicin N/mL of F4 (K88) dose did not allow any increase over the initial inoculum in the number of CFU/mL after 3 hours of incubation (Table 1). With this dose there was greater than a 3 log reduction in the F4 (K88) CFU/mL

after 3 hours incubation, when compared to the control. After 3 hours of incubation with 50 ~.g of Colicin N/mL of media, there was approximately one log less Fl 8 CFU/mL than in the initial inoculum (Table 1), and greater than three log less F18 CFUImL
than compared to the untreated culture after 3 hours incubation.
s Discussion Both of the E. coli strains considered primarily responsible for post-weaning diarrhea and edema disease in swine were sensitive to Colicin E1 and Colicin N. While both colicins inhibit the growth of these strains, their efficacy varied substantially. Colicin N was dramatically more effective than Colicin E1 against E. coli F4 (K88).
Significant to reductions in growth of E. coli F4 (K88) were seen with 10 fold less Colicin N than Colicin E1 (FIGS. 1 and 2). With equal dosage (50 ~g/mL), approximately 1 log fewer CFU/mL
were seen with Colicin N than with Colicin E1, after three hours of incubation (Table 1).
Colicin N was also more effective in reducing the growth of F4 (K88) than F18 (FIGS. 3 and 4). To completely inhibit the growth of F18 required approximately 10 fold more 15 Colicin N than was needed for F4 (K88). The effectiveness of Colicin E1 against these E.
coli strains was opposite that of Colicin N.
Colicin E1 was more effective against F18 than against F4 (K88), requiring approximately 50 fold less to completely inhibit the growth of F18 (FIGS. 1 and 2).
Colicin E1 was highly effective against F18 with as little as .25 ~,g/mL
dramatically 2o inhibiting its growth. Complete inhibition of growth, for six hours, was obtained with only 1 ~,g/mL, while greater than 2,5 ~,g Colicin N/mL was required for the complete inhibition of growth. Although growth was completely inhibited at this dosage, even a 100 fold increase in Colicin D1 could not eliminate all of the viable F18 in culture.
Approximately x 104 CFU/mL remained after 3 hours of incubation with 100 ~,g of Colicin E1/mL
25 (original inoculum was 1 x 10~ CFU/mL).

Inhibition of Growth of Esclzericlzia coli 0157:H7 In Vitro 3o MATERIALS AND METHODS
Colicin Production and Purification Each colicin was produced from a specific colicin-producing E. coli K-12 strain (NC50129-O1 containing plasmid pColA-CA31, NC50132-O1 containing plasmid pColEl-K53, and NC50145-O1 containing plasmid pColN-284) obtained from the National Collection of Type Cultures (Public Health Laboratory Service, London, UK).
Cultures were inoculated into Luria-Bertani (LB) broth to an initial optical density of 600 nm (ODsoo) of approximately 0.1 and incubated in a shaker at 37°C. When the cultures reached OD600 = 0.9, colicin production was induced by the addition of 0.2 U
of mitomycin C per ml of culture (Sigma Chemicals, St. Louis, Mo.). The cell-free supernatant was obtained by centrifugation 5.5 h later and concentrated by ultrafiltration in to a stir-cell apparatus (Amicon, Millipore, Bedford, Mass.) across a regenerated cellulose membrane with a 30-kDa cut-off (Millipore). The concentrated sample was then desalted against 10 mM Tris-Cl, pH 8, and purified by ion exchange chromatography. The desalted samples were applied to a column containing Q Sepharose (Amersham Biosciences, Piscataway N.J.), equilibrated with 10 mM Tris-Cl, pH 8.0 (equilibration buffer), and i5 exhaustively washed with the equilibration buffer. The bound protein was eluted with a continuous NaCI gradient on an AKTAprime chromatography system (Amersham Bioscience). The fractions containing the highest concentrations of colicin, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomasie blue staining, were pooled and concentrated by ultrafiltration. The protein concentrations of 2o these samples were determined in each pooled sample (20), and percent colicin was determined by densitometry with the use of a 16-bit megapixel charge-coupled device camera (FluorChem 8800, Alpha Innotech, San Leandro, Calif.) and Fluor Chem software (Alpha Innotech).
Bacterial Strains and Culture Conditions 25 E. coli 0157:H7 strains 933 (ATCC 43895) and 86-24 were obtained from the Food and Feed Safety Research Unit culture collection (U.S. Department of Agriculture/Agricultural Research Service, College Station, Tex.); both strains were originally isolated from human hemorrhagic colitis outbreaks. E. coli Ol 57:H7 strain 933 was naturally resistant to 25 ~,g/ml novobiocin and were made resistant to 20 ~,glml 30 nalidixic acid by repeated transfer and selection. E. coli 0157:H7 86-24 was made resistant to streptomycin (100 ~g/ml) by repeated transfer and selection. Differences in growth rates and antibiotic resistance profiles (other than for specifically selected and closely related antibiotics) were not detected between these antibiotic-resistant strains and wild-type parental strains (data not shown).
Esclaer-ichia coli 0157:H7 strains 933 (ATCC 43895) and 86-24 were anaerobically s (90% NZ, 5% H2, 5% C02 atmosphere) incubated at 39°C in anoxic tryptic soy broth (Difco Laboratories, Detroit, Mich.) to ensure colicin activity under anaerobic conditions similar to those within the gastrointestinal tract. Growth rates (n = 2) were estimated via measurement of absorbance changes with a Spectronic 20D spectrophotometer (600 nm, Thermo Spectronic Inc., Madison, Wis.); growth rate was calculated with the formula (In 1o OD2 - In OD1)/OT. Final optical densities after 24 h of incubation were measured with a Gilford 2600 spectrophotometer (600 nm, 1-cm cuvette). Cultures with optical densities greater than 0.7 OD units were appropriately diluted in 0.9% NaCl.
Quantitative Bacterial Enumeration Samples were taken from incubations at 6 and ~4 h to determine the effect of is colicins on populations of E. coli 0157:H7. Samples were serially diluted (in 10-fold increments) in phosphate-buffered saline (pH 7.0) and subsequently plated on MacConkey's agar (supplemented with 25 ~,g/ml novobiocin and 20 ~g/ml nalidixic acid for E. coli 0157:H7 strain 933 or with 100 ~,g/ml streptomycin for E. coli 0157:H7 strain 86-24) and incubated at 37°C overnight for direct counting (CFU per milliliter).
2o Colicin Addition To initially evaluate the effectiveness of these colicins against E. coli 0157:H7 strains, cultures were inoculated into tryptic soy broth tubes containing equivalent concentrations (4.1 ~.g/ml) of each of the individual colicins tested (A, E1, and N). To determine the effective range of doses for use in more complex mixed culture and in vivo 2s studies, freshly inoculated tryptic soy broth was added (5 ml) to culture tubes containing concentrations (0 to 40.8 p,g of each colicin per ml) of colicin A, colicin E1, and colicin N.
The total volume of each colicin addition, including the 0 ~,g/ml colicin control, was 175 ~.1; the volume of the colicin dose was made constant by the addition of sterile, anoxic 10 mM Tris, pH 8. To determine the lowest effective dose of colicin E1 against both E. coli 30 0157:H7 strains, cultures were grown in the presence of 0, 0.016, 0.032, 0.064, 0.128, 0.255, 0.51, 1.02, 2.04, 4.1, 7.7, 15.4, 28.8, and 40.8 ~.g of colicin E1 per ml.

Statistical analysis Experiments were performed in duplicate, and the values presented are means.
Students' t test was used to determine significance of differences between means.
Chemicals Unless specifically mentioned, all chemicals were obtained from Sigma Chemical Company.
RESULTS
Effect of Colicins on E. coli 0157:H7 E. coli 0157:H7 grew rapidly in tryptic soy broth, but the addition of colicins 1o affected growth (Figs. 5 and 6). Colicin E1 significantly (P < 0.05) reduced growth of both E. coli 0157:H7 strains 933 and 86-24 (Figs. 5 and 6). Colicin N did not affect the growth rate of E. coli 0157:H7 strain 933 (FIG. 5) but did reduce the OD or growth rate of E. coli 0157:H7 strain 86-24 (FIG. 6). Colicin A did not affect the OD of either strain of E. coli 0157:H7. Colicin El significantly (P < 0.05) reduced the specific growth rate of both strains of E. coli 0157:H7 examined at low concentrations (FIGS. 7 and 8).
Colicin N was nearly as effective as E1 against strain 86-24 but was not (P > 0.10) effective against strain 933.
Regardless of the colicin dose used, all E. coli 0157:H7 cultures eventually grew overnight,; therefore, final (24 h) optical densities were not significantly reduced in either 2o strain by any of the colicins (data not shown).
Bacterial populations of both E. coli 0157:H7 strains were unaffected by colicin A
(FIG. 9 and 10). Treatment with colicin El significantly (P < 0.05) reduced E.
c~li 0157:H7 populations by at least 4 log units for both strains tested, whereas colicin N
significantly (P < 0.05) reduced populations only for E. coli 0157:H7 strain 85-24 (FIG.
10). Because of the sensitivity of both strains of E. coli 0157:H7 to colicin E1, the efficacy of very low doses against these strains was examined. Colicin E1 reduced (P <
0.05) the specific growth rate of both strains of E. coli 0157:H7 at concentrations below 0.1 ~.g/ml.
(FIG. 11). In a negative control, Salmonella Typlzinauf°ium culture growth rates, final OD, and populations were not affected by any colicin treatment (data not shown).
3o DISCUSSION

In this study, colicin El was the most effective colicin. This result agrees with previous data indicating that colicin E1 displayed antimicrobial activity against several EHEC strains, not just 0157:H7. Other E-type colicins were found to be suitable ca~ididates as "biopreservatives" against E. coli 0157:H7. However, other studies have indicated that the sensitivity of E. coli 0157:H7 strains to any single colicin can be highly variable. For example, only 1 of 18 colicins examined inhibited all 540 E.
coli 0157:H7 strains screened. Because some E. coli 0157:H7 strains are colicinogenic and produce specific concomitant immunity proteins, they can be resistant to certain colicins or even a broad category of colicins. Therefore, simultaneous administration of a mixture of several 1o categories of colicins should be considered as a treatment concept to reduce E. coli 0157:H7 (and other EHEC) in the gastrointestinal tract of food animals.

Recombinant Expression of Colicins Yeast expression vectors were constructed and verified by sequencing for Colicin A, B, E1, la, and N. Expression and secretion of an active Colicin A by Pichia pastoris were obtained, and confirmed by spot testing of cell-free supernatant of Pichia pastoris expressing Colicin A against E. eoli DHSa. In the functional assay (spot testing) 10 ~.1 of 2o cell-free supernatant from transformed P. pasto~is showed a clearing of approximately the same size as 1 ~,g of purified Colicin A from E. coli. Spot 2 showed 1 ~,1 of cell-free supernatant from P. pastoris. Spot 3 showed 5 ~,1 of cell-free supernatant from P. pastoris, and spot 4 showed 10.1 of cell-free supernatant from P. pastoris.
The prophylactic use of antibiotics in animal agriculture has been greatly scrutinized in recent years, due to concerns regarding its role in contributing to antibiotic resistance. This scrutiny has led to increased regulation over the use of antibiotics in animal agriculture, and will likely continue towards a zero tolerance for the use of 3o prophylactic or growth promoting antibiotic use in animals. With this regulatory milieu in mind, it is essential for the sustainability of animal agriculture to examine alternatives to conventional antibiotics to improve animal health and production efficiency.
Because of their efficacy against E. coli, colicins are a viable alternative to conventional antibiotics in swine production. The present invention demonstrates that colicins may be economically and readily synthesized using recombinant techniques.
For the above-stated reasons, it is submitted that the present invention accomplishes at least all of its stated obj ectives.
Having described the invention with reference to particular compositions and methods, theories of effectiveness, and the like, it will be apparent to those of skill in the art that it is not intended that the invention be limited by such illustrative embodiments or to mechanisms, and that modifications can be made without departing from the scope or spirit of the invention, as defined by the appended claims. It is intended that all such obvious modifications and variations be included within the scope of the present invention as defined in the appended claims. The claims are meant to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended.

Claims (28)

1. An isolated expression vector construct for the production of a recombinant colicin comprising: (a) a DNA sequence encoding a colicin; (b) DNA sequences allowing for the expression of the colicin; said colicin being selected from the group consisting of colicin A, colicin B, colicin Ia, and colicin N; and (c) a yeast vector.

2. The vector construct of claim 1 wherein the DNA sequence encoding the colicin is isolated from E. coli.

3. The vector construct of claim 1 further including a promoter in reading frame with the coding sequence.

4. The vector construct of claim 3 whereby the promoter is a yeast promoter.

5. The vector construct of claim 3 whereby the promoter is selected from the group consisting of glyceraldehyde-3-phosphate dehydrogenase and alcohol oxidase 1.

6. The vector construct of claim 1 further including a signal peptide in reading frame with the coding sequence.

7. The vector construct of claim 6 whereby the signal peptide is an alpha-factor signal peptide of Sacchromyces cerevisiae.

8. The vector construct of claim 7 whereby the signal peptide is upstream from the coding sequence for the colicin.

9. A prokaryotic or eukaryotic host cell transformed strain transformed with the vector construct of claim 1.

10. The cell of claim 9 that is a strain of P. pastoris.

11. The cell of claim 10 that is Pichia pastoris.

12. A recombinant colicin expressed by the vector construct of claim 1.

13. A method for producing a recombinant colicin comprising: growing a bacterial or yeast strain transformed with a vector construct of claim 1 in a medium; and isolating the recombinant colicin.

14. The method of claim 13 further including the step of purifying the recombinant colicin.

15. A method of inhibiting the growth of pathogenic bacteria in animals comprising:

introducing to an animal a bacterial growth inhibiting effective amount of a recombinant colicin of claim 12.

16. The method of claim 15 whereby the pathogenic bacteria is selected from one or more of the group consisting of E. coli and Salmonella.

17. The method of claim 16 whereby the pathogenic bacteria is an enterohemorrhagic strain of E. coli.

18. The method of claim 17 whereby the E.coli is selected from the group consisting of E. coli F4 and E. coli F18.

19. A method for the production of a colicin from a recombinant microorganism comprising: transforming a suitable host microorganism with one or more transformation cassettes each of which comprises a gene encoding a colicin, wherein the gene is introduced into the host microorganism; culturing the transformed host microorganism under suitable conditions; and recovering the colicin, said colicin being selected from the group consisting ofcolicin A, colicin B, colicin El, colicin Ia, and colicin N, and further providing that the suitable host microorganism is yeast.

20. The method of claim 19 wherein the suitable host microorganism is Pichia.

21. The method of claim 19 further including the step of purifying the recovered colicin.

22. A yeast host cell transformed with a gene encoding a colicin, said colicin selected from the group consisting of A, B, E1, 1a, and N.

23. A method of reducing pathogenic bacteria on food comprising: applying a recombinant colicin selected from the group consisting of A, B, E1, 1a, and N
to the food.

24. The method of claim 23 whereby the colicin is applied to meat.

25. A method of reducing pathogenic bacteria on animal carcasses comprising:

applying a recombinant colicin according to claim 14 to an animal carcass.

26. The method of claim 25 whereby the pathogenic bacteria is selected from one or more of the group consisting of E. coli and Salmonella.

27. A method of reducing pathogenic bacteria in animals comprising: feeding the animal a recombinant colicin according to claim 12.

28. The method of claim 27 whereby the pathogenic bacteria is selected from one or more of the group consisting of E. coli and Salmonella.