Patent Grant
7211426
The present invention relates to phage isolates having a strong lytic activity against pathogenic Enteriobacteriaceae such as Escherichia coli and Salmonella strains and their use in various human or pet food products for the treatment or prevention of bacterial diseases caused by pathogenic Enteriobacteriaceae such as Escherichia coli or Salmonella, in particular for phage therapy of pediatric gastroenteritis. It also relates to human or pet food products prepared thereof.
In the past, antibiotics have been used to treat various infections. The work of Selman Waksman in the introduction and production of Streptomycetes, and Dr. Fleming's discovery of penicillin, as well as the work of numerous others in the field of antibiotics, are well known. Over the years, there have been additions and chemical modifications to the “basic” antibiotics in attempts to make them more powerful, or to treat people allergic to these antibiotics.
However, as more antibiotics have been prescribed or used at an ever-increasing rate for a variety of illnesses, increasing numbers of bacteria have developed a resistance to antibiotics. Larger doses of stronger antibiotics are now being used to treat ever more resistant strains of bacteria. Consequently, multiple antibiotic resistant bacteria have been developed and some hospital-acquired infections with Gram-positive bacteria have already become untreatable. The use of more antibiotics and the number of bacteria showing resistance has led to increases in the amount of time that the antibiotics need to be used. Broad, non-specific antibiotics, some of which have detrimental effects on the patient, are now being used more frequently. Additionally, the number of people showing allergic reactions to antibiotics appears to be increasing.
Consequently, other efforts have been sought to first identify and then kill bacteria.
Attempts have been made to treat bacterial diseases with the use of bacteriophages. These are a heterogeneous group of viruses that infect bacteria that were first discovered in the early part of the 20th Century (d'Herelle, F., The bacteriophage. Its role in immunity, translated by Smith, G. H., Williams & Wilkins Co., Baltimore (1922)). Bacteriophages presently are widely used in scientific research, such as in molecular biology (e.g. as genetic vectors) and in medical diagnostics (e.g. phage typing of bacteria). Insofar as phages naturally infect and kill bacteria, it traditionally was suggested that they could be utilized in medical therapeutics, because bacteria are a major cause of disease.
Indeed, potential therapeutic uses of phage in experimental systems have been reported extensively in the literature.
For example, U.S. Pat. No. 5,688,501 (Merril, et al.) discloses a method for treating an infectious bacterial disease with lytic or non-lytic bacteriophages that are specific for particular bacteria.
Also, U.S. Pat. No. 4,957,686 (Norris) discloses a procedure of improved dental hygiene which comprises introducing into the mouth bacteriophages parasitic to bacteria which possess the property of readily adhering to the salivary pellicle.
Despite the recognized value and importance of new antimicrobial therapies, the potential of phage therapy has not been accomplished in any practical sense that is used in modem therapeutics, nor have methods, compositions, or other uses been defined for nontoxic and efficacious therapies of this type with practical and effective delivery. In particular for efficient prevention and treatment of infections related to E. coli, such as diarrhea diseases.
In fact, E. coli is an extremely well-investigated bacterium, with two completely sequenced members, the non-pathogenic strain K-12 (Blattner, F. R., et al., 1997 Science 277, 1453–1474) and the pathogenic strain 0157:H7 (Perna, N. T. et al., 2001, Nature 409, 529–533; Hayaslis et al., 2001, DNA Research 8, 11–22) which resembles enteropathogenic E. coli (EPEC) strains.
Although, two E. coli phages (lambda and T4) are the most carefully characterized biological systems (Karam, J. D., 1994, Molecular biology of bacteriophage T4. ASM Press, Washington, D.C.), there still exists a need to develop new and useful phage therapeutics which can be used to prevent or to treat infectious disorders caused by such bacterial microorganisms, such as E. coli serotypes associated with diarrhea of children and the traveler.
There also exists a need to develop compositions including phage therapeutics in acceptable vehicles that can be administered to subjects infected with bacterial microorganisms.
Finally, there exists a need to develop a bacteriophage preparation that is virulent and non-toxic thereby rendering it useful in treating bacterial infections related to pathogenic E. coli or Salmonella.
It is therefore an object of the present invention to develop new and useful phage therapeutics which can be used to prevent or to treat infectious disorders caused by pathogenic Enteriobacteriaceae such as E. coli infections, in particular diarrheal or urinary diseases in humans or animals, or Salmonella infections.
Another object of the invention is to provide isolated bacteriophage preparations with substantial lytic potential towards pathogenic Enteriobacteriaceae such as E. coli and/or Salmonella strains that is virulent and non-toxic.
It is also an object of the present invention to provide the use of such bacteriophage preparations in various nutritional compositions, pet food products, supplements or pharmaceutical compositions for the treatment or prevention of infections caused by pathogenic E. coli and/or Salmonella in humans or animals.
It is a further object to provide a nutritional composition, a pet food product, a supplement, or a pharmaceutical composition comprising at least one phage as described above.
It is yet another object of the present invention to provide a method of prevention or treatment of infections caused by pathogenic Enteriobacteriaceae such as E. coli and/or Salmonella in humans or animals using phage therapeutics in nutritional compositions, pet food, supplements or pharmaceutical preparations.
A major advantage of the present invention is that the bacteriophage preparations selected according to the invention have a broad host range on causing-causing E. coli strains, they are non toxic, so that they may be used safely in food compositions for an efficient prevention or treatment of infections caused by pathogenic E. coli. The phages according to the invention can also be used for industrial applications. They can efficiently be propagated on a non-pathogenic E. coli strain while retaining a broad host range. The phages kill efficiently the host cell and the phages can be highly purified by a standard virus isolation method.
Within the following description, “NCC” designates Nestle Culture Collection (Nestle Research Center (NRC), Vers-chez-les-Blanc, Lausanne, Switzerland).
According to a first aspect, a broad-host range Myoviridae phage isolates with substantial lytic potential towards pathogenic Enteriobacteriaceae such as E. coli and/or Salmonella strains have been isolated from human stool samples and the environment.
Stool samples from children hospitalized with acute diarrhea were screened with the plaque assay for the presence of bacteriophages directed against E. coli. Different source of water has also been screened for the presence of bacteriophages. These waters (tap, sewage, drain, pond, canal, street and market water) are from Dhaka/Bangladesh and from Lausanne (sewage water). About 45 myoviridae phages were isolated and amplified on the non-pathogenic laboratory E. coli K-12. These isolates showed a broad host range on a large collection of pathogenic E. coli strains. Phages could be purified by standard methods of virus purification. All phages investigated in detail turned out to be T4-like phages (obligate virulent phages with contractile tail). The diagnosis was based on the large genome size (170 kb), modification of the DNA (resistance against digestion with restriction endonucleases) and morphology.
They can efficiently be propagated on a non-pathogenic E. coli strain while retaining a broad host range on diarrhea-causing E. coli strains. The T4-like phages kill efficiently the host cell. They are the prototype virulent phages that destroy the bacterial genome during the infection process. This property adds substantially to the safety of the phage therapy approach.
In a preferred embodiment, the phages are selected from the group comprising NCCJS9, NCC-JS31, NCC-JS102.2, NCC-JS150, NCC-JS1, NCC-JS10(renamed NCC-JS4) (CNCM I-2763), NCC-JS22, NCC-JS32, NCC-JS114.1, NCC-JS140.1, NCC-JS146.1, NCC-JS65.1, NCC-JS65.2′, NCC-JS76.2′, NCC-JS12, NCC-JS152, NCC-JS66, NCC-JS102.1, NCC-JS140.2, NCC-JS146.2, NCC-JS61.2, NCC-JS61.3, NCC-JS114.3, NCC-JS35 (renamed NCC-JS94.1) (CNCM I-2764), NCC-JS94.2, NCC-JS98, NCC-JS104, NCC-JS94.3, NCC-JS110.1, NCC-JS110.2, NCC-JS110.3, NCC-JS1.4 (renamed NCC-JSD.1) (CNCM I-2765), NCC-JSD.3, NCC-JSD.2, NCC-JSD.4, NCC-JSD.5, NCC-JS142, NCC-JS148 and NCC-JSL.1, NCC-JSL.2, NCC-JSL.3, NCC-JSL.4, NCC-JSL.5, NCC-JSF (renamed NCC-JSL.6) (CNCM I-2766).
In a most preferred embodiment, the phages NCC-JS10, NCC-JS35, NCC-JS1.4 and NCC-JSF have been deposited by way of example at the Institut Pasteur, 28 rue du Docteur Roux, F-75024 Paris cédex 15, FRANCE, on Dec. 11, 2001, under the deposit number CNCM I-2763, CNCM I-2764, CNCM I-2765 and CNCM I-2766.
Advantageously, the phages deposited, for example, are able to lyse 42 percent of the diarrhea-causing E. coli Nestlé Research Center collection composed of 50 strains. This collection represents the major E. coli serotypes of childhood diarrhea in developing countries and traveler's diarrhea. With the phage material according to the invention, coverage of 60 percent can be obtained with an increase of individual phage isolates included into the cocktail.
There is, however, no risk to other commensal gut bacteria outside of the species E. coli. The ecological equilibrium of the gut is maintained (see example 3,
With the phage material according to the invention, only less than 50 percent of nonpathogenic human gut E. coli serotypes are attached.
Thus, according to a further aspect, the invention relates to the use of at least one phage isolate as described above, in any food or pharmaceutical product, or a nutritional supplement or a composition for oral administration. Examples for food or pharmaceuticals products are milk, yogurt, curd, cheese, fermented milks, milk based fermented products, ice-creams, fermented cereal based products, milk based powders, infant formulae or tablets, liquid suspensions, dried oral supplement, wet oral supplement, dry-rube-feeding, pet food products. The composition for oral administration may be in capsules, soft capsules, tablets, pastes or pastilles, gums, or drinkable solutions or emulsions. Methods for preparing them are common knowledge.
The composition according to the invention may also comprise usual excipients, in particular sweeteners, flavoring agents or preservatives. It can further comprise a prebiotic and/or a probiotic micro-organism. The compositions of the invention may be formulated according to anyone of a number of techniques that are well known to this art.
In one embodiment, a pharmaceutical composition containing at least one of the bacteriophages in an amount sufficient to achieve the desired effect in an individual can be prepared. This composition may be a tablet, a liquid, a dried oral supplement, a wet oral supplement, dry tube-feeding, wet tube-feeding etc. The pharmaceutical composition will further contain carriers and excipients that are suitable for delivering the respective active molecule of different nature to the target tissue. The kind of the carrier/excipient and the amount thereof will depend on the nature of the substance and the mode of drug delivery and/or administration contemplated. It will be appreciated that the skilled person will, based on his own knowledge, select the appropriate components and galenic form.
In another embodiment, a food composition for human consumption is prepared. This composition may be a nutritional complete formula, a dairy product, a chilled or shelf stable beverage, water, a soup, a dietary supplement, a meal replacement, a nutritional bar or a confectionery.
The nutritional formula is preferably enterally administrable; for example in the form of a powder, a liquid concentrate, or a ready-to-drink beverage. If it is desired to produce a powdered nutritional formula, the homogenized mixture is transferred to a suitable drying apparatus such as a spray drier or freeze drier and converted to powder.
In a preferred embodiment, phages may be added to a milk formula with lukewarm water and could be of benefit to children suffering from E. coli gastroenteritis or Salmonella infections, or are at risk to acquire such infections. Pediatric E. coli diarrhea is rare in industrial countries of the Northern hemisphere, however, these infections are still very prevalent in countries like Mexico, Brazil, India and China.
Also, phages may be used in the prevention or treatment of traveler's diarrhea. European tourists spending their holidays in Northern Africa or US American tourists traveling to Central America or the Caribbean are at high risk to acquire ETEC infections.
Also, E. coli infections are a frequent cause of urinary infections in humans, including industrial countries. The O serotypes of extra-intestinal E. coli isolates differ from those of intestinal E. coli strains. However, the broad host-range of many T4-like phages leave no doubt that many of the urinary E. coli strains can be lysed with T4 phages isolated from stool samples.
In another embodiment, a usual food product may be enriched with at least one phage isolate according to the present invention. For example, a fermented milk, a yogurt, a fresh cheese, a renneted milk, article of confectionery, for example a sweet or sweetened beverage, a confectionery bar, breakfast cereal flakes or bars, drinks, milk powders, soy-based products, non-milk fermented products or nutritional supplements for clinical nutrition.
The bacteriophages according to the present invention may thus be used in an amount of at least 1.104 pfu (plaque forming unit)/ml and more preferably 106 pfu/ml.
In a further embodiment, pet food products may be prepared. In fact, E. coli is the major cause of diarrhea in dogs. The serotypes of EPEC and ETEC strains isolated from dogs partially overlap those of humans. Dogs harbor in their intestine E. coli serotypes that frequently lead to smear infections in the urinary tract. This canine urinary tract E. coli isolates showed close genetic relationships with human extra-intestinal E. coli isolates. The pet food formulation is preferably a complete and nutritionally balanced pet food. It can also be a dietary supplement for pets or in the form of a pharmaceutical composition. The nutritionally complete pet food formulation according to the invention may be in any suitable form, for example a powder, a dried kibble, or pellet or other dried form, extruded form, semi-moist or wet form, such as a chunk or loaf or pudding. It may be chilled or provided as a shelf stable product. This pet food may be produced by conventional methods.
In another embodiment, dietary adjuncts may be prepared so as to improve pet food quality. As dietary adjuncts, they may be encapsulated or may be provided in powder form and packaged in conjunction with or separately from a main meal, be it wet or dry. By way of example, a powder containing bacteriophages according to the invention may be packed in sachets in a powder form or in a gel or lipid or other suitable carrier. These separately packaged units may be provided together with a main meal or in multi-unit packs for use with a main meal or treat, according to user instructions.
Also, oral application of phages according to the invention or spraying of phages in the household could thus benefit both the dog and the pet owner.
In a last embodiment, phages may be of value in food production to decrease the degree of contamination of meat with E. coli and/or Salmonella.
The following examples are given by way of illustration only and in no way should be construed as limiting the subject matter of the present application. All percentages are given by weight unless otherwise indicated. The examples are preceded by a brief description of the figures.
Material and Methods
Preparation of Stools Samples
The stool samples were stored frozen at ˜20° C. at the hospital and were transported on dry ice to Lausanne. The thawed stool sample (˜10 g) was resuspended in TS (8.5 g/l NaCl, 1 g/l tryptone) to a final volume of 30 ml. The resuspended stool samples were then centrifuged for 15 minutes at 10.000 rpm in 50 ml Falcon tubes. One ml of each stool was filtered through a Millex AP20 prefilter followed by a 0.45 μm Minisart filter. After resuspension all stool samples were stored at +4° C. since T4 phages are sensitive to repeated cycles of freezing/thawing.
Bacterial Culture
All E. coli strains were propagated on Hershey broth containing 2 ml of thiamine (2 mg/ml) per liter in a glass-tube in a final volume of 5 ml under agitation (240 rpm) at 37° C. for overnight incubation. After growth, the strains were streaked on a Petri dish (Hershey agar) in order to obtain single colonies. To avoid serial passages in broth (which could select for mutants), a new culture was initiated from 3 single colonies each time when needed. The stock cultures were kept as stab-cultures at 4° C.
Plaque Assay
Phage plaques were obtained on Hershey agar supplemented with thiamine overlayed by 3.5 ml of Hershey top-agar inoculated with 200 μl of a fresh grown overnight culture and 100 μl of stool sample or cell lysate. The Petri dishes were incubated overnight at 37° C.
Isolation of Plaques and Phage Amplification
Single plaques were picked with toothpicks and inoculated in 5 ml of Hershey broth supplemented with thiamine were inoculated with 1% of a freshly grown culture and 1% of phage lysate. Incubation was performed under agitation at 240 rpm at 37° C. for approximately 5 hours. When the lysis occurred, 3 drops of chloroform was added by gentle mix. The lysate was left overnight at room temperature followed by a centrifugation at 10,000 rpm for 10 minutes. To obtain phage stocks the supernatant was recovered by carefully avoiding the chloroform and transferred into a screw-cap glass tube. Three drops of fresh chloroform were added and the phage stock was stored at 4° C. The strains used for phage amplification were either K-12 or O127:K63 (B74-9). The following nomenclature was used for the different phage isolates: JS (the initials of Josette Sidoti who conducted the phage survey) followed by the patient ID number. In case of multiple isolates from the same patient, this number was followed by a further number (e.g. JS61.2). When the second isolate came from a second (convalescent) stool sample of the patient, the second number was primed (e.g. JS65.2′). For phages isolated from environmental water the initials were followed by a D (Dhaka) or L (Lausanne), respectively, and an identifier for the phage isolate (e.g. JSL4).
Spot-test
A mixture of 3.5 ml Hershey top-agar with 200 μl of a freshly grown E. coli culture was poured on a bottom layer of Hershey agar supplemented with thiamine. When the top-agar was solidified, 10 μl of filtered extracted stool was put on it as 8 spots in clockwise distribution. Incubation was done overnight at 37° C.
Lysis in Tubes
5 ml of Hershey broth inoculated with 1% of a freshly grown culture. Incubation was continued for 3 to 5 hours when the uninfected control cells reached the stationary growth phase. The optical density in the phage-inoculated cell was compared visually with the optical density in the mock-infected control cell.
Phage Purification
For purification of the T4-like phages, one liter of Hershey medium containing thiamine was inoculated 1% with K-12, cells were grown to an OD of 0.1 and then infected with a moi (multiplicity of infection) in excess of 1. NaCl was added to the lysate yielding a final concentration of 0.5M and incubated one hour at 4° C. After centrifugation at 10,000 rpm (16 min at 4° C.) in a Sorvall RC5B centrifuge, polyethylenglycol PEG-6000 was added to the supernatant to a final concentration of 10%. The lysate was incubated overnight at 4° C. under gentle stirring. PEG-precipitated phages were collected by centrifugation at 10,000 rpm for 16 min, the resulting pellets were resuspended in 3 ml of phage buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 10 mM MgSO4). The resuspended phage pellet was applied onto a discontinuous CsCl gradient (1.35 g/ml CsCl, 1.53 g/ml, 1.65 g/ml). The gradients were centrifuged in a SW55 rotor at 40,000 rpm for 3 h at 4° C. with Beckman L860M ultracentrifuge.
Results
Isolation of Phages
E. coli phages can be isolated from clinical material (e.g. stool) or environmental samples (e.g. sewage). Previous work suggested that phages are only rarely isolated from stool samples of healthy subjects, phage titers were low and most isolates were temperate phages (Furuse, K., 1987, Distribution of coliphages in the environment: general considerations. In: Phage Ecology, pp. 87–124, J. Wiley, New York). Stool samples from diarrhea patients, in contrast, yielded phages with higher incidence and higher titer than stool samples from healthy controls and most isolates were virulent phages. To increase the likelihood for T4-like phage isolation we worked with stool samples from children hospitalized with acute diarrhea in the general ward of a large diarrhea clinic in Dhaka/Bangladesh. The statistical aspects of the phage screening are summarized in Table 1.
Shigella infections
Stool samples from 157 acute diarrhea patients were taken over a three-week time period. The patients were unselected and the sampling was done according the availability of the nurses to take and store a stool sample. For 52 patients a second stool sample was obtained at the discharge from the hospital. Seventeen stool samples were excluded since an E. coli diarrhea could be excluded: 5 patients had cholera (as demonstrated by dark-field microscopy of vibrios in rice stools) and 12 had Shigella dysentery (diagnosed on clinical grounds: bloody, mucous stools). Together both pathogens account to 11 percent of the hospitalized patients. This figure corresponds to the average detection rate of these pathogens in diarrhea patients from this hospital (Vibrio cholerae 7%, Shigella spp. 4%).
With a detection limit of 30 pfu (plaque forming units)/g feces, 19 percent of the stool samples scored positive when tested on the non-pathogenic laboratory E. coli strain K-12. The highest amount of phage shed into the stool by any of the patients was 5×104 pfu/g feces. However, only 4 percent of the patients showed such high stool phage titers. A somewhat lower percentage of patients showed phages that could be detected on the pathogenic EPEC strain O127:K63 (14% detection rate). Only four stool samples were doubly positive for phages on the laboratory and pathogenic E. coli strain, while 14 were positive only for K-12 and 14 other only for EPEC strain O127:K63. Apparently we detected with the two different E. coli indicator cells two distinct phage populations with minimal overlap in host range. Interestingly, the phage detection rate of 14% on the pathogenic E. coli indicator cell corresponded closely to the average detection rate of pathogenic E. coli strains in the stool samples from diarrhea patients in Bangladesh (15%).
Different sources of water (tap, drain, refuse, canal, pond, sewage, market, street, river, ditch waters) from Dhaka/Bangladesh has also been screened for the presence of phages. From almost a hundred samples (about 10 of each source of water), six different bacteriophages have been obtained and amplified. One of them was from tap water, three from drain water and one from sewage water. Sewage waters from Lausanne (Switzerland) have also yielded to six phage isolates.
On the laboratory E. coli strain, the detection rate of phages was lower in the convalescent than in the acute stool sample (4 vs. 11 positive samples, 52 paired stool samples studied). On the pathogenic E. coli strain comparable numbers of phages were isolated from convalescent and acute stool samples (5 vs. 4 positive samples, 16 stool samples studied).
All plaques detected on K-12 cells could be amplified in liquid culture growth on K12. Phage titers ranged from 2×104 to 4×1010 pfu/ml. The median titer was 109 pfu/ml. There was no correlation between the amplified titer and plaque size (large, medium, small or pinpoint), plaque transparency (turbid, clear) or initial stool titer. Similarly, plaques detected on the EPEC strain O127:K63 could be amplified to 1–5×108 pfu/ml on this pathogenic E. coli strain.
Target Population of Causing-causing E. coli Strains
The phage isolates were selected with the broadest host range on 50 pathogenic E. coli strains from the major serotypes isolated worldwide from pediatric diarrhea patients. Table 2 shows some of these strains. This set of strains covers 14 different somatic O antigens reflecting distinct carbohydrate side chains of the lipopolysaccharide antigen (LPS) and also 14 different capsular antigens K reflecting again different polysaccharide compositions of the bacterial capsular material. In addition, we possess a collection of the major enterotoxigenic E. coli (ETEC) serotypes isolated worldwide from both pediatric gastro-enteritis patients and adult traveler's diarrhea (Table 2). These ETEC strains represent 14 further distinct O antigens and 13 different H antigens. The latter antigen is a protein antigen from the bacterial flagellum. The ETEC strains could be further subdivided according to their production of enterotoxins. Eight strains produced heat labile (LT) and heat stabile (ST) enterotoxins. Two strains produced only the heat labile; four strains only the heat stabile enterotoxin.
E. coli strains from the Nestec collection
Host Range Screening of the Isolated Phages
In the first round 32 phages were isolated from the stool samples of the diarrhea patients. These phages were systematically tested on the 50 causing-causing E. coli strains from our combined cell collection. In a second round further phage isolates with broad host range were obtained.
As the host range testing involves a large number of phage-cell combinations (n=32×50=1600 on 50 E. coli strains), we used the spot test for screening. In this test, eight different amplified phage lysates can be tested at the same time by spotting a drop of the amplified phage lysate on the bacterial lawn in a Petri dish. The results are read after overnight incubation and are scored as positive when the growth of the bacteria was compromised or impeded over the spotted area. The results of some phage isolates are presented in Table 3 and are summarized in Table 4.
E. coli
When distinct phage isolates from the same patient were compared, nearly identical results were obtained. For example, two different plaques were amplified from the stool sample of patient 146. Phage NCC-JS146.1 scored positive on 35 cells, while phage NCC-JS 146.2 infected 36 out of the 50 tested phages. With one exception, there was a perfect overlap between both phages with respect to the host range (Table 3). Similar overlaps were seen for two phages isolated from the acute and the convalescent stool sample of a single patient: phages NCC-JS65.1 and NCC-JS65.2′ of patient 65 infected 26 and 25 E. coli strains, respectively. Again with one exception, there was a perfect overlap in host range (Table 3). This high correlation of results between independent phage isolates from the same patient proves the reliability of the spot test.
The host range was drastically different for phages propagated on the non-pathogenic laboratory strain K-12 or the pathogenic strain O127:K63. The former phages could grow on a median of 26 different pathogenic E. coli strains, while the latter grew only on a median of 1 pathogenic strain in addition to the propagating cell (Table 4). This is illustrated for the stool sample of patient 114 which was one of the few stool samples (n=3) that yielded phages on both K-12 and O127:K63. The K-12-amplified phage infected 24 pathogenic strains, while the O127:K63-amplified phage infected only a single strain (Table 4).
A number of phages isolated from distinct patients were closely related with respect to host range (e.g. isolates NCC-JS.4, NCC.JS.146.1 and NCC-JS66 infected up to 35 cells with a maximum difference of 4 cells; isolates NCC-JS76.2′ and NCC-JS12 showing only 4 differences). However, phages with identical host range were only found in those phages having a very restricted host range (isolates NCC-JS83.1, NCC-JS89, NCC-JS114.2, NCC-JS83.2 and NCC-JS86 infecting only strains AD-24304 and O127:K63).
Lytic Potential of the Isolated Phages
Cell lysis was assessed visually as lack of turbidity in the tube incubated with the E. coli strain and the phage. The test was read when the uninfected E. coli strain had reached maximal turbidity. For example, phage isolates NCC-JS4, NCC-JS146.1, NCC-JS66 and NCC-JS146.2 which showed in the spot test a broad and very similar host range on the pathogenic E. coli strains, differed significantly in their capacity to lyse the strains (Table 5). Phage NCC-JS4 lysed 13 out of the 50 tested E. coli cells, while phages NCC-JS146.1, NCC-JS66 and NCC-JS146.2 lysed only 4 to 5 E. coli strains which were a subgroup of the strains lysed by phage NCC-JS 10.
E. coil
The growth of uninfected and phage-infected K-12 cells was followed by OD readings (
Purification of the Phages
One liter volumes of K-12 cultures were inoculated with six different phages yielding phage titers between 6×107 and 1010 plaque forming units (pfu)/ml. The phages from the lysate were precipitated with polyethylenglycol and the resulting phage pellet was further purified by CsCl density gradient centrifugation. The phage bands localized in the CsCl gradient were recovered and dialyzed to free them from the salt solution.
The following Table documents the efficiency of the phage purification procedure as monitored by the amount of the recovery of viral infectivity. The highest starting titers of phage infectivity were observed with phage NCC-JSI0, however this phage was also the most difficult to purify. Phage recovery ranged only from 0.3 to 5% of the starting infective material. For the other phages—at least 10% recovery was !be rule and the recovery could be as high as 40% of the initial infectivity in the crude lysate.
Materiel and Methods
Pulse Field Electrophoresis (pFGE)
PFGE blocks of 1% agarose containing 106 plaque-forming units of the indicated phage were incubated overnight in a lysis buffer (0.05M EDTA, 10 mM Tris pH 8.0, 1% SDS and 50 μg of proteinase K) and washed three times for one hour in 10 ml of TE (10 mM Tris pH 8.0, 1 mM EDTA). These blocks were analyzed by electrophoresis in 1% agarose low melting gel (0.5% TBE: 45 mM Tris-borate, 1 mM EDTA) for 20 hours at 6V/cm, 14° C. with a pulse time of 1–20 sec. The gel was stained for 30 min. with ethidium bromide (0.5 μg/ml).
DNA Purification
Purified phages were treated with proteinase K at a final concentration of 1 mg/ml for 2 h at 37° C. and 3 M sodium acetate pH 4.3 was added. DNA was extracted twice by phenol-chloroform and precipitated with 2 volumes of ethanol. After centrifugation, pellets were washed with 70% ethanol and resuspended in 50 μl of TE. DNA was digested with restriction enzymes according to the indications provided by the manufacturer.
Electron Microscopy
A drop of the phages suspension was applied to formvar-carbon coated copper grids for 5 min, the suspension was removed with a pipette and immediately replaced by a mixture of solutions A and B (A: 2% ammonium molybdate at pH 7.0, B: 7.5 mg bacitracine in distilled water). After 1 min the liquid was removed with filter paper. The grids were examined in a Philips CM12 transmission electron microscope at 80 kV (magnification: 176'000× or 224 '000×).
Results
Twelve stool samples from different patients that showed phages with a 170-kb genome were selected for further characterization. We investigated the morphology of these phages by electron microscopy and all of them were Myoviridae with long contractile tails. The close morphological similarity with the well-known coliphage T4 is obvious. For example the elongated head measured about 110 nm by 75 nm. A collar separated the head from the tail sheath. The tail sheath by an annular substructure measured about 95 nm in length and 18 nm in width. The tail is terminated by a baseplate structure to which both short tail spikes and long tail fibers are attached. The tail fibers are about 150 nm long and are kinked at a knee joint approximately in the middle of the fiber. Phages with a 40 nm contracted tail sheath were observed. In these phages, the internal tail tube now extends beyond the contracted tail sheath. Different negative staining procedures selectively revealed various morphological features of these phages. For example, the annular structure of the tail sheath and its structural changes after tail contraction was best visualized by uranyl acetate staining, whereas the tail fibers were better seen with phosphotungstic acid negative contrast. Occasionally, whiskers could be seen at the collar.
DNA-based techniques were used to differentiate the T4-like phages isolated in the current survey. T4 phage contains glucosylated hydroxymethyl cytosine instead of cytosine. This modification makes T4 DNA resistant to digestion with most restriction enzymes. Only a few enzymes are known to cut T4 DNA (EcoRV, NdeI, PacI, SwaI, SspI, and SphI). However, from nine T4-like phage isolates selected from different patients, none was digested by EcoRV, NdeI, SwaI or SphI, and only two were digested by SspI, while all were at least partially cut by PacI.
In the next step, we probed the PacI-digested phage DNA on Southern blots with labeled T4 DNA. Some isolates showed extensive cross-hybridization with the T4 probe, while others showed weaker cross-hybridization or lacked DNA homology with T4 at the high stringency hybridization conditions.
These Southern hybridizations were confirmed by PCR using a primer pair (FR60 and FR61) from the T4 gene 32 sequence that are diagnostic (Repoila et al 1994 genomic polymorphism in the T-even bacteriophages, EMBO J. 13:4181–4192) for the T-even subgroup of the T4-type phage. Phages that cross-hybridized with T4 DNA on the blots gave a clearly positive PCR result in this assay, while phage isolates that did not cross-hybridize with T4 gave no, or weak PCR bands. The phage isolates negative for PCR product for gene 32 were then retested with a PCR assay based on a set of primers (primer MziaI and caps8) in T4 gene 23 sequence which allows the detection of the pseudo T-even and schizo T-even subgroups of the T4-type phage as well as the T-evens (Tetard et al 2001, Phylogeny of the major head and tail genes of the wide-ranging T4-type bacteriophages. J. Bacteriol. 183 (1): 358–366. All but two phage isolates yielded the diagnostic g23 amplification product (Table 7). The exceptions were the stool isolate JS98 and the Swiss sewage isolate JSL.3. Both phages showed a rather conventional T4-like morphology in electron microscopy. In Southern blots JSL.3 cross-hybridized strongly with another Swiss sewage isolate JSL.5, but only weakly with a few other phages, including T4. JSL.5 cross-reacted weakly with a number of isolates, but notably not with T4.
Materiel and Methods
Experimental Animals:
Eight-week-old C3H male mice (in groups of 5, otherwise noted) were used for infection experiments. Each mouse was kept in a cage with a filtered-top. The drinking water used was sterile Vittel®. Feces were taken once a day and resuspend in one ml PBS (phosphate-buffered saline). To enumerate E. coli, decimal dilutions of the resuspended feces were plated on Drigalski agar (Biorad). Phages were added to the drinking water. 0.5 ml of resuspended feces was filtered and phages were enumerated. Mice were sacrificed when phages were still present in the water and an autopsy was performed. Different organs were taken (colon, duodenum, ileum, jejunum, lymph nodes and liver) and tested for the presence of E. coli and phages.
In Vivo Phage Mix Activity in a Mouse Model:
In the first two weeks, normal water was used then sterile Vittel®. In the third week, a mix of four T4-like phages (JS94.1, JSD.1, JS4 and JSL.6) was added to the drinking water at a final concentration of 103 plaque forming units (pfu)/ml in the first two days then 105 pfu/ml until the end of the week. In the fourth week sterile Vittel® water was given. Phages were added a second time to the water at a final concentration of 105 pfu/ml, then sterile water was given, then finally 107 pfu/ml phages. Phages were monitored all along the trial.
Dose Response Effect of the Phages
As previously, the same phages were given alternatively with water, four mice were used each one receiving a single concentration of phages. The first mice received phages at a final concentration of 103 pfu/ml, the second had the concentration of 104 pfu/ml, and the third received 105 pfu/ml and the last 106 pfu/ml phages. Phages were monitored all along the trial.
Phage Infection Using Axenic Mice
Eight-week-old C3H axenic male mice were used. Sterile Vitte® was used all along the trial as the drinking water. Four mice were used. Two of them were force-fed with E. coli K12 (0.5 ml at a concentration of 108 cfu/ml). Feces were taken once a day and E. coli cells were enumerated on Drigalski plates. One week after, phages were given at a concentration of 105 pfu/ml. Feces were recovered four times a day. Dilutions of the resuspended feces were plated on Drigalski and after filtration, phages were enumerated. Three days later water was given and in the beginning of the next week, a second force-feeding was performed with E. coli ECOR5. Phages were given in the same conditions and the resuspended feces were treated as previously.
Results
In a first step, the E. coli normal flora was investigated. Normal water was given to five mice for two weeks then sterile Vittel® was used. Feces were recovered every day and after resuspension, E. coli cells were counted in a selective media. In conventional mice we found that the normal flora of E. coli vary from 105 to 107 cfu/g (
Lytic Activity in Axenic Mice
The infectiosity of the phages has been tested in an axenic mouse model. Two eight-week-old C3H axenic male mice were colonized with E. coli K12. A level of 108 cfu/g was obtained which is more than in conventional mice but normal for axenic mice (Poulsen et al). When adding phages in the drinking water at a concentration of 105 pfu/ml, within half a day, phages were recovered in the feces at a really high concentration showing that phages lysed K-12 in the intestine and multiplied. Within this half a day, the level of E. coli dropped dramatically. One day later, phages were still at a high level but E. coli could still survive and maintained at a concentration of 105 cfu/g. Even if the phages were removed from water they were still present in the feces and a decline of two logs have been observed within 3 days.
After a force-feeding with ECOR5, which is an E. coli isolated from human normal flora, mice were colonized again at a high level of E. coli (about 1010 cfu/g). ECOR5 is not affected by phage J894.1 already used in this test, and for this reason, colonization was quick and stable. Even if phages were again added to the drinking water, cell count remained the same whereas phage titer reached the concentration given to the mice in the water.
When phages were not any longer present in the intestine, an autopsy was performed and results are shown in
Different Phages Survives the Gut-intestine
Alternatively, sterile Vittel® water was given a week and phages at a final concentration of 107 pfu/ml were given the next week. Four different phages were tested (JS4, JSD.1, JSL.6 and JS94.1 respectively). Four mice were treated with phages and the last mouse was kept as a control and received only sterile-Vittel® water. Phages were monitored all along the trial.
In this experiment, we have given to five mice the T4-like phages composing the mix of the previous trial one by one. All phages reacted in the same manner. Phages were added to the water and a day after they were recovered in the feces at the same concentration as given. When phages were removed from the water, no more phages were found in the feces after 24 hours. Whatever the phage used in the drinking water, the same results are obtained.
If four different concentrations of these phages are given to four different mice, a dose response effect can be noticed. When a very low concentration (103 pfu/ml) is used, phages are present in the feces but not in a stable manner. When increasing this concentration with one log, phages were present in the feces all along the treatment and recovered at a higher level. With one and two more logs the same observations were done.
A feed mixture is made up of about 58% by weight of corn, about 5.5% by weight of corn gluten, about 22% by weight of chicken meal, 2.5% dried chicory, 106 pfu/ml bacteriophage cocktail, salts, vitamins and minerals making up the remainder. The fed mixture is fed into a preconditioner and moistened. The moistened feed is then fed into an extruder-cooker and gelatinized. The gelatinized matrix leaving the extruder is forced through a die and extruded. The extrudate is cut into pieces suitable for feeding to dogs, dried at about 110° C. for about 20 minutes, and cooled to form pellets.
This dry dog food is able to prevent or to treat infectious disorders caused by pathogenic Enteriobacteriaceae such as E. coli or Salmonella infections, such as gastroenteritis, diarrheal or urinary diseases in pets.
A nutritional composition is prepared, and which contains for 100 g of powder: 15% of protein hydrolysate, 25% of fats, 55% carbohydrates (including maltodextrin 37%, starch 6%, sucrose 12%), traces of vitamins and oligoelements to meet daily requirements, 2% minerals and 3% moisture and 106 pfu/ml bacteriophage cocktail
13 g of this powder is mixed in 100 ml of water. The obtained formula is particularly intended for preventing gastroenteritis, diarrhea or urinary diseases in humans.
| Number | Date | Country | Kind |
|---|---|---|---|
| 01204879 | Dec 2001 | EP | regional |
This application is a continuation of International application no. PCT/EP02/12184, filed on Oct. 30, 2002, the entire content of which is expressly incorporated herein by reference thereto.
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| 4957686 | Norris | Sep 1990 | A |
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| 5952033 | Anantharaman et al. | Sep 1999 | A |
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| 6461608 | Averback et al. | Oct 2002 | B1 |
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| Number | Date | Country |
|---|---|---|
| WO 9847521 | Oct 1998 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20040223954 A1 | Nov 2004 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP02/12184 | Oct 2002 | US |
| Child | 10864604 | US |
