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Title:
BACTERIOPHAGE COCKTAIL FOR LISTERIA MONOCYTOGENES AND ESCHERICHIA COLI
Document Type and Number:
WIPO Patent Application WO/2021/080520
Kind Code:
A1
Abstract:
The present invention relates to a bacteriophage cocktail for eliminating Listeria monocytogenes and Escherichia coli Ol57:H7 to be used in food, beverage, food additive, food supplement, feed, antibiotics and disinfectant contents.

Inventors:
GÖNCÜOĞLU MUAMMER (TR)
AYAZ NAIM DENIZ (TR)
EROL İRFAN (TR)
ÇUFAOĞLU GIZEM (TR)
Application Number:
PCT/TR2019/000078
Publication Date:
April 29, 2021
Filing Date:
November 12, 2019
Export Citation:
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Assignee:
ANKARA UENIVERSITESI REKTOERLUEGUE (TR)
International Classes:
A23B4/22; A61K35/76; C12N7/00
Attorney, Agent or Firm:
YAZIR AKSAKALLI, Özlem (TR)
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Claims:
CLAIMS

1. Bacteriophage cocktail for eliminating Listeria monocytogenes and Escherichia coli 0157:H7 to be used in food, beverage, food additive, food supplement, feed, antibiotics and disinfectant contents, characterized in that;

- phages Ml 17, Ml 19 and M135 are used against isolates L. monocytogenes

- bacteriophage M8AEC16 is used against isolates E. coli 0157:H7.

Description:
BACTERIOPHAGE COCKTAIL FOR LISTERIA MONOCYTOGENES AND

ESCHERICHIA COLI

Related Technical Field of the Invention

The present invention relates to a bacteriophage cocktail for eliminating Listeria monocytogenes and Escherichia coli 0157:H7 to be used in food, beverage, food additive, food supplement, feed, antibiotics and disinfectant contents.

State of the Art

Bacteriophages were defined as viruses eating bacteria in early 1900 for the first time. Bacteriophages that are in great amounts (10 3 °-10 32 ) and diversity were isolated from various sources such as sea water, fresh water, soil, gastrointestinal and Genito-urinary tract of animals and humans, leather and milk. Bacteriophages, of which extracellular form (virion) is nonviable and of which sizes are under 1 gm (that can pass through Millipore filters of 0,22 gm that bacteria cannot pass) comprises DNA or RNA in a protein shell (capsid). Most of them comprise a tail functioning to transmit the genetic material to the bacteria cells. Compared to antibacterial agents such as antibiotics and antiseptics, what makes bacteriophages different and functional is that it can be implemented without damaging any living tissues because of the highly selective toxicity. Two types of bacteriophages with a very high host capacity (a species or a few species in the same genus) were presented. One of those is the type that is known as temperate bacteriophages and stays in the prophage form subsequent to transmitting its genetic material to the bacteria and multiplies as the bacteria multiplies and exhibits lysogenic cycle. The latter type is phages that are called as lytic type (virulent to bacteria), transmits its genetic material holding on to the bacteria, multiplies in the bacteria and leaves and lyses the bacteria approximately in 20-60 minutes and infects new bacteria.

Various chemical additives are used nowadays for the purpose of destruction of factors specified in foods nowadays and prevention of their development. Those additives are used in predetermined limits within the scope of regulations because of their structures. On the other hand, people show reactions against food additives and chemical preservatives. Further, those additives may constitute health risks because of their potential that they accumulate on organism.

Various chemical agents are used for the purpose of disinfecting tools, equipment and surfaces. The most important disadvantages of those chemicals are that they destroy bacteria in the medium that are not desired to be destroyed, because they are not factor-specific, that they cause corrosion on surface by forming residue thereon, that they also may cause allergic reaction on sensitive individuals and even may cause serious health problem like cancer.

Antibiotics are used in the treatment of diseases caused by bacterial factors. Intense and wrong use of antibiotics both on humans and in veterinaries causes that bacteria develop a rapid and very powerful resistance to antibiotics, and new antibiotics are not developed, resulting in limiting treatment opportunity. Furthermore, this is interpreted nowadays that the post-antibiotic period has started. Another disadvantage of antibiotics are allergic reactions developing depending on the increase in the number of sensitive individuals. Since antibiotics are not host-specific like bacteriophages subject of the present invention, they destroy also microbiota for a certain extent along with the target bacteria in livings, on which it is to be applied, in other words, they may kill beneficial and critical microorganisms for livings along with harmful ones. Separate phage preparations for L. monocytogenes and E. coli 0157:H7 developed in this area may be found in some countries, excluding Turkey. In addition to this, said preparation are not combined as mentioned above, and they only affect on a single factor. Moreover, when considering the host capacity of the bacteriophages, their effects on local strains in Turkey may be limited.

Consequently, there is no other product that is similar to the product provided by the present invention. In general, phages used around the world can be used for a single microorganism. A phage preparation that affects on two different pathogen bacteria simultaneously has not been used so far. Furthermore, it is detected that cocktails-wise mixtures, in which more than one phage are used in different matrices such as disinfectant, feed and food, are not used commercially.

In addition to this, the inventive product is a cocktail product in which phages against both factors exist together, wherein there is no similar preparation around the world. Moreover, the present product is the first domestic and national phage. There is no other patent relating the product specified in the present invention specific to both L. monocytoegens and E. coli 0157:H7 Further, every bacteriophage is characteristic, wherein every novel bacteriophage is a novel patent subject. The bacteriophages used in this cocktail are new phages isolated within the scope of the present invention.

Technical Problems Intended to be Solved with the Present Invention

There is a bacteriophage use in different mediums and all studies and results obtained are performed over a single microorganism. There is no other phage mixture specific to two different microorganisms like in this invention. By means of this product both a Gramnegative and a Gram-positive microorganism is simultaneously targeted.

There is no use of the phages in the related sector in Turkey. However, lytic phages were included by FDA (Food and Drug Administration) in the class GRAS (Generally Recognize as Safe). Therefore, there is use of the bacteriophages with its specified purposes around the world. As mentioned above, the present phage preparations few in number are specific to a single type of bacteria. Further, various chemical additives are used in eliminating bacteria from foods. Antibiotics are used in the treatment of those diseases caused by the bacteria in humans and animals. Disinfectants are used in eliminating bacteria from surfaces. Problems pertaining to the specified current technique is listed below;

- Reactions shown by people against food additives and chemical preservatives,

- Health risks caused by chemical food additives,

Corrosion caused by chemical disinfectants,

- Residue and environmental pollution, health risks and allergic irritations etc. caused by chemical disinfectants,

Antimicrobial resistance developed in bacteria because of use of antibiotics,

- Health risks developed against antibiotics, allergic reactions, cancer, kidney-lung damages,

- Destruction of microbiota for a certain extent along with target bacteria in livings because of use of antibiotics, in other words, killing beneficial and critical microorganisms for livings in additions to harmful ones,

- Causing kidney damage, called as hemolytic uremic syndrome (HUS) by stimulating the toxin release particularly in children because of use of antibiotics in the treatment of E. coli 0157:H7 on humans, and non-preference because of renal impairment caused thereby.

On the contrary, the inventive phage preparation is an absolutely natural product, wherein it comprises no chemical additives. Lytic bacteriophages are included by FDA in the list of GRAS, wherein it is considered as safe in terms of human, animal and environment life. Further, it was observed no negative findings in terms of health in in vivo acute toxicity analysis carried out in this invention. In the infections E. coli 0157:H7, in which use of antibiotics is considered as contraindicated, it was developed for the purpose of providing treatment opportunity by means of lysing the factor by use of bacteriophage. Bacteriophages are host-specific, wherein it exhibits a lytic effect on only target microorganism species. It constitutes no positive or negative effect on other bacteria in the medium. Antibiotic resistance limits use of antibiotics day by day, wherein the antibiotics, to which resistance is developed, cannot be replaced with new ones. At this point, alternative treatment opportunities are needed. The present phage preparation is developed as an alternative treatment opportunity to the antibiotics in L. monocytogenes and E. coli 0157:H7 infections. The inventive phage preparation is an absolutely natural product, wherein it comprises no chemical additives. It is safe in terms of human, animal and environment life. The inventive bacteriophages cause no corrosion on surfaces.

Description of the Invention

The present invention relates to joint use of lytic bacteriophages specific to L monocytogenes and E. coli 0157:H7 that constitute a crucial importance for public health. Thanks to the product obtained, it is ensured to eliminate said bacteria on food, feed, beverage and different surfaces. It can be used for the treatment by means of killing causes of disease in living organism in case of diseases on humans and animals caused by said bacteria.

Consequently, this mixture can be used directly and/or as additives in food and beverage industry, feed and animal feeding industry, slaughterhouses, every sort of hand and facial cleansing materials and even in pharmaceutical industry. The present invention relates to a cocktail (mixture) comprised of 4 bacteriophages intended for the destruction of L. monocytogenes and E. coli 0157:H7 to be used in the specified contents. Detailed Description of the Invention:

It is determined the specificity of phages by means of all genome sequence analyses to be performed on bacteriophages. In the method; all genome sequence analyses of the present bacteriophages were performed, and a cocktail-mixture were prepared by combining phages determined for each bacterium. The obtained cocktail can be used in the content of food, beverage, food additive, food supplement, feed, antibiotics, disinfectants. It was detected that the inventive phages were lytic effective in the widest range on L. monocytogeneses and had no acute effect as a result of in-vivo examination and the bacteriophages were of the family Myoviridae and the order Caudovirales in the TEM (Transmission Electron Microscope) imagings. In the preliminary studies conducted so as to determine the phage efficiency; a very significant level of reduction like respectively 2,14 log cuf/g and 3,30 log cuf/ml in the number of L. monocytogenes in food models of meatball and chicken leg was achieved by means of two phage cocktails prepared from the specified phages.

- It was detected that they were lytic effective on E. coli 0157:H7 and had no acute effect as a result of in-vivo examination and the bacteriophages were of the family Myoviridae and the order Caudovirales in the TEM (Transmission Electron Microscope) imagings. In the preliminary studies conducted within this scope, it was specified that the bacteria reduction at a level of 2,14 log cuf/g was achieved.

By means of this product, the reduction of both Gram-negative and Gram-positive microorganism to 3 logarithms can be achieved. There is a high host-target specification. It can show its effect without damaging any living tissue. It constitutes no toxic effect on human, animals and environment. There is no risk of residue, mainly including chemical substances. It can be used repeatedly. There is no time limitation on its use. There is no dosing limitation. It can be used in every stage in the related areas. It can be used on different products and its range of use is pretty wide.

Existence of L. monocytogenes lytic bacteriophage in slaughterhouse waste waters and plaque characterization 16 different lysteriophage out of 11 of 160 slaughterhouse waste waters collected in twenty mounts were isolated, wherein the reference stains, on which they showed lytic effect, were determined.

It was analyzed the dilution and plaque appearance of the bacteriophage isolate forming plaque on a double-layer petri dish by means of spot cultivation technique. It was determined that morphologies of plaques formed as a result of the cultivation to the double-layer agars so as to detect the sensitivity of L. monocytogeneses to bacteriophages were clear zone or weak zone fields in a diameter of 1-2 mm. 8 phages (LMF-M61, 63, 83, 116, 117, 119, 131 and 135) forming a clear zone in diameter of 1-2 mm were selected so as to be used in biocontrol studies and the characterization process was continued (Table 1). Table 1. Lytic-effective bacteriophages to L. monocytogenes isolated from slaughterhouse waste waters.

Reference on which they show lytic

Sample effect

Slaughter Bacteriophag Plaque

L. monocytogenes strains house e isolate code characteristics

521 47 47

Period No (9111N71557644 N7143 4 5 6

LMF-M5-1 z - z

July-August M5 Kinkkale (A) weak

LMF-M5-2 z - z

LMF-M61-1 clear zone in + z + + + +

M61 Kinkkale (F) diameter of 1-2

December- LMF-M61-2 + z + + + + mm

January-

LMF-M63-1 clear zone in + + + + +

February

M63 Kinkkale (F) diameter of 1-2

LMF-M63-2 + + + + + mm

LMF-M82-1 z

M82 Kinkkale (F) weak

LMF-M82-2 z - -

March-April- LMF-M83-1 clear zone in + + + + + +

May M83 Kinkkale (F) diameter of 1-2

LMF-M83-2 + + + + + + mm

M86 Kinkkale (F) LMF-M86-1 weak z clear zone in

Ml 16 Bolu (G) LMF-M116 diameter of 1-2 z + + + + + mm clear zone in

June Ml 17 Bolu (G) LMF-M117 diameter of 1-2 + z + + + + + mm clear zone in

Ml 19 Bolu (G) LMF-M119 diameter of 1-2 - z + mm clear zone in

Ml 31 Bolu (G) LMF-M131 diameter of 1-2 + + + z + + z mm

September clear zone in

Ml 35 Bolu (G) LMF-M135 diameter of 1-2 + + + z + + z mm M Slaughterhouse waste water; 1911: L. monocytogenes ATCC 19111 (serotype l/2a); N7155: L. monocytogenes N7155 (serotype l/2b); 7644: L. monocytogenes ATCC 7644 (serotype l/2c); N7143: L. monocytogenes N7143 (serotype 3a); 5214: L. monocytogenes NCTC 5214 (serotype 4a), 475: L. monocytogenes RSKK 475 (serotype 4b); 476: L. monocytogenes RSKK 476 (serotype 4c); z: weak lytic effect

Comparison between restriction enzymes and Phage DNA It was specified in the present invention that phage DNAs treated with Clal and Sad enzymes (NEB, New England Biolabs, Ipswich, MA, USA) and imaged on the electrophoresis gel comprised 3 different profiles. Accordingly, LMF-M61, 63, 83, 116 and 117 were determined as profile 1, LMF-M119 were determined as profile 2 and 135 was determined as profile 3. Major gene profiles from bacteriophages

In the examination of the lysteriophage isolates performed with real time PCR, hlyA, actA, inlA, inlB, inlC, inlJ, plcA, plcB, fbpA and fri were detected in all of them, wherein vip was not determined in LM-M83 numbered phage, even though it could be found in all phages. Data pertaining to gene profiles of phages selected so as to tested on food models are presented on Table 2. Table 2. Major gene profiles of bacteriophages selected for phage cocktail

App. Cq values

Gene Ml 17 Ml 19 M135 M61 M83 actA 19,87 16,73 19,45 18,32 19,53 jbpA 25,89 22,65 21,71 23,87 24,94 fri 28,50 24,96 27,55 28,44 28,37 hlyA 18,07 16,75 18,04 17,07 18,12 inlA 18,84 15,26 17,38 16,90 18,09

MB 18,33 15,61 17,24 16,26 17,57

MC 18,58 15,95 16,88 16,67 17,71

MJ 17,82 15,94 17,11 16,33 18,21 plcA 17,29 14,83 16,54 15,67 17,55 plcB 23,00 20,33 20,85 21,44 22,06 spoG 18,30 16,30 17,14 16,43 18,42 vip 32.10 26.33 27.14 39.56 N/A

Toleration characteristics of bacteriophages to acidic pH and various temperature values

In order to determine the toleration capability of the LMF-M61, M63, M83, Ml 16, Ml 17, M119, M131 v3 M135 numbered bacteriophages to the acidic pH values, wherein they were determined to have a high lytic effect, pure bacteriophage suspensions were adjusted to pH values of 3,0 and 4,0 with lactic acid, kept in the room temperature for an hour and spot cultivations were performed at 0th, 10th, 30th and 60th minutes of the experiment. Accordingly, bacteriophages were detected to be losing their liveliness rapidly at both pH values as of minute 10 mark and. therefore, they were determined to be incapable of tolerating pH values of 3.0 and 4.0.

In order to determine the toleration capabilities of bacteriophages to various temperatures, pure bacteriophage suspensions were subjected to the temperatures of 50 and 60°C for an hour and bacteriophage count at 0th, 10th. 30th. and 60th minutes was performed on the double-layer LB agar. Accordingly, it was observed that all of the bacteriophages lost rapidly their liveliness as of 10th minutes of incubation of 60°C. However, it was detected that all phages kept their activities at 10th minutes of the incubation of 50°C and formed a plaque on the double-layer petri, and that the LMF-M63, M83, Ml 17, Ml 19 and M135 numbered phages kept exhibiting the lytic effect by tolerating said temperature. It was detected that no phage formed the plaque on the petri at 60th minutes of the incubation and therefore the M63, M83, Ml 17, Ml 19 and M135 numbered phages tolerated the temperature of 50°C hardly at 30th minutes.

Determination of bacteriophage host speciflty and lytic effect spectrum

In the present invention, 31 units of strains of Z. monocytogenes isolated from sheep carcass swab and chicken neck skin samples and 7 units of reference strains (Z. monocytogenes ATCC 19111 [serotype l/2a], N7155 [serotype l/2b], ATCC 7644 [serotype l/2c], N7143 [serotype 3a], NCTC 5214 [serotype 4a], RSKK 475 [serotype 4b] and RSKK 476 [serotype 4c]) of L. monocytogenes belonging to 32 units of L. monocytogenes (serotype l/2a, l/2b, l/2c and 4b) and further 70 units of L. monocytogenes in total, including reference strains of E. coli 0157:H7, E. faecium, E. faecalis, S. Typhimurium, A. hydrophila, Y. enterocolitica, R. equi, C. perfringens and S. aureus and 9 units of non-Z. monocytogenes were used so as to determine the host specifities and lytic effect profiles of the bacteriophages isolated in the study. None of the bacteriophages obtained showed any lytic effect on the reference strains of non-Z. monocytogenes. It was detected that the bacteriophages had lytic effect profiles on Z. monocytogeneses used in the study in a different range. In the light of those data, it was detected that LMF-M61, M83, Ml 17, Ml 19 and Ml 35 had the lytic effect on Z. monocytogeneses used in the study in the widest spectrum.

Development of nalidixic acid resistance in the L. monocytogenes ATCC 19111 strain, evaluation of the phage sensitivity and preparation of inoculum

L. monocytogenes ATCC 19111 strain to give nalidixic acid resistance that increases as a selective feature (so as to give a characteristic feature to the strain used in food models) was given resistance to nalidixic acid of 50 pg/ml with passages carried out on the LB (Tryptone [pancreatic digest of casein] 10 g/1, yeast extract 5 g/1, NaCl 5 g/1, agar 15 g/1) agar comprising nalidixic acid in the increasing concentration ((10, 20, 30, 40 and 50 pg/ml). The reference Z. monocytogenes ATCC 19111 (NA-LM19111), that was made acid-resistant to the nalidixic acid enriched for a night at 37°C in TSB was counted on the MOX agar (NA- MOX) both comprising 50 pg/ml NA and not comprising 50 pg/ml NA and its resistance developed was confirmed. Accordingly, it was determined that L. monocytogenes colony was reproduced in the same number (2,2 x 10 10 cuf/ml) as a result of the incubation on the both petris and therefore, the resistance developed was approved (Table 3). Table 3. Reproduction potential of L. monocytogenes isolate that developed an increasing resistance to nalidixic acid on the NA-MOX and MOX agars.

NA-LM 19111 counting (cuf/ml) nt curves of the bacteriophages selected for the phage cocktail

The in-vitro lytic activities of the candidate phages specified for the cocktail to the host NA-LM19111 strain was evaluated with performing countings of phage and bacteria from samples taken in different periods (0., 30., 60. and 90. min. and 3., 6., 24. and 48. hours) with adding (Mol, Multiplicity of Infection, phage number/bacteria) with different multiplicities of infection to the L. monocytogenes strain in the log-reproduction phase, enriched for a night at 37°C in TSB. Within this scope, L. monocytogenes cultures diluted at the level of 10 2 , 10 4 and 10 6 cuf/ml with TSB in line with OD600 values were infected such that their final titre was 10 8 pob /ml (plaque forming unit/ml) and were incubated for 48 hours at 37°C. Accordingly, counting results of L. monocytogenes are shown in Table 4 and 5, and counting results of the bacteriophage are shown in Table 6.

Table 4. OD values of NA-LM 19111 in 600 nm according to serial dilutions performed

Dilution rate 600nm OD NA-LM 19111 numbers (cuf/ml)

0,0 1.128 6,6 x 10 10

0,1 0.232 6,6 x 10 9

0,01 0.092 6,6 x 10 8

0,001 0.018 6,6 x 10 7

0,0001 0.001 6,6 x 10 6 Table 5. Counting L. monocytogenes throughout the preservation period as a result of two different phage cocktail applied to different bacteria concentration in TSB

Period 2 phages (M61+M83) applied 3 phages (M117+M119+M135) applied

NA-LM 19111 numbers (cuf/ml)

Oth min. 1,9 x 10 2 1,5 x 10 4 8,0 x 10 6 1,9 x 10 2 1,5 xlO 4 8,0 x 10 6 30 th min 3,0 x 10 1 5,0 x 10 4 5,0 x 10 1 2,2 x 10 4 60th min 2,0 x 10 1 2,7 xlO 3 1,0 x 10 3 90th min 1,0 x 10 1 2,1 x 10 3 3rd hour 2,0 x 10 1 6,0 x 10 2 6th hour 1,0 xlO 1 2,0x10' 24th hour 4,0 x 10 2 6,6 x 10 3 48th hour 2,0 x 10 6 3,0 x 10 6

No detection

Table 6. Bacteriophage countings throughout the preservation period in different bacteria concentrations in TSB

NA-LM 19111 Bacteriophage counting (cuf/mi)

3 phages (LMF-M117, LMF-M119

2 phages (LMF-M61 and LMF-Ms83) and LMF-M135)

LM ATCC 19111 (cuf/ml)

Oth min. 5x 10 8 5x 10 8 5 x 10 8 7x 10 8 7x 10 8 7 x 10 s 30th min. 5x 10 8 5 x 10 8 5 x 10 8 7x 10 8 7x 10 s 7x 10 8 60th min. 3x 10 9 5x 10 9 7x 10 9 4x 10 9 6 x 10 9 7x 10 9 90th min. 6x 10 9 8x 10 9 9x 10 9 6x 10 9 8x 10 9 9 x 10 9 3rd h. 1 x 10'° 1,5 x 10'° 3 x 10 10 1 x 10'° 1,6 x 10 10 3 x 10 10

6th h. 4x 10 10 2x 10'° 3 x 10 10 4x 10‘° 2x 10 10 3 x 10'° 24th h. 5x 10 10 4x 10'° 6 x 10 10 5x 10 10 5 x 10 10 6x 10 10 48th h. 3 x 10 10 2 x 10*° 4 x 10 10 3 x 10 10 4x 10'° 5 x 10 10 Detection of phage-insensitive mutant colonies as a result of bacteriophage sensitivity test of L. monocytogenes isolates

The spot cultivations were performed on the double-layer agars in the result of determination analyses of bacterio-sensitivity profiles, and the number of the phage-insensitive mutant L. monocytogenes strains that developed resistance to bacteriophage with which it was cultivated was detected, and then it was observed that there was no difference between their colony morphologies, when comparing their colony morphologies with L. monocytogenes strains that did not develop the resistance.

Determination of the morphology of bacteriophages with TEM

Lysate (>10 9 pob/ml) of the phages determined so as to form the cocktail was centrifuged in 21.000 x g in ammonium acetate of 0,1 mol/L for two hours and the phages suspended in SM buffer were dyed with aqueous uranyl acetate solution of 2 % in carbonized copper grids. The dyed bacteriophages were observed at 800 kY with Transmission Electron Microcopy (TEM) and then they were photographed. It was detected that the bacteriophages were comprised of head, tail and holding plaque and they did not comprise pili. In the imaging performed, it was detected the bacteriophage in two different morphologies. It was determined that head diameter of the phages in the first morphology was of 67,96 and 93,32 nm and the tail length was of 190,08 - 304,15 nm and the tail width was of 19,95-26,42. It was also determined that the phages characterizing the latter morphology were similar with the first phage in terms of structure, that its head diameter was almost the same with the first type, however its tail length was approximately half of the other type (150,88 nm) and its tail width was thicker (30,17 nm). It was detected in the performed imaging that the order Caudovirales was from the family Myoviridae according to the morphological characteristics exhibited by the bacteriophages.

In-vivo toxicity test

It was detected that LMF-M61, M83, Ml 17, Ml 19 and M135 phages applied separately on the animals as a result of the analysis did not exhibit any acute toxic effects. It was further reported in the pathological examination that it was encountered no pathological results on esophagus, stomach, duodenum, jejunum, ileum, cecum and colon of animals.

Preparation of the bacteriophage cocktail

It was decided to prepare two different phage cocktails with LMF-M61, M83, Ml 17, Ml 19 and Ml 35 numbered phages that exhibited the lytic effect in the widest spectrum to the L. monocytogenes isolates analyzed as a result of the phage sensitivity profile tests carried out and that were determined to comprise no acute toxic effect in the oral toxicity test. The cocktail comprised of LMF-M61 and M83 obtained from waste waters of the cattle and sheep slaughterhouses was prepared to be used in the meatball model and the cocktail comprised of LMF-M117, Ml 19 and Ml 35 numbered phages isolated from waste waters of poultry slaughter houses was prepared to be used in food model. Each phage selected for this was multiplied in L. monocytogenes host cell cultures enriched (Nucleus EN120) at 37°C in TSB one night before, was incubated at 37°C for one night, was filtered through sterile Millipore filter in diameter of 0,22 pm the day after and was transferred into sterile tubes. Each selected phage was enriched separately in the host cell and was mixed immediately before the use stage in food model as a biocontrol agent such that each was approximately of the number of 10 9 pob/ml and of the equal sizes.

Biocontrol of L. monocytogenes with the bacteriophage cocktail in food models

It was observed in the chicken leg food model that LM regressed as of 1 st hour in the group contaminated at a level of 2,32 log cuf/ml such that it could not be detected; underwent a reduction at a level of 2,34 log cuf/ml as of 1st hour in the group contaminated at a level of 4,6 log cuf/ml; the highest reduction was of 3,30 log cuf/ml as of 3rd hours in the group contaminated at a level of 6,6 log cuf/ml. It was detected in the study that a significant reduction was achieved in the number of LM compared to the initial contamination by submerging the chicken legs in the cooling water with bacteriophage and it was not observed a significant re-increase in the number of bacteria in preservation at 4°C. It was concluded within this scope that the cocktail comprised of three different phages were effective in LM biocontrol. Amount of the reduction seen on L. monocytogenes in the chicken leg food model and bacteria counting results in the course of preservation are presented in Table 7 and 8.

Table 7. Number of L. monocytogenes according to the preservation periods in the control and experiment groups in the chicken leg food model

Period Number of L. monocytogenes - log (cuf/g)

Control Group Group with phage

10 2 10 4 10 6 10 2 10 4 10 6

0th min. 2,32 4,60 6,60

1st Hour 2,30 4,34 5,30 <2,00* 2,00 2,30 3rd hour 2,70 3,20 5,30 < 2,00* 2,00 2,00

6th hour 2,78 3,00 5,84 < 2,00* 2,30 3,00

24th hour 3,00 3,00 5,84 2,3 2,30 3,04

*: No detection. Detection limit: log 2,00 cuf/ml

Table 8. Amount of the reduction seen in L. monocytogenes in the chicken leg food model.

Initial Initial Mol Initial Temper Average reduction

Bacteriophag (log [pob/cuf]/g) Number of ature (log cuf/g) e bacteria (°C) l h 3 h 6 h 24 h

(log pob/g) (log cuf/g)

8/78 6 232 0,3-2,32* 0,7-2,70* 0,78-2,78* 0,7

8,78 4,18 4.60 2,34 1,20 0,7 0,7

8,78 2,18 6.60 3,00 3.30 2,84 2,80

*: Log 2,00 kob/ml being the detection limit of the minimum reduction value as a bad scenario for the undetected bacteria countings was used and the number of bacteria in the control group as a good scenario was used, wherein the reduction was carried out between said values.

As a result of testing data obtained through phage tests of the chicken leg food model by One Way Analysis of Variance (ANOVA) and Tukey HSD test; when the reduction in the number of L. monocytogenes in groups of Mol 6,46, Mol 4,18 and Mol 2,18, in which bacteriophage cocktail was added, was examined based on the preservation periods in different hours, it was found that the reduction difference between hours was statistically significant (p<0,05). Accordingly, the highest reduction was achieved as 3,30 log cuf/g as a result of preservation for 3 hours at 4°C in Mol 2,18. However, it was determined that the difference between Mol values in the reduction achieved in the number of L. monocytogenes depending on the bacteriophage application in the chicken leg food model was insignificant (p>0.05).

As a result of the study pertaining to the meatball food model, it was determined that LM fell down below the detection limit as of 1 st hour in the group contaminated with the bacteria of 2,84 log cuf/g; the reduction of 2,14 log cuf/g was realised as of 1st hour in the group contaminated with the bacteria of 4,14 log cuf/g; the highest reduction of 1,96 log cuf/g was performed as of 3rd hours in the group contaminated with the bacteria of 6,9 log cuf/g. In the study, the reduction of 2,14 log cuf/g in the number of LM were achieved by means of two different bacteriophages, wherein it was detected that the phage cocktail used constituted an effect in terms of biocontrol. It was detected that the reduction observed was associated with the number of bacteriophages and LM in the medium and the reduction in the number of LM in meatball was increased with the MOI increase. Amount of the reduction seen on L. monocytogenes in the meatball food model and bacteria counting results in the course of preservation were detected.

Table 9. Number of L. monocytogenes according to the preservation periods in the control and experiment groups in the meatball food model

Period Number of L. monocytogenes - log (cuf/g)

Control Group Group with phage 10 2 10 4 10 6 10 2 10 4 10 6

0th min. 2J84 6,90 1st Hour 2,78 4,14 6,20 <1,00* 2,00 4,81 3rd hour 2,70 4,07 6,28 <1,00* 2,39 4,32 6th hour 3,11 4,04 6,30 <1,00* 2,39 4,36 24th hour 3,28 4,52 6,46 2,84 2,81 4,70

*: No detection. Detection limit: log 1,00 cuf/ml Table 10. Amount of the reduction seen in L. monocytogenes in the meatball food model.

Initial Initial Mol Initial Temper Average reduction

Bacterioph (log [pob/cuf]/g) Number ature (log cuf/g) age of bacteria (°C) 1 h 3 h 6 h 24 h

(log pob/g) (log cuf/g)

8,78 5^94 2M 1,78-2,78* 1,70-2,70* 2,11-3,11* (f44

4

8,78 4,64 4.14 2,14 1,68 1,65 1,71

8,78 1,88 6.90 1,39 1.96 1,94 1,76

*: Log 1,00 cuf/ml being the detection limit of the minimum reduction value as a bad scenario for the undetected bacteria countings was used and the number of bacteria in the control group as a good scenario was used, wherein the reduction was carried out between said values.

As a result of testing data obtained through phage tests of the meatball food model by One Way Analysis of Variance (ANOVA) and Tukey HSD test; when the reduction in the number of L. monocytogenes in groups of Mol 5,94, Mol 4,64 and Mol 1,88, in which bacteriophage cocktail was added, was examined based on the preservation periods in different hours, it was found that the reduction difference between hours was statistically significant (p<0,05), wherein the statistical analyses of the comparison between hours were stated on Tables 22, 23, 24, 25, 26 and 27. Accordingly, the highest reduction was achieved as 2,14 log cuf/g as a result of preservation for 1 hours at 4°C in Mol 4,64. However, it was determined that the difference between Mol values in the reduction achieved in the number of L. monocytogenes depending on the bacteriophage application in the meatball food model was insignificant (p>0.05).

Existence of the lytic bacteriophages E. coli 0157 :H7 in the slaughterhouse waste waters, profiles of bacteriophage sensitivity of the isolates E. coli 0157:H7 and detection of morphologies of the bacteriophages on TEM

It was performed a lytic bacteriophage isolation specific to E. coli 0157:H7 from the samples of slaughterhouse waste from. To this end, it was used the reference strains E. coli 0157:H7 ATCC 43895, E. coli 0157:H7 ATCC 43888, E. coli 0157:H7 ATCC 35150, E. coli 0157:H7 NCTC 12900 and E. coli 0157:H7 ECl. In the invention, thirty-one bacteriophages virulent (lytic) to E. coli 0157:H7 in total were isolated from 9 (37,5%) of 24 slaughterhouse waste waters in total. When the existence of bacteriophage in the slaughterhouse waste waters was evaluated seasonally, it was detected that the prevalence in warm months (May-October) (5/12; 41,7%) was higher than cold months (4/12; 33,3%) (Table 11). 31 bacteriophages obtained were used in determining profiles of the bacteriophage sensitivity of the isolates E. coli 0157.H7 subsequent to their purification. Results of the bacteriophage sensitivity test were analyzed, wherein the bacteriophages Ml AEC88, M23A-CEC16 and M4A-BEC88, respectively, did not demonstrate a lytic effect against the bovine and slaughterhouse waste water isolates 12, 13 and 19 E. coli 0157:H7 Furthermore, the bacteriophages M 12A-BEC 16 showed a more lytic effect on the two isolate E. coli 0157:H7 along with the isolate E. coli 0157:H7- coded 210KB. The other 23 bacteriophages obtained in the study demonstrated a more lytic effect on one isolate E. coli 0157:H7, four of those demonstrated a more lytic effect on the isolate 1 E. coli 0157:H7- in addition to the isolate 1 E. coli 0157:H7. Therefore, considering also the reference strains in addition to the bovine and slaughterhouse waste water isolates, it was convenient that the bacteriophages M1AEC00, M8AEC16, M9AEC95 and M12AEC50 were used in preparing the bacteriophage cocktail demonstrating the lytic effect in the widest spectrum against the isolates E. coli 0157:H7. According to results of the bacteriophage sensitivity test, it was approved that the cocktail comprised of 4 bacteriophages obtained demonstrated the lytic effect against all isolates E. coli 0157:H7+/H7-. It was detected that the bacteriophages were of the family Myoviridae and the order Caudovirales in the TEM (Transmission Electron Microscope) imagings.

Table 11. Bacteriophages isolated from the samples of slaughterhouse waste water and reference strains used in their isolation, wherein bacteriophages demonstrated a lytic activity

Sample no Month Bacteriophage Reference strain it demonstrated a lytic code effect

MAS 1 July M1AEC88 E. coli 0157:H7 ATCC 43888

M1AEC00 E. co/r 0157:H7 NCTC 12900

M1BECOO

MAS 4 October M4AEC88 E. coli 0157:H7 ATCC 43888

M4BEC88

MAS 8 February M8AEC16 E. co/i 0157:H7 EC16

M8BEC16

M8CEC16

M8DEC16

M8EEC16

MAS 9 March M9AEC95 E. coli 0157:H7 ATCC 43895

M9BEC95

M9CEC95

M9DEC95

M9EEC95

MAS 11 May Ml 1AEC16 E. coli 0157:H7 EC16

Ml 1BEC16

Ml 1CEC16

Ml 1DEC16

MAS 12 June M12AEC00 E. coli 0157:H7 NCTC 12900

M12BEC00

M12AEC16 E. coli 0157:H7 EC16

M12BEC16 M12AEC50 E. coli 0157:H7 ATCC 35150

MAS 18 December M18AEC00 E. coli 0157:H7 NCTC 12900

M18BEC00

M18CEC00

MAS 21 March M21AEC00 E. coli 0157:H7 NCTC 12900

MAS 23 May M23AEC16 E. coli 0157:H7 EC16

M23BEC16

M23CEC16 Table 12. Profiles of bacteriophage sensitivity of E. coli 0157:H7 + /H7 isolated from samples of bovine and slaughterhouse waste water

The strain E. coli 0157:H7 ATCC 43895 (EC95)-effective bacteriophage numbered M8AEC were used so as to be employed in the experimental studies. The activity of the bacteriophage to the strain E. coli 0157:H7 ATCC 43895 were evaluated in-vitro, and to this end, NAEC95 cultures diluted at the level of 10 4 and 10 6 cuf/ml with TSB in line with OD600 values were infected such that their final titre was 10 8 pob /ml (plaque forming unit/ml) and were incubated for 48 hours at 37°C. 10 fold dilutions prepared subsequent to determining the reductions in the values Oϋ ό oo from samples taken in the specified periods for NAEC95 counting were cultivated in NA-CT-SMAC through the double serial drop plaque method and petris were incubated at 37°C for a night. Subsequent to the incubation, colonies were counted and the reductions in cuf/ml were recorded.

Samples taken in the specified periods for the phage counting were counted with double serial spot cultivations on layered agars prepared with NAEC95 by means of filtering through sterile Millipore filters in diameter of 0.22 pm, as stated above. It was observed an increase in the phage amount as a result of countings.

Biocontrol of E. coli 0157:H7' with the bacteriophage cocktail in food models

Raw meatball samples of 25 gr prepared with "meatball mix (set)" and bovine ground meat without fat as two groups, namely with phage (Group F) and without phage (Group K), were contaminated with NAEC95 as being 10 4 or 10 6 cuf/g. As a result of the countings, different amounts of reductions were observed in the number of bacteria samples in the control group in comparison with the bacteriophage-added group in accordance with the parameters of initial multiplicity of infection (MOI), preservation temperature and preservation periods. Accordingly, as a result of the countings, the highest reduction up to 2,09 log was observed in the analysis of 5th hours in the sample, to which 5,59 MOI bacteriophage was applied in the group F and which was preserved at 22°C. Even though it was understood that different preservation temperature and infection multiplicity of the phage did not comprise a statistical significance, it was detected (p < 0.05) that the effect of M8AEC16 on E. coli 0157:H7 significantly increased statistically in comparison with the lower multiplicities of infection (4,17 log and 2,38 log), in case the initial infection multiplicity of the phage was high (5,59 log)· Table 13. Decrease in the number of bacteria according to the infection multiplicity, preservation temperature and preservation period formed in the experiment samples prepared.

Initial Multiplicity of Preservation Overall decrease (log cuf/g) Infection (Mol) temperature lh 3h 5h 24h 48h

(log [pob/cufj/g) (°C)

5^59 4 1,79 1,85 1,96 1,72

22 1,85 1,82 2,09 1,41

4,17 4 1,00 1,02 0,87 1,11

22 0,74 1,52 1,47 0,91

2,38 4 0,70 0,69 0,05 0,84

22 0,73 0,82 0,91 0,98

The fact that raw meatballs are made with products such as raw bovine meat, fresh vegetables and spices capable of contaminating with E. coli 0157:H7 puts this traditional food in a critical situation in terms of public health. Use of bacteriophage specific to E. coli 0157:H7 in making raw meatballs provides an obvious effect, particularly when the contamination level of raw meatballs is low. In addition to this, the development of bacteriophage-resistant colonies limits this effect particularly because of the effect of storage conditions. In recent years, it was found that the isolates E. coli 0157:H7 demonstrated a significant resistance to antibiotics. Moreover, the antibiotic therapy may increase the risk of HUS formation in human due to the fact that cells are lysed, the gene expression is increased because of stress and Shiga toxin release is increased in intestine channel. Therefore, it is crucial to develop an alternative therapy to antibiotics and protection precautions in infections E. coli 0157:H7.

In the present invention, it was determined through in-vitro studies so as to research opportunities to use bacteriophages for the purpose of biocontrol in foods that it had a lytic effect on E. coli 0157:H7 in the widest spectrum, and the activity of the phage M8AEC16 was researched in the biocontrol of Escherichia coli 0157:H7 in raw meatballs, thereby achieving successful results.

This mixture can be used directly and/or as additives in food and beverage industry, feed and animal feeding industry, slaughterhouses, every sort of hand and facial cleansing materials and even in pharmaceutical industry by means of eliminating the above-mentioned drawbacks originated from chemical additive substances, disinfectants and antibiotics. By means of the present invention, the reduction of both Gram-negative ( E . coli 0157:H7) and Gram-positive (L. monocytogenes ) microorganism to 3 logarithms in very various mediums- matrices can be achieved.

The present invention has a high host-target specification. It can show its effect without damaging any living tissue. It constitutes no toxic effect on human, animals and environment. There is no risk of residue, mainly including chemical substances. It can be used repeatedly. There is no time limitation on its use. It can be used repeatedly. Its effect may be observed in 30 minutes after its application. There is no dosing limitation. It can be used in every stage in the related areas. It can be used on different products. Its range of use is pretty wide. It is absolutely natural and comprises no chemical substance.