CN115927205A

CN115927205A - Cross-species schizolysis type Klebsiella pneumoniae phage and application thereof

Info

Publication number: CN115927205A
Application number: CN202210898150.3A
Authority: CN
Inventors: 杜新永; 刘长太; 崔耐; 马如霞; 李先胜
Original assignee: Qingdao Runda Biotechnology Co ltd
Current assignee: Qingdao Runda Biotechnology Co ltd
Priority date: 2022-07-28
Filing date: 2022-07-28
Publication date: 2023-04-07

Abstract

The invention belongs to the technical field of biology, and particularly relates to a cross-species lytic bacteriophage and application thereof. A cross-species schizolysis type Klebsiella pneumoniae is Klebsiella pneumoniae RDP-EC-20056 which is preserved in China general microbiological culture Collection center (CGMCC) at 20 months in 2022 with the preservation number of CGMCC No.45090. The Klebsiella pneumoniae RDP-EC-20056 disclosed by the invention has extremely strong cracking property on Klebsiella pneumoniae, escherichia coli and salmonella of different species, has higher temperature tolerance, can be used for efficiently treating Escherichia coli and salmonella diseases caused by pathogenic bacteria in livestock and poultry breeding, can be used as a disinfectant, and provides a phage source for industrialized, large-scale and programmed production of phages and treatment of livestock and poultry breeding bacterial diseases caused by Escherichia coli and salmonella.

Description

Cross-species schizolysis type Klebsiella pneumoniae phage and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a cross-species lytic bacteriophage and application thereof.

Background

Klebsiella pneumoniae is a saprophytic bacterium of human and other mammals, is distributed in gastrointestinal tract, skin and nasopharynx, is facultative anaerobe, is a resident flora or transient flora (particularly in gastrointestinal tract), and is widely present in various environmental ecoparts (soil, water, etc.). It is one of the most common clinical important conditioned pathogenic bacteria and iatrogenic infectious bacteria, and the infection caused by Klebsiella pneumoniae has the characteristic of high mortality rate, and the mortality rate is still as high as about 50% under the antibacterial treatment. The Klebsiella pneumoniae is very easy to mutate, so that the drug resistance is stronger.

Coli is a normal colonizer in the intestinal tract of animals, a small proportion of which causes disease under certain conditions. The serotype of escherichia coli can cause gastrointestinal tract infections in humans or animals, mainly caused by infections with specific pilus antigens, pathogenic toxins, and the like. Colibacillosis in animals can occur in a variety of domestic animals, poultry, farm animals, and other terrestrial and some aquatic animals, with pigs and chickens being the most susceptible and serious hazards. Colibacillosis is common in newborn animals and young animals, and is manifested by enteritis, enterotoxemia, septicemia or tissue organ inflammation, and the like, so that the growth and development of the livestock are blocked and the livestock die, and great economic loss is caused.

Salmonella is a common food-borne pathogenic bacterium, widely distributed in nature and often inhabited in humans and animals, particularly in the intestines of poultry, livestock and pets. Infection of poultry and livestock with salmonella can cause corresponding infectious diseases, such as hog cholera, pullorum disease, etc. When the resistance of animals is reduced due to illness, weakness, malnutrition and fatigue, salmonella in the intestinal tract can enter blood through mesenteric lymph nodes and lymphatic tissues to cause systemic infection and even death. The salmonellosis belongs to common diseases of livestock and poultry, and has high morbidity and great harm. In nature, however, poultry constitutes the largest individual storage host for salmonella. The most commonly reported salmonella in all animals is derived from poultry and poultry products.

After the phage infects host bacteria, new phage can be released during cracking, and a small amount of phage can play a role in preventing and treating. The action target of the phage is on the cell wall of the prokaryotic microorganism, and no corresponding action site exists on animal and plant cells, so that the use safety of the phage can be ensured. The bacteriophage and the pathogenic bacteria can evolve together to prevent the occurrence of bacterial drug resistance. Therefore, the utilization of the phage to solve bacteria is a biological control approach with wide prospects.

Germplasm resources with the characteristics of high phage titer, high temperature resistance, acid and alkali resistance and the like are more available, but few germplasm resources capable of performing cross-species cracking on klebsiella pneumoniae, escherichia coli and salmonella are available. The species and the number of the intestinal phages directly influence the intestinal flora and the physiological state thereof, and compared with the phages which only can infect single species of bacteria, the phages which can cross species of bacteria can have more complex action in the intestinal tract and have larger influence on the intestinal flora and the intestinal health.

Disclosure of Invention

The invention aims to provide a cross-species lytic Klebsiella pneumoniae bacteriophage to replace antibiotics in the prevention and treatment of livestock breeding, and solve the problems of ineffective administration of livestock and poultry caused by drug resistance of pathogenic bacteria and the like.

In order to achieve the purpose, the invention adopts the following technical scheme: a cross-species schizolysis type Klebsiella pneumoniae is disclosed, the phage RDP-EC-20056 is classified as Klebsiella pneumoniae, and is preserved in China general microbiological culture Collection center (CGMCC No. 45090) at 20/4 in 2022.

Preferably, the bacteriophage has a strong lytic effect on Klebsiella pneumoniae, escherichia coli, or Salmonella.

Preferably, the invention also provides the application of the bacteriophage in preparing a medicament for preventing and/or treating diseases caused by any one or more than two of klebsiella pneumoniae, escherichia coli and salmonella.

Preferably, the invention also provides the application of the bacteriophage in preparing feed, additive or health care product for preventing diseases caused by escherichia coli and/or salmonella.

Further, the invention also provides a sterilization composition of Klebsiella pneumoniae, escherichia coli or Salmonella, which comprises the cross-species schizolysis type Klebsiella pneumoniae phage.

Further, the invention also provides a medicament for preventing and/or treating diseases caused by escherichia coli and salmonella, which comprises the cross-species lytic klebsiella pneumoniae.

Further, the invention also provides a feed, a feed additive or a health care product for preventing diseases caused by escherichia coli and/or salmonella, which comprises the cross-species lytic klebsiella pneumoniae.

Furthermore, the invention also provides a cleaning agent or a disinfectant for a livestock and poultry breeding environment, which comprises the cross-species lytic Klebsiella pneumoniae.

Compared with the prior art, the invention has the beneficial effects that:

(1) The phage provided by the invention takes escherichia coli as a host, can crack klebsiella pneumoniae, can crack escherichia coli and salmonella in cross species, has strong cracking activity, provides a material source for industrial production of the phage, and has good application prospect in prevention and control of livestock and poultry pathogenic escherichia coli and salmonella.

(2) The bacteriophage provided by the invention can still keep stable titer after being acted for 30min in a water bath at the temperature of 50 ℃, has higher titer which can be more than 10^8pfu/mL, has certain high-temperature tolerance, higher titer, safety and effectiveness. The optimal temperature of the phage RDP-EC-20056 is 37 ℃, and the titer reaches 8 × 10^9pfu/mL at most.

(3) The phage provided by the invention is a virulent phage test phage separated from nature and does not contain virulence genes or undesirable genes; the present invention does not involve any genetic modification of the test phage.

Drawings

FIG. 1 is an electron micrograph of bacteriophage RDP-EC-20056 in an example of the present invention;

FIG. 2 shows the result of the lysis experiment of the phage RDP-EC-20056 on the host bacterium BE-20055 RTD in the embodiment of the present invention;

FIG. 3 is a schematic representation of the thermal stability of phage RDP-EC-20056 in an example of the invention;

FIG. 4 is a schematic representation of the genetic stability of bacteriophage RDP-EC-20056 in accordance with an embodiment of the present invention.

Detailed Description

The invention is further illustrated with reference to the following figures and examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the concept of the invention. All falling within the scope of the invention.

The invention separates Escherichia coli BE-20055 from poultry manure of a comet group farm, determines pathogenicity and drug resistance of the Escherichia coli BE-20055 through experiments, separates a lytic Klebsiella pneumoniae phage from the poultry manure by taking the Escherichia coli BE-20055 as a host, and determines the genotype of the phage through a whole genome sequence.

Example 1 isolation and identification of pathogenic bacteria and phages

(1) Isolation and identification of host Escherichia coli BE-20055

From poultry dung, adopting an aseptic operation method, carrying out streak separation on a Kemajia large intestine chromogenic culture medium, culturing at 37 ℃ for 18-24h, and then presenting blue or blue-green colonies with neat edges on a large intestine chromogenic plate, wherein the diameter is about 1-2 mm; then selecting a typical colony to streak on a Macconkey culture medium, culturing for 18-24h at 37 ℃, then presenting pink colony on the Macconkey culture medium and having choline salt sediment around the Macconkey culture medium, selecting the typical colony to continuously streak and purify for 3 times, then selecting a single colony to inoculate in 5mL of LB broth culture medium, and culturing for 4h at 37 ℃ and 200rpm in a shaking way to obtain a uniform and turbid bacterial suspension. And identifying by 16sRNA molecule to obtain Escherichia coli, and storing one strain of the Escherichia coli BE-20055 at-80 deg.C in refrigerator.

(2) Phage RDP-EC-20056 isolation and identification

The poultry excrement sample for the test is collected from China comet group farms in 2020 and is used as a sample for phage separation.

Sample treatment: soaking appropriate amount of fowl feces in 20 mL LB broth, centrifuging at 10000rpm for 10min, removing larger impurities and most bacteria, and filtering with 0.22 μm microporous membrane for sterilization.

Preparing a mixed bacterial suspension: 2 mL of the filtrate and 1mL of the suspension of the host bacterium were added to 27 mL of the autoclaved LB broth, and then placed in a 37 ℃ incubator for overnight culture. After the culture, 3mL of the solution is taken, centrifuged at 10000rpm for 5 minutes, filtered and sterilized by a 0.22 mu m microporous filter, and the filtrate is collected for later use, namely the stock solution which is supposed to contain the phage.

Phage separation: separating phage by double plate method, mixing the filtrate 0.1mL of mixed bacterial suspension and 0.1mL of Escherichia coli suspension, adding into nutrient agar heated to 45-54 deg.C, 3mL and 4%, spreading double plates, culturing in 37 deg.C incubator for 6-8h, and observing result to obtain transparent plaque on the plate if there is phage. Soaking plaques in 1mL of physiological saline for 10min, adding 0.1mL of leachate and 0.1mL of bacterial suspension into 4% nutrient agar heated to 45-54 ℃ and 3mL, spreading a double-plate, culturing in a 37 ℃ incubator for 4-6h for purification, and purifying for 2-3 times according to the steps until the plaques are uniform in size, thereby obtaining a phage strain and naming the phage strain as Klebsiella pneumoniae phage. The plaque diameter formed by the phage RDP-EC-20056 on the double plate is 0.1mm-0.5mm, and the round shape is transparent, as shown in FIG. 2.

(3) Phage RDP-EC-20056 potency assay

Laying a double-layer plate on the purified phage, taking a single plaque, adding the plaque into an LB liquid culture medium, simultaneously adding a host bacterium suspension, after the phage is multiplied, centrifuging the multiplication liquid at 10000rpm for 5 minutes, filtering by using a 0.22-micrometer microporous filter, performing 10-fold gradient dilution on the filtrate to 10-fold, taking 0.1mL of the phage dilution liquid with the last 3 dilutions and 0.1mL of the host bacterium suspension, laying the double-layer plate, and determining the titer of the phage. Phage titer (pfu/mL) = plaque number ×. Dilution factor ÷ 0.1. The result shows that the plaque has consistent shape, size and definition and has typical lytic phage characteristics; the titer of the phage can reach more than 10^9pfu/mL at most.

(4) Preservation of phage RDP-EC-20056

Phage multiplication fluid was mixed with 60% glycerol according to 1:1, and storing in liquid nitrogen.

EXAMPLE 2 Electron microscopy of phage RDP-EC-20056

Dropping 20 μ L of liquid containing crude phage particles on a copper net, naturally precipitating for 15 min, sucking off excessive liquid from the side by using filter paper, adding a drop of 2% phosphotungstic acid (PTA) on the copper net to dye the phage for 10min, sucking off the staining solution from the side by using filter paper, and observing the form of the phage by using an electron microscope after a sample is dried.

As can be seen from FIG. 1, the phage RDP-EC-20056 is a long-tailed phage, the head is a regular icosahedron, the diameter of the head is about 60nm, and the length of the tail is 182nm.

Example 3 phage RDP-EC-20056 genomic sequencing and organism preservation

After enrichment culture of a single phage, centrifuging for 15 minutes at 8000 r under the condition of 4 ℃, adding 10 percent of PEG8000 and 0.5M NaCl, standing overnight, adding equal amount of chloroform, mixing uniformly, standing, layering, centrifuging for 10 minutes at 5000 r, removing a chloroform layer and a PEG layer, adding restriction endonuclease for digestion treatment, suspending the phage under the condition of gradient density cesium chloride, dialyzing for 3 times at the later stage by TM buffer solution, and sequencing the whole genome by Huada gene biosequencing company after 30 minutes each time. The result is shown in a sequence table SEQ ID No.1, and the genome has the full length of 60913bp. And in 20 days in 2022, 4 months, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No.45090. The address of the preservation unit is as follows: xilu No.1 Hospital No. 3, beijing, chaoyang, beicheng.

Example 4 determination of optimal multiplicity of infection for bacteriophage RDP-EC-20056

The phage multiplication fluid and the host (e.coli BE-20055) were added to LB at a ratio of multiplicity of infection of 100. After shaking culture at 37 ℃ and 200rpm for 8h, centrifuging at 12000 r/min at normal temperature for 5min, and spreading the supernatant on a double-plate to determine the titer. The results are shown in Table 1.

TABLE 1 determination of optimal multiplicity of infection for bacteriophage RDP-EC-20056

Pipe number	BE-20055 number of Escherichia coli (cfu/mL)	RDP-EC-20056 phage number (pfu/mL)	Multiplicity of infection	8h phage titer (pfu/mL)
					1	10^8	10^6	100:1	8.1*10^8
2	10^8	10^7	10:1	1.5*10^8
					3	10^8	10^8	1:1	5*10^8
4	10^8	10^9	1:10	2.9*10^8
					5	10^7	10^9	1:100	1*10^8
6	10^6	10^9	1:1000	1*10^8

As can be seen from the data in Table 1, when the multiplicity of infection is 100, the proliferation fluid is relatively clear after 8h of culture, and the titer is the highest and can reach 8.1X 10^8, which shows that the optimal multiplicity of infection of the Klebsiella pneumoniae RDP-EC-20056 of the invention is 100.

Example 5 lysis profiling experiment of bacteriophage RDP-EC-20056

The lysis capacity of Klebsiella pneumoniae RDP-EC-20056 is determined by a dropping method, bacteria are evenly coated on an LB flat plate, 5 mu L of phage suspension is dropped in the center of the coating range after drying, and the bacteria can be cracked if bright empty spots appear on lawn after incubation for 6h at 37 ℃.4 strains of O18, 2 strains of O2, 4 strains of O45, 1 strain of O1 and 1 strain of O145 are selected to carry out a lysis spectrum experiment on 5 serotypes of escherichia coli to the Klebsiella pneumoniae RDP-EC-20056, and the lysis capacity is shown in table 2. Enteritis, typhus, typhoid, paratyphoid a, oritarolin, alonway, indiana, rison, rengen, fuderburg, nieuwland, kentucky, stanley, duck salmonellae, blonsbury, thopson, cotebsiella pneumoniae RDP-EC-20056 are subjected to lysis spectrum experiments with 17 different serotypes of salmonella, the lysis capacities of which are shown in table 3.

TABLE 2 lysis of E.coli of different serotypes by the bacteriophage RDP-EC-20056

Serial number	Serotype	Cracking ability
				1	O18	++
2	O18	++
			3	O18	++
4	O18	++
			5	O2	+
6	O2	-
			7	O45	++
8	O45	++
			9	O45	++
10	O45	++
			11	O1	++
12	O145	-

TABLE 3 lysis of different serotypes of Salmonella by the bacteriophage RDP-EC-20056

Serial number	Serotype	Cracking ability
				1	Enteritis (enteritis)	+
2	Enteritis (enteritis)	+
			3	Mouse and typhoid	++
4	Typhoid fever	-
			5	Paratyphoid A	-
6	Olita vine	++
			7	'Zhengwei' medicine	++
8	Indiana	++
			9	Lisen	++
10	Ragen	++
			11	Mountain fudenburg	++
12	New Zealand	++
			13	New year lily	++
14	Kentucky foot	++
			15	Kentucky foot	++
16	Stanley	++
			17	Stanley	++
18	Duck salmonellae	++
			19	Bloom burley	++
20	Thompson	++
			21	Cotebutus sp	++

Note: "+ +" is strongly cleaved, "+" is cleavable, and "-" is not cleaved

Example 6 determination of the thermostability of phage RDP-EC-20056

The phage stock solution was dispensed into EP tubes, incubated at 40 deg.C, 50 deg.C, 60 deg.C and 70 deg.C for 30min, diluted with 10-fold physiological saline, and then plated on double plates to determine the titer, the results of which are shown in FIG. 3. As can be seen from FIG. 3, the activity of the phage is kept stable within 50 ℃, and the titer is more than 10^ 8pfu/mL.

Example 7 phage RDP-EC-20056 stability genetic experiment

Continuously passaging the phage RDP-EC-20056 for 15 times, adding the same amount of RDP-EC-20056 and BE-20055 into each generation of culture medium, culturing for 6h for each generation, then paving double plates, and measuring the titer. The results are shown in FIG. 4.

As can be seen from FIG. 4, after continuously transmitting phage RDP-EC-20056 for 10 generations, the titer of the phage is determined, and the titer is stabilized at more than 10^9pfu/mL, which indicates that the phage has better genetic stability and is suitable for industrial production.

Example 8 phage RDP-EC-20056 lysis Rate experiments

Under aseptic conditions, 1mL of sample and 1mL of host bacterium solution (1X 10) ⁶ CFU/mL), incubating for 15 minutes at 37 ℃, mixing uniformly, and diluting to 10 with physiological saline ^-1 -10 ^-3 Each gradient was plated at 100. Mu.L onto nutrient agar plates and incubated at 37 ℃ for 24 hours, and each gradient was repeated twice. Simultaneously taking 1mL of physiological saline and 1mL of host bacterium liquid (1X 10) ⁶ CFU/mL) as a blank, repeat the above steps. Plates with 30-300 colonies were picked for enumeration. The test was repeated 3 times and the average was taken.

Phage lysis rate = (1-number of treated group colonies/number of control group colonies) × 100%.

Through calculation, the lysis rate of the phage RDP-EC-20056 reaches 98.8%, the phage has a good lysis effect on a host, and the phage is suitable for being used in a culture process.

Example 9 RTD experiment of phage RDP-EC-20056

The plates were divided into zones, a drop of host bacteria (about 10. Mu.L) was added to the center of each zone, air dried, a drop of phage was added at different dilutions to the spot of host bacteria, air dried, and incubated overnight at 37 ℃. According to the growth condition of the host bacteria, the CL concentration is called as the CL concentration (the CL concentration is the concentration of the host bacteria in the environment which can be completely lysed, and can be used for guiding the dilution scheme of the production practice).

The phage RDP-EC-20056 still has better lysis effect on host bacteria after being diluted by-3 times.

Example 10 method for Large Scale production of bacteriophage RDP-EC-20056

The process flow is simple, reliable and effective, and is favorable for industrial production.

The specific steps

1. Seed preparation

(1) Preparing a host seed: under aseptic conditions, single colonies were picked and streaked onto nutrient agar medium and incubated overnight at 37 ℃. Then, the cultured single colony is picked up and put into 5mL LB broth medium, and after shaking culture at 37 ℃ and 180rpm for 6h, the single colony is inoculated into the LB broth medium with the inoculum size of 1%, and shaking culture at 37 ℃ and 180rpm for 4h. The prepared seed liquid is stored in a refrigerator at 4 ℃ for later use.

(2) Preparing a phage seed: diluting the preserved phage seeds, placing 100 μ L and 100 μ L host bacteria in the upper layer culture medium, pouring the double-layer plate, culturing at 37 deg.C for 4-6h, scraping with sterile coating rod, centrifuging at 10000rpm and 5min, sterilizing with 0.22 μm filter membrane, and storing the collected filtrate in a refrigerator at 4 deg.C.

The phage and the host are respectively inoculated in LB broth culture medium in the proportion of 1 percent and 5 percent, and cultured for 6-8h at 37 ℃ and 180rpm until the proliferation liquid is clear and a large amount of floccules appear.

2. And (3) phage multiplication: inoculating 2% of host bacteria in a seeding tank, culturing for 2-3h, then inoculating 0.6-0.8 OD600, then inoculating 1% of the inoculum size to phage seeds, culturing for 6-8h, until the dissolved oxygen is reversely increased and the pH value tends to be stable.

Inoculating 2% of host bacteria in a seeding tank, culturing for 4-5h until dissolved oxygen begins to slowly decrease.

Inoculating the prepared host bacteria into a large fermentation tank with the inoculation amount of 5%, culturing for 2-3h until dissolved oxygen begins to slowly decrease, then inoculating phage seeds with the inoculation amount of 5%, increasing the ventilation amount to 1vvm, increasing the rotation speed to 150rpm, culturing for 8-10h until the dissolved oxygen reversely increases and the pH value increases, and finishing proliferation.

Note: fermentation tank parameters: the seeding tank has 200-300rpm,37 deg.c and ventilation amount of 0.6-1vvm. Fermentation tank: 120-150pm,37 ℃ and an aeration rate of 0.6-1vvm.

The components of the culture medium: 0.5% of yeast extract powder, 1% of peptone, 0.1% of beef extract, 0.5% of glycerol, 0.5% of sodium chloride, 0.1% of monopotassium phosphate, 0.1% of dipotassium phosphate, 20ppm of calcium chloride and 20ppm of magnesium chloride.

3. Phage post-treatment process

(1) And (3) filtering: and (3) respectively adopting 800nm and 500nm ceramic membranes for filtration to remove cell fragments and host bacteria which are not cracked, wherein the filtration pressure is less than 0.4mpa, and the damage of shearing force to the bacteriophage is reduced.

(2) Concentration: hollow fiber membranes are used for concentration to improve the titer of the phage.

(3) A stabilizer: adding 5% of glycerol, 3% of trehalose, 3% of polysorbate, 3% of sodium dihydrogen phosphate and 2% of sodium phosphate, and stirring until the mixture is completely dissolved.

(4) And (3) degerming: three-stage filtration (glass fiber 0.45 μm, glass fiber 0.2 μm, polyethersulfone 0.22 μm) was used for sterilization.

(5) The filter-sterilized phage liquid was stored at 4 ℃.

It should be noted that the specific embodiments are merely representative examples of the present invention, and it is obvious that the technical solution of the present invention is not limited to the above examples, and many variations are possible. Those skilled in the art, having the benefit of this disclosure and the benefit of this written description, will appreciate that other embodiments can be devised which do not depart from the specific details disclosed herein.

Claims

1. A cross-species schizolysis type Klebsiella pneumoniae phage is characterized in that: the bacteriophage RDP-EC-20056 is classified as Klebsiella pneumoniae, is preserved in China general microbiological culture Collection center (CGMCC) at 20/4 in 2022, and has a preservation number of CGMCC No.45090.

2. The transspecies lytic klebsiella pneumoniae phage of claim 1, wherein: the bacteriophage has strong lytic effect on Klebsiella pneumoniae, escherichia coli or Salmonella.

3. Use of a transspecies lytic klebsiella pneumoniae phage according to claim 1 or 2, wherein: the bacteriophage is used for preparing a medicine for preventing and/or treating diseases caused by any one or more of Klebsiella pneumoniae, escherichia coli and Salmonella.

4. Use of a transspecies lytic klebsiella pneumoniae bacteriophage according to claim 1 or 2, wherein: the bacteriophage is used for preparing feed, additive or health product for preventing and/or treating diseases caused by Escherichia coli and/or Salmonella infection.

5. A sterilization composition of Klebsiella pneumoniae, escherichia coli or salmonella, which is characterized in that: comprising an effective amount of a transspecies lytic Klebsiella pneumoniae bacteriophage of claim 1 or 2.

6. A medicament for preventing and/or treating diseases caused by Escherichia coli and Salmonella, characterized by: comprising a transspecies lytic Klebsiella pneumoniae bacteriophage according to claim 1 or 2.

7. A feed, additive or health product for preventing diseases caused by Escherichia coli and/or Salmonella, which is characterized in that: comprising a transspecies lytic Klebsiella pneumoniae bacteriophage according to claim 1 or 2.

8. A cleaning agent or disinfectant for livestock and poultry breeding environment is characterized in that: comprising the transspecies lytic Klebsiella pneumoniae phage of claim 1 or 2.