CN115322973B - A strain of Pseudomonas aeruginosa phage HCZ001 and its application - Google Patents
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Abstract
The invention discloses a pseudomonas aeruginosa bacteriophage HCZ001, wherein the pseudomonas aeruginosa bacteriophage (Pseudomonas aeruginosa phage) HCZ has a preservation number of CCTCC M2022666, and the pseudomonas aeruginosa bacteriophage HCZ001 is applied to treating canine pyoderma caused by pseudomonas aeruginosa. The pseudomonas aeruginosa phage HCZ001 has the capability of cross-species range cracking, good alkali resistance stability and good thermal stability, can be stored for a long time under room temperature conditions and can be kept at a high titer level, and has good treatment effect on the pseudomonas aeruginosa infected by canine skin.
Description
Technical Field
The invention relates to the technical field of pseudomonas aeruginosa phages, in particular to a pseudomonas aeruginosa phage HCZ001 and application thereof.
Background
Pseudomonas aeruginosa is a gram negative pathogenic bacterium which is widely distributed in nature, exists in soil, water, skin, respiratory tract, intestinal tract and the like of normal people, and can cause serious infection of human body or animal body tissues when the body resistance is reduced or the immune system is damaged. Pseudomonas aeruginosa has a capsule, flagella and pili, no spores, active movement, and is an obligate aerobic bacterium. The culture medium has good survival state on a nutrient agar culture medium, the optimal growth pH value is 5-7, the optimal toxigenic temperature is 26 ℃, the optimal growth temperature is 36 ℃, the metabolism is slow at the low temperature of 4 ℃, the growth is stagnant, but the growth can be continued at the high temperature of 42 ℃. Can produce water-soluble pigment pyocin and pyocin, and can make the culture medium become bright green. The bacteria can grow in a turbid manner in the liquid culture medium, and a bacterial film can be formed on the surface of the culture medium. Pseudomonas aeruginosa can decompose glucose, produce acid, does not produce gas, can not decompose mannitol, maltose, sucrose, lactose and other substances, can decompose urea, is positive in oxidase, and does not form indole. Pseudomonas aeruginosa is a common infectious agent in infections in human nosocomial infections, severe burns, cancer, transplantation, AIDS and other immunocompromised patients. Infection with pseudomonas aeruginosa is also common in companion animals and can cause major skin, respiratory, urinary tract infections in dogs and cats.
Phage (bacteriophage, phage) is a generic term for viruses that infect microorganisms such as bacteria, fungi, algae, actinomycetes, and spirochetes, and is called a phage because some of them cause lysis of host bacteria. Phages replicate using the replication system of the bacterial host, destroying the host cell (lytic cycle), or reside in the bacterial genome (lysogenic state). To initiate their biological infection cycle, phages attach to specific bacterial receptors, and thus each phage has a specific bacterial host. After attachment, the phage injects its genome into the cell and exerts a bacterial replication mechanism to produce phage genomes and proteins, which assemble within the host unit. Since phages are much smaller than their host cells, one bacterial cell can have hundreds or thousands of new phages. The newly generated phage lyses the host cells and is released into the extracellular environment, and the pathogenic bacteria accumulate and become embedded in the extracellular polymeric substance matrix called the biofilm. Biofilm formation may make bacteria better resistant to the action of antibiotics and bactericides. However, phage act on pathogenic bacteria irrespective of the presence of a biofilm, they penetrate the biofilm, replicate within bacterial cells, and produce enzymes that destroy extracellular polymeric substances, thereby destroying the bacteria.
Phage have been used to treat patients with bacterial infections since the beginning of discovery, but there have been few reports of their clinical use in companion animals, and phage therapy has been very promising for the treatment of bacterial infectious diseases in companion animals according to established practices. Although companion animals are independent of the human food chain, they can also host multi-drug resistant bacteria, and their proximity to humans makes them a potential focus of zoonotic bacterial cross-transmission. Canine pyoderma has high clinical incidence rate and is easy to relapse, and is always a disaster area for veterinary clinical discovery of multi-drug resistant bacteria. Aiming at the problems, the phage can be used as a powerful tool for resisting multiple drug-resistant bacteria, the possibility of the phage in treatment of the canine pyoderma is explored, the effect of the phage is evaluated, and a new idea is provided for treatment of the canine pyoderma.
Disclosure of Invention
Aiming at the technical problems, the invention provides a bacteriophage capable of cracking multi-drug resistant pseudomonas aeruginosa, and aims to provide a new treatment scheme for the canine pyoderma caused by the pseudomonas aeruginosa.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
Pseudomonas aeruginosa phage HCZ001, wherein the Pseudomonas aeruginosa phage (Pseudomonas aeruginosa phage) HCZ has a preservation number of CCTCC M2022666 and a preservation date: 2022, 5, 19, deposit address: the university of Wuhan China center for type culture collection of Wuhan in Wuhan district of Hubei province.
The application of the pseudomonas aeruginosa phage HCZ001 in preparing medicines for treating the canine pyoderma caused by pseudomonas aeruginosa.
A phage composition comprising pseudomonas aeruginosa phage (Pseudomonas aeruginosa phage) HCZ001 as described above.
A phage pharmaceutical formulation comprising as an active ingredient pseudomonas aeruginosa phage (Pseudomonas aeruginosa phage) HCZ001 as described above or a phage composition as described above.
Wherein the phage pharmaceutical preparation also comprises a pharmaceutically acceptable carrier, the dosage form is solution or gel, and the administration route is injection or external use.
The pseudomonas aeruginosa phage (Pseudomonas aeruginosa phage) HCZ001 has a good disinfection effect on host pseudomonas aeruginosa VHP002 separated from pustule dogs, has a strong disinfection effect on escherichia coli from canine feces, and can be applied to preparation of medicaments for treating pseudomonas aeruginosa infection.
Compared with the prior art, the invention has the following beneficial technical effects:
The pseudomonas aeruginosa phage HCZ001 has the capability of cross-species range cracking, good alkali resistance stability and good thermal stability, can be stored for a long time under room temperature conditions and can be kept at a high titer level, and has good treatment effect on the pseudomonas aeruginosa infected by canine skin.
Description of preservation information
Pseudomonas aeruginosa phage (Pseudomonas aeruginosa phage) HCZ001 was preserved in China center for type culture collection (CCTCC for short) with a preservation number of CCTCC M2022666 at 2022, 5 and 19 days.
Drawings
FIG. 1 is a photograph of a plaque of the Pseudomonas aeruginosa phage (Pseudomonas aeruginosa phage) HCZ001 of the present invention.
FIG. 2 is a transmission electron microscope image of a Pseudomonas aeruginosa bacteriophage (Pseudomonas aeruginosa phage) HCZ001 according to the present invention.
FIG. 3 is a graph of the multiplicity of infection optimal for Pseudomonas aeruginosa phage (Pseudomonas aeruginosa phage) HCZ001 according to the present invention.
FIG. 4 is a graph of one-step growth of Pseudomonas aeruginosa phage (Pseudomonas aeruginosa phage) HCZ001 according to the present invention.
FIG. 5 is a schematic representation of the effect of temperature on the activity of Pseudomonas aeruginosa phage (Pseudomonas aeruginosa phage) HCZ001 according to the present invention.
FIG. 6 is a schematic representation of the effect of pH on the activity of Pseudomonas aeruginosa phage (Pseudomonas aeruginosa phage) HCZ001 according to the present invention.
FIG. 7 is a schematic representation of the bacteriostatic effects of Pseudomonas aeruginosa phage (Pseudomonas aeruginosa phage) HCZ001 medium according to the present invention.
FIG. 8 is a graphical representation of the skin infection effect score of Pseudomonas aeruginosa phage HCZ001 control Pseudomonas aeruginosa dogs according to the present invention.
FIG. 9 is a schematic representation of the actual effect of the Pseudomonas aeruginosa phage HCZ001 of the present invention on controlling skin infection in Pseudomonas aeruginosa dogs.
Detailed Description
The following detailed description, in conjunction with the accompanying drawings, describes in detail, but it is to be understood that the scope of the invention is not limited to the specific embodiments. The raw materials and reagents used in the examples were commercially available unless otherwise specified. The experimental methods used in the following examples are conventional methods unless otherwise specified. The quantitative experiments in the following examples were all performed in triplicate, and the results were averaged.
The host bacteria used in the experiment are multi-drug resistant clinical strains, namely pseudomonas aeruginosa clinical strain VHP002, which is separated from skin swabs of pustular patients in Guangdong Shenzhen animal hospital, and are preserved in China Center for Type Culture Collection (CCTCC) M2022666 together with pseudomonas aeruginosa phage HCZ 001.
LB (Luria broth) liquid Medium (1L): 10g of peptone, 5g of yeast powder, 10g of NaCl, adding ddH 2 O to 1L, adjusting the pH to 7.0, 121 ℃ and autoclaving for 20 min.
0.6% LB semisolid medium (1L): 10g of peptone, 5g of yeast powder, 10g of NaCl, 6g of agar powder, adding ddH 2 O to 1L, adjusting the pH to 7.0, and sterilizing at 121 ℃ for 20 min.
1.2% LB solid Medium (1L): 10g of peptone, 5g of yeast powder, 10g of NaCl, 12g of agar powder, adding ddH 2 O to 1L, adjusting the pH to 7.0, sterilizing at 121 ℃ for 20min under high pressure, cooling to 50 ℃, pouring the flat plate, cooling to solidify, and inverting for later use.
SM buffer (1L): 6.055g of Tris-HCI (pH 7.5) was weighed to a volume of 100ml, 5.800g NaCl,2.000g MgSO 4 was added thereto, and ddH 2 O was added thereto to a volume of 1L.
DNase I, RNase A, PEG8000, phosphotungstic acid (PTA, 2% w/v), chloroform were commercially available.
Example 1
Isolation of Pseudomonas aeruginosa phage HCZ001
The sample is collected from the sewage in the Ruikang hospital in Guangxi nan Ning, the sample is centrifuged for 10min at the temperature of 4 ℃ and at the speed of 12000rpm, the supernatant is centrifuged for 3 times again, and the final supernatant is filtered by a filter membrane of 0.45 mu m and a filter membrane of 0.22 mu m respectively; taking 5mL of filtrate, adding 0.1mL of pseudomonas aeruginosa clinical strain VHP002 host bacterium, adding 5mL of 2 XLB liquid culture medium, placing the mixture at 37 ℃ for culturing for 14-16 h, culturing for 10min after the next day, at 4 ℃ and 12000rpm, centrifuging the obtained culture for 14-16 h, filtering and sterilizing the supernatant by using a 0.22 mu m filter membrane, and obtaining stock solution containing phage, namely phage suspension for later use.
The clinical strain VHP002 host strain of pseudomonas aeruginosa is streaked and inoculated on 1.2% LB solid medium, cultured overnight, and then selected and inoculated on 5mL LB (Luria broth) liquid medium, and shake-cultured for 8 hours at 37 ℃ to be used as a host strain culture for standby.
Dividing 1.2% LB solid culture medium into 2 areas, sucking the above standby host bacteria culture 0.1mL and 3mL of 0.6% LB semisolid culture medium, uniformly mixing, spreading on 1.2% LB solid culture medium, air drying, dripping 10 μl of the above standby phage suspension into one of the areas, naturally air drying, culturing in a 37 ℃ incubator, observing whether plaque formation occurs in the dripping phage area, and if plaque formation occurs, proving that phage exists.
Taking another 0.1ml of the standby phage suspension for continuous 10-fold dilution, taking out 0.1ml of 10 -2、10-4、10-6 dilution liquid respectively, adding 0.1ml of pseudomonas aeruginosa clinical strain VHP002 host bacteria, standing for 15min, adding 3.5ml of 0.6% LB semisolid culture medium at about 45 ℃, uniformly spreading on 1.2% LB solid culture medium, culturing at 37 ℃ for 8h, and observing plaque growth; single transparent halo-free plaque with uniform size and neat edges is picked up and placed in an EP tube containing 0.1mL of standby host bacteria culture and LB liquid medium, and co-cultured overnight at 37 ℃; the following day, the co-culture is taken for centrifugal filtration, the filtrate is diluted 10 times by SM buffer solution and double-layered with 0.1mL of pseudomonas aeruginosa clinical strain VHP002 host bacteria, and phage with uniform plaque size can be obtained by repeating the steps about 10 times, and the phage is preserved at 4 ℃ for standby.
The above standby phage was examined by a double-layer plate method (i.e., double-layer agar plate method), and as a result, as shown in FIG. 1, the phage could form a needlepoint transparent plaque in an agar medium, without a halo around, with clear and regular edges, which is a typical lytic phage.
Example 2
Amplification purification of Pseudomonas aeruginosa phage HCZ001
Taking 0.1ml of phage reserved in the embodiment 14 ℃ and 0.1ml of host bacteria culture reserved in the embodiment 1, acting for 15min in a test tube, adding 10ml of LB liquid medium, culturing for 6h at 37 ℃, centrifuging for 20min at 12000rpm, taking the supernatant, filtering with a 0.22 μm filter membrane, and obtaining the filtrate as phage lysate.
PEG purification: DNase I and RNase A are added into phage lysate to a final concentration of 1 mug/ml, incubated for 30min at 37 ℃, naCl ice bath with a final concentration of 1M is added (i.e. sodium chloride is added to make the final concentration of sodium chloride in the mixed solution 1M), centrifugation is carried out at 4 ℃ and 12000rpm for 10min, PEG8000 with a final concentration of 10% is taken out from the supernatant, overnight at 4 ℃ and 12000rpm for 10min, the supernatant is discarded, inverted for 5min, redundant water is removed as much as possible, SM buffer solution is added into the rest solid matters for heavy suspension, chloroform with an equal volume is added and shaking is carried out for 30s, and centrifugation is carried out at 5000rpm for 15min to separate an organic phase and a hydrophilic phase, and a hydrophilic phase containing phage particles is recovered, thus obtaining purified phage suspension.
Phage titers were detected by double-layer plate method: performing 10-time gradient dilution on the purified phage suspension, taking 0.1ml of phage dilution liquid of each gradient and 0.1ml of pseudomonas aeruginosa clinical strain VHP002 host bacterial liquid, fully mixing, paving double-layer agar plates, culturing at a constant temperature of 37 ℃ for about 6-10 hours, counting plaques on each agar plate, selecting plates with about 30-300 plaques, and calculating the initial concentration of the obtained phage according to the dilution multiple to obtain phage titer, wherein the phage titer (PFU/ml) =dilution multiple×the number of plaques×10, and the phage titer is 3×10 9 PFU/ml.
Example 3
Transmission electron microscope observation of Pseudomonas aeruginosa phage HCZ001
Performing electron microscope observation on the phage suspension purified in the example 2, dripping the phage suspension purified in the example 2 on a copper sheet, naturally precipitating for 5-10 min, sucking redundant liquid by using filter paper, dripping 2% phosphotungstic acid (PTA, 2% w/v) for dyeing, drying at room temperature, and observing by using a transmission electron microscope; as a result of observation, as shown in FIG. 2 (100 kV), the phage had a head with a regular icosahedron, a head diameter of about 150nm and a tail of 100nm, and was designated HCZ001 as belonging to the myotail virus family (Myoviridae) according to the eighth report of the International Commission on viral Classification-International Classification of viruses (ICTV) published 2015.
Example 4
Determination of optimal multiplicity of infection for Pseudomonas aeruginosa phage HCZ (multiplicity of infection is the ratio of the number of phages at the initial stage of infection to the number of host bacteria)
The culture of the host bacteria prepared in example 1 was prepared, the concentration was adjusted to 1X 10 9 CFU/mL, the phage prepared in example 1 and the culture of the host bacteria prepared in example 1 were added in the ratios of the multiplicity of infection of 1000, 100, 10, 1, 0.1, 0.01 and 0.001, respectively, LB liquid medium was added to make the total volume of the culture system the same, the culture was allowed to stand still at 37℃for 6 hours, centrifugation was carried out at 10000rpm for 10 minutes, the supernatant was collected and diluted to an appropriate concentration, and the titer was measured by a bilayer method, and the result was shown in FIG. 3, the optimum multiplicity of infection of Pseudomonas aeruginosa phage HCZ001 was 1.
Example 5
Determination of one-step growth curve of Pseudomonas aeruginosa phage HCZ001
The host bacterial cultures prepared in example 1 were mixed with excess phage prepared in example 1 (MOI >10, ensuring adsorption of all bacteria to phage), centrifuged at 12000rpm for 1min after incubation at 37℃for 15min, the supernatant (unadsorbed phage) was discarded, the pellet was washed 1 time with LB liquid medium, resuspended in 10ml of pre-warmed LB liquid medium, rapidly placed in a shaking table at 37℃for shaking culture, 120. Mu.l of culture was taken every 10min, removed by centrifugation at 4℃for 2min at 10000rpm, the supernatant was diluted to the appropriate concentration (appropriate concentration, i.e. concentration of 30-300 plaques formed on plates), phage titers were determined by the plate bilayer method, 120min, sampled 13 times altogether, the log of phage titers was plotted on the ordinate with the sampling time as abscissa, and the latency, flash-out period, burst size of phage were obtained by plotting one-step growth curves. The result of the one-step growth curve is shown in FIG. 4, the incubation period of the infected host bacteria is 10min, the burst period is 40min, and the burst amount is52 PFU/cell.
Example 6
Temperature and pH tolerance test of Pseudomonas aeruginosa phage HCZ001
Taking 10 sterile EP tubes, adding 0.5ml of phage prepared in example 1, respectively, allowing to act at 30deg.C, 40deg.C, 50deg.C, 60deg.C, 70deg.C, 80deg.C for 60min, taking 100 μl every 10min, immediately cooling in water bath, and measuring phage titer; the detection results are shown in fig. 5: the phage can withstand high temperature of 50 ℃, the titer is basically stable within 50min, the phage titer is obviously reduced with time when the phage is larger than 60 ℃, the titer is basically stable within 10min in 60 ℃, and then the phage begins to be reduced to be inactivated, and the phage is rapidly inactivated in the environment of 70 ℃ and 80 ℃.
Taking 11 parts of 0.1ml of phage prepared in example 1, respectively placing the phage into SM buffer solutions (0.9 ml) with pH of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12 and 13, allowing the phage to act at 37 ℃ for 1-2 hours, and then measuring the titer of the phage after reaction by a plate bilayer method; the detection results are shown in fig. 6: the potency of the pseudomonas aeruginosa phage HCZ001 in the environment with the pH value of 6-8 is less in change, and the activity is basically unchanged; when the environmental pH >8 or pH <6, the titer of phage decreases with increasing acid, base; when pH >13 or pH <3, the phage titer was 0 and all were inactivated, so that the optimum pH of the phage was found to be 6 to 8.
Example 8
Antibacterial effect of Pseudomonas aeruginosa phage HCZ001 in culture medium
The experiment was divided into 5 test groups, with concentrations of 1X 10 6、1×107、1×108 phage groups (phage used in example 1), positive control group, negative control group, respectively. Each group was subjected to 3 replicates. 100. Mu.L of phage solution and 100. Mu.L of LB broth were added to each well of phage group, and 200. Mu.L of LB broth was added to each of positive control group and negative control group. Except for the negative control group, all test wells were inoculated with 5. Mu.L of Pseudomonas aeruginosa clinical strain VHP 002. After the sample is added, the initial value of OD450 is tested on an enzyme-labeled instrument, then the 96-well plate is sent into a 37 ℃ incubator for culture for 16 hours, the change of the tested OD450 value is taken out every 1 hour, the test is repeated 3 times each time, then a graph is drawn, and the result is shown in figure 7, so that the higher the concentration of phage is, the better the bacteriostasis effect is.
Example 9
Pseudomonas aeruginosa phage HCZ001 control Pseudomonas aeruginosa canine skin infection experiment
The experiment is carried out in animal houses of university of Guangxi, the experimental subjects are experimental dogs, 2 animals are mixed blood beagle dogs about 1 year old, the weight is about 5kg, two female dogs are not sterilized, and the immunity and insect expelling are complete. The dogs were divided into 2 groups, one for each group, phage treatment and positive control, all drugs in the experiment were administered by subcutaneous injection.
The method comprises the steps of firstly using pseudomonas aeruginosa to carry out pyoderma modeling, selecting 3 pieces of skin with the area of 5 multiplied by 5cm for each dog to shave hair, brushing and sterilizing the skin with alcohol and chlorhexidine, then using a microneedle roller to dip bacterial liquid to inoculate bacteria on the skin, inoculating once every 24 hours for 5 days, and observing for 72 hours until the skin has no sign of improvement, and then starting treatment.
Taking the phage for standby in the example 1, adjusting the concentration of the phage to 10 9 PFU/mL, and giving 1mL to each molding part once a day; from the beginning of the modeling, the experimental sites were scored every 24 hours according to the itching scoring scale (table 1), the cytological bacterial count scale (table 2) and the apparent skin lesions scale (table 3).
Table 1 pruritus scoring scale
TABLE 2 cytological bacterial count scale
TABLE 3 apparent skin lesions scale
The scores of the three tables are combined after the experiment is finished, and a graph is drawn, and the result is shown in fig. 8.
Photographs of the molded skin site were taken after successful molding and after the end of treatment, as shown in fig. 9, it was seen that the eye was greatly improved in a short period of time in the skin treated with phage, while there was little indication of improvement in the skin not treated with phage.
The foregoing descriptions of specific exemplary embodiments of the present invention are presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application to thereby enable one skilled in the art to make and utilize the invention in various exemplary embodiments and with various modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
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