CN100413962C - A bacteriophage acting on extended-spectrum β-lactamase-producing Escherichia coli - Google Patents

Abstract

The invention discloses an extended-span-lactamase (ESBLs) coliphage acting on extended-span-lactamase, and relates to an extended-span-lactamase (ESBLs) coliphage capable of producing extended-span-lactamase with a wide host spectrum. The bacteriophage is a specific bacterial virus, and the lytic bacteriophage can enable host bacteria to be lysed and killed. The phage has strict host specificity, and the broad phage with host spectrum specificity mutation can crack host bacteria and susceptible host bacteria, can be used for treating the ultra-broad spectrum beta-lactamase Escherichia coli infection, and can be prepared into a clinical biological preparation for resisting bacterial infection.

Description

A bacteriophage acting on extended-spectrum β-lactamase-producing Escherichia coli

【Technical field】

The invention relates to a broad host spectrum producing extended-spectrum β-lactamase Escherichia coli phage, which belongs to the field of microbial bioengineering.

【Background technique】

With the widespread and irrational use of antimicrobial drugs by humans, a large number of drug-resistant bacteria have emerged, and the generation and spread of multi-drug resistant bacteria has become a problem of great concern to the world. People try to deal with the increasingly complex drug resistance of bacteria by continuously developing new antibacterial drugs, but after a new antibacterial drug is clinically used, it will still face the emergence of bacterial drug resistance.

β-lactam antibiotics refer to a large class of antibiotics containing a β-lactam ring in their molecular structure, including penicillins, cephalosporins and non-yellow β-lactams. β-lactam antibiotics play a therapeutic role by inhibiting penicillin binding proteins (PBPs), preventing the synthesis of bacterial cell wall mucopeptides (Mucopiptide), preventing the cross-linking of mucopeptide chains, so that bacteria cannot form cell walls, resulting in bacterial dissolves and dies.

Extended-spectrum beta-lactamases (ESBLs) refer to bacterial plasmid-mediated antibiotics capable of hydrolyzing oxyimino β-lactams, which can make third-generation cephalosporins (such as ceftazidime, cephalosporins, etc.) Azoxime, ceftriaxone) and monobactamycin (such as aztreonam) inactivate a class of enzymes. Since the German scholar Knothe first reported ESBLs in 1983, many countries have successively discovered the emergence of ESBL-producing bacteria. At present, the isolation rate of ESBL-producing bacteria in clinical specimens in the world has an increasing trend. ESBLs are mainly produced by Enterobacteriaceae, represented by Escherichia coli and Klebsiella pneumoniae, and 10%-20% of the strains produce ESBLs. The generation of ESBLs makes treatment more difficult and narrows the selection of antimicrobial agents. Greatly reducing the choice of antibacterial drugs often leads to failure of clinical treatment, recurrence of infection, and increased risk of death.

Since British bacteriologist Twort in 1915 and French bacteriologist d'Herelle observed phages in 1917, people have been studying phages more and more deeply. Phage is a kind of specific bacterial virus, there are two types of virulent phage and temperate phage. The virulent phage replicates and proliferates in the host bacterium, and lyses the host bacterium. State-owned scientists in the United States, Britain, France, Australia, Poland, and the former Soviet Union have been studying the use of lytic phages to treat bacterial infections. In the former Soviet Union, phage preparations have been widely used for the control of diarrhea in the army for a long time and achieved remarkable results. In Eastern European countries (such as Poland) and France, phages have also been used for a long time as a means of treating infections and there are finished preparations on the market.

In recent years, it has been noticed that the detection rate of ESBL-producing bacteria is high, and the drug resistance produced by it has become a global clinical treatment problem. ESBL-producing bacterial infections lead to prolonged hospitalization, increased costs and increased mortality. In this severe situation, all countries have also adopted measures such as rational use of antibiotics, reduction of selectivity and blindness in antibiotic application, strengthening monitoring of bacterial resistance rates, and regular reporting of bacterial resistance. However, ESBLs can transfer and disseminate between strains mediated by plasmids, and it is difficult to control nosocomial cross-infection and bacterial spread outside the hospital. ESBLs strains are not only resistant to most β-lactam antibiotics, but also resistant to aminoglycosides, fluoxanolones and sulfonamides. At present, the use of β-lactamase inhibitors can also achieve a certain effect on ESBL-producing bacterial infections in clinical practice, but Enterobacteriaceae are prone to develop resistance to β-lactamase inhibitors. The analysis of drug resistance of ESBL-producing bacteria shows that imipenem has the strongest antibacterial effect and can be used as the first choice for clinical treatment of ESBL-producing bacterial infections. The emergence of drug resistance. Therefore, actively facing the worldwide problem of drug resistance of ESBL-producing bacteria, and applying phages to the treatment of ESBL-producing bacterial infections has a very broad prospect.

【Content of invention】

After extensive research, the present inventor has isolated and obtained a broad-spectrum extended-spectrum β-lactamase-producing Escherichia coli phage φ9882, which can effectively kill extended-spectrum β-lactamase-producing Escherichia coli in vivo and in vitro φ9882. The inventors found in further research that the phage can lyse multiple strains of clinically isolated extended-spectrum β-lactamase-producing Escherichia coli in vitro and in vivo, and has a broad host spectrum.

Therefore, a kind of extended-spectrum β-lactamase-producing Escherichia coli phage φ9882 of the present invention can effectively kill clinically isolated extended-spectrum β-lactamase-producing Escherichia coli in vivo and in vitro.

The second purpose of the present invention is to confirm the superiority and feasibility of broad-spectrum phage as a therapeutic substance in the clinical anti-extended-spectrum β-lactamase-producing Escherichia coli infection treatment. - Lactamase could be an effective biologic agent in the development and application of treatment of E. coli infection.

According to the present invention, the extended-spectrum beta-lactamase-producing coliphage φ9882 of broad-host spectrum can form larger clear plaques on the petri dish, and the collected 30 strains of extended-spectrum beta-lactamase-producing colon Eleven of the clinical isolates of Bacillus have a lytic effect, and the cleavage rate is 36.67%. The clinical isolates that can be lysed are as follows: E. , 9882, 9730, 9739, 9914, 1068. When the phage of the present invention is used for the treatment of mouse bacteremia, all mice can be cured when MOI≥0.0001 (multiple of infection, multiplicity of infection). Even when the treatment was delayed for 3 hours (at this time, the mice had obvious symptoms of systemic infection such as inactivity, curly hair, arched back, accumulation of purulent exudate around the eyes, etc.), there was still a 20% curative effect. Both in vivo and in vitro experiments prove that the phage of the present invention is an effective anti-infection substance and has potential pharmaceutical value for clinical application.

The process of isolating and obtaining the bacteriophage of the present invention is as follows: take 1 L of untreated sewage from the hospital sewage treatment center, add 58 g of NaCl, and centrifuge at 10,000×g for 10 min. Take the supernatant, add PEG-g000 to a final concentration of 10% (w/v), put it in a refrigerator at 4°C overnight, and centrifuge at 10,000×g for 20min at 4°C. Resuspend the pellet with 5ml SM Buffer. Extract with an equal volume of chloroform once. 300 μl of treated sewage and 200 μl of extended-spectrum β-lactamase-producing Escherichia coli φ9882 cultured overnight, incubated at 37°C for 20 minutes, mixed with 3ml of melted top layer agar at 50°C, spread the plate, cooled and inverted at 37°C for overnight culture . On the next day, several clear plaques of different sizes were found on the petri dish. Remove the largest plaque with a tip, dissolve it in 2ml of LB culture solution, add extended-spectrum β-lactamase-producing E. Turn and shake for 4.5 to 5 hours. Centrifuge at 12000 rpm for 5 minutes, take 80% supernatant and store at 40°C. Take the supernatant and co-culture with extended-spectrum β-lactamase-producing Escherichia coli φ9882 on solid medium, and pick larger plaques with a tip. Repeat this 3 times to obtain plaques of uniform size. The plaques were taken out, and a small amount of amplification was carried out according to the above method to obtain a liquid phage amplification solution. The overnight cultured host bacteria were diluted 1:100, and cultured to the early logarithmic growth phase. Add 10 μl of the aforementioned concentrated phage, continue to incubate for 5 hours, add 1/10 volume of chloroform, continue to shake for 10 minutes, add DNase I and RNase A to a final concentration of 1 μg/ml, place at room temperature for 30 minutes, and centrifuge to remove bacterial debris . Add 1/6 volume of PEG/NaCl, overnight at 4°C. The next day, centrifuge at 12,000×g for 20 minutes at 4°C. Resuspend the pellet with 1ml SM Buffer. Add 1/6 volume of PEG/NaCl to precipitate again, incubate on ice for 1 hour, centrifuge at 12,000×g for 20 minutes at 4°C. Resuspend the pellet with 200 μl SM Buffer to obtain the amplified phage, which can reach the required titer after repeated amplification.

A broad-host spectrum producing extended-spectrum β-lactamase E. coli phage φ9882 of the present invention has been preserved in the φ National Microorganism Collection Center (Wuhan, China, Wuhan University, Budapest Treaty Depository Unit) on May 16, 2005 , and received deposit registration number CCTCC NO: M205044.

The present invention is an Escherichia coli phage φ9882CCTCC NO: M205044 acting on the extended-spectrum β-lactamase-producing coli, which has a relatively broad host spectrum.

The host cell of the wide host spectrum of the present invention is the extended-spectrum β-lactamase-producing E. coli phage φ9882, which contains the biological preparation of the above-mentioned coli phage.

The invention is an application of the extended-spectrum β-lactamase-producing colibacillus phage φ9882 in the preparation of anti-infection biological preparations.

The medium used in the present invention has the following composition: LB medium: 1% tryptone, 0.5% Yeast extract and 1% NaCl. Solid agar: 1% tryptone, 0.5% Yeastextract and 1% NaCl and 1.5% Agar. Top agar: 1% tryptone, 0.5% Yeastextract and 1% NaCl and 0.7% Agarose. SM Buffer is prepared by 0.1M NaCl, 0.01MMgSO ₄ ·7H ₂ O, 0.05M Tris-HCl (pH7.5) and 0.01% gelatin (gelatin). PEG/NaCl was formulated with 20% PEG-8000 and 2.5M NaCl.

A kind of broad host spectrum of the present invention produces extended-spectrum β-lactamase coliphage ph9882 (CCTCC NO: M205044) has the following microbiological characteristics:

1. Morphological characteristics:

Phosphotungstic acid negative staining electron microscope observation, electron microscope ultrastructure showed that the phage was a microspherical particle with a diameter of 70nm, with inconspicuous edges and corners, and a long tail axis of 100nm was visible (as shown in Figure 1).

2. Cultivation characteristics

The phage strain of the present invention can form transparent plaques on the solid medium of the host bacterium Escherichia coli φ9882 producing extended-spectrum β-lactamase, the diameter of the plaques is 2-3mm, and there is no halo around.

3. Biological characteristics:

The phage strain of the present invention can be quickly adsorbed on host bacteria Escherichia coli φ9882 producing extended-spectrum β-lactamase, and the adsorption rate reaches 98% within 5 minutes (as shown in FIG. 2 ). The one-step growth curve of the phage (as shown in FIG. 3 ), from which it can be seen that the incubation period of the phage infection host bacteria is 30-40 minutes, and the average lysis amount is 110. The short incubation period and large amount of lysis indicated that the broad phage had a strong lytic effect.

A broad-spectrum extended-spectrum β-lactamase-producing Escherichia coli phage φ9882 of the present invention can simultaneously crack 11 strains of clinically isolated Escherichia coli strains producing extended-spectrum β-lactamase, and its wide-phagocytosis rate is 36.67% , and the titer was 8.1×10 ¹⁸ pfu/ml. At the same time, the extended-spectrum β-lactamase-producing Escherichia coli phage φ9882 with broad host spectrum cannot crack Staphylococcus aureus, Pseudomonas aeruginosa, A-hemolytic streptococcus and B-hemolytic streptococcus and other bacterial strains.

The present inventors have confirmed that the isolated extended-spectrum β-lactamase-producing coliphage φ9882 can effectively treat probiotics in animal experiments (BlAB/c mice, female, body weight 20.0±0.5g). Infection of extended-spectrum β-lactamase Escherichia coli φ9853. A broad-host-spectrum extended-spectrum β-lactamase-producing Escherichia coli phage φ9882 of the present invention can treat mouse extended-spectrum β-lactamase-producing Escherichia coli infection, and has obvious therapeutic effect on susceptible host bacterial infection .

The invention will be explained in more detail by means of the following examples. The following examples are illustrative only, and it should be noted that the present invention is not limited by these examples.

【Description of drawings】

Figure 1: Electron micrograph of φ9882, the bacteriophage has a polyhedral head structure with a diameter of 70nm and a tail shaft mechanism. The tail axis is 100nm long.

Figure 2: The absorption curve of bacteriophage φ9882, the adsorption rate reaches 98% within 5 minutes (as shown in Figure 2).

Figure 3: One-step growth curve of phage φ9882, the incubation period of the phage infection host bacteria is 30-40 minutes, and the average lysis amount is 110. The short incubation period and large amount of lysis indicated that the phage had a strong lytic effect.

Figure 4: Screening of broad phages: use extended-spectrum β-lactamase-producing Escherichia coli φ9561 as the host bacteria to make a uniform lawn, and then divide 20 small squares on the back of the petri dish, dropwise add Phages, lytic phages can form clear plaques.

Figure 5 Determination of the minimum lethal dose of extended-spectrum β-lactamase-producing Escherichia coli φ9853 infecting mice. The bacterial solution of 3×10 ⁷ -1×10 ⁸ CFU can cause the death of all mice, while 1×10 ⁷ - 2×10 ⁷ CFU of bacterial solution could not cause death of all mice. Therefore, 3×10 ⁷ CFU is the minimum lethal dose to cause death of mice.

Fig. 6 Accurate phase analysis of the minimum lethal dose, the bacterial solution of MLD was intraperitoneally injected into 10 mice, and the mice died within 8-14 hours.

Fig. 7 Treatment effect of different doses of phage, all mice can survive and eventually recover when the dose of phage is MOI ≥ 0.0001.

Fig. 8 Phage delays the treatment effect, and all dying mice can be cured within 40 minutes of the delay. When the treatment was delayed for more than 1 hour, due to the poor state of the mice at this time, obvious symptoms of infection such as curly hair, arched back, and accumulation of exudate around the eyes, etc., the effect of the phage decreased, but it still had Statistically significant (P<0.01).

【Detailed ways】

Example 1:

Take 1L of sewage from the hospital sewage treatment center, add 58g of NaCl, and centrifuge at 10,000×g for 10min. Take the supernatant, add PEG-8000 to a final concentration of 10% (w/v), put it in a refrigerator at 4°C overnight, and centrifuge at 10,000×g for 20min at 4°C. Resuspend the pellet with 5ml SM Buffer. Extract with an equal volume of chloroform once. Mix 300 μl of treated sewage with 200 μl of overnight cultured host bacteria, incubate at 37°C for 20 minutes, mix with 3ml of melted top layer agar at 50°C, spread the plate, cool and invert at 37°C for overnight culture. Remove the largest plaque with a tip, dissolve it in 2ml of LB culture solution, add extended-spectrum β-lactamase-producing E. Turn and shake for 4.5 to 5 hours. Centrifuge at 12,000 rpm for 5 minutes, and take 80% of the supernatant and store it at 40°C. Take the supernatant and co-culture with extended-spectrum β-lactamase-producing Escherichia coli φ9882 on solid medium, and pick larger plaques with a tip. Repeat this 3 times to obtain plaques of uniform size. The plaques were taken and amplified in liquid according to the method mentioned above to obtain concentrated phage liquid.

Example 2:

The extended-spectrum β-lactamase-producing Escherichia coli φ9882 cultured overnight was diluted 1:100, and cultured to the early logarithmic growth phase. Add 10 μl of the concentrated phage in Example 1, continue culturing for 5 h, add 1/10 volume of chloroform, continue shaking for 10 minutes, add DNase I and RNase A to the final concentration of 1 μg/ml, and place at room temperature for 30 minutes. Bacterial debris was removed by centrifugation. Add 1/6 volume of PEG/NaCl, overnight at 4°C. The next day, centrifuge at 12,000×g for 20 minutes at 4°C. Resuspend the pellet with 1ml SM Buffer. Add 1/6 volume of PEG/NaCl to precipitate again, incubate on ice for 1 hour, centrifuge at 12,000×g for 20 minutes at 4°C. Resuspend the pellet with 200 μl SM Buffer to obtain the amplified phage, which can reach the required titer after repeated amplification.

Example 3:

A uniform lawn was made on LB medium with 30 diluted extended-spectrum β-lactamase-producing Escherichia coli. Divide and mark 16-20 squares on the back of the culture medium, then drop phage φ9882 solution into the corresponding squares, and after the droplets are dry, place them upside down and incubate at 37°C for 12-16 hours to observe the results (Figure 4). It can be seen that φ9882 forms plaques on 11 strains, as follows:

1. Take 100ul of bacterial culture grown overnight, put it into a Erlenmeyer flask with 10ml medium, shake it at 370°C for 2-2.5 hours, and count it under the microscope, the cells can reach more than 10 ⁸ /ml, dilute with medium, Prepared to 5×10 ⁷ /ml.

2. Measure the titer of phage one day before the experiment, and then dilute it to make 5×10 ⁹ PFU/ml.

3. Balance the above two in a 37°C water bath, add 1ml of phage liquid to 9ml3 of bacterial suspension according to MOI=10, mix well, put back in the water bath to keep warm or shake to keep warm.

4. Take samples at a certain time interval (1-5min), dilute 1000 times, put them in ice, and stop the adsorption.

5. Take 1ml of the diluted sample and centrifuge it in an Ep tube for 3 minutes or 4000rpm for 5 minutes to settle the bacteria and the cells that adsorb the phage.

6. After appropriate dilution (10-100 times), the free phage in the supernatant was measured by the double-layer agar method, and recorded after cultivation.

Example 4:

1. Dilute the phage liquid to 5×10 ⁷ PFU/ml.

2. Dilute the bacterial solution to 5×10 ⁷ CFU/ml.

3. Take 20 Ep tubes and add 900ul LB solution to each tube.

4. Take 20 Ep tubes and add 200ul of bacterial solution to each tube.

5. Take 20 test tubes, add 3ml of melted top glue to each tube, and keep warm in a 50-degree water bath.

6. Incubate the above-mentioned phage liquid and bacterial liquid at 37 degrees.

7. Add 0.1ml of phage liquid into 0.9ml of bacterial suspension, and absorb for 5 minutes.

8. Take 0.1ml of the mixed solution and add it to 9.9ml of ice TSBM solution, mix well and take 1ml each.

9. Take 0.1ml of the resuspension solution and add it to 9.9ml of LB solution, shake and culture at 370°C, during which time take 0.1ml at the 0th, 5th, 10th, 20th, 30th, 40th, 50th, 60th, 70th, 80th, 90th, and 100th minutes The co-cultivation solution was mixed with 200ul bacterial solution, plated after 5 minutes, and cultured at 370°C respectively. From the number of plaques at the above points-time curve (Figure 3), it can be obtained that the incubation period of the phage is 30-40min, and the lysis amount is 112.

Example 5:

To establish a systemic infection model, culture the extended-spectrum β-lactamase-producing Escherichia coli φ9853 overnight and dilute it in 100ml LB at a ratio of 1:100, and continue to culture until the early logarithmic growth phase (OD ₆₀₀ value is about 0.5), 4°C , centrifuged at 8000g for 5 minutes, discarded the supernatant, resuspended the precipitate with an equal volume of sterilized normal saline, centrifuged again, and dissolved the precipitate in 5ml of normal saline. Extended-spectrum β-lactamase-producing Escherichia coli φ9853 bacteria solution serially diluted with normal saline was injected intraperitoneally (ip) into 11 groups of mice, resulting in one group (5 mice/group unless otherwise specified) The minimum dose for all mice to die is the minimal lethal dose (MLD). After serially diluted bacterial solution was injected into mice, the bacterial solution of 3×10 ⁷ -1×10 ⁸ CFU could cause the death of all mice, but the bacterial solution of 1×10 ⁷ -2×10 ⁷ CFU could not cause all mice to die. Mice died (as shown in Figure 5). Therefore, 3×10 ⁷ CFU is the minimum lethal dose to cause death of mice. The extended-spectrum β-lactamase-producing Escherichia coli φ9853 bacterial solution of MLD was intraperitoneally injected into 10 mice, and the mice died within 6 to 14 hours (as shown in Figure 6).

Embodiment 6:

The effect of different phage doses on the curative effect: 9 groups of mice were taken, and the bacterial solution of MLD dose was injected intraperitoneally on one side, and different doses of phage φ9882 preparations (diluted in LB) were injected intraperitoneally on the other side immediately, and one group was injected i.p. with LB culture medium as a control. Observe the health status of the mice for more than 20 days. As shown in Figure 7, all mice can survive and eventually recover when the phage dose is MOI ≥ 0.0001. There was a significant statistical difference in the survival rate of mice under the conditions of MOI=0 and MOI≥0.0001 (P<0.01).

Embodiment 7:

Delayed treatment effect monitoring: 7 groups of mice were taken, all infected with MLD dose of bacterial solution, and injected with 500 μl of higher titer phage φ9882 solution (MOI =200) i.p injection, observed for more than 20 days. A corresponding control group was set up for each group of mice (i.p. injection of the same amount of LB culture solution). As shown in Figure 8, in the case of a delay of 40 minutes, all mice could be saved from death. When the treatment was delayed for more than 1 hour, due to the poor state of the mice at this time, obvious symptoms of infection such as curly hair, arched back, and accumulation of exudate around the eyes, etc., the effect of the phage decreased, but it still had Statistically significant (P<0.01).

Claims (3)

1. A bacteriophage φ9882CCTCC NO: M205044 that acts on extended-spectrum β-lactamase-producing Escherichia coli. 2. A biological preparation containing coliphage according to claim 1. 3. the coliphage described in claim 1 is being used in the preparation of the purposes in anti-infective biological preparation.

Images