WO2021082512A1

WO2021082512A1 - Use of blue-light activated blebbistatin analogues in killing drug-resistant bacteria

Info

Publication number: WO2021082512A1
Application number: PCT/CN2020/100528
Authority: WO
Inventors: Nai-Kei Wong; Yong-li XIE; Jing Yuan; Yang Zhang; Ziyuan ZHOU; Mingde Li; Lei Liu
Original assignee: Shenzhen Third People's Hospital
Priority date: 2019-11-01
Filing date: 2020-07-07
Publication date: 2021-05-06
Also published as: CN110812481A; CN110812481B

Abstract

Provided herein is the use of biologically inert compounds that can be photoactiveted by blue light at a specific wavelength to remove or kill drug-resistant bacteria. The photoactivatable compounds include blebbistatin analogues such as (R)-blebbistatin. (R)-blebbistatin has no mammalian protein targets and can be photoactivated by blue light to exert significant bactericidal effects. Also provided herein is a method for killing pathogenic bacteria of varied antimicrobial resistance profiles including Staphylococcus aureus, Escherichia coli, Enterococcus spp., Pseudomonas aeruginosa, and Acinetobacter baumannii. Bacterial killing efficiency increases with increasing illumination time, and there is no risk of inducing drug resistance. (R) -blebbistatin is safe to zebrafish embryos after 60 min treatment in vivo. The method is not limited by antibiotic resistance in bacteria, which provides foundation for treating infections by multi-drug resistant pathogens including but not limited to bacteria.

Description

USE OF BLUE-LIGHT ACTIVATED BLEBBISTATIN ANALOGUES IN KILLING DRUG-RESISTANT BACTERIA

TECHNICAL FIELD

The invention relates to the field of biomedicine, and in particular relates to the use of blue-light activated blebbistatin analogues in killing drug-resistant bacteria and a method thereof.

BACKGROUND

Pathogenic bacteria commonly found in hospital-acquired infections (i.e., nosocomial infections) include Gram-positive bacteria (e.g., Staphylococcus aureus, Enterococcus spp. ) and Gram-negative bacteria (e.g., Pseudomonas aeruginosa, Acinetobacter baumannii, E. coli) . Staphylococcus aureus is a common food-borne pathogenic microorganism, often parasitic in the skin, nasal cavity, throat, gastrointestinal, suppurative sores, and easily lead to food poisoning. Enterococcus is widely distributed in the gastrointestinal (GI) tract of human beings and is an important opportunistic pathogen in nosocomial infections, capable of causing urinary tract infections, skin and soft tissue infections, abdominal infections, meningitis, etc. However, due to intrinsic drug resistance of Enterococcus, incidence of infection and mortality rate associated with its infections have increased, adding to treatment costs and disease burden. Pseudomonas aeruginosa is widely distributed in the natural environment, normal human skin, intestines, and respiratory tract. It is one of the major opportunistic pathogens encountered in the clinic, with close to 10%of hospital infections being caused by this bacterium. Moreover, Pseudomonas aeruginosa is naturally resistant to most antibiotics and can rapidly acquire drug resistance. Except for quinolone class of antibiotics, other antibiotics need to be used via an injection route. Acinetobacter baumannii is commonly isolatable from the environment as well as the human skin, digestive system, urinary system, etc. The main population vulnerable to its infections is patients from intensive care unit (ICU) , elderly people, immuno-compromised patients. Acinetobacter baumannii also opportunistically cause infections in surgical and invasive treatments, including use of mechanical ventilators. Phenotypic character of multidrug-resistant Acinetobacter baumannii is very prominent, typically involving extensive resistance to carbapenem and quinolone classes of antibiotics. Tigecycline and colistin antibiotics have become the last line of defense against clinical infections by Acinetobacter baumannii, but these two types of drugs tend to have major side-effects and relatively low efficacy. Escherichia coli is a common nosocomial pathogen. Due to uncritical and excessive use of broad-spectrum antibiotics, the spread of multidrug-resistant E. coli has greatly increased the challenge of anti-infective treatment. It has even reached a point that there are no effective anti-infective medicine available. In recent years, 80%of clinical antibacterial drugs have been made ineffective due to a menacing of drug resistance among bacterial pathogens.

At present, due to a critical lack of incentives for research and development of new antibiotics, it is extremely urgent to search for alternative antimicrobial strategies to address the serious shortages of usable antibiotics. Blue light-dependent photochemotherapies are currently an attractive and promising therapeutic approach, which is directly and precisely delivered by controllable photochemical reactions; photoactivatable agents do not cause genetic mutations or resistance in bacteria in the absence of light. The synergistic bactericidal effects of photoactivatable agents and blue light increase with time, and there is no risk of inducing drug resistance.

Aaron F. Straight et al. (Aaron F. Straight, Dissecting Temporal and Spatial Control of Cytokinesis with a Myosin II Inhibitor, Science, 2003) disclosed the chemical structure and biological characteristics of levorotatory blebbistatin (that is, (S) -blebbistatin) and dextrorotatory blebbistatin (that is, (R) -blebbistatin) . In particular, (R) -blebbistatin has been shown to be devoid of biological targets and is biologically active in mammalian cells. There have been no studies on (R) -blebbistatin biological activity or its biological or medical applications to this date.

SUMMARY

One object of the invention is to provide the use of blue-light activated blebbistatin analogues in killing drug-resistant bacteria and a method thereof, which display an extremely efficient bactericidal activity under blue light, without risks for bacterial mutation or inducing drug resistance during the bacterial killing process, and thereby solve the problems of prior art.

One aspect of the present invention is the use of blue-light activated blebbistatin analogues in killing drug-resistant bacteria.

Preferably, the structural formulas of the blebbistatin analogues are:

One of R ₁, R ₂, R ₃, R ₄ and R ₅ may be one of H, CN, NH ₂, F, Cl, Br, I, alkyl, haloalkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, alkylamino, arylamino, dialkylamino, alkarylamino, aryloxy, arylalkoxy, acyloxy, carbamoyl, trifluoromethyl, trifluoromethoxy, phenoxy, benzyloxy, phosphonyl, phosphate, sulfonic, sulfonate, and sulfonamide; when one of R ₁, R ₂, R ₃, R ₄ and R ₅ is the above group, the remaining four groups are all H.

Preferably, the structural formulas of the blebbistatin analogues are:

One of X ₁, X ₂, X ₃, X ₄ and X ₅ may be one of H, CN, CF ₃, F, Cl, Br, and I, and when one of X ₁, X ₂, X ₃, X ₄ and X ₅ is the above group, the remaining four groups are H.One of R ₁, R ₂, R ₃, R ₄ and R ₅ may be one of H, CN, NH ₂, F, Cl, Br, I, alkyl, haloalkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkaryl, heterocyclic, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, alkylamino, arylamino, dialkylamino, alkarylamino, aryloxy, arylalkoxy, acyloxy, carbamoyl, trifluoromethyl, trifluoromethoxy, phenoxy, benzyloxy, phosphonyl, phosphate, sulfonic, sulfonate, and sulfonamide, and when one of R ₁, R ₂, R ₃, R ₄ and R ₅ is the above groups, the remaining four groups are H.

More preferably, one of the blebbistatin analogues is (R) -blebbistatin.

Preferably, the drug-resistant bacteria include Staphylococcus aureus, Escherichia coli, Enterococcus spp., Pseudomonas aeruginosa, and Acinetobacter baumannii.

Preferably, the blue light excitation wavelength is 420 nm.

Another aspect of the present invention is a method of killing drug-resistant bacteria by using a blue-light activated blebbistatin analogue, which mainly comprises: mixing a liquid culture of exponential-phase drug-resistant bacteria having a 2-fold working density and a blebbistatin analogue having a 2-fold working concentration in a ratio of 1: 1. After mixing, the blue light with an excitation wavelength of 420 nm was used for irradiating the bacterial liquid culture for 30-60 min. To evaluate the killing effects of the compounds, an inoculum of the bacterial liquid culture was gently spotted vertically on the surface of an agar plate, and the plate was inverted and cultured for 12 hours for observation of any surviving colonies, based on which the bacterial killing efficiency was to be evaluated. The absence or presence of residual regrowth of bacterial colonies on an inoculated agar plate provides a measure of bactericidal activity of blue-light activated blebbistatin analogues. The agar plate assay is a sensitive screening test.

Preferably, the blue light source is placed at 7.2 cm above liquid culture of exponential-phase drug-resistant bacteria.

Preferably, the working density of the liquid culture of exponential-phase drug-resistant bacteria is 10 ⁸ cfu/mL or lower.

Preferably, the working concentrations of blebbistatin analogues are from 2 μM to 10 μM.

Preferably, the liquid culture of exponential-phase drug-resistant bacteria may be prepared by the following steps:

(1) Drug-resistant bacteria stored at -80 ℃ were streaked multiple times on an agar plate, and monoclonal bacterial colonies were obtained after 12 h culture on the plate;

(2) The monoclonal bacterial colonies were picked and added to an LB (Luria-Bertani) medium and /or MH (Müller-Hinton) medium, and cultured for 1.5 h in a bacterial culture shaker to obtain the liquid culture of exponential-phase drug-resistant bacteria.

Preferably, the 2-fold working concentration of the blebbistatin analogue may be prepared with DMF (dimethylformamide) as a solvent to give a stock solution, which was then diluted to an intermediate concentration of 2-fold with phosphate buffered saline (PBS) . The composition of phosphate buffer (PBS) includes NaCl, KCl, Na ₂HPO ₄, and KH ₂PO ₄.

Compared with prior art, the invention can be used to effectively eradicate drug-resistant bacteria such as Staphylococcus aureus, Escherichia coli, Enterococcus spp., Pseudomonas aeruginosa, and Acinetobacter baumannii by the combined use of blue light (excitation at 420 nm) and (R) -blebbistatin, which has no known mammalian protein targets and is therefore biologically inactive. The invention produces a strong bactericidal effect. As an embodiment of the invention, (R) -blebbistatin treatment was safe for 60 min treatment in vivo (in zebrafish embryo model) , and its bacterial sterilization efficiency is improved with increasing irradiation time. There is no risk of inducing drug resistance when blue light is absent. The application scope of the invention is clearly not restricted by bacterial resistance, and provides a solid and powerful technical framework for the treatment of clinical multi-drug resistant bacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: A photochemical reaction platform with a blue LED light source in the invention.

Figure 2: Acinetobacter baumannii of high densities (10 ⁸ cfu/mL and 10 ⁷ cfu/mL) was killed effectively by (R) -blebbistatin across a concentration gradient under blue light (excitation at 420 nm) , and thereafter any residual bacterial growth was detected on MH agar plates, with IC ₅₀ value and MIC value reported for evaluation.

Figure 3: Acinetobacter baumannii of high densities (10 ⁸ cfu/mL to 10 ³ cfu/mL) was killed by (R) -blebbistatin under blue light (excitation at 420 nm) , whose residual bacterial growth was monitored on MH agar plates.

Figure 4: Acinetobacter baumannii of high density (10 ⁸ cfu/mL) was killed by (R) -blebbistatin under blue light of different wavelengths, whose residual bacterial growth was evaluated on MH agar plates to give a corresponding histogram reporting residual growth.

Figure 5: Different strains of Acinetobacter baumannii of high density (10 ⁸ cfu/mL) was killed by (R) -blebbistatin under blue light (excitation at 420 nm) , whose residual bacterial growth was monitored on MH agar plates.

Figure 6: Staphylococcus aureus of high density (10 ⁸ cfu/mL) was killed by (R) -blebbistatin under blue light (excitation at 420 nm) , whose residual bacterial growth was evaluated on MH agar plates to give a corresponding histogram reporting residual growth.

Figure 7: Enterococcus faecium of high density (10 ⁸ cfu/mL) was killed by (R) -blebbistatin under blue light (excitation at 420 nm) , whose residual bacterial growth was evaluated on MH agar plates to give a corresponding histogram reporting residual growth.

Figure 8: E. coli of high density (10 ⁸ cfu/mL) was killed by (R) -blebbistatin under blue light (excitation at 420 nm) , whose residual bacterial growth was evaluated on MH agar plates to give a corresponding histogram reporting residual growth.

Figure 9: Pseudomonas aeruginosa of high density (10 ⁸ cfu/mL) was killed by (R) -blebbistatin under blue light (excitation at 420 nm) , whose residual bacterial growth was evaluated on MH agar plates to give a corresponding histogram reporting residual growth.

Figure 10: Klebsiella pneumoniae of high density (10 ⁸ cfu/mL) was treated with (R) -blebbistatin under blue light (excitation at 420 nm) , whose residual bacterial growth was evaluated on MH agar plates to give a corresponding histogram reporting residual growth.

Figure 11: Microscopic imaging of the effects of (R) -blebbistatin and (S) -blebbistatin on zebrafish embryos in vivo (20× magnification) .

Figure 12: Bacterial killing effects of (R) -blebbistatin and (S) -blebbistatin across a concentration gradient under blue light (excitation at 420 nm) on Acinetobacter baumannii at relatively high density (10 ⁷ cfu/mL) , as evaluated on MH agar plates.

Figure 13: Heatmap of antimicrobial susceptibility test (AST) data (MIC values) for various drug-resistant bacteria tested in the embodiments. R: resistant; S: sensitive; I: intermediate.

DETAILED DESCRIPTION OF THE EMBODIMENTS

To clarify the purpose, technical solution and advantages of the invention, the present invention is further described herein in detail with reference to the accompanying drawings. In addition to a variety of clinical pathogens such as Acinetobacter baumannii, the rest of the equipment and reagents involved in the embodiments are commercially available.

Example 1

A method ( “concentration gradient sterilization” ) for killing clinical isolates of drug-resistant Acinetobacter baumannii (strain GD0302) by a blebbistatin analogue, (R) -blebbistatin, activated under blue light at 420 nm, comprising the following steps:

1. Streak a clinical isolate of drug-resistant Acinetobacter baumannii on a culture plate to obtain a monoclonal clone.

1.1) Retrieve the clinical isolate of drug-resistant Acinetobacter baumannii (strain GD0302) stored at -80 ℃. After briefly standing the bacterial stock on ice, use a circular inoculation needle to draw 1 μL of the bacterial stock and inoculate the LB agar plates by streaking multiple lines.

1.2) Invert an inoculated plate and leave it in a bacterial incubator (37℃) for 12 h to obtain a bacterial lawn from a monoclonal strain.

2. Use the purified monoclonal strain to inoculate fresh culture medium to obtain a liquid culture of Acinetobacter baumannii in logarithmic growth phase.

2.1) Pick 1-4 bacterial colonies of monoclonal strains from LB agar plates and add them to a culture tube containing 3 mL of LB medium for overnight incubation with shaking (220 rpm) .

2.2) The next morning, take 1/10 of the overnight bacterial culture and add it to a new culture tube containing 3 mL LB medium for 1-1.5 h incubation with shaking (220 rpm) . This typically yields a liquid culture of Acinetobacter baumannii in logarithmic growth phase.

2.3) Turbidity or growth can be validated by measuring optic density or absorbance (600 nm) in a Nanodrop instrument. The desired range of bacterial harvest is such that OD _600 nm = 0.6-1.0, which corresponds to logarithmic growth phase of bacterial culture.

2.4) Transfer 3 mL of the bacterial culture to a 15 mL centrifuge tube and spin down the culture at 4000×g for 10 min to collect a bacterial cell pellet.

2.5) Prepare PBS, and wash the bacteria twice with 3 mL PBS, followed by centrifugation at 4000×g for 5 min.

2.6) Re-suspend the bacteria in 3 mL PBS, and use Nanodrop to re-obtain accurate OD _600 nm values for the bacterial suspension in PBS.

3. (R) -blebbistatin in PBS is to be mixed with the bacterial suspension by doing a 2-fold dilution, and then placed at a certain distance under the blue light source for irradiation to allow bacterial killing to occur.

3.1) According to the OD values obtained in step 2, convert a bacterial culture in logarithmic growth phase to a 2-fold working density, typically as 2 × 10 ⁸ cfu/mL or 2 × 10 ⁷ cfu/mL.

3.2) Weigh 5 mg of (R) -blebbistatin into the organic solvent DMF to prepare a 100 mM stock solution. Store this at -20 ℃ until use. On the day of application, prepare intermediate stock solutions in PBS at 2 × working concentration of the compound: 2 × 0.4 μM, 2 × 2 μM, 2 × 6 μM.

3.3) Pipette 500 μL of 2× bacterial suspension in PBS and add it to 500 μL of 2×(R) -blebbistatin in PBS in a 1.5 mL Eppendorf tube, followed by mixing to give a desired working concentration (i.e., 1 × 10 ⁸ cfu/mL or 1 × 10 ⁷ cfu/mL) .

3.4) Assemble the blue LED light source platform (see Figure 1. This blue-light platform consists of a rectangular box with a hollow circular hole at the top that can fit in the LED lamp. The LED is coupled to a small fan acting as a heat sink for heat dissipation to reduce the errors generated by heat. The Eppendorf tube containing the bacterial suspension to be killed by the compound is left to stand upright in a transparent plastic rack, so that the liquid level of the bacterial suspension and the LED lamp is separated by a distance of 7.2 cm.

3.5) Proceed with blue light (420 nm) irradiation for 60 min. At the end of the photochemical reaction, collect the bacterial suspension in Eppendorf tube and immediately placed it on ice to avoid further reaction.

4. Assess the bactericidal effect and determine if there are residual colonies of bacteria surviving the photochemical reaction, after regrowth in a sufficiently long period of time on an MH agar plate.

4.1) Based on the (R) -blebbistatin treated bacterial suspension obtained in step 3, by means of a pipetman, about 6 μL of the undiluted suspension was vertically spotted as an inoculum on MH agar plates. The agar plates were incubated in an inverted position in an incubator for 12 hours for visualizing any bactericidal effects.

5. Results

In this example, a clinical isolate of drug-resistant Acinetobacter baumannii (strain GD0302) was used for testing bactericidal effects at a working density of 10 ⁸ cfu/mL or 10 ⁷ cfu/mL. After mixing with different concentrations of (R) -blebbistatin, the blue LED light source was set as 420 nm to deliver irradiation for 60 min. It was observed that 2 μM (R) -blebbistatin can kill a large number (10 ⁸ cfu/mL) of drug-resistant Acinetobacter baumannii, and that (R) -blebbistatin at a concentration above 2 μM can completely eradicate drug-resistant Acinetobacter baumannii. For 10 ⁷ cfu/mL of Acinetobacter baumannii, (R) -blebbistatin at 2 μM or more can completely eradicate drug-resistant Acinetobacter baumannii. The corresponding MICs (minimum inhibitory concentration) are 2.52 μg/mL (for 10 ⁸ cfu/mL) , and 1.84 μg/mL (for 10 ⁷ cfu/mL) (See Figure 2) .

Example 2

A method for killing (density-gradient sterilization) the clinical isolate of drug-resistant Acinetobacter baumannii (strain GD0302) by a blue-light activated blebbistatin analogue, (R) -blebbistatin, comprising the following steps:

The culture materials, instruments and processes of this embodiment are basically the same as those of the first embodiment, and the difference lies in:

(1) Unlike 3.1) in Example 1, the working density of the bacteria was changed to cover the range of 10 ⁸ cfu/mL-10 ³ cfu/mL. Prior to photochemical reactions, bacteria were serially diluted in PBS with a 10-fold density gradient to give 2 × working densities, i.e. : 2 × 10 ⁸ cfu/mL, 2 × 10 ⁷ cfu/mL, 2 × 10 ⁶ cfu/mL, 2 × 10 ⁵ cfu/mL, 2 × 10 ⁴ cfu /mL, 2 × 10 ³ cfu/mL.

(2) Results

This example uses a clinical isolate of drug-resistant Acinetobacter baumannii (strain GD0302) with a working density of 10 ⁸ cfu/mL, 10 ⁷ cfu/mL, 10 ⁶ cfu/mL, 10 ⁵ cfu/mL, 10 ⁴ cfu/mL, or 10 ³ cfu/mL. After mixing with 6 μM of (R) -blebbistatin, the bacterial suspension was exposed to blue light (420 nm) for 60 min. It was observed that 6 μM of (R) -blebbistatin was able to eradicate drug-resistant Acinetobacter baumannii at all densities tested (Figure 3) .

Example 3

A blue-light activated blebbistatin analogue (R) -blebbistatin kills the clinical isolate of drug-resistant Acinetobacter baumannii (strain GD0302) under blue light at different wavelengths, including the following steps:

(1) Unlike 3.1) in Example 1, the density of the drug-resistant Acinetobacter baumannii in Example 3 was 10 ⁸ cfu/mL, that is, its 2 × density was 2×10 ⁸ cfu/mL.

(2) Unlike 3.2) in Example 1, the concentration of (R) -blebbistatin used in Example 3 is 0 μM, 1 μM, and 6 μM, that is, their 2 × concentrations were 2×0 μM, 2×1 μM, 2 × 6 μM.

(3) Unlike Example 3.5) , Example 3 uses three different wavelengths of blue light sources, such as 360 nm, 420 nm, and 460 nm.

(4) Unlike 4.1 in Example 1, based on the (R) -blebbistatin treated bacterial suspension obtained in Step 3, 100 μL of MH medium (4 wells) and 10 μL of the bacterial solution was added to a well (in at least 4 replicates) in a 96-well microplate, and the culture was allowed to stand in a bacterial incubator. After culturing overnight, the OD _600 nm absorbance value was measured by an absorbance reader for microplates to assess any bactericidal effects.

(5) Results

In this example, a clinical isolate of drug-resistant Acinetobacter baumannii (strain GD0302) was used at a working density of 10 ⁸ cfu/mL. After mixing with 0 μM, 1 μM, and 6 μM (R) -blebbistatin, blue light irradiation proceeded at 360 nm, 420 nm or 460 nm for 60 min. It was observed that a 460 nm wavelength with (R) -blebbistatin had a weak bactericidal activity; a 360 nm wavelength with (R) -blebbistatin had no bactericidal effects; and a 420 nm wavelength with (R) -blebbistatin could stop growth of drug-resistant Acinetobacter baumannii at 6 μM. The strain was completely killed (Figure 4) .

Example 4

A method for killing different clinical subtypes of Acinetobacter baumannii by blebbistatin analogue (R) -blebbistatin activated by blue light, comprising the following steps:

(1) Unlike 1.1) in Example 1, this time, Acinetobacter baumannii isolates of different sequence types (STs) were used, including: GD0302 of sequence type 195, ATCC19606 of sequence type 931, GD0311 of sequence type 208, AYE of sequence type 231, and GD0304 of sequence type 195.

(2) Unlike the case of different subtypes of drug-resistant A. baumannii used in example 4, 3.1) in Example 1, the working densities of 10 ⁸ cfu/mL, 10 ⁷ cfu/mL, and 10 ⁶ cfu/mL were used, which were diluted from 2-fold densities: 2 × 10 ⁸ cfu/mL, 2 ×10 ⁷ cfu/mL, and 2 × 10 ⁶ cfu/mL.

(3) Unlike 4.1) in Example 1, in Example 4, the inoculum spotted on the MH agar plates was of a bacterial liquid density of 10 ⁸ cfu/mL;

(4) Results

In this example, Acinetobacter baumannii isolates of different sequence types of drug-resistant (strains GD0302, ATCC19606, GD0311, AYE, and GD0304) were used, and the working density was 10 ⁸ cfu/mL. After mixing with 6 μM (R) -blebbistatin, blue light irradiation proceeded at a wavelength of 420 nm for 60 min. As visualized on MH agar plates, 6 μM of (R) -blebbistatin under 420 nm blue light could completely kill drug-resistant Acinetobacter baumannii of different sequence types without residual colonies (Figure 5) . The antimicrobial susceptibility test (AST) profiles of drug-resistant Acinetobacter baumannii of different sequence types are as shown in Table 1.

Table 1: Antimicrobial susceptibility test (AST) results of drug-resistant Acinetobacter baumannii of different sequence types

AMP: ampicillin; AMC: amoxicillin/clavulanate; CSL: cefoperazone/sulbactam; IMP: imipenem; MEM: meropenem; CIP: ciprofloxacin; AMK: amikacin; COL: colistin;

Antibiotic categories: I: penicillins; II: cephalosporins; III: penems; IV: quinolones; V: aminoglycosides; VI: lipopeptides.

Example 5

A method for killing clinical S. aureus by a blue-light activated blebbistatin analogue (R) -blebbistatin, comprising the following steps:

(1) Unlike 1.1) in example 1, this time the bacterium Staphylococcus aureus (strain Sau16162) was used.

(2) Unlike 3.1) in example 1, the S. aureus working density used in example 5 was 10 ⁸ cfu/mL, and whose 2× density was thus 2×10 ⁸ cfu/mL.

(3) Unlike the 3.2) in the example 1, the working concentration of (R) -blebbistatin used in this example was 10 μM.

(4) Unlike 4.1) in example 1, based on the (R) -blebbistatin-treated bacterial suspension obtained in the step 3, after 50,000× dilution, 100 μL of the bacterial suspension was aspirated and added as an inoculum to MH agar plates. Bactericidal effects were observed after incubation overnight.

(5) Unlike 4.1) in example 1, based on the (R) -blebbistatin-treated bacterial suspension obtained in step 3, 100 μL of MH medium (4 wells) and 10 μL of the bacterial solution were added to a well (in at least 4 replicates) in a 96-well microplate, and the culture was allowed to stand. After culture for overnight, the OD _600 nm absorbance value was measured by a microplate reader to assess any bactericidal effects.

(6) Results

In this example, Staphylococcus aureus (strain Sau16162) was used, whose working density was 10 ⁸ cfu/mL. After mixing with 10 μM of (R) -blebbistatin, irradiation proceeded under 420 nm blue light for 60 min. It was found that 10 μM of (R) -blebbistatin under blue light completely kills S. aureus on MH agar plates, with no residual colonies. Staphylococcus aureus also did not re-grow in liquid culture (see Figure 6) .

The heatmap for antimicrobial susceptibility test (AST) results (MIC value) of S. aureus with respect to clinical antibiotics of the embodiment is as shown in figure 13.

Example 6

A method for killing a clinical isolate of drug-resistant Enterococcus faecalis by a blue-light activated blebbistatin analogue (R) -blebbistatin, comprising the following steps: the culture materials, equipment and processes of the embodiment are substantially the same as those of the first embodiment, the difference being:

(1) Unlike 1.1) in example 1, this time the bacterium Enterococcus faecium (strain Ef36950) was used.

(2) Unlike 3.1) in example 1, the density of Enterococcus faecalis used in example 6 was 10 ⁸ cfu/mL, whose 2× density was 2×10 ⁸ cfu/mL.

(3) Unlike 3.2) in example 1, the working concentration of (R) -blebbistatin used in this example was 10 μM.

(4) Unlike 4.1) in example 1, based on the (R) -blebbistatin-treated bacterial suspension obtained in step 3, after 50,000× dilution, 100 μL of the bacterial suspension was aspirated and spotted as an inoculum onto MH agar plates. Bactericidal effects were assessed following overnight culture.

(5) Unlike 4.1) in example 1, based on the (R) -blebbistatin-treated bacterial suspension obtained in step 3, 100 μL of MH medium (4 wells) and 10 μL of the bacterial suspension were added to a well (in at least 4 replicates) in 96-well microplate, and the culture was allowed to stand overnight. After overnight culture, OD _600 nm absorbance value was measured by a microplate reader to assess any bactericidal effects.

(6) Results

In this example, Enterococcus faecium (strain Ef36950) was used, with a working density of 10 ⁸ cfu/mL. After mixing with 10 μM of (R) -blebbistatin, the mix was irradiated by blue light at 420 nm for 60 min. It was found that 10 μM of (R) -blebbistatin under blue light can completely eradicate Enterococcus faecium without residual colonies on MH agar plates and without regrowth in liquid culture (Figure 7) .

Heatmap of antimicrobial susceptibility test (AST) results (MIC value) for Enterococcus faecium used in the present embodiment is as shown in figure 13.

Example 7

A method for killing a clinical isolate of drug-resistant Escherichia coli by a blue-activated blebbistatin analogue (R) -blebbistatin, comprising the following steps:

(1) Unlike 1.1) in example 1, this time the bacterium E. coli (strain Eco16351) was used.

(2) Unlike the 3.1) in example 1, the working density of the drug-resistant Escherichia coli used in example 7 was 10 ⁸ cfu/mL, and thus its 2× density was 2×10 ⁸ cfu/mL.

(3) Unlike the 3.2) in the examples, the working concentration of (R) -blebbistatin used in this example was 10 μM.

(4) Unlike example 4.1) , based on the (R) -blebbistatin treated bacterial suspension obtained in step 3, after 50,000× dilution, 100 μL of the bacterial liquid was aspirated and spotted as an inoculum onto MH agar plates, and the bactericidal effects thereof were assessed following overnight culture.

(5) Unlike 4.1) in example 1, based on the (R) -blebbistatin treated bacterial suspension obtained in step 3, 100 μL of MH medium (4 pairs of wells) and 10 μL of the bacterial solution were added to a well (in at least triplicates) in a 96-well microplate, and the culture was allowed to stand overnight. After overnight culture, the OD _600 nm absorbance value was determined by a microplate reader to assess any bactericidal effect.

(6) Results

In this example, a clinical isolate of drug-resistant Escherichia coli (strain Eco16351) was used, whose working density was 10 ⁸ cfu/mL. After mixing with 10 μM of (R) -blebbistatin, blue light irradiation proceeded at 420 nm for 60 min. The results show that 10 μM of (R) -blebbistatin in combination with blue light completely eradicated drug-resistant E. coli leaving no residual colonies on MH agar plates, and no regrowth in liquid culture (Figure 8) .

Heatmap of antimicrobial susceptibility test (AST) results (MIC value) for Escherichia coli used in the example is as shown in figure 13.

Example 8

A method for killing a clinical isolate of Pseudomonas aeruginosa by a blue-light activated blebbistatin analogue (R) -blebbistatin, comprising the following steps:

(1) Unlike 1.1) in example 1, this time the bacterium Pseudomonas aeruginosa (strain Pae6709) was used.

(2) Unlike 3.1) in example 1, Pseudomonas aeruginosa used in example 8 had a working density of 10 ⁸ cfu/mL, and thus its 2× density is 2 × 10 ⁸ cfu/mL.

(3) Unlike 3.2) in example 1, working concentration of (R) -blebbistatin used in this example was 10 μM.

(4) Unlike the 4.1) in example 1, based on the (R) -blebbistatin treated bacterial suspension obtained in step 3, after 50,000× dilution, 100 μL of the bacterial suspension was aspirated and spotted as an inoculum onto MH agar plates. Bactericidal effects were assessed following overnight culture.

(5) Unlike 4.1) in example 1, based on the (R) -blebbistatin treated bacterial suspension in step 3, 100 μL of MH medium (4 wells) and 10 μL of the bacterial solution were added into a well (in at least 4 replicates) in a 96-well microplate, and the culture was allowed to stand overnight. After overnight culture, the OD _600nm absorbance value was determined by a microplate reader to assess the bactericidal effects.

(6) Results

In this example, a clinical isolate of Pseudomonas aeruginosa (strain Pae6709) was used, whose working density was 10 ⁸ cfu/mL. After mixing with 10 μM of (R) -blebbistatin, irradiation of blue light proceeded at 420 nm for 60 min. The results showed that 10 μM of (R) -blebbistatin with blue light can completely eradicate Pseudomonas aeruginosa leaving no residual colonies on MH agar plates, and no regrowth in liquid culture (figure 9) .

Heatmap of antimicrobial susceptibility test (AST) results (MIC values) of Pseudomonas aeruginosa used in this example is as shown in figure 13.

Example 9

A method for killing Klebsiella pneumoniae by a blue-light activated blebbistatin analogue (R) -blebbistatin, comprising the following steps:

(1) Unlike 1.1) in example 1, this time the bacterium Klebsiella pneumoniae (strain Kpn1096) was used.

(2) Unlike 3.1) in example 1, the working density of Klebsiella pneumoniae used in example 9 was 10 ⁸ cfu/mL, and thus its 2× density was 2 × 10 ⁸ cfu/mL.

(4) Unlike the 4.1) in example 1, based on the (R) -blebbistatin treated bacterial suspension obtained in the step 3, after 50,000× dilution, 100 μL of the bacterial suspension was aspirated and spotted as an inoculum onto MH agar plates. The bactericidal effects thereof were observed following overnight culture.

(5) Unlike 4.1 in example 1, based on the (R) -blebbistatin treated bacterial suspension obtained in step 3, 100 μL of MH medium (4 wells) and 10 μL of the bacterial suspension was added into a well (in at least 4 replicates) in a 96-well microplate, and the culture was allowed to stand. After culturing overnight, the OD ₆₀₀ _nm absorbance value was measured by a microplate reader to assess bactericidal effects.

(6) Results

In this example, a clinical isolate of drug-resistant Klebsiella pneumoniae (strain Kpn1096) was used, whose working density was 10 ⁸ cfu/mL. After mixing with 10 μM of (R) -blebbistatin, irradiation of blue light proceeded at 420 nm blue light for 60 min. However, it was found that 10 μM of (R) -blebbistatin with blue light was ineffective against Klebsiella pneumoniae on MH plates and could not kill Klebsiella pneumoniae to a significant extent (figure 10) .

Heatmap of antimicrobial susceptibility test (AST) results (MIC value) of Klebsiella pneumoniae used in the present embodiment as shown in figure 13.

Example 10

(S) -blebbistatin, a known myosin II inhibitor is expected to elicit cellular toxicity, whereas (R) -blebbistatin, a compound with no known biological targets in mammalian cells, is expected to elicit low or no toxicity in dark conditions.

Z3 Q3Comparisons of the properties of the blebbistatin analogues (R) -blebbistatin and (S) -blebbistatin:

1.Comparison of intrinsic toxicity in dark between (R) -blebbistatin and (S) -blebbistatin

(1) In this example, zebrafish embryos at post-fertilization day 0 (pfd 0) were treated (R) -blebbistatin and (S) -blebbistatin at 0 μM and 10 μM. After treatment for 6 h, 12 h, 24 h, and 48 h without blue light, in vivo toxicity was assessed by means of an optical microscope (magnification = 20×) to observe whether the development and morphology of zebrafish embryos were altered.

(2) Results

As shown in Figure 11, following treatment of the compounds in zebrafish embryos for 6 h, 12 h, 24 h, 48 h, 10 μM in the absence of blue light, (R) -blebbistatin treatment group elicited no effects on the morphological development of zebrafish embryos, indicating that (R) -blebbistatin generated no toxicity in vivo. Zebrafish embryos cannot develop further after 6 h treatment of (S) -blebbistatin. Therefore, the intrinsic toxicity of (R) -blebbistatin is much lower than that of (S) -blebbistatin.

2. Comparison of bacterial killing efficiency between (R) -blebbistatin and (S) -blebbistatin

(1) Bacterial killing assay

1) Unlike 3.1) in example 1, the working density of the bacteria (Acinetobacter baumannii) was changed to 10 ⁷ cfu/mL;

2) Unlike 3.2) in example 1, the compounds used in the experiment were (R) -blebbistatin at 2 μM and 10 μM; and (S) -blebbistatin at 2 μM and 10 μM;

3) Unlike 4.1) in example 1, based on the blebbistatin-treated bacterial suspension obtained in the step 3, after 10,000× dilution, 100 μL of the bacterial suspension was sampled and spotted as an inoculum onto MH agar plates, and the bactericidal effects thereof were assessed following overnight culture.

(2) Results

This example uses a clinical isolate of drug-resistant Acinetobacter baumannii (strain GD0302) with a density of 10 ⁷ cfu/mL, mixed with 0 μM, 2 μM, 10 μM of (R) -blebbistatin and (S) -blebbistatin. After that, blue light irradiation was performed at a wavelength of 420 nm for 60 min. It was observed that under blue light, (R) -blebbistatin and (S) -blebbistatin completely eradicated the bacteria at 10 μM, while at 2 μM (R) -blebbistatin under blue light killed the bacterium with less bacterial regrowth than the case of (S) -blebbistatin. It was found that (R) -blebbistatin has better bacterial killing efficiency than does (S) -blebbistatin under the same conditions.

Example 11

Experiment on optimizing the separation between light source and bacterial object: A method for optimizing the blue light delivery efficiency in killing a clinical isolate of drug-resistant Acinetobacter baumannii (strain GD0302) by the blebbistatin analogue (R) -blebbistatin.

(1) Unlike 3.4) in example 1, and the assembly of the blue light source platform (Figure 1) in 3.4) , the Eppendorf tube was placed on a transparent plastic rack, so that the separation between the liquid surface of bacterial suspension and the blue LED light source was varied: 8.2, 10, 12 cm.

(2) Results

When the separation between the liquid surface of the bacterial suspension and the blue LED light source was increased from 8.2, 10, to 12 cm, the sterilization efficiency became weaker.

Example 12

A method for killing a clinical isolate of drug-resistant Acinetobacter baumannii (strain GD0302) by blue-light activated blebbistatin analogues.

The (R) -blebbistatin solution of example 1 was replaced with other blebbistatin analogues, and the killing effects against drug-resistant Acinetobacter baumannii (strain GD0302) were similar to that of example 1.

The structural formulas of the blebbistatin analogues include:

The structural formulas of the blebbistatin analogues also include:

One of X ₁, X ₂, X ₃, X ₄ and X ₅ may be one of H, CN, CF ₃, F, Cl, Br, and I, and when one of X ₁, X ₂, X ₃, X ₄ and X ₅ is the above group, the remaining four groups are H.One of R ₁, R ₂, R ₃, R ₄ and R ₅ may be one of H, CN, NH ₂, F, Cl, Br, I, alkyl, haloalkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkaryl, heterocyclic, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, alkylamino, arylamino, dialkylamino, alkarylamino, aryloxy, arylalkoxy, acyloxy, carbamoyl, trifluoromethyl, trifluoromethoxy, phenoxy, benzyloxy, phosphonyl, phosphate, sulfonic, sulfonate and sulfonamide, and when one of R ₁, R ₂, R ₃, R ₄ and R ₅ is the above group, the remaining four groups are all H.

Claims

Use of blue-light activated blebbistatin analogues in killing drug-resistant bacteria.
The use according to claim 1, characterized in that, the blebbistatin analogues have the following structural formulas:

wherein one of R ₁, R ₂, R ₃, R ₄ and R ₅ is selected from H, CN, NH ₂, F, Cl, Br, I, alkyl, haloalkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, alkylamino, arylamino, dialkylamino, alkarylamino, aryloxy, arylalkoxy, acyloxy, carbamoyl, trifluoromethyl, trifluoromethoxy, phenoxy, benzyloxy, phosphonyl, phosphate, sulfonic, sulfonate, and sulfonamide; when one of R ₁, R ₂, R ₃, R ₄ and R ₅ is the above group, the four remaining groups are all H.
The use according to claim 1, characterized in that, the blebbistatin analogues have the following structural formulas:

wherein one of X ₁, X ₂, X ₃, X ₄ and X ₅ is selected from H, CN, CF ₃, F, Cl, Br, and I, and when one of X ₁, X ₂, X ₃, X ₄ and X ₅ is the above group, the remaining four groups are H;

one of R ₁, R ₂, R ₃, R ₄ and R ₅ is selected from H, CN, NH ₂, F, Cl, Br, I, alkyl, haloalkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkaryl, heterocyclyl, cycloalkyl, cycloalkenyl, cycloalkynyl, hydroxyalkyl, aminoalkyl, alkylamino, arylamino, dialkylamino, alkarylamino, aryloxy, arylalkoxy, acyloxy, carbamoyl, trifluoromethyl, trifluoromethoxy, phenoxy, benzyloxy , phosphonyl, phosphate, sulfonic, sulfonate, and sulfonamide, and when one of R ₁, R ₂, R ₃, R ₄ and R ₅ is the above group, the remaining four groups are all H.
The use according to claim 1, characterized in that, the drug-resistant bacteria include Staphylococcus aureus, Escherichia coli, Enterococcus spp., Pseudomonas aeruginosa, and Acinetobacter baumannii.
The use according to claim 1, characterized in that, a wavelength of the blue light is 420 nm.
A method of killing drug-resistant bacteria using a blue-light activated blebbistatin analogue, characterized in that, the method comprises: mixing a liquid culture of exponential-phase drug-resistant bacteria having a 2-fold working density and a blebbistatin analogue having a 2-fold working concentration in a ratio of 1: 1, and subjecting to irradiation by blue light of 420 nm for 30-60 min.
The method according to claim 6, characterized in that, a light source of the blue light is placed at 7.2 cm above the liquid culture of exponential-phase drug-resistant bacteria.
The method according to claim 6, characterized in that, the liquid culture of exponential-phase drug-resistant bacteria has a working density of 10 ⁸ cfu/mL or less, and the blebbistatin analogue has a working concentration of 2 μM to 10 μM.
The method according to claim 6, characterized in that, a method for preparing the liquid culture of exponential-phase drug-resistant bacteria comprises:

(1) streaking the drug-resistant bacteria stored at -80 ℃ multiple times on an agar plate, and inverting the plate to allow culture for 12 h to obtain monoclonal bacterial colonies of the drug-resistant bacteria;

(2) picking the monoclonal bacterial colonies of the drug-resistant bacteria and adding to a Luria-Bertani medium and/or Mueller-Hinton medium, and culturing for 1.5 h in a bacterial culture shaker to obtain the liquid culture of exponential-phase drug-resistant bacteria.
The method according to claim 6, characterized in that, the blebbistatin analogue having a 2-fold working concentration is prepared by reconstituting a blebbistatin analogue in dimethylformamide to give a stock solution, and then diluting in a phosphate buffer saline to the 2-fold working concentration.