CN115501324A - Composite antibacterial agent and application thereof - Google Patents

Abstract

The invention discloses a composite antibacterial agent and application thereof, wherein the composite antibacterial agent comprises the following components in parts by mass: 0.05 to 1.6 parts of Nisin and 1.6 to 51.2 parts of berberine hydrochloride, which are used for inhibiting the growth of MRSA, and the combination of Nisin and berberine hydrochloride has a synergistic antibacterial effect on MRSA, so that the use concentration of the Nisin and the berberine hydrochloride can be reduced, the sterilization time is shortened, the mature biological membrane can be efficiently broken, the formation of the biological membrane can be inhibited, and the wound healing can be promoted more strongly.

Description

Composite antibacterial agent and application thereof

Technical Field

The invention relates to the field of microbial medicines, in particular to a composite antibacterial agent and application thereof.

Background

MRSA is the most frequently isolated bacterium in chronic wounds. Treatment options for MRSA-induced wound infections are limited because these strains are resistant to the entire class of beta-lactam antibiotics. Vancomycin was once considered the last line of defense in treating MRSA infections, but there are also increasing reports of vancomycin resistance in vitro. In addition, vancomycin must be administered intravenously, and wound repair is a long-term process, which makes it difficult to administer vancomycin outside of the hospital and burdens the patient. Topical antimicrobial agents such as silver ions, iodine, etc. are non-selective against microorganisms and are cytotoxic to normal cells, potentially damaging granulation tissue and epithelialization on the wound bed. The use of antibiotics may also lead to the development of new drug resistant species and, therefore, there is an increasing interest in finding alternative therapies.

With the intensive research on the skin's micro-ecology, micro-ecological agent therapy is in the focus. It has been found by examining the data that Nisin (Nisin bacteriocin, hereinafter Nisin) produced by streptococcus lactis is the only commercially produced bacteriocin at present. Nisin has a long history of use as a food preservative with little evidence of stable drug resistant mutants in food products that have been used Nisin for nearly 50 years. At present, the use of Nisin has been extended to the biomedical field, with G being resistant to various drugs including MRSA ⁺ All the bacteria have inhibiting effect. Nisin is a bacteriocin with high safety, but Nisin cannot be artificially and chemically synthesized, and the production cost is high. Therefore, nisin alone may result in excessive amounts, thereby increasing treatment costs. In addition, nisin has a narrow antibacterial spectrum and weak antibacterial effect on gram-negative bacteria, and the single use of Nisin as a therapeutic drug is far from sufficient because MRSA infected wounds often combine with increased colonization of flora associated with secondary infection of the wounds.

Disclosure of Invention

The invention provides a composite antibacterial agent and application thereof, which are used for solving the technical problems of high treatment cost and weak antibacterial action on specific strains of the existing microecologics.

In order to solve the technical problems, the invention adopts the following technical scheme:

the composite antibacterial agent comprises the following components in parts by mass:

0.05 to 1.6 portions of nisin and 1.6 to 51.2 portions of berberine hydrochloride.

The design idea of the technical scheme is that the inventor finds that berberine hydrochloride has broad-spectrum antibacterial property and has strong inhibiting effect on staphylococcus aureus, tubercle bacillus, pneumococcus and other bacteria, the main substance of harmful bacteria inhibited by lactobacillus is bacteriocin, and Nisin and berberine hydrochloride both have the function of destroying bacterial biofilms, so that the berberine hydrochloride and Nisin have synergistic effect in the aspect of antibacterial effect. The combined use can reduce the use concentration of the antibacterial agent and the antibacterial agent, reduce the toxicity and the effective dose of the antibacterial agent, further shorten the acting time of the antibacterial agent, accelerate the sterilization speed and reduce the drug resistance of bacteria to the maximum extent; meanwhile, the inventor also finds that the combination of nisin and berberine hydrochloride promotes the healing of the wound.

As a further preferable mode of the above technical solution, the composite antibacterial agent comprises the following components in parts by mass:

0.1 to 0.4 portion of nisin and 6.4 to 12.8 portions of berberine hydrochloride.

Based on the same technical concept, the invention also provides application of the composite antibacterial agent, wherein the composite antibacterial agent is used for inhibiting the growth of MRSA.

As a further preferred mode of the technical scheme, when the composite antibacterial agent is used for inhibiting the growth of MRSA, the composite antibacterial agent is coated on the MRSA bacterial colony to form an antibacterial system, the concentration of nisin in the antibacterial system is 5-16 mug/mL, and the concentration of berberine hydrochloride in the antibacterial system is 16-512 mug/mL.

Compared with the prior art, the invention has the advantages that:

the combination of Nisin and berberine hydrochloride in the composite antibacterial agent has a synergistic antibacterial effect on MRSA, can reduce the use concentration of the Nisin and the berberine hydrochloride, quickens the sterilization time, can efficiently break mature biomembranes and inhibit the formation of the biomembranes, and has a stronger promotion effect on wound healing.

Drawings

FIG. 1 is a graph showing the antibacterial effect of Nisin at various concentrations in example 1;

FIG. 2 is a graph showing the antibacterial effects of berberine hydrochloride at different concentrations in example 1;

FIG. 3 is a time-sterilization curve for different groups of agents of example 1;

FIG. 4 is a graph showing the effect of different groups of agents on the destruction of MRSA biofilms in example 1;

FIG. 5 is a comparison of the four groups of agents of FIG. 4 for the destruction of MRSA biofilm;

FIG. 6 is a graph of the inhibitory effect of different groups of agents on the growth of MRSA biofilms in example 1;

FIG. 7 is a comparison of the inhibitory effect of the four groups of agents of FIG. 6 on the growth of MRSA biofilm;

FIG. 8 is a graph showing the body weight growth curves of different groups of mice in example 1;

FIG. 9 shows the healing of the wound surface in different groups of mice in example 1;

FIG. 10 is a graph of the rate of wound healing over time for different groups of mice in example 1;

FIG. 11 is a 10D pathological tissue HE section of different groups of mice in example 1 (species A is berberine hydrochloride group, B is Nisin group, C is berberine hydrochloride + Nisin group, D is physiological saline group, and E is gentamicin group).

Detailed Description

The present invention will be described in further detail with reference to the following examples, in which the main reagents are shown in Table 1 and the main instruments are shown in Table 2, and the experimental animals used in the examples are SPF-grade BALB/c male mice, aged 4W and weighing 18. + -.2 g, purchased from Shanghai Si Ricker laboratory animals, inc., license number: SCXK (Shanghai) 2017-0005.

TABLE 1 examples Primary reagents

TABLE 2 examples Main Equipment

Example 1:

this example investigated the inhibitory effect of the complex antibacterial agent on MRSA.

1. In vitro antibacterial experiments

1. Preparation of test reagents

MH broth (CAMHB): 21g of MH broth powder was dissolved in 1000ml of triple distilled water and autoclaved at high temperature for 15min (temperature 121 ℃).

Nisin stock solution: nisin powder was stored at 4 ℃ in the dark. 10mg Nisin powder was dissolved in 6.25mL DMSO to prepare a mother solution of 1600ug/mL, which was then diluted with MH broth.

Berberine hydrochloride: storing the berberine hydrochloride powder at normal temperature in the dark, weighing 10mg of berberine hydrochloride powder, dissolving in 2mL of DMSO, and preparing into mother solution with the concentration of 5 mg/mL; diluted with MH broth to prepare 512ug/ml drug-containing medium.

2. Bacterial culture and preparation of bacterial liquid

The strain preserved in glycerol at-20 deg.C is placed at room temperature for 30min for resuscitation. Scraping glycerol bacteria with inoculating loop, streaking on MH plate, and culturing the plate at 37 deg.C for overnight; picking monoclonal bacteria with inoculating loop, inoculating into MH broth, and shaking in shaking table at constant temperature of 37 deg.C overnight; the next day, the bacterial solution was aspirated by a pipette and diluted to 1X10 with MH medium ⁵ CFU/ml, inoculation within 30 min.

3. Determination of Minimum Inhibitory Concentration (MIC)

In 96-well plates, a total of 12 groups of 3 multiple wells were set, with 200ul sterile distilled water sealed off the edges. 100ul and 10 ul of diluted drug solution are added into each group of holes of the 1 st to 10 th groups in turn ⁵ 100ul of cfu/ml fresh bacterium liquid, and 100ul and 10 of sterile MH broth are added into each hole of 11 th group ⁵ cfu/ml fresh bacterial solution 100ul was used as a negative control. Sterile MH broth 100ul and distilled water 100ul were added to each well of group 12, respectively, as a blank control.

The final Nisin concentrations were 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.06, 0.03ug/ml, and the OD of each well was measured and recorded using a microplate reader at a wavelength of 600 nm. The method steps are the same, so that the concentration of the berberine hydrochloride is respectively 512, 256, 128, 64, 32, 16, 8, 4, 2 and 1ug/ml.

And (4) judging a result: measuring each pore OD by using enzyme-labeling instrument before and after 24h of culture ₆₀₀ And calculating the difference between the two readings, wherein the working concentration of the medicament in the hole with the difference of the measured OD values smaller than 0.1 is the MIC of the medicament.

The above experimental procedure was performed 3 times and the average was taken.

As shown in FIGS. 1-2, the MIC of Nisin to MRSA-ATCC 43300 was 2. Mu.g/mL, and the MIC of berberine hydrochloride to MRSA-ATCC 43300 was 64. Mu.g/mL.

Determination of combined anti-MRSA activity of Nisin and berberine hydrochloride

And (3) determining the combined inhibitory concentration of Nisin and berberine hydrochloride on MRSA by adopting a micro chessboard method. Nisin and berberine hydrochloride are respectively diluted by MH culture medium for standby. Sterile 96-well plates were removed, sealed at the edges with 200ul distilled water, and 100ul MH medium was added to each well in column 2.Nisin with the concentration of 8 × MIC, 4 × MIC, 2 × MIC, 1 × MIC and 1/2 × MIC is added into each column of 3-7 columns in turn, and berberine hydrochloride with the concentration of 4 × MIC, 2 × MIC, 1/2 × MIC, 1/4 × MIC and 1/8 × MIC is added into each row of B-G rows in turn, wherein each dose is 50ul.

In the above-mentioned holes, 100ul of sterile distilled water was added to B2, C2 and D2 holes, and 10 ul of sterile distilled water was added to the remaining holes ⁵ CFU/mL bacterial suspension 100ul, so that finally each hole is 200ul. The final concentrations of Nisin in the 3 rd to 7 th columns are 2 × MIC, 1/2 × 0MIC, 1/4 × MIC and 1/8 × MIC respectively. The final concentrations of the hydrochloric acid cleaved alkali in the B-G lines are 1 × MIC, 1/2 × MIC, 1/4 × MIC, 1/8 × MIC, 1/16 × MIC and 1/32 × MIC respectively. B2, C2 and D2 wells are blank controls, and E2, F2 and G2 wells are negative controls. Measuring the OD value of each hole of each group by using a microplate reader at the wavelength of 600nm, recording the reading, culturing at the constant temperature of 37 ℃ for 24h, and detecting the OD value again.

OD value determination method determines the combined bacteriostasis concentration of Nisin and berberine hydrochloride. Calculating the FICI value: FICI = MIC _{First connection} /MIC _{Nail sheet} +MICb _{Second connection} /MICb _{B sheet} (ii) a The judgment basis is as follows: (1) the FICI is less than or equal to 0.5, and the cooperation is realized; (2) 0.5<FICI is not more than 4 as irrelevant; (3) FICI>4 is antagonistic.

The above experimental procedure was performed 3 times and the average was taken.

As a result, as shown in Table 3, when Nisin was used in combination with berberine hydrochloride, the concentration of D6 well, i.e., the well at column D and column 6 in the table was MIC for the combination, and the MIC combination of Nisin in D6 well was 0.5. Mu.g/mL (1/4 × MIC). The MIC combination of berberine hydrochloride is 16 μ g/mL (1/4 MIC), so FICI =1/4+1/4=0.5, suggesting that Nisin and berberine hydrochloride have a combined effect.

TABLE 3. Example 1 Microcheckerboard method for well concentration and OD differences

5. Time-kill experiment

The suspension concentration was adjusted to 10 with MH medium ⁵ cfu/ml. According to the single-drug MIC result, nisin with sub-inhibitory concentrations of 1/4MIC and 1MIC and berberine hydrochloride with sub-inhibitory concentrations of 1/4MIC and 1MIC or a combination of the two are selected to be respectively incubated with the MRSA strain, and blank MH broth containing bacteria with the same concentration is taken as a control group. Each group of the liquid was incubated at 37 ℃ and 100. Mu.l of the bacterial solution was aspirated in stages of 0,1,4,6, 12, and 24 hours, diluted with MH broth, and the diluted liquid was spread on MH agar plates, which were incubated at a constant temperature of 37 ℃ and colonies were counted after about 18 hours. The above experimental steps were performed 3 times, and the average value was taken. The time-sterilization curve is drawn according to the experimental results, as shown in FIG. 3, the sterilization curve of berberine hydrochloride used in combination with Nisin to MRSA-ATCC 43300 shows that Nisin and berberine hydrochloride only have an inhibitory effect on MRSA when the concentrations of Nisin and berberine hydrochloride are 1/4MIC respectively, and the sterilization effect is achieved within 12h when Nisin and berberine hydrochloride with 1/4MIC are used in combination. When 1/4MIC Nisin and 1/4MIC berberine hydrochloride are combined with the other drug at the concentration of 1MIC, the sterilization effect is achieved within 6 h. When 1MIC Nisin and 1MIC berberine hydrochloride are used together, the effect of killing within 4h is achievedAnd (4) performing bacterial action.

2. Anti-biofilm experiment

Establishment of MRSA biofilm model

Preparing bacterial liquid: 30g of TSB broth powder and 7.5g of glucose (2.5 g in TBS) were weighed, 1000ml of triple distilled water was added, and the mixture was autoclaved for 15min to prepare TSBg medium containing 1% glucose for use. The recovered bacteria were inoculated on a plate and cultured at 37 ℃ for 24 hours. The following day, single colonies were collected with an inoculating loop and shaken in TSBg medium (50 rpm) at 37 ℃. Detecting OD600 value, adjusting OD value of bacterial suspension to 1 with TSBg culture medium, namely 1 × 10 ⁸ CFU/ml. In the super clean bench, the bacterial liquid is diluted with TSBg medium to a concentration of 1x10 ⁶ CFU/ml。

Inoculation: the 96-well plate was added with MRSA group 200ul and TSBg medium group 200ul (6 duplicate wells per group). Placing in a constant temperature incubator at 37 ℃ for 24h.

Washing the plate: the medium was aspirated and washed 3 times with PBS, 3min each time.

Fixing: adding 200ul methanol (concentration of 99%) per well, fixing the bacteria to form membrane, standing at room temperature for 15min, discarding the liquid in the well, and standing at room temperature for 10min to fully volatilize methanol.

Dyeing: 200. Mu.l of 0.1% crystal violet solution was added to each well and allowed to stand at room temperature for 5min.

Washing the plate: and (4) pouring the solution in the 96-well plate, flushing the solution for 5min under running water, and naturally drying the solution.

And (3) decoloring: 200ul 95% ethanol solution was added to each well, and the plates were shaken on a shaker for 1h. Measuring its OD value at 595nm

2. Investigating the Effect of the Individual drug and the combination drug on the formation of a biofilm

Inoculating MRSA bacterial solution into a sterile 96-well plate, wherein the bacterial concentration is 1 × 10 ⁶ CFU/ml. Mu.l of the bacterial suspension was added to each well, and the mixture was incubated at 37 ℃ for 24 hours. Then discarding the solution in the wells, washing 3 times with PBS, and washing off the free bacteria; the culture medium regulates the concentration of each medicament as follows: 1/2MIC, 1/20MIC, 1/200MIC (6 replicates per well), 100. Mu.l each was added. The final concentration of Nisin was: 1 mug/mL, 0.1 mug/mL, 0.01 mug/mL; the final concentration of berberine hydrochloride is: 32ug/mL, 3.2. Mu.g/mL, 0.32. Mu.g-mL; the combined drug concentration is as follows: 1 mu g/mL Nisin +32ug/mL berberine hydrochloride, 0.1 mu g/mL Nisin +3.2 mu g/mL berberine hydrochloride, 0.01 mu g/mL Nisin +0.32 mu g/mL berberine hydrochloride; setting negative control group and blank control group, culturing at 37 deg.C for 24h; the solution in the wells was discarded, and the OD value was measured using a microplate reader (595 nm). Each set of experiments was performed in triplicate.

The OD value test results are shown in fig. 4 and fig. 5, and the results show that the two drugs have no obvious effect of removing the biological membrane under the concentration of 1/200MIC and the concentration of 1/20MIC respectively, and when the two drugs are applied in combination, the combined drug group under the concentration of 1/20MIC has certain effect on the MRSA biological membrane (P is less than 0.001); nisin and berberine hydrochloride were able to disrupt the formed biofilm both at 1/2MIC concentrations alone and in combination (P < 0.001), and the combination showed a stronger effect (P < 0.001) than either alone.

3. Examination of the Effect of Individual and combination drugs on MRSA biofilm formation

The inoculation concentration in sterile 96-well plates was 1X10 ⁶ CFU/ml MRSA bacterial liquid, each hole is 100 mul; according to the above experimental results, the drug concentration was adjusted using TSBg medium as follows: 1/2MIC, 1/20MIC, 1/200MIC (6 duplicate wells each), 100. Mu.l of drug was added to each well; the final concentration of Nisin was: 1 mug/mL, 0.1 mug/mL, 0.01 mug/mL; the final concentration of berberine hydrochloride is: 32ug/mL,3.2 ug/mL, 0.32 ug/mL; the concentration of the combined medicine is as follows: 1 mug/mL Nisin +32ug/mL berberine hydrochloride, 0.1 mug/mL Nisin +3.2 mug/mL berberine hydrochloride, 0.01 mug/mL Nisin +0.32 mug/mL berberine hydrochloride; setting negative control group and blank control group, culturing at 37 deg.C for 24h; OD was measured using a microplate reader (595 nm). Each set of experiments was performed in triplicate.

OD value test results are shown in FIG. 6 and FIG. 7, nisin and berberine hydrochloride have almost no inhibition effect on the biofilm formation process of MRSA at the concentrations of 1/20MIC and 1/200MIC, nisin can inhibit the biofilm formation of 28.75% under the condition of 1/2MIC concentration, and 1/2MIC berberine hydrochloride can inhibit the biofilm formation of 20.91% compared with the untreated group (P < 0.001), and the statistical difference (P < 0.001) is compared with the untreated group. 1/20MIC nisin +1/20MIC berberine group can inhibit the formation of a 15.08 percent biomembrane. Under the combined action of 1/2MIC Nisin +1/2MIC berberine, the inhibition rate on the biological membrane is 37.75 percent, the inhibition rate is higher than Nisin and berberine hydrochloride which are used independently under the condition of 1/2MIC concentration and have statistical difference (P <0.05

4. Statistical analysis

The data obtained were statistically processed using SPSS17.0 software in steps 2 and 3 above. Statistical methods the one-way analysis of variance was used, dunnett-t test was used to compare each group to the negative control group, LSD test was used to compare the combination group to the individual group, P <0.05 indicated that the difference was statistically significant.

3. Animal experiments

1. Establishment of infection model

The mice are bred adaptively for 7 days, anesthetized by using 1.5 percent pentobarbital, and after anesthesia is successful, the skin on the backs of the mice is depilated and disinfected to manufacture wound surfaces with the size of about 1cm multiplied by 1cm and the depth of the wound surfaces reaches a fascia layer. To a concentration of 10 ⁸ CFU/mL MRSA bacterial liquid is smeared on the wound surface, and about 20 mu L is obtained. And repeating the operation for re-inoculation after 24h, checking the wound condition after 24h, observing that the periphery of the wound presents red, swelling and other inflammatory reactions and has faint yellow exudate, culturing the exudate to show that MRSA strains grow, and thus establishing the mouse MRSA infected wound model.

2. Grouping of laboratory animals

75 male mice were randomly divided into 5 groups: berberine hydrochloride group, nisin group, berberine hydrochloride + Nisin group, normal saline control group, and gentamicin positive control group.

3. Method of administration

After the model is successfully made, the medicine is administrated every day, the medicine is uniformly smeared on the wound surface by adopting a smearing method until the medicine is completely absorbed, the environment of the wound surface is kept moist, and the medicine is continuously fed until the wound surface is completely healed.

(1) Berberine hydrochloride group: the berberine hydrochloride solution (2 μ g/ml) 20 μ l is applied on the mouse wound.

(2) Nisin group: nisin solution (64. Mu.g/ml) 20. Mu.l was applied to the mouse wound surface.

(3) Berberine hydrochloride + Nisin group: the mixed solution (berberine hydrochloride 2 mug/ml + Nisin 64 mug/ml) 20 mug is smeared on the mouse wound surface.

(4) Physiological saline group: the physiological saline 20 mul is smeared on the wound surface of a mouse.

(5) Gentamicin group: gentamicin solution (1 mg/ml) 20. Mu.l was applied to the wound surface of mice.

4. Detecting the index

(1) General conditions

The general conditions (mental, dietary, excretory and active) of the mice were continuously observed, and the body weights of the mice were measured at 0d, 2d, 5d, 10d and 15 d. On the treatment day, mice in the Nisin group, the berberine hydrochloride group, the Nisin + berberine hydrochloride group and the gentamicin group have normal mental state, diet, excretion, activity and the like, and mice in the normal saline group have poor mental state and reduced activity. As shown in fig. 8, the body weight of each group of mice was observed to increase with time at 2d, 5d, 10d, and 15d after treatment, and there was no significant statistical difference.

(2) Wound healing conditions

The wound conditions include size, red swelling, and exudate. Photographs of the wounds were taken at 0d, 2d, 5d, 10d, 15d using a digital camera. And measuring the sizes of the wound surfaces at 0d, 5d, 10d, 15d, 18d and 21d, and calculating the wound surface closure rate. Wound closure rate = (wound area on day-wound area on nth day)/wound area on day × 100%. As shown in FIG. 9, the wound surface appeared red after the mice of each group had successfully modeled. Congestion and edema around the wound surface of mice in the fifth 2d group can be seen, and skin edema around the wound surface of the normal saline group is heavier. In the 5 th day, the wound surfaces of the Nisin and berberine hydrochloride groups and the gentamicin group are obviously reduced, the wound edges are slightly red and swollen, and no obvious seepage exists. The wound surface of the mice in the Nisin group and the berberine hydrochloride group is reduced, the wound margin is red and swollen, a small amount of faint yellow exudate can be seen, the wound margin of the mice in the normal saline group is red and swollen obviously, the exudate is more, and the wound surface is not reduced obviously. In the 10 th day, the wound surface of the Nisin and berberine hydrochloride group mouse is reduced and hardened in a large range, and the color is dark brown. The mouse ulcer surface of the Nisin group, the berberine hydrochloride group and the gentamicin group becomes small and shallow, and the red swelling and the exudate do not exist, and the mouse of the normal saline group can be slightly red swelling. And 15d, the mouse wound surfaces of the Nisin group, the berberine hydrochloride group and the normal saline group are seen to have fallen off, the wound surfaces are obviously reduced, a small range of unhealed wound surfaces is left, the mouse wound surfaces of the Nisin + berberine hydrochloride group and the gentamicin group are nearly completely healed, and the healing speed is obviously accelerated compared with that of the Nisin group and the berberine hydrochloride group. As shown in fig. 10, each group of histograms in fig. 10 sequentially shows the wound healing rates of the berberine hydrochloride group, the nisin group, the union group, the normal saline group, and the gentamicin group from left to right, and the wound healing rates show: at 5d, 10d and 15d, the healing rate of each drug group was statistically different (P < 0.05) compared to the normal saline group, indicating that the treatment was effective. At 5d, 10d and 15d, the healing rate of the Nisin and berberine hydrochloride combined group is higher than that of the Nisin group and the berberine hydrochloride group, and the Nisin and berberine hydrochloride combined group has statistical difference (P is less than 0.05) and has no statistical difference (P is more than 0.05) with the gentamicin group. At 18d, the wound surfaces of the mice in the Nisin and berberine hydrochloride combined group and the gentamicin group are completely healed, and at 21d, the wound surfaces of the other groups of mice are completely healed.

(3) Bacterial load of wound surface

And (3) respectively taking wound surface tissue homogenate at 2d, 5d, 10d and 15d (after 3 mice in each group die suddenly at each time point, taking the wound surface tissue for bacterial observation), diluting by PBS to a proper multiple, inoculating the diluted homogenate into an MRSA chromogenic medium and an MH sheep blood agar culture plate, culturing for 24h at a constant temperature of 37 ℃, and counting. Results are expressed as log of colony units per gram of tissue (CFU/g). The bacterial culture results of the wound tissue are shown in table 4, and no significant statistical difference exists between the bacterial loads of the groups at 2d after treatment. The wound surface bacterial load of mice of the 5d, 10d and 15d berberine hydrochloride groups, nisin + berberine hydrochloride groups and gentamicin groups is obviously lower than that of the normal saline group, the statistical difference is obvious (P < 0.01), the treatment effect of each group is proved, and the bacterial load of the Nisin + berberine hydrochloride groups is lower than that of the Nisin groups and the berberine hydrochloride groups and has the statistical difference (P < 0.01). At 10d and 15d, the bacterial load of the Nisin and berberine hydrochloride group is lower than that of the gentamicin group, and the statistical difference is realized (P is less than 0.05).

In conclusion, the drug groups effectively reduce the bacterial load of the wound surface, and compared with the single drug group, the Nisin and berberine hydrochloride group inhibits the proliferation of the bacteria on the MRSA infected wound surface earlier, thereby preventing the infection from further spreading and developing.

TABLE 4 wound bacterial load results (mean SD) Lg (CFU/g tissue) for each group of mice in the animal experiments of example 1

(4) Histological observation

Wound tissues of each group of mice were collected 10d after the injury, collected specimens were fixed in PBS at 4 ℃, fractionated dehydrated, paraffin-embedded, and then hematoxylin-eosin (HE) stained. As shown in FIG. 11, the HE stained section showed less inflammatory cells in the 10d berberine hydrochloride group and Nisin group, and fibroblasts, neovascularization, and the like were observed. A large number of fibroblasts and new vessels can be seen in the Nisin + berberine hydrochloride group and the gentamicin group. In the normal saline group, a large amount of inflammatory cell infiltration and necrotic pus cells are seen in local tissues of the wound surface of the mouse, and the regenerated granulation tissue is less. And (4) prompting: compared with the single drug group, the Nisin and berberine hydrochloride group has obvious promotion effect on wound healing.

(5) Statistical analysis

The data were processed using the SPSS17.0 software in steps (1) to (4) above, and the results are expressed as mean. + -. SD. And the difference comparison among multiple groups is carried out by adopting one-factor variance analysis, the difference comparison between two groups is carried out by adopting t test, and the difference is statistically significant when P is less than 0.05.

4. Analysis of results

Antibacterial effect of combination of Nisin and berberine hydrochloride on MRSA

The combined use of antibacterial drugs can reduce the drug resistance of bacteria to the maximum extent and reduce the toxicity and effective dose of the antibacterial drugs. Drug interactions include synergy, additivity, and antagonism. Synergistic effects are preferred because the use of lower concentrations of antibiotics can reduce the cost, toxicity of the final treatment and delay the development of bacterial resistance. The experiment mainly aims to research the combined antibacterial effect of Nisin and berberine hydrochloride on MRSA, and in vitro and in vivo experiments are respectively carried out. In the first step, the MIC of Nisin and berberine hydrochloride to MRSA is determined, the MIC of Nisin to MRSA-43300 is 2 mug/mL, and the MIC of Nisin to MRSA-43300 is 16 mug/mL by Mataraci. The results of this example show that the MIC value of Nisin is low, which may be related to the high purity Nisin used in this experiment. The MIC result of berberine hydrochloride to MRSA-43300 in this example was 64. Mu.g/mL. The preparation of the solution found that the MIC of berberine hydrochloride to MRSA-ATCC33591 in vitro was 32 μ g/ml < MIC ≤ 64 μ g/ml, which was consistent with the test results. The fluctuation difference of the MIC value range of the berberine hydrochloride is possibly related to the type and PH difference of the culture medium, and researches report that the pH difference of the culture medium can influence the conformation of the berberine, so that the berberine shows stronger antibacterial effect under the neutral condition. And secondly, verifying the feasibility of combined medication of Nisin and berberine hydrochloride by adopting a trace chessboard method, wherein the result shows that the FICI value of the combined medication of the Nisin and the berberine hydrochloride is 0.50, which shows that the two medicaments have synergistic effect on the inhibition of the MRSA. Although MIC measurement is a gold standard for measuring the activity of the antibacterial agent, the MIC measurement cannot provide an aging curve of the antibacterial agent, so that a time-sterilization experiment is further performed, and the result shows that Nisin and berberine hydrochloride have a certain inhibition effect on MRSA at sub-lethal concentration, but cannot kill bacteria. When the two medicines are used together, the synergistic effect is achieved, the concentration dependence is presented, and the bactericidal effect can be achieved in a short time. The results of the embodiment show that Nisin and berberine hydrochloride have synergistic effect, and the combined use can reduce the use concentration of the Nisin and the berberine hydrochloride, further shorten the drug action time and accelerate the sterilization speed.

Action of combination of Nisin and berberine hydrochloride on resisting MRSA biofilm

Bacteria can be classified into planktonic bacteria and bacteria in a biofilm state. Biofilms are microbial communities that attach to the surface of an object, differentiate to multiply and produce a cohesive matrix containing Extracellular Polymers (EPS), their EPS matrix consisting mainly of polysaccharides, proteins and eDNA. Biofilms are a form of protection for bacteria in harsh environments, can form on the surface of human bodies or objects, and are widely found in nature and hospital environments. However, biofilm is also one of the important mechanisms of bacterial resistance, and it can protect the bacteria in the biofilm from the antibacterial drugs and release them out of the biofilm at the right time, resulting in recurrence of infection and delayed healing. Kwon et al have reported that the biofilm positivity of MRSA is significantly higher than that of methicillin-sensitive strains. Therefore, a good bactericide should have a certain killing effect on bacteria in a biofilm state having a stronger resistance in addition to the killing effect on planktonic bacteria. Cleriane Andre microscopically observed that the form of the biofilm structure of Staphylococcus aureus changed under the action of Nisin. The composition of the biofilm matrix (polysaccharides, proteins and Extracellular DNA (EDNA)) was also altered as measured spectrophotometrically. The results of this example show that Nisin and berberine hydrochloride have a certain destructive effect on the formed biofilm and the combined effect is stronger, so we speculate that Nisin and berberine hydrochloride can achieve the bactericidal effect by destroying the biofilm and the combined effect is better.

On the surface of a human body or an object, the formation of a biological film is mainly divided into four steps: (1) adhering; (2) aggregate proliferation; (3) the biological membrane is mature; (4) fall off and spread. Linjian finds that berberine hydrochloride can inhibit synthesis of Polysaccharide Intercellular Adhesin (PIA) and release of EDNA in a staphylococcus aureus biomembrane through experimental research, so that the formation of the biomembrane is inhibited. The study of Liujun shows that berberine hydrochloride has an inhibiting effect on amyloid fibers aggregated by phenol soluble regulatory Proteins (PSMs), which are main structures promoting biofilm formation. The results of the embodiment show that both Nisin and berberine hydrochloride have certain inhibition effect on the activity of the MRSA biomembrane, and the lower the generation rate of the MRSA biomembrane along with the increase of the concentration, which indicates that both Nisin and berberine hydrochloride have enhanced inhibition effect on the MRSA biomembrane along with the increase of the concentration. When the two drugs are used in combination, the anti-biofilm effect is stronger than that of each drug which is used independently under the same concentration, and the two drugs have statistical difference.

In conclusion, the Nisin and berberine hydrochloride are considered to have the bactericidal effect on MRSA and also have the effects of breaking and inhibiting the formation of biofilms, and the antibacterial effect is better when the two medicines are used in combination. This indicates that the application in the early stage of infection inhibits the formation of MRSA biofilm and can effectively control infection.

Action of combination of Nisin and berberine hydrochloride on wound healing

When the skin is wounded, the skin barrier is broken and the skin's resistance to external microorganisms is reduced, so that wound infection is easily generated. MRSA is one of the most frequently separated bacteria in bacteria infected on wound surfaces, accounts for 60% -70% of staphylococcus aureus infection in hospitals, is the most invasive bacteria in all antibiotic-resistant bacteria, can cause bacteremia, pneumonia, endocarditis and the like in severe infection, brings great challenges to clinical treatment, and also becomes one of the hot problems of current research. Healing of MRSA infected wounds relies to a large extent on the inhibition of pathogenic bacteria, so controlling infection becomes a critical link in the treatment of wounds.

According to the in vitro experimental results of this example, nisin and berberine hydrochloride, MRSA, respectively, have been demonstrated to have antibacterial activity. Nisin has antibacterial activity, immunoregulation function and can promote wound healing. Hensis et al used an electrospun nanofiber wound dressing containing Nisin to diffuse active Nisin onto skin wounds and studied the efficacy of Nisin in mouse skin infection models. The Nisin-containing wound dressing can obviously reduce the colonization of staphylococcus aureus and accelerate the healing of wounds. The Mouritzen study found that Nisin significantly affected the migration of Human Umbilical Vein Endothelial Cells (HUVEC) and human keratinocytes (HaCaT) by reducing the levels of the proinflammatory cytokines tumor necrosis factor-alpha, interleukin-6 and interleukin-8. Nisin is a bacteriocin with high safety. Not only has the functions of bacteriostasis and immunoregulation, but also can promote the healing of the wound surface, and is an excellent choice for treating MRSA infectious wound surfaces. On the basis, the berberine hydrochloride and the Nisin have the synergistic effect of antibiosis, and also have certain anti-inflammatory effect and the function of expanding blood vessels, thereby increasing the blood supply and the oxygen supply at the base part of the wound surface and accelerating the healing of the wound surface.

In this example, a mouse MRSA infectious wound surface model is established, and the influence of Nisin and berberine hydrochloride on the healing of MRSA infectious wound surfaces is explored, the wound surfaces of mice in the Nisin + berberine hydrochloride group 5d begin to become dry and crusted, the wound surfaces shrink obviously, the redness and swelling are slight, almost no obvious exudate exists, and the bacterial load of local tissues of the mouse wound surfaces is lower than those of the Nisin group and the berberine hydrochloride group. The wound surface of mice in the 10d Nisin and berberine hydrochloride group is obviously reduced, and the healing rate is not obviously different from that of the gentamicin group (P > 0.05). The pathological result of the 10 th day shows that the Nisin and berberine hydrochloride inflammatory cells are obviously reduced, and a large amount of proliferation and neovascularization of fibroblasts can be seen. Animal experiments prove that the Nisin and the berberine hydrochloride can effectively reduce the number of bacteria on the wound surface, inhibit the growth of the bacteria, control infection and promote the healing of the wound surface. In conclusion, the combined medicament of Nisin and berberine hydrochloride has great application prospect in the treatment of MRSA infectious wound surfaces.

The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-described embodiments. Modifications and variations that may occur to those skilled in the art without departing from the spirit and scope of the invention are to be considered as within the scope of the invention.

Claims (4)

1. The composite antibacterial agent is characterized by comprising the following components in parts by mass:

0.05 to 1.6 portions of nisin and 1.6 to 51.2 portions of berberine hydrochloride.

2. The composite antibacterial agent according to claim 1, comprising the following components in parts by mass:

0.1 to 0.4 portion of nisin and 6.4 to 12.8 portions of berberine hydrochloride.

3. Use of the composite antimicrobial agent of claim 1 or 2, for inhibiting the growth of MRSA.

4. The use of the composite antibacterial agent according to claim 3, wherein when the composite antibacterial agent is used for inhibiting the growth of MRSA, the composite antibacterial agent is coated on the MRSA bacterial colony to form an antibacterial system, the concentration of nisin in the antibacterial system is 5-16 μ g/mL, and the concentration of berberine hydrochloride is 16-512 μ g/mL.

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