An antibacterial composition, application and drug

Abstract

This invention belongs to the field of antibiotics and discloses the use of a combination of trans-cinnamaldehyde and antibiotics in the preparation of medicaments for the prevention and treatment of drug-resistant bacterial infections. This antibacterial composition exhibits good inhibitory or bactericidal effects against multidrug-resistant *Streptococcus pleuropneumoniae*, *Streptococcus suis*, and *Staphylococcus suis*, can reverse drug-resistant antibiotics into sensitive ones, significantly reduce the use of commonly used antibiotics, improve efficacy, and simultaneously avoid antibiotic residues in pork products, ensuring food safety. Furthermore, this invention also provides applications of this antibacterial composition and related pharmaceutical formulations.

Description

Technical Field

[0001] This invention belongs to the field of antibiotics, and more specifically, relates to an antibacterial composition, application, and medicament. Background Technology

[0002] Exudative epidermitis in piglets is primarily caused by Staphylococcus hyicus, resulting in a systemic exudative inflammation of the skin. The lesions are characterized by the release of oily exudate and can also lead to bacteremia, septicemia, and arthritis in piglets. Ultimately, piglets die from dehydration due to fluid loss through the skin, and those who recover exhibit growth retardation. The morbidity rate varies greatly, reaching as high as 80%. Staphylococcus hyicus has been found on the skin, nasal mucosa, conjunctiva, and genitals (vagina in sows, prepuce in boars) of asymptomatic carrier pigs, as well as in the rearing environment. It tends to proliferate rapidly in humid and warm conditions, and can also cause exudative epidermitis in piglets with weakened immune systems. This disease has become one of the most common, prevalent, and significant infectious diseases affecting piglet growth in current pig farming practices.

[0003] Up to 20% of porcine bacterial pneumonia cases are caused by *Actinobacillus pleuropneumoniae*, a Gram-negative, non-motile, spontaneously transforming, coccidial-like facultative anaerobic bacterium. Clinical symptoms caused by infection include respiratory distress in sows, frothy bloody oral discharge, fever, anorexia, mild diarrhea, cyanosis, lethargy, and abortion. Pigs that do not die from the disease may retain highly virulent porcine bacterial pneumonia even after recovery. *Actinobacillus pleuropneumoniae* has developed resistance to various antibiotics, including aminoglycosides, β-lactams, fluoroquinolones, macrolides, sulfonamides, and tetracyclines, as widely reported. This pathogen is also a common cause in pig farming, having a profound economic impact on pork production and pig farmers.

[0004] Streptococcal disease in pigs, caused by infection with *Streptococcus suis*, is considered a major infectious disease in the swine industry, clinically characterized by meningitis, septicemia, or arthritis. It causes significant economic losses to the global swine industry annually. *Streptococcus suis* is prevalent in almost all countries with large-scale pig farming and is also a zoonotic disease, capable of transmission from pigs to humans. Infection with *Streptococcus suis* can lead to severe meningitis, septicemia, endocarditis, and deafness. The widespread use of antibiotics for treatment and prophylaxis in swine farming has led to increasingly serious antibiotic resistance in *Streptococcus suis*, making it a significant host for the spread of antibiotic resistance genes. Resistance to tetracyclines, macrolides, aminoglycosides, β-lactams, sulfonamides, chloramphenicol, and quinolones has been reported in *Streptococcus suis*. *Streptococcus suis* is increasingly becoming a reservoir host for antibiotic resistance genes, promoting the accelerated spread of these genes among different bacterial species, and posing a serious threat to public health.

[0005] The long-term overuse of antibiotics has led to increased resistance in bacteria such as *Streptococcus pleuropneumoniae*, *Streptococcus suis*, and *Staphylococcus suis*. These bacteria have been found to be resistant to many antibiotics. This necessitates the use of higher doses of antibiotics for treatment, significantly increasing the risks of drug residues in livestock farming and food safety. Furthermore, excessive antibiotic overuse can lead to the emergence of highly drug-resistant bacteria, posing a significant threat to livestock and public health. Currently, reducing antibiotic use and finding alternative antibiotic strategies are widely accepted in the prevention and control of bacterial infections.

[0006] Cinnamaldehyde is a naturally occurring small molecule, also known as (E)-3-phenyl-2-propenal, with the molecular formula C9H. 8O Molecular weight 132.16, CAS Registry Number: 14371-10-9, Structural formula:

[0007]

[0008] Naturally occurring cinnamaldehyde exists in its trans-form, which holds some biological importance. Studies have shown that trans-cinnamaldehyde possesses certain antibacterial and antioxidant activities. It exerts its antibacterial effect by interacting with bacterial cell membranes, disrupting their integrity, and also exerts its antioxidant effect by scavenging free radicals and reducing oxidative stress. Trans-cinnamaldehyde is a permitted synthetic flavoring agent for food use according to GB2760-2014, and can be used to prepare flavorings for meat, condiments, oral care products, chewing gum, and confectionery. Local standard DB37 / T 3614-2019 permits its use as a feed additive. Therefore, its safety and application in animals are relatively widespread.

[0009] For information on the antimicrobial application of trans-cinnamaldehyde, see: CN103391774B: Pharmaceutical compositions containing trans-cinnamaldehyde and their use in the treatment of infections, which specifically mentions cinnamon essential oil, particularly cinnamon essential oil containing trans-cinnamaldehyde, or trans-cinnamaldehyde itself as a known effective antimicrobial agent.

[0010] Regarding the combined use of trans-cinnamaldehyde and other antibiotics, see CN113855680B: Application of cinnamaldehyde in combination with ceftriaxone sodium. It discloses that when cinnamaldehyde at a sub-inhibitory concentration is used in combination with 512 μg / mL of ceftriaxone sodium, Salmonella showed virtually no growth within 24 hours, and that the combination of cinnamaldehyde at a concentration of 1 / 4 MIC and ceftriaxone sodium at a concentration of 1 / 8 MIC could inhibit the proliferation of Salmonella. Summary of the Invention

[0011] The main objective of this invention is to provide an antibacterial composition that has a good inhibitory or killing effect on multidrug-resistant Streptococcus pleuropneumoniae, Streptococcus suis, and Staphylococcus suis. It can reverse drug-resistant antibiotics into sensitive antibiotics, significantly reduce the use of commonly used antibiotics, improve quality and efficiency, and at the same time avoid antibiotic residues in pork products, thus ensuring food safety.

[0012] In addition, the present invention also provides the application of this antibacterial combination and the drug thereof.

[0013] The technical solution of the present invention is as follows:

[0014] The use of the combination of trans-cinnamaldehyde and antibiotics in the preparation of drugs for the prevention and treatment of drug-resistant bacterial infections;

[0015] The antibiotic is one or more of amoxicillin, penicillin G, ciprofloxacin, erythromycin, and lincomycin.

[0016] In the above-mentioned uses, the ratio of trans-cinnamaldehyde to antibiotic is 1–1.56 μM : 1.56–400 μg / ml.

[0017] In the above-mentioned uses, the drug-resistant bacteria are one or more of Bacillus pleuropneumoniae, Streptococcus suis, and Staphylococcus suis.

[0018] Meanwhile, the present invention discloses an antibacterial composition comprising trans-cinnamaldehyde and an antibiotic; the antibiotic being one or more of amoxicillin, penicillin G, ciprofloxacin, erythromycin, and lincomycin.

[0019] In the above-mentioned antibacterial composition, the ratio of trans-cinnamaldehyde to antibiotic is 1-1.56 μM : 1.56-400 μg / ml.

[0020] Finally, the present invention also discloses a drug comprising the antibacterial combination described above.

[0021] In the aforementioned drugs, the dosage form of the drugs is oral liquid, injection, tablet, granule or microcapsule.

[0022] One of the above-described technical solutions of the present invention has at least one of the following advantages or beneficial effects:

[0023] The compositions of the present invention have good inhibitory or killing effects on multidrug-resistant Streptococcus pleuropneumoniae, Streptococcus suis, and Staphylococcus suis.

[0024] The composition of this invention can reverse resistant antibiotics into sensitive antibiotics, significantly reduce the use of commonly used antibiotics, improve quality and efficiency, and at the same time avoid antibiotic residues in pork products, thus ensuring food safety. Attached Figure Description

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0026] Figure 1A This is a statistical table showing the results of MIC and MBC determination of trans-cinnamaldehyde against multidrug-resistant Staphylococcus aureus strain SG-7.

[0027] Figure 1B Photographs showing the MIC and MBC results of trans-cinnamaldehyde against multidrug-resistant Staphylococcus aureus strain SG-7;

[0028] Figure 2 The groups included a blank control group, a Streptococcus suis infection group, and a Streptococcus suis infection group combined with cinnamaldehyde administration. Photos of mice after infection are also included.

[0029] Figure 3 The groups included a blank control group, a Streptococcus suis infection group, a Streptococcus suis infection group, and a cinnamaldehyde-treated group. Survival curves of mice after infection are shown in the table. Detailed Implementation

[0030] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0031] The *Staphylococcus suis* SG-7 strain used in this embodiment of the invention was obtained from the Animal Health Research Institute of Guangdong Academy of Agricultural Sciences. It is a common strain well-known to those skilled in the art. *Staphylococcus suis* strains obtained from other sources are also applicable to this invention.

[0032] Implementation Case 1

[0033] Ten antibacterial agents (Table 1) and trans-cinnamaldehyde were used as test drugs to perform the following performance verifications:

[0034] 1. Bacterial culture

[0035] Staphylococcus suis SG-7 strain was inoculated into Luria Broth medium (LB medium), shaken overnight at 37°C and 220 rpm, and the bacteria were collected by centrifugation.

[0036] 2. Antimicrobial susceptibility testing of bacteria to commonly used antibiotics

[0037] Drug stock solution preparation and dilution were performed according to the guidelines developed by the Clinical and Laboratory Standards Institute (CLSI). Heavily bacterial cells were suspended in 3 ml of physiological saline, mixed thoroughly, and compared with a turbidity test tube. Using a white paper with black writing as a background, the turbidity was adjusted to match that of the turbidity test tube (0.5 McFarland units). A sterile cotton swab was dipped into the adjusted bacterial suspension, and excess suspension was squeezed out onto the tube wall. The swab was then repeatedly spread three times on an LB agar plate, rotating the plate 60° each time, and finally spreading two circles around the perimeter of the plate to ensure even distribution. After incubating at room temperature for 3-5 minutes, corresponding standard drug sensitivity test strips (6 mm in diameter) were attached, and the plate was incubated at 37°C for 18-24 hours. The diameter of the inhibition zone was measured using calipers. According to CLSI standards, an area with no visible growth was considered an inhibition zone. Extremely small colonies were negligible. Reports were submitted as sensitive [S], intermediate [I], or resistant [R]. The drug susceptibility results are as follows: strain SG-7 is only sensitive to ampicillin / sulbactam, cefotaxime and cefazolin, tends to be between sensitive and resistant to ciprofloxacin and vancomycin, and is resistant to the other tested drugs. It can be seen that SG-7 is a highly drug-resistant strain.

[0038] Table 1. Results of drug susceptibility testing of Staphylococcus aureus strain SG-7.

[0039]

[0040]

[0041] 3. Determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of trans-cinnamaldehyde against bacteria.

[0042] The minimum inhibitory concentration of the drug was determined using the micro-dilution method, and the specific procedure is as follows:

[0043] Add 190 μL of culture medium to the first well of a 96-well plate, and 100 μL of culture medium to each of the remaining wells. Add 10 μL of 1000 mM trans-cinnamaldehyde stock solution to the first well and mix well. Then, aspirate 100 μL from the first well and add it to the second well and mix well. Repeat this process until the 10th well. Wells 11 and 12 are left untreated as blank controls. Take a cultured Staphylococcus suis SG-7 bacterial suspension with a concentration equivalent to 0.5 McFarland standard, dilute it 1:1000 with culture medium, and add 100 μL to each well. Set up three replicates for each well, and include a control to avoid interference from drug color. Incubate at 37°C in a standard air incubator for 16-20 hours and then evaluate the results. The lowest drug concentration well with clearly visible, non-sterile colonies and a large volume represents the minimum inhibitory concentration (MIC) against Staphylococcus aureus SG-7. OD was also measured simultaneously. 600 Readings to aid in judgment. Mix the bacterial suspension in the wells of the above-mentioned plate by pipetting. Pipette 100 μL from each well (MIC and higher concentrations) onto an LB agar plate. Spread the bacterial suspension using a spreader until the plate is completely covered. Incubate overnight at 37°C in a standard air incubator, and record the results. The lowest dilution of the drug with fewer than 5 colonies on the plate is the minimum bactericidal concentration (MBC). Results are as follows: Figure 1A and Figure 1B As shown.

[0044] The minimum inhibitory concentration (MIC) of trans-cinnamaldehyde against Staphylococcus aureus SG-7 was determined to be 1.6 mM (211.4 μg / ml), and the minimum bactericidal concentration (MBC) was 6.3 mM (832.6 μg / ml). It still exhibits some antibacterial activity at a concentration of MIC / 2.

[0045] In addition, the same assay method was used to determine the MIC and MBC of trans-cinnamaldehyde against Staphylococcus aureus (ATCC 6538), Streptococcus suis, Actinobacillus pleuropneumoniae, and Haemophilus parasuis. The results are shown in Table 2.

[0046] Table 2. Minimum inhibitory concentration (MIC) of trans-cinnamaldehyde combined with antibacterial drugs against SG-7 resistant bacteria.

[0047]

[0048] The results showed that trans-cinnamaldehyde had good antibacterial and bacteriostatic effects on Staphylococcus aureus (ATCC 6538), Streptococcus suis, and Actinobacillus pleuropneumoniae, but had no significant effect on Haemophilus parasuis.

[0049] Implementation Case 2

[0050] The antibacterial performance of trans-cinnamaldehyde against Staphylococcus aureus was verified by combining it with different antibacterial drugs.

[0051] Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of trans-cinnamaldehyde in combination with antimicrobial agents against SG-7 resistant bacteria.

[0052] Medium containing trans-cinnamaldehyde was prepared in advance at concentrations of 1000 μM, 400 μM, 200 μM, 100 μM, 10 μM, and 1 μM, with a blank control without trans-cinnamaldehyde. For each concentration, 190 μL of medium containing trans-cinnamaldehyde was added to the first well of each row of a 96-well plate, and 100 μL of medium was added to each of the remaining wells. Amoxicillin was diluted using the micro-two-fold dilution method as described in step 2 of Example 1, with a maximum concentration of 50 μg / ml. The remaining procedures were the same, and the inhibitory effects of different concentrations of trans-cinnamaldehyde combined with different antimicrobial drug combinations on the multidrug-resistant Staphylococcus aureus strain SG-7 were determined. The results are shown in Tables 3 and 4. At a concentration of 1000 μM, amoxicillin reduced the MIC concentration of the SG-7 resistant strain by 32-fold and the MBC concentration by 4-fold.

[0053] Following the method described in step 1 of Implementation Case 1, the inhibitory effects of antibiotics—penicillin G, ciprofloxacin, erythromycin, and lincomycin—combined with trans-cinnamaldehyde on multidrug-resistant Staphylococcus aureus strain SG-7 were also determined. The results are shown in Tables 2 and 3.

[0054] Table 3. Minimum inhibitory concentration (MIC) of trans-cinnamaldehyde combined with antibacterial drugs against SG-7 resistant bacteria.

[0055]

[0056]

[0057] Table 4. Minimum bactericidal concentration (MBC) of trans-cinnamaldehyde in combination with various antibacterial agents against SG-7 resistant bacteria.

[0058]

[0059] In Tables 3 and 4, the dosage of each antibiotic is expressed in μg / ml.

[0060] The results show that:

[0061] 1. Trans-cinnamaldehyde can reduce the MIC and MBC values ​​of various types of antimicrobial drugs, increase the sensitivity of SG-7 resistant strains to various antimicrobial drugs, and significantly reduce the amount of antibiotics used.

[0062] 2. As shown in Table 3, when using 1 / 4 MIC of trans-cinnamaldehyde, it requires 1 / 16 MIC of amoxicillin, 1 / 8 MIC of penicillin G, 1 / 8 MIC of ciprofloxacin, 1 / 32 MIC of erythromycin, and 1 / 16 MIC of lincomycin.

[0063] When using 1 / 8 MIC of trans-cinnamaldehyde, the following dosages are required: 1 / 8 MIC of amoxicillin, 1 / 4 MIC of penicillin G, 1 / 4 MIC of ciprofloxacin, 1 / 8 MIC of erythromycin, and 1 / 4 MIC of lincomycin.

[0064] When using 1 / 16 MIC of trans-cinnamaldehyde, it requires 1 / 4 MIC of amoxicillin, 1 / 4 MIC of penicillin G, 1 / 4 MIC of ciprofloxacin, 1 / 4 MIC of erythromycin, and 1 / 2 MIC of lincomycin.

[0065] This indicates that trans-cinnamaldehyde can produce a synergistic effect with many antibiotics in inhibiting SG-7 resistant bacteria. In particular, the dosage of trans-cinnamaldehyde at 1 / 4 MIC and 5 / 8 MIC can significantly reduce the amount of antibiotics used, with a reduction of up to 64 times.

[0066] 3. As can be seen from Table 4, when the dosage of trans-cinnamaldehyde is 4 / 63 MBC, it requires 1 / 2 MBC of amoxicillin, 1 / 8 MBC of penicillin G, 1 / 2 MBC of ciprofloxacin, <1 / 4 MBC of erythromycin, and <1 / 2 MBC of lincomycin.

[0067] When using 2 / 63MBC trans-cinnamaldehyde, 1MBC amoxicillin, 1 / 4MBC penicillin G, 1 / 2MBC ciprofloxacin, and 1 / 2MBC erythromycin are required.

[0068] When using 1 / 64 MBC trans-cinnamaldehyde, it requires 1 MBC amoxicillin, 1 / 2 MBC penicillin G, and 1 / 2 MBC ciprofloxacin.

[0069] This indicates that trans-cinnamaldehyde can produce synergistic effects with many antibiotics in the treatment of SG-7 resistant bacteria. In particular, the dosage of trans-cinnamaldehyde at 1 / 4 MIC and 5 / 8 MIC can significantly reduce the amount of antibiotics used, with a reduction of up to 16 times.

[0070] Implementation Case 3

[0071] The role of cinnamaldehyde in combating Streptococcus suis infection in vivo was evaluated by constructing a mouse meningitis animal model.

[0072] The preparation of Streptococcus suis culture and its infection in mice were performed as follows: Streak the challenge bacteria in three zones on a TSA plate, then incubate upside down at 37°C for 18 hours. Using forceps, pick a single colony from the TSA plate with a sterile pipette tip and place it in 5 mL of 5% bovine serum TSB broth. Incubate on a shaker (37°C, 170 rpm) for 18 hours until the liquid in the tube changes from clear to turbid; this indicates the fresh bacterial culture. Dilute the fresh bacterial culture 10-fold with 5% serum TSB broth to a suitable gradient. Pipette 100 μL of the diluted culture onto a standard lower-layer plate (three replicates), spread evenly with a sterile spreader, and after the plate surface dries, incubate upside down at 37°C for 18 hours. Count the number of colonies on the plate and calculate the bacterial concentration [Calculation formula: Bacterial concentration (CFU / mL) = Average colony count × Dilution factor × 10]. The bacterial culture was diluted with PBS to 1x10^9 / ml and injected intramuscularly into mice. Each mouse in the infection group was injected intramuscularly with 100μL of S. suis 2 bacterial culture, and the blank control group was injected with an equal volume of PBS.

[0073] Cinnamaldehyde was administered at a dosage of 24 mg / kg per mouse, with an average mouse weight of 25 g. 20 mg of cinnamaldehyde was dissolved in 16.7 ml of PBS to prepare a concentration of 1.2 mg / ml cinnamaldehyde. Each mouse was intraperitoneally injected with 0.5 ml of the 1.2 mg / ml cinnamaldehyde solution.

[0074] Animals were divided into three groups: a blank control group, a Streptococcus suis infection group, and a Streptococcus suis infection group & cinnamaldehyde treatment group. Mice were observed for three days post-infection, and their condition was recorded and survival curves were calculated. Results are as follows: Figure 2 and Figure 3 As shown.

[0075] The results showed that cinnamaldehyde could reduce cerebral congestion in mice and significantly reduce the mortality rate of mice infected with Streptococcus suis.

Claims

1. Use of the combination of trans-cinnamaldehyde and antibiotics in the preparation of medicaments for the prevention and treatment of drug-resistant bacterial infections; The ratio of trans-cinnamaldehyde to antibiotic is: 100–400 μM: 3.13–12.5 μg / ml; The antibiotic in question is amoxicillin; The drug-resistant bacteria is Staphylococcus suis SG-7.

2. Use of the combination of trans-cinnamaldehyde and antibiotics in the preparation of medicaments for the prevention and treatment of drug-resistant bacterial infections; The ratio of trans-cinnamaldehyde to antibiotic is: 100–400 μM: 12.5–25 μg / ml; The antibiotic is penicillin G; The drug-resistant bacteria is Staphylococcus suis SG-7.

3. Use of the combination of trans-cinnamaldehyde and antibiotics in the preparation of medicaments for the prevention and treatment of drug-resistant bacterial infections; The ratio of trans-cinnamaldehyde to antibiotic is: 100-400 μM: 25-50 μg / ml; The antibiotic is ciprofloxacin; The drug-resistant bacteria is Staphylococcus suis SG-7.

4. Use of the combination of trans-cinnamaldehyde and antibiotics in the preparation of medicaments for the prevention and treatment of drug-resistant bacterial infections; The ratio of trans-cinnamaldehyde to antibiotic is: 100–400 μM: 12.5–100 μg / ml; The antibiotic is erythromycin; The drug-resistant bacteria is Staphylococcus suis SG-7.

5. Use of the combination of trans-cinnamaldehyde and antibiotics in the preparation of medicaments for the prevention and treatment of drug-resistant bacterial infections; The ratio of trans-cinnamaldehyde to antibiotic is: 200-400 μM: 25-100 μg / ml; The antibiotic is lincomycin; The drug-resistant bacteria is Staphylococcus suis SG-7.

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