N-terminal fatty acid modified antimicrobial peptide analogs and uses thereof

Abstract

The application provides an N-terminal fatty acid modified antibacterial peptide analogue and application thereof. Specifically, the antibacterial peptide analogue of the application has a fatty acid (dodecanoic acid, tetradecanoic acid or octadecanoic acid) modification at the N terminus of a super short peptide Lys-Gly-Lys to form a lipopeptide substance. The N-terminal fatty acid modified antibacterial peptide analogue described in the application can inhibit a plurality of pathogenic bacteria, in particular a plurality of mastitis pathogenic bacteria, has high inhibitory activity, and has a unique antibacterial mechanism, is not easy to induce drug resistance of pathogenic bacteria, can more safely inhibit bacteria, and can also solve the problem of antibiotic resistance easily produced in the treatment process of mastitis; further, the antibacterial peptide analogue of the application can also down-regulate the expression of body pro-inflammatory factors (for example, TNF-alpha, IL-6), and accelerate the repair of infected site tissues. The antibacterial peptide analogue of the application can effectively improve the cure rate of dairy cows and reduce economic losses.

Description

Technical Field

[0001] This invention relates to the field of biochemistry, specifically to an N-terminal fatty acid-modified antimicrobial peptide analog and its uses. Background Technology

[0002] Currently, clinical treatment of mastitis in dairy cows mainly relies on antibiotics, such as penicillins and cephalosporins. However, the long-term and widespread use of antibiotics has led to numerous problems. On the one hand, the increasing drug resistance of pathogens has gradually reduced the effectiveness of antibiotic treatment. The drug residues resulting from the extensive use of antibiotics and the increased drug resistance of pathogenic microorganisms have seriously threatened food safety, further exacerbating consumer concerns about the quality and safety of dairy products. Therefore, the development of new, safe, and effective drugs for treating mastitis in dairy cows is urgently needed.

[0003] Antimicrobial peptides, due to their unique chemical structures, exhibit excellent antibacterial, antiviral, and immunomodulatory biological activities, making their potential applications in the pharmaceutical field widely recognized. However, antimicrobial peptides with different structures show significant differences in activity and safety, making the screening of antimicrobial peptides that are highly effective and safe for treating bovine mastitis of significant practical importance. Summary of the Invention

[0004] To address the problems of existing technologies, this invention provides an N-terminal fatty acid-modified antimicrobial peptide analog. Specifically, the antimicrobial peptide analog of this invention has a fatty acid modification at the N-terminus of the ultrashort peptide Lys-Gly-Lys to form a lipopeptide (e.g., C12-KGK, C14-KGK, C16-KGK, and C18-KGK). For example, the C18-KGK structure consists of a fatty acid chain (C18) linked with L-lysine (Lys, K) and L-glycine (Gly, G) via an amide bond, and an amide group is capped at the C-terminus of the short peptide chain, represented as C18-Lys-Gly-Lys-NH2, with the chemical formula C... 32 H 64 N6O4, molecular structural formula as follows Figure 1 As shown.

[0005] This invention is the first to discover that lipopeptides obtained by modifying the N-terminus of the ultrashort peptide Lys-Gly-Lys with fatty acids can effectively inhibit bacteria, particularly those causing mastitis in dairy cows. Therefore, in another aspect, this invention also provides the use of the N-terminal fatty acid-modified antimicrobial peptide analogs described herein, particularly in the preparation of antimicrobial drugs / formulations or in the preparation of drugs / formulations for treating mastitis (e.g., bovine mastitis).

[0006] Furthermore, the N-terminal fatty acid-modified antimicrobial peptide analogs described in this invention offer hope and opportunity as novel antimicrobial agents against drug-resistant bacteria. Numerous studies have demonstrated that positive charge and hydrophobicity are essential for the bioactivity and primary mechanism of action of antimicrobial peptides. Positive charge promotes electrostatic interactions between the antimicrobial peptide and the negatively charged bacterial membrane, while hydrophobicity facilitates the insertion of the antimicrobial peptide into the bacterial membrane bilayer, causing membrane disruption and permeability, ultimately leading to leakage of bacterial content and bacterial death.

[0007] In this regard, the technical solutions of the present invention include, but are not limited to, the following:

[0008] In one aspect, the present invention provides an N-terminal fatty acid-modified antimicrobial peptide analog with the structural formula: Cn-Lys-Gly-Lys-NH2, wherein Cn is a fatty acid linked to the α-amino group of an N-terminal lysine residue, said fatty acid being selected from dodecanoic acid (i.e., CH3-(CH2)9-CH2-COOH) and tetradecanoic acid (i.e., CH3-(CH2)). 11 -CH2-COOH) or octadecanoic acid (i.e., CH3-(CH2)). 15 -CH2-COOH).

[0009] In one aspect, the fatty acid described in this invention is selected from dodecanoic acid (i.e., CH3-(CH2)9-CH2-COOH) or octadecanoic acid (i.e., CH3-(CH2)9-CH2-COOH). 15 -CH2-COOH), preferably, the fatty acid is selected from octadecanoic acid (i.e., CH3-(CH2)). 15 -CH2-COOH).

[0010] Preferably, in one aspect, the fatty acid described in this invention is octadecanoic acid (i.e., CH3-(CH2)). 15 -CH2-COOH).

[0011] In one aspect, the present invention provides an N-terminal fatty acid-modified antimicrobial peptide analog with the structural formula: Cn-Lys-Gly-Lys-NH2, wherein Cn is a fatty acid linked to the α-amino group of an N-terminal lysine residue, and said fatty acid is octadecanoic acid (i.e., CH3-(CH2)). 15 -CH2-COOH).

[0012] In one aspect, the present invention provides an N-terminal fatty acid-modified antimicrobial peptide analog, the structural formula of which is:

[0013] In one aspect, the amino acids in the N-terminal fatty acid-modified antimicrobial peptide analogs of the present invention are all natural amino acids.

[0014] In another aspect, the present invention provides the use of the N-terminal fatty acid-modified antimicrobial peptide analogs described herein in the preparation of antimicrobial drugs / formulations.

[0015] In one aspect, the antibacterial drug / preparation of the present invention is an anti-Gram-positive bacteria drug / preparation or an anti-Gram-negative bacteria drug / preparation.

[0016] In one aspect, the anti-Gram-positive bacteria drug / preparation of the present invention is selected from anti-streptococcus agalactiae drugs / preparations and anti-staphylococcus aureus drugs / preparations. Preferably, the anti-Gram-positive bacteria drug / preparation of the present invention is selected from anti-streptococcus agalactiae GS032 drug / preparation and anti-staphylococcus aureus GS1311 drug / preparation.

[0017] In one aspect, the anti-Gram-positive bacteria drug / preparation of the present invention is an anti-Streptococcus agalactiae drug / preparation. Preferably, the anti-Gram-positive bacteria drug / preparation of the present invention is an anti-Streptococcus agalactiae GS032 drug / preparation.

[0018] In one aspect, the anti-Gram-negative bacteria drug / preparation of the present invention is selected from anti-Escherichia coli drugs / preparations. Preferably, the anti-Gram-negative bacteria drug / preparation of the present invention is an anti-Escherichia coli YN001-2 drug / preparation.

[0019] In another aspect, the present invention provides the use of the N-terminal fatty acid-modified antimicrobial peptide analogs described herein in the preparation of medicaments / formulations for the treatment of mastitis.

[0020] In one aspect, the mastitis described in this invention is mastitis caused by Streptococcus agalactiae, Staphylococcus aureus, or Escherichia coli.

[0021] In one aspect, the mastitis described in this invention is mastitis caused by Streptococcus agalactiae.

[0022] Preferably, the mastitis described in this invention is mastitis caused by Streptococcus agalactiae GSO32.

[0023] In one aspect, the mastitis described in this invention is bovine mastitis.

[0024] In one aspect, the mastitis described in this invention is bovine mastitis caused by Streptococcus agalactiae, Staphylococcus aureus, or Escherichia coli. Preferably, the mastitis described in this invention is bovine mastitis caused by Streptococcus agalactiae. More preferably, the mastitis described in this invention is bovine mastitis caused by Streptococcus agalactiae GS032.

[0025] In another aspect, the present invention provides the use of the N-terminal fatty acid-modified antimicrobial peptide analogs of the present invention in the preparation of medicaments for reducing inflammation in the body, preferably, the inflammation being caused by Streptococcus agalactiae, Staphylococcus aureus, or Escherichia coli.

[0026] In one aspect, the inflammation described in this invention is caused by Streptococcus agalactiae, Staphylococcus aureus, or Escherichia coli.

[0027] In one aspect, the inflammation described in this invention is caused by *Streptococcus agalactiae*. Preferably, the inflammation described in this invention is caused by *Streptococcus agalactiae* GSO32.

[0028] In one aspect, the reduction of systemic inflammation described in this invention is manifested as a reduction in the expression of pro-inflammatory factors (e.g., TNF-α and / or IL-6) in body tissues.

[0029] In one aspect, the reduction of inflammation in the body as described in this invention also manifests as a reduction of inflammatory cells in the body's tissues.

[0030] In one aspect, the reduction of bodily inflammation described in this invention also manifests as promoting the healing or recovery of inflamed tissues in the body.

[0031] The beneficial effects of this invention are:

[0032] In summary, the N-terminal fatty acid-modified antimicrobial peptide analogs of this invention exhibit highly efficient inhibitory activity against a variety of pathogens, particularly various pathogens causing bovine mastitis. Furthermore, their unique antimicrobial mechanism makes them less likely to induce drug resistance in pathogens, thus solving the problem of antibiotic resistance that easily arises during the treatment of bovine mastitis. In addition, the antimicrobial peptide analogs of this invention are naturally derived, biodegradable in animals, and leave no drug residues, ensuring the safety of dairy products. The antimicrobial peptide analogs of this invention can effectively improve the cure rate of dairy cows and reduce economic losses.

[0033] Specifically, this invention successfully constructed an N-terminal fatty acid-modified antimicrobial peptide analog that possesses both high antibacterial activity and low risk of drug resistance. Given that the target specificity of traditional antibiotics can lead to rapid development of resistance, while the multi-target mechanism of antimicrobial peptides makes it more difficult for bacteria to develop resistance, this invention constructs a functional molecule with an amphiphilic structure through precise coupling of hydrophobic alkyl chains (e.g., C12, C14, C16, and C18 alkyl chains) with cationic KGK peptide sequences. This demonstrates significant advantages in terms of mechanism of action, safety, and application potential. Furthermore, the antimicrobial peptide analog of this invention not only possesses antibacterial function but also modulates the expression of host inflammatory factors. The antimicrobial peptide analog of this invention (e.g., C18-KGK) can not only kill bacteria but also downregulate the expression of pro-inflammatory factors (e.g., TNF-α, IL-6), accelerating tissue repair at the site of infection. In addition, the antimicrobial peptide analog of this invention is composed of natural amino acids and fatty acids, making it easily decomposed by microorganisms in the environment, while traditional antibiotics tend to remain in soil and water for extended periods, inducing the spread of environmental resistance genes. Attached Figure Description

[0034] Figure 1 The structural formula is C18-KGK.

[0035] Figure 2 This is the mass spectrum of C18-KGK.

[0036] Figure 3 This is the mass spectrum of C12-KGK.

[0037] Figure 4 This is the mass spectrum of C14-KGK.

[0038] Figure 5 This is the mass spectrum of C16-KGK.

[0039] Figure 6 This is the mass spectrum of C18-KK.

[0040] Figure 7 The hemolysis rate curves are for lipopeptides with different fatty acid chains.

[0041] Figure 8 To establish a mouse mastitis model, the following groups were established: N group (control group): mice received no treatment; P group (mastitis group): mice infected with Streptococcus agalactiae GS032 developed mastitis and received no treatment; KGK group (antimicrobial peptide analog treatment group): mice infected with Streptococcus agalactiae GS032 developed mastitis and treated with C18-KGK antimicrobial peptide analog via mammary duct injection; Cefotaxime group (cefotaxime treatment group): mice infected with Streptococcus agalactiae GS032 developed mastitis and treated with antibiotic (cefotaxime) via mammary duct injection.

[0042] Figure 9 HE staining results of mouse mammary gland tissue pathological sections.

[0043] Figure 10 The results are statistical analysis of the bacterial load assay in mouse mammary tissue.

[0044] Figure 11 The results of statistical analysis of IL-6 and TNF-α factors in the detection of inflammatory factors in mouse mammary tissue. Detailed Implementation Plan

[0045] Example 1: Preparation of C18-KGK antimicrobial peptide analogs

[0046] The antimicrobial peptide analogs involved in this invention were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. (Shanghai, China) using solid-phase synthesis technology. Specifically, using resin as a solid-phase carrier, Fmoc-K(Fmoc)-OH, Fmoc-G-OH, and Fmoc-K(Fmoc)-OH amino acids were sequentially coupled according to the Fmoc (9-fluorenylmethoxycarbonyl) amino acid protection strategy. Then, the Fmoc protecting group was removed with piperidine, followed by a condensation reaction with C18 fatty acids. Finally, the resin was cleaved with trifluoroacetic acid to obtain the crude product. The crude product was purified by high-performance liquid chromatography (HPLC) to obtain C18-KGK antimicrobial peptide analogs with a purity greater than 95%. The final product is in lyophilized powder form, wherein the antimicrobial peptide analog C18-KGK is represented as C18-Lys-Gly-Lys-NH2, and its molecular structure is as follows: Figure 1 As shown, the mass spectrum is as follows Figure 2 As shown. The antimicrobial peptide was prepared into a stock solution with a final concentration of 10 mg / mL using sterile PBS and stored at -80°C for later use.

[0047] Example 2: Minimum inhibitory concentration (MIC) of antimicrobial peptide analogs

[0048] C12-KGK, C14-KGK, C16-KGK, and C18-KK were synthesized according to the method in Example 1. The mass spectra are as follows: Figure 3 , Figure 4 , Figure 5 , Figure 6 As a control for C18-KGK, overnight cultures of *Streptococcus agalactiae* GS032, *Staphylococcus aureus* GS1311, and *Escherichia coli* YN001-2 were inoculated at a ratio of 1:50 into TSB (tryptic soy broth), shaken at 220 rpm until the OD600 reached 0.6–0.8, and the bacterial cells were collected. The TSB was then diluted to a concentration of 1 × 10⁻⁶. 5The CFU / mL concentration of the synthesized lipopeptides was adjusted using a two-fold serial dilution method to final concentrations of 400 μg / mL, 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.12 μg / mL, 1.5 μg / mL, 0.75 μg / mL, 0.37 μg / mL, 0.18 μg / mL, 0.09 μg / mL, and 0.045 μg / mL. Sterile PBS was used as a positive control group. 100 μL of the prepared bacterial suspension was added to each concentration, and the mixture was incubated at 37℃ and 220 rpm for 12–16 h to observe the results.

[0049] In this embodiment, *Streptococcus agalactiae* GS032, *Staphylococcus aureus* GS1311, and *Escherichia coli* YN001-2 were all isolated from milk samples produced by cows suffering from mastitis in the inventors' laboratory. These were previously reported in the inventors' published articles, for example, *Streptococcus agalactiae* GS032 (named LZ032 in that article) was disclosed in Peng J et al., *Biochem Biophys Res Commun. 2022 Apr 23; 601:153-159*, and *Staphylococcus aureus* GS1311 was disclosed in Peng J et al., *Int J Antimicrob Agents. 2023 Sep; 62(3):106916*. Furthermore, the specific isolation methods for *Streptococcus agalactiae* GS032, *Staphylococcus aureus* GS1311, and *Escherichia coli* YN001-2 can be found in Liu Xuming et al., *Animal Husbandry and Veterinary Medicine*, 2021, 53(11):117-122.

[0050] Table 1. MIC of different fatty acid chain lipopeptides

[0051]

[0052] Example 3: Hemolytic activity of C18-KGK

[0053] Take a clean 1.5 mL centrifuge tube, label it, add 200 μL of serially diluted lipopeptide in sterile PBS and an equal volume of 1% mouse red blood cell suspension, and simultaneously treat with 1% Triton-X100 as a positive control. Incubate at 37℃ for 1 h, centrifuge at 1000 rpm for 10 min, collect 100 μL of supernatant, and detect the absorbance of the sample at 540 nm to plot the hemolysis curve. The hemolysis percentage is calculated as follows: Hemolysis percentage (%) = (ODS - ODB) / (ODP - ODB), where ODS represents the absorbance of the sample, ODB represents the absorbance of the blank control (PBS buffer), and ODP represents the absorbance of the positive control. The results are as follows. Figure 7 As shown.

[0054] Example 4: Establishment of a mouse mastitis model

[0055] (1) Primiparous and lactating female mice, 4-6 days postpartum, were anesthetized by inhalation, placed in a supine position, and their abdominal mammary glands and surrounding skin were disinfected with 75% alcohol. Streptococcus agalactiae GS032 was infused into each mammary gland using a microsyringe via the mammary ducts, with 5 × 10⁻⁶ doses injected. 5 An infection model of Streptococcus agalactiae GS032 was constructed using CFU / mL Streptococcus agalactiae resuspension.

[0056] (2) Twenty-four hours after injection of *Streptococcus agalactiae* GS032, the antimicrobial peptide analog C18-KGK was administered. Before C18-KGK treatment, the mother mice and pups were separated for two hours. After the mammary glands were full of milk, the mother mice were anesthetized, and 10 μg of the antimicrobial peptide analog diluted with sterile PBS (C18-KGK solution) was injected through the mammary duct. Two hours after injection, the mother mice and pups were placed in the same cage. The medication was administered once daily for seven days, followed by a two-day rest period after the treatment trial. The mice's condition was observed daily.

[0057] (3) Four mice were used in each group, and the experimental groups were as follows:

[0058] (i) Group N (control group): Mice received no treatment;

[0059] (ii) P group (mastitis group): a mouse mastitis model without Streptococcus lactis GS032 infection and without any treatment;

[0060] (iii) KGK group (antimicrobial peptide analog treatment group): a mouse mastitis model without Streptococcus lactis GS032 infection, and treated with C18-KGK antimicrobial peptide analog via mammary duct injection.

[0061] (iv) Cefotaxime group: a mouse mastitis model infected with Streptococcus agalactiae GS032 and treated with antibiotics (cefotaxime, a broad-spectrum antibiotic commonly used to treat mastitis in dairy cows) via mammary duct injection.

[0062] (4) Mice were infected with Streptococcus agalactiae GS032 through the nipple root. N group mice were more active, with smooth fur, and ate and drank more frequently; P group mice became more sluggish, with messy fur, and drank and ate less frequently; Cefotaxime group and KGK group mice drank and ate more frequently than P group mice, and were more active overall.

[0063] In addition, such as Figure 8The results show that mice in group N were in normal condition and did not develop mastitis; mice in group P developed mastitis; mice in group KGK developed mastitis, and their symptoms were alleviated after treatment. Mice in group Cefotaxime (cefotaxime treatment group) also developed mastitis, and their symptoms were alleviated after treatment. This indicates that the C18-KGK antimicrobial peptide analog provided by this invention has a therapeutic effect on mastitis and can achieve an effect at least equivalent to that of antibiotics.

[0064] Example 5: Pathological section analysis of breast tissue

[0065] This embodiment performs pathological analysis on the mouse mastitis model constructed in Example 2. The specific experimental steps are as follows:

[0066] 1. Dewaxing paraffin sections to water: Place the sections in xylene twice, each time with fresh xylene for 20 min, then place them in anhydrous ethanol twice, each time with fresh anhydrous ethanol for 10 min, followed by the following treatments: 95% ethanol (5 min), 90% ethanol (5 min), 80% ethanol (5 min), 70% ethanol (5 min), and wash with distilled water (10 s).

[0067] 2. Staining: Immerse the sections in hematoxylin solution for 5 minutes, rinse with tap water, differentiate and rinse with 1% hydrochloric acid alcohol for 5 seconds, and stop when the sections turn light red. Place them under running water to restore the blue color, and then stain with eosin for 3 minutes.

[0068] 3. Dehydration and mounting: Immerse the sections in 95% alcohol twice, each time using fresh 95% alcohol for 5 minutes. Then immerse them in anhydrous ethanol twice, each time using fresh anhydrous ethanol for 5 minutes. Next, immerse them in xylene twice, each time using fresh xylene for 5 minutes, to dehydrate and clear the sections. Remove the sections from the xylene and allow them to dry slightly. Mount them with neutral resin and observe and photograph them under a microscope.

[0069] The results are as follows Figure 9 As shown, no obvious inflammatory symptoms were observed in the mammary tissue sections of mice in group N; obvious inflammatory symptoms were observed in the mammary tissues of mice in group P, with a large amount of serous fluid and inflammatory cells in the mammary alveoli, and the alveolar structure was incomplete; fewer inflammatory cells were found in the mammary alveoli of mice in group KGK, with clear boundaries, relatively intact structure, and no fibrosis in the stroma; fewer inflammatory cells were found in the mammary alveoli of mice in group Cefotaxime, with clear boundaries, relatively intact structure, and no fibrosis in the stroma.

[0070] Example 6: Determination of bacterial load in breast tissue

[0071] This embodiment measures the bacterial load of the mouse mastitis model constructed in Example 2.

[0072] The specific experimental steps are as follows: 0.1g of mammary gland tissue was weighed from experimental mice and homogenized in 0.9mL of sterile PBS solution. The tissue homogenate was serially diluted, and 100μL of each dilution was spread onto TSB plates. The inoculated culture dishes were inverted and incubated overnight at 37℃. The colonies growing on the plates were counted and statistically analyzed.

[0073] The results are as follows Figure 10 As shown, the number of bacteria in the mammary tissue of mice treated with C18-KGK was significantly lower than that in the untreated mastitis group (i.e., the P group) and the antibiotic-treated group (Cefotaxime group).

[0074] Example 7: Measurement of Inflammatory Factors in Breast Tissue

[0075] This embodiment measures the inflammatory factors of the mouse mastitis model constructed in Example 2.

[0076] The specific experimental steps are as follows: Real-time quantitative polymerase chain reaction (qPCR) was used to detect the expression of inflammatory factors in breast tissue. Total RNA was extracted from mouse breast tissue using TRIzol reagent according to the manufacturer's instructions. cDNA was synthesized via reverse transcription using PrimeScript™ reverse transcriptase mixture (manufactured by Nanjing Novizan Materials Technology Co., Ltd.) according to the manufacturer's instructions. Green Master Mix (High ROX Premixed) (produced by Nanjing Novizan Materials Technology Co., Ltd.) was used to detect the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in breast tissue by real-time quantitative PCR (qRT-PCR) to determine the degree of inflammation. Primer sequences are shown in Table 2. The reaction system was as follows: Green Master Mix 10 μL; reverse primer 0.4 μL; forward primer 0.4 μL; cDNA 5 μL; double-distilled water 4.2 μL. The reaction conditions were as follows: pre-denaturation, 95℃, 5 min; 40 cycles, 95℃ for 10 s, 60℃ for 30 s. The comparison Ct method (2) was used. -ΔΔct The calculation results were normalized to the internal reference level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

[0077] Table 2 Primer sequences for real-time quantitative PCR

[0078]

[0079] The results are as follows Figure 11 As shown, compared with the mammary tissue of the P group mice, the expression levels of IL-6 and TNF-α in the mammary tissue of the KGK group mice were significantly reduced.

[0080] The above embodiments confirm that the C18-KGK antimicrobial peptide analog prepared in this invention has excellent antibacterial effects. Furthermore, by constructing a mouse mastitis model and treating it with the C18-KGK antimicrobial peptide analog of this invention, it was found that the C18-KGK antimicrobial peptide analog can also promote the healing of inflamed tissues. Pathological section observation, bacterial load determination, and inflammatory cytokine assays confirmed that the C18-KGK antimicrobial peptide has an inhibitory effect on inflammatory factors, thereby promoting the recovery of inflamed tissues.

Claims

1. An N-terminally fatty acid-modified analog of an antibacterial peptide, characterized in that, The antibacterial peptide analogue has the formula Cn-Lys-Gly-Lys-NH2, wherein Cn is a fatty acid attached to the alpha amino group of the N-terminal lysine, said fatty acid being selected from octadecanoic acid.

2. Use of the N-terminally fatty acid modified antibacterial peptide analogue according to claim 1 for the manufacture of an antibacterial medicament / preparation.

3. Use according to claim 2, characterized in that, The antibacterial medicament / preparation is an anti-Gram-positive or an anti-Gram-negative medicament / preparation.

4. Use according to claim 3, characterized in that, The anti-Gram-positive medicament / preparation is selected from an anti-Streptococcus agalactiae or an anti-Staphylococcus aureus medicament / preparation.

5. Use according to claim 3, characterized in that, The anti-Gram-negative medicament / preparation is selected from an anti-Escherichia coli medicament / preparation.

6. Use of the N-terminally fatty acid modified antibacterial peptide analogue according to claim 1 for the manufacture of a medicament / preparation for the treatment of mastitis caused by Streptococcus agalactiae, Staphylococcus aureus or Escherichia coli.

7. Use according to claim 6, characterized in that, The mastitis is mastitis in dairy cattle.

8. Use of the N-terminally fatty acid modified antibacterial peptide analogue according to claim 1 for the manufacture of a medicament for the reduction of inflammation in the body caused by Streptococcus agalactiae, Staphylococcus aureus or Escherichia coli.

Images