Composition against acinetobacter baumannii

Abstract

The present invention provides a disclosure of a composition involving an extract from lactic acid bacteria. The composition involves an extract from Lactobacillus plantarum L001 strain isolated from kimchi. The lactic acid bacteria strain has a novel gene sequence which is currently reported for first time using 16S rRNA method and the gene sequence is currently disclosed. The purification method used to isolate the strain involves MRS broth. The strain is processed and turned into a methanol extract. The methanol extract is filtered to obtain a feed. The feed is evaporated to obtain the extract which has efficacy to inhibit Acinetobacter baumannii. The extract can be used as a pharmaceutical composition, a food product, a food supplement or a veterinary composition.

Description

[0001] COMPOSITION AGAINST ACINETOBACTER BAUMANNII

[0002] The present invention provides an extract by a strain of lactic acid bacteria. The following comprises the composition of said extract, the use and the method to produce thereof.

[0003] BACKGROUND ART

[0004] Multidrug resistance is the ability of some microorganisms to resist the effect of antimicrobial drugs. The outbreak was reported in several involving Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterococcus faecalis.

[0005] A. baumannii has developed multidrug resistance to extremely drug resistance from antibiotics such as ureidopenicillin, aminopenicillin, cephalosporin, cefoxitin, and chloramphenicol. It resulted in low efficacy in treatment caused by A. baumannii infection. The limited antibiotics efficacy against A. baumannii creates the need for alternative treatment.

[0006] Lactic acid bacteria are a group of bacteria that produce lactic acid as the main product of carbohydrate fermentation. The bacteria are gram-positive, acid tolerant and produce natural antimicrobial substance. It is gaining interest as an alternative for antibiotic treatment. Generally, lactic acid bacteria can survive in gastrointestinal tract.

[0007] Lactobacillus plantarum is a type of lactic acid bacteria commonly found in fermented foods and anaerobic plant matter. Antimicrobial metabolites were produced by lactic acid bacteria including L. plantarum. The antimicrobial substances are primary or by-products of lactic fermentation. There are various antifungal agents and bacteriocins reported.

[0008] Strom isolated L. plantarum from grass silage that produces antifungal compounds. Fusarium sporotrichiodes and Aspergillus fumigatus were sensitive among the moulds and Kluyveromyces marxianus was sensitive among yeast. US2014 / 0328814A1 disclosed an antimicrobial probiotic composition involving L. plantarum Tensia DSM 21380. The strain is derived from child faecal. The bacteriocin inhibits Listeria monocytogenes.

[0009] US874786B2 describes a composition against A. baumannii. The composition comprises an isolated antibody or antigen binding fragment. The composition is used as a vaccine.

[0010] The present inventor found that L. plantarum has great promise to inhibit A. baumannii. Lactic acid bacteria is generally regarded as safe. As such, the present invention is made using lactic acid bacteria to provide antibiotics efficacy against A. baumannii.

[0011] SUMMARY OF INVENTION

[0012] The inventors have obtained an extract produced by Lactobacillus plantarum L 001 strain. The bacteria, isolated from kimchi, comprises 16S rRNA sequence of SEQ ID NO 1 for use in treatment that inhibits Acinetobacter baumannii. The extract can be used as a pharmaceutical composition, a food product, a food supplement or a veterinary composition.

[0013] A method to prepare the extract is also disclosed. After isolating the L. plantarum culture from kimchi, the culture is grown in MRS broth. Then, the culture is centrifuged to obtain the supernatant. The supernatant is neutralized and freeze dried. A methanol extract is prepared from the supernatant. The methanol extract is filtered to obtain the feed. The feed is evaporated to obtain the extract.

[0014] DESCRIPTION OF EMBODIMENTS

[0015] Hereinafter, the present invention is described in the following detailed description in some embodiments, presented as an example and without limitation.

[0016] The present invention describes an extract from Lactobacillus plantarum L001 strain. This lactic acid bacteria have a novel gene sequence which has been reported for first time using 16S rRNA method and the gene sequence is shown in SEQ ID NO:1 . The sequence listing that accompany the specification was prepared according to WIPO Standard ST.26 “Recommended Standard for the Presentation of Nucleotide and Amino Acid Sequence Listings Using XML (Extensible Markup Language)”, version 1 .6, 2022 revision. The sequence listing was prepared with WIPO sequence version 2.3.0.

[0017] A base sequence assay for identifying and classifying Lactobacillus plantarum in 16S rRNA is performed. The base sequence of a 16S rRNA gene of Lactobacillus plantarum strain L001 is shown at SEQ ID NO 1. The extract can be used as a pharmaceutical composition, a food product, a food supplement or a veterinary composition.

[0018] Specific morphological and physiological properties of L. plantarum L001 were analyzed using conventional process and shown in Table 1 .

[0019] Table 1 .

[0020] The lactic acid bacteria can be obtained from commonly available kimchi. The process to obtain the strain is repeatable. The lactic acid bacteria are sufficiently disclosed by disclosing the 16S rRNA sequence and process to obtain the strain. A person having ordinary skilled in the art can repeat the process to obtain the strain.

[0021] Hence, it is not necessary to deposit the lactic acid bacteria recognized deposit authority. Nevertheless, a sample of the bacteria can be requested from the inventor.

[0022] Now, the process of preparing Lactobacillus plantarum L001 comprising the 16S rRNA sequence of SEQ ID NO 1 is described.

[0023] Kimchi is a Korean dish that comprise of fermented cabbage. The recipe to prepare kimchi is widely available.

[0024] Kimchi is mixed with glycerol and MRS broth. MRS broth is an acronym for authors that created the broth, De Man JD, Rogosa and Sharpe. Mixing is performed with kimchi weight to broth volume ratio of 1 :10. The MRS broth is incubated overnight at 37 °C. Then, streaking is performed onto the MRS agar. The step of incubating and streaking is repeated to obtain pure L001 culture. Presumptive colonies were randomly selected to obtain pure culture. Bioassay testing is performed on the pure culture.

[0025] Next, the process of preparing the extract of Lactobacillus plantarum L001 comprising the 16S rRNA sequence of SEQ ID NO 1 is described.

[0026] The L. plantarum culture is grown in modified 20 ml MRS broth. The culture is incubated for 24 to 36 hours in 37 °C using 5% carbon dioxide incubator. Then, the culture is transferred overnight to increase volume and incubated in 37 °C for another 24 to 36 hours.

[0027] The culture is centrifuged to obtain a supernatant. Next, the supernatant acid is neutralized to pH7. The supernatant hydrogen peroxide is then neutralized with 30 ng / ml catalase. Next, the supernatant is freeze dried to obtain supernatant concentrate.

[0028] A methanol extract is created with the weight ratio of supernatant over methanol of 1 :10. The extraction was made in 48 hours in room temperature. The methanol extract is left for 24 hours in room temperature. Methanol is filtered from the extract twice using filter paper. The feed is evaporated with rotary evaporator until methanol is evaporated to obtain the extract.

[0029] The extract is tested in antagonistic activity against 30 A. baumannii strains classified as Extremely Drug Resistance (XDR). It was found that 20 out of 30 XDR A. baumannii strains were inhibited. The common compound found in the active isolates against A. baumannii was 3-lsobutyl-2,3,6,7,8,8a-hexahydropyrrolo[1 ,2- a]pyrazine-1 ,4-dione, a known potential diketopiperazine group. The extract can be used in a compound for pharmaceutical composition, food product, food supplement or veterinary composition.

[0030] Accordingly, an extract produced by strain Lactobacilus plantarum L0001 isolated from kimchi was produced in MRS broth. From this finding, further development of the extract produced by L. plantarum can be expedited specifically targeted at novel antibiotics in this era of increasing antibiotic resistance.

[0031] Non-patent citations:

[0032] Stoyanova, L. G., Ustyugova, E. A., & Netrusov, A. I. (2012). Antibacterial metabolites of lactic acid bacteria: their diversity and properties. Applied Biochemistry and Microbiology, 48, 229-243.

[0033] Strom, K., Sjogren, J., Broberg, A., & Schnurer, J. (2002). Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo (L-Phe-L-Pro) and cyclo (L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid. Applied and environmental microbiology, 68(9), 4322-4327.

[0034] Yap, P. C., Ayuhan, N., Woon, J. J., Teh, C. S. J., Lee, V. S., Azman, A. S., ... & Lee, H. Y. (2021 ). Profiling of Potential Antibacterial Compounds of Lactic Acid Bacteria against Extremely Drug Resistant (XDR) Acinetobacter baumannii.

[0035] Molecules, 26(6), 1727.

[0036] 1 / 1

[0037] Sequence Listing Information :

[0038] DTD Vers ion : Vl_3

[0039] Fi le Name : Plantaruml . xml

[0040] Software Name : WI PO Sequence Software Vers ion : 2 . 3 . 0 Production Date : 2023 - 08 - 29 General Information :

[0041] Current application / Applicant file reference : BI P230022 Applicant name : Univers iti Malaya Applicant name / Language : en Inventor name : Lee Hai Yen Inventor name / Language : en

[0042] Invention title : Compos ition Agains t Acinetobacter Baumanni i ( en Sequence Total Quantity : 1

[0043] Sequences :

[0044] Sequence Number ( ID) : 1

[0045] Length : 1077

[0046] Molecule Type : RNA

[0047] Features Location / Quali f iers :

[0048] - source , 1 . . 1077

[0049] > mol_type , rRNA

[0050] > organism, Lactobacillus plantarum Re s idues : ctatacatgc aagtcgaacg aactctggta ttgattggtg cttgcatcat gatttacatt 60 tgagtgagtg gcgaactggt gagtaacacg tgggaaacct gcccagaagc gggggataac 120 acctggaaac agatgctaat accgcataac aacttggacc gcatggtccg agtttgaaag 180 atggcttcgg ctatcacttt tggatggtcc cgcggcgtat tagctagatg gtgaggtaac 240 ggctcaccat ggcaatgata cgtagccgac ctgagagggt aatcggccac attgggactg 300 agacacggcc caaactccta cgggaggcag cagtagggaa tcttccacaa tggacgaaag 3 60 tctgatggag caacgccgcg tgagtgaaga agggtttcgg ctcgtaaaac tctgttgtta 420 aagaagaaca tatctgagag taactgttca ggtattgacg gtatttaacc agaaagccac 480 ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggatttat 540 tgggcgtaaa gcgagcgcag gcggtttttt aagtctgatg tgaaagcctt cggctcaacc 600 gaagaagtgc atcggaaact gggaaacttg agtgcagaag aggacagtgg aactccatgt 660 gtagcggtga aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg ctgtctggtc 720 tgtaactgac gctgaggctc gaaagtatgg gtagcaaaca ggattagata ccctggtagt 780 ccataccgta aacgatgaat gctaagtgtt ggagggtttc cgcccttcag tgctgcagct 8 40 aacgcattaa gcattccgcc tggggagtac ggccgcaagg ctgaaactca aaggaattga 900 cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagctacgcg aagaacctta 960 ccaggtcttg acatactatg caaatctaag agattagacg ttcccttcgg ggacatggaa 1020 acaggtgtgc atggttgtcc tcacctccgg tcgggaaant tgggttaagt cccgcaa 1077

[0051] END

Claims

CLAIMS1 . A composition comprising: an extract from Lactobacillus plantarum L001 strain comprising the 16S rRNA sequence of SEQ ID NO 1 for use in treatment to inhibit Acinetobacter baumannii.

2. The composition for use according to claim 1 , wherein said composition is a pharmaceutical composition, a food product, a food supplement or a veterinary composition.

3. An extract from Lactobacillus plantarum L001 strain comprising the 16S rRNA sequence of SEQ ID NO 1 for use in treatment characterized in that the extract inhibits Acinetobacter baumannii.

4. A method to prepare an extract from Lactobacillus plantarum L001 strain comprising: isolating the Lactobacillus plantarum L001 culture from kimchi, wherein the Lactobacillus plantarum L0001 comprising the 16S rRNA sequence of SEQ ID NO 1 ; growing the culture in MRS broth; centrifuging the culture to obtain the supernatant; neutralizing the supernatant; freeze drying the supernatant; preparing a methanol extract of the supernatant; filtering the methanol extract to obtain the feed; evaporating the feed to obtain the extract.

5. The method of claim 4, wherein the step of isolating of strain from kimchi is performed, comprising: mixing kimchi with MRS broth; incubating the MRS broth; streaking onto the MRS agar; incubating and streaking the MRS agar to obtain pure culture of Lactobacillus plantarum L001 comprising the 16S rRNA sequence of SEQ ID NO 1 .

6. The method of claim 5, wherein the step of mixing kimchi with MRS broth is performed with kimchi weight to broth volume ratio of 1 :10.

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