Rheinheimera aquimaris strain from ocean sludge and application thereof

A Haimeria, marine technology, applied in the field of marine microorganisms, to achieve the effect of simple fermentation and culture conditions, simple and easy-to-control preparation process

Active Publication Date: 2015-09-23
CHINA UNIV OF PETROLEUM (EAST CHINA)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In recent years, the problem of bacterial drug resistance has become increasingly prominent. Traditional antibacterial drugs can easily cause bacterial drug resistance due to survival pressure. Therefore, antibacterial drugs obtained with traditional targets and screening methods cannot fundamentally solve bacterial infections. disease problem

Method used

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  • Rheinheimera aquimaris strain from ocean sludge and application thereof
  • Rheinheimera aquimaris strain from ocean sludge and application thereof
  • Rheinheimera aquimaris strain from ocean sludge and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Strains R. aquimaris Enrichment screen for QSI02.

[0021] 1.1 Sample collection: In September 2010, seawater sludge samples were collected from the Qingdao waters of the Yellow Sea and stored in sterile bottles.

[0022] 1.2 Enrichment culture: Take 2g of the sample and add it to 150mL denitrifying bacteria enrichment medium (KNO 3 2g, K 2 HPO 4 0.5g, MgSO 4 5g, sodium acetate 9.5g, H 2 O 1L, pH 7.0-7.4) in a 250mL Erlenmeyer flask, cultured in a shaker at 30°C and 150r / min, sampled every 3-4 days and tested for NO with Griess reagent 2 - changes. Choose NO 2 - The highly variable denitrifying bacteria were introduced into fresh medium. After repeated operations several times, take 1 mL of the bacterial suspension and place it in a test tube filled with 9 mL of sterile water, mix well to make a dilution of 10 -1 For the bacterial suspension, do 5 more dilutions in the same way.

[0023] 1.3 Isolation and purification of bacterial strains: Tak...

Embodiment 2

[0024] Example 2: Strains R. aquimaris QSI02 identification and preservation.

[0025] 2.1 Morphological characteristics: strains R. aquimaris QSI02 was milky white with neat edges on LB medium, and it was Gram-negative bacteria. Scanning results by transmission electron microscopy showed that ( figure 1 ), the bacteria are short rod-shaped, about 1.5-2.0 in size mu m wide, 7.0-9.0 mu m long, contains many flagella, and has transparent inclusions in the cell.

[0026] 2.2 Identification and preservation of strain 16S rDNA: 16S rDNA sequence analysis was performed by colony PCR method, primers were fD1: 27F 5'-AGAGTTTGATCMTGGCTCAG-3' and rD1: 1492R 5'-TACGGYTACCTTGTTACGACTT-3', the PCR reaction conditions were as follows: 94°C Denaturation for 3min, 30 cycles at 94°C for 30s, 55°C for 1min, 72°C for 90s; extension at 72°C for 10min. After PCR amplification, the PCR products were analyzed by 1.0% agarose gel electrophoresis. The PCR product purified by the gel recove...

Embodiment 3

[0032] Example 3: Strains R. aquimaris Quorum sensing inhibitory activity assay of QSI02 metabolites.

[0033] 3.1 Strains R. aquimaris Preparation of QSI02 metabolites: take activated marine bacteria R. aquimaris QSI02, inoculated into the fermentation medium for shaker culture, after the fermentation, centrifuged, concentrated in vacuum at low temperature to a paste, leached with methanol to obtain the methanol extract, and the methanol extract was mixed with 0.22 mu The m organic phase filter was used for biological activity detection after filtration.

[0034] 3.2 Detection of quorum sensing inhibition activity: coating 200 on LB plate mu l diluted 10 times C. violaceum For the fermentation broth, paste a filter paper sheet dipped in 0.5% DMSO aqueous solution in the center as a control, and paste filter paper sheets dipped in the extract of the bacteria to be tested around it in turn, and observe whether bacteria grow around the filter paper sheet but no purp...

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Abstract

The invention relates to a rheinheimera aquimaris strain QSI02 from ocean sludge and the application of the rheinheimera aquimaris strain QSI02 to the aspect of bacterial quorum sensing inhabiting and screening. The strain is preserved in China Center for Type Culture Collection on April 23rd, 2015, and the preservation serial number is CCTCC No: M2015245. The fact that the Rheinheimera type strain has the bacterial quorum sensing inhabiting activity is reported for the first time. A metabolite prepared through a fermentation liquor of the strain has the double inhibiting functions on chromobacterium violaceum and pseudomonas aeruginosa quorum sensing system and does not have the double inhibiting functions on generation of screening model chromobacterium violaceum CV026 within an effective concentration range. A strain extract can remarkably lower the yield of purpurin of chromobacterium violaceum and the swarming motility of pseudomonas aeruginosa and weaken the drug resistance of pathogenic bacterium, and therefore the rheinheimera aquimaris strain QSI02 has broad application prospects on the aspects of solving a bacterial drug resistance problem and researching and developing novel antibacterial agents.

Description

technical field [0001] The invention belongs to the field of marine microorganisms, and in particular relates to a strain of Rhineheimeria water derived from marine sludge ( Rheinheimera aquimaris ) The application of QSI02 strain and its metabolites in the screening of bacterial quorum sensing inhibition. Background technique [0002] Quorum sensing (QS) is a phenomenon of information exchange that widely exists among bacteria. Recent studies have found that bacteria can transmit and communicate with each other by producing, recognizing, and sensing one or more soluble chemical signaling molecules. Although a species of marine Vibrio fischeri ( Vibrio fischeri ), but the concept of bacterial quorum sensing was not proposed until 1994 (J. Bacteriol., 1994, 176:269-275). Many bacteria can synthesize and release a signal molecule called auto inducer (AI). When the number of bacteria in a specific environment increases sharply, the concentration of the signal molecule secre...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12Q1/02C12R1/01
CPCC12Q1/02C12N1/205C12R2001/01
Inventor 朱虎孙世伟李慧孙郊周万龙
Owner CHINA UNIV OF PETROLEUM (EAST CHINA)
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