Acinetobacter LDH6-2 and its application in degradation of tetracycline antibiotics

Abstract

The present invention discloses a strain of Acinetobacter spp. LDH6-2 and its application in degrading tetracycline antibiotics. The LDH6-2 strain was deposited in Guangdong Province Microbial Culture Collection Center on November 23, 2023, with a deposit number of GDMCC NO: 64034. The Acinetobacter spp. LDH6-2 of the present invention is different from other reported strains at the genomic level. It is a new type of Acinetobacter species, which has tetracycline antibiotic degradation ability and can significantly degrade tetracycline, minocycline and tigecycline within 24 h, and the degradation rate of the newly listed eravacycline reaches (75.8 ± 2.9)%, and has good application prospects in the degradation of residual tetracycline antibiotics in water and soil environments.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to an Acinetobacter sp. LDH6-2 and an application thereof in degrading tetracycline antibiotics. Background Art

[0002] Tetracycline antibiotics are a class of broad-spectrum antibiotics produced and synthesized by actinomycetes. Their common chemical structure is tetracene. These include first-generation (e.g., tetracycline), second-generation (e.g., minocycline), third-generation (e.g., tigecycline), and fourth-generation (e.g., eravacycline) drugs, commonly used to treat infections caused by Gram-negative and Gram-positive bacteria, mycoplasmas, chlamydiae, and rickettsiae in humans and animals. The widespread use and residual use of tetracycline antibiotics has severely damaged the environment on which humans depend and posed significant challenges to human and animal health. Therefore, addressing the issue of antibiotic residues is urgent.

[0003] At present, the degradation of residual antibiotics in the environment mainly includes non-biological degradation and biological degradation. Among them, the disadvantages of non-biological degradation are low degradation efficiency and very little bioavailable part after degradation. Biodegradation can be divided into plant degradation and microbial degradation. Among them, plant degradation is mostly concentrated on using a certain plant to repair a specific polluted environment, so it has great limitations. Microbial degradation has the advantages of high efficiency and simple operation in reducing antibiotics. However, the strains with high efficiency in degrading tetracycline antibiotics and their functions are currently limited. Chinese patent application CN111088192A discloses a Chryseobacterium jejuense DDW42, which degrades 48.4% of tetracycline after 16 hours. Chinese patent application CN115232758A discloses a single Acinetobacter (Acinetobacter soil) AC-tetX, which has a degradation rate of more than 69% for tetracycline, chlortetracycline, oxytetracycline and doxycycline after 4 days. Chinese patent application CN107058156A discloses an Acinetobacter calcoaceticus JZB42C005, which has a degradation rate of oxytetracycline of 67.8% after 5 days. Summary of the Invention

[0004] The present invention provides an Acinetobacter sp. LDH6-2 strain having the ability to degrade tetracycline antibiotics.

[0005] The inventors screened a strain with tetracycline degradation ability from chicken manure samples by adding tigecycline to the culture medium. After whole genome sequencing and digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analysis, the strain was most closely related to Acinetobacter variabilis, but below the intraspecific threshold, indicating that it is a new type of Acinetobacter species. The strain was deposited in the Guangdong Provincial Microbial Culture Collection on November 23, 2023. The deposit address is 5th Floor, Dayuan Laboratory Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the deposit number is GDMCC NO: 64034.

[0006] The present invention also provides a method for culturing the above-mentioned Acinetobacter LDH6-2, which specifically comprises: inoculating Acinetobacter LDH6-2 into LB agar or LB broth culture medium, culturing at a pH of 5 to 11, a temperature of 20° C. to 40° C., and a sodium chloride concentration of 0% to 4%, for culturing.

[0007] Furthermore, the present invention also provides the use of the above-mentioned Acinetobacter LDH6-2 in the degradation of tetracycline antibiotics.

[0008] In the above application, the specific application method is: inoculating Acinetobacter LDH6-2 into a solution containing tetracycline antibiotics to degrade the tetracycline antibiotics.

[0009] In the above application, the tetracycline antibiotic is tetracycline (TC), minocycline (MIN), tigecycline (TGC) or eravacycline (ERA).

[0010] The present invention discovered a strain of Acinetobacter, which was identified as a new Acinetobacter species based on dDDH and ANI analysis, namely Acinetobacter LDH6-2, which has the ability to degrade tetracycline antibiotics and can significantly degrade tetracycline, minocycline, tigecycline and eravacycline within 24 hours, with a degradation rate of eravacycline as high as (75.8±2.9)%. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A genome-wide phylogenetic tree based on single nucleotide polymorphisms (SNPs).

[0012] Figure 2 Figure 1 is the result of the punching experiment, where A is the degradation result of tetracycline, B is the degradation result of minocycline, C is the degradation result of tigecycline, D is the statistical analysis of the diameter of the tetracycline inhibition zone, E is the statistical analysis of the diameter of the minocycline inhibition zone, and F is the statistical analysis of the diameter of the tigecycline inhibition zone.

[0013] Figure 3This is a diagram showing the liquid chromatography-tandem mass spectrometry (LC-MS / MS) determination results of eravacycline. DETAILED DESCRIPTION

[0014] The present invention will be further described below in conjunction with specific embodiments and accompanying drawings.

[0015] In the following examples, the culture medium used was composed as follows:

[0016] LB agar medium: Each liter contains 10.0 g peptone, 5.0 g yeast extract powder, 5.0 g sodium chloride, 1.0 g glucose and 15.0 g agar, and is sterilized by autoclaving at 121°C for 20 min.

[0017] LB broth medium: Each liter contains 10.0 g peptone, 5.0 g yeast extract powder, 5.0 g sodium chloride and 1.0 g glucose, and is sterilized by autoclaving at 121°C for 20 min.

[0018] MH agar medium: Each liter contains 300.0 g of beef (from which extract powder is extracted), 1.5 g of soluble starch, 17.5 g of casein hydrolyzate and 17.0 g of agar, and is sterilized by high pressure at 121°C for 20 minutes.

[0019] MH broth medium: Each liter contains 300.0g beef (from which extract powder is extracted), 1.5g soluble starch and 17.5g casein hydrolyzate, and is sterilized by high pressure at 121℃ for 20min.

[0020] 1× M9 complete medium: Contains 11.28 g of M9 minimal salts, 0.24 g of magnesium sulfate, and 11.1 mg of calcium chloride per liter. Autoclave at 121°C for 20 min. Subsequently, add 0.22 μM-filtered glucose solution (final concentration 9 g / L), thiamine solution (final concentration 100 mg / L), and leucine solution (final concentration 100 mg / L).

[0021] Example 1

[0022] 1. Sample Collection and Strain Isolation

[0023] In October 2021, chicken manure samples were collected from a chicken farm in Yangzhou, Jiangsu Province. 0.1 g of fresh sample was vortex-mixed with 1 mL of 0.9% saline. A sterile cotton swab was used to apply the suspension and streak onto LB agar containing 4 μg / mL tigecycline. The mixture was incubated inverted at 35°C for 20 hours. A single colony was picked using a 1 μL disposable inoculation loop and purified again under the same conditions to obtain the isolate LDH6-2.

[0024] 2. Whole Genome Sequencing and 16S rDNA Analysis

[0025] The LDH6-2 strain was sent to Annoroad Gene Technology (Beijing) Co., Ltd. for genome extraction and high-throughput sequencing using the Illumina NovaSeq 6000 platform. The complete genome sequence of the LDH6-2 strain was obtained using SPAdes version 3.13.1 software. Its 16S rDNA is shown in SEQ ID No. 1. Using the National Center for Biotechnology Information (NCBI) database, a blastn comparison of the 16S rDNA of the LDH6-2 strain with that of the model strain revealed that the strain had the highest similarity with the source strain, Acinetobacter rudis, at approximately 97.19%.

[0026] 3. Bacterial species identification based on dDDH and ANI analysis

[0027] Analysis by dDDH (formula d4, https: / / tygs.dsmz.de / user_requests / new) showed that the strain had the highest similarity with Acinetobacter variegatus (24.4%), which was far below the intraspecific threshold (>70%). ANI analysis of the whole genome sequence of the LDH6-2 strain was performed using IPGA version 1.09, and a phylogenetic tree was constructed based on SNPs. The results also showed that the strain was most closely related to Acinetobacter variegatus (80.8%), but below the intraspecific threshold (>95%). In summary, the LDH6-2 strain is a new species of Acinetobacter. For details of the phylogenetic tree, see Figure 1 .

[0028] 4. Determination of Physiological and Biochemical Characteristics

[0029] The physiological and biochemical indicators to be tested mainly include the following aspects:

[0030] (1) Morphological observation. The LDH6-2 strain was streaked onto a blank LB agar medium in three zones and incubated upside down at 35°C for 24 h. The single colonies were round, with smooth, full surfaces and regular edges.

[0031] (2) Maximum growth temperature. A single colony of the LDH6-2 strain was picked and streaked onto a blank LB agar plate. The plate was then inverted and cultured at 20°C, 25°C, 30°C, 35°C, 37°C, 40°C, and 41°C for 24 h. The maximum growth temperature of the strain was 40°C.

[0032] (3) pH and sodium chloride. Pipette 10 μL of fresh bacterial suspension of the logarithmic phase LDH6-2 strain into 4 mL of LB broth with a pH of 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a sodium chloride concentration of 0%, 1%, 2%, 3%, 4%, or 5%. Incubate at 35°C and 200 rpm for 24 h. The pH and sodium chloride concentrations suitable for the growth of the LDH6-2 strain are 5-11 and 0%-4%, respectively.

[0033] (4) Hemolysis. A single colony of the LDH6-2 strain was picked and streaked onto LB agar medium containing 5% sheep blood. The culture was inverted at 35°C for 24 h. No hemolytic zone was produced.

[0034] (5) Motility. A single colony of the LDH6-2 strain was picked and inoculated onto a LB semisolid agar plate containing 0.4% agar. The plate was inverted and cultured at 35°C for 24 h. No proliferation or growth was observed.

[0035] (6) Using a non-fermenting bacterial identification tube (purchased from Guangdong Huankai Microbial Technology Co., Ltd.), the LDH6-2 strain was subjected to oxidase, glucose oxidation and fermentation (OF) tests, DNA, citrate, maltose, arginine dihydrolase, mannitol, acetamide, nitrate reduction, and xylose assays according to the instructions. Detailed physiological and biochemical identification results are shown in Table 1.

[0036] Table 1 Physiological and biochemical characteristics of Acinetobacter LDH6-2

[0037]

[0038]

[0039] 5. Evaluation of the Degradation Activity of Acinetobacter LDH6-2 Using the Agar Well Degradation Model

[0040] (1) Preparation of MH agar plates. Use an electric pipette to draw up 20 mL of autoclaved MH agar medium and transfer it to a 9 cm diameter disposable sterile plate. After the agar medium solidifies, evenly spread 100 μL of logarithmic-phase Escherichia coli (sensitive to tetracycline antibiotics) ATCC 25922 on the agar surface. Use a 4 mm diameter sterile hole punch to punch three holes, and add 10 μL of MH agar medium to each well to seal the bottom. Set aside.

[0041] (2) Co-culture of LDH6-2 strain with tetracycline antibiotics. Use a 10 μL disposable inoculation loop to scrape a mung bean-sized piece of fresh LDH6-2 bacterial moss, vortex mix it with 1 mL of tetracycline (64 μg / mL), minocycline (80 μg / mL) and tigecycline (10 μg / mL) MH broth medium, and incubate it at 35°C for 24 hours. Three parallel cultures were set up for each group, and MH broth medium containing the corresponding antibiotics and blank MH broth medium were used as positive and negative controls.

[0042] (3) Determination of inhibition zone. The co-culture after incubation was centrifuged at 6000 rpm for 3 minutes, 15 μL was drawn and added to the prepared agar well, and incubated at 40 ° C for 16 hours. The diameter of the inhibition zone was measured using a vernier caliper, and the unpaired T-test method was used for statistical analysis. Compared with the tetracycline, minocycline and tigecycline control groups, the diameter of the inhibition zone of LDH6-2 strain after incubation with each antibiotic was significantly reduced, indicating that the strain can significantly degrade tetracycline (P = 0.0009), minocycline (P < 0.0001) and tigecycline (P < 0.0001). Specific measurement results are shown in Figure 2 .

[0043] 6. LC-MS / MS Determination of Eravacycline Degradation

[0044] A single colony of Acinetobacter baumannii (LDH6-2) was transferred to 1 mL of 1×M9 complete medium and incubated at 35°C, 200 rpm, for 8 h. A 40 μL aliquot was then added to M9 medium containing 2 μg / mL eravacycline (final volume 4 mL) and incubated at 35°C, 200 rpm, in the dark for 24 h. Subsequently, the cells were centrifuged at 12,000 rpm for 2 min and filtered through a 0.22 μm filter. The supernatant was diluted 10-fold or 100-fold, and eravacycline was quantitatively determined by LC-MS / MS using a QTRAP 5500 liquid chromatography-mass spectrometry system. Acinetobacter baumannii ATCC 19606 was used as a control group, with six replicates per group. The results showed that the degradation rate of eravacycline by LDH6-2 reached 75.8 ± 2.9% within 24 h.

Claims

1. Acinetobacter ( Acinetobacter sp.)LDH6-2, deposited with GDMCC NO: 64034.

2. The method for culturing Acinetobacter spp. LDH6-2 according to claim 1, comprising inoculating Acinetobacter spp. LDH6-2 into LB agar or LB broth medium at a pH of 5 to 11, a temperature of 20°C to 40°C, and a sodium chloride concentration of 0% to 4%, for culturing.

3. The use of Acinetobacter LDH6-2 according to claim 1 in the degradation of tetracycline antibiotics, characterized in that The tetracycline antibiotics are tetracycline, minocycline, tigecycline or eravacycline.

4. The use according to claim 3, characterized in that The specific application method is: inoculating Acinetobacter LDH6-2 into a solution containing tetracycline antibiotics to degrade the tetracycline antibiotics.

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