Pseudomonas sp. and application thereof in degrading abacavir

Abstract

The application discloses an abacavir degrading bacterium. The degrading bacterium is obtained from activated sludge in an aerobic tank of a sewage treatment plant in Guangzhou, Guangdong, and is obtained through artificial domestication culture and separation and purification. The name of the bacterium is Pseudomonas sp. abacavir-1, and the bacterium is preserved in the Guangdong Microbial Culture Collection Center of the Guangdong Institute of Microbiology, located at No. 59, Building 5, Guangzhou Martyrs' Road, Guangzhou, on September 23, 2022, and the preservation number is GDMCC NO: 62829. The strain of the application has the ability to degrade single abacavir under oligotrophic conditions, can be used for degrading abacavir in the environment or a sewage treatment plant, and provides a new germplasm resource for abacavir treatment.

Description

Technical Field

[0001] This invention belongs to the field of biological treatment technology for environmental organic pollutants, and specifically relates to a strain of Pseudomonas and its application in the degradation of abacavir. Background Technology

[0002] Abacavir is a synthetic antiviral drug that works by inhibiting the growth of pathogens and is widely used in the treatment of HIV infection. Due to the widespread use of azithromycin at concentrations of ng / L in HIV-infected individuals (Paredes AJ, Volpe-Zanutto F, Vora LK, Tekko IA, Permana AD, Picco CJ, McCarthy HO, Donnelly RF. Systemic delivery often ofovir alafenamide using dissolving and implantable microneedlepatches. Mater Today Bio. 2022, 13:100217.) and incomplete absorption and metabolism (Ncube S, Madikizela LM, Chimuka L, Nindi MM. Environmental fate and ecotoxicological effects of antiretrovirals: a current global status and future perspectives. Water Res. 2018, 145:231–247.), and the limited removal efficiency of traditional wastewater treatment processes, azithromycin at concentrations of ng / L has been widely detected in wastewater treated by wastewater treatment plants and in the surrounding receiving environment (Aminot Y, Litrico X, Chambolle M, Arnaud). C, Pardon P, Budzindki H. Development and application of a multi-residue method for the determination of 53pharmaceuticals in water, sediment, and suspended solids using liquidchromatography-tandem mass spectrometry. Anal Bioanal Chem. 2015, 407(28):8585-8604.).Currently, limited research indicates that abacavir poses a potential threat to aquatic ecosystems and can accumulate in aquatic organisms (Fernández LP, Brasca R, Repetti MR, Attademo AM, Peltzer PM, Lajmanovich RC, Culzoni MJ. Bioaccumulation of abacavir and efavirenz in Rhinella arenarumtadpoles after exposure to environmentally relevant. Chemosphere. 2022, 301:134631.). With the increasing number of HIV / AIDS patients, the detection rate and concentration of abacavir in the environment are also expected to rise. Therefore, seeking efficient technologies to prevent and control abacavir pollution is of great significance to human health and ecological environmental safety.

[0003] The control of contamination from antiviral drugs has only recently gained attention, with research on the degradation and removal of abacavir being particularly scarce. Although wastewater treatment plants have some removal effect on abacavir, widespread detection in the surrounding receiving environment indicates a lack of a targeted method for its removal. Microbial degradation is currently recognized as an environmentally friendly, low-cost, and highly efficient method for removing organic matter from the environment. However, there are currently no reports, either domestically or internationally, on the degradation of abacavir by bacterial strains, highlighting the urgent need for further research. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of Pseudomonas.

[0005] Another object of the present invention is to provide the application of the above-mentioned Pseudomonas bacteria in the degradation of abacavir.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A strain of Pseudomonas sp. was obtained from activated sludge in a wastewater treatment plant in Guangzhou, Guangdong Province, through artificial enrichment culture, isolation and purification, and was named Pseudomonas sp. abacavir-1.

[0008] The aforementioned Pseudomonas bacteria, with accession number GDMCC No: 62829, was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on September 23, 2022. The depository address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The colonies of the aforementioned Pseudomonas bacteria on LB solid plates are round, milky white, with a raised, glossy surface and a viscous texture.

[0010] The aforementioned Pseudomonas bacteria have the ability to degrade abacavir.

[0011] The nucleotide sequence of the 16S rDNA of the aforementioned Pseudomonas is shown in SEQ ID NO: 1.

[0012] A method for degrading abacavir includes the following steps:

[0013] (1) Take Pseudomonas aeruginosa and inoculate it into the culture medium to activate it, and culture it to the logarithmic growth phase;

[0014] (2) Add Pseudomonas aeruginosa cultured to the logarithmic growth phase to the system containing abacavir to degrade the abacavir in the system.

[0015] The culture medium is LB medium.

[0016] Abacavir was added to the culture medium to a concentration of 4 mg / L.

[0017] The application of the aforementioned Pseudomonas in the degradation of abacavir.

[0018] The present invention has the following advantages and effects compared with the prior art:

[0019] 1. This invention has the ability to degrade abacavir. In Minimal Salt Medium (MSM) culture medium, with an initial concentration of 1 mg / L of abacavir, more than 80% of abacavir can be degraded after 228 hours.

[0020] 2. The strain of this invention can degrade the antiviral drug abacavir to its half-life in a short time, providing a new germplasm resource for the biological treatment of wastewater containing abacavir. Attached Figure Description

[0021] Figure 1 This is a graph showing the test results of the activated sludge degradation experiment in Example 1.

[0022] Figure 2 This is a colony diagram of Pseudomonas abacavir-1 grown on LB medium.

[0023] Figure 3 This is a scanning electron microscope image of Pseudomonas abacavir-1 in the field.

[0024] Figure 4 This is a graph showing the degradation effect of abacavir-1 by Pseudomonas abacavir in MSM medium. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0026] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0027] MSM medium formulation:

[0028] 1L mini-Q water, 9g K2HPO4·3H2O, 3g KH2PO4, 0.5g NaCl, 1g NH4Cl, 0.25g MgSO4·7H2O, 0.014g anhydrous CaCl2, 1mL trace element solution (0.636g / L NaHCO3, 0.1g / L ZnSO4·7H2O, 0.05g / L CuSO4·5H2O, 0.01g / L (NH4)6Mo7O) 24 ·4H2O, 0.25g / L CoCl2·6H2O, 0.25g / L FeSO4·7H2O, 0.15g / L MnCl2·4H2O), 1mL vitamin stock solution (20mg / L biotin, 20mg / L floic acid, 100mg / L pyridoxine hydrochloride, 50mg / L riboflavin, 50mg / L thiamine, 50mg / L nicotinic acid, 50mg / L pantothenic acid, 1mg / L vitamin B12, 50mg / L p-aminobenzoic acid, 50mg / L thioctic acid).

[0029] Example 1

[0030] (1) Activated sludge was collected from the aerobic tank of a wastewater treatment plant in Guangzhou, Guangdong Province. After mixing, 60 mL of the sludge was added to a 1 L Erlenmeyer flask containing 240 mL of MSM medium. Five antiviral drugs (lamivudine, terbivudine, abacavir, zidovudine, and nevirapine) were added as a mixed carbon source for co-culture. The initial concentration of each antiviral drug in the culture system was 1 mg / L. The flask was sealed with breathable sealing film, wrapped with aluminum foil to protect it from light, and then cultured in a shaker at 30℃ and 150 rpm. Samples were taken every 12 h after the start of culture, and the degradation level of the drugs in the samples was detected by LC-MS / MS. After 96 h of culture, the results are as follows: Figure 1 As shown in Table 1, activated sludge was found to have a good degradation effect on abacavir.

[0031] Table 1. Degradation of different antiviral drugs by activated sludge in a mixed carbon source medium.

[0032]

[0033]

[0034] Note: To make the data more intuitive, Ct values ​​below the instrument's lowest detection line are treated as 0 in the charts, which does not represent an actual concentration of 0; Ct / CO greater than 100% may be due to experimental errors such as sludge adsorption or uneven mixing of samples before sampling.

[0035] (2) Take 30 mL of the culture medium obtained by shaking after culturing in step (1), add it to a 1 L conical flask containing 120 mL of MSM medium, and add a higher concentration of abacavir as a single carbon source for acclimatization culture. Shake the medium well, seal it with a breathable sealing film, wrap it with aluminum foil to protect it from light, and then culture it in a shaker at 30℃ and 150 rpm. Take samples regularly and use HPLC to detect the degradation effect of the drug in the samples.

[0036] (3) After the concentration of abacavir in the culture medium is lower than 80%, repeat step (2) and repeatedly acclimatize multiple times until the 10th generation, to obtain an acclimatization solution enriched with strains capable of degrading abacavir. At this time, add abacavir to the culture medium to a concentration of 4 mg / L.

[0037] Example 2

[0038] Concentrate 3 mL of acclimatization solution to 1 mL, then perform serial dilutions and spread the solution onto sterile, cooled LB abacavir medium containing 4 mg / L abacavir (dilution gradient: 1, 1 × 10⁻⁶). -1 1×10 -2 1×10 -3 1×10 -4 1×10 -5 1×10 -6 1×10 -7The culture was incubated at 30℃ for 24 hours. Single colonies of different colors, shapes, and odors were selected and purified twice by streaking on sterile LB solid medium. Single colonies were then inoculated into LB liquid medium and incubated for 12 hours. 30 mL of the bacterial culture was then inoculated into a 250 mL Erlenmeyer flask containing 120 mL of MSM medium, and abacavir was added to achieve an initial concentration of 1 mg / L. Samples were taken every 24 hours to detect abacavir residues in the MSM medium. Two single bacteria, abacavir-1 and abacavir-2, capable of degrading abacavir were screened. Abacavir-1 showed better degradation of abacavir. The bacterial strains were preserved using glycerol broth (1 mL of bacterial culture was mixed with 0.5 mL of 60% glycerol in 2 mL of sterile preservation tubes, and then subjected to gradient freezing, with the final strain stored at -80℃). The remaining bacterial strains were sent for sequencing to identify the strains.

[0039] After DNA extraction from the bacterial strain, PCR amplification was performed using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGACTTAACCCCAATCGC-3'). The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 54℃ annealing for 30 s, and 72℃ extension for 1.5 min for a total of 35 cycles. After a final extension at 72℃ for 10 min, the sample was stored at 4℃ and sent to BGI for sequencing. BLAST homology comparison of the sequencing results on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ) revealed that the 16S rRNA sequencing results of the abacavir-degrading strain abacavir-1 were 99% identical to those of Pseudomonas sp. The colonies were observed to be round, milky white, smooth, relatively moist, and sticky, with a slightly convex center. The colony photograph is shown below. Figure 2 As shown, the SEM image is as follows Figure 3 As shown; combined with the 16S rRNA results, abacavir-1 was preliminarily identified as Pseudomonas sp.

[0040] DNA from strain abacavir-1 was further extracted using the Tiangen Bacterial DNA Extraction Kit and sent to Shanghai Meiji Biotechnology Co., Ltd. for whole-genome sequencing. The alignment results are shown in Table 1 (sequences were aligned using SSU-Align software). Among the sequences, abacavir-1 showed the highest similarity (99.413%) to strain Pseudomonas_knackmussii. Because its whole-genome is not completely similar to Pseudomonas_knackmussii and it possesses the ability to degrade abacavir, it was named *Pseudomonas sp.* abacavir-1. Its preservation information is as follows: Preservation Institution: Guangdong Provincial Microbial Culture Collection Center (GDMCC); Preservation Date: September 23, 2022; Preservation Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Institute of Microbiology; Preservation Number: GDMCC NO: 62829.

[0041] Table 2. Details of whole genome alignment of strain abacavir-1

[0042] Sample Name Hit ID Hit species Identity(%) Coverage(%) Evalue Score Pseudomonas_abacavir_1 GCF_000689415.1 Pseudomonas_knackmussii 99.413 99.93 0 2778 Pseudomonas_abacavir_1 GCF_900187975.1 Pseudomonas_delhiensis 99.217 99.93 0 2761 Pseudomonas_abacavir_1 GCF_000349845.1 Pseudomonas_sp000349845 98.89 99.93 0 2734 Pseudomonas_abacavir_1 GCF_009189165.1 Pseudomonas_sp009189165 98.695 99.93 0 2717 Pseudomonas_abacavir_1 GCF_000955805.1 Pseudomonas_sp000955805 98.629 99.93 0 2712 Pseudomonas_abacavir_1 GCF_002091755.1 Pseudomonas_nitroreducens 98.433 99.93 0 2695 Pseudomonas_abacavir_1 GCF_002304225.1 Pseudomonas_E_sp002304225 97.327 99.8 0 2601 Pseudomonas_abacavir_1 GCF_000017205.1 Pseudomonas_aeruginosa_A 97.135 99.67 0 2584 Pseudomonas_abacavir_1 GCF_000467105.1 Pseudomonas_E_alcaligenes 97.068 99.74 0 2579 Pseudomonas_abacavir_1 GCF_014490745.1 Pseudomonas_E_alcaligenes_C 96.873 99.74 0 2562 Pseudomonas_abacavir_1 GCF_000341615.1 Pseudomonas_A_stutzeri_G 96.678 99.74 0 2545 Pseudomonas_abacavir_1 GCF_900100495.1 Pseudomonas_E_benzenivorans 96.54 99.93 0 2534 Pseudomonas_abacavir_1 GCF_0135 ​ 96.489 99.54 0 2529 ​ ​ ​ 96.415 99.8 0 2523 ​ ​ ​ 96.359 99.54 0 2519 Pseudomonas_abacavir_1 GCF_004801855.1 Pseudomonas_K_sp004801855 96.352 99.74 0 2518 Pseudomonas_abacavir_1 GCF_003052585.1 Pseudomonas_E_mangrovi 96.287 99.74 0 2512 Pseudomonas_abacavir_1 GCF_015291885.1 Pseudomonas_A_stutzeri_AC 96.287 99.74 0 2512 Pseudomonas_abacavir_1 GCF_004521985.1 Pseudomonas_E_khazarica 96.224 99.67 0 2507 Pseudomonas_abacavir_1 GCF_900114065.1 Pseudomonas_A_kunmingensis 96.089 99.8 0 2495

[0043] Example 3: Identification of the degradation effect of Pseudomonas abacavir-1 on abacavir

[0044] Add 450 mL and 500 mL of MSM culture medium to 1 L Erlenmeyer flasks respectively, seal with breathable sealing film, sterilize at high temperature, and cool for later use. The experiment was divided into three groups:

[0045] (1) In the experimental group, a single colony of purified Pseudomonas abacavir-1 from LB solid medium was picked up using a disposable inoculation loop and inoculated into 60 mL of LB liquid medium. Abacavir was added to bring the initial concentration to 4 mg / L. OD was measured by sampling. 600 The bacteria were found to have entered the logarithmic growth phase, OD 600 When the concentration is around 1.3, centrifuge 50 mL of bacterial culture (8000 rpm, 2 min) to obtain the supernatant, then resuspend and wash with 50 mL of MSM culture medium. Repeat the washing twice. Inoculate 50 mL of the resuspended solution into 450 mL of MSM culture medium, add abacavir to make the initial concentration 1 mg / L, and mix well.

[0046] (2) Degradation effect comparison group: Following the method of the experimental group, abacavir-2 strain was inoculated into a consistent culture system and mixed evenly;

[0047] (3) Aseptic control group: Abacavir was added to an Erlenmeyer flask containing 500 mL of MSM medium to make the initial concentration 1 mg / L, and no bacterial strain was inoculated.

[0048] All conical flasks were wrapped in aluminum foil to protect them from light and then placed in a shaker for incubation (150 rpm, 30℃). Three replicates were taken from both the experimental and control groups. The residual amount of abacavir in the culture medium was measured, and the degradation ability of *Pseudomonas abacavir-1* strain on abacavir was observed and compared. The results are as follows: Figure 4 As shown, the degradation efficiency of abacavir in the abacavir-2 experimental system was only 26.2% after 228 hours of experimentation. In contrast, the degradation efficiency of abacavir in the Pseudomonas abacavir-1 experimental system was greater than 80%, and the degradation half-life of abacavir was 81.5 hours.

[0049] Table 3 Kinetic parameters during the degradation process

[0050]

[0051] This example fully demonstrates that Pseudomonas abacavir-1 has a good degradation ability for 1 mg / L abacavir under oligotrophic conditions.

[0052] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas, characterized by: The specimen, named Pseudomonas sp. abacavir-1, with accession number GDMCC No: 62829, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 23, 2022. The depository address is the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The Pseudomonas bacillus according to claim 1, characterized in that: The colonies of the aforementioned Pseudomonas bacteria on LB solid plates are round, milky white, with a raised, glossy surface and a viscous texture.

3. The Pseudomonas bacillus according to claim 1, characterized in that: The aforementioned Pseudomonas bacteria have the ability to degrade abacavir.

4. The Pseudomonas bacillus according to claim 1, characterized in that: The nucleotide sequence of the 16S rDNA of the aforementioned Pseudomonas is shown in SEQ ID NO:

1.

5. The use of the Pseudomonas aeruginosa according to any one of claims 1 to 4 in the degradation of abacavir.

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