一种假单胞菌对土壤中土霉素的降解

doi:10.1016/j.jia.2024.11.007

摘要/Abstract

摘要：

土霉素（OTC）在畜牧业中被广泛使用并以不同形式进入土壤，对环境造成严重危害。前人研究表明，假单胞菌属可能具有降解土壤中抗生素的能力；同时，抗生素的初始浓度、降解温度等对抗生素降解菌的降解效率有显著影响，但是关于环境因素对假单胞菌降解效率的影响鲜有报道。本实验中，我们研究了不同OTC初始浓度、降解温度和土壤灭菌处理对假单胞菌T4降解效率的影响，还重点研究了加入T4菌后OTC的微生物降解途径、降解过程中抗性基因（ARGs）以及微生物群落的变化。结果表明，在未灭菌的土壤中，OTC初始浓度为2.5 mg kg^-1、降解温度为30℃时，T4菌对OTC的降解效果最好，63天后OTC的降解率达到69.53%。加入T4菌后OTC的降解途径包括脱水、去甲基化、脱胺化、羟基化、氧化和环裂解。拟杆菌门、变形菌门和酸杆菌门对土壤中OTC的生物降解起关键作用。同时发现tet(G)在13种常见四环素ARGs中检出频率最高。本研究中观察到的微生物群落变化可为土壤中OTC的生物降解提供新的思路。

Abstract:

Oxytetracycline (OTC) is used extensively in animal husbandry and enters the soil in different forms, causing severe environmental pollution. Previous studies have shown that the genus Pseudomonas can potentially degrade antibiotics in the soil environment. Environmental conditions, such as the initial concentration of antibiotics, incubation temperature and others, have significant impacts on the activity of antibiotic-degrading bacteria. However, few reports have clarified the environmental impacts on the effectiveness of Pseudomonas spp. In the present study, we investigated the effects of different initial concentrations of OTC and incubation temperatures, as well as soil sterilization, on OTC degradation by Pseudomonas strain T4. We also focused on the microbial degradation pathways of OTC, and variations in both antibiotic resistance genes (ARGs) and microbial communities with T4 functioning under optimal conditions. The results showed that the most effective degradation occurred under an initial OTC concentration of 2.5 mg kg^–1 at 30°C in unsterilized soil spiked with T4. These conditions yielded an OTC degradation rate of 69.53% within 63 days. The putative degradation pathways of OTC in the presence of T4 included dehydration, demethylation, deamination, hydroxylation, oxidation and ring opening. Bacteroidetes, Proteobacteria and Acidobacteria played key roles in the biodegradation of OTC with T4 in the soil. The results also showed that tet(G) was the most frequently detected ARGs among the 13 common tetracycline ARGs that were investigated. The bacterial community shift observed in this study may provide new insights into the microbial degradation of OTC in soil.

Key words: oxytetracycline , ARGs , Pseudomonas , biodegradation , soil

. 一种假单胞菌对土壤中土霉素的降解[J]. Journal of Integrative Agriculture, 2025, 24(5): 2002-2014.

Xueqi Guo, Weining Qi, Yao Feng, Zhaojun Li. Degradation of oxytetracycline in soil by a Pseudomonas strain[J]. Journal of Integrative Agriculture, 2025, 24(5): 2002-2014.

参考文献

Cadena M, Durso L M, Miller D N, Waldrip H M, Castleberry B L, Drijber R A, Wortmann C. 2018. Tetracycline and sulfonamide antibiotic resistance genes in soils from nebraska organic farming operations. Frontiers in Microbiology, 9, 1–10.

Cai M M, Ma S T, Hu R Q, Tomberlin J K, Yu C, Huang Y P, Zhan S, Li W, Zheng L Y, Yu Z N, Zhang J B. 2018. Systematic characterization and proposed pathway of tetracycline degradation in solid waste treatment by Hermetia illucens with intestinal microbiota. Environmental Pollution, 242, 634–642.

Cao J, Ji D G, Wang C. 2015. Interaction between earthworms and arbuscular mycorrhizal fungi on the degradation of oxytetracycline in soils. Soil Biology and Biochemistry, 90, 283–292.

Cao L J, Zhang J Y, Zhao R X, Deng Y, Liu J, Fu W, Lei Y, Zhang T, Li X, Li B. 2019. Genomic characterization, kinetics, and pathways of sulfamethazine biodegradation by Paenarthrobacter sp. A01. Environment International, 131, 104961.

Cao X, Zhao W, Zhang H, Lin J, Hu J, Lou Y, Wang H, Yang Q, Pan H, Zhuge Y. 2022. Individual and combined contamination of oxytetracycline and cadmium inhibited nitrification by inhibiting ammonia oxidizers. Frontiers in Microbiology, 13, 1–15.

Chen G X, He W W, Wang Y, Zou Y D, Liang J B, Liao X D, Wu Y B. 2014. Effect of different oxytetracycline addition methods on its degradation behavior in soil. Science of the Total Environment, 479–480, 241–246.

Chen J F, Ke Y C, Zhu Y, Chen X L, Xie S G. 2023. Deciphering of sulfonamide biodegradation mechanism in wetland sediments: From microbial community and individual populations to pathway and functional genes. Water Research, 240, 120132.

Chen Y S, Wang Z J, Shen Z J, Ou Z L, Xu D C, Yuan Z X, Zhou S B. 2017. Effects of oxytetracycline on growth and chlorophyll fluorescence in rape (Brassica campestris L.). Polish Journal of Environmental Studies, 26, 995–1001.

Cheng Q P, Cheung Y C, Liu C Y, Xiao Q J, Sun B, Zhou J H, Chan E W C, Zhang R, Chen S. 2021. Structural and mechanistic basis of the high catalytic activity of monooxygenase Tet(X4) on tigecycline. BMC Biology, 19, 262.

Chessa L, Pusino A, Garau G, Mangia N P, Pinna M V. 2016. Soil microbial response to tetracycline in two different soils amended with cow manure. Environmental Science and Pollution Research, 23, 5807–5817.

Chopra I, Roberts M. 2001. Tetracycline antibiotics: Mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and Molecular Biology Reviews, 65, 232–260

Cunningham C J, Kuyukina M S, Ivshina I B, Konev A I, Peshkur T A, Knapp C W. 2020. Potential risks of antibiotic resistant bacteria and genes in bioremediation of petroleum hydrocarbon contaminated soils. Environmental Science (Processes and Impacts), 22, 1110–1124.

Cycoń M, Żmijowska A, Piotrowska-Seget Z. 2014. Enhancement of deltamethrin degradation by soil bioaugmentation with two different strains of Serratia marcescens. International Journal of Environmental Science and Technology, 11, 1305–1316.

Elder F C T, Pascoe B, Wells S, Sheppard S K, Snape J, Gaze W H, Feil E J, Kasprzyk-Hordern B. 2022. Stereoselective metabolism of chloramphenicol by bacteria isolated from wastewater, and the importance of stereochemistry in environmental risk assessments for antibiotics. Water Research, 217, 118415.

Fang H, Han L X, Cui Y L, Xue Y F, Cai L, Yu Y L. 2016. Changes in soil microbial community structure and function associated with degradation and resistance of carbendazim and chlortetracycline during repeated treatments. Science of the Total Environment, 572, 1203–1212.

Fiaz A, Zhu D C, Sun J Z. 2021. Environmental fate of tetracycline antibiotics: Degradation pathway mechanisms, challenges, and perspectives. Environmental Sciences Europe, 33, 64.

Gasparrini A J, Markley J L, Kumar H, Wang B, Fang L T, Irum S, Symister C T, Wallace M, Burnham C A D, Andleeb S, Tolia N H, Wencewicz T A, Dantas G. 2020. Tetracycline-inactivating enzymes from environmental, human commensal, and pathogenic bacteria cause broad-spectrum tetracycline resistance. Communications Biology, 3, 241.

Guo X H, Xie C Y, Wang L J, Li Q F, Wang Y. 2019. Biodegradation of persistent environmental pollutants by Arthrobacter sp. Environmental Science and Pollution Research, 26, 8429–8443.

Halling-Sørensen B, Lykkeberg A, Ingerslev F, Blackwell P, Tjørnelund J. 2003. Characterisation of the abiotic degradation pathways of oxytetracyclines in soil interstitial water using LC-MS-MS. Chemosphere, 50, 1331–1342.

Hansen M L, Wibowo M, Jarmusch S A, Larsen T O, Jelsbak L. 2022. Sequential interspecies interactions affect production of antimicrobial secondary metabolites in Pseudomonas protegens DTU9.1. The ISME Journal, 16, 2680–2690.

Hasan T, Allen M, Cooperman B S. 1985. Anhydrotetracycline is a major product of tetracycline photolysis. Journal of Organic Chemistry, 50, 1755–1757.

Hong X X, Zhao Y C, Zhuang R D, Liu J Y, Guo G T, Chen J M, Yao Y M. 2020. Bioremediation of tetracycline antibiotics-contaminated soil by bioaugmentation. RSC Advances, 10, 33086–33102.

Kim S H, Kim G E, Jho E H. 2023. Effect of soil pH and temperature on the biodegradation of an agricultural antibiotic oxolinic acid. Korean Journal of Environmental Agriculture, 42, 8–13.

Koch N, Islam N F, Sonowal S, Prasad R, Sarma H. 2021. Environmental antibiotics and resistance genes as emerging contaminants: Methods of detection and bioremediation. Current Research in Microbial Sciences, 2, 100027.

Kong W D, Zhu Y G, Fu B J, Marschner P, He J Z. 2006. The veterinary antibiotic oxytetracycline and Cu influence functional diversity of the soil microbial community. Environmental Pollution, 143, 129–137.

Kumar K, Gupta C S, Chander Y, Singh A K. 2005. Antibiotic use in agriculture and its impact on the terrestrial environment. Advances in Agronomy, 87, 1–54.

Le V V, Tran Q G, Ko S R, Lee S A, Oh H M, Kim H S, Ahn C Y. 2023. How do freshwater microalgae and cyanobacteria respond to antibiotics? Critical Reviews in Biotechnology, 43, 191–211.

Li Y, Yu X, Bian B B, Peng S S, Zhang G. 2020. The level of oxytetracycline uptake in the presence of copper ions and the physiological response of Brassica chinensis L. Polish Journal of Environmental Studies, 29, 1229–1236.

Li Y X, Yang T, Lin X J, Huang J F, Zeng J W, Cai Q Y, Zhang Y L, Rong J N, Yu W D, Qiu J R, Pang Y W, Zhou J L. 2024. Isolation, identification, and optimization of conditions for the degradation of four sulfonamide antibiotics and their metabolic pathways in Pseudomonas stutzeri strain DLY–21. Heliyon, 10, e29123.

Lin B K, Lyu J L, Lyu X, Yu H, Hu Z, Lam J C W, Lam P K S. 2015. Characterization of cefalexin degradation capabilities of two Pseudomonas strains isolated from activated sludge. Journal of Hazardous Materials, 282, 158–164.

Lin Z Q, Zhang W P, Pang S M, Huang Y H, Mishra S, Bhatt P, Chen S H. 2020. Current approaches to and future perspectives on methomyl degradation in contaminated soil/water environments. Molecules, 25, 1–16.

Liu Y Q, He X X, Duan X D, Fu Y S, Fatta-Kassinos D, Dionysiou D D. 2016a. Significant role of UV and carbonate radical on the degradation of oxytetracycline in UV-AOPs: Kinetics and mechanism. Water Research, 95, 195–204.

Liu Y Q, He X X, Fu Y S, Dionysiou D D. 2016b. Degradation kinetics and mechanism of oxytetracycline by hydroxyl radical-based advanced oxidation processes. Chemical Engineering Journal, 284, 1317–1327.

Liu Y W, Chang H Q, Li Z J, Zhang C, Feng Y, Cheng D M. 2016. Gentamicin removal in submerged fermentation using the novel fungal strain Aspergillus terreus FZC3. Scientific Reports, 6, 3–12.

Ma T T, Pan X, Chen L K, Liu W X, Christie P, Luo Y M, Wu L H. 2016. Effects of different concentrations and application frequencies of oxytetracycline on soil enzyme activities and microbial community diversity. European Journal of Soil Biology, 76, 53–60.

Macauley J J, Adams C D, Mormile M R. 2007. Diversity of tet resistance genes in tetracycline-resistant bacteria isolated from a swine lagoon with low antibiotic impact. Canadian Journal of Microbiology, 53, 1307–1315.

Marti R, Scott A, Tien Y C, Murray R, Sabourin L, Zhang Y, Topp E. 2013. Impact of manure fertilization on the abundance of antibiotic-resistant bacteria and frequency of detection of antibiotic resistance genes in soil and on vegetables at harvest. Applied and Environmental Microbiology, 79, 5701–5709.

Martinez J L. 2009. Environmental pollution by antibiotics and by antibiotic resistance determinants. Environmental Pollution, 157, 2893–2902.

Meng Y H, Feng Y, Li X F, Liu Y W, Li Z J. 2018. Isolation of an oxytetracycline-degrading bacterial strain and its biodegradation characteristics. Journal of Plant Nutrition and Fertilizers, 24, 720–727. (in Chinese)

Millet S, Maertens L. 2011. The European ban on antibiotic growth promoters in animal feed: From challenges to opportunities. Veterinary Journal, 187, 143–144.

Nas B, Argun M E, Dolu T, Ateş H, Yel E, Koyuncu S, Dinç S, Kara M. 2020. Occurrence, loadings and removal of EU-priority polycyclic aromatic hydrocarbons (PAHs) in wastewater and sludge by advanced biological treatment, stabilization pond and constructed wetland. Journal of Environmental Management, 268, 110580.

Pan M, Chu L M. 2017. Leaching behavior of veterinary antibiotics in animal manure-applied soils. Science of the Total Environment, 579, 466–473.

Peng S, Wang Y M, Zhou B B, Lin X G. 2015. Long-term application of fresh and composted manure increase tetracycline resistance in the arable soil of eastern China. Science of the Total Environment, 506–507, 279–286.

Piotrowska M, Rzeczycka M, Ostrowski R, Popowska M A. 2017. Diversity of antibiotic resistance among bacteria isolated from sediments and water of carp farms located in a Polish nature reserve. Polish Journal of Environmental Studies, 26, 239–252.

Qi M Y, Liang B, Zhang L, Ma X D, Yan L, Dong W C, Kong D Y, Zhang L Y, Zhu H Z, Gao S H, Jiang J D, Liu S J, Corvini P F X, Wang A J. 2021. Microbial interactions drive the complete catabolism of the antibiotic sulfamethoxazole in activated sludge microbiomes. Environmental Science & Technology, 55, 3270–3282.

Qi W N, Long J, Feng C Q, Feng Y, Cheng D M, Liu Y W, Xue J M, Li Z J. 2019. Fe3+ enhanced degradation of oxytetracycline in water by pseudomonas. Water Research, 160, 361–370.

Rico A, Zhao W K, Gillissen F, Lürling M, Van den Brink P J. 2018. Effects of temperature, genetic variation and species competition on the sensitivity of algae populations to the antibiotic enrofloxacin. Ecotoxicology and Environmental Safety, 148, 228–236.

Saha N, McGaughy K, Held M A, Reza M T. 2020. Hydrothermal degradation of β-estradiol and oxytetracycline at selective reaction severities. SN Applied Sciences, 2, 1–9.

Sanow S, Kuang W Q, Schaaf G, Huesgen P, Schurr U, Roessner U, Watt M, Arsova B. 2023. Molecular mechanisms of Pseudomonas-assisted plant nitrogen uptake: Opportunities for modern agriculture. Molecular Plant-Microbe Interactions, 36, 536–548.

Santás-Miguel V, Arias-Estévez M, Díaz-Raviña M, Fernández-Sanjurjo M J, Álvarez-Rodríguez E, Núñez-Delgado A, Fernández-Calviño D. 2020a. Effect of oxytetracycline and chlortetracycline on bacterial community growth in agricultural soils. Agronomy, 10, 1–14.

Santás-Miguel V, Arias-Estévez M, Díaz-Raviña M, Fernández-Sanjurjo M J, Álvarez-Rodríguez E, Núñez-Delgado A, Fernández-Calviño D. 2020b. Interactions between soil properties and tetracycline toxicity affecting to bacterial community growth in agricultural soil. Applied Soil Ecology, 147, 103437.

Shao S C, Hu Y Y, Cheng C, Cheng J H, Chen Y C. 2018. Simultaneous degradation of tetracycline and denitrification by a novel bacterium, Klebsiella sp. SQY5. Chemosphere, 209, 35–43.

Shao S C, Hu Y Y, Cheng J H, Chen Y C. 2019. Action of oxytetracycline (OTC) degrading bacterium and its application in Moving Bed Biofilm Reactor (MBBR) for aquaculture wastewater pre-treatment. Ecotoxicology and Environmental Safety, 171, 833–842.

Shentu J L, Zhang K, Shen D S, Wang M Z, Feng H J. 2015. Effect from low-level exposure of oxytetracycline on abundance of tetracycline resistance genes in arable soils. Environmental Science and Pollution Research, 22, 13102–13110.

Shi Y K, Lin H, Ma J W, Zhu R R, Sun W C, Lin X Y, Zhang J, Zheng H B, Zhang X. 2021. Degradation of tetracycline antibiotics by Arthrobacter nicotianae OTC-16. Journal of Hazardous Materials, 403, 123996.

Sun L, Zhang G H, Luan L L, Liu F. 2016. Temporal variation in soil resistance to flowing water erosion for soil incorporated with plant litters in the Loess Plateau of China. Catena, 145, 239–245.

Tan H, Kong D L, Ma Q Y, Li Q Q, Zhou Y Q, Jiang X, Wang Z Y, Parales R E, Ruan Z Y. 2022. Biodegradation of tetracycline antibiotics by the yeast strain Cutaneotrichosporon dermatis M503. Microorganism, 10, 565.

Tan Z W, Chen J C, Liu Y L, Chen L, Xu Y Q, Zou Y X, Li Y T, Gong B N. 2021. The survival and removal mechanism of Sphingobacterium changzhouense TC931 under tetracycline stress and its’ ecological safety after application. Bioresource Technology, 333, 125067.

Thiele-Bruhn S. 2003. Pharmaceutical antibiotic compounds in soils - A review. Journal of Plant Nutrition and Soil Science, 166, 145–167.

Tian J C, Chen C, Lartey-Young G, Ma L M. 2023. Biodegradation of cefalexin by two bacteria strains from sewage sludge. Royal Society Open Science, 10, 220442.

Visca A, Barra Caracciolo A, Grenni P, Patrolecco L, Rauseo J, Massini G, Mazzurco Miritana V, Spataro F. 2021. Anaerobic digestion and removal of sulfamethoxazole, enrofloxacin, ciprofloxacin and their antibiotic resistance genes in a full-scale biogas plant. Antibiotics, 10, 502.

Wang J, Shen X L, Wang J, Yang Y P, Yuan Q P, Yan Y J. 2018. Exploring the promiscuity of phenol hydroxylase from Pseudomonas stutzeri OX1 for the biosynthesis of phenolic compounds. ACS Synthetic Biology, 7, 1238–1243.

Wang J W, Long Y N, Yu G L, Wang G L, Zhou Z Y, Li P Y, Zhang Y M, Yang K, Wang S T. 2022. A review on microorganisms in constructed wetlands for typical pollutant removal: Species, function, and diversity. Frontiers in Microbiology, 13, 845725.

Wang X C, Chen Z L, Kang J, Zhao X, Shen J M. 2018. Removal of tetracycline by aerobic granular sludge and its bacterial community dynamics in SBR. RSC Advances, 8, 18284–18293.

Wei Z S, Chen X L, Huang Z S, Jiao H Y, Xiao X L. 2022. Insights into the removal of gaseous oxytetracycline by combined ozone and membrane biofilm reactor. Environmental Engineering Research, 27, 210460–210469.

Wu N, Qiao M, Zhang B, Cheng W D, Zhu Y G. 2010. Abundance and diversity of tetracycline resistance genes in soils adjacent to representative swine feedlots in China. Environmental Science & Technology, 44, 6933–6939.

Wu X L, Gu Y C, Wu X Y, Zhou X Y, Zhou H, Amanze C, Shen L, Zeng W M. 2020. Construction of a tetracycline degrading bacterial consortium and its application evaluation in laboratory-scale soil remediation. Microorganisms, 8, 292.

Yang C W, Liu C E, Chang B V. 2020. Biodegradation of amoxicillin, tetracyclines and sulfonamides in wastewater sludge. Water, 12, 2147.

Yang Q L, Gao Y, Ke J, Show P L, Ge Y H, Liu Y H, Guo R X, Chen J Q. 2021. Antibiotics: An overview on the environmental occurrence, toxicity, degradation, and removal methods. Bioengineered, 12, 7376–7416.

Yang Q X, Zhang J, Zhu K F, Zhang H. 2009. Influence of oxytetracycline on the structure and activity of microbial community in wheat rhizosphere soil. Journal of Environmental Sciences, 21, 954–959.

Yang W R, Moore I F, Koteva K P, Bareich D C, Hughes D W, Wright G D. 2004. TetX is a flavin-dependent monooxygenase conferring resistance to tetracycline antibiotics. Journal of Biological Chemistry, 279, 52346–52352.

Yuan F, Hu C, Hu X X, Wei D B, Chen Y, Qu J H. 2011. Photodegradation and toxicity changes of antibiotics in UV and UV/H2O2 process. Journal of Hazardous Materials, 185, 1256–1263.

Zhang C F, Yang Z G, Jin W, Wang X, Zhang Y K, Zhu S J, Yu X, Hu G, Hong Q. 2017. Degradation of methomyl by the combination of Aminobacter sp. MDW-2 and Afipia sp. MDW-3. Letters in Applied Microbiology, 64, 289–296.

Zhang H B, Zhou Y, Huang Y J, Wu L H, Liu X H, Luo Y M. 2016. Residues and risks of veterinary antibiotics in protected vegetable soils following application of different manures. Chemosphere, 152, 229–237.

Zhang J X, Zhang Y B, Quan X. 2015. Bio-electrochemical enhancement of anaerobic reduction of nitrobenzene and its effects on microbial community. Biochemical Engineering Journal, 94, 85–91.

Zhang Z J, Shen J G, Wang H, Liu M, Wu L H, Ping F, He Q, Li H Y, Zheng C F, Xu X H. 2014. Attenuation of veterinary antibiotics in full-scale vermicomposting of swine manure via the housefly larvae (Musca domestica). Scientific Reports, 4, 6844.

Zhang Z K, He H H, Han T, Tian X M, Pang J Y, Lambers H. 2023. Soil oxytetracycline alters the effects of phosphate fertilisation and Bacillus amyloliquefaciens on the bacterial community of Medicago sativa rhizosphere. Applied Soil Ecology, 187, 104861.

Zheng J Y, Zhang J X, Gao L, Kong F Y, Shen G M, Wang R, Gao J M, Zhang J G. 2020. The effects of tetracycline residues on the microbial community structure of tobacco soil in pot experiment. Scientific Reports, 10, 1–10.

Zhong C Q, Zhou Y P, Fu J F, Qi X Y, Wang Z, Li J Q, Zhang P P, Zong G L, Cao G X. 2022. Cadmium stress efficiently enhanced meropenem degradation by the meropenem- and cadmium-resistant strain Pseudomonas putida R51. Journal of Hazardous Materials, 429, 128354.

Zhou S Y D, Zhu D, Giles M, Daniell T, Neilson R, Yang X R. 2020. Does reduced usage of antibiotics in livestock production mitigate the spread of antibiotic resistance in soil, earthworm guts, and the phyllosphere? Environment International, 136, 105359.

Zhu Y G, Johnson T A, Su J Q, Qiao M, Guo G X, Stedtfeld R D, Hashsham S A, Tiedje J M. 2013. Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proceedings of the National Academy of Sciences of the United States of America, 110, 3435–3440.

Zhu Y G, Zhao Y, Zhu D, Gillings M, Penuelas J, Ok Y S, Capon A, Banwart S. 2019. Soil biota, antimicrobial resistance and planetary health. Environment International, 131, 105059.

Zou S B, Zhang Q Q, Zhang X L, Dupuy C, Gong J. 2020. Environmental factors and pollution stresses select bacterial populations in association with protists. Frontiers in Marine Science, 7, 659.