阿莫西林降解菌的筛选及降解机制研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-0287

摘要/Abstract

摘要：

目的筛选生猪粪污中阿莫西林（AMX）高效降解菌并优化其降解条件，明确AMX的降解产物及途径，为清除环境中抗生素残留提供依据和参考。方法采用抗生素驯化法和高效液相色谱-串联质谱（HPLC-MS/MS）法对猪粪中AMX高效降解菌进行筛选，通过形态学观察及16S rDNA测序对高效降解菌进行菌种鉴定。采用单因素试验法对AMX高效降解菌的培养条件进行优化。探究了培养温度、pH值、接种量、抗生素初始浓度等因素对菌株降解AMX的影响。利用超高效液相色谱-串联质谱（UPLC-MS/MS）对阿莫西林降解过程中的产物进行了鉴定，以推测阿莫西林的降解途径。通过转录组测序，结合基因组学和生物信息学方法，对菌株AMX-1的DEGs进行了功能注释。结果分离筛选得到一株AMX高效降解菌，命名为菌株AMX-1，经形态学观察及分子生物学鉴定，菌株AMX-1被鉴定为大肠杆菌（Escherichia coli）。菌株AMX-1降解AMX的最佳条件为温度为35 ℃，pH为7.0，AMX初始浓度为50 mg/L，接种量为6%。在此条件下，48 h内AMX降解率达到100%。对AMX的降解产物进行鉴定，共鉴定出8种中间产物，提出了AMX的降解途径。转录组测序结果显示，786个降解必需基因通过头孢噻呋钠的水解而上调，基因注释结果表明AMX的降解与多种生物过程相关。结论成功从猪粪中筛选并鉴定出一株阿莫西林高效降解菌——大肠杆菌AMX-1。该菌株在优化条件下，48 h内对AMX的降解率可达100%。菌株AMX-1展现出的高效降解能力及明确的降解机制，为环境中阿莫西林残留的清除提供了重要的菌种资源和理论依据。

关键词: 阿莫西林, 大肠杆菌, 分离鉴定, 转录组测序, 降解机制

Abstract:

Objective This study is aimed to screen highly efficient amoxicillin (AMX) -degrading bacteria from pig manure sewage, optimize their degradation conditions, identify the degradation products and pathways of AMX, which may provide a basis and reference for removing antibiotic residues in the environment. Method Antibiotic domestication method and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) were used to screen highly efficient AMX-degrading bacteria from pig manure. The highly efficient degrading bacteria were identified by morphological observation and 16S rDNA sequencing. The single-factor experiment method was adopted to optimize the culture conditions of AMX-degrading bacteria. The effects of culture temperature, pH value, inoculum size, and initial antibiotic concentration on the degradation of AMX by the strain were investigated. Ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to identify the products during the degradation of amoxicillin, so as to speculate the degradation pathway of amoxicillin. Through transcriptome sequencing, combined with genomics and bioinformatics methods, functional annotation of differentially expressed genes (DEGs) of strain AMX-1 was carried out. Result A highly efficient AMX-degrading bacterium was isolated and screened, named strain as AMX-1. Via morphological observation and molecular biological identification, strain AMX 1 was identified as Escherichia coli. The optimal conditions for strain AMX-1 to degrade AMX were as follows: Temperature was 35 ℃, pH was 7.0, the initial concentration of AMX was 50 mg/L, and the inoculum size was 6%. Under these conditions, the degradation rate of AMX reached 100% within 48 h. Eight intermediate products were identified during the degradation of AMX, and the degradation pathway of AMX was proposed. Transcriptome sequencing results showed that 786 essential degradation genes were up-regulated by the hydrolysis of ceftiofur sodium. Gene annotation results indicated that the degradation of AMX was related to a variety of biological processes. Conclusion In this study, a strain of E. coli AMX-1 with highly-efficient amoxicillin degradation is successfully screened and identified from pig manure. Under optimized conditions, the degradation rate of AMX by this strain can reach 100% within 48 h. The highly-efficient degradation ability and clear degradation mechanism demonstrated by strain AMX-1 provide important microbial resources and theoretical basis for the removal of amoxicillin residues in the environment.

Key words: amoxicillin, Escherichia coli, separation and identification, transcriptome sequencing, degradation mechanism

闫梦阳, 梁晓阳, 戴君昂, 张妍, 关团, 张辉, 刘良波, 孙志华. 阿莫西林降解菌的筛选及降解机制研究[J]. 生物技术通报, 2025, 41(9): 314-325.

YAN Meng-yang, LIANG Xiao-yang, DAI Jun-ang, ZHANG Yan, GUAN Tuan, ZHANG Hui, LIU Liang-bo, SUN Zhi-hua. Screening of Amoxicillin-degrading Bacteria and Study on Its Degradation Mechanisms[J]. Biotechnology Bulletin, 2025, 41(9): 314-325.

图/表 8

表1 RT-qPCR引物序列信息

Table 1 RT-qPCR primer sequence information

基因名称

Gene

引物名称

Primer

引物序列

Sequence （5′-3′）

产物长度

Product length （bp）

nlpC

nlpC-F

nlpC-R

ATGCTGGAACTACTTTTTGTAATTGG

GCCCTGATCACCCACAC

188

aac6-Ib

aac6-Ib-F

aac6-Ib-R

AGGGCCTTTAGTGGTCG

GCTTCTGTTCAGCACGT

203

astE

astE-F

astE-R

CAACTCAAACTACCACCGAGA

GGTGACGGCGAATAGC

195

astB

astB-F

astB-R

ATCAAAGGCATACTGGCAGG

ATCACCCGAACTAAATCTTGTTG

113

图1 菌株AMX-1的鉴定a-b ：分别为菌株 AMX-1 在MSM筛选培养基生长图及革兰氏镜检图； c ：菌株 AMX-1 的系统发育树

Fig. 1 Identification of strain AMX-1a-b: Growth images of strain AMX-1 in MSM screening medium and Gram staining microscopic images, respectively. c: Phylogenetic tree of strain AMX-1

图2 菌株AMX-1的生长曲线

Fig. 2 Growth curves of strain AMX-1

图3 环境条件对AMX降解率的影响A-D：分别为温度、pH 值、AMX 初始浓度和接种量对菌株AMX-1降解AMX的影响；E：最佳条件下AMX的降解曲线。不同字母代表差异显著（P<0.05）

Fig. 3 Influence of environmental conditions on the degradation rate of AMXA-D: Effects of temperature, pH, initial concentration of AMX, and inoculum amount on AMX degradation by strain AMX-1, respectively; E: degradation curves of AMX under optimal conditions. Different letters indicate significant difference (P<0.05)

图4 AMX生物降解产物及降解途径

Fig. 4 Biodegradation products and degradation pathways of AMX

图5 AMX-1 转录本的注释结果A：差异表达基因火山图；B：菌株AMX-1转录组的 GO 功能注释；C：AMX-1 转录组的 KEGG 功能注释

Fig. 5 Annotations of AMX-1 transcriptsA: Volcanic maps of differentially expressed genes; B: GO functional annotation of the transcriptome of strain AMX-1; C: KEGG functional annotation of the AMX-1 transcriptome

表2 AMX降解菌AMX-1中的差异表达基因（DEGs）

Table 2 Differentially expressed genes in the AMX-degrading bacterium AMX-1

Gene name	Gene description	FC（a/b）	Log₂FC（a/b）	P value
yhbO	General stress protein	12.696	3.666 350 812	3.15×10 ^-¹²⁴
lepB	Signal peptidase	7.871	2.976 563 759	2.46×10 ^-⁶⁶
aac6-Ib	Aminoglycoside 6-adenylyltransferase	5.194	2.376 838	9.44×10 ^-⁸⁶
nlpC	Endopeptidase	7.125	2.832 941	4.06×10 ^-⁶⁶
astE	Succinylglutamate desuccinylase	17.152	4.100 312 827	4.54×10 ^-⁴¹
astB	Succinylarginine dihydrolase	9.291	3.215 854 657	3.64×10 ^-²⁶

图6 转录组结果验证A：RT-qPCR结果验证；B：上调基因的功能验证

Fig. 6 Validation of transcriptome resultsA: Validation of RT-qPCR results; B: functional validation of upregulated genes. *** P<0.001

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