Screening of Amoxicillin-degrading Bacteria and Study on Its Degradation Mechanisms

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

Abstract

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

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.

Figures/Tables 8

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

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

Fig. 2 Growth curves of strain AMX-1

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)

Fig. 4 Biodegradation products and degradation pathways of AMX

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

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

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

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