头孢菌素类抗生素因其抗菌谱广、抑菌效果好和毒性低等优点，被广泛应用于人类及动物医学。然而，过量使用导致头孢菌素在各环境介质中频繁检出，去除环境中头孢菌素残余对消除抗生素污染、延缓抗生素耐药性的产生和发展具有重要意义。生物降解是实现抗生素去除的有力手段，但目前头孢菌素类抗生素的生物降解机制仍有待进一步阐明。本研究利用头孢匹罗（第四代头孢菌素）从污水处理厂好氧活性污泥中富集高效降解菌群，分离头孢匹罗降解功能菌株，应用（宏）基因组学方法并整合基于质谱（HPLC-QTOF-MS）的代谢产物解析，系统阐述了头孢匹罗的好氧生物降解机制，为环境中头孢菌素的高效去除提供了研究基础。活性污泥经过三阶段的长期驯化后形成高度富集的头孢匹罗降解菌群，在96小时内可完全降解100 mg/L头孢匹罗。16S rRNA扩增子测序揭示驯化后期菌群的核心菌属为贪噬菌属（Variovorax）、生丝微菌属（Hyphomicrobium）、潘多拉菌属（Pandoraea）及鞘氨醇单胞菌属（Sphingomonas）等。从菌群中分离得到5株头孢匹罗降解菌Sphingomonas sp. LPL1、Sphingomonas sp. LPL2、Sphingomonas sp. P33、Bosea sp. MPL1、Sphingomonas sp. MIX_Y和1株非降解菌Achromobacter sp. MIX_W。降解性能测试结果表明菌群和菌株P33均可利用头孢匹罗作为唯一碳源、氮源及能源，对头孢匹罗具有较强的耐受和降解性能。外加碳源促进菌群对头孢匹罗的降解和生物量增长，但对菌株P33降解头孢匹罗产生不利影响；氨氮是菌株P33降解头孢匹罗的首选氮源，菌群则表现出对亚硝态氮的偏好性。基于HPLC-QTOF-MS分析鉴定了8种头孢匹罗降解产物，并根据代谢产物的信息构建了6条头孢匹罗生物转化途径，反应类型涵盖吡啶环脱落、β-内酰胺键断裂、脱羧、内酯化及其他侧链修饰过程，其中有3条是本研究首次报道的头孢匹罗生物降解途径。宏基因组物种注释结果表明硫杆菌（Thiobacillus sp001897705）、贪噬菌（Variovorax soli）、甲基变形菌（Methyloversatilis discipulorum_A）、鞘氨醇单胞菌（Sphingomonas sp000797515）和博斯氏菌（Bosea sp.）是参与头孢匹罗生物降解的关键功能菌株。整合宏基因组功能注释结果、菌株P33全基因组测序数据和酶学特性分析预测了多个参与头孢匹罗生物转化的关键功能基因，其中以B类金属β-内酰胺酶为代表的β-内酰胺酶和以青霉素酰化酶为代表的酰胺水解酶是负责头孢匹罗降解过程中β-内酰胺键断裂和侧链修饰的关键酶。
Due to the merits of broad-spectrum antibacterial spectrum, effective bactericidal performance and low toxicity, cephalosporins are extensively applied in human and veterinary medicines. However, the overuse of cephalosporins has resulted in their frequent detection in multiple environmental compartments, whereas the removal of cephalosporin residues from the environment contributes significantly to eliminate antibiotic contamination and alleviate the development and spread of antibiotic resistance. Biodegradation is a robust process for effective depletion of cephalosporin contamination, nevertheless, there are still many research gaps regarding the biodegradation mechanism of cephalosporin antibiotics to be filled up. In this work, an efficient degrading consortium was enriched from aerobic activated sludge fed with the fourth-generation cephalosporin, cefpirome, as the substrate. Cefpirome-degrading pure cultures were then isolated from the enriched culture. An integrated approach combining (meta-)genomic and metabolic analysis based on the mass spectrum (HPLC-QTOF-MS) were enrolled to systematically decipher the biodegradation mechanism of cefpirome. Results of this work provide theoretical guidance for effective removal of cephalosporins from environment.A high-performance cefpirome-degrading consortium was obtained after three stages of long-term domestication, which was able to completely degrade 100 mg/L of cefpirome within 96 h. The analysis of 16S rRNA gene amplicon sequencing showed that the genera Variovorax, Hyphomicrobium, Pandoraea and Sphingomonas were the dominant bacteria among the enriched culture. Five cefpirome-degrading strains including Sphingomonas sp. LPL1, Sphingomonas sp. LPL2, Sphingomonas sp. P33, Bosea sp. MPL1, Sphingomonas sp. MIX_Y and one non-degrading strain Achromobacter sp. MIX_W were isolated from the consortium. Batch experiment concerning the degradation capacity of the enriched culture and isolated strains were conducted under distinct nutrient conditions, including elevated initial cefpirome concentrations, different additional carbon and nitrogen sources. Results illustrated that both the consortium and strain P33 could subsist on cefpirome as the sole carbon, nitrogen and energy source, possessing a strong ability to resist and degrade cefpirome under high cefpirome-loadings. The supplementation of extra carbon sources boosted cefpirome biodegradation and bacterial proliferation of the enriched culture, whereas an adverse effect was observed on the degradation performance of strain P33. Ammonium nitrogen was the preferred nitrogen source for strain P33 to degrade cefpirome, while the consortium showed a preference for nitrite nitrogen.A total of 8 biodegradation products were detected by HPLC-QTOF-MS, and six biodegradation pathways were proposed based on the information of these metabolites, including the detachment of pyridine moiety, cleavage of β-lactam ring, decarboxylation, lactonization and other side chain modification processes. Of which, three biodegradation pathways were firstly reported in this work. Species annotation of metagenomics revealed that Thiobacillus sp001897705, Variovorax soli, Methyloversatilis discipulorum_A, Sphingomonas sp000797515, and Bosea sp. were the key functional strains participated in the biodegradation of cefpirome. Multiple functional genes involved in cefpirome biotransformation were predicted via integrating the results of metagenomic functional annotation, whole genome sequencing analysis of Sphingomonas sp. P33 and enzymatic properties analysis, of which the β-lactamase represented by class B metallo-β-lactamase and hydrolases represented by Penicillin G acylase were found to be the key enzymes responsible for β-lactam bond cleavage and side-chain modifications, respectively.
