近年来,细菌抗生素耐药性导致的年全球死亡人数达到了495万人,被认为是21世纪亟需解决的关键公共卫生问题之一。生物降解是去除环境中抗生素的有力手段,开展抗生素生物降解研究对于降低细菌抗生素耐药性产生和传播风险具有重要意义。林可霉素是我国常用的一种抗生素,然而目前针对林可霉素的生物降解机理有待进一步阐明。本研究从市政污水厂活性污泥中富集得到林可霉素的降解菌群,并分离出一株林可霉素降解菌Conexibacter sp. LD01,应用(宏)基因组学与宏转录组学方法并结合基于质谱(HPLC-QTOF-MS)的代谢产物解析,对林可霉素的生物降解机理进行了系统地研究,主要结论有:16S rRNA扩增子测序揭示富集菌群的优势菌属为贪噬菌属(Variovorax)、无色菌属(Achromobacter)、苯基杆菌属(Phenylobacterium)、康奈氏菌属(Conexibacter)及生丝微菌属(Hyphomicrobium)。全基因组测序分析结果显示林可霉素降解菌LD01属于放线菌门(Acfinobacteria)、康奈氏菌属(Conexibacter)。本研究首次发现放线菌门的菌株具有林可霉素降解能力,在门水平上拓展了对林可霉素降解菌的认识。比较基因组分析表明Conexibacter sp. LD01与现有参考基因组的相似度较低,是康奈氏菌属的新菌种。本研究测试了菌群和菌株在不同抗生素浓度、不同外加碳源及氮源条件下对林可霉素的降解能力,结果表明无论是菌群还是菌株都可以利用林可霉素作为唯一的碳、氮源进行生长,表现出较强的林可霉素耐受和降解能力,且其最适氮源为氨氮。基于HPLC-QTOF-MS分析,本研究鉴定了17种林可霉素生物降解产物,其中有9种中间产物是本研究中首次发现的林可霉素生物降解产物。基于代谢产物信息,本研究构建了5条林可霉素的生物降解途径,即N-去甲基化、酰胺键断裂、吡咯烷环氧化、亚砜化及丙胺侧链氧化。宏基因组与宏转录组的联合分析表明,ermE、AmiB2与msrA基因可能分别是负责林可霉素N-去甲基化、酰胺键断裂和亚砜化的关键基因。值得一提的是,本研究通过宏转录组学分析发现林可霉素N-去甲基化和酰胺键断裂后的产物可能通过脯氨酸代谢(Proline metabolism)和糖酵解途径(Glycolysis pathway)进入三羧酸循环。此外,宏转录组分析表明群落微生物可能存在营养互作关系,且共培养降解实验证明了大部分非林可霉素降解菌可以显著促进菌株LD01对林可霉素的降解(p < 0.01)。
Antimicrobial resistance (AMR) has been a severe global public health issue that caused approximately 4.95 million deaths over the world in 2019. Biodegradation is a robust process for the remediation of antibiotic-contaminated environments. Thus, researching the biodegradation of antibiotics is beneficial for dealing with the development and spread of antibiotic resistance. Lincomycin, as an effective antibiotic to treat human and animal diseases, has a large usage in China. Nevertheless, there are still many research gaps about the biodegradation of lincomycin to be filled up. Herein, an enriched culture was obtained from a municipal wastewater treatment plant via a long term enrichment process and a lincomycin-subsisting strain was isolated from the enriched culture, namely Conexibacter sp. LD01. An integrated multi-omics approach including metabolomics, (meta-)genomics, and metatranscriptomic, was enrolled in this research to systematically study the biodegradation mechanism of lincomycin. The main results are as followings:The analysis of 16S rRNA genes amplicon sequencing showed that the genus Variovorax, Achromobacter, Phenylobacterium, Conexibacter, and Hyphomicrobium were the dominant bacteria among the enriched culture. The isolated lincomycin-subsisting strain (i.e., Conexibacter sp. LD01) was assigned to the phylum of Actinobacteria which significantly expanded the knowledge boundary of the affiliation of lincomycin-subsisting bacteria. The experiments regarding the biodegradation capacity of enriched culture and isolated strain were conducted under various nutrition conditions, including a range of initial concentrations of lincomycin, and various additional carbon and nitrogen sources. The outcomes illustrated that both enriched culture and isolated strain could subsist on lincomycin as sole carbon and nitrogen source, but ammonia nitrogen would be the preferred nitrogen source for growth and lincomycin degradation. The results indicated that the bacteria possessed a strong ability to resist and degrade lincomycin after long-term enrichment under high selected pressure of lincomycin. A total of 17 biodegradation products were detected by HPLC-QTOF-MS, and 9 of them were firstly reported in this study. Five biodegradation pathways were constructed based on these products, including N-demethylation, breakage of amido bond, sulfoxidation, oxidation of pyrrolidine ring and propylamino chain. Through the combined analysis of metagenomics and metatranscriptomics, genes ermE, AmiB2, and msrA were speculated to be the key genes that were probably responsible for the N-demethylation, breakage of amido bond, sulfoxidation of lincomycin, respectively. Notably, though the downstream products (i.e., proline and α-D-glucose) belonging to the pathways of N-demethylation and breakage of amido bond could not be detected by HPLC-QTOF-MS, they were predicted via the analysis of metatranscriptomics. It was speculated that the products of lincomycin finally entered the TCA cycle through the proline metabolism and glycolysis pathways. Additionally, the analysis of metatranscriptomics indicated the potential metabolic interaction between the compositions of microbial community and the biodegradation efficiency of lincomycin was improved significantly when Conexibacter sp. LD01 was co-cultured with most non-degrading strains (p < 0.01).