Abstract:

In order to screen medicinal plant endophytes with good antibacterial activity against super drug-resistant Escherichia coli that simultaneously carry New Delhi metal-β lactamase （NDM） gene NDM1/NDM5 and NDM4/NDM5， the endophytes of Forsythia suspensa that had antagonistic effects on the super drug-resistant E. coli carrying NDM1/NDM5 and NDM4/NDM5 were isolated and screened by surface disinfection method and plate confrontation method. Morphological and molecular biological identification of these endophytes were performed， and their antibacterial activities were investigated by genome-wide analysis. The results showed that 23 strains of endophytes were isolated from root， stem， leaf and fruit different of Forsythia suspensa. A Gram-positive endophyte with good antibacterial effect was obtained from 23 endophyte strains by plate confrontation assay. It was identified as Paenibacillus spby morphology and molecular identification， and named as HNYJ2209J. HNYJ2209J could effectively inhibit the super drug-resistant E. coli. Whole genome sequencing analysis showed that the genome size of HNYJ2209J was 5 827 342 bp， the content of GC was 46.22%， and the number of coding genes was 5 232. There were 5 229， 3 648， 4 107， 3 493 and 2 495 annotated genes by NR， Swiss-Prot， COG， GO and KEGG databases， respectively， and 8 gene clusters related to secondary metabolite synthesis were predicted by antiSMASH. The main secondary metabolites were cyclolipopeptide antibiotics （fusaricidin B）， tridecaptin， polymyxin B， paenibacillin， marthiapeptide A， paeninodin， paenilipoheptin and aurantinin B/C/D. Among them， fusaricidin B， tridecaptin， polymyxin B and paenibacillin showed 100%， 100%， 100% and 90% homology between the predicted synthetic gene cluster and the known gene cluster. To sum up， HNYJ2209J had the potential to develop new drugs against super drug-resistant E. coli carrying NDM1/NDM5 and NDM4/NDM5 and as a feedable microorganism.

Key words: Paenibacillus sp., Forsythia suspensa, endophytes, feeding microorganism, antibacterial effect, Escherichia coli, super drug-resistant

摘要：

为筛选对同时携带新德里金属β-内酰胺酶（New Delhi metal-β-lactamase，NDM）基因NDM1/NDM5和NDM4/NDM5的超级耐药大肠杆菌抑菌活性较好的药用植物内生菌，采用表面消毒法和平板对峙法分离筛选对同时携带NDM1/NDM5和NDM4/NDM5的超级耐药大肠杆菌有拮抗作用的连翘内生菌，通过形态学和分子生物学鉴定，并结合全基因组学分析探究其抗菌活性物质。结果表明，从连翘根、茎、叶和果中分离得到23株内生菌。采用平板对峙法抑菌试验，从23株内生菌中获得1株抗菌效果较好的革兰氏阳性内生菌HNYJ2209J，经形态学鉴定和分子鉴定为类芽孢杆菌（Paenibacillus sp），该菌能有效抑制超级耐药大肠杆菌的活性。对HNYJ2209J菌株进行全基因组测序分析，其基因组大小为5 827 342 bp，GC含量为46.22%，预测其编码基因为5 232个，其中被NR、Swiss-Prot、COG、GO、KEGG数据库注释的基因数分别为5 229、3 648、4 107、3 493和2 495。通过antiSMASH预测到8个与次级代谢产物合成相关的基因簇；主要次级代谢产物有环脂肽类抗生素、十三肽菌素、多粘菌素B、抗菌肽、噻唑环肽类化合物、拉索肽、脂质庚肽和多烯聚酮类化合物。其中，脂肽类抗生素、十三肽菌素、多粘菌素B和抗菌肽的预测合成基因簇与已知基因簇的同源性分别为100%、100%、100%和90%。综上表明，HNYJ2209J菌株具有开发针对同时携带NDM1/NDM5和NDM4/NDM5超级耐药大肠杆菌的新药和作为可饲用微生物的潜力。

关键词: 类芽孢杆菌, 连翘, 内生菌, 饲用微生物, 抑菌效果, 大肠杆菌, 超级耐药