类芽孢杆菌对携带NDM基因的超级耐药大肠杆菌的抑菌活性分析

doi:10.13304/j.nykjdb.2024.0457

摘要/Abstract

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

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

中图分类号:

S968.22

李海利, 杨帆, 陈建豪, 杨康, 杨夷平, 段进刚, 李斌, 张婉琪, 马春江. 类芽孢杆菌对携带NDM基因的超级耐药大肠杆菌的抑菌活性分析[J]. 中国农业科技导报, 2025, 27(8): 119-131.

Haili LI, Fan YANG, Jianhao CHEN, Kang YANG, Yiping YANG, Jingang DUAN, Bin LI, Wanqi ZHANG, Chunjiang MA. Analysis of Bacteriostasis Activity of Paenibacillus sp. Against Super Drug-resistant Escherichia coli Carrying NDM Genes[J]. Journal of Agricultural Science and Technology, 2025, 27(8): 119-131.

图/表 13

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功能分类 Functional classification	基因数 Number of genes
辅助氧化还原酶（Auxiliary activities，AAs）	2
碳水化合物结合模块（Carbohydrate-binding modules，CBMs）	26
碳水化合物酯酶（Carbohydrate esterases，CEs）	32
糖苷水解酶（Glycoside hydrolases，GHs）	125
糖基转移酶（Glycosyl transferases，GTs）	33
多糖裂合酶（Polysaccharide lyases，PLs）	1

功能分类 Functional classification	基因数 Number of genes
辅助氧化还原酶（Auxiliary activities，AAs）	2
碳水化合物结合模块（Carbohydrate-binding modules，CBMs）	26
碳水化合物酯酶（Carbohydrate esterases，CEs）	32
糖苷水解酶（Glycoside hydrolases，GHs）	125
糖基转移酶（Glycosyl transferases，GTs）	33
多糖裂合酶（Polysaccharide lyases，PLs）	1

类型 Type	簇ID Cluster ID	基因位置 Genome location	最相似的基因簇 Most similar known cluster	序列一致性 Similarity of sequence/%
非核糖体多肽合成酶Non-ribosomal peptide synthetase	Cluster 1	68 140~130 725	环脂肽类抗生素B Fusariciden B	100
	Cluster 2	341 060~362 265	－	－
	Cluster 7	1 433 971~1 494 636	噻唑环肽类化合物AMarthiapeptide A
	Cluster 9	2 042 324~2 103 728	－	－
	Cluster 10	2 469 235~2 562 320	十三肽菌素Tridecaptin	100
	Cluster 11	2 720 521~2 845 826	脂质庚肽Paenilipoheptin	19
	Cluster 12	2 944 761~2 997 159	－	－
	Cluster 17	5 001 683~5 082 857	多粘菌素BPolymyxin B	100
羊毛硫肽类化合物Lanthipeptide	Cluster 3	994 616~1 020 919	细菌抗菌肽Paenibacillin	90
羊毛硫肽类化合物Lanthipeptide	Cluster 16	4 869 313~4 892 543		－
TransAT-PKS	Cluster 4	1 042 831~1 119 499	－	－
Proteusin	Cluster 5	1 220 795~1 241 031	－	－
套索肽Lassopeptide	Cluster 6	1 356 777~1 380 842	拉索肽Paeninodin	40
循环-内酯Cyclic-lactone	Cluster 8	1 800 731~1 819 131	－	－
循环-内酯Cyclic-lactone	Cluster 13	3 140 538~3 160 684	－	－
TransAT-PKS-like	Cluster 14	3 639 549~3 741 439	Aurantinin B/C/D	35
Ranthipeptide	Cluster 15	4 502 063~4 527 449		－

类型 Type	簇ID Cluster ID	基因位置 Genome location	最相似的基因簇 Most similar known cluster	序列一致性 Similarity of sequence/%
非核糖体多肽合成酶Non-ribosomal peptide synthetase	Cluster 1	68 140~130 725	环脂肽类抗生素B Fusariciden B	100
	Cluster 2	341 060~362 265	－	－
	Cluster 7	1 433 971~1 494 636	噻唑环肽类化合物AMarthiapeptide A
	Cluster 9	2 042 324~2 103 728	－	－
	Cluster 10	2 469 235~2 562 320	十三肽菌素Tridecaptin	100
	Cluster 11	2 720 521~2 845 826	脂质庚肽Paenilipoheptin	19
	Cluster 12	2 944 761~2 997 159	－	－
	Cluster 17	5 001 683~5 082 857	多粘菌素BPolymyxin B	100
羊毛硫肽类化合物Lanthipeptide	Cluster 3	994 616~1 020 919	细菌抗菌肽Paenibacillin	90
羊毛硫肽类化合物Lanthipeptide	Cluster 16	4 869 313~4 892 543		－
TransAT-PKS	Cluster 4	1 042 831~1 119 499	－	－
Proteusin	Cluster 5	1 220 795~1 241 031	－	－
套索肽Lassopeptide	Cluster 6	1 356 777~1 380 842	拉索肽Paeninodin	40
循环-内酯Cyclic-lactone	Cluster 8	1 800 731~1 819 131	－	－
循环-内酯Cyclic-lactone	Cluster 13	3 140 538~3 160 684	－	－
TransAT-PKS-like	Cluster 14	3 639 549~3 741 439	Aurantinin B/C/D	35
Ranthipeptide	Cluster 15	4 502 063~4 527 449		－

基因ID Gene ID	毒力因子ID编号 VFDB ID	毒力因子名称 Vfs	序列一致性 Identity/%
Gene0185	VFG000077（gb\|NP_465991）	ClpP（VF0074）	69.05
Gene01193	VFG050186（gb\|AJI07989.1）	BpsC （CVF891）	71.58
Gene01194	VFG050185（gb\|AJI07977.1）	BpsD （CVF891）	63.73
Gene01214	VFG050185（gb\|AJI07977.1）	BpsD （CVF891）	61.31
Gene01261	VFG012095（gb\|YP_001086664）	GroEL （VF0594）	70.71
Gene01364	VFG002158（gb\|NP_464456）	LpIAI （VF0347）	61.63
Gene01805	VFG039282（gb\|NP_819314）	CBU （CVF803）	63.01
Gene02451	VFG000077（gb\|NP_465991）	ClpP （VF0074）	64.17
Gene03327	VFG006717（gb\|NP_465159）	Lap （VF0444）	63.74
Gene03414	VFG048830（gb\|YP_002920353.1）	Capsule （VF0560）	68.59
Gene04258	VFG000077（gb\|NP_465991）	ClpP （VF0074）	65.82
Gene04825	VFG046465（gb\|YP_169203.1）	EF-Tu （VF0460）	72.84
Gene04856	VFG000079（gb\|NP_463763）	ClpC （VF0072）	76.20
Gene04883	VFG050186（gb\|AJI07989.1）	BpsC （CVF891）	64.77