金银花、连翘及其药对对马链球菌马亚种耐药基因和毒力基因的影响

doi:10.16431/j.cnki.1671-7236.2022.02.033

摘要/Abstract

摘要： 【目的】研究金银花、连翘及金银花-连翘药对(金银花-连翘1∶1)对北疆地区携带fneB毒力基因的马链球菌马亚种(Streptococcus equi subsp. equi, SEE)耐药基因和毒力基因的影响。【方法】首先对1 g/mL的金银花、连翘及金银花-连翘药对(金银花-连翘1∶1)水提物进行中药配比浓度梯度试验, 然后与携带fneB毒力基因的L1菌株、lytA+fneB+ply毒力基因的D1菌株和qnrA+bla_TEM+fneB基因的Y1菌株3种SEE菌株共培养, 检测中药对SEE菌株耐药基因bla_TEM、qnrA及毒力基因ply、fneB的影响; 将192只SPF级昆明小鼠均分为16组: 空白对照组(生理盐水)、金银花组、连翘组、金银花-连翘1∶1组、阴性对照组(Y1、L1和D1菌株组)及3种菌株分别与金银花、连翘和金银花-连翘1∶1共培养组, 各组药物或菌液经腹腔注射0.5 mL进行小鼠体内抑菌试验, 检测药物对小鼠的致病性和fneB毒力基因的影响。【结果】中药最适配比浓度为中药水提物∶THB培养基∶待测菌液(D_{600 nm}值均为0.6)为1 000 μL∶500 μL∶20 μL; 与中药水提取物共培养的3株SEE菌株均未检出耐药基因和毒力基因, 且菌株形态结构均未发生改变。小鼠致病性试验结果显示, 阴性对照组的小鼠成活率分别为16.7%、8.3%和0;而L1+连翘、L1+金银花共培养组小鼠存活率分别为83.3%、75.0%, 金银花-连翘1∶1药对与3株SEE菌株共培养组小鼠成活率分别为41.7%、16.7%、50.0%;小鼠病理解剖结果显示, 除接种SEE菌株的小鼠肝脏肿大淤血、边缘钝圆外, 其余各组肝脏均正常。小鼠体内fneB毒力基因检测结果显示, Y1+金银花、Y1+连翘、Y1+金银花-连翘1∶1、L1+金银花、L1+金银花-连翘1∶1、D1+金银花、D1+连翘、D1+金银花-连翘组均携带fneB毒力基因, L1+连翘组未检出fneB毒力基因, 表明小鼠体内SEE菌株有重新获得fneB毒力基因的能力, 出现菌种反毒复壮, 其机制有待进一步研究。【结论】金银花、连翘及金银花-连翘药对能够减弱SEE菌株的毒力基因和耐药基因, 从而对马腺疫疾病的防治具有指导意义, 为减抗、替抗提供理论支撑。

关键词: 金银花; 连翘; 马链球菌马亚种; 耐药基因; 毒力基因

Abstract: 【Objective】 This study was to investigate the effects of honeysuckle, forsythia and their drug pair (honeysuckle-forsythia 1∶1) on drug resistance genes and virulence genes of Streptococcus equi subsp. equi (SEE) carrying fneB virulence gene in Northern Xinjiang.【Method】 The water extract of 1 g/mL honeysuckle, forsythia and honeysuckle, forsythia drug pair 1∶1 (honeysuckle-forsythia 1∶1) were tested by traditional Chinese medicine ratio concentration gradient test.The concentration gradient test of traditional Chinese medicine was carried out, and the three strains were co-cultured with L1 strain carrying fneB virulence gene, D1 strain carrying lytA+fneB+ply virulence genes and Y1 strain carrying qnrA+bla_TEM+fneB genes.The effects of traditional Chinese medicine on drug resistance genes bla_TEM, qnrA and virulence genes ply, fneB of SEE strains were detected.192 SPF Kunming mice were divided into 16 groups: Control group (normal saline), honeysuckle group, forsythia group, honeysuckle-forsythia 1∶1 group, negative control group (Y1, L1 and D1 strain groups) and three strains were co-cultured with honeysuckle, forsythia and honeysuckle-forsythia 1∶1 respectively.The drugs or bactering fluid in each group were injected intraperitoneally with 0.5 mL per mouse for antibacterial test in vivo, and the effects of drugs on pathogenicity and fneB virulence gene in mice were detected.【Result】 The optimal concentration of Chinese herbal medicine was as follows∶Water extract of Chinese herbal medicine∶THB medium∶bacteria solution (D_{600 nm} value was 0.6) was 1 000 μL∶500 μL∶20 μL.The drug resistance gene and virulence gene were not detected in the three SEE strains co-cultured with the aqueous extract of traditional Chinese medicine, and their morphological structure did not change.The results of mouse pathogenicity test showed that the survival rates of negative control group were 16.7%, 8.3% and 0, respectively.The survival rate of mice in the co-culture of L1 strain+forsythia group and L1 strain+honeysuckle group were 83.3% and 75.0%, respectively.The survival rate of mice in the co-culture group of honeysuckle-forsythia 1∶1 and 3 SEE strains were 41.7%, 16.7% and 50.0%, respectively.The results of pathological anatomy of mice showed that the livers of other groups were normal except that the livers of mice inoculated with the original SEE strain was swollen and congestion, and the edge was blunt and round.The results of fneB virulence gene detection showed that Y1+honeysuckle, Y1+forsythia, Y1+honeysuckle-forsythia 1∶1, L1+honeysuckle, L1+honeysuckle-forsythia 1∶1, D1+honeysuckle, D1+forsythia and D1+honeysuckle-forsythia groups carried fneB virulence gene, but not in L1+forsythia group, which indicated that the SEE strains in mice had the ability to regain fneB virulence gene again, and the strain had anti-virus rejuvenation, and its mechanism needed to be further studied.【Conclusion】 honeysuckle, forsythia and their drug pair could weak the virulence gene and eliminate the drug resistance gene of SEE strain, which had guiding significance for the prevention and treatment of horse adenosis disease, and provided theoretical support for reducing and replacing resistance.

Key words: honeysuckle; forsythia; Streptococcus equi subsp. equi; drug resistance genes; virulence genes

中图分类号:

S859.3

买占海, 卢亚宾, 李建龙, 赵璐, 孙丹岚, 佟盼盼, 况玲. 金银花、连翘及其药对对马链球菌马亚种耐药基因和毒力基因的影响[J]. 中国畜牧兽医, 2022, 49(2): 709-717.

MAI Zhanhai, LU Yabin, LI Jianlong, ZHAO Lu, SUN Danlan, TONG Panpan, KUANG Ling. Effects of Honeysuckle, Forsythia and Their Drug Pair on Resistance Genes and Virulence Genes of Streptococcus equi subsp. equi[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 709-717.

参考文献

[1] 王雨朦. 马链球菌马亚种的分离鉴定、致病性检测和抗菌药物筛选[D]. 乌鲁木齐: 新疆农业大学, 2017.
WANG Y M. Isolation, identification, pathogenicity detection and screening antibacterial drugs of Streptococcus equi subspecies equi[D]. Urumqi: Xinjiang Agricultural University, 2017. (in Chinese)
[2] 买占海, 沙娅·奴尔兰, 王德超, 等. 马腺疫链球菌fneB基因LAMP检测方法的建立及应用[J]. 中国动物传染病学报, 2021, 3: 1-7.
MAI Z H, SHA Y, WANG D C, et al. Establishment and application of LAMP detection method for fneB gene of Streptococcus equi[J]. Chinese Journal of Animal Infectious Diseases, 2021, 3: 1-7. (in Chinese)
[3] NEWTON J R, VERHEYEN K, TALBOT N C, et al. Control of strangles outbreaks by isolation of guttural pouch carriers identified using PCR and culture of Streptococcus equi[J]. Equine Veterinary Journal, 2000, 32(6): 515-526.
[4] MAY J P, WALKER C A, MASKELL D J, et al. Development of an in vivo Himar1 transposon mutagenesis system for use in Streptococcus equi subsp. equi[J]. FEMS Microbiology Letters, 2004, 238(2): 401-409.
[5] 柯春林, 乔德亮, 曾晓雄. 兽疫链球菌荚膜多糖对免疫抑制鼠的抗氧化和免疫调节活性研究[J]. 中国微生态学杂志, 2011, 23(8): 684-688.
KE C L, QIAO D L, ZENG X X. Antioxidant and immunomodulatory acitivity of the capsule polysaccharides from Streptococcus equi subsp. Zooepidemicus in immunosuppressed mice[J]. Chinese Journal of Microecology, 2011, 23(8): 684-688. (in Chinese)
[6] BOYLE A G, RANKIN S C, DUFE L. et al. Streptococcus equi detection polymerase chain reaction assay for equine nasopharyngeal and guttural pouch wash samples[J]. Journal of Veterinary Internal Medicine, 2016, 30(1): 276-281.
[7] 王雨朦, 何国文, 买占海, 等. 马链球菌马亚种的分离鉴定及fneB基因序列分析[J]. 动物医学进展, 2017, 38(10): 8-12.
WANG Y M, HE G W, MAI Z H, et al. Isolation, identification and fneB gene sequencing of Streptococcus equi subspecies equi[J]. Progress in Veterinary Medicine, 2017, 38(10): 8-12. (in Chinese)
[8] 买占海. 马腺疫、马急腹症的临床病案分析[D]. 乌鲁木齐: 新疆农业大学, 2015.
MAI Z H. Clinical analysis of cases about horse strangles and acute abdominal disease[D]. Urumqi: Xinjiang Agricultural University, 2015. (in Chinese)
[9] 吴娇, 王聪, 于海川. 金银花中的化学成分及其药理作用研究进展[J]. 中国实验方剂学杂志, 2019, 25(4): 225-234.
WU J, WANG C, YU H C. Chemical constituents and pharmacological effect of Lonicerae Japonicae Flos[J]. Chinese Journal of Experimental Traditional Medical Formulae, 2019, 25(4): 225-234. (in Chinese)
[10] 宋亚玲, 王红梅, 倪付勇, 等. 金银花中酚酸类成分及其抗炎活性研究[J]. 中草药, 2015, 46(4): 490-495.
SONG Y L, WANG H M, NI F Y, et al. Study on anti-inflammatory activities of phenolic acids from Lonicerae Japonicae Flos[J]. Chinese Traditional and Herbal Drugs, 2015, 46(4): 490-495. (in Chinese)
[11] 赵英妹, 乔昀. 8种中草药临床常见菌株体外抑菌活性分析[J]. 检验医学, 2019, 34(11): 987-990.
ZHAO Y M, QIAO Y. Antibacterial activities of 8 kinds of Chinese herbal medicines to clinical common isolates in vitro[J]. Laboratory Medicine, 2019, 34(11): 987-990. (in Chinese)
[12] 高铎, 马峰涛, 孙鹏. 金银花提取物的生物学功能及其在养殖中的应用[J]. 动物营养学报, 2019, 31(5): 2045-2051.
GAO D, MA F T, SUN P. Biological function of honeysuckle extract and its application in breeding industry[J]. Chinese Journal of Animal Nutrition, 2019, 31(5): 2045-2051. (in Chinese)
[13] 翟云逸, 姜胜男, 冉彬, 等. 金银花的药理作用及其在兽医临床中的应用[J]. 动物医学进展, 2019, 40(4): 128-131.
ZHAI Y Y, JIANG S N, RAN B, et al. Pharmcological action of Lonicera japonica and its application in veterinary clinic[J]. Progress in Veterinary Medicine, 2019, 40(4): 128-131. (in Chinese)
[14] YANG B, MENG Z, MAY, et al. One new conjugate of a secoiridoid glucoside with a sesquiterpene glucoside from the flower buds of Lonicera japonica[J]. Natural Product Communication, 2015, 10(9): 1499-1500.
[15] 王清, 朱萱萱, 张赤兵, 等. 金银花提取物抗菌作用的实验研究[J]. 中国医药导刊, 2008, 10(9): 1428-1430.
WANG Q, ZHU X X, ZHANG C B, et al. Experimental study on honeysuckle extract against bacteria[J]. Chinese Journal of Medicinal Guide, 2008, 10(9): 1428-1430. (in Chinese)
[16] 齐丽娜, 陈炫好, 金华, 等. 中药连翘化学成分及药理活性研究进展[J]. 天津中医药大学学报, 2021, 40(2): 168-175.
QI L N, CHEN X H, JIN H, et al. Research progress on the chemical constituents and pharmacological activities of Forsythiae fructus[J]. Journal of Tianjin University of Traditional Chinese Medicine, 2021, 40(2): 168-175. (in Chinese)
[17] 杜会枝, 马红. 连翘对神经系统的药理作用研究进展[J]. 中国药学杂志, 2021, 56(7): 526-530.
DU H Z, MA H. Research progress in pharmacological effects of Forsythia suspensa (Thunb. ) vahl on the nervous system[J]. Chinese Pharmaceutical Journal, 2021, 56(7): 526-530. (in Chinese)
[18] 沈霞, 徐蓉蓉, 裴丽珊, 等. 基于网络药理学连翘清热解毒功效的分子机制研究[J]. 药学学报, 2018, 53(11): 1834-1842.
SHEN X, XU R R, PEI L S, et al. Molecular mechanism of heat-clearing and detoxifying efficacy of Forsythia suspensa based on network pharmacology[J]. Acta Pharmaceutica Sinica, 2018, 53(11): 1834-1842. (in Chinese)
[19] 孔宪琴, 梁航, 杨国胜. 五味消毒饮对猪链球菌病病原体的体外抑菌试验[J]. 中兽医学杂志, 2009, 148(3): 10-13.
KONG X Q, LIANG H, YANG G S, et al. Antibacterial effect of Wuwei Xiaodu Decoction on Streptococcus suis in vitro[J]. Chinese Journal of Traditional Veterinary Science, 2009, 148(3): 10-13. (in Chinese)
[20] 孙丹岚, 买占海, 佟盼盼, 等. 北疆地区马链球菌分离鉴定及耐药性和毒力基因分析[J]. 中国动物传染病学报, 2021, 5: 1-9.
SUN D L, MAI Z H, TONG P P, et al. Isolation and identification and analysis of resistance and virulence genes of Streptococcus equi on Northern Xinjiang[J]. Chinese Journal of Animal Infectious Diseases, 2021, 5: 1-9. (in Chinese)
[21] 陈士林, 刘安, 李琦, 等. 中药饮片标准汤剂研究策略[J]. 中国中药杂志, 2016, 41(8): 1367-1375.
CHEN S L, LIU A, LI Q, et al. Research strategies in standard decoction of medicinal slices[J]. China Journal of Chinese Materia Medica, 2016, 41(8): 1367-1375. (in Chinese)
[22] 熊晓顺, 林祥芳, 张楠, 等. 肺炎链球菌毒力基因表达差异性及对大环内酯类耐药性分析[J]. 中华医院感染学杂志, 2020, 30(5): 641-647.
XIONG X S, LIN X F, ZHANG N, et al. Differences of virulence gene expression in Streptococcus pneumonia and analysis of resistance to macrolide[J]. Chinese Journal of Nosocomiology, 2020, 30(5): 641-647. (in Chinese)
[23] 王琳, 杨阳, 张一颖, 等. 中药抗菌作用文献研究现状[J]. 世界中西医结合杂志, 2017, 12(12): 1663-1665.
WANG L, YANG Y, ZHANG Y Y, et al. Literature research of the anti-bacterial effects of Chinese herbal medicines[J]. World Journal of Integrated Traditional and Western Medicine, 2017, 12(12): 1663-1665. (in Chinese)
[24] 陶飞. 5种中药提取物对蛇源性细菌耐药基因影响的初步研究[D]. 南宁: 广西大学, 2018.
TAO F. Study on the preliminary effect of five kinds of traditional Chinese medicine extracts on resistance genes of snake-derived bacteria[D]. Nanning: Guangxi University, 2018. (in Chinese)
[25] 陈俭清, 周永辉, 杨艳北, 等. 中药水提物对体外猪链球菌生物被膜作用的试验[J]. 中国兽医杂志, 2016, 52(11): 14-16.
CHEN J Q, ZHOU Y H, YANG Y B, et al. Screening of Chinese herbal extracts against Streptococcus suis biofilm formation[J]. Chinese Journal of Veterinary Medicine, 2016, 52(11): 14-16. (in Chinese)
[26] 王嵩. 中草药抗细菌感染的研究[J]. 北京中医, 2002, 21(4): 249-251.
WANG S. Study on anti bacterial infection of Chinese herbal medicine[J]. Beijing Journal of Traditional Chinese Medicine, 2002, 21(4): 249-251. (in Chinese)
[27] 魏振装. 中草药抗感染作用机理探讨[J]. 四川中医, 1991, 9(4): 19-20.
WEI Z Z, Discussion on anti infection mechanism of Chinese herbal medicine[J]. Journal of Sichuan of Traditional Chinese Medicine, 1991, 9(4): 19-20. (in Chinese)
[28] MINAMI M, TAKASE H, NAKAMURA M, et al. Methanol extract of Lonicera caerulea var. emphyllocalyx fruit has antibacterial and anti-biofilm activity against Streptococcus pyogenes in vitro[J]. Bioscience Trends, 2019, 13(2): 145-151.
[29] MINAMI M, TAKASE H, NAKAMURA M, et al. Methanol extract of Lonicera caerulea var. emphyllocalyx fruit has anti-motility and anti-biofilm activity against enteropathogenic Escherichia coli[J]. Drug Discoveries & Therapeutics, 2019, 13(6): 335-342.