硒化甘草多糖、甘草多糖及其联合抗生素对无乳链球菌体外抗菌活性及机制分析

doi:10.6048/j.issn.1001-4330.2024.02.024

新疆农业科学 ›› 2024, Vol. 61 ›› Issue (2): 469-478.DOI: 10.6048/j.issn.1001-4330.2024.02.024

• 微生物·生理生化·畜牧兽医 • 上一篇下一篇

硒化甘草多糖、甘草多糖及其联合抗生素对无乳链球菌体外抗菌活性及机制分析

张梦圆¹(), 朱晓庆¹(), 谷新利¹(), 王燕¹, 杨慧莹¹, 刘雨然¹, 王蒙蒙¹, 孙国结¹, 连科迅¹, 李效振²

1.石河子大学动物科技学院,新疆石河子 832000
2.乌鲁木齐海关技术中心,乌鲁木齐 830000

收稿日期:2023-06-18 出版日期:2024-02-20 发布日期:2024-03-19
通信作者: 朱晓庆(1981-),女,甘肃金塔人,讲师,博士,研究方向为中药免疫药理学,(E-mail)zxhqqmy@126.com；
谷新利(1963-),男,河南许昌人,教授,博士,硕士生/博士生导师,研究方向为中兽药开发与应用,(E-mail)xlgu@shzu.edu.cn
作者简介:张梦圆(1996-),女,安徽阜阳人,硕士研究生,研究方向为中药免疫药理学, (E-mail)330941027@qq.com
基金资助:
国家自然科学基金项目(32060814)

Study on antibacterial activity and mechanism of selenium glycyrrhiza polysaccharide, glycyrrhiza polysaccharide and their combined antibiotics against Streptococcus agalactiae in vitro

ZHANG Mengyuan¹(), ZHU Xiaoqing¹(), GU Xinli¹(), WANG Yan¹, YANG Huiying¹, LIU Yuran¹, WANG Mengmeng¹, SUN Guojie¹, LIAN Kexun¹, LI Xiaozhen²

1. College of Animal Science and Techoology, Shihezi University, Shihezi Xinjiang 832003, China
2. Urumqi Customs Technical Center, Urumqi 830000, China

Received:2023-06-18 Published:2024-02-20 Online:2024-03-19
Supported by:
National Natural Science Foundation of China(32060814)

摘要/Abstract

摘要：

【目的】分析硒化乌拉尔甘草多糖(selenium glycyrrhiza polysaccharide,SeGUP)、乌拉尔甘草多糖(glycymhiza polyacchiade,GUP)对无乳链球菌体外抗菌活性及机制,研究其联合的抗菌效果。【方法】通过药敏纸片筛选出对无乳链球菌敏感抗生素,采用最小二倍稀释法和棋盘法测得最小抑菌浓度(minimal inhibit concentration,MIC)、最小杀菌浓度(minimum bactericidal concentration,MBC)和联合抑菌指数(FICI index,FICI);将无乳链球菌与120、60、30 mg/mL的SeGUP、GUP及抗生素分别等量混合后绘制细菌生长速率图;通过ELISA法检测细菌培养液中碱性磷酸酶、β-半乳糖苷酶活性、蛋白质含量及胞内DNA浓度,研究SeGUP、GUP及联合抗生素对细菌细胞膜、细胞壁及DNA的影响;采用结晶紫染色法检测SeGUP、GUP及联合抗生素对无乳链球菌生物被膜形成的影响。【结果】无乳链球菌对SeGUP 480 mg/mL、GUP 480 mg/mL均呈现中度敏感,抑菌圈直径为14.0、11.0 mm;无乳链球菌对盐酸卡那霉素、盐酸多西环素、头孢曲松钠、氟苯尼考极敏;SeGUP和GUP分别对肺炎链球菌的MIC、MBC为120、240 mg/mL和240、480 mg/mL;SeGUP与盐酸卡那霉素、盐酸多西环素、头孢曲松钠、氟苯尼考的FICI为1.5、0.75、1、0.5,SeGUP与盐酸卡那霉素之间关系为无关,与盐酸多西环素、头孢曲松钠之间为相加关系,与氟苯尼考间为协同关系,GUP与盐酸卡那霉素、盐酸多西环素、头孢曲松钠、氟苯尼考的FICI为1.5、1、1.5、0.75,GUP与盐酸卡那霉素、头孢曲松钠之间关系为无关,与盐酸多西环素、氟苯尼考间为相加关系;SeGUP、GUP明显抑制细菌的生长,其中SeGUP 120 mg/mL效果最强,SeGUP、GUP与盐酸多西环素、头孢曲松钠、氟苯尼考联合使用后增强了抑制效;SeGUP、GUP及联合抗生素使用后均能显著升高培养液中碱性磷酸酶(AKP)、β-半乳糖苷酶(β-gal)、蛋白质含量(P<0.05),显著降低胞内DNA含量(P<0.05),显著抑制无乳链球菌生物被膜的形成(P<0.05)。【结论】SeGUP能够通过损伤细菌细胞壁、细胞膜完整性,影响DNA含量对无乳链球菌产生明显的抑制作用,且其抗菌活性优于GUP。SeGUP与抗生素联合使用后能够增强抗生素的抗菌活性,并且其作用效果强于单独使用SeGUP、GUP和抗生素。

关键词: 乌拉尔甘草多糖; 硒化乌拉尔甘草多糖; 联合抗菌; 无乳链球菌; 生物膜

Abstract:

【Objective】 To study the antibacterial activity and mechanism of selenium glycyrrhiza polysaccharide(SeGUP)and glycyrrhiza polysaccharide(GUP)against Streptococcus agalactiae in vitro, and determine whether the combination of them can enhance the antibacterial effect of antibiotics.【Methods】 Antibiotics sensitive to Streptococcus agalactis were screened by drug sensitive paper.The minimum inhibitory concentration(MIC), minimum bactericidal concentration(MBC)and joint inhibitory index(FICI)were measured by the minimum double dilution method and chessboard method; Mixed Streptococcus agalactis with 120, 60, 30 mg/mL SeGUP, GUP and antibiotics in equal amounts respectively were used to draw the bacterial growth rate diagram; Meanwhile, detection of alkaline phosphatase β-Galactosidase activity, protein content and intracellular DNA concentration were studied to find the effects of SeGUP, GUP and combined antibiotics on bacterial cell membrane, cell wall and DNA; Finally, the effect of SeGUP, GUP and combined antibiotics on the biofilm formation of Streptococcus agalactis was detected by crystal violet staining.【Results】 Streptococcus agalactis was moderately sensitive to SeGUP 480 mg/mL and GUP 480 mg/mL, and the diameter of the inhibition zone was 14.0 mm and 11.0 mm; Streptococcus agalactis is extremely sensitive to kanamycin hydrochloride, doxycycline hydrochloride, ceftriaxone sodium and florfenicol; MIC and MBC of SeGUP and GUP against Streptococcus pneumoniae were 120, 240 mg/mL and 240, 480 mg/mL respectively; The FICIs of SeGUP with kanamycin hydrochloride, doxycycline hydrochloride, ceftriaxone sodium, and florfenicol were 1.5, 0.75, 1, 0.5, indicating that the relationship between SeGUP and kanamycin hydrochloride was irrelevant, the relationship between SeGUP and doxycycline hydrochloride, ceftriaxone sodium was additive, and the relationship between SeGUP and florfenicol was synergistic.The FICIs of GUP with kanamycin hydrochloride, doxycycline hydrochloride, ceftriaxone sodium, and florfenicol were 1.5, 1, 1.5, 0.75, The results showed that GUP had no relationship with kanamycin hydrochloride and ceftriaxone sodium, but additive relationship with doxycycline hydrochloride and florfenicol; SeGUP and GUP significantly inhibited the growth of bacteria, of which SeGUP 120 mg/mL had the strongest effect.The combination of SeGUP and GUP with doxycycline hydrochloride, ceftriaxone sodium and florfenicol has enhanced the inhibition effect; SeGUP, GUP and combination of antibiotics could significantly increase alkaline phosphatase(AKP)β-Galactosidase(β-gal), protein content(P<0.05), significantly reduced the content of intracellular DNA(P < 0.05), significantly inhibited the formation of biofilm of Streptococcus agalactis(P < 0.05)【Conclusion】 SeGUP can significantly inhibit Streptococcus agalactis by damaging the integrity of bacterial cell wall and cell membrane and affect DNA content, and its antibacterial activity is superior to that of GUP.The combination of SeGUP and antibiotics can enhance the antibacterial activity of antibiotics, and its effect is stronger than those of SeGUP, GUP and antibiotics used alone.

Key words: glycymhiza polyacchiade; selenium glycyrrhiza polysaccharide; combined antibacterial; Streptococcus agalactiae; biofilm

中图分类号:

S188

张梦圆, 朱晓庆, 谷新利, 王燕, 杨慧莹, 刘雨然, 王蒙蒙, 孙国结, 连科迅, 李效振. 硒化甘草多糖、甘草多糖及其联合抗生素对无乳链球菌体外抗菌活性及机制分析[J]. 新疆农业科学, 2024, 61(2): 469-478.

ZHANG Mengyuan, ZHU Xiaoqing, GU Xinli, WANG Yan, YANG Huiying, LIU Yuran, WANG Mengmeng, SUN Guojie, LIAN Kexun, LI Xiaozhen. Study on antibacterial activity and mechanism of selenium glycyrrhiza polysaccharide, glycyrrhiza polysaccharide and their combined antibiotics against Streptococcus agalactiae in vitro[J]. Xinjiang Agricultural Sciences, 2024, 61(2): 469-478.

图/表 7

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药物名称 Drug name	抑菌圈直径 Diameter of bacteriostatic ring(mm)
阿莫西林Amoxicillin	17.0
头孢曲松钠Ceftriaxone sodium	27.0
盐酸卡那霉素Kanamycin hydrochloride	22.0
盐酸多西环素Doxycycline hydrochloride	26.0
链霉素streptomycin	1.5
氟苯尼考Florfenicol	28.0
SeGUP 120 mg/mL	9.0
SeGUP 240 mg/mL	12.0
SeGUP 480 mg/mL	14.0
GUP 120 mg/mL	6.0
GUP 240 mg/mL	9.0
GUP 480 mg/mL	11.0

药物名称 Drug name	抑菌圈直径 Diameter of bacteriostatic ring(mm)
阿莫西林Amoxicillin	17.0
头孢曲松钠Ceftriaxone sodium	27.0
盐酸卡那霉素Kanamycin hydrochloride	22.0
盐酸多西环素Doxycycline hydrochloride	26.0
链霉素streptomycin	1.5
氟苯尼考Florfenicol	28.0
SeGUP 120 mg/mL	9.0
SeGUP 240 mg/mL	12.0
SeGUP 480 mg/mL	14.0
GUP 120 mg/mL	6.0
GUP 240 mg/mL	9.0
GUP 480 mg/mL	11.0

药物名称 Drug name	单用MIC Signle use MIC	MBC	与SeGUP 联用后抗生素浓度 Antibiotic concentration afer combination with SeGUP	联用后 SeGUP 浓度 Concentration of SeGUP after combinet use	FICI	关系 Relation- ship	与GUP联用后抗生素浓度 Antibiotic concentration afer combination with GUP	联用后 GUP浓度 Concentration of GUP after combinet use	FICI	关系 Relation- ship
盐酸卡那霉素 Kanamycin hydrochloride (μg/mL)	31.25	62.5	15.63	120	1.5	无关	15.63	240	1.5	无关
盐酸多西环素 Doxycycline hydrochloride (μg/mL)	15.63	31.25	3.91	60	0.75	相加	7.81	120	1	相加
头孢曲松钠 Ceftriaxone sodium (μg/mL)	62.5	125	31.25	60	1	相加	31.25	240	1.5	无关
氟苯尼考 Florfenicol μg/mL	31.25	62.5	7.81	30	0.5	协同	7.81	120	0.75	相加
SeGUP (mg/mL)	120	120
GUP (mg/mL)	240	480

药物名称 Drug name	单用MIC Signle use MIC	MBC	与SeGUP 联用后抗生素浓度 Antibiotic concentration afer combination with SeGUP	联用后 SeGUP 浓度 Concentration of SeGUP after combinet use	FICI	关系 Relation- ship	与GUP联用后抗生素浓度 Antibiotic concentration afer combination with GUP	联用后 GUP浓度 Concentration of GUP after combinet use	FICI	关系 Relation- ship
盐酸卡那霉素 Kanamycin hydrochloride (μg/mL)	31.25	62.5	15.63	120	1.5	无关	15.63	240	1.5	无关
盐酸多西环素 Doxycycline hydrochloride (μg/mL)	15.63	31.25	3.91	60	0.75	相加	7.81	120	1	相加
头孢曲松钠 Ceftriaxone sodium (μg/mL)	62.5	125	31.25	60	1	相加	31.25	240	1.5	无关
氟苯尼考 Florfenicol μg/mL	31.25	62.5	7.81	30	0.5	协同	7.81	120	0.75	相加
SeGUP (mg/mL)	120	120
GUP (mg/mL)	240	480

药物Drug	AKP(ng/L)	β-gal(pg/mL)	蛋白质 Protein(μg/mL)	DNA(ng/μL)
SeGUP 120 mg/mL	202.97±4.32^b	105.43±2.51^e	640.40±4.62^e	58.63±2.06^j
SeGUP 60 mg/mL	173.07±3.67^e	94.70±2.34^f	532.70±7.41^gh	81.87±2.47^h
SeGUP 30 mg/mL	143.37±2.14^j	75.37±2.05^gh	471.57±8.21^j	91.60±1.65^g
GUP 120 mg/mL	182.43±2.56^d	92.80±1.87^f	542.47±2.77^f	82.37±2.04^h
GUP 60 mg/mL	151.70±0.75ⁱ	74.67±1.20^ghi	477.73±4.54^j	113.47±4.21^de
GUP 30 mg/mL	104.70±2.80^k	53.67±2.91l	384.43±6.96l	153.37±3.95^b
1/2盐酸卡那霉素MIC Kanamycin hydrochloride	158.40±4.20^gh	66.33±2.06^k	457.53±5.81^k	93.73±4.21^g
1/2盐酸多西环素MIC Doxycycline hydrochloride	151.43±2.10ⁱ	74.00±1.97hi	471.67±7.75^j	108.67±2.78^e
1/2头孢曲松钠MIC Ceftriaxone sodium	162.70±4.11^g	66.20±2.17^k	527.43±6.29^h	120.37±1.72^c
1/2氟苯尼考MIC Florfenicol	153.63±3.65^hi	78.20±1.85ⁱ	546.47±5.14^f	83.40±1.66^h
1/2(盐酸卡那霉素MIC+SeGUP MIC)	163.23±5.58^fg	76.50±1.83^gh	504.00±4.97ⁱ	100.27±6.12^f
1/2(盐酸多西环素MIC+SeGUP MIC)	217.43±3.48^a	123.83±2.12^bg	748.70±4.28^b	47.30±1.87^k
1/2(头孢曲松钠MIC+SeGUP MIC)	202.60±3.54^b	121.00±2.35^bc	705.57±4.15^c	51.70±2.15^k
1/2(氟苯尼考MIC+SeGUP MIC)	219.43±2.61^a	129.57±2.10^a	789.37±1.46^a	57.40±2.40^j
1/2(盐酸卡那霉素MIC+GUP MIC)	168.93±1.65^ef	68.30±1.90^jk	462.60±5.20^k	117.70±2.91^cd
1/2(盐酸多西环素MIC+GUP MIC)	197.90±5.45^bc	119.57±2.06^c	679.93±1.38^d	61.37±2.23^ij
1/2(头孢曲松钠MIC+GUP MIC)	155.27±1.88^hi	71.07±2.70^ij	539.03±1.76^fg	118.87±2.90^cd
1/2(氟苯尼考MIC+GUP MIC)	193.13±5.05^c	115.50±3.10^d	713.23±3.35^c	66.30±2.95ⁱ
空白对照组	29.00±1.15l	31.67±1.96^m	102.00±3.25^m	215.00±4.56^a

硒化甘草多糖、甘草多糖及其联合抗生素对无乳链球菌体外抗菌活性及机制分析

Study on antibacterial activity and mechanism of selenium glycyrrhiza polysaccharide, glycyrrhiza polysaccharide and their combined antibiotics against Streptococcus agalactiae in vitro

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