CN113604535A - 消除大肠杆菌药物耐药性的中药组方及其筛选方法与应用 - Google Patents
消除大肠杆菌药物耐药性的中药组方及其筛选方法与应用 Download PDFInfo
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Abstract
本发明提供了一种组成消除耐药中药组方及筛选方法，试验选择4株多重耐药大肠杆菌作为研究对象，分别以亚抑菌浓度(1/2MIC)中药与4株菌分别共培养72h。然后采用凝胶电泳法、双纸片法、荧光定量PCR和生物膜阳性筛选方法检测细菌培养物，根据体外消除细菌耐药效果，确定消除耐药效果较好单味中药组成组方；再通过动物试验，检查各组心肝组织病理学变化，并统计各组治愈率、有效率、死亡率，并验证组方体内消除大肠杆菌耐药的效果。本发明组方山楂黄连组方+抗生素效果优于传统组方三黄汤，说明利用此种方法组成的组方治疗效果优于根据中药辩证论治组成的传统组方。
Description
技术领域
本发明属于中药制剂技术领域，具体涉及消除大肠杆菌药物耐药性的中药组方及其筛选方法。
背景技术
近年来，大肠杆菌病在国内养禽场广泛流行，发病率和死亡率居高不下，严重威胁着养禽业发展，造成巨大经济损失。长期以来，在治疗该病过程中，抗菌药物发挥极其重要作用。但抗菌药物广泛、持续和不合理使用，加大耐药大肠杆菌尤其是产超广谱β-内酰胺酶大肠杆菌的产生和扩散，造成药物治疗效果不理想，已成为阻碍养禽业发展的难题之一。由此可见，细菌产生耐药性是导致上述结果的主要原因。因此降低大肠杆菌耐药性，恢复对部分或全部抗生素的敏感性对于临床治疗细菌感染及防止细菌耐药性传播具有重要意义。降低细菌耐药性有多种途径，其中低毒、成本低廉的中药和天然植物备而受关注，因此，利用中药消除鸡源大肠杆菌耐药性成为一个可深入探索的研究方向。
中药通过消除R质粒、抑制ESBLs活性、影响细菌生长速度和蛋白表达量、抑制主动外排系统和细菌生物膜形成等作用机制，使细菌恢复对抗菌素敏感性。研究表明，中药可改变大肠杆菌特异性耐药基因和非特异性耐药基因的转录和翻译水平，从根本上逆转细菌的耐药性。然而细菌多重耐药机制证明多重耐药是由多种因素决定的，单一的逆转制剂不可能抑制所有的耐药机制，多种逆转制剂联合应用针对多重耐药机制可能更有效。通过这种策略，即组合单体或复合天然提取物可以发挥协同对抗作用并且影响药物多靶点。目前中药配伍遵循中药辩证论治结合中兽医传统理论，而本发明针对不同耐药机制，可全方位、多角度的消除细菌多重耐药性，延长抗生素使用寿命。
发明内容
本发明的目的是为了解决现有技术中存在的缺点，提供一种安全有效地用来消除对鸡源大肠杆菌β-内酰胺类药物耐药的中药组合物及其筛选方法。
本发明采用的技术方案为：一种消除大肠杆菌药物耐药性的中药组方筛选方法，包括以下步骤：
A、根据消除大肠杆菌耐药机制筛选单味中药，所述耐药机制主要包括R质粒介导、产生ESBLs、通过主动外排系统和形成生物膜；
B、采用凝胶电泳方法筛选消除R质粒的单味中药；
C、利用双纸片法筛选抑制ESBLs活性的单味中药；
D、利用荧光定量PCR方法筛选降低外排系统基因Acra-mRNA、Acrb-mRNA表达量的单味中药；
E、利用生物膜阳性筛选方法，筛选出降低生物膜黏附性的单味中药；
F、将步骤B-E中筛选出的单味中药组成中药组合物。
进一步地，所述筛选方法还包括：
A-1、中药水提物制备：将单味中药浸泡30min，文火熬制2次，合并浓缩、过滤除菌，浓度为1g/mL以生药计，得到单味中药水提物；
A-2、大肠杆菌菌液制备：将冷冻保存菌液接种于LB液体培养基中，37℃培养过夜，将活化菌液划线接种于麦康凯琼脂平板，37℃倒置培养过夜，挑取单菌落接种于LB液体培养基中，37℃培养过夜，得到大肠杆菌液。
一种消除大肠杆菌药物耐药性的中药组方，所述中药组合物为山楂1份和黄连1份，所述中药组合物与抗生素联合使用，所述抗生素为头孢噻肟，所述抗生素与中药组合物的重量份数比为(2～5)：10。
一种中药组方的应用，所述中药组方用于消除鸡源大肠杆菌β-内酰胺类药物耐药性。
本发明获得的有益效果为：本发明试验选择4株多重耐药大肠杆菌作为研究对象，采用梯度稀释法测定6种中药水提物对4株耐药大肠杆菌最低抑菌浓度(MIC)，分别以亚抑菌浓度(1/2MIC)与4株菌分别共培养72h；然后采用凝胶电泳法、双纸片法、荧光定量PCR和生物膜阳性筛选方法检测培养物，根据体外消除细菌耐药效果，确定消除耐药效果较好单味中药组成组方；(山楂、舌草；山楂、黄连)；再通过动物试验，检查各组心肝组织病理学变化，并统计各组治愈率、有效率、死亡率，并检测组方体内消除大肠杆菌耐药效果。本发明筛选的山楂黄连组方+头孢噻肟治疗有效率高达93.3、治愈率达90％、死亡率为6.7％；治疗效果最佳，优于山楂舌草组方+抗生素和传统组方三黄汤。
附图说明
图1a为单味中药处理第1株鸡源大肠杆菌前后质粒电泳图谱；
图1b为单味中药处理第2株鸡源大肠杆菌前后质粒电泳图谱；
图1c为单味中药处理第3株鸡源大肠杆菌前后质粒电泳图谱；
图1d为单味中药处理第4株鸡源大肠杆菌前后质粒电泳图谱；
图2为4株鸡源大肠杆菌经中药水提物处理后生物膜粘附性分析结果；
图3为4株鸡源大肠杆菌经中药水提物处理前后外排泵基因AcrA-mRNA相对表达量(2—△△CT)；
图4为4株鸡源大肠杆菌经中药水提物处理前后外排泵基因AcrB-mRNA相对表达量(2—△△CT)；
图5为鸡病理剖检图；
图6为分离菌株菌落形态图；
图7为分离菌株细菌形态图；
图8为健康鸡心脏H.E.染色(10×10)；
图9为患病鸡心脏H.E.染色(10×10、20×10)；
图10为健康鸡肝脏H.E.染色(10×10)；
图11为患病鸡肝脏H.E.染色(10×10)；
图12为分离菌株16S rDNA基因扩增产物电泳结果；
其中图1a-1d中a：黄连处理的菌株；b：舌草处理的菌株；c：蒲公英处理的菌株d：大黄处理的菌株；e：黄芩处理的菌株；f：地锦草处理的菌株。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。
实施例1消除鸡源大肠杆菌R质粒单味中药筛选
(1)中药水提物制备：黄芩、黄连、大黄、地锦、蒲公英、舌草均购自同仁堂(邯郸)，浸泡30min，文火熬制2次，合并浓缩，过滤除菌，浓度以生药计(1g/mL)。
(2)鸡源大肠杆菌菌液制备：将冷冻保存菌液接种于LB液体培养基中，37℃培养过夜。将活化菌液划线接种于麦康凯琼脂平板，37℃倒置培养过夜。挑取单菌落接种于LB液体培养基中，37℃过夜培养。
(3)中药水提物MIC测定：采用二倍稀释法测定黄芩、黄连、大黄、地锦、蒲公英、舌草对4株鸡源大肠杆菌最低抑菌浓度，以无菌生长的最低稀释度即定为MIC值。
表1 6种中药水提物对4株大肠杆菌MIC测定(mg/mL)
根据表1结果显示黄连、大黄、黄芩对4株大肠杆菌MIC为30～60mg/mL，蒲公英、舌草、地锦草对4株大肠杆菌MIC均大于60mg/mL。
(4)中药水提物处理菌液制备：在浓度为1×106cfu/mL菌液试管中加入终浓度1/2MIC中药液，37℃摇动培养18～24h后，将此菌液重新接种至LB液体培养基中，37℃摇动培养4-4.5h。重复以上步骤连续作用72h。
(5)中药水提物对大肠杆菌R质粒影响：按照南京诺唯赞生物科技有限公司质粒抽提试剂盒说明书操作提取质粒，检测中药水提物处理菌液前后耐药质粒消除情况。如图1a-1d所示，4株大肠杆菌均携带2300、3200、21000三个质粒条带，E-1、E-2、E-3、E-4经黄连处理后与处理前相比质粒条带减少1-3条，E-4经舌草处理后质粒条带减少2条，且消除范围在2300-21000bp。说明与其它中药相比舌草和黄连消除R质粒效果更佳。
实施例2抑制鸡源大肠杆菌ESBLs活性单味中药筛选
鸡源大肠杆菌菌液制备、中药水提物制备、中药水提物处理菌液制备、中药水提物MIC测定同
实施例1
采用K-B法测定中药水提物对ESBLs活性影响
结果判定：若CAZ/CA比CAZ抑菌圈直径≥5mm，或CTX/CA比CTX抑菌圈直径≥5mm，则判断为ESBLs阳性。
表2 E-1菌对头孢噻肟、头孢噻肟/克拉维酸、头孢他啶、头孢他啶/克拉维酸抑菌圈
注：肩标代表差异显著，字母相同代表差异不显著(P＜0.05)，下同。
表3 E-2菌对头孢噻肟、头孢噻肟/克拉维酸、头孢他啶、头孢他啶/克拉维酸抑菌圈
表4 E-3菌对头孢噻肟、头孢噻肟/克拉维酸、头孢他啶、头孢他啶/克拉维酸抑菌圈
表5 E-4菌对头孢噻肟、头孢噻肟/克拉维酸、头孢他啶、头孢他啶/克拉维酸抑菌圈
表2可见：含酶抑制剂纸片比单药纸片抑菌圈直径＜5mm，E-1判定为产ESBLs阴性菌。表3可见：E-2含酶抑制剂纸片比单药纸片抑菌圈直径≥5判定为产ESBLs阳性菌，与对照组相比CAZ和CAZ/CA、CTX和CTX/CA两组药敏纸片抑菌圈直径增大，且黄连肩标字母与对照组不同差异显著，说明黄连可抑制ESBLs活性。由表4可见：含酶抑制剂纸片比单药纸片抑菌圈直径＜5mm，E-3判定为产ESBLs阴性菌。由表5可见：E-4含酶抑制剂纸片比单药纸片抑菌圈直径≥5判定为产ESBLs阳性菌，与对照组相比CAZ和CAZ/CA、CTX和CTX/CA两组药敏纸片抑菌圈直径增大，且黄连组肩标字母与对照组不同，差异显著，说明黄连可抑制ESBLs活性。由以上结果可以看出，与其它中药相比黄连抑制ESBLs活性更佳。
实施例3影响鸡源大肠杆菌生物膜黏附性单味中药筛选
鸡源大肠杆菌菌液制备、中药水提物制备、中药水提物处理菌液制备、中药水提物MIC测定同
实施例1
采用生物膜阳性筛选方法测定中药水提物对大肠杆菌生物膜黏附性影响，计算和结果判定：测定值-空白值＞0.12判为生物膜阳性，临界值(AC):空白对照组平均OD600值+3×标准差；不黏附(-)A＜AC，弱黏附性(+)AC＜A＜2AC，中等黏附性(++)2AC＜A＜4AC，强黏附性(+++)A＞4AC。
如图2所示，E-1测定值-空白值<0.12，判定为生物膜阴性菌；E-2经黄连、舌草处理后由中等黏附性(++)变为弱黏附性(+)；E-3经黄连、舌草处理后由强黏附性(+++)变为弱黏附性(+)、经蒲公英、地锦草处理后变为中等黏附性(++)；E-4经黄连、舌草处理后由中等黏附性(++)变为弱黏附性(+)。从以上结果可以看出，黄连、舌草可至4株菌生物膜粘附性下降，而地锦草、蒲公英可至E-3生物膜粘附性下降。
实施例4抑制鸡源大肠杆菌主动外排系统单味中药筛选
鸡源大肠杆菌菌液制备、中药水提物制备、中药水提物处理菌液制备、中药水提物MIC测定同
实施例1
利用荧光定量PCR方法检测中药水提物对大肠杆菌主动外排系统(Acra-mRNA、Acrb-mRNA)影响。如图3所示与中药处理前相比，黄连可降低4株菌AcrA-mRNA表达量，且差异显著(*P<0.05)。舌草处理后，E-3、E-4AcrA-mRNA表达量下降，且差异显著(*P<0.05)。地锦草处理后E-1、E-4AcrA-mRNA表达量下降，且差异显著(*P<0.05)。如图4所示，与中药处理前相比，黄连可降低4株菌AcrB-mRNA表达量，且E-2、E-3、E-4差异显著(*P<0.05)。舌草作用后E-3、E-4AcrB-mRNA表达量下降，且差异显著(*P<0.05)。地锦草处理后，E-1AcrB-mRNA表达量下降，且差异显著(*P<0.05)。综上所述，黄连、舌草、地锦草可降低鸡源大肠杆菌主动外排系统耐药基因表达量。
实施例5消除鸡源大肠杆菌β-内酰胺类药物中药组方确定
综上所述：单味中药(黄连、舌草)消除耐药大肠杆菌R质粒的效果好；单味中药(黄连)抑制耐药大肠杆菌ESBLs活性的效果好；单味中药(黄连、舌草、地锦草)抑制耐药大肠杆菌主动外排系统的效果好；单味中药(黄连、舌草、蒲公英、地锦草)降低耐药大肠杆菌生物膜黏附性的效果好。前期研究证实，山楂在7味中药(山楂、黄芩、黄连、大黄、地锦草、蒲公英、舌草)中消除大肠杆菌β-内酰胺药物耐药性效果最佳。因此组成两组组方，试验组一：山楂、黄连(1：1)、试验组二：山楂、舌草(1：1)
(1)FIC验证组方配伍效果
采用棋盘法检测中药、西药的联合作用。分别测定中药黄连、山楂；舌草、山楂；头孢噻肟、山楂+黄连；头孢噻肟、山楂+舌草；头孢噻肟、三黄汤间联合用药的效果。
表6组方中药水提物FIC测定结果(mg/mL)
由表6可知，山楂、黄连；山楂、舌草；头孢噻肟与山楂舌草组方；头孢噻肟与三黄汤组方间0.5＜FIC指数≤1.0判定结果均为相加作用。头孢噻肟与山楂黄连组方间FIC指数≤0.5判定结果为协同作用，说明各药物配伍间效果较好。
(2)大肠杆菌动物模型建立
选择1日龄蛋雏鸡120只，正常饲养6天，7日龄时用血清型为O78且对头孢噻肟耐药的鸡源大肠杆菌攻毒。记录48h内死亡率，按照改良寇氏法计算半数致死量。剖检死亡雏鸡，观察脏器病理变化。剖检攻毒后的蛋雏鸡，无菌操作从病变脏器中分离细菌，进行生化试验，确定细菌种属，通过玻片凝集试验鉴定血清型，检测分离菌株和攻毒菌株是否一致。组织病理学检查：剖检病死鸡并记录病理变化，取心脏、肝脏组织，制作病理切片，记录结果。
表7大肠杆菌最佳攻毒剂量确定(cfu/mL)
| 空白组 | 1×10<sup>9</sup> | 1×10<sup>8</sup> | 1×10<sup>7</sup> | 生理盐水组 | |
| 起始数(只) | 15 | 15 | 15 | 15 | 15 |
| 死亡数(只) | 0 | 12 | 7 | 4 | 1 |
| 存活数(只) | 15 | 3 | 8 | 11 | 14 |
由表7可知，根据改良寇氏法LD50＝lg-1[Xm-i(∑p-(3-Pm-Pn)/4)]，得出大肠杆菌O78对雏鸡LD50＝1.102×108cfu/mL。即1.102×108cfu/mL、0.5mL为最佳的攻毒剂量。
剖检后，心脏、肝脏表面有黄色纤维素性渗出物，包膜肥厚易剥离，与周围组织粘连(图5)。无菌操作，将上述病料接种于麦康凯琼脂平板，置于37℃恒温箱培养24h，形成中等大小、突起、光滑潮润的圆形粉红色菌落(图6)。显微镜下观察到两端钝圆、粉红色的革兰氏阴性短杆菌(图7)。
H.E.染色心脏组织切片结果如图8、9所示。患病蛋雏鸡的心脏纤维组织结构稀疏，间隙增大，组织间出现水肿(图9Ba)；心外膜、心肌局部组织存在炎性浸润(图9Ba、Bb)。如图11所示，病死鸡肝小叶结构不清晰，肝细胞索间隙变大，出现水肿。
表8分离菌株生化反应
生化试验结果显示，大肠杆菌接近度为99.8％(表8)。通过PCR验证，分离菌株在1467bp位置上出现清晰条带(图12)，并将其PCR扩增产物送至上海生工生物工程有限公司进行测序，测序结果于GenBank上进行序列比对，同源性在99.9％以上，表明分离株为大肠杆菌，且玻片凝集试验出现明显凝集现象，说明分离菌株与攻毒菌株为同一菌株，大肠杆菌致病模型建立成功。
(3)中药临床应用效果观察
选择1日龄蛋雏鸡120只，随机分成6组，适应性饲养6天。7日龄时，用浓度为1.102×108cfu/mL O78型且对头孢噻肟大肠杆菌攻毒。从接种时开始，采用饮水给药的治疗方式。对患病临床症状严重，不能正常饮水的鸡人工灌服，剂量为20mL/kg，2次/日。记录试验组鸡的发病情况，统计治愈率、有效率、死亡率。
表9中药组方对鸡大肠杆菌病临床治疗效果
由表9可知，中药方剂联合抗生素使用可增强抗生素对大肠杆菌的治疗作用，且试验组间表现出不同程度的治疗效果。阳性对照组1(头孢噻肟)的治疗鸡群治愈率为40％、有效率为66.6％、死亡率为33.3％；阳性对照组2(头孢噻肟+三黄汤)的治疗鸡群治愈率为66.6％、有效率为80％、死亡率为20％。试验组1，即经山楂、黄连联合抗生素头孢噻肟治疗的鸡群，治愈率达90％，有效率为93.3％、死亡率为6.7％；试验组2，即山楂、舌草联合抗生素头孢噻肟治疗的鸡群，治愈率为80％、有效率为83.3％、死亡率为13.3％。综上所述，与其它试验组相比，山楂、黄连与头孢噻肟联合使用效果最佳。
以上结果可以看出，山楂、黄连组方效果优于传统组方(三黄汤)和山楂、舌草组方，说明山楂、黄连组方治疗鸡源耐药大肠杆菌病效果最佳。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
Claims (4)
1.一种消除大肠杆菌药物耐药性的中药组方筛选方法，其特征在于：包括以下步骤：
A、根据消除大肠杆菌耐药机制筛选单味中药，所述耐药机制主要包括R质粒介导、产生ESBLs、通过主动外排系统和形成生物膜；
B、采用凝胶电泳方法筛选消除R质粒的单味中药；
C、利用双纸片法筛选抑制ESBLs活性的单味中药；
D、利用荧光定量PCR方法筛选降低外排系统基因Acra-mRNA、Acrb-mRNA表达量的单味中药；
E、利用生物膜阳性筛选方法，筛选出降低生物膜黏附性的单味中药；
F、将步骤B-E中筛选出的单味中药组成中药组合物。
2.根据权利要求1所述一种消除大肠杆菌药物耐药性的中药组方筛选方法，其特征在于：所述筛选方法还包括：
A-1、中药水提物制备：将单味中药浸泡30min，文火熬制2次，合并浓缩、过滤除菌，浓度为1g/mL以生药计，得到单味中药水提物；
A-2、大肠杆菌菌液制备：将冷冻保存菌液接种于LB液体培养基中，37℃培养过夜，将活化菌液划线接种于麦康凯琼脂平板，37℃倒置培养过夜，挑取单菌落接种于LB液体培养基中，37℃培养过夜，得到大肠杆菌菌液。
3.一种消除大肠杆菌药物耐药性的中药组方，其特征在于：所述中药组合物为山楂1份和黄连1份，所述中药组合物与抗生素联合使用，所述抗生素为头孢噻肟，所述抗生素与中药组合物的重量份数比为(2～5)：10。
4.一种中药组方的应用，其特征在于：所述中药组方用于消除鸡源大肠杆菌β-内酰胺类药物耐药性。
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| CN109042765A (zh) * | 2018-09-29 | 2018-12-21 | 河北工程大学 | 一种绿色环保环境消毒剂的药物组合及其制备方法 |
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| CN116396880A (zh) * | 2021-12-27 | 2023-07-07 | 青岛润达生物科技有限公司 | 一种利用噬菌体降低细菌耐药性的方法及其应用 |
| CN116396880B (zh) * | 2021-12-27 | 2024-05-14 | 青岛润达生物科技有限公司 | 一种利用噬菌体降低细菌耐药性的方法及其应用 |
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