CN115322934B - 抗生素耐受性细菌及其培养方法和应用 - Google Patents
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Abstract
本发明提供了一种抗生素耐受性细菌及其培养方法和应用,培养方法包括:将野生型细菌的菌落接种在培养基中培养,再次接种得到对数期生长菌液;加入抗生素溶液,继续培养,离心,洗涤,细菌冻存备用,剩余细菌进入下一暴露循环;将暴露循环的细菌和未处理的野生型细菌分别稀释,并加入抗生素培养;将暴露循环的细菌和未处理的野生型细菌使用抗生素纸片放置于该细菌平板上培养,结束后去除抗生素纸片,使用含葡萄糖溶液的纸片在平板上继续培养;本发明培养筛选得到的抗生素耐受性细菌可在没有抗生素耐药性出现的前提下表现出明显的抗生素耐受性,所获得的耐受性细菌对于耐受性细菌相关难治性感染、细菌耐药性解决、感染药物研发均有重要的支撑作用。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种抗生素耐受性细菌及其培养方法和应用。
背景技术
以抗生素为中心的抗感染治疗是目前应对细菌感染相关疾病的主要手段。抗生素耐药细菌的出现与流行使得这一主要治疗方法与人群健康面临严峻挑战。因此,阻断细菌抗生素耐药性进化是防治抗生素耐药细菌相关感染疾病的重要方法之一。
近年来,细菌抗生素耐受性的发现为探明细菌耐药进化过程提供新的突破口。与抗生素耐药性不同,抗生素耐受性指细菌在抗生素环境中通过下调代谢活动等方式增加细菌在抗生素暴露中的存活时间。研究发现,细菌抗生素耐受充当细菌抗生素耐药性进化的过渡阶段,并促进细菌抗生素耐药性的获得。针对抗生素耐受性的新型诊疗方法开发是抗感染类药物的重要发展方向。因此,稳定的抗生素耐药细菌培养方法对抗生素耐药性细菌研究起到重要基础支撑作用。
目前,抗生素耐药细菌的来源主要有患者来源于体外培养两种方法提供。患者感染细菌的异质性与不统一的体外培养方法难以为抗生素耐受性研究的广泛开展提供可靠的基础研究工具。因此,有必要开发一种更稳定的标准化抗生素耐受细菌的培养方法。
发明内容
针对现有技术中的不足,本发明提供了一种抗生素耐受性细菌及其培养方法和应用。即为细菌抗生素耐受性相关的生物基础实验研究与临床药物疗效研究,提供一种简单可标准化的抗生素耐受性细菌的培养方法。
为达到上述目的,本发明的解决方案是:
第一方面,本发明提供了一种抗生素耐受性细菌的培养方法,其包括如下步骤:
(1)、将ATCC公司的野生型细菌的菌落接种在无菌CAMHB培养基中过夜培养,然后再次接种在无菌CAMHB培养基中至OD600值为0.02,得到对数期生长菌液;在对数期生长菌液中加入抗生素溶液,继续培养;
培养结束后,离心收集细菌,并洗涤数次,再加入新鲜的CAMHB培养基过夜培养细菌;再次离心收集,2/3细菌冻存备用,1/3细菌进入下一暴露循环;
(2)、使用无菌CAMHB培养基将暴露循环的细菌和未处理的野生型细菌分别稀释,并加入不同浓度的抗生素培养,观察最小抑菌浓度;
将暴露循环的细菌和未处理的野生型细菌均匀涂布于MH平板上,使用抗生素纸片放置于该细菌平板上培养,结束后去除抗生素纸片,使用含葡萄糖溶液的纸片放置在该细菌平板上继续培养,观察细菌存活情况。
优选地,步骤(1)中,过夜培养的温度为35-37℃,优选为37℃。针对大多数人体寄生细菌,所用培养温度范围为35-37℃。针对特殊种类细菌,其培养温度由此菌种最适培养温度决定。
优选地,步骤(1)中,抗生素溶液中的抗生素包括但不限于β-内酰胺类抗生素、多肽类抗生素和抗结合类抗生素中的一种以上。抗生素溶液主要为具有繁殖期杀菌作用的抗生素。
其中,β-内酰胺类抗生素选自青霉素、头孢菌素、碳青霉烯类抗生素或β-内酰胺酶抑制剂。
多肽类抗生素选自多黏菌素B、多黏菌素E、万古霉素、去甲万古霉素或壁霉素。
部分抗结合类抗生素选自异烟肼或乙胺丁醇。
优选地,步骤(1)中,继续培养的温度为37℃,时间为30-300min,优选为120min。120min时间在10×MIC浓度抗生素条件下,对于低浓度对数期生长细菌有60-70%杀灭作用,可较好的保持种群性的细菌耐受性,可与持留菌较好区分(在大于99%的细菌杀灭率背景下,个别细菌对抗生素敏感性降低导致持留菌出现)。
优选地,步骤(1)中,离心的转速为800-3000rpm,优选为1200rpm;时间为1-20min,优选为10min。经优化的离心机转速可适配绝大多数50mL水平离心机转速范围,又可有效将液体内细菌离心沉淀并收集。
优选地,步骤(2)中,加入抗生素后,培养的温度为35-37℃,时间为16-20h。针对大多数人体寄生细菌,所用培养温度范围为35-37℃。针对特殊种类细菌,其培养温度由此菌种最适培养温度决定。
其中,不同浓度抗生素:一般应包含8-10个抗生素浓度,至少涵盖美国临床和实验室标准协会(CLSI)所列标准质控菌株的敏感至耐药的最小抑菌浓度范围(MIC)。
优选地,步骤(2)中,使用对应种类的抗生素纸片,培养的温度为37℃,时间为2-24h,优选为18h。18h的处理时间可使得抑菌环范围内的非抗生素耐受菌落充分杀灭,而仍使抗生素耐受性菌落存活,利于区分抗生素耐受性菌落。
优选地,步骤(2)中,继续培养的时间为12-48h,优选为24h。24h处理时间可避免因处理时长过短而导致增殖较为缓慢的抗生素耐受菌落未充分增殖而获得假阴性的抗生素非耐受性结果。24h也可满足绝大多数种类细菌增殖至可肉眼明显观察到菌落形成所需的时间。
第二方面,本发明提供了一种抗生素耐受性细菌,其由上述的培养方法得到。
第三方面,本发明提供了一种上述的抗生素耐受性细菌在耐受性细菌药效材料评估中的应用。
由于采用上述方案,本发明的有益效果是:
本发明培养筛选得到的抗生素耐受性细菌可在没有抗生素耐药性出现的前提下表现出明显的抗生素耐受性,即在细菌某特定抗生素最小抑菌浓度(MIC)未改变的情况下,获得在杀灭剂量抗生素浓度处理下存活时间明显延长的特定抗生素耐受性细菌。所获得的耐受性细菌对于耐受性细菌相关难治性感染、细菌耐药性解决、感染药物研发均有重要的支撑作用。
附图说明
图1为本发明的抗生素耐受性细菌循环处理示意图。
图2为本发明的实施例1中野生型与筛选获得耐受性细菌抗生素耐受性评估示意图。
具体实施方式
本发明提供了一种抗生素耐受性细菌及其培养方法和应用。
本发明通过细菌体外循环抗生素暴露培养与细菌体外抗生素耐药性筛选两个步骤获得具有抗生素耐受性表型的细菌。如图1所示,细菌先进行离心,然后到对数期,再进行抗生素暴露,最后筛选鉴定。
下面将结合本发明实施例和附图,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。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1:
本实施例的抗生素耐受性细菌的培养方法包括如下步骤:
(1)、循环抗生素暴露:将待筛选的野生型细菌的菌落接种在无菌CAMHB培养基中,于摇床37℃过夜培养获得新鲜菌液。然后再次接种在无菌CAMHB培养基中,37℃培养至OD600值为0.02时,得到对数期生长菌液;在对数期生长菌液中加入抗生素溶液,使培养基抗生素浓度达到10×MIC浓度,于37℃继续培养120min。
培养结束后,离心(1200rpm,10min)收集细菌。使用新鲜的CAMHB洗涤细菌沉淀两次。再加入新鲜的CAMHB培养基过夜培养细菌;第二天将细菌离心收集,2/3细菌冻存备用,1/3细菌进入下一暴露循环;每次循环处理后的菌种冻存备用。初次筛选可循环暴露10次。
(2)、细菌耐受性与耐药性鉴定:在CAMHB培养基中复苏每次循环所获得的细菌与未处理的野生型细菌,37℃过夜培养。对于抗生素耐药性的鉴定,使用无菌CAMHB培养基将暴露循环的细菌和未处理的野生型细菌分别稀释至5.0×105CFUs/ml密度,加入抗生素。于摇床37℃培养18h。观察不同循环处理所获得细菌的最小抑菌浓度(MIC),与未处理野生菌的MIC进行对比。
如图2所示,对于抗生素耐受性评估,将暴露循环的细菌和未处理的野生型细菌(即标准质控细菌)菌落以每平板107CFUs均匀涂布于MH平板上,使用抗生素纸片放置于该细菌平板上,37℃培养18h。结束后去除抗生素纸片,使用含10μL、40%葡萄糖溶液的纸片放置在该细菌平板上继续培养24h,观察对比各循环处理细菌存活菌落数量情况。确定待筛选野生型细菌的基本耐药性与耐受性表型,筛选出MIC未有明显增加(耐药性未有明显变化)但存活菌落数量增多的菌种(耐受性增强)。
上述对实施例的描述是为了便于该技术领域的普通技术人员能理解和使用本发明。熟悉本领域技术人员显然可以容易的对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中,而不必经过创造性的劳动。因此,本发明不限于上述实施例。本领域技术人员根据本发明的原理,不脱离本发明的范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (1)
1.一种抗生素耐受性细菌的培养方法,其特征在于,包括如下步骤:
(1)循环抗生素暴露:将野生型细菌的菌落接种在无菌CAMHB培养基中35-37℃过夜培养,再次接种在无菌CAMHB培养基中至OD600值为0.02,得到对数期生长菌液;在所述对数期生长菌液中加入抗生素溶液,使培养基抗生素浓度达到10×MIC浓度,于37℃继续培养30-300min;
培养结束后,800-3000rpm离心1-20min,收集细菌,并洗涤数次,再加入新鲜的CAMHB培养基过夜培养细菌;再次离心收集,2/3细菌冻存备用,1/3细菌进入下一暴露循环;每次循环处理后的菌种冻存备用,循环暴露10次;
(2)细菌耐受性与耐药性鉴定:在CAMHB培养基中复苏每次循环所获得的细菌与未处理的野生型细菌,37℃过夜培养;
对于抗生素耐药性的鉴定:使用无菌CAMHB培养基将暴露循环的细菌和未处理的野生型细菌分别稀释至5.0×105CFUs/mL密度,加入不同浓度的抗生素,35-37℃摇床培养16-20h,观察不同循环处理所获得细菌的MIC,与未处理野生菌的MIC进行对比,其中不同浓度抗生素:包含8-10个抗生素浓度,至少涵盖美国临床和实验室标准协会所列标准质控菌株的敏感至耐药的MIC;
对于抗生素耐受性的评估:将暴露循环的细菌和未处理的野生型细菌以每平板107CFUs均匀涂布于MH平板上,使用杀灭剂量抗生素浓度的纸片放置于该细菌平板上,37℃培养18h,结束后去除抗生素纸片,使用含10μL、40%葡萄糖溶液的纸片放置在该细菌平板上继续培养24h,观察对比各循环处理细菌存活菌落数量情况,筛选出MIC未有明显增加但存活菌落数量增多的菌种;
所述抗生素溶液中的抗生素选自β-内酰胺类抗生素、多肽类抗生素和抗结合类抗生素中的一种以上;其中:
所述β-内酰胺类抗生素选自青霉素、头孢菌素、碳青霉烯类抗生素或β-内酰胺酶抑制剂;
所述多肽类抗生素选自多黏菌素B、多黏菌素E、万古霉素、去甲万古霉素或壁霉素;
所述抗结合类抗生素选自异烟肼或乙胺丁醇。
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