CN108330157A - 用于检测活组织上的生物膜的组合物 - Google Patents
用于检测活组织上的生物膜的组合物 Download PDFInfo
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- CN108330157A CN108330157A CN201810229737.9A CN201810229737A CN108330157A CN 108330157 A CN108330157 A CN 108330157A CN 201810229737 A CN201810229737 A CN 201810229737A CN 108330157 A CN108330157 A CN 108330157A
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0041—Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
一种染色组合物,其用于使活组织上的生物膜可检测到,其中所述组合物优先将生物膜染色,并包含有效地将所述生物膜染色并使它可检测到的量的染色剂。
Description
本申请是国际申请日为2011年11月30日的发明名称为“用于检测活组织上的生物膜的组合物”、国家申请号为201180066375.2的发明专利申请的分案申请。
本发明涉及一种组合物,其可以施用于例如慢性创伤(例如腿溃疡、压力性溃疡、糖尿病性足溃疡)、急性创伤(例如割伤、擦伤、烧伤)、皮肤和骨等活组织,以用于检测作为组织处于感染风险的早期警报指示的微生物生物膜。更具体地,本发明涉及一种组合物,其能够使活组织上的生物膜可见,同时避免创伤和皮肤刺激和愈合滞后。本发明的另一个实施方案涉及一种套件,其用于检测活组织上的生物膜。
活组织经常被多种微生物群居,其中的一些微生物可造成感染。日益变得接受的是,群居在创伤上的微生物的存在会损害慢性和急性伤口愈合。令人信服的证据显示,这些微生物可以主要以生物膜的形式存在于创伤中。在群居过程中,细菌和其它微生物(例如酵母和真菌类)牢固地附着于组织,并通过聚合物质的细胞外基质的分泌而形成生物膜。由于周围的基质,该生长模式会以物理保护的形式在一定程度上保护所述生物膜内的微生物免于局部和全身性抗微生物剂的作用。还有人认为,在生物膜内的微生物与它们的自由泳动的、浮游的对应物相比改变了表型和基因型。已知生物膜微生物在代谢上不太活跃,所以这也可以提供对已知是针对代谢活跃的细菌起作用的常规抗微生物方法(例如抗生素)的一定程度的抗性。此外,生物膜在创伤中的存在也会阻碍炎症性微生物清除中的宿主免疫系统以及正常伤口愈合过程的肉芽形成和再生上皮形成阶段。
所以,需要开发在选择的治疗方案之前和之后快速地检测生物膜在活组织中的存在的方法或装置。这会帮助研究人员理解,微生物是否以生物膜状态生活在皮肤上和创伤内部,并且允许他们跟踪这样的生物膜群落的成熟或清除。生物膜检测方法或装置也允许研究人员开发有效的抗生物膜策略和有效的伤口愈合护理方案,并且在临床实践中,它会指导适当的创伤敷料的选择,并辅助监测由创伤护理从业人员施用的治疗方案的有效性。
生物膜通常由被包围在外聚合物(exopolymeric substance)(或基质) 内的细菌构成,所述外聚合物由具有例如1,3-或1,4-β-连接的己糖键等复合键的长链多糖(一些常见的生物膜多糖的例子是磷壁酸、缩酮连接的丙酮酸酯(pryruvate)N-乙酰氨基葡萄糖和糖醛酸:D-古罗糖醛酸、D-半乳糖醛酸和甘露糖醛酸)、蛋白质(其中的一些可能起结构作用)、DNA (细胞外的,其中的一些可能具有结构作用)、脂质、金属离子(Ca2+、Mg2+、Fe3+等)和水组成。生物膜也可能与失活的宿主组织(例如腐肉和坏死组织)有关。
生物膜细胞外聚合物质(EPS) 也被称作生物膜细胞外基质或细菌衍生的组织。尽管生物膜按重量计主要是水并且细菌可能仅构成生物膜体积的10-20%,但是EPS构成生物膜的生物质/干重的大部分。
生物膜(例如以牙斑形式在牙齿上发现的那些)由于它们的厚度、颜色和它们借以形成的基质的性质而经常易于用肉眼观察到。由于在创伤中存在的颜色和创伤的内含物,在例如慢性或急性创伤中的生物膜观察并不简单。慢性和急性创伤经常以除了含有细菌和生物膜以外还含有死的或失活的组织(腐肉)、渗出物、脓、血液、药剂、敷料组分的方式而复杂化。所以,可能难以检测生物膜在创伤中的存在,因为用肉眼难以观察创伤生物膜。因此,需要辅助检测生物膜的方法,例如利用能够优先将创伤生物膜染色以使得它们可以被观察到的组合物。一旦观察到,可以适当地处理生物膜。
我们已经令人惊讶地发现,通过使用特定组合物可能优先将生物膜染色,所述组合物包含允许检测生物膜的染色剂。
因此,本发明的第一方面提供了一种染色组合物,其用于使活组织上的生物膜可检测到,其中所述组合物优先将生物膜染色,并包含在溶液中的有色的或荧光的染色剂,其量可有效地将所述生物膜染色并使它可检测到。
优选地,使得所述生物膜可被肉眼、或在与照明和/或光学装置结合的情况下检测到。
优选地,所述染色剂是选择性地结合EPS/细菌且不结合宿主活组织的染料。所述染色剂应当不会将下述物质显著染色,所述物质是在例如慢性或急性创伤(例如创伤组织)中的非生物膜组分,周围皮肤,腐肉(死的或失活的组织),渗出物,血液,脓,炎症细胞(嗜中性粒细胞、巨噬细胞),在愈合过程中涉及的细胞(纤维原细胞、内皮细胞、角化细胞),或可能保留在创伤中的药剂或敷料组分。这样的染色剂优选地是具有要求的大小和结构的分子;例如,能够穿过EPS和细菌细胞膜而扩散的低分子量的、紧凑的、平面分子。所述染色剂可以具有产生颜色的阴离子基团和可能的荧光性能;并且可以具有用于与带负电荷的生物膜EPS多糖和细菌细胞壁进行电荷相互作用的阳离子基团。这些带电荷的基团应当优选地是永久电荷,使得它们不受施加的制剂的pH或活组织/生物膜环境影响。
这样的染色剂优选地具有合适的颜色或亮度,使得被染色的生物膜可能用肉眼直接观察到。但是,由于创伤的复杂的和高度着色的性质,染色剂的一些颜色可能难以观察到。例如,在坏死性创伤、腐烂性创伤或出血性创伤中,可能存在黑色、褐色、红色、黄色等色调。尽管蓝色或绿色染料在这里可被视作优选的,但如果位于褐色、红色或黄色着色的组织附近或其上面,所述染料可能显示为暗色或黑色。并非单独地依赖于观察人员能够用肉眼检测染色剂,优选的是,使用可以发荧光的染色剂,使得它可更容易地检测。许多类别的作为潜在染色剂的化合物也会发荧光,因为它们能够吸收某些波长的光的光子并变得电子性地激发,从而以荧光形式发出该光能。使用滤光器可能检测这样的荧光,所述滤光器针对相应分子的荧光发射光谱而设计,使得观察人员仅看到与荧光波长相对应的狭窄光谱带。例如,具有特定滤光器的透镜(例如在激光防护中使用的那些)可以与适当特异性的光源相结合以检测荧光,由此允许检测活组织上的生物膜。该生物膜检测方法可能具有胜过使用肉眼的检测的优点,因为荧光检测对于甚至非常薄的生物膜(其可能是仅仅几层微生物细胞厚)而言是可能的,且不唯一依赖于染色剂本身是可见的。
优先染色是指,染色剂选择性地结合生物膜,而不是宿主活组织。以此方式,染色剂通过揭示生物膜的存在和位置而可以简单地用于检测生物膜。染色剂结合或吸附至细胞外的生物膜基质分子,以及被生物膜细菌细胞、而不是创伤的组织结合或吸附和/或吸收。优选地,染色剂对生物膜的染色通过使它可被肉眼检测到而揭示生物膜的存在。就一些染色剂而言,可以使被染色的生物膜发荧光,例如通过用光源照明。所述荧光可以使被染色的生物膜更加可见。选择所述光源以发射适当波长的光,以致于染色剂吸收光子形式的光能以激发该染色剂并造成它发荧光。使用排除染色剂的非荧光波长的适当滤光器(例如以光学过滤透镜的形式)可以增强荧光的观察。
根据本发明的第一方面的组合物包含一种或多种能够优先将生物膜染色的染色剂。优选地,所述染色剂是染料,在可见区、更优选380 nm至720 nm、甚至更优选500 nm至600 nm中具有最大吸收的生物膜染色染料,因为这会使得宽范围的光源适合与本发明的组合物一起使用或包含在本发明的套件中。
优选地,所述染色剂会产生足够的用于检测的荧光,使得适合激发的波长范围以及适合荧光发射检测的独特的且更高的波长范围可以由照明装置释放并用光学过滤透镜装置检测到。
适合用于本发明的组合物中的染色剂最优选地选自基于有机化合物的那些,更可能是含有芳族环结构(例如苯)或扩展共轭(例如在卟啉类化合物或脱镁叶绿酸(pheophorbide)中)的那些,更可能是具有稠合的多环烃(例如萘、蒽、菲咯啉(phenanthraline)和芘)的那些,且最可能是也含有杂环芳族结构(其中,一个或多个杂环原子是氧、氮或硫或其混合物,例如呋喃、噻吩、吡咯、吡喃、吡啶和噁嗪)的那些。
这些染色剂也可以表现出荧光性能。最适当的是,吸收和发射在可见电磁波谱中的波长(380 nm-720 nm)的光的那些。这样的试剂可能具有含有扩展的共轭区域的化学结构,例如荧光酮及其衍生物(也称作为呫吨和若丹明)例如曙红、赤藓红、玫瑰红、5-异硫氰酸荧光素、5-氯甲基荧光素、6-羧基荧光素、2,7-二-(羧基乙基)-5(6)-羧基荧光素;若丹明B、若丹明6G、若丹明123、若丹明碘乙酰胺、磺酰若丹明B、磺酰若丹明101、四甲基若丹明或德克萨斯红。花青染料的衍生物例如3,3’-双十六烷基吲哚羰花青(3,3’-dihexadecylindocarbocyanines)、3,3’-二丙基氧杂二羰花青(3,3’-dipropyloxadicrbocyanine)、二磺酸铝酞菁(aluminium phthalocyaninedisulphonate)、四磺基铝酞菁(aluminium tetrasulphophthalocyanine)、铝酞菁、锌酞菁、萘酞菁(napthalocyanine)或粘多糖染色剂阿尔新蓝(mucopolysaccharide stainAlcian Blue)。吖啶及其衍生物例如吖啶黄、氨基吖啶、2-氨基吖啶或9-氨基吖啶。最后,适当的试剂可以选自下述类别:噁嗪衍生物例如尼罗蓝、尼罗红、Fura Red或Fura-2;喹诺酮及其衍生物;腺苷衍生物例如2’3’-O-(2,4,6-三硝基-亚环己二烯基(cyclohexadienylidine))腺苷5’-三磷酸或3’-O-(N-甲基氨茴酰基)腺苷5’-三磷酸;三芳基甲烷例如专利蓝V、结晶紫、亮蓝或固绿;吩噻嗪鎓例如亚甲蓝或甲苯胺蓝O及其衍生物例如1-甲基亚甲蓝或1,9-二甲基亚甲蓝;菲啶衍生物例如溴乙非啶或氢化乙啶(hydroethidine);脱镁叶绿酸衍生物例如脱镁叶绿酸钠);和卟啉衍生物例如二氢卟酚e6(chlorin e6)、苯并卟啉衍生物、卟吩(porphines)、内消旋-四卟吩(meso-tetraporphines)、血卟啉或原卟啉。
所述染色剂优选地以0.0001重量%至1重量%、更优选地0.0025重量%至0.025重量%、甚至更优选地0.0025重量%至0.01重量%的水平包含在所述组合物中。
本发明的组合物可以处于轻微地附着于组织上的形式,且可以在短的持续时间以后容易地冲洗掉,以辅助被染色的生物膜的显像。粘稠的流体,例如基于水或甘油的凝胶(以凝胶涂敷器(applicator)、喷雾(spray)或薄片(sheet)的形式)、起泡摩丝、乳膏或软膏,会提供与创伤层的亲密接触。或者,可以使用薄的可溶性的浇铸膜或冻干的、溶解的圆片(wafer)来提供与创伤层的亲密接触。在任何这样的递送系统中,优选地,使用标准冲洗剂(例如盐水)冲洗数秒,可以容易地从活组织冲洗掉制剂。
处于凝胶形式的组合物还可以包含增粘剂,例如纤维素衍生物如羟乙基纤维素(HEC)、羧甲基纤维素或羟丙基纤维素;树胶;糖/醇衍生物例如甘油、山梨醇、麦芽糖醇、甘露醇、麦芽糖糊精或聚葡萄糖;天然的聚合物例如明胶、果胶、聚氨基葡萄糖或海藻酸盐;合成的聚合物例如聚羰乙烯、聚乙烯吡咯烷酮、聚乙酸乙烯酯、聚乙烯醇、聚丙烯酸酯、聚甲基丙烯酸酯、聚乙二醇或泊洛沙姆。优选地,处于凝胶形式的组合物可以包含1-5重量%的增粘剂,且最优选HEC。
本发明的组合物可以处于凝胶形式,且还可以包含湿润剂例如丙二醇(PG)、甘油、聚乙二醇、聚葡萄糖、山梨醇、三乙醇胺(triethanlolamine)、环甲硅油、乳酸铵或甘油酯。优选地,所述组合物包含5重量%至15重量%的湿润剂,且最优选PG。
优选地,本发明的组合物包含赋形剂以优化染色剂与生物膜的结合。例如,本发明的组合物还可以包含:其水平为0.1-2.0重量%的金属螯合剂例如乙二胺四乙酸四钠(EDTA)、柠檬酸、地拉罗司、去铁酮、去铁胺、deferazoxane、乙二醇四乙酸、葡糖酸(gluonicacid)、次氮基三乙酸或柠檬酸三钠,或者其水平为0.1-1.0重量%的帮助染色剂向生物膜中穿透的试剂,例如表面活性剂例如吐温80、司盘20或椰油酰氨基丙基甜菜碱(coamidopropylbetaine,CAPD)和尤其是阳离子季铵表面活性剂例如二烷基二甲基氯化铵、烷基吡啶鎓氯化物、杀藻铵(BaCl)、苄索氯铵(BeCl)、椰油酰两性基二乙酸二钠(disodium cocamphodiacetate)、鲸蜡基吗啉鎓乙硫酸盐或烷基三甲基氯化铵。表面活性剂的添加也可以充当起泡剂。例如,表面活性剂BeCl、吐温80、司盘20、CAPD、C14-16烯烃磺酸钠或Softisan 649可以充当起泡剂以提供起泡摩丝的制剂特征。
优选地,本发明的组合物具有在5至7的范围内、最优选约5.5的pH。
优选地,本发明的组合物呈粘稠流体的形式,且包含:增粘剂例如HEC、湿润剂例如PG、金属螯合剂例如EDTA、表面活性剂例如BeC1和水,尤其是2.0% w/v羟乙基纤维素、10.0%w/v丙二醇、0.5% EDTA、0.5% BeC1和大约87% v/v无菌蒸馏水。或者,本发明的组合物可以呈下述形式:从注射器、小袋、雾化瓶、气雾剂瓶、无推进剂泵瓶、刷子向创伤施用的溶液或凝胶薄片、膜或溶解的圆片。
通过高压灭菌或γ照射可将制剂最终灭菌。或者,所述制剂可以是储存溶液,其含有例如防腐剂例如DMDM乙内酰脲、或对羟基苯甲酸酯例如对羟基苯甲酸甲酯、对羟基苯甲酸乙酯或对羟基苯甲酸丙酯。
本发明的组合物主要用于这样的活组织上:其表现出临床感染的迹象(炎症、恶臭、脓性的渗出物、组织缺氧等),可能处于感染风险中,似乎具有腐肉(宿主衍生的组织)或生物膜(细菌衍生的组织)存在,或通常是顽固性的。所述组合物也可在敷料更换时使用,以便检测生物膜,以及监测治疗方式的效力和通过检测到的生物膜的减少而指导将来的治疗。
本发明的另一个方面涉及用于检测活组织上的生物膜的部件的套件,所述套件包括:组合物,其包含优先将生物膜染色的染色剂,和
光源,其能够造成所述染色剂发出荧光。
优选地,所述套件进而包括眼镜或集成滤光器形式的使得荧光能够被特异性地检测到的滤光器。
所述光源可以是白光源,例如穿过“窄带”滤光片的钨丝灯、卤素灯或脉冲氙灯。优选地,所述光源是不需要滤光或衰减的单色或狭窄光谱源,例如具有与染色剂的光谱特征密切相关的输出的发光二极管。这样,所述光源优选地是小型的、便携的、手持式的,且不产生或产生可忽略的热。所述光源可以是多次使用的或完全一次性的。
优选地,所述套件进而包括眼镜,或所述光源含有集成透镜,所述眼镜或透镜用于检测生物膜,其中所述眼镜或透镜含有滤光器以排除低于染色剂的吸收最大值(Absmax) 的所有波长的光。以此方式辅助用户检测存在于生物膜中的染色剂的荧光。更优选地,所述眼镜或透镜滤光器会有效地传输与染色剂的荧光发射光谱相对应的波长的光。所述眼镜可以是多次使用的或完全一次性的(光源中的集成透镜也是如此)。
优选地,所述套件另外包括创伤冲洗溶液,其用于在光源照明之前和/或之后冲洗活组织。
在典型应用的一个实施例中,将根据本发明的组合物施用于整个创伤或所需的区域,以便获得薄的、但是一致的组合物层,例如0.1-0.5 cm的厚度。将所述组合物放置在适当的位置0.1-15分钟、更优选地0.5-2分钟。在任何照明之前,可以将制剂放置在适当的位置,或者更优选地,使用合适的创伤冲洗溶液从创伤冲洗掉多余的制剂。冲洗掉多余的制剂,可以使得染色能够显示出生物膜在创伤中的位置,从而可以处理(例如用刮匙)这些区域。
然后针对优先被染色的生物膜的存在来检查创伤。这可以用肉眼实现,或者可以用光源在1-50 cm范围内的距离照明创伤。优选地,所述光源是在5-20 cm的距离。将光源光谱输出选择成与染色剂的荧光发射光谱相对应;例如,就具有575 nm的荧光最大值的玫瑰红而言,合适的光源可能发射550-600nm的波长。这会造成被染色的生物膜中的试剂发荧光。优选地,所述用户佩戴眼镜来观察创伤,或通过光源中的集成透镜来观察创伤。所述眼镜或透镜具有滤光系统,所述滤光系统排除低于染色剂的荧光发射范围的波长的光,并允许任何荧光被非常清楚地和特异性地看到,并从而检测存在的任何生物膜。
通常,应当在随后的敷料更换时进行治疗。可以针对生物膜的存在和减少,用肉眼或用光源和眼镜或集成透镜进一步检查创伤。然后可以用适当的主要敷料和次要敷料裹敷创伤。
下面是附图和表格的简要描述:
图1. 使用在CDFF(n=6)中培养的生物膜,对作为生物膜染色剂的选择的染料的初期筛选。
图2a. 使用在CDC生物膜反应器(n=9)中培养的生物膜,对作为生物膜染色剂的选择的染料的筛选。吸收数据。
图2b. 使用在CDC反应器(n=9)中培养的生物膜,对作为生物膜染色剂的选择的染料的筛选。浓度数据。
图3. 含有混合的金黄色葡萄球菌(S. aureus)和铜绿假单胞菌(P. aeruginosa)生物膜的肉的、玫瑰红+ EDTA和BaCl (RBEB) 染色(A-C)的样品和对照(D-E) 的样品。
图4. 金黄色葡萄球菌生物膜膜龄vs染料浓度。
图5. 铜绿假单胞菌生物膜膜龄vs玫瑰红浓度。
下述实施例是本发明的示例。
实施例1
使用恒定深度生物膜发酵桶评估生物膜染色剂
使用恒定深度生物膜发酵桶(CDFF) 培养金黄色葡萄球菌和铜绿假单胞菌的混合生物膜4天。简而言之,在环绕可转动的旋转钢圈的15个PTFE盘内的凹陷处形成生物膜,在所述PTFE盘上稳定地滴落接种物或无菌生长培养基。随着可转动的钢圈的旋转,刮棒将细菌接种物或培养基分布在盘上,从而维持生物膜在稳定的深度。每个盘含有5个可去除的直径为4 mm的塞子,所述塞子插入至300 μm的深度。在塞子凹陷处生长的得到的生物膜在外观和微生物组成方面是可再现的,并且可以使用无菌仪器通过取样端口从CDFF取出。从CDFF一式两份地取出含有生物膜的盘,通过在无菌盐水中浸泡5秒来冲洗1次,然后在10 ml体积的下述潜在生物膜染色剂(除非说明,否则在100 μM浓度下、在去离子水中)中在暗处培育2分钟:赤藓红;玫瑰红;固绿;玫瑰红+ 2% w/v EDTA和1% w/v杀藻铵(BaCl) (RBEB);玫瑰红+2% w/v EDTA四钠和1% w/v杀藻铵/2% w/v羟乙基纤维素和10% w/v丙二醇凝胶(凝胶-RBEB)。在盐水中冲洗另外1次后,对于每种染色剂,将6个塞子在6ml体积的2% 十二烷基硫酸钠(SDS)水溶液中在37℃培育过夜,以进行消化。然后将样品以13,000 rpm旋转10分钟,以使上清液与消化的细胞碎片分离。然后在分光光度计中测量抽吸的上清液的吸收光谱,并将这些光谱与已知浓度(100 μM)的每种染色剂在2% SDS中的光谱进行对比。
图1表明,固绿似乎是在CDFF中培养的生物膜的最有效染色剂,紧随其后的是液体形式的玫瑰红+EDTA和BaCl。EDTA和BaCl赋形剂的添加似乎会增强玫瑰红向生物膜中的摄取。
实施例2
使用CDC生物膜反应器评估生物膜染色剂
使用疾控中心(Centre for Disease Control,CDC) 生物膜反应器来培养金黄色葡萄球菌和铜绿假单胞菌的混合生物膜48小时。简而言之,在反应器杆上的取样管(coupon)上形成生物膜,所述反应器杆被保持在反应器罐内,所述反应器罐含有在35℃连续混合的金黄色葡萄球菌和铜绿假单胞菌培养物。从所述杆取出含有生物膜的取样管,通过在无菌盐水中浸泡5秒来冲洗1次,然后在10 ml体积的下述生物膜染色剂(在100 μM浓度在去离子水中)中在暗处培育2分钟:赤藓红;玫瑰红;阿尔新蓝;若丹明B;若丹明123;玫瑰红+ 2% w/vEDTA和1% w/v BaCl (RBEB)。在盐水中冲洗另外1次后,对于每种染色剂,将9个取样管各自加入2 ml体积的2% SDS中,用于在“高”设置(setting)消化(stomaching)1分钟,然后在暗处在37℃培育过夜进行消化。然后将样品以13,000 rpm旋转5分钟,以使上清液与消化的细胞碎片分离。然后在分光光度计中测量抽吸的上清液的吸收光谱,并将这些光谱与已知浓度(100 μM)的在2% SDS中的每种染色剂的光谱进行对比。
图2a显示了以绝对吸收或“亮度”计的方式,RBEB是最有效的生物膜染色剂,其次是碳水化合物染色剂阿尔新蓝。EDTA和/或BaCl似乎会使玫瑰红的摄取增加超过60%。当将同样数据表示为测得的吸收:在100 μM (即浓度)处的吸收的比例时,阿尔新蓝似乎是最有效的生物膜染色剂。
实施例3
使用猪胸肉生物膜模型评估生物膜染色剂
使用猪胸肉生物膜模型来进一步评估生物膜染色剂。使用20 mm钻孔器切割猪腰肉块,并使用6 mm钻头来在样品中央建立凹入部位。然后通过γ照射将样品灭菌。给样品接种10μl体积的约1 x 107 cfu/ml的金黄色葡萄球菌和铜绿假单胞菌的混合混悬液,然后在石蜡膜密封的陪替氏培养皿中在35℃培育72-96小时。然后仅对似乎在中央钻孔中具有可见生物膜的样品如下染色:将所述样品浸入10 ml体积的100 μM玫瑰红+2% w/v EDTA和1% w/vBaCl (RBEB) 中保持2分钟,并在染色之前和之后在盐水中冲洗。对照样品未被染色,因此仅浸入盐水中保持2分钟。然后将样品拍摄照片,其实例显示在图3中。显示的3个被RBEB染色的样品(AtoC) 清楚地证实了被包含在凹入部位内的生物膜对染色剂的选择性摄取。3个对照样品表明,在没有该染色存在下,难以确定生物膜是否存在于样品中和存在于何处。
实施例4
使用膜滤器生物膜模型培养的生物膜的玫瑰红染色
使用膜滤器生物膜模型来研究玫瑰红浓度对生物膜染色的效力的影响。简而言之,将5μl体积的5 x 105 cfu/ml的金黄色葡萄球菌或铜绿假单胞菌的混悬液加入无菌膜滤片(孔径0.2 μm; Anodisc, Whatman) 的中央,所述膜滤片放在盖了盖的6-孔板内的7 ml体积的无菌胰蛋白酶大豆肉汤(Tryptic Soya Broth)上。将样品培育4小时(未成熟的生物膜)和24和48小时(成熟的生物膜),并从过滤器冲洗掉多余的浮游生物细胞。如下将过滤器上的生物膜染色:吸量2 ml体积的玫瑰红(60 μM或300 μM)或盐水(阴性对照) 并保持30秒,然后冲洗。如下从生物膜样品回收玫瑰红:在2% 十二烷基硫酸钠中消化(stomaching)和过夜消化,离心,然后在紫外可见分光光度计中测量吸收光谱。通过将吸收值与玫瑰红在2% SDS中的标准曲线进行对比,确定每个样品的玫瑰红摄取。
表1和2表明,成熟(mature)的金黄色葡萄球菌和铜绿假单胞菌生物膜以染料浓度和生物膜膜龄依赖性的方式摄取玫瑰红。仅在300 μM的最高浓度,未成熟的4-小时生物膜才显得被染色,尽管这可能是由于过滤器本身的一些染色。图4和5显示了成熟的48-小时的金黄色葡萄球菌和铜绿假单胞菌生物膜摄取比24-小时生物膜更多的玫瑰红,并且300 μM玫瑰红导致与60 μM玫瑰红相比显著更多的生物膜染色。这种简单的定量生物膜对玫瑰红的摄取的方法利用了染料的吸收光谱。使用吸收光谱来定量染料,与使用具有滤光器的光源检测荧光类似。然后可替换地如下定性地观察摄取:使用肉眼,或通过与光源和滤光器结合地观察玫瑰红的荧光发射。
表1.
膜龄(h) | [RB] (uM) | 1 | 2 | 3 | 均值 | 标准差 |
4 | 0 | 0 | 0 | 0 | 0 | 0 |
4 | 60 | 0 | 0 | 0 | 0 | 0 |
4 | 300 | 1.59 | 1.45 | 1.64 | 1.56 | 0.10 |
24 | 0 | 0 | 0 | 0 | 0 | 0 |
24 | 60 | 1.66 | 1.72 | 2.17 | 1.85 | 0.28 |
24 | 300 | 4.82 | 4.55 | 3.95 | 4.44 | 0.45 |
48 | 0 | 0 | 0 | 0 | 0 | 0 |
48 | 60 | 1.22 | 1.12 | 1.37 | 1.24 | 0.12 |
48 | 300 | 6.26 | 5.18 | 5.71 | 5.72 | 0.54 |
表2
膜龄(h) | [RB] (uM) | 1 | 2 | 3 | 均值 | 标准差 |
4 | 0 | 0 | 0 | 0 | 0 | 0 |
4 | 60 | 0 | 0 | 0 | 0 | 0 |
4 | 300 | 1.52 | 1.76 | 1.88 | 1.72 | 0.18 |
24 | 0 | 0 | 0 | 0 | 0 | 0 |
24 | 60 | 2.04 | 1.76 | 2.02 | 1.94 | 0.15 |
24 | 300 | 5.05 | 5.84 | 4.41 | 5.10 | 0.72 |
48 | 0 | 0 | 0 | 0 | 0 | 0 |
48 | 60 | 3.15 | 1.66 | 2.37 | 2.39 | 0.74 |
48 | 300 | 6.20 | 9.01 | 6.69 | 7.30 | 1.50 |
Claims (8)
1.染色组合物,其用于在活组织上使生物膜可检测到,其中所述组合物是非胶凝液体且优先将生物膜、而不是活组织染色,且包含防腐剂、EDTA、表面活性剂并以有效地将所述生物膜染色并使它可检测到的量包含染色剂,其中所述染色剂选自玫瑰红、阿尔新蓝、若丹明123、若丹明B、固绿和赤藓红,所述表面活性剂选自杀藻胺或苄索氯铵。
2.根据权利要求1所述的组合物,其特征在于,所述组合物通过选择性地结合生物膜,优先将生物膜染色,所述组合物任选地能够通过荧光优先将生物膜染色,并用肉眼或使用光源和与染色剂的荧光发射光谱相对应的滤光器进行目视检测。
3.根据权利要求1或权利要求2所述的组合物,其特征在于,所述组合物允许通过优先将生物膜染色并使该生物膜显示、优选地对于肉眼显示,而检测所述生物膜。
4.根据权利要求1或2所述的组合物,其特征在于,所述染色剂吸收波长为380 nm至720nm的光。
5.在任一项前述权利要求中所述的组合物,其特征在于,所述组合物允许通过优先将生物膜染色并在用光照射该生物膜时使该生物膜显示,而检测所述生物膜,所述光包括引起所述生物膜发荧光的波长。
6.在任一项前述权利要求中所述的组合物,其特征在于,所述组合物是以涂敷器、喷雾或薄片的形式的、基于水或甘油的非胶凝液体溶液。
7.在任一项前述权利要求中所述的组合物,其特征在于,所述组合物包含0.0001重量%至1重量%、更优选0.0025重量%至0.025重量%的一种或多种染色剂。
8.根据权利要求1所述的组合物,其进而包含羟乙基纤维素、丙二醇、和无菌蒸馏水。
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US20190216953A1 (en) | 2019-07-18 |
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HUE032884T2 (hu) | 2017-11-28 |
RU2650803C2 (ru) | 2018-04-17 |
JP2017062238A (ja) | 2017-03-30 |
AU2011334682B2 (en) | 2016-11-17 |
JP2014500495A (ja) | 2014-01-09 |
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CN103328651A (zh) | 2013-09-25 |
CA2819303A1 (en) | 2012-06-07 |
RU2013129866A (ru) | 2015-01-10 |
PT2646566T (pt) | 2017-01-26 |
AU2017201084B2 (en) | 2019-04-18 |
JP2018189655A (ja) | 2018-11-29 |
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