CN105358141A - 治疗细菌感染的方法 - Google Patents
治疗细菌感染的方法 Download PDFInfo
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- CN105358141A CN105358141A CN201480037821.0A CN201480037821A CN105358141A CN 105358141 A CN105358141 A CN 105358141A CN 201480037821 A CN201480037821 A CN 201480037821A CN 105358141 A CN105358141 A CN 105358141A
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Abstract
本发明包括治疗和预防受试者中的细菌感染的方法,制备用于治疗和预防受试者中的细菌感染的药物的方法,以及用于所述方法中时的药物和兽药抗菌组合物。
Description
技术领域
本发明涉及治疗和预防受试者中的细菌感染的方法,制备用于治疗和预防受试者中的细菌感染的药物的方法,以及用于所述方法中时的药物和兽药抗菌组合物。
背景技术
在致病革兰氏阳性(G+ve)(金黄色葡萄球菌(Staphylococcusaureus)、肠球菌属物种(Enterococcusspp.)和肺炎链球菌(Streptococcuspneumoniae))和革兰氏阴性(G-ve)病原体(大肠杆菌(Escherichiacoli)、肠杆菌属物种(Enterobacterspp.)、沙门氏菌属物种(Salmonellaspp.)、鲍氏不动杆菌(Acinetobacterbaumannii)、肺炎克雷伯氏菌(Klebsiellapneumoniae)和铜绿假单胞菌(Pseudomonasaeruginosa))中多药物耐性盛行的显著增加与全球新型抗感染药物投资空前减少一致。目前几乎没有多药物耐性(MDR)细菌感染的注册替代药物,迫使临床医生考虑老一代具有窄谱和相当大的潜在毒性副作用的药物例如粘菌素。此外,很少新型抗感染治疗剂类别从头到尾走完药物开发流水线。
从2000年起,在几乎15年的时间期内,美国FDA仅批准了5种新型作用模式(MOA)抗菌剂–2000年批准的利奈唑胺(linezolid)(噁唑烷酮)、2003年批准的达托霉素(daptomycin)(脂肽)、2007年批准的瑞他莫林(retapamulin)(截短侧耳素)、2011年批准的非达霉素(fidaxomicin)(大环内酯台勾霉素)以及2012年批准的贝达喹啉(bedaquiline)(二芳基喹啉)。值得注意的是,这些药剂对革兰氏阴性菌均无显著活性。2013年未批准新型MOA抗菌剂,迄今2014年为止,在美国仅两种现有类别的类似物泰迪唑胺(tedizolid)和达巴万星(dalbavancin)被推荐批准。虽然在各种开发阶段有超过300种抗感染药物,但大多数这些药物为此前批准的正在进行新型适应症研究的抗菌化合物或其衍生物。
此外,动物特异性病原体中多药物耐性的盛行连同注册规则的加强和抗微生物剂在动物中的使用,使得兽医越来越依赖于传统类别的抗微生物剂。MDR人畜共患病生物从动物至人转移的风险还要求进一步限制一些最近注册抗菌药物例如氟喹诺酮类和第三代和第四代先锋霉素的使用。
人和动物病原体中抗菌剂耐性发展的流行病学
许多耐性发展由关键MDR生物的流行病学变化推动。一旦仅限制于人类医院和老年护理设施,耐甲氧西林金黄色葡萄球菌(MRSA)菌株就以惊人的比例从社区中分离。此外,社区获得性MRSA菌株很可能携带潘顿-瓦伦丁杀白细胞素(PVL)毒素,其为皮肤和软组织病灶以及具有显著相关死亡率的快速、爆发性、坏死性肺炎相关的毒性因子。最近的MRSA菌株在多个重要动物物种包括牲畜、马和伴侣动物中具有宿主适应性,并且人至动物和动物至人转移的常规病例有所记录。对于菌株传播和公共健康,这具有很大的重要性。最近对751名澳大利亚兽医的MRSA鼻腔携带调查发现,显著地21.4%的马科兽医为MRSA阳性,相比之下4.9%的小动物兽医、0.9%很少接触动物的兽医为MRSA阳性。MRSA的这些生态转移,连同针对MRSA特别开发的新型药物例如利奈唑胺的耐性的出现确认,迫切需要新型MRSA抗感染药物。此外,使用万古霉素治疗MRSA然后必须对付其患者中耐万古霉素肠球菌(VRE)感染爆发的医院,对替代性抗微生物剂的选择再次受到限制。
高毒性MDR革兰氏阴性(G-ve)菌例如大肠杆菌O25b:ST131在社区内的整体出现和传播确认,细菌病原体的毒性和耐性决定子二者可同时进化。根据最近的MRSA流行病学,人类中的尿道和血流感染的主要致因大肠杆菌O25b:ST131目前已从伴侣动物和家禽中的肠外感染分离。具有对氟喹诺酮类和广谱β-内酰胺类和碳青霉烯类的组合耐性的大肠杆菌O25b:ST131和其它MDR肠杆菌科(Enterobacteriaceae)的重要性增加是另一个令人担心的趋势,尤其是考虑到最近除碳青霉烯类家族的逐渐推进之外,很少有G-ve谱抗感染药物开发的突破。
世界卫生组织(WorldHealthOrganisation)认定,抗生素耐性是未来全球健康的三个主要威胁之一。美国疾病控制与预防中心(USCentersforDiseaseControlandPrevention(CDC))最近的报告预计,“在美国,每年超过两百万人患上抗生素耐性感染,至少23,000人因此而死亡。”仅在美国,治疗和管理抗生素耐性感染单个病例相关的额外医疗成本预计在每年18,588美元和29,069美元之间,导致美国医疗系统每年的总体直接成本超过200亿美元。此外,就生产力损失而言美国家庭每年的成本预计超过350亿美元。尽管很多EU国家具有世界最佳实践医院监督和感染控制策略,但在欧盟(EU)每年仍有25000名患者死于MDR细菌感染。EU在MDR感染相关的保健花费和生产力损失的成本预计为至少每年15亿欧元。
补充和代替现有抗菌剂的新型作用机制不能满足临床抗菌剂需要,其功效逐渐被抗菌剂耐性机制破坏。治疗多药物耐性细菌感染仍然需要替代抗菌剂。然而,据美国传染病学会(InfectiousDiseasesSocietyofAmerica)和欧洲疾病控制与预防中心(EuropeanCentreforDiseaseControlandPrevention)报道,几乎未开发出提供比现有治疗更满意结果的新药(InfectiousDiseasesSocietyofAmerica2010,ClinicalInfectiousDiseases,50(8):1081-1083)。
本发明的目的是克服现有技术的至少一个缺点。
上述背景技术的讨论仅仅旨在促进对本发明的理解。该讨论不是承认或认可任何所述材料在本专利申请的优先日期均为一般常识的部分。
发明内容
根据本发明的一个方面,提供了治疗或预防受试者中细菌定植或感染的方法,所述方法包括以下步骤:将治疗有效量的氯苯胍或其治疗学上可接受的盐施用给所述受试者。在此方面,所述细菌定植或感染由菌剂(bacterialagent)引起。
根据本发明的另一个方面,提供了氯苯胍或其治疗学上可接受的盐在制造用于治疗受试者中细菌定植或感染的药物中的用途。在此方面,所述细菌定植或感染由菌剂引起。
所述受试者可为能够被细菌定植或感染的任何受试者。所述受试者可为哺乳动物,或可为鱼或鸟。优选地,所述受试者选自但不限于人、犬科动物、猫科动物、牛、绵羊、山羊、其它反刍类物种、猪、马、鸟或鱼。
如本文所用,术语氯苯胍(也称为1,2-双[(E)-(4-氯苯基)亚甲基氨基]胍,或如本说明书所述NCL812)是指具有以下化学结构的化合物:
氯苯胍可以选自0.1mg/kg至250mg/kg体重,优选地1mg/kg至100mg/kg体重,更优选地5mg/kg至50mg/kg体重的剂量施用给所述受试者。氯苯胍可使用选自以下的给药计划施用给所述受试者:每小时、每天三次;每天两次;每天;每隔一天;每周两次;每周一次;隔周一次;每月一次;每两月一次或以恒定速率或可变速率注入。优选地,氯苯胍施用直到定植或感染的病征和症状至少部分治愈或减轻。
在一个实施方案中,在治疗后所述受试者的血液中氯苯胍(或氯苯胍代谢物)的浓度在选自但不限于:2小时0.1和10ug/mL之间、12小时后1和200ug/mL之间、24小时后0.1和5ug/mL之间、48小时后0.01和2ug/mL之间、72小时后0.0001和1ug/mL之间的范围内。优选地,浓度选自但不限于:12小时后小于200ug/mL、24小时后小于5ug/mL、48小时后小于1ug/L和72小时后小于0.5ug/mL。
导致所述细菌感染的作用剂是菌剂。在一个优选的实施方案中,所述作用剂不是原生动物物种。在一个优选的实施方案中,所述作用剂不是球虫原生动物。更优选地,所述作用剂不是产气荚膜梭菌(Clostridiumperfringens),也不是存在于Hansen等从丹麦Jyndevad收集的土壤样品中的异养生物菌种,如以下论文中所讨论:Hansen等2012,Chemosphere,86:212-215和Hansen等2009,EnvironmentalPollution157:474-480。
在另一个实施方案中,所述菌剂是革兰氏阴性的。在另一个实施方案中,所述菌剂是革兰氏阳性的。在另一个实施方案中,所述菌剂无细胞壁。在另一个实施方案中,所述细菌感染由至少两种选自以下的作用剂的混合物引起:革兰氏阴性、革兰氏阳性和无细胞壁的菌剂。
导致所述细菌感染的所述菌剂可为选自但不限于:葡萄球菌属物种(Staphylococcusspp)、链球菌(Streptococci)、肠球菌属物种、明串珠菌属物种(Leuconostocspp)、棒状杆菌属物种(Corynebacteriumspp)、隐秘杆菌属物种(Arcanobacteriaspp)、隐秘杆菌属物种(Trueperellaspp)、红球菌属物种(Rhodococcusspp)、芽孢杆菌属物种(Bacillusspp)、厌氧球菌(AnaerobicCocci)、厌氧革兰氏阳性不形成孢子杆菌、放线菌属物种(Actinomycesspp)、梭菌属物种(Clostridiumspp)、诺卡菌属物种(Nocardiaspp)、丹毒丝菌属物种(Erysipelothrixspp)、李斯特菌属物种(Listeriaspp)、盖球菌属物种(Kytococcusspp)、支原体属物种(Mycoplasmaspp)、尿原体属物种(Ureaplasmaspp)和分枝杆菌属物种(Mycobacteriumspp)的革兰氏阳性菌剂。
在一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于葡萄球菌属物种。葡萄球菌属物种的实例包括表皮葡萄球菌(Staphylococcusepidermidis)、溶血葡萄球菌(Staphylococcushaemolyticus)、路邓葡萄球菌(Staphylococcuslugdunensis)、腐生葡萄球菌(Staphylococcussaprophyticus)、耳葡萄球菌(Staphylococcusauricularis)、头状葡萄球菌(Staphylococcuscapitis)、山羊葡萄球菌(Staphylococcuscaprae)、肉葡萄球菌(Staphylococcuscarnosus)、科氏葡萄球菌(Staphylococcuscohnii)、人葡萄球菌(Staphylococcushominis)、巴氏葡萄球菌(Staphylococcuspasteuri)、佩氏葡萄球菌(Staphylococcuspettenkoferi)、普氏葡萄球菌(Staphylococcuspulvereri)、解糖葡萄球菌(Staphylococcussaccharolyticus)、模仿葡萄球菌(Staphylococcussimulans)、施氏葡萄球菌(Staphylococcusschleiferi)、沃氏葡萄球菌(Staphylococcuswarneri)、木糖葡萄球菌(Staphylococcusxylosus)、阿尔莱特葡萄球菌(Staphylococcusarlettae)、溶酪葡萄球菌(Staphylococcuscaseolyticus)、产色葡萄球菌(Staphylococcuschromogenes)、香料葡萄球菌(Staphylococcuscondimenti)、海豚葡萄球菌(Staphylococcusdelphini)、马胃葡萄球菌(Staphylococcusequorum)、猫葡萄球菌(Staphylococcusfelis)、福氏葡萄球菌(Staphylococcusfleurettii)、鸡葡萄球菌(Staphylococcusgallinarum)、猪葡萄球菌(Staphylococcushyicus)、中间葡萄球菌(Staphylococcusintermedius)、克氏葡萄球菌(Staphylococcuskloosii)、缓慢葡萄球菌(Staphylococcuslentus)、水獭葡萄球菌(Staphylococcuslutrae)、苍蝇葡萄球菌(Staphylococcusmuscae)、尼泊尔葡萄球菌(Staphylococcusnepalensis)、鱼发酵葡萄球菌(Staphylococcuspiscifermentans)、伪中间葡萄球菌(Staphylococcuspseudintermedius)、松鼠葡萄球菌(Staphylococcussciuri)、猿猴葡萄球菌(Staphylococcussimiae)、琥珀葡萄球菌(Staphylococcussuccinus)和牛葡萄球菌(Staphylococcusvitulinus)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于链球菌属物种(Streptococcusspp)。链球菌属物种的实例包括无乳链球菌(Streptococcusagalactiae)、非解乳糖链球菌(Streptococcusalactolyticus)、咽峡炎链球菌(Streptococcusanginosus)、犬链球菌(Streptococcuscanis)、星座链球菌(Streptococcusconstellatus)、大鼠链球菌(Streptococcuscricetus)、嵴链球菌(Streptococcuscristatus)、汗毛链球菌(Streptococcusdownei)、停乳链球菌停乳亚种(Streptococcusdysgalactiaesubsp.dysgalactiae)、停乳链球菌似马亚种(Streptococcusdysgalactiaesubsp.equisimilis)、马链球菌马亚种(Streptococcusequisubsp.equi)、马链球菌兽瘟亚种(Streptococcusequisubsp.zooepidemicus)、野生链球菌(Streptococcusferus)、解没食子酸链球菌解没食子酸亚种(Streptococcusgallolyticussubsp.gallolyticus)(过去称为牛链球菌(Streptococcusbovis)生物型i)、解没食子酸链球菌巴氏亚种(Streptococcusgallolyticussubsp.pasteurianus)(过去称为牛链球菌(Streptococcusbovis)生物型ii/2)、格氏链球菌(Streptococcusgordonii)、猪肠链球菌(Streptococcushyointestinalis)、生殖道链球菌(Streptococcushyovaginalis)、婴儿链球菌(Streptococcusinfantarius)、婴儿链球菌婴儿亚种(Streptococcusinfantariussubspinfantarius)、婴儿链球菌(Streptococcusinfantis)、海豚链球菌(Streptococcusiniae)、中间链球菌(Streptococcusintermedius)、巴黎链球菌(Streptococcuslutetiensis)(过去称为牛链球菌(Streptococcusbovis)生物型ii.1)、猴链球菌(Streptococcusmacaccae)、缓症链球菌(Streptococcusmitis)、变异链球菌(Streptococcusmutans)、口腔链球菌(Streptococcusoralis)、鼠口腔链球菌(Streptococcusorisratti)、副血链球菌(Streptococcusparasanguinis)、栖口腔链球菌(Streptococcusperoris)、肺炎链球菌、豕链球菌(Streptococcusporcinus)、伪中间链球菌(Streptococcuspseudintermedius)、化脓链球菌(Streptococcuspyogenes)、大鼠链球菌(Streptococcusratti)、唾液链球菌(Streptococcussalivarius)、血链球菌(Streptococcussanguinis)、表兄链球菌(Streptococcussobrinus)、猪链球菌(Streptococcussuis)、嗜热链球菌(Streptococcusthermophilus)、前庭链球菌(Streptococcusvestibularis)以及营养变异(缺陷)链球菌(软弱贫养菌(Abiotrophiadefectiva)、毗邻颗粒链菌(Granulicatellaadiacens)、细长颗粒链菌(Granulicatellaelegans)和副毗邻颗粒链菌(Granulicatellapara-adiacens))和相关物种例如粘滑罗斯菌(Rothiamucilaginosa)(过去称为粘滑口腔球菌(Stomatococcusmucilaginosus))和片球菌(Pediococcus)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于肠球菌属物种。肠球菌属物种的实例包括粪肠球菌(Enterococcusfaecalis)、屎肠球菌(Enterococcusfaecium)、鹑鸡肠球菌(Enterococcusgallinarum)、耐久肠球菌(Enterococcusdurans)、鸟肠球菌(Enterococcusavium)、棉子糖肠球菌(Enterococcusraffinosus)、苍白肠球菌(Enterococcuspallens)、浅黄肠球菌(Enterococcusgilvus)、盲肠肠球菌(Enterococcuscecorum)、病臭肠球菌(Enterococcusmalodoratus)、意大利肠球菌(Enterococcusitalicus)、血栖肠球菌(Enterococcussanguinicola)、芒地肠球菌(Enterococcusmundtii)、铅黄/黄色肠球菌(Enterococcuscasseliflavus/flavescens)、殊异肠球菌(Enterococcusdispar)、海氏肠球菌(Enterococcushirae)、类鸟肠球菌(Enterococcuspseudoavium)和牛链球菌(Enterococcusbovis)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于明串珠菌属物种。明串珠菌属物种的实例包括肠膜明串珠菌(Leuconostocmesenteroides)、假肠膜明串珠菌(Leuconostocpseudomesenteroides)、类肠膜明串珠菌(Leuconostocparamesenteroides)、柠檬明串珠菌(Leuconostoccitreum)和乳明串珠菌(Leuconostoclactis)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于棒状杆菌属物种。棒状杆菌属物种的实例包括非亲脂性、发酵棒状杆菌属物种例如溃疡棒状杆菌(Corynebacteriumulcerans)、假结核棒状杆菌(Corynebacteriumpseudotuberculosis)、结膜干燥棒状杆菌(Corynebacteriumxerosis)、纹带棒状杆菌(Corynebacteriumstriatum)、极小棒状杆菌(Corynebacteriumminutissimum)、无枝菌酸棒状杆菌(Corynebacteriumamycolatum)、解葡萄糖苷棒状杆菌(Corynebacteriumglucuronolyticum)、银色棒状杆菌(Corynebacteriumargentoratense)、马氏棒状杆菌(Corynebacteriummatruchotii)、芮氏棒状杆菌(Corynebacteriumriegelii)、混乱棒状杆菌(Corynebacteriumconfusum)、膀胱炎棒状杆菌(Corynebacteriumcystidis)、白喉棒状杆菌(Corynebacteriumdiphtheria)、模仿棒状杆菌(Corynebacteriumsimulans)、Corynebacteriumsundvallense、Corynebacteriumthomssensii、弗雷尼棒状杆菌(Corynebacteriumfreneyi)和黏金色棒状杆菌(Corynebacteriumaurimucosum),非亲脂性、非发酵棒状杆菌属物种例如非发酵棒状杆菌非发酵亚种(Corynebacteriumafermentansafermentans)、耳棒状杆菌(Corynebacteriumauris)、假白喉棒状杆菌(Corynebacteriumpseudodiphtheriticum)和丙酸棒状杆菌(Corynebacteriumpropinquum)以及亲脂性棒状杆菌属物种例如杰氏棒状杆菌(Corynebacteriumjeikeium)、解脲棒状杆菌(Corynebacteriumurealyticum)、非发酵棒状杆菌嗜脂亚种(Corynebacteriumafermentanslipophilum)、邻居棒状杆菌(Corynebacteriumaccolens)、麦利金氏棒状杆菌(Corynebacteriummacginleyi)、Corynebacteriumtuberculostearum、克罗彭施泰特棒状杆菌(Corynebacteriumkroppenstedtii)、库彻氏棒状杆菌(Corynebacteriumkutscheri)、多毛棒状杆菌(Corynebacteriumpilosum)、牛棒状杆菌(Corynebacteriumbovis)、CDC棒状杆菌群F-1和G和黄色嗜脂棒状杆菌(Corynebacteriumlipophiloflavum)以及其它棒状杆菌属物种例如苏黎士菌属(Turicella)、节杆菌属(Arthrobacter)、短杆菌(Brevibacterium)、皮杆菌属(Dermabacter)、罗氏菌属(Rothia)、厄氏菌属(Oerskovia)、微杆菌属(Microbacterium)和水生雷弗森菌(Leifsoniaaquatica)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于隐秘杆菌属物种。隐秘杆菌属物种的实例包括溶血隐秘杆菌(A.haemolyticum)、化脓隐秘杆菌(A.pyogenes)(现在称为化脓隐秘杆菌(Trueperellapyogenes),最初称为化脓放线菌(Actinomycespyogenes))和伯纳德隐秘杆菌(A.bernardiae)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于红球菌属物种。红球菌属物种的实例包括马红球菌(Rhodococcusequi)、红串红球菌(Rhodococcuserythropolis)、束红球菌(Rhodococcusfasciens)和玫瑰红红球菌(Rhodococcusrhodochrous)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于戈登氏菌属物种(Gordoniaspp)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于束村氏菌属物种(Tsukamurellaspp)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于无胆甾原体属物种(Acholeplasmaspp)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于放线菌(Actinobacteria)例如马克洛斯氏菌(Crossiellaequi)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于芽孢杆菌属物种。芽孢杆菌属物种的实例包括炭疽芽孢杆菌(Bacillusanthracis)、蜡状芽孢杆菌(Bacilluscereus)、环状芽胞杆菌(Bacilluscirculans)、地衣芽孢杆菌(Bacilluslicheniformis)、巨大芽胞杆菌(Bacillusmegaterium)、短小芽孢杆菌(Bacilluspumilus)、球形芽孢杆菌(Bacillussphaericus)、枯草芽孢杆菌(Bacillussubtilis)、短短芽孢杆菌(Brevibacillusbrevis)、侧孢短芽孢杆菌(Brevibacilluslaterosporus)和蜂房类芽孢杆菌(Paenibacillusalvei)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于厌氧球菌。厌氧球菌的实例包括莫道克厌氧球菌(Anaerococcusmurdochii)、普氏厌氧球菌(Anaerococcusprevotii)、四联厌氧球菌(Anaerococcustetradius)、八叠厌氧球菌(Anaerococcusoctavius)、产氢厌氧球菌(Anaerococcushydrogenalis)、解乳厌氧球菌(Anaerococcuslactolyticus)、阴道厌氧球菌(Anaerococcusvaginalis)、极小阿托波氏菌(Atopobiumparvulum)、大芬戈尔德菌(Finegoldiamagna)、巴尔涅斯孪生球菌(Gallicolabarnesae)、不解糖卟啉孪生球菌(Gemellaasaccharolytica)、博格孪生球菌(Gemellabergeri)、兔孪生球菌(Gemellacuniculi)、溶血孪生球菌(Gemellahaemolysans)、麻疹孪生球菌(Gemellamorbillorum)、Gemellapalaticanis、血色孪生球菌(Gemellasanguinis)、微小微单胞菌(Parvimonasmicra)、黑色消化球菌(Peptococcusniger)、不解糖嗜胨菌(Peptoniphilusasaccharolyticus)、戈巴赫嗜胨菌(Peptoniphilusgorbachii)、吲哚嗜胨菌(Peptoniphilusindolicus)、兔嗜胨菌(Peptoniphilusharei)、艾弗嗜胨菌(Peptoniphilusivorii)、泪腺嗜胨菌(Peptoniphiluslacrimalis)、奥尔森嗜胨菌(Peptoniphilusolsenii)、口炎消化链球菌(Peptostreptococcusstomatis)、厌氧消化链球菌(Peptostreptococcusanaerobius)、产生瘤胃球菌(Ruminococcusproductus)、还原天芥菜碱斯奈克氏菌(Slackiaheliotrinireducens)和解糖葡萄球菌。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于厌氧革兰氏阳性不形成孢子杆菌。厌氧革兰氏阳性不形成孢子杆菌的实例包括异斯卡多维亚菌(Alloscardoviaomnicolens)、阿托波氏菌属物种(例如微小阿托波氏菌(Atopobiumminutum)、龈裂阿托波氏菌(Atopobiumrimae)、极小阿托波氏菌(Atopobiumparvulum)和阴道阿托波氏菌(Atopobiumvaginae))、双歧杆菌属(Bifidobacteria)(例如青春双歧杆菌(Bifidobacteriaadolescentis)、齿双歧杆菌(Bifidobacteriadentium)、斯卡尔多维双歧杆菌(Bifidobacteriascardovii))、Catabacterhongkongensis、产气柯林斯菌(Collinsellaaerofaciens)、埃格特菌(Eggerthella)(例如迟缓埃格特菌(Eggerthellalenta)、香港埃格特菌(Eggerthellahongkongensis)和中国埃格特菌(Eggerthellasinensis))、真杆菌属(Eubacterium)和相关物种(例如缠结真杆菌(Eubacteriumnodatum)、纤细真杆菌(Eubacteriumtenue)、短真杆菌(Eubacteriumbrachy)、娇弱真杆菌(Eubacteriuminfirmum)、微小真杆菌(Eubacteriumminutum)、缠结真杆菌、隐藏真杆菌(Eubacteriumsaphenum)、沟迹真杆菌(Eubacteriumsulci)、龈沟产线菌(Filifactoralocis)、胆怯艰难杆菌(Mogibacteriumtimidum)、弱小艰难杆菌(Mogibacteriumvescum)、非解乳假枝杆菌(Pseudoramibacteralactolyticus)、缓慢布雷德菌(Bulleidiaextructa)和Solobacteriummoorei)、乳杆菌属(Lactobacillus)物种(例如鼠李糖乳杆菌(Lactobacillusrhamnosus)、干酪乳杆菌(Lactobacilluscasei)、发酵乳杆菌(Lactobacillusfermentum)、戈氏乳杆菌(Lactobacillusgasseri)、植物乳杆菌(Lactobacillusplantarum)、嗜酸乳杆菌(Lactobacillusacidophilus)、惰性乳杆菌(Lactobacillusiners)和Lactobacillusultunensis)、动弯杆菌属(Mobiluncus)物种(例如柯氏动弯杆菌(Mobiluncuscurtisii)、羞怯动弯杆菌(Mobiluncusmulieris))、Moryellaindoligenes、欧氏菌属(Olsenella)口腔物种(例如齿龈欧氏菌(Olsenellauli)和Olsenellaprofuse)、Oribacteriumsinus、丙酸杆菌属(Propionibacterium)(例如疮疱丙酸杆菌(Propionibacteriumacnes)和丙酸丙酸杆菌(Propionibacteriumpropionicum))、Slackiaexigua和Turicibactersanguine。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于放线菌属物种。放线菌属物种的实例包括衣氏放线菌(Actinomycesisraelii)、内氏放线菌(Actinomycesnaeslundii)、粘性放线菌(Actinomycesviscosus)、溶齿放线菌(Actinomycesodontolyticus)、麦尔放线菌(Actinomycesmeyeri)和戈氏放线菌(Actinomycesgerencseriae)(过去称为衣氏放线菌(Actinomycesisraelii)血清型II)、欧洲放线菌(Actinomyceseuropaeus)、纽氏放线菌(Actinomycesneuii)、罗氏放线菌(Actinomycesradingae)、格雷费尼茨放线菌(Actinomycesgraevenitzii)、大麦伤口放线菌(Actinomyceshordeovulneris)、图列茨放线菌(Actinomycesturicensis)、乔氏放线菌(Actinomycesgeorgiae)、化脓隐秘杆菌(放线菌(Actinomyces))、伯纳德隐秘杆菌(放线菌)、芬氏放线菌(Actinomycesfunkei)、Actinomyceslingnae、休斯顿放线菌(Actinomyceshoustonensis,)和卡迪夫放线菌(Actinomycescardiffensis)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于梭菌属物种。梭菌属物种的实例包括巴氏梭菌(Clostridiumbaratii)、双酶梭菌(Clostridiumbifermentans)、肉毒梭菌(Clostridiumbotulinum)、肉毒梭菌(A、B、C、D、E、F、G型)、丁酸梭菌(Clostridiumbutyricum)、艰难梭菌(Clostridiumdifficile)、溶组织梭菌(Clostridiumhistolyticum)、诺氏梭菌(Clostridiumnovyi)(A型)、诺氏梭菌(B型)、产气荚膜梭菌、产气荚膜梭菌(A-E型)、多枝梭菌(Clostridiumramosum)、败毒梭菌(Clostridiumsepticum)、索氏梭菌(Clostridiumsordelli)、楔形梭菌(Clostridiumsphenoides)、第三梭菌(Clostridiumtertium)和破伤风梭菌(Clostridiumtetani)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于诺卡菌属物种。诺卡菌属物种的实例包括星状诺卡氏菌(Nocardiaasteroides)、巴西诺卡氏菌(Nocardiabrasiliensis)、皮疽诺卡氏菌(Nocardiafarcinica)、新星诺卡氏菌(Nocardianova)、豚鼠耳炎诺卡氏菌(Nocardiaotitidiscaviarum)和南非诺卡氏菌(Nocardiatransvalensis)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于丹毒丝菌属物种,例如猪丹毒丝菌(Erysipelothrixrhusiopathiae)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于李斯特菌属物种,例如单核细胞增生李斯特菌(Listeriamonocytogenes)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于盖球菌属物种,例如许氏盖球菌(Kytococcusschroeteri)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于分枝杆菌属物种。分枝杆菌属物种的实例包括脓肿分枝杆菌(Mycobacteriumabscessus)、Mycobacteriumarupense、亚洲分枝杆菌(Mycobacteriumasiaticum)、欧巴涅分枝杆菌(Mycobacteriumaubagnense)、鸟分枝杆菌群(Mycobacteriumaviumcomplex)、博氏分枝杆菌(Mycobacteriumbolletii)、博氏分枝杆菌、德氏分枝杆菌(Mycobacteriumbranderi)、卡内蒂分枝杆菌(Mycobacteriumcanettii)、山羊分枝杆菌(Mycobacteriumcaprae)、隐藏分枝杆菌(Mycobacteriumcelatum)、龟分枝杆菌(Mycobacteriumchelonae)、嵌合分枝杆菌(Mycobacteriumchimaera)、哥伦比亚分枝杆菌(Mycobacteriumcolombiense)、康赛医院分枝杆菌(Mycobacteriumconceptionense)、出众分枝杆菌(Mycobacteriumconspicuum)、象分枝杆菌(Mycobacteriumelephantis)、鼻疽分枝杆菌(Mycobacteriumfarcinogenes)、佛罗伦萨分枝杆菌(Mycobacteriumflorentinum)、偶然分枝杆菌群(Mycobacteriumfortuitumgroup)、日内瓦分枝杆菌(Mycobacteriumgenavense)、古地分枝杆菌(Mycobacteriumgoodii)、嗜血分枝杆菌(Mycobacteriumhaemophilum)、半岛分枝杆菌(Mycobacteriumheckeshornense)、海德堡分枝杆菌(Mycobacteriumheidelbergense)、休斯顿分枝杆菌(Mycobacteriumhoustonense)、免疫原分枝杆菌(Mycobacteriumimmunogenum)、居间分枝杆菌(Mycobacteriuminterjectum)、胞内分枝杆菌(Mycobacteriumintracellulare)、塞内加尔分枝杆菌(Mycobacteriumsenegalense)、非洲分枝杆菌(Mycobacteriumafricanum)、鸟分枝杆菌类结核亚种(Mycobacteriumaviumsubspparatuberculosis)、堪萨斯分枝杆菌(Mycobacteriumkansasii)、洛克司分枝杆菌(Mycobacteriumlacus)、缓黄分枝杆菌(Mycobacteriumlentiflavum)、麻风分枝杆菌(Mycobacteriumleprae)、鼠麻风分枝杆菌(Mycobacteriumlepraemurium)、马德里分枝杆菌(Mycobacteriummageritense)、玛尔摩分枝杆菌(Mycobacteriummalmoense)、海洋分枝杆菌(Mycobacteriummarinum)、马赛分枝杆菌(Mycobacteriummassiliense)、田鼠分枝杆菌(Mycobacteriummicroti)、摩特弗分枝杆菌(Mycobacteriummontefiorense)(鳗鱼)、莫娜分枝杆菌(Mycobacteriummoracense)、产粘液分枝杆菌(Mycobacteriummucogenicum)、内布拉斯加分枝杆菌(Mycobacteriumnebraskense)、新金色分枝杆菌(Mycobacteriumneoaurum)、新城分枝杆菌(Mycobacteriumnovocastrense)、沼泽分枝杆菌(Mycobacteriumpalustre)、帕尔门塞分枝杆菌(Mycobacteriumparmense)、草分枝杆菌(Mycobacteriumphlei)、弗卡分枝杆菌(Mycobacteriumphocaicum)、鳍脚亚目动物分枝杆菌(Mycobacteriumpinnipedii)、猪分枝杆菌(Mycobacteriumporcinum)、假分枝杆菌(Mycobacteriumpseudoshottsii)(鱼)、假结核分枝杆菌(Mycobacteriumpseudotuberculosis)、萨斯喀彻温分枝杆菌(Mycobacteriumsaskatchewanense)、瘰病分枝杆菌(Mycobacteriumscrofulaceum)、Mycobacteriumsenuense、败血分枝杆菌(Mycobacteriumsepticum)、猴分枝杆菌(Mycobacteriumsimiae)、包皮垢分枝杆菌(Mycobacteriumsmegmatis)、苏加分枝杆菌(Mycobacteriumszulgai)、土分枝杆菌/无色分枝杆菌群(Mycobacteriumterrae/chromogenicumcomplex)、三重分枝杆菌(Mycobacteriumtriplex)、肺结核分枝杆菌(Mycobacteriumtuberculosis)、托斯卡纳分枝杆菌(Mycobacteriumtusciae)、溃痕分枝杆菌(Mycobacteriumulcerans)、沃氏分枝杆菌(Mycobacteriumwolinskyi)和蟾分枝杆菌(Mycobacteriumxenopi)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于隐秘杆菌属物种。隐秘杆菌属物种的实例包括Trueperellaabortisuis、伯氏隐秘杆菌(Trueperellabernardiae)、Trueperellabialowiezensis、Trueperellabonasi、化脓隐秘杆菌(Trueperellapyogenes)。
在另一个实施方案中,所述菌剂是革兰氏阳性的、革兰氏阴性的或无细胞壁,并且选自但不限于牲畜病原体。牲畜病原体的实例包括猪放线杆菌(Actinobaculumsuis)、牛放线菌(Actinomycesbovis)、化脓隐秘杆菌、炭疽芽孢杆菌、蜡状芽孢杆菌、地衣芽孢杆菌、短小芽孢杆菌、产黑色素芽孢杆菌(Bacillusmelaninogenicus)、枯草芽孢杆菌、肉毒梭菌、气肿疽梭菌(Clostridiumchauvoei)、溶血梭菌(Clostridiumhaemolyticum)、诺氏梭菌、产气荚膜梭菌、败毒梭菌、索氏梭菌、破伤风梭菌、鹌鹑梭菌(Clostridiumcolinum)、假结核棒状杆菌、牛肾盂炎棒状杆菌(Corynebacteriumrenale)、刚果嗜皮菌(Dermatophiluscongolensis)、肠球菌属物种(例如粪肠球菌、屎肠球菌、耐久肠球菌、鸟肠球菌、海氏肠球菌)、猪丹毒丝菌、伊万诺夫李斯特菌(Listeriaivanovii)、格雷李斯特菌(Listeriagrayi)、无害李斯特菌(Listeriainnocua)、西尔李斯特菌(Listeriaseeligeri)、威尔逊李斯特菌(Listeriawelshimeri)、单核细胞增生李斯特菌、鸟分枝杆菌、牛分枝杆菌(Mycobacteriumbovis)、类结核分枝杆菌(Mycobacteriumparatuberculosis)(副结核性肠炎(Johne'sDisease))、支原体属(例如山羊支原体山羊肺炎亚种(Mycoplasmacapricolumsubsp.capripneumoniae)、山羊支原体山羊亚种(Mycoplasmacapricolumsubsp.capricolum)、霉状支原体霉状亚种(M.mycoidessubspmycoides)、无乳支原体(M.agalactiae)、羊肺炎支原体(M.ovipneumoniae)、结膜支原体(M.conjunctivae)、精氨酸支原体(M.arginini)、牛支原体(M.bovis)和腐败支原体(M.putrefaciens))、牛支原体、殊异支原体(Mycoplasmadispar)、霉状支原体霉状亚种(例如牛传染性胸膜肺炎(CBPP))鸡败血支原体(Mycoplasmagallisepticum)(MG)、衣阿华火鸡支原体(Mycoplasmaiowaemeleagridis)(MM)、关节液支原体(Mycoplasmasynoviae)(MS)、猪嗜血支原体(Mycoplasmahaemosuis)(过去称为猪附红血球体(Eperythrozoonsuis))、产碱支原体(Mycoplasmaalkalescens)、牛生殖道支原体(Mycoplasmabovigenitalum)、牛鼻支原体(Mycoplasmabovirhinis)、牛眼支原体(Mycoplasmabovoculi)、加利福尼亚支原体(Mycoplasmacalifornicum)、加拿大支原体(Mycoplasmacanadense)、犬支原体(Mycoplasmacynos)、马生殖道支原体(Mycoplasmaequigenitalium)、猫支原体(Mycoplasmagateae)、犬嗜血支原体(Mycoplasmahaemocanis)、猫嗜血支原体(Mycoplasmahaemofelis)、猪肺炎支原体(Mycoplasmahyopneumoniae)、猪鼻支原体(Mycoplasmahyorhinis)、猪关节液支原体(Mycoplasmahyosynoviae)、衣阿华支原体(Mycoplasmaiowae)、利氏支原体(Mycoplasmaleachii)、火鸡支原体(Mycoplasmameleagridis)、霉状支原体山羊亚种(Mycoplasmamycoidessubspcapri)、温氏支原体(Mycoplasmawenyonii)、猪支原体(Mycoplasmasuis)、马红球菌、表皮葡萄球菌、模仿葡萄球菌、猫葡萄球菌、木糖葡萄球菌、产色葡萄球菌、沃氏葡萄球菌、溶血葡萄球菌、松鼠葡萄球菌、腐生葡萄球菌、人葡萄球菌、山羊葡萄球菌、科氏葡萄球菌科氏亚种(Staphylococcuscohniisubsp.cohnii)、科氏葡萄球菌解脲亚种(Staphylococcuscohniisubsp.urealyticus)、头状葡萄球菌头状亚种(Staphylococcuscapitissubsp.capitis)、头状葡萄球菌解脲亚种(Staphylococcuscapitissubsp.urealyticus)、猪葡萄球菌、金黄色葡萄球菌、伪中间葡萄球菌、海豚葡萄球菌、施氏葡萄球菌凝结亚种(Staphylococcusschleiferisubsp.coagulans)、金黄色葡萄球菌厌氧亚种(Staphylococcusaureussubsp.anaerobius)、乳房链球菌(Streptococcusuberis)、犬链球菌、无乳链球菌、停乳链球菌、化脓链球菌、牛链球菌、马链球菌兽瘟亚种、马肠链球菌(Streptococcusequinus)、马链球菌(Streptococcusequi)(马链球菌马亚种)、似马链球菌(Streptococcusequisimilis)(停乳链球菌似马亚种)、豕链球菌、猪链球菌、兽瘟链球菌(Streptococcuszooepidemicus)、兽瘟链球菌(马链球菌兽瘟亚种)、停乳链球菌似马亚种、疮疱丙酸杆菌、颗粒丙酸杆菌(Propionibacteriumgranulosum)、真杆菌属、吲哚消化球菌(Peptococcusindolicus)和厌氧消化链球菌;以及以下革兰氏阴性属的各物种:放线杆菌属(Actinobacillus)、气单胞菌属(Aeromonas)、无形体属(Anaplasma)、弓形菌属(Arcobacter)、禽杆菌属(Avibacterium)、类杆菌属(Bacteroides)、巴尔通氏体属(Bartonella)、博德特氏杆菌属(Bordetella)、包柔氏螺旋体属(Borrelia)、短螺旋体属(Brachyspira)、布氏杆菌属(Brucella)、弯曲杆菌属(Campylobacter)、二氧化碳噬纤维菌属(Capnocytophaga)、衣原体属(Chlamydia)、嗜衣原体属(Chlamydophila)、金黄杆菌属(Chryseobacterium)、考克斯氏体属(Coxiella)、噬纤维菌属(Cytophaga)、偶蹄形菌属(Dichelobacter)、爱德华氏菌属(Edwardsiella)、埃利希氏体属(Ehrlichia)、埃希氏菌属(Escherichia)、黄杆菌属(Flavobacterium)、弗朗西斯氏菌属(Francisella)、梭杆菌属(Fusobacterium)、鸡杆菌属(Gallibacterium)、嗜血杆菌属(Haemophilus)、嗜组织菌属(Histophilus)、克雷伯氏菌属(Klebsiella)、指甲花属(Lawsonia)、钩端螺旋体属(Leptospira)、曼氏杆菌属(Mannheimia)、巨球形菌属(Megasphaera)、摩拉克氏菌属(Moraxella)、新立克次氏体属(Neorickettsia)、Nicoletella、鸟杆菌属(Ornithobacterium)、巴斯德氏菌属(Pasteurella)、发光杆菌属(Photobacterium)、鱼衣原体属(Piscichlamydia)、鱼立克次氏体属(Piscirickettsia)、红棕色单胞菌属(Porphyromonas)、普雷沃氏菌属(Prevotella)、变形杆菌属(Proteus)、假单胞菌属(Pseudomonas)、立克次氏体属(Rickettsia)、立默氏菌属(Riemerella)、沙门氏菌属(Salmonella)、链杆菌属(Streptobacillus)、黏着杆菌属(Tenacibaculum)、弧菌属(Vibrio)和耶尔森氏菌属(Yersinia)。
在另一个实施方案中,所述菌剂是革兰氏阳性的,并且选自但不限于伴侣动物物种例如猫、狗和马的病原体。此类病原体的实例包括马病原体,例如马链球菌、兽瘟链球菌、马红球菌、艰难梭菌、产气荚膜梭菌、假结核棒状杆菌、毛状梭菌(Clostridiumpiliforme)、牛放线菌、金黄色葡萄球菌、β溶血链球菌属物种、刚果嗜皮菌、破伤风梭菌和肉毒梭菌。另外的实例包括狗和猫的病原体,例如葡萄球菌属物种、链球菌属物种、梭菌属物种、放线菌属物种、肠球菌属物种、诺卡菌属物种、支原体属物种和分枝杆菌属物种。
在另一个实施方案中,所述菌剂是革兰氏阴性的,并且选自以下代表性科和种:醋酸杆菌科(Acetobacteraceae):-颈玫瑰单胞菌(Roseomonascervicalis)、费氏玫瑰单胞菌(Roseomonasfauriae)、吉氏玫瑰单胞菌(Roseomonasgilardii)--气单胞菌科(Aeromonadaceae):-异常嗜糖气单胞菌(Aeromonasallosaccharophila)、嗜水气单胞菌(Aeromonasaquariorum)、豚鼠气单胞菌(Aeromonascaviae)、嗜水气单胞菌(Aeromonashydrophila)(和亚种)、杀鲑气单胞菌(Aeromonassalmonicida)、舒伯特气单胞菌(Aeromonasshubertii)、维罗纳气单胞菌温和生物变型(Aeromonasveroniibiovarsobria)(温和气单胞菌(Aeromonassobria))--产碱杆菌科(Alcaligenaceae):-木糖氧化无色杆菌(Achromobacterxylosoxidans)、粪产碱杆菌(Alcaligenesfaecalis)、Bordetellaansorpii、鸟博德特氏杆菌(Bordetellaavium)、支气管炎博德特氏杆菌(Bordetellabronchiseptica)、欣茨博德特氏杆菌(Bordetellahinzii)、霍氏博德特氏杆菌(Bordetellaholmesii)、副百日咳博德特氏杆菌(Bordetellaparapertussis)、百日咳博德特氏杆菌(Bordetellapertussis)、彼得里博德特氏杆菌(Bordetellapetrii)、创口博德特氏杆菌(Bordetellatrematum)、解脲寡源杆菌(Oligellaureolytica)、尿道寡源杆菌(Oligellaurethralis)--无浆体科(Anaplasmataceae):-嗜吞噬细胞无浆体(Anaplasmaphagocytophilum)、扁平无浆体(Anaplasmaplatys)、牛无浆体(Anaplasmabovis)、红血球内生无浆体(Anaplasmacentrale)、边缘无浆体(Anaplasmamarginale)、奥氏无浆体(Anaplasmaodocoilei)、羊无浆体(Anaplasmaovis)、犬埃里希氏体(Ehrlichiacanis)、恰菲埃里希氏体(Ehrlichiachaffeensis)、尤因埃里希氏体(Ehrlichiaewingii)、小鼠埃里希氏体(Ehrlichiamuris)、羊埃里希氏体(Ehrlichiaovina)、反刍兽埃里希氏体(Ehrlichiaruminantium)、Neoehrlichialotoris、米库尔新埃里希氏体(Neoehrlichiamikurensis)、蠕虫新立克次氏体(Neorickettsiahelminthoeca)、立氏新立克次氏体(Neorickettsiaristicii)、腺热新立克次体(Neorickettsiasennetsu)、尖音库蚊沃尔巴克氏体(Wolbachiapipientis)--装甲菌科(Armatimonadaceae):-玫瑰装甲菌(Armatimonasrosea)--拟杆菌科(Bacteroidaceae):-福赛斯拟杆菌(Bacteroidesforsythus)、脆弱拟杆菌(Bacteroidesfragilis)、产黑色素拟杆菌(Bacteroidesmelaninogenicus)、赤拟杆菌(Bacteroidesruber)、解脲拟杆菌(Bacteroidesurealtyicus)--巴尔通氏体科(Bartonellaceae):-阿尔萨斯巴尔通氏体(Bartonellaalsatica)、澳大利亚巴尔通氏体(Bartonellaaustralis)、杆状巴尔通氏体(Bartonellabacilliformis)、Bartonellabirtlesii、牛巴尔通氏体(Bartonellabovis)、卡普里巴尔通氏体(Bartonellacapreoli)、Bartonellachomelii、克氏巴尔通氏体(Bartonellaclarridgeiae)、多氏巴尔通氏体(Bartonelladoshiae)、伊丽莎白巴尔通氏体(Bartonellaelizabethae)、格氏巴尔通氏体(Bartonellagrahamii)、汉氏巴尔通氏体(Bartonellahenselae)、克勒巴尔通氏体(Bartonellakoehlerae)、跳鼠巴尔通氏体(Bartonellaperomysci)、Bartonellaphoceensis、五日热巴尔通氏体(Bartonellaquintana)、Bartonellarattimassiliensis、罗卡利巴尔通氏体(Bartonellarochalimae)、Bartonellaschoenbuchensis、鼹鼠巴尔通氏体(Bartonellatalpae)、Bartonellatamiae、泰勒氏巴尔通氏体(Bartonellataylorii)、特利波契巴尔通氏体(Bartonellatribocorum)、文森氏巴尔通氏体博格霍夫亚种(Bartonellavinsoniisubspberkhoffii)、文森氏巴尔通氏体阿鲁潘亚种(Bartonellavinsoniisubsp.arupensis)、文森氏巴尔通氏体文森氏亚种(Bartonellavinsoniisubsp.vinsonii)--蛭弧菌科(Bdellovibrionaceae):-蛭弧菌属物种(Bdellovibriospp.)--短螺旋体科(Brachyspiraceae):-短螺旋体属物种(Brachyspiraspp),包括汉普森短螺旋体(Brachyspirahampsonii)、猪痢疾短螺旋体(Brachyspirahyodysenteriae)、莫道克短螺旋体(Brachyspiramurdochii)、结肠菌毛样短螺旋体(Brachyspirapilosicoli)--布鲁氏菌科(Brucellaceae):-流产布鲁氏菌(Brucellaabortus)、犬布鲁氏菌(Brucellacanis)、鲸型布鲁氏菌(Brucellaceti)、马尔他布鲁氏菌(Brucellamelitensis)、羊布鲁氏菌(Brucellaovis)、鳍型布鲁氏菌(Brucellapinnipedialis)、猪布鲁氏菌(Brucellasuis)、人苍白杆菌(Ochrobactrumanthropi)、中间苍白杆菌(Ochrobactrumintermedium)--伯克氏菌科(Burkholderiaceae):-Burkholderiaaboris、须芒草伯克氏菌(Burkholderiaambifaria)(基因组变型VII)、Burkholderiaanthina(基因组变型VIII)、新洋葱伯克氏菌(Burkholderiacenocepacia)(基因组变型III)、洋葱伯克氏菌(Burkholderiacepacia)(基因组变型I)、Burkholderiadiffusa、多罗萨伯克氏菌(Burkholderiadolosa)(基因组变型VI)、Burkholderialatens、鼻疽伯克氏菌(Burkholderiamallei)、Burkholderiametallica、多噬伯克氏菌(Burkholderiamultivorans)(基因组变型II)、类鼻疽伯克氏菌(Burkholderiapseudomallei)、吡咯伯克氏菌(Burkholderiapyrrocinia)(基因组变型IX)、Burkholderiaseminalis、稳定伯克氏菌(Burkholderiastabilis)(基因组变型IV)、尤伯纳伯克氏菌(Burkholderiaubonensis)(基因组变型X)、越南伯克氏菌(Burkholderiavietnamiensis)(基因组变型V)、罕见贪铜菌(Cupriaviduspauculus)、吉氏贪铜菌(Cupriavidusgilardii)、皮氏罗尔斯顿菌(Ralstoniapickettii)、解甘露醇罗尔斯顿菌(Ralstoniamannitolilytica)、Sphaerotilushippei、蒙大拿球衣菌(Sphaerotilusmontanus)、浮游球衣菌(Sphaerotilusnatans)--弯曲菌科(Campylobacteraceae):-弓形菌属物种(Arcobacterspp),包括斯氏弓形菌(Arcobacterskirrowii)、大肠弯曲杆菌(Campylobactercoli)、简明弯曲杆菌(Campylobacterconcisus)、曲形弯曲杆菌(Campylobactercurvus)、胚胎弯曲杆菌(Campylobacterfetus)、纤细弯曲杆菌(Campylobactergracilis)、瑞士弯曲杆菌(Campylobacterhelveticus)、人弯曲杆菌(Campylobacterhominis)、豚肠弯曲杆菌(Campylobacterhyointestinalis)、海象弯曲杆菌(Campylobacterinsulaenigrae)、空肠弯曲杆菌(Campylobacterjejuni)、拉尼尔弯曲杆菌(Campylobacterlanienae)、红嘴鸥弯曲杆菌(Campylobacterlari)、海鸟弯曲杆菌(Campylobacterlaridis)、粘膜弯曲杆菌(Campylobactermucosalis)、直肠弯曲杆菌(Campylobacterrectus)、昭和弯曲杆菌(Campylobactershowae)、痰液弯曲杆菌(Campylobactersputorum)、乌普萨拉弯曲杆菌(Campylobacterupsaliensis)--Candidatus:-鲑鱼衣原体(Piscichlamydiasalmonis)--心杆菌科(Cardiobacteriaceae):-人心杆菌(Cardiobacteriumhominis)、瓣膜心杆菌(Cardiobacteriumvalvarum)、节瘤偶蹄形菌(Dichelobacternodosus)--衣原体科(Chlamydiaceae):-衣原体属物种(Chlamydiaspp),包括鸟衣原体(Chlamydiaavium)、家禽衣原体(Chlamydiagallinacea)、鼠沙眼衣原体(Chlamydiamuridarum)、猪衣原体(Chlamydiasuis)、沙眼衣原体(Chlamydiatrachomatis);嗜衣原体属物种(Chlamydophilaspp),包括肺炎嗜衣原体(Chlamydophilapneumoniae)、家畜嗜衣原体(Chlamydophilapecorum)、鹦鹉热嗜衣原体(Chlamydophilapsittaci)、流产嗜衣原体(Chlamydophilaabortus)、豚鼠嗜衣原体(Chlamydophilacaviae)和猫嗜衣原体(Chlamydophilafelis)--Chthonomonadaceae:-Chthonomonascalidirosea.--丛毛单胞菌科(Comamonadaceae):-睾丸酮丛毛单胞菌(Comamonastestosteroni);Verminephrobacterspp.--柯克斯体科(Coxiellaceae):-伯氏柯克斯体(Coxiellaburnetii)--噬纤维菌科(Cytophagaceae):-柱状噬纤维菌(Cytophagacolumnaris)、哈氏噬纤维菌(Cytophagahutchinsonii)、Flexibacterechinicida、华美屈挠杆菌(Flexibacterelegans)、易挠屈挠杆菌(Flexibacterflexilis)、海滨屈挠杆菌(Flexibacterlitoralis)、多形屈挠杆菌(Flexibacterpolymorphus)、玫瑰屈挠杆菌(Flexibacterroseolus)、红屈挠杆菌(Flexibacterruber)--脱硫弧菌科(Desulfovibrionaceae):-沃氏嗜胆菌(Bilophilawadsworthia)、细胞内劳索尼亚氏菌(Lawsoniaintracellularis)--肠杆菌科(Enterobacteriaceae):-戴氏西地西菌(Cedeceadavisae)、拉氏西地西菌(Cedecealapagei)、奈氏西地西菌(Cedeceaneteri)、无丙二酸柠檬酸杆菌(Citrobacteramalonaticus)、差异柠檬酸杆菌(Citrobacterdiversus)、弗氏柠檬酸杆菌(Citrobacterfreundii)、柯氏柠檬酸杆菌(Citrobacterkoseri)、康帝蒙提克罗诺杆菌(Cronobactercondimenti)、都柏林克罗诺杆菌(Cronobacterdublinensis)、瑞士克罗诺杆菌(Cronobacterhelveticus)、丙二酸克罗诺杆菌(Cronobactermalonaticus)、穆汀斯克罗诺杆菌(Cronobactermuytjensii)、Cronobacterpulveris、阪崎克罗诺杆菌(Cronobactersakazakii)、图列茨克罗诺杆菌(Cronobacterturicensis)、尤尼沃斯克罗诺杆菌(Cronobacteruniversalis)、苏黎世克罗诺杆菌(Cronobacterzurichensis)、鲶鱼爱德华氏菌(Edwardsiellaictaluri)、迟钝爱德华氏菌(Edwardsiellatarda)、产气肠杆菌(Enterobacteraerogenes)、成团肠杆菌(Enterobacteragglomerans)、阴沟肠杆菌(Enterobactercloacae)、Enterobactercowanii、阿尔伯蒂埃希氏菌(Escherichiaalbertii)、大肠杆菌包括AIEC=粘附侵袭性大肠杆菌、EaggEC=肠凝集性大肠杆菌、EHEC=肠出血性大肠杆菌、EIEC=肠侵袭性大肠杆菌、EPEC=肠致病性大肠杆菌、ETEC=产肠毒素大肠杆菌、ExPEC=肠外致病性大肠杆菌、NMEC=新生儿脑膜炎大肠杆菌、NTEC=坏死性毒素大肠杆菌、UPEC=尿路致病性大肠杆菌、弗格森埃希氏菌(Escherichiafergusonii)、美洲爱文氏菌(Ewingellaamericana)、蜂房哈夫尼菌(Hafniaalvei)、副蜂房哈夫尼菌(Hafniaparalvei)、肉芽肿克雷伯氏菌(Klebsiellagranulomatis)、产酸克雷伯氏菌(Klebsiellaoxytoca)、肺炎克雷伯氏菌、抗坏血酸克吕沃尔氏菌(Kluyveraascorbata)、栖冷克吕沃尔氏菌(Kluyveracryocrescens)、摩氏摩根氏菌(Morganellamorganii)、成团泛菌(Pantoeaagglomerans)(过去称为成团肠杆菌(Enterobacteragglomerans))、非共生发光杆菌(Photorhabdusasymbiotica)、类志贺邻单胞菌(Plesiomonasshigelloides)、奇异变形杆菌(Proteusmirabilis)、彭氏变形杆菌(Proteuspenneri)、普通变形杆菌(Proteusvulgaris)、产碱普罗威登斯菌(Providenciaalcalifaciens)、雷氏普罗威登斯菌(Providenciarettgeri)、斯氏普罗威登斯菌(Providenciastuartii)、Raoultellaelectrica、解鸟氨酸拉乌尔菌(Raoultellaornithinolytica)、植生拉乌尔菌(Raoultellaplanticola)、土生拉乌尔菌(Raoultellaterrigena)、邦戈尔沙门菌(Salmonellabongori)、肠沙门氏菌肠亚种(Salmonellaentericasubspeciesenterica)(多个血清型)、液化沙雷氏菌(Serratialiquifaciens)、粘质沙雷氏菌(Serratiamarcesans)、鲍氏志贺氏菌(Shigellaboydii)、痢疾志贺氏菌(Shigelladysenteriae)、弗氏志贺氏菌(Shigellaflexneri)、索氏志贺氏菌(Shigellasonnei)、小肠结肠炎耶尔森氏菌(Yersiniaenterocolitica)、鼠疫耶尔森氏菌(Yersiniapestis)、假结核耶尔森氏菌(Yersiniapseudotuberculosis)、鲁氏耶尔森氏菌(Yersiniaruckeri)--伞单胞菌科(Fimbriimonadaceae):-Fimbriimonasginsengisoli.--黄杆菌科(Flavobacteriaceae):-动物溃疡伯杰氏菌(Bergeyellazoohelcum)、狗咬二氧化碳噬纤维菌(Capnocytophagacanimorsus)、犬咬二氧化碳噬纤维菌(Capnocytophagacynodegmi)、牙龈二氧化碳噬纤维菌(Capnocytophagagingivalis)、颗粒二氧化碳噬纤维菌(Capnocytophagagranulosa)、溶血二氧化碳噬纤维菌(Capnocytophagahaemolytica)、Capnocytophagaleadbetteri、黄褐二氧化碳噬纤维菌(Capnocytophagaochracea)、生痰二氧化碳噬纤维菌(Capnocytophagasputigena)、产吲哚金黄杆菌(Chryseobacteriumindologenes)、鱼金黄杆菌(Chryseobacteriumpiscicola)、脑膜炎败血伊丽莎白金菌(Elizabethkingiameningoseptica)、嗜鳃黄杆菌(Flavobacteriumbranchiophilum)、柱状黄杆菌(Flavobacteriumcolumnare)、小鳟鱼黄杆菌(Flavobacteriumoncorhynchi)、杀鱼黄杆菌(Flavobacteriumpiscicida)、嗜冷黄杆菌(Flavobacteriumpsychrophilum)、气味类香菌(Myroidesodoratus)、拟香味类香菌(Myroidesodoratimimus)、鼻腔鸟杆菌(Ornithobacteriumrhinotracheale)、鸭瘟立默氏菌(Riemerellaanatipestifer)、哥伦比亚立默氏菌(Riemerellacolumbina)、鸽咽喉立默氏菌(Riemerellacolumbipharyngis)、Tenacibaculumdicentrarchi、脱色黏着杆菌(Tenacibaculumdiscolour)、Tenacibaculumgallaicum、海洋黏着杆菌(Tenacibaculummaritimum)、鳎鱼黏着杆菌(Tenacibaculumsoleae)、有毒威克斯氏菌(Weeksellavirosa)--弗朗西斯氏菌科(Francisellaceae):-土拉热弗朗西斯氏菌土拉热亚种(Francisellatularensissubsp.tularensis)、土拉热弗朗西斯氏菌全北区亚种(Francisellatularensissubsp.holarctica)、土拉热弗朗西斯氏菌新凶手亚种(Francisellatularensissubsp.novicida)、蜃楼弗朗西斯氏菌(Francisellaphilomiragia)、诺神弗朗西斯氏菌(Francisellanoatunensis)、诺神弗朗西斯氏菌东方亚种(Francisellanoatunensissubsp.orientalis)(也称为亚洲弗朗西斯氏菌(Francisellaasiatica))--梭杆菌科(Fusobacteriaceae);-梭杆菌属物种(Fusobacteriumspp.),包括坏死梭杆菌(Fusobacteriumnecrophorum)、具核梭杆菌(Fusobacteriumnucleatum)、多形梭杆菌(Fusobacteriumpolymorphum)--螺杆菌科(Helicobacteraceae):-同性恋螺杆菌(Helicobactercinaedi)、芬纳尔螺杆菌(Helicobacterfennelliae)、幽门螺杆菌(Helicobacterpylori)--军团菌科(Legionellaceae):-嗜肺军团菌(Legionellapneumophila)和其它物种,包括茴香军团菌(Legionellaanisa)、伯明瀚军团菌(Legionellabirminghamensis)、博杰曼军团菌(Legionellabozemannii)、辛辛那提军团菌(Legionellacincinnatiensis)、杜莫夫军团菌(Legionelladumoffii)、费勒氏军团菌(Legionellafeeleii)、戈曼军团菌(Legionellagormanii)、哈开里军团菌(Legionellahackeliae)、约旦军团菌(Legionellajordanis)、兰斯格军团菌(Legionellalansingensis)、长滩军团菌(Legionellalongbeachae)、马氏军团菌(Legionellamaceachernii)、麦氏军团菌(Legionellamicdadei)、橡树岭军团菌(Legionellaoakridgensis)、巴黎军团菌(Legionellaparisiensis)、赫伦荒原军团菌(Legionellasainthelens)、图森军团菌(Legionellatusconensis)、沃氏军团菌(Legionellawadsworthii)、沃斯利军团菌(Legionellawaltersii)--钩端螺旋体科(Leptospiraceae):-亚历山大钩端螺旋体(Leptospiraalexanderi)(包括亚历山大钩端螺旋体七日热血清变型(LeptospiraalexanderiserovarHebdomadis)、亚历山大钩端螺旋体曼耗群3血清变型(LeptospiraalexanderiserovarManhao3))、Leptospiraalstoni(包括LeptospiraalstoniserovarPingchang、LeptospiraalstoniserovarSichuan)、双曲钩端螺旋体(Leptospirabiflexa)(包括双曲钩端螺旋体安科纳血清变型(LeptospirabiflexaserovarAncona)、双曲钩端螺旋体卡内拉血清变型(LeptospirabiflexaserovarCanela))、博氏钩端螺旋体(Leptospiraborgpetersenii)(包括博氏钩端螺旋体哈德乔血清变型(LeptospiraborgpeterseniiserovarHardjo)、博氏钩端螺旋体哈德乔-牛血清变型(LeptospiraborgpeterseniiserovarHardjo-bovis)、博氏钩端螺旋体波莫纳血清变型(LeptospiraborgpeterseniiserovarPomona)、博氏钩端螺旋体塔拉索夫血清变型(LeptospiraborgpeterseniiserovarTarassovi))、布鲁姆钩端螺旋体(Leptospirabroomii)(包括布鲁姆钩端螺旋体赫斯布里治血清变型(LeptospirabroomiiserovarHurstbridge))、弗氏钩端螺旋体(Leptospirafainei)(包括弗氏钩端螺旋体赫斯布里治血清变型(LeptospirafaineiserovarHurstbridge))、Leptospiraidonii、稻田氏钩端螺旋体(Leptospirainadai)(包括稻田氏钩端螺旋体莱姆血清变型(LeptospirainadaiserovarLyme)、稻田氏钩端螺旋体马来亚血清变型(LeptospirainadaiserovarMalaya))、问号钩端螺旋体(Leptospirainterrogans)(包括问号钩端螺旋体澳大利亚血清变型(LeptospirainterrogansserovarAustralis)、问号钩端螺旋体秋生血清变型(LeptospirainterrogansserovarAutumnalis)、问号钩端螺旋体布拉迪斯拉发血清变型(LeptospirainterrogansserovarBratislava)、问号钩端螺旋体犬血清变型(LeptospirainterrogansserovarCanicola)、问号钩端螺旋体感冒伤寒血清变型(LeptospirainterrogansserovarGrippotyphosa)、问号钩端螺旋体哈德乔血清变型(LeptospirainterrogansserovarHardjo)、问号钩端螺旋体哈德乔-牛血清变型(LeptospirainterrogansserovarHardjo-bovis)、问号钩端螺旋体出血性黄疸血清变型(LeptospirainterrogansserovarIcterohaemorrhagiae)、问号钩端螺旋体波莫纳血清变型(LeptospirainterrogansserovarPomona)、问号钩端螺旋体致热血清变型(LeptospirainterrogansserovarPyrogenes)、问号钩端螺旋体塔拉索夫血清变型(LeptospirainterrogansserovarTarassovi))、基施纳钩端螺旋体(Leptospirakirschneri)(包括基施纳钩端螺旋体保加利亚血清变型(LeptospirakirschneriserovarBulgarica)、基施纳钩端螺旋体蝙蝠血清变型(LeptospirakirschneriserovarCynopteri)、基施纳钩端螺旋体感冒伤寒血清变型(LeptospirakirschneriserovarGrippotyphosa))、克氏钩端螺旋体(Leptospirakmetyi)、黎氏钩端螺旋体(Leptospiralicerasiae)、迈氏钩端螺旋体(Leptospirameyeri)(包括迈氏钩端螺旋体索非亚血清变型(LeptospirameyeriserovarSofia))、野口氏钩端螺旋体(Leptospiranoguchii)(包括野口氏钩端螺旋体巴拿马血清变型(LeptospiranoguchiiserovarPanama)、野口氏钩端螺旋体波莫纳血清变型(LeptospiranoguchiiserovarPomona))、圣地罗斯钩端螺旋体(Leptospirasantarosai)、Leptospiraterpstrae、Leptospiravanthielii、韦氏钩端螺旋体(Leptospiraweilii)(包括韦氏钩端螺旋体塞尔东尼血清变型(LeptospiraweiliiserovarCelledoni)、韦氏钩端螺旋体沙敏血清变型(LeptospiraweiliiserovarSarmin))、沃尔氏钩端螺旋体(Leptospirawolbachii)、沃夫钩端螺旋体(Leptospirawolffii)、柳川钩端螺旋体(Leptospirayanagawae)--纤毛菌科(Leptotrichiaceae):-口腔纤毛菌(Leptotrichiabuccalis)、念珠状链杆菌(Streptobacillusmoniliformis)--甲基杆菌科(Methylobacteriaceae):-外链甲基杆菌群(Methylobacteriumextorquensgroup)、藤泽氏甲基杆菌(Methylobacteriumfujisawaense)、嗜中温甲基杆菌(Methylobacteriummesophilicum)、扎氏甲基杆菌(Methylobacteriumzatmanii)--摩拉克氏菌科(Moraxellaceae):-鲍氏不动杆菌(基因组种2)、贝氏不动杆菌(Acinetobacterbaylyi)、布氏不动杆菌(Acinetobacterbouvetii)、乙酸钙不动杆菌(Acinetobactercalcoaceticus)(基因组种1)、格尔纳不动杆菌(Acinetobactergerneri)、格氏不动杆菌(Acinetobactergrimontii)、溶血不动杆菌(Acinetobacterhaemolyticus)(基因组种4)、约氏不动杆菌(Acinetobacterjohnsonii)(基因组种7)、琼氏不动杆菌(Acinetobacterjunii)(基因组种5)、鲁氏不动杆菌(Acinetobacterlwoffi)(基因组种8/9)、帕氏不动杆菌(Acinetobacterparvus)、抗辐射不动杆菌(Acinetobacterradioresistens)(基因组种12)、申氏不动杆菌(Acinetobacterschindleri)、潭氏不动杆菌(Acinetobactertandoii)、特氏不动杆菌(Acinetobactertjernbergiae)、汤氏不动杆菌(Acinetobactertowneri)、邬氏不动杆菌(Acinetobacterursingii)、威尼斯不动杆菌(Acinetobactervenetianus)、亚特兰大摩拉克氏菌(Moraxellaatlantae)、鲍氏摩拉克氏菌(Moraxellaboevrei)、牛摩拉克氏菌(Moraxellabovis)、牛眼摩拉克氏菌(Moraxellabovoculi)、犬摩拉克氏菌(Moraxellacanis)、山羊摩拉克氏菌(Moraxellacaprae)、卡他摩拉克氏菌(Moraxellacatarrhalis)、豚鼠摩拉克氏菌(Moraxellacaviae)、兔摩拉克氏菌(Moraxellacuniculi)、马摩拉克氏菌(Moraxellaequi)、结膜炎摩拉克氏菌(Moraxellalacunata)、林肯摩拉克氏菌(Moraxellalincolnii)、猴摩拉克氏菌(Moraxellamacacae)、不液化摩拉克氏菌(Moraxellanonliquefaciens)、Moraxellaoblonga、奥斯陆摩拉克氏菌(Moraxellaosloensis)、羊摩拉克氏菌(Moraxellaovis)、苯丙酮酸摩拉克氏菌(Moraxellaphenylpyruvica)、多动物摩拉克氏菌(Moraxellapluranimalium)、猪摩拉克氏菌(Moraxellaporci)--摩替亚氏菌科(Moritellaceae):-深渊摩替亚氏菌(Moritellaabyssi)、茶山站摩替亚氏菌(Moritelladasanensis)、日本摩替亚氏菌(Moritellajaponica)、海洋摩替亚氏菌(Moritellamarina)、深海摩替亚氏菌(Moritellapro-funda)、粘摩替亚氏菌(Moritellaviscosa)、雅氏摩替亚氏菌(Moritellayayanosii)--奈瑟氏菌科(Neisseriaceae):-紫色色杆菌(Chromobacteriumviolaceum)、啮蚀艾肯氏菌(Eikenellacorrodens)、反硝化金氏菌(Kingelladenitrificans)、金氏金氏菌(Kingellakingae)、口金氏菌(Kingellaoralis)、波塔新金氏杆菌(Kingellapotus)、灰色奈瑟氏菌(Neisseriacinerea)、长奈瑟氏菌(Neisseriaelongata)、浅黄奈瑟氏菌(Neisseriaflavescens)、淋病奈瑟氏菌(Neisseriagonorrhoeae)、乳糖奈瑟氏菌(Neisserialactamica)、脑膜炎奈瑟氏菌(Neisseriameningitidis)、粘液奈瑟氏菌(Neisseriamucosa)、多糖奈瑟氏菌(Neisseriapolysaccharea)、干燥奈瑟氏菌(Neisseriasicca)、微黄奈瑟氏菌(Neisseriasubflava)、Neisseriaweaver、透明颤菌属物种(Vitreoscillaspp)--亚硝化单胞菌科(Nitrosomonadaceae):-真养亚硝化单胞菌(Nitrosomonaseutropha)、嗜盐亚硝化单胞菌(Nitrosomonashalophila)、寡养亚硝化单胞菌(Nitrosomonasoligotropha)--巴斯德氏菌科(Pasteurellaceae):-伴放线放线杆菌(Actinobacillusactinomycetemcomitans)、马驹放线杆菌(Actinobacillusequuli)、李氏放线杆菌(Actinobacilluslignieresii)、大叶性肺炎放线杆菌(Actinobacilluspleuropneumoniae)、精液放线杆菌(Actinobacillusseminis)、产琥珀酸放线杆菌(Actinobacillussuccinogenes)、脲放线杆菌(Actinobacillusureae)、伴放线凝聚杆菌(Aggregatibacteractinomycetemcomitans)、惰性凝聚杆菌(Aggregatibactersegnis)、嗜沫凝聚杆菌(Aggregatibacteraphrophilus)、鸟禽杆菌(Avibacteriumavium)、禽心内膜炎杆菌(Avibacteriumendocarditidis)、鸡禽杆菌(Avibacteriumgallinarum)、副鸡禽杆菌(Avibacteriumparagallinarum)、敏捷禽杆菌(Avibacteriumvolantium)、Bibersteiniatrehalose、鸭源鸡杆菌(Gallibacteriumanatis)、鸡杆菌(Gallibacterium)基因组种1、鸡杆菌基因组种2、鸡杆菌基因组种3、鸡杆菌群V(GallibacteriumgroupV)、虎皮鹦鹉鸡杆菌(Gallibacteriummelopsittaci)、输卵管炎鸡杆菌(Gallibacteriumsalpingitidis)、海藻糖发酵鸡杆菌(Gallibacteriumtrehalosifermentans)、埃及嗜血杆菌(Haemophilusaegyptius)、鸟嗜血杆菌(Haemophilusavium)、杜克雷嗜血杆菌(Haemophilusducreyi)、溶血嗜血杆菌(Haemophilushaemolyticus)、流感嗜血杆菌(Haemophilusinfluenzae)、副溶血嗜血杆菌(Haemophilusparahaemolyticus)、副流感嗜血杆菌(Haemophilusparainfluenzae)、副猪嗜血杆菌(Haemophilusparasuis)、睡眠嗜组织菌(Histophilussomni)、豚鼠曼海姆菌(Mannheimiacaviae)、葡糖苷曼海姆菌(Mannheimiaglucosida)、肉芽肿曼海姆菌(Mannheimiagranulomatis)、溶血曼海姆菌(Mannheimiahaemolytica)、反刍兽曼海姆菌(Mannheimiaruminalis)、Mannheimiavarigena、Nicoletellasemolina、产气巴斯德氏菌(Pasteurellaaerogenes)、贝氏巴斯德氏菌(Pasteurellabettyae)、马巴斯德氏菌(Pasteurellacaballi)、犬巴斯德氏菌(Pasteurellacanis)、达可马巴斯德氏菌(Pasteurelladagmatis)、多杀巴斯德氏菌(Pasteurellamultocida)(多杀亚种(multocida)、败毒亚种(septicum)、败血亚种(gallicida))、侵肺巴斯德氏菌(Pasteurellapneumotropica)、口巴斯德氏菌(Pasteurellastomatis)、海藻巴斯德氏菌(Pasteurellatrehalosi)--鱼立克次氏体科(Piscirickettsiaceae):-鲑鱼立克次氏体(Piscirickettsiasalmonis)--邻单胞菌科(Plesiomonadaceae):-类志贺邻单胞菌--多囊菌科(Polyangiaceae):-纤维堆囊菌(Sorangiumcellulosum)--紫单胞菌科(Porphyromonadaceae):-Dysgonomonascapnocytophagoides、Dysgonomonasgadei、Dysgonomonashofstadii、Dysgonomonasmossii、Dysgonomonasoryzarvi、Dysgonomonaswimpennyi、牙龈红棕色单胞菌(Porphyromonasgingivalis)--普雷沃氏菌科(Prevotellaceae):-普雷沃氏菌属物种(Prevotellaspp.),包括中间普雷沃氏菌(Prevotellaintermedia)、产黑色素普雷沃氏菌(Prevotellamelaninogenica)--假单胞菌科(Pseudomonadaceae):-浅黄金色单胞菌(Chryseomonasluteola)、铜绿假单胞菌、浅黄假单胞菌(Pseudomonasluteola)、荧光假单胞菌(Pseudomonasfluorescens)、恶臭假单胞菌(Pseudomonasputida)、施氏假单胞菌(Pseudomonasstutzeri)、栖稻假单胞菌(Pseudomonasoryzihabitans)--根瘤菌科(Rhizobiaceae):-根癌农杆菌(Agrobacteriumtumefaciens)、放射根瘤菌(Rhizobiumradiobacter)--立克次氏体科(Rickettsiaceae):-Orientiachuto、恙虫病东方体(Orientiatsutsugamushi)、埃氏立克次氏体(Rickettsiaaeschlimannii)、非洲立克次氏体(Rickettsiaafricae)、小蛛立克次氏体(Rickettsiaakari)、Rickettsiaargasii、亚洲立克次氏体(Rickettsiaasiatica)、澳大利亚立克次氏体(Rickettsiaaustralis)、贝利立克次氏体(Rickettsiabellii)、加拿大立克次氏体(Rickettsiacanadensis)、康氏立克次氏体(Rickettsiaconorii)、Rickettsiacooleyi、猫立克次氏体(Rickettsiafelis)、黑龙江立克次氏体(Rickettsiaheilongjiangensis)、瑞士立克次氏体(Rickettsiahelvetica)、Rickettsiahonei、胡氏立克次体(Rickettsiahoogstraalii)、虎林立克次氏体(Rickettsiahulinensis)、Rickettsiahulinii、日本立克次氏体(Rickettsiajaponica)、Rickettsiamarmionii、Rickettsiamartinet、马赛立克次氏体(Rickettsiamassiliae)、摩纳立克次氏体(Rickettsiamonacensis)、蒙大拿立克次氏体(Rickettsiamontanensis)、Rickettsiamonteiroi、Rickettsiamoreli、帕氏立克次氏体(Rickettsiaparkeri)、皮氏立克次氏体(Rickettsiapeacockii)、Rickettsiaphilipii、普氏立克次氏体(Rickettsiaprowazekii)、劳氏立克次氏体(Rickettsiaraoultii)、扁头蜱立克次氏体(Rickettsiarhipicephali)、立氏立克次氏体(Rickettsiarickettsii)、西伯利亚立克次氏体亚群(Rickettsiasibiricasubgroup)、斯洛伐克立克次氏体(Rickettsiaslovaca)、田村氏立克次氏体(Rickettsiatamurae)、斑疹伤寒立克次氏体(Rickettsiatyphi)--希瓦氏菌科(Shewanellaceae):-腐败希瓦氏菌(Shewanellaputrefaciens)--鞘脂单胞菌科(Sphingomonadaceae):-多食鞘氨醇杆菌(Sphingobacteriummultivorum)、食神鞘氨醇杆菌(Sphingobacteriumspiritivorum)、少动鞘氨醇单胞菌(Sphingomonaspaucimobilis)--螺菌科(Spirillaceae):-减少螺菌(Spirillumminus)、迂回螺菌(Spirillumvolutans)、维氏螺菌(Spirillumwinogradskyi)--螺旋体科(Spirochaetaceae):-阿氏疏螺旋体(Borreliaafzelii)、鹅疏螺旋体(Borreliaanserina)、比塞蒂疏螺旋体(Borreliabissettii)、博氏疏螺旋体(Borreliaburgdorferi)、质革疏螺旋体(Borreliacoriaceae)、达氏疏螺旋体(Borreliaduttonii)、嘎氏疏螺旋体(Borreliagarinii)、赫氏疏螺旋体(Borreliahermsii)、西班牙疏螺旋体(Borreliahispanica)、日本疏螺旋体(Borreliajaponica)、Borrelialonestari、卢西塔尼亚疏螺旋体(Borrelialusitaniae)、宫本疏螺旋体(Borreliamiyamotoi)、扁虱疏螺旋体(Borreliaparkeri)、波斯疏螺旋体(Borreliapersica)、回归热疏螺旋体(Borreliarecurrentis)、斯氏疏螺旋体(Borreliaspielmanii)、特里蜱疏螺旋体(Borreliaturicatae)、特里蜱疏螺旋体、法雷斯疏螺旋体(Borreliavalaisiana)、斑点病密螺旋体(Treponemacarateum)、苍白密螺旋体地方亚种(Treponemapallidumssp.endemicum)、苍白密螺旋体苍白亚种(Treponemapallidumssp.pallidum)、苍白密螺旋体细长亚种(Treponemapallidumssp.pertenue)--琥珀酸弧菌科(Succinivibrionaceae):-厌氧螺菌属物种(Anaerobiospirillumspp.)--萨特氏菌科(Sutterellaceae):-萨特氏菌属物种(Sutterellaspp),包括华德萨特氏菌(Sutterellawadsworthia)--栖热菌科(Thermaceae):-亚栖热菌属物种(Meiothermusspp.)--栖热袍菌科(Thermotogaceae):-新阿波罗栖热袍菌(Thermotoganeapolitana)--韦荣氏球菌科(Veillonellaceae):-戴阿利斯特杆菌属物种(Dialisterspp)、巨单胞菌属物种(Megamonasspp)、巨球形菌属物种(Megasphaeraspp)、梳状菌属物种(Pectinatusspp)、Pelosinusspp、Propionisporaspp、鼠孢菌属物种(Sporomusaspp)、韦荣氏球菌属物种(Veillonellaspp.)、嗜酵母菌属物种(Zymophilusspp.)--弧菌科(Vibrionaceae):-美人鱼发光杆菌(Photobacteriumdamselae)、适应弧菌(Vibrioadaptatus)、解藻酸弧菌(Vibrioalginolyticus)、Vibrioazasii、坎氏弧菌(Vibriocampbellii)、霍乱弧菌(Vibriocholera)、美人鱼弧菌(Vibriodamsel)、河流弧菌(Vibriofluvialis)、弗尼斯弧菌(Vibriofurnisii)、霍利斯弧菌(Vibriohollisae)、麦奇尼科夫氏弧菌(Vibriometchnikovii)、最小弧菌(Vibriomimicus)、副溶血弧菌(Vibrioparahaemolyticus)、创伤弧菌(Vibriovulnificus)--沃尔巴克氏体科(Wolbachieae):-沃尔巴克氏体属物种(Wolbachiaspp.)--黄单胞菌科(Xanthomonadaceae):-Luteimonasaestuarii、水生藤黄色单胞菌(Luteimonasaquatica)、食物堆肥藤黄色单胞菌(Luteimonascomposti)、Luteimonaslutimaris、海洋藤黄色单胞菌(Luteimonasmarina)、怪味藤黄色单胞菌(Luteimonasmephitis)、Luteimonasvadosa、Pseudoxanthomonasbroegbernensis、日本假黄单胞菌(Pseudoxanthomonasjaponensis)、嗜麦芽糖寡养单胞菌(Stenotrophomonasmaltophilia)、亚硝酸盐还原寡养单胞菌(Stenotrophomonasnitritireducens)。
最优选地,导致所述细菌感染的所述菌剂是革兰氏阴性的,并且选自:不动杆菌属(Acinetobafcter)物种、嗜水气单胞菌、柠檬酸杆菌属(Citrobacter)物种、肠杆菌属物种、大肠杆菌、肺炎克雷伯氏菌、摩氏摩根氏菌、铜绿假单胞菌和嗜麦芽糖寡养单胞菌。
在另一个优选的实施方案中,导致所述细菌定植或感染的所述菌剂具有对用于治疗所述定植或感染的常规抗生素的耐性。在一个优选的实施方案中,所述菌剂具有对选自以下的化合物的耐性:氨基糖苷类(例如庆大霉素、妥布霉素、阿米卡星或奈替米星)、抗MRSA先锋霉素类(例如头孢洛林)、抗假单胞性青霉素+β-内酰胺酶抑制剂(例如替卡西林-棒酸或哌拉西林-他唑巴坦)、碳青霉烯类(例如厄他培南、亚胺培南、美罗培南或多尼培南)、非广谱先锋霉素类:第一代和第二代先锋霉素(例如头孢唑林或头孢呋辛)、广谱先锋霉素类:第三代和第四代先锋霉素(例如头孢噻肟或头孢曲松)、头霉素类(例如头孢西丁或头孢替坦)、氟喹诺酮类(例如环丙沙星)、叶酸途径抑制剂(例如三甲氧苄二氨嘧啶-磺胺甲噁唑)、甘氨酰环素类(例如替加环素)、单环内酰胺类(例如氨曲南)、青霉素类(例如氨苄青霉素)、青霉素+β-内酰胺酶抑制剂(例如阿莫西林-棒酸或氨苄青霉素-舒巴坦)、苯丙醇类(例如氯霉素)、膦酸类(例如磷霉素)以及多粘菌素类(例如粘菌素)、四环素类(例如四环素、强力霉素或米诺环素)中的一者或多者。优选地,具有对这些化合物的耐性的所述菌剂是革兰氏阴性的。
优选地,所述菌剂具有对选自以下的化合物的耐性:青霉素类、先锋霉素类、碳青霉烯类、单环内酰胺类及其它β-内酰胺抗生素、夫西地烷类、氨基糖苷类、氟喹诺酮类、链阳性菌素类、四环素类、甘氨酰环素类、氯霉素及其它苯丙醇类、大环内酯类和酮内酯类、林可酰胺类、噁唑烷酮类、氨基环醇类、多粘菌素类、糖肽类、脂肽类、杆菌肽、莫匹罗星、截短侧耳素类、利福霉素类、磺胺类以及三甲氧苄二氨嘧啶。更优选地,所述化合物选自:β-内酰胺类、糖肽类、脂肽类、大环内酯类、噁唑烷酮类以及四环素类。优选地,当所述化合物在选自:0.001μg/mL-10,000μg/mL、0.01μg/mL-1000μg/mL、0.10μg/mL-100μg/mL和1μg/mL-50μg/mL的浓度范围内时,所述菌剂具有对所述化合物的耐性。
最优选地,导致所述细菌感染的所述菌剂选自但不限于革兰氏阳性菌。所述菌剂为最优选地选自金黄色葡萄球菌、伪中间葡萄球菌、肺炎链球菌、化脓链球菌、无乳链球菌、乳房链球菌、屎肠球菌、粪肠球菌和艰难梭菌的革兰氏阳性菌剂。
在一个优选的实施方案中,所述菌剂无细胞壁。优选地,所述菌剂选自:支原体属物种、无乳支原体、产碱支原体、Mycoplasmaamphoriforme、精氨酸支原体、牛生殖道支原体、牛鼻支原体、牛支原体、牛眼支原体、颊支原体(Mycoplasmabuccale)、加利福尼亚支原体、加拿大支原体、山羊支原体山羊亚种、山羊支原体山羊肺炎亚种、结膜支原体、犬支原体、殊异支原体、马生殖道支原体、咽喉支原体(Mycoplasmafaucium)、狸猫支原体(Mycoplasmafelis)、发酵支原体(Mycoplasmafermentans)(incognitus株)、鸡败血支原体(Mycoplasmagallisepticum)(MG)、猫支原体、生殖道支原体(Mycoplasmagenitalium)、犬嗜血支原体(Mycoplasmahaemocanis)、猫嗜血支原体(Mycoplasmahaemofelis)、猪嗜血支原体(Mycoplasmahaemosuis)(过去称为猪附红血球体(Eperythrozoonsuis))、人型支原体(Mycoplasmahominis)、猪肺炎支原体、猪鼻支原体、猪关节液支原体、衣阿华火鸡支原体(MM)、衣阿华支原体、利氏支原体、嗜脂支原体(Mycoplasmalipophilum)、火鸡支原体、霉状支原体山羊亚种、霉状支原体霉状亚种、霉状支原体霉状亚种(例如牛传染性胸膜肺炎(CBPP))、口腔支原体(Mycoplasmaorale)、羊肺炎支原体、牛支原体、穿透支原体(Mycoplasmapenetrans)、梨支原体(Mycoplasmapirum)、肺炎支原体(Mycoplasmapneumoniae)、灵长类支原体(Mycoplasmaprimatum)、腐败支原体(Mycoplasmaputrefaciens)、唾液支原体(Mycoplasmasalivarium)、嗜精子支原体(Mycoplasmaspermatophilum)、猪支原体、关节液支原体(MS)、温氏支原体,支原体、尿原体属物种、微小尿原体(Ureaplasmaparvum)、解脲尿原体(Ureaplasmaurealyticum)、尿原体和差异尿原体(Ureoplasmadiversum)。
在另一个最优选的实施方案中,所述菌剂是金黄色葡萄球菌。
在另一个优选的实施方案中,所述菌剂具有对选自以下的化合物的耐性:氨基糖苷类(例如庆大霉素)、安莎霉素类(例如利福平)、抗MRSA先锋霉素类(例如头孢洛林)、抗葡萄球菌性β-内酰胺类(或头霉素类)(例如苯唑西林或头孢西丁)、碳青霉烯类(例如厄他培南、亚胺培南、美罗培南或多尼培南)、非广谱先锋霉素类:第一代和第二代先锋霉素(例如头孢唑林或头孢呋辛)、广谱先锋霉素类:第三代和第四代先锋霉素(例如头孢噻肟或头孢曲松)、头霉素类(例如头孢西丁或头孢替坦)、氟喹诺酮类(例如环丙沙星或莫西沙星)、叶酸途径抑制剂(例如三甲氧苄二氨嘧啶-磺胺甲噁唑)、夫西地烷类(例如梭链孢酸)、糖肽类(例如万古霉素、替考拉宁或特拉万星)、甘氨酰环素类(例如替加环素)、林可酰胺类(例如克林霉素)、脂肽类(例如达托霉素)、大环内酯类(例如红霉素)、噁唑烷酮类(例如利奈唑胺或泰迪唑胺)、苯丙醇类(例如氯霉素)、膦酸类(例如磷霉素)、链阳性菌素类(例如奎奴普丁-达福普丁)以及四环素类(例如四环素、强力霉素或米诺环素)中的一者或多者。优选地,具有对这些化合物的耐性的所述菌剂是革兰氏阳性的。
在另一个最优选的实施方案中,所述菌剂是肺炎链球菌。所述肺炎链球菌可以是具有对β-内酰胺类和大环内酯类中的一者或多者的耐性的菌株。
在另一个最优选的实施方案中,所述菌剂是化脓链球菌。
在另一个最优选的实施方案中,所述菌剂是无乳链球菌。
在另一个最优选的实施方案中,所述菌剂是屎肠球菌或粪肠球菌。所述屎肠球菌或粪肠球菌可以是具有氨基糖苷类(例如庆大霉素(高水平)或链霉素(例如链霉素(高水平))、碳青霉烯类(例如亚胺培南、美罗培南或多尼培南)、氟喹诺酮类(例如环丙沙星、左氧氟沙星或莫西沙星)、糖肽类(例如万古霉素或替考拉宁)、甘氨酰环素类(例如替加环素)、脂肽类(例如达托霉素)、噁唑烷酮类(例如利奈唑胺)、青霉素类(例如氨苄青霉素)、链阳性菌素类(例如奎奴普丁-达福普丁)、四环素(例如强力霉素或米诺环素)中的一者或多者的耐性的菌株。
在另一个最优选的实施方案中,所述菌剂是艰难梭菌。
所述受试者中的所述细菌感染可导致的疾病选自但不限于:金黄色葡萄球菌(MDR、XDR、PDR或甲氧西林敏感或耐甲氧西林菌株)导致的医院获得性肺炎,或侵袭性肺炎球菌疾病例如肺炎、支气管炎、急性窦炎、中耳炎、结膜炎、脑膜炎、菌血症、败血病、骨髓炎、脓毒性关节炎、心内膜炎、腹膜炎、心包炎、蜂窝织炎,和肺炎链球菌(包括多药物耐性菌株[MDRSP],例如具有对β-内酰胺类和大环内酯类耐性的那些)导致的脑脓肿,金黄色葡萄球菌(甲氧西林敏感和耐甲氧西林菌株)、化脓链球菌或无乳链球菌导致的复杂性皮肤和皮肤结构感染,包括糖尿病足感染,有或无伴发性骨髓炎,金黄色葡萄球菌(甲氧西林敏感和耐甲氧西林菌株)或化脓链球菌导致的单纯性皮肤和皮肤结构感染,肺炎链球菌(包括多药物耐性菌株[MDRSP]或金黄色葡萄球菌(甲氧西林敏感和耐甲氧西林菌株)导致的社区获得性肺炎,包括具有并发性菌血症的病例,以及甲氧西林敏感和耐甲氧西林分离株导致的金黄色葡萄球菌血流感染(菌血症),包括具有右心感染性心内膜炎的那些,耐万古霉素肠球菌感染,包括具有并发性菌血症的病例,以及艰难梭菌相关腹泻(CDAD)的治疗。
革兰氏阴性生物是人和其它动物物种中多种传染病的重要致因。骨和关节感染(革兰氏阴性生物或混合菌种是脊椎骨髓炎和脓毒性关节炎的重要致因)、心血管系统感染(包括HACEK群-副流感嗜血杆菌、嗜沫嗜血杆菌(Haemophilusaphrophilus)、伴放线凝聚杆菌、人心杆菌、啮蚀艾肯氏菌、金氏金氏菌导致的心内膜炎)、中枢神经系统感染(细菌性脑膜炎的最常见的致因是脑膜炎奈瑟氏菌、肺炎链球菌,并且在未接种的幼童中乙型流感嗜血杆菌(Haemophilusinfluenzae类型b)(Hib),在年龄小于3个月的初生儿和婴儿中无乳链球菌(B型链球菌),大肠杆菌及其它需氧革兰氏阴性杆菌是重要的病原体,脑脓肿或硬脑膜下积脓,感染的生物体随潜在的致病因素而变化,但其中最可能的起源部位是耳,通常涉及肠革兰氏阴性杆菌)、眼部感染(常见的病原体包括流感嗜血杆菌、淋病奈瑟氏菌或沙眼衣原体)、胃肠道感染(涉及一系列广泛的病原体,包括产肠毒素大肠杆菌(ETEC)、沙门氏菌属、弯曲杆菌属、志贺氏菌属(Shigella)、霍乱弧菌和小肠结肠炎耶尔森氏菌)、生殖道感染(细菌性阴道炎是高浓度的厌氧(如动弯杆菌属物种)和其它苛养菌(包括阴道加德纳氏菌(Gardnerellavaginalis)和阴道阿托波氏菌)以及人型支原体导致的多种微生物临床综合征;非性获得性盆腔炎(PID)通常由混合阴道菌群包括厌氧微生物、兼性革兰氏阴性菌和人型支原体导致,而性获得性PID通常由沙眼衣原体或淋病奈瑟氏菌引起,越来越多的证据显示生殖道支原体感染涉及的病例显著较少)、腹腔内感染(由于内脏穿孔而导致的腹膜炎通常是需氧和厌氧菌肠群的多种微生物感染的,而自发性细菌腹膜炎(SBP)通常由肠革兰氏阴性杆菌例如大肠杆菌和克雷伯氏菌属物种导致,逐步认为肺炎克雷伯氏菌是肝脓肿的致因)、社区获得性肺炎(肺炎支原体、肺炎嗜衣原体(肺炎衣原体)(Chlamydophila(Chlamydia)pneumoniae)、鹦鹉热嗜衣原体(鹦鹉热衣原体)(Chlamydophila(Chlamydia)psittaci)、流感嗜血杆菌、需氧革兰氏阴性杆菌包括肺炎克雷伯氏菌、铜绿假单胞菌、鲍氏不动杆菌、类鼻疽伯克氏菌)、外耳炎(包括急性弥散性外耳炎)(细菌培养物通常产生铜绿假单胞菌、金黄色葡萄球菌和变形杆菌属以及克雷伯氏菌属(Klebsiella)物种)、中耳炎(包括急性中耳炎)(常见的细菌病原体包括肺炎链球菌、流感嗜血杆菌和卡他摩拉克氏菌)、败血症(包括严重败血症)(包括鲍氏不动杆菌、弥散淋球菌性败血症、肠革兰氏阴性菌、脑膜炎奈瑟氏菌(脑膜炎球菌性败血症)和铜绿假单胞菌)、全身性感染(斑疹热(立克次氏体属)和恙虫病(东方体属(Orientia))、普鲁氏菌病、猫抓病和其它巴尔通氏体感染、钩端螺旋体病、莱姆病、类鼻疽、Q热、伤寒和副伤寒(肠热病)、泌尿道感染(急性膀胱炎、急性肾盂肾炎、复发性泌尿道感染和导尿管相关菌尿和泌尿道感染)。
在人中,革兰氏阴性菌是腹腔内感染(IAI)、泌尿道感染(UTI)、医院获得性肺炎和菌血症的常见致因。大肠杆菌(E.coli)、肺炎克雷伯氏菌(K.pneumoniae)和铜绿假单胞菌(P.aeruginosa)在医院环境中是重要的病原体,占导致健康相关感染的所有病原体的27%和所有革兰氏阴性病原体的70%[SievertDM、RicksP、EdwardsJR等,Antimicrobial-resistantpathogensassociatedwithhealthcare-associatedinfections:summaryofdatareportedtotheNationalHealthcareSafetyNetworkattheCentersforDiseaseControlandPrevention,2009-2010.InfectControlHospEpidemiol.2013;34:1-14.]。
革兰氏阴性菌显示出对当前疗法的耐药率增加。广谱β-内酰胺酶(ESBL)的产生是常见的耐性机制。产生ESBL的大肠杆菌和肺炎链球菌的耐药率显著增加,结果是这些细菌对广泛使用的抗微生物剂的耐性增加。
在美国,铜绿假单胞菌是导致医院获得性肺炎的最常见革兰氏阴性致因和导致导尿管相关UTI的第二最常见致因。
大肠杆菌是UTI的最常见致因。产生ESBL的大肠杆菌和肺炎克雷伯氏菌以及铜绿假单胞菌包括MDR菌株导致的UTI病例增加。产生ESBL的大肠杆菌和肺炎克雷伯氏菌通常也从患有复杂性IAI(cIAI)的患者分离。
铜绿假单胞菌是可导致人的常见感染例如医院获得性肺炎、UTI、IAI和血流感染的临床挑战和毒性病原体。铜绿假单胞菌是导致呼吸器相关肺炎的最常见革兰氏阴性生物和导尿管相关UTI的第二常见致因。
革兰氏阴性菌导致的感染数量增加通过耐药率提高来实现。满足该挑战的治疗选项逐渐受到限制。迫切需要满足患者目前和将来需要的新型抗生素。
在另一个优选的实施方案中,氯苯胍或其治疗学上可接受的盐与移除或基本上移除或降低所述菌剂的所述细胞壁完整性的化合物或药剂一起施用。例如,所述化合物选自:β-内酰胺类、磷霉素、溶菌酶、多粘菌素类和螯合剂例如乙二胺四乙酸(EDTA)。例如,所述药剂是降低所述细胞壁的完整性的免疫剂(例如抗体或疫苗)。在一个优选的实施方案中,氯苯胍或其治疗学上可接受的盐与移除或基本上移除或削弱革兰氏阴性或阳性菌剂的所述外细胞壁的完整性的化合物一起施用。
根据本发明的另一个方面,提供了包含治疗有效量的氯苯胍或其治疗学上可接受的盐的抗菌药物组合物。
根据本发明的另一个方面,提供了包含治疗有效量的氯苯胍或其治疗学上可接受的盐的抗菌兽药组合物。
所述治疗或预防受试者中的细菌感染或定植的方法还可包括施用本发明的药物或兽药组合物。
所述药物组合物可任选地包括药学上可接受的赋形剂或载体。所述兽药组合物可任选地包括兽药学上可接受的赋形剂或载体。
本发明的所述药物或兽药组合物优选地包含浓度选自:1mg/g至500mg/g、5mg至400mg/g、10mg/g至200mg/g、20mg/g至100mg/g、30mg/g至70mg/g和40mg/g至60mg/g的氯苯胍或药学上可接受的盐。
在另一个实施方案中,所述药物或兽药组合物包含杂质,其中杂质的量以组合物的总重量的百分比计选自:小于20%杂质(以组合物的总重量计)、小于15%杂质、小于10%杂质、小于8%杂质、小于5%杂质、小于4%杂质、小于3%杂质、小于2%杂质、小于1%杂质、小于0.5%杂质、小于0.1%杂质。在一个实施方案中,所述药物或兽药组合物包含微生物杂质或次级代谢物,其中微生物杂质的量以组合物的总重量的百分比计选自:小于5%、小于4%、小于3%、小于2%、小于1%、小于0.5%、小于0.1%、小于0.01%、小于0.001%。在一个实施方案中,所述药物或兽药组合物是无菌的,并且保存在密封和无菌容器中。在一个实施方案中,所述药物或兽药组合物包含不可检测水平的微生物污染物。
优选地,所述氯苯胍为药物或兽药级。合成商用量氯苯胍的方法在本领域中广泛使用。商用量药物或兽药级氯苯胍得自ZhejiangEsigmaAnimalHealthCo.,Ltd,HainingCity,PeoplesRepublicofChina。
本发明的药物或兽药组合物可包含另外的抗微生物剂。所述另外的抗微生物剂可以是抗真菌剂或抗菌剂。所述治疗或预防受试者中细菌感染或定植的方法还可包括氯苯胍与另外的抗微生物剂一起施用。
在一个实施方案中,所述抗真菌剂选自但不限于:天然存在的药剂,包括棘球白素(阿尼芬净、卡泊芬净、米卡芬净)、聚烯(两性霉素B、杀假丝菌素、菲律宾菌素、制霉色基素(戊霉素)、曲古霉素、哈霉素、鲁斯霉素、美帕曲星、游霉素、制霉菌素、培西洛星、表霉素),以及其它天然存在的抗真菌剂,包括灰黄霉素、寡霉素、硝吡咯菌素、干蠕孢菌素和绿毛菌素。所述抗真菌剂可以是选自但不限于:烯丙胺类(布替萘芬、奈替芬、特比萘芬)咪唑类(联苯苄唑、布康唑、氯咪唑、甘宝素、氯康唑(克罗可唑)、克霉唑、依柏康唑、益康唑、恩康唑、芬替康唑、氟曲马唑、磷氟康唑、异康唑、酮康唑、拉诺康唑、卢立康唑、咪康唑、奈康唑、奥莫康唑、硝酸奥昔康唑、帕康唑、舍他康唑、硫康唑、噻康唑)、硫代氨基甲酸酯类(利拉萘酯、托西拉酯、托林达酯、托萘酯)、三唑类(氟康唑、艾沙康唑、伊曲康唑、泊沙康唑、雷夫康唑、沙康唑、特康唑、伏立康唑),以及其它合成剂例如吖啶琐辛、阿莫罗芬、溴柳氯苯胺(溴氯水杨酰苯胺)、丁氯柳胺、丙酸钙、氯苯甘油醚、环吡酮、氯羟喹(5-氯-8-羟基喹啉)、科帕腊芬内特、依沙酰胺、氟胞嘧啶、卤普罗近、合克替啶、氯氟卡班、硝呋太尔、硝呋甲肟、吡罗克酮、碘化钾、丙酸、巯基吡啶氧化物、N-水杨酰苯胺、对氯苯甲酸钠、丙酸钠、舒苯汀、替诺尼唑、甘油三乙酸酯、三甲曲沙、十一碳烯酸(十一烯酸)和丙酸锌的合成化合物。
本发明的组合物可包含选自但不限于:β-内酰胺酶抑制剂(阿维巴坦、棒酸、舒巴坦、舒他西林、他唑巴坦)、肾二肽酶抑制剂(西司他丁)以及肾保护剂(倍他米隆)的抗生素助剂。
在一个实施方案中,本发明的组合物包含选自但不限于:2,4-二氨基嘧啶类,包括巴喹普林、溴莫普林、艾拉普林、奥美普林、乙胺嘧啶、四氧普林、三甲氧苄二氨嘧啶;氨基香豆素类,包括新生霉素;氨基环醇类,包括壮观霉素;氨基糖苷类,包括阿米卡星、阿普拉霉素、阿贝卡星、卡那霉素B、丁苷菌素、地贝卡星、二氢链霉素、依替米星、福提霉素类(阿司米星)、弗氏菌丝素、庆大霉素、潮霉素B、异帕米星、卡那霉素、小诺霉素、新霉素、奈替米星、巴龙霉素、Plazomicin、核糖霉素、西索米星、链霉素、妥布霉素、威达米星;氨基美他环素类,包括奥玛环素;酰胺醇类,包括叠氮氯霉素、氯霉素、氟苯尼考、甲砜氯霉素;安莎霉素类,包括利福布汀、利福酰胺、利福平(甲哌利福霉素)、利福霉素、利福喷汀、利福昔明;防腐剂类,包括吖啶衍生物(包括吖啶黄、氨基吖啶、依沙吖啶、普罗黄素)、双吡啶类(包括奥替尼啶二盐酸盐)、溴化水杨酰苯胺(包括溴胺杀)、氯己定、酚衍生物(包括百里酚和三氯生)、季铵化合物(包括烷基二甲基乙基苄基氯化铵、苯扎氯铵、十六烷基氯化吡啶鎓、氯化苄乙氧铵、十六烷基三甲铵);抗结核药,包括环丝氨酸、迪拉马尼、乙胺丁醇、乙硫磷酰胺、异烟肼(异烟腙)、吗啉酰吡嗪、对氨基水杨酸(PAS)、丙硫异烟胺、吡嗪酰胺、特立齐酮、胺苯硫脲、硫卡利特;砷剂类,包括阿散酸、洛克沙砷;杆菌素类,包括乳链菌肽、Brilacidin(PMX-30063);β-内酰胺碳头孢烯类,包括氯碳头孢;Β-内酰胺碳青霉烯类,包括比阿培南、多尼培南、厄他培南、法罗培南、亚胺培南、美罗培南、帕尼培南、阿祖培南、利替培南、硫培南、泰比培南、托莫培南;Β-内酰胺先锋霉素类,包括头孢乙腈、头孢克洛、头孢羟氨苄、头孢氨苄、头孢来星、头孢罗宁、头孢利定、头孢菌素、头孢孟多、头孢匹林、羟胺唑头孢菌素、头孢氮氟、头孢西酮、头孢唑林、头孢卡品、头孢地尼、头孢妥仑、头孢吡肟、头孢他美、头孢克肟、头孢甲肟、头孢地嗪、头孢尼西、头孢哌酮、头孢雷特、头孢噻利、头孢噻肟、头孢替安、头孢维星、头孢唑兰、头孢咪唑、头孢匹胺、头孢匹罗、头孢泊肟、头孢丙烯、头孢喹肟、头孢拉啶、头孢沙定、头孢磺啶、头孢洛林、头孢他啶、头孢特仑、头孢替唑、头孢布烯、头孢噻呋、头孢唑肟、头孢吡普、Ceftolozane、头孢拉定、头孢替唑、头孢曲松、头孢沙定、头孢呋辛、头孢唑南、Pivcefalexin;Β-内酰胺头霉素类,包括头孢拉宗、头孢美他唑、头孢米诺、头孢替坦、头孢西丁;Β-内酰胺单环内酰胺类,包括氨曲南、卡芦莫南、替吉莫南;Β-内酰胺氧头孢烯类,包括氟氧头孢、拉氧头孢、羟酸氧酰胺菌素;Β-内酰胺青霉素类,包括阿德诺西林(美西林)、阿莫西林、氨苄青霉素、阿帕西林、阿扑西林、叠氮西林、阿洛西林、巴氨西林、羧苄青霉素、卡茚西林、环己西林、氯咪唑青霉素、氯甲西林、氯唑西林、环青霉素、双氯西林、依匹西林、芬贝西林、氟氯青霉素(氟氯西林)、海他西林、仑氨苄西林、甲亚胺青霉素、美坦西林、甲氧西林钠、美洛西林、萘夫西林、苯唑西林、培那西林、青霉素G二乙氨乙酯氢碘酸盐、青霉素G、苄星青霉素G、普鲁卡因青霉素G、青霉素N、青霉素O、青霉素V、苯氧乙基青霉素钾、哌拉西林、匹氨西林、匹美西林、苯丙西林、喹那西林、磺苄西林、舒他西林、酞氨西林、替莫西林、替卡西林;双环霉素类,包括二环霉素;含硼抗菌剂,包括AN3365(氨基甲基苯并硼唑类)、GSK2251052(亮氨酰-tRNA合成酶抑制剂);环状酯类,包括磷霉素;脂肪酸合成抑制剂(FabI)、AFN-1252、MUT056399、FAB-001;氟喹诺酮类,包括Avarofloxacin、巴洛沙星、贝西沙星、昌欣沙星、西诺沙星、环丙沙星、克林沙星、达诺沙星、德拉沙星、双氟沙星、依诺沙星、恩氟沙星、非那沙星、氟罗沙星、氟甲喹、加雷沙星、加替沙星、吉米沙星、格帕沙星、依巴沙星、左氧氟沙星、洛美沙星、马波沙星、米洛沙星、莫西沙星、那氟沙星、诺氟沙星、氧氟沙星、奥比沙星、帕珠沙星、培氟沙星、普多沙星、普卢利沙星、罗索沙星、芦氟沙星、沙氟沙星、西他沙星、斯帕沙星、替马沙星、妥舒沙星、曲伐沙星、扎波沙星;夫西地烷类,包括梭链孢酸;糖脂缩肽,包括雷莫拉宁;糖肽类,包括阿佛帕星、达贝万星、去甲万古霉素、奥利万星、替考拉宁、替拉万星、万古霉素;糖磷脂类,包括班贝霉素类(班贝霉素、默诺霉素类、黄磷脂素);甘氨酰环素类,包括替加环素;混合药物、卡达唑胺(噁唑烷酮-喹诺酮)、TD-1792(糖肽-先锋霉素);林可酰胺类,包括克林霉素、林肯霉素、吡利霉素;脂肽类,包括达托霉素、Surotomycin;大环内酯类,包括阿奇霉素、碳霉素、赛红霉素、克拉霉素、地红霉素、红霉素、非达霉素、氟红霉素、加米霉素、交沙霉素、北里霉素、柱晶白霉素、麦白霉素、麦迪霉素类、蜜柑霉素、米罗沙霉素、竹桃霉素、伯霉素、罗他霉素、蔷薇霉素、罗红霉素、西地霉素、索利霉素、螺旋霉素、泰利霉素、特卡霉素、泰地罗新、替米考星、醋竹桃霉素、托拉菌素、泰乐菌素、泰伐洛星;硝基呋喃类,包括呋喃他酮、呋喃烯啶、呋喃唑酮、呋唑氯铵、硝呋拉太、硝呋复林、硝呋酚酰肼、硝呋吡醇、硝呋妥醇、硝呋噻肼、呋喃西林、硝基呋喃妥英、硝基糠腙;硝基咪唑类,包括迪美唑、甲硝唑、奥硝唑、洛硝哒唑、塞克硝唑、磺甲硝咪唑;寡糖类,包括阿维霉素、扁枝衣霉素;其它抗菌剂,包括Auriclosene、氯喔星、氯喹那多、氯碘喹啉、氯福克酚、哈喹诺、洛替利星、扁桃酸、乌洛托品(六胺)、醋胺硝唑、硝羟喹啉、Perchlozone、牛磺罗定、噻吩甲酸、异冰片二甲酚;噁唑烷酮类,包括依哌唑胺、利奈唑胺、泼斯唑来、雷得唑来、Sutezolid、特地唑胺(泰迪唑胺);肽去甲酰酶抑制剂,包括GSK1322322;肽类,包括奥米加南、培西加南;截短侧耳素类,包括瑞他莫林、泰妙菌素、伐奈莫林;聚醚离子载体,包括莱特洛霉素、拉沙里菌素、马杜霉素、莫能菌素、甲基盐霉素、盐霉素、生度米星;多粘菌素类,包括粘菌素、多粘菌素B;多肽类,包括安福霉素、杆菌肽、卷曲霉素、持久杀菌素、持久霉素、恩维霉素、夫沙芬近、短杆菌肽类、依色加南、爪蟾抗菌肽类、诺西肽、瑞斯托菌素、硫链丝菌肽、结核放射菌素、短杆菌酪肽、短杆菌素、紫霉素;假单胞菌类,包括莫匹罗星;喹诺酮类,包括萘啶酸、奈诺沙星、噁喹酸、奥泽沙星、吡哌酸、吡咯米酸;喹喔啉类,包括卡巴多司、喹乙醇;氯苯吩嗪类,包括氯苯吩嗪;斯达汀类,包括阿托伐他汀、氟伐他汀、洛伐他汀、美伐他汀、匹伐他汀、普伐他汀、罗苏伐他汀、辛伐他汀;链阳性菌素类,包括达福普丁、氟洛普丁、Linopristin、原始霉素、奎奴普丁、维及尼霉素;链丝菌素类,包括诺尔丝菌素;磺胺类,包括磺胺乙酰甲氧吡嗪、氯胺-B、氯胺-T、二氯胺T、甲醛磺胺噻唑、磺胺米隆、N4-磺胺酰磺胺、诺丙磺胺、N-磺胺酰-3,4-二甲苯甲酰胺、Ormaosulfathiazole、邻苯二甲酰磺乙酰胺、邻苯二甲酰磺胺噻唑、水杨基偶氮磺胺二甲嘧啶、琥珀酰磺胺噻唑、苯酰磺胺、磺胺脲、乙酰磺胺、磺胺氯哒嗪、磺胺柯衣酸、磺胺氯吡嗪、磺胺乙胞嘧啶、磺胺嘧啶、磺胺戊烯、碘胺二甲氧哒嗪、磺胺二甲嘧啶、周效磺胺、磺胺乙二唑、磺胺胍、磺胺胍诺、磺胺林、磺胺洛西酸、磺胺二甲基嘧啶、磺胺对甲氧嘧啶、磺胺甲嘧啶、磺胺甲噻二唑、磺胺甲氧甲嘧啶、磺胺甲噁唑、磺胺甲氧哒嗪、磺胺甲基噻唑、磺胺甲氧吡嗪、磺胺美曲、磺胺柯衣定、磺胺莫托辛、磺胺噁唑、对氨基苯磺酰胺、磺胺酰脲、磺胺培林、磺胺苯吡唑、磺胺普罗林、磺胺吡嗪、磺胺嘧啶、磺胺喹啉、磺胺噻唑、磺胺硫脲、磺胺曲沙唑、磺胺索嘧啶、磺胺异噁唑(磺胺二甲异噁唑);砜类,包括醋地砜、氨苯砜、葡萄糖砜钠、对磺胺酰基苄胺、琥珀氨苯砜、磺胺酸、阿地砜钠、噻唑砜;四环素类,包括氯四环素、氯莫环素、地美环素、强力霉素、Eravacycline、胍甲环素、赖甲四环素、甲氯环素、甲烯土霉素、米诺环素、土霉素、青哌环素、匹哌环素、罗列环素、Sarecycline、四环素的另外的抗生素。
本发明的组合物还可包含选自但不限于:粘合剂和压缩助剂、涂层和膜、染色剂、稀释剂和媒介物崩解剂、乳化和增溶剂、调味剂和甜味剂、拒斥剂、助流剂和润滑剂、增塑剂、防腐剂、推进剂、溶剂、稳定剂、悬浮剂和增粘剂的赋形剂。
根据本发明的另一个方面,提供了用于治疗或预防所述受试者中细菌感染的方法的医疗装置。
根据本发明的另外方面,提供了包括本发明所述的组合物的医疗装置。本发明所述的组合物可以是任何缓释形式,和/或所述医疗装置的涂层形式。
所述医疗装置可以是选自以下的形式:应用于受试者中的细菌感染的植入物、膏药、绷带和其它敷料。
根据本发明的另外方面,提供了杀灭细菌的方法,所述方法包括用氯苯胍或其治疗学上可接受的盐接触所述细菌的步骤。
根据本发明的另外方面,提供了使用氯苯胍或其治疗学上可接受的盐杀灭细菌的用途,所述用途包括用氯苯胍或其治疗学上可接受的盐接触所述细菌的步骤。
除非另外指明,本文所用的术语将具有本领域中通常的意义。
附图简述
本发明的另外特征将在以下其若干非限制性实施方案的描述中更完整地描述。该描述出于示例本发明的目的单独并入。其不应理解为对上文所示的本发明的广泛发明内容、公开或具体实施方式进行限制。具体实施方式将结合以下附图说明,其中:
图1示出了根据实施例1的单个金黄色葡萄球菌分离株的最小抑制浓度表;
图2示出了根据实施例1的单个肠球菌分离株的最小抑制浓度表;
图3示出了根据实施例1的单个肺炎链球菌分离株的最小抑制浓度表;
图4示出了根据实施例1的MRSA、VRE和肺炎链球菌的澳大利亚分离株的NCL812MIC50、MIC90、MIC众数和MIC范围表。氨苄青霉素的比较MIC值以括号示出;
图5示出了根据实施例2的NCL812(氯苯胍)和利奈唑胺对金黄色葡萄球菌ATCC29213的最小抑制浓度值表;
图6示出了根据实施例2的NCL812对金黄色葡萄球菌中DNA大分子合成的影响曲线图;
图7示出了根据实施例2的NCL812对金黄色葡萄球菌中RNA大分子合成的影响曲线图;
图8示出了根据实施例2的NCL812对金黄色葡萄球菌(ATCC29213)中蛋白质大分子合成的影响曲线图;
图9示出了根据实施例2的NCL812对金黄色葡萄球菌(ATCC29213)中细胞壁大分子合成的影响曲线图;
图10示出了根据实施例2的NCL812对金黄色葡萄球菌(ATCC29213)中脂质大分子合成的影响曲线图;
图11示出了汇总根据实施例2的NCL812对金黄色葡萄球菌(ATCC29213)中大分子合成的影响曲线图;
图12示出了根据实施例3的NCL812对ATP从金黄色葡萄球菌(ATCC29213)释放的影响曲线图;
图13示出了根据实施例4所用的金黄色葡萄球菌克隆/分离株名称、类型、来源、抗菌谱、克林霉素耐性状态、多基因座序列类型(MLST)、葡萄球菌盒染色体(SCCmec)类型、克隆复合体、Panton-Valentine杀白细胞素状态(PVL)以及分离株的spa类型表。MSSA;甲氧西林敏感金黄色葡萄球菌。HA-MRSA;医院获得性耐甲氧西林金黄色葡萄球菌。CA-MRSA;社区相关性耐甲氧西林金黄色葡萄球菌。M.B;M.Barton(南澳大学)。G;Gribbles病理学系(南澳大利亚)。J.P.;J.Perry(阿德莱德大学)。VIMP;兽医免疫学、微生物学和公共健康系(阿德莱德大学)学生的鼻孔。S.P.;S.Polyak(阿德莱德大学)。G.C.;GeoffCoombs(西澳大利亚PathWest实验医学)。Em;红霉素。Ci;环丙沙星。Gn;庆大霉素。Tm;三甲氧苄二氨嘧啶。Te;四环素。FA;梭链孢酸。Rf;利福平。Mp;莫匹罗星;
图14示出了根据实施例4报告为选择表型和基因型测试阳性的推定鉴定的金黄色葡萄球菌分离株的百分比表。HA-MRSA;医院获得性金黄色葡萄球菌。CA-MRSA;社区相关性金黄色葡萄球菌。金黄色葡萄球菌分离株鉴定为蛋白A乳胶凝集(蛋白A)、玻片凝固酶、Voges-Proskauer和耐多粘菌素B测试阳性,以及spa基因聚合酶链式反应(PCR)和实时PCR扩增测试阳性。耐甲氧西林金黄色葡萄球菌分离株鉴定为上述标准测试阳性以及mecA基因PCR和实时PCR阳性分离株;
图15示出了根据实施例4的使用Kirby-Bauer纸片扩散法测定的金黄色葡萄球菌分离株的抗菌剂耐性表。HA-MRSA;医院获得性耐甲氧西林金黄色葡萄球菌。CA-MRSA;社区相关性耐甲氧西林金黄色葡萄球菌;
图16示出了根据实施例4的归类为耐甲氧西林的20个金黄色葡萄球菌菌株中鉴定mec基因复合物的数量和百分比表。图中示出了表达苯唑西林和头孢替坦表型耐性的各个葡萄球菌盒染色体(SCCmec)复合物和类型,以及实时mecA状态和得自mecA基因实时PCR的解链温度分析的平均负dF/dT峰值。图中括号表示百分比;
图17示出了耐甲氧西林金黄色葡萄球菌分离株中mecA基因实时聚合酶链式反应后负导数曲线–dF/dT图的平均解链温度峰值曲线图,以mec基因复合物分组,A(n=4)、B(n=10)、C2(n=4)和未归类(n=2)。根据实施例4,以不同上标标出的组具有显著性差异(P<0.05);
图18示出了根据实施例4的抗菌NCL812和β-内酰胺抗菌氨苄青霉素特性表,其详细示出了初步研究确定的和目前研究期间确定的二甲基亚砜(DMSO)的抗菌溶解度、阳离子调节Mueller-HintonII肉汤(CAMHB)的溶解度以及耐甲氧西林金黄色葡萄球菌(MRSA)的平均最小抑制浓度(MIC)(μg/ml24-h)。ATCC49775;甲氧西林敏感金黄色葡萄球菌分离株和ATCC对照菌。MRSA580;耐甲氧西林金黄色葡萄球菌分离株#580。MRSA698;耐甲氧西林金黄色葡萄球菌分离株#698;
图19示出了根据实施例4的新型抗菌NCL812和β-内酰胺抗菌氨苄青霉素对金黄色葡萄球菌临床分离株的体外活性表。HA-MRSA;医院获得性耐甲氧西林金黄色葡萄球菌。CA-MRSA;社区相关性耐甲氧西林金黄色葡萄球菌。MIC;最小抑制浓度(μg/ml)。MBC;最小杀菌浓度(μg/ml)。MIC/MBC范围;所有分离株的最小和最大MIC/MBC。MIC/MBC50;其中抑制50%分离株的MIC/MBC。MIC/MBC90;其中抑制90%分离株的MIC/MBC;
图20示出了根据实施例4的使用微量肉汤稀释法测定的未补充生长对照、氨苄青霉素和不同浓度抗菌剂NCL812对甲氧西林敏感金黄色葡萄球菌与ATCC49775的光密度曲线图。NCL812浓度以MIC测试,在温育最多24-h后为测试条件下测定的MIC的四倍。氨苄青霉素以MIC测试。在0-、1-、2-、4-、8-、12-和24-h测试抗菌剂的杀菌活性;
图21示出了根据实施例4的甲氧西林敏感金黄色葡萄球菌与ATCC49775的杀菌动力学曲线图,其示出了使用临床和实验室标准协会(ClinicalandLaboratoryStandardsInstitute)常量稀释法在10-ml试剂瓶中测定的NCL812杀菌活性。所测试的抗菌剂浓度为测试条件下测定的MIC的1×和4×。杀菌活性在添加抗菌剂0-、1-、2-、4-、8-、12-和24-h后测定。杀菌活性定义为存活细菌的数量从初始接种量减少3-log10(99.9%);
图22示出了根据实施例5的20个肺炎链球菌分离株对六种不同抗菌剂的抗菌敏感性表;
图23示出了根据实施例5的宏量肉汤稀释测定法测定的肺炎链球菌菌株D39暴露至4μg/mlNCL812和0.0023μg/ml氨苄青霉素期间pH改变的曲线图;
图24示出了根据实施例5的NCL812处理48小时的肺炎链球菌菌株D39时间杀菌曲线图;
图25示出了根据实施例5的NCL812处理14小时的肺炎链球菌菌株D39时间杀菌曲线图;
图26示出了根据实施例5的氨苄青霉素处理14小时的肺炎链球菌菌株D39时间杀菌曲线图;
图27示出了根据实施例5的从图40收集的NCL812处理12小时的肺炎链球菌菌株D39时间杀菌曲线图;
图28示出了根据实施例5的氨苄青霉素处理48小时的肺炎链球菌菌株D39时间杀菌曲线图;
图29示出了根据实施例5的红霉素处理48小时的肺炎链球菌菌株D39时间杀菌曲线图;
图30示出了根据实施例5的NCL812和5%氯化胆碱处理48小时的肺炎链球菌菌株D39时间杀菌曲线图;
图31示出了根据实施例5的NCL812和5%氯化胆碱处理12小时的肺炎链球菌菌株D39时间杀菌曲线图;
图32示出了根据实施例5的氨苄青霉素处理48小时时间期的肺炎链球菌菌株D39相对最小杀菌浓度(MBC)曲线图;
图33示出了根据实施例5的红霉素处理48小时时间期的肺炎链球菌菌株D39相对MBC曲线图;
图34示出了根据实施例5的宏量肉汤稀释测定的NCL812处理24小时的肺炎链球菌菌株D39的时间杀菌存活计数(log10CFU/ml)曲线图;
图35示出了根据实施例5的宏量肉汤稀释测定的氨苄青霉素处理24小时的肺炎链球菌菌株D39的时间杀菌存活计数(log10CFU/ml)曲线图;
图36示出了根据实施例5的处理和未处理的D39的平均细胞膜厚度条形图;
图37示出了根据实施例5的处理(16μg/mlNCL812)和未处理的D39样品的周质空间平均宽度条形图;
图38示出了根据实施例6的伪中间葡萄球菌分离株测试表;
图39示出了根据实施例6的伪中间葡萄球菌分离株的抗生素耐性测试曲线表。
图40示出了根据实施例7的NCL812对革兰氏阴性大肠杆菌原生质球的效应曲线图;
图41示出了根据实施例9的NCL812从制剂B的累积释放曲线图;
图42示出了根据实施例11的金黄色葡萄球菌KC01在不同NCL812浓度下温育最多24h的杀菌动力学测定结果;以及
图43示出了根据实施例11的粪肠球菌USA01在不同NCL812浓度下温育最多24h的杀菌动力学测定结果。
实施方案描述
概述
在详细描述本发明之前,应当理解本发明不限于本文所公开的具体示例方法或组合物。还应当理解,本文所用的术语只是为了描述本发明的具体实施方案,并非旨在进行限制。
本文涉及的所有出版物,包括专利和专利申请,全文以引用方式并入。然而,本文涉及的专利申请仅仅是出于描述和公开结合本发明使用的出版物中涉及的程序、方案和试剂的目的。本文涉及的任何出版物的引用不应解释为承认本发明不能因为是在先发明而先于此类公开揭示。
此外,除非另外指明,本发明的实施使用本领域内的常规微生物学技术。此类常规技术是技术人员已知的。
本文中和随附的权利要求书中所用的单数形式“一”、“一个”和“所述”包括复数,除非上下文清楚表明不是如此。
除非另外指明,否则本文所用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同的意义。虽然与本文所述类似或等同的任何材料和方法可用于实施本发明,但本文描述了优选的材料和方法。
本文描述的发明可包括一个或多个数值范围(如尺寸、浓度、剂量等)。数值范围将理解为包括范围内的所有数值,包括定义范围的数值,以及产生与紧邻定义范围边界的数值相同或基本上相同结果的邻近范围的数值。
本发明的药物或兽药组合物可以多个单位剂量施用,取决于施用方法、目标部位、患者的生理状态以及所施用的其它药物。例如,适于经口施用的单位剂型包括固体剂型例如粉剂、片剂、丸剂和胶囊剂,以及液体剂型例如酏剂、糖浆、溶液和悬浮液。活性成分还可以无菌液体剂型非肠道施用。胶囊剂可包含活性成分和非活性成分,例如粉末载体、葡萄糖、乳糖、蔗糖、甘露糖醇、淀粉、纤维素或纤维素衍生物、硬脂酸镁、硬脂酸、糖精钠、滑石、碳酸镁等等。
如本文所用,术语“治疗有效量”是指足以抑制细菌感染或定植相关的细菌生长的量。即,提及根据本发明的方法或组合物施用治疗有效量的氯苯胍是指其中基本上杀菌或抑菌活性致使细菌感染大量抑制的治疗效应。如本文所用,术语“治疗有效量”是指足以提供所需的生物、治疗和/或预防结果的组合物的量。所需的结果包括消除细菌感染或定植或减少和/或减轻疾病的病征、症状或致因,或生物系统的任何其它所需改变。在任何个别情况下,有效量可由本领域的普通技术人员使用日常实验确定。相对于药物或兽药组合物,有效量可以是疾病状态或其病征或症状调整推荐的剂量。有效量根据所用的组合物和所用的施用途径而不同。有效量考虑药代动力学和药效动力学特性以及具体患者的各种因素例如年龄、体重、性别等和疾病或致病微生物影响的区域而例行优化。
如本文所用,术语“治疗”是指完全或部分移除病症的症状和病征。例如,在细菌感染或定植的治疗中,治疗完全或部分移除感染的病征。优选地,在感染的治疗中,治疗减少或消除导致微生物治愈的传染性细菌病原体。
如本文所用,术语“细菌”是指原核微生物的广泛域成员。通常,长度为几微米的细菌具有多种形状,从球形到杆状和球体,并且可以单个细胞存在或以可变数量和形状的直链或簇存在。优选地,术语“细菌”及其形容词“细菌的”是指例如革兰氏阳性葡萄球菌属物种、链球菌属物种、芽胞杆菌属物种(Bacillusspp)、肠球菌属物种、李斯特菌属物种、支原体属物种和厌氧细菌;革兰氏阴性大肠杆菌、肠杆菌属物种、克雷伯氏菌属物种和假单胞菌属物种的细菌;以及无细胞壁的细菌例如支原体属物种和尿原体属物种。该术语可以指抗生素敏感或耐抗生素菌株。在一个优选的实施方案中,该术语是指MRSA或MRSP。在另一个优选的实施方案中,该术语是指MDR葡萄球菌属物种、链球菌属物种、肠球菌属物种、艰难梭菌、大肠杆菌、肠杆菌属物种、克雷伯氏菌属物种和假单胞菌属物种。
如本文所用,术语“耐甲氧西林细菌”(例如耐甲氧西林葡萄球菌)是指显示出在任何剂量下对所有β-内酰胺包括青霉素类、碳青霉烯类和第一代至第四代先锋霉素类具有耐性,但对第五代抗MRSA先锋霉素类(例如头孢洛林)没有耐性的细菌分离株。多药物耐性(MDR)定义为对三种或更多种抗微生物剂类别中的至少一种药剂的获得性不敏感性,广泛耐药性(XDR)定义为除两种或更少的抗微生物剂类别之外的所有药剂中的至少一种药剂的不敏感性(即细菌分离株仅对一种或两种类别保持敏感),以及泛耐药性(PDR)定义为现有的所有抗微生物剂类别中的所有药剂的不敏感性。
敏感、MDR、XDR和PDR细菌的实例包括:野生型、未暴露至抗菌剂、可能对所有以下抗菌剂类别(和药剂)敏感的金黄色葡萄球菌分离株:氨基糖苷类(例如庆大霉素)、安莎霉素类(例如利福平)、抗MRSA先锋霉素类(例如头孢洛林)、抗葡萄球菌性β-内酰胺类(或头霉素类)(例如苯唑西林或头孢西丁)、碳青霉烯类(例如厄他培南、亚胺培南、美罗培南或多尼培南)、非广谱先锋霉素类:第一代和第二代先锋霉素(例如头孢唑林或头孢呋辛)、广谱先锋霉素类:第三代和第四代先锋霉素(例如头孢噻肟或头孢曲松)、头霉素类(例如头孢西丁或头孢替坦)、氟喹诺酮类(例如环丙沙星或莫西沙星)、叶酸途径抑制剂(例如三甲氧苄二氨嘧啶-磺胺甲噁唑)、夫西地烷类(例如梭链孢酸)、糖肽类(例如万古霉素、替考拉宁或特拉万星)、甘氨酰环素类(例如替加环素)、林可酰胺类(例如克林霉素)、脂肽类(例如达托霉素)、大环内酯类(例如红霉素)、噁唑烷酮类(例如利奈唑胺或泰迪唑胺)、苯丙醇类(例如氯霉素)、膦酸类(例如磷霉素)、链阳性菌素类(例如奎奴普丁-达福普丁)以及四环素类(例如四环素、强力霉素或米诺环素)中的一者或多者。对超过三种抗微生物剂类别中的不止一种药剂不敏感的分离株归类为MDR(所有MRSA,例如满足MDR的定义)。对除一种或两种抗微生物剂类别之外的所有药剂中的不止一种药剂不敏感的分离株归类为XDR。对所有列出的抗菌剂不敏感的分离株为PDR。
药学上和兽药学上可接受的盐包括保持本公开的化合物的生物有效性和性质并且为非生物学或换句话讲不期望的盐。在多种情况下,本文所公开的化合物由于氨基和/或羧基或其类似基团的存在能够形成酸式和/或碱式盐。可接受的碱式盐可从无机和有机碱制备。衍生自无机碱的盐包括,仅以举例的方式,钠、钾、锂、铵、钙和镁盐。衍生自有机碱的盐包括但不限于伯胺、仲胺和叔胺盐,例如仅以举例的方式,烷基胺、二烷基胺、三烷基胺、取代烷基胺、二(取代烷基)胺、三(取代烷基)胺、烯基胺、二烯基胺、三烯基胺、取代烯基胺、二(取代烯基)胺、三(取代烯基)胺、环烷基胺、二(环烷基)胺、三(环烷基)胺、取代环烷基胺、二取代环烷基胺、三取代环烷基胺、环烯基胺、二(环烯基)胺、三(环烯基)胺、取代环烯基胺、二取代环烯基胺、三取代环烯基胺、芳基胺、二芳基胺、三芳基胺、杂芳基胺、二杂芳基胺、三杂芳基胺、杂环胺、二杂环胺、三杂环胺,其中胺上的至少两个取代基不同并且选自烷基、取代烷基、烯基、取代烯基、环烷基、取代环烷基、环烯基、取代环烯基、芳基、杂芳基、杂环等等的二胺和三胺混合物。还包括其中两个或三个取代基连同氨基氮一起形成杂环杂芳基的胺。
药学上和兽药学上可接受的酸式盐可从无机和有机酸制备。可使用的无机酸包括,仅以举例的方式,盐酸、氢溴酸、硫酸、硝酸、磷酸等等。可使用的有机酸包括,仅以举例的方式,乙酸、丙酸、乙醇酸、丙酮酸、草酸、苹果酸、丙二酸、琥珀酸、马来酸、延胡索酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸、水杨酸等等。
可用于本公开的化合物的药学上或兽药学上可接受的盐可通过常规化学方法从母体化合物合成,该母体化合物包含碱性或酸性部分。一般来讲,此类盐可通过使这些化合物的离酸或碱形式与化学计量的适当碱或酸在水或有机溶剂中,或二者的混合物中反应来制备;一般来讲,无水介质如醚、乙酸乙酯、乙醇、异丙醇或乙腈是优选的。合适盐的列表可见于Remington'sPharmaceuticalSciences.第17版,MackPublishingCompany,Easton,Pa.(1985),第1418页中,该文献的公开内容以引用的方式并入本文。此类合适盐的实例为碘化物、乙酸盐、苯乙酸盐、三氟乙酸盐、丙烯酸盐、抗坏血酸盐、苯甲酸盐、氯代苯甲酸盐、二硝基苯甲酸盐、羟基苯甲酸盐、甲氧基苯甲酸盐、甲基苯甲酸盐、邻乙酰氧基苯甲酸盐、萘-2-苯甲酸盐、溴化物、异丁酸盐、苯丁酸盐、γ-羟丁酸盐、β-羟基丁酸盐、丁炔-1,4-二酸盐、己炔-1,4-二酸盐、己炔-1,6-二酸盐、己酸盐、辛酸盐、氯化物、肉桂酸盐、柠檬酸盐、癸酸盐、甲酸盐、延胡索酸盐、乙醇酸盐、庚酸盐、马尿酸盐、乳酸盐、苹果酸盐、马来酸盐、羟基马来酸盐、丙二酸盐、扁桃酸盐、甲磺酸盐、烟酸盐、异烟酸盐、硝酸盐、草酸盐、邻苯二甲酸盐、对苯二甲酸盐、磷酸盐、磷酸氢盐、磷酸二氢盐、偏磷酸盐、焦磷酸盐、丙炔酸盐、苯丙酸盐、水杨酸盐、癸二酸盐、琥珀酸盐、辛二酸盐、硫酸盐、硫酸氢盐、焦硫酸盐、亚硫酸盐、亚硫酸氢盐、磺酸盐、苯磺酸盐、对溴苯磺酸盐、氯苯磺酸盐、丙磺酸盐、乙磺酸盐、2-羟基乙磺酸盐、甲磺酸盐、萘-I-磺酸盐、萘-2-磺酸盐、对甲苯磺酸盐、二甲苯磺酸盐、酒石酸盐等等。
本发明的药物或兽药组合物可根据需要以常规方式与其它药学上可接受的赋形剂一起配制成适于经口、肠胃外或局部施用的形式。施用模式可包括肠胃外施用,例如肌内、皮下和静脉内施用、经口施用、局部施用和直接施用至感染部位例如眼内、耳内、子宫内、鼻内、乳房内、腹膜内和病灶内。
本发明的药物或兽药组合物可配制用于经口施用。可添加传统的非活性成分以提供所需的颜色、味道、稳定性、缓冲能力、分散性或其它已知的所需特征。实例包括红氧化铁、硅胶、月桂硫酸钠、二氧化钛、可食用白色墨水等等。常规的稀释剂可用于制备压缩片剂。片剂和胶囊剂二者可制备为药物随时间推移连续释放的持续释放组合物。压缩片剂可为糖衣或膜包衣片剂,或用于在胃肠道中选择性崩解的包肠溶衣片剂的形式。供经口施用的液体剂型可包含染色剂和/或调味剂以增加患者的顺从性。例如,包含NCL812的口服制剂可为包含以下赋形剂中的任一者或其组合的片剂:无水磷酸氢钙、微晶纤维素、乳糖、羟丙基甲基纤维素和滑石。
本文所述的组合物可为液体制剂的形式。优选的液体组合物的实例包括溶液、乳液、注射溶液、容纳于胶囊剂中的溶液。液体制剂可包括包含溶解于溶剂中的治疗剂的溶液。一般来讲,可使用具有所需效果的任何溶剂,其中治疗剂溶解于溶剂中并且可施用给受试者。一般来讲,可使用具有所需效果的任何浓度治疗剂。在一些变型形式中,制剂为不饱和、饱和或过饱和溶液。该溶剂可为纯溶剂,或可为液体溶剂组分的混合物。在一些变型形式中,所形成的溶液为原位凝胶制剂。可用的溶剂和溶液类型是该药物递送技术领域的技术人员熟知的。
本文所述的组合物可为液体悬浮液形式。液体悬浮液可根据本领域已知的标准程序制备。液体悬浮液的实例包括微乳液、配混化合物和稳定悬浮液的形式。液体悬浮液可为未稀释或浓缩形式。口服使用的液体悬浮液可包含合适的防腐剂、抗氧化剂以及本领域已知的作为分散剂、悬浮剂、增稠剂、乳化剂、润湿剂、增溶剂、稳定剂、调味和甜味剂、染色剂等中的一者或多者的其它赋形剂。液体悬浮液可包含甘油和水。
本文所述的组合物可为口服糊剂形式。口服糊剂可根据本领域已知的标准程序制备。
本文所述的组合物可为用于注射例如肌肉内注射的液体制剂形式,并且使用本领域已知的方法制备。例如,液体制剂可包含聚乙烯基吡咯烷酮K30和水。
本文所述的组合物可为局部用制剂形式。局部用制剂可为使用本领域已知的方法制备的洗剂或霜剂形式。例如,洗剂可用水性或油性基料配制,并且可包括一种或多种本领域已知的作为增粘剂、乳化剂、芳香剂或香料、防腐剂、螯合剂、pH调节剂、抗氧化剂等的赋形剂。例如,包含NCL812的局部用制剂可为包含以下赋形剂中的任一者或其组合的凝胶:PEG4000、PEG200、甘油、丙二醇。NCL812化合物可使用SoluPlus(BASF,www.soluplys.com)进一步配制为固态分散体,并且使用以下赋形剂中的任一者或其组合配制:PEG4000、PEG200、甘油、丙二醇。
对于气雾剂施用,本发明的组合物以细分形式连同无毒表面活性剂和推进剂一起提供。表面活性剂优选地溶解于推进剂中。此类表面活性剂可包括脂肪酸酯或偏酯。
本发明的组合物可使用纳米药物递送技术例如本领域已知的那些另外配制。基于纳米技术的药物递送体系具有改善生物利用率、患者的顺从性以及减少副作用的优点。
本发明组合物的配制包括根据化合物溶解度以纳米悬浮液或纳米乳液的形式制备纳米粒子。纳米悬浮液是通过自底向上或自顶向下技术制备并且用合适的赋形剂稳定的纳米尺度药物颗粒分散体。该方法可应用到水溶性和脂溶性较差的氯苯胍,以增加饱和溶解度和改善溶解特性。该技术的实例在Sharma和Garg(2010)(Puredrugandpolymer-basednanotechnologiesfortheimprovedsolubility,stability,bioavailability,andtargetingofanti-HIVdrugs.AdvancedDrugDeliveryReviews,62:第491-502页)中示出。饱和溶解度将理解为取决于温度、溶解介质的性质和粒度(<1–2μm)的化合物特异性常数。
本发明的组合物可以纳米悬浮液形式提供。对于纳米悬浮液,表面积的增加可导致饱和溶解度增加。纳米悬浮液是由小于1μm的颗粒组成的胶体药物递送体系。本发明的组合物可为纳米悬浮液形式,包括纳米晶悬浮液、固体脂质纳米粒子(SLN)、聚合物纳米粒子、纳米胶囊剂、聚合物胶束和树枝状体。纳米悬浮液可使用自顶向下法制备,其中大颗粒可通过本领域已知的多种技术包括湿磨和高压均化减小至纳米尺度。或者,纳米悬浮液可使用自底向上技术制备,其中颗粒的控制沉淀可从溶液中进行。
本发明的组合物可以纳米乳液形式提供。纳米乳液通常为澄清的水包油或油包水双相体系,液滴尺寸在100–500nm范围内,并且所关注的化合物以疏水相存在。纳米乳液的制备可提高本文所述的氯苯胍的溶解度,使其生物利用率提高。纳米尺度悬浮液可包括静电或空间稳定剂,例如聚合物和表面活性剂。SLN形式的组合物可包括可生物降解的脂质,例如甘油三酯、类固醇、蜡和乳化剂,例如大豆卵磷脂、蛋黄卵磷脂和泊洛沙姆。SLN制剂的制备可涉及在熔融脂质中溶解/分散药物,然后是热或冷均化。如果使用热均化,则熔融脂质相可分散于水相,制备成乳液。这可通过冷却固化得到SLN。如果使用冷均化,则脂质相可在液氮中固化并研磨成微米尺寸。所得的粉末可在表面活性剂水溶液中经受高压均化。
本文所述的氯苯胍可溶解于油/液体脂质中并稳定成乳液制剂。纳米乳液可使用高能和低能液滴减小技术制备。高能法可包括高压均化、超声破碎和微射流。如果使用低能法,则溶剂扩散和转相将自发生成纳米乳液。用于纳米乳液的脂质可选自甘油三酯、大豆油、红花油和芝麻油。还可添加其它组分,例如乳化剂、抗氧化剂、pH调节剂和防腐剂。
组合物可为控释制剂形式,可包括可降解或不可降解的聚合物、水凝胶、有机凝胶,或修改聚醚离子载体释放的其它物理构建体。应当理解,此类制剂可包括另外的非活性成分,其添加可提供所需的颜色、稳定性、缓冲能力、分散性或其它已知的所需特征。此类制剂还可包括脂质体,例如乳液、泡沫、胶束、不溶性单层、液晶、磷脂分散体、片状层等等。本发明所用的脂质体可从标准囊泡形成脂质形成,通常包括中性和带负电的磷脂和甾醇,例如胆固醇。
本发明的制剂可具有提高NCL812的溶解度和/或稳定性的优点,尤其是那些使用纳米技术制备的制剂。此类NCL812稳定性和/或稳定性提高可提高生物利用率,并且增加口服和/或肠胃外剂型的药物暴露量。
在整个说明书中,除非上下文要求相反,单词“包括”或变型形式例如“包括”或“包含”将理解为意味着包含指定的整数或整数集,但不排除任何其它整数或整数集。
实施例
实施例1:耐甲氧西林金黄色葡萄球菌(MRSA)、耐万古霉素肠球菌属物种(VRE)和肺炎链球菌中NCL812的最小抑制浓度(MIC)
在本说明书的该实施例和其它实施例中,术语NCL812用于表示氯苯胍。
本研究进行新型抗菌剂NCL812的最小抑制浓度(MIC)测定。该抗菌剂代表潜在的新型药物类别,其具有理解的窄谱抗菌活性和新作用机制。本研究关注人的三种主要机会病原体的最近分离株:耐甲氧西林金黄色葡萄球菌(MRSA)、耐万古霉素肠球菌属物种(VRE)和肺炎链球菌,其中对现有抗菌剂类别的抗菌剂耐性发展是有问题的。
材料和方法
细菌分离株收集和鉴定
六十一个测试分离株来源于临床诊断微生物学实验室。MRSA分离株最初在选择性BrillianceMRSA显色琼脂(Oxoid)上培养。可疑菌落根据其在该琼脂上的菌落外观选择,并且金黄色葡萄球菌的鉴定使用非选择性绵羊血琼脂上的菌落特性和表型特性例如革兰氏染色、过氧化氢酶测试阳性、凝固酶测试阳性(试管凝固酶测试使用兔血浆进行)和凝集因子(凝集使用OxoidStaphytect乳胶测试进行)、VogesProskauer测试阳性以及从海藻糖生成酸的能力确定。耐头孢西丁筛选阳性确定分离株为MRSA。所有肠球菌属分离株经受标准生物化学鉴定。生物化学表达谱将四种VRE分离株暂时鉴定为粪肠球菌,其余为屎肠球菌,然而对于人肠球菌属菌株这并非100%可靠,使用API-ZYM进行完全生物化学表达谱可100%确定种类。所有肺炎链球菌分离株根据标准生物化学表达谱鉴定。
抗微生物剂的制备
获得具有1000mg/g(即100%)的定义效价的分析级NCL812(批号20081214)。该粉末在-20℃的温度下保存于研究地点的锁闭冰箱中。原液(25.6mg/ml)的等分试样(1ml)在DMSO中制备,保存于-80℃,在使用前立即解冻。
最小抑制浓度测定
最小抑制浓度测试根据CLSI标准(CLSI2008)进行。将90μL测试化合物溶液或氨苄青霉素中的一者添加到96孔板的最后一列,该96孔板的每个孔包含90μLCAMHB。然后跨行系列稀释溶液,留下2列作为阳性和阴性对照。细菌悬浮液通过将来自绵羊血琼脂(SBA)过夜培养物的新鲜菌落添加至9.1g/L盐水溶液来制备。将该悬浮液调整至4x108和5x108CFU/mL之间的浓度。悬浮液的浓度通过使用分光光度计测定600nm波长下光密度(OD)来确定,其中校正浓度确定为具有1.00和1.20之间的光密度。在加入所有孔之前将一毫升该悬浮液加入9mL生理盐水,排除阴性对照孔,每孔中10μL体积得到4x105和5x105CFU/mL之间的终浓度。然后在37℃下温育测试24小时,使用肉眼和600nm波长下微板读数器的OD读数二者评估。这些测试一式两份,如果观察到差异则重复进行。
使用肉眼和OD读数二者确定最小抑制浓度(MIC)为抑制细菌生长的最小抗生素浓度。测试化合物和氨苄青霉素之间的直接统计比较不可按照机密信息限制进行,例如限制公开化合物结构相关的信息,例如分子量。相反,整理MIC值并用于确定对50%和90%分离株有效的每个化合物最小浓度,分别称为MIC50和MIC90。然后将这些值以及MIC值的范围用于测试化合物之间的直接比较以及与氨苄青霉素的一般比较。
结果
根据CLSI推荐预期得自ATCC对照菌株的氨苄青霉素MIC值在正常范围内。每个分离株的NCL812和氨苄青霉素MIC值在图1(MRSA分离株)、图2(VRE分离株)和图3(肺炎链球菌分离株)中示出。
每个测试细菌菌种的NCL812MIC50、MIC90、MIC众数和MIC范围合并表格示于图4中。
三个菌种之内和每个之间的NCL812MIC值显著一致。MRSA、VRE和肺炎链球菌分离株的MIC50和MIC90值均相等(4μg/ml),小于10%的分离株显示出MIC值小1-2个稀释度或只有一个稀释度大于该数值。
实施例2:NCL812对金黄色葡萄球菌大分子合成的影响
材料和方法
测试化合物
测试化合物NCL812在环境温度条件下运输至实验装置,然后保存于2-8℃直到进行测试。原液通过将NCL812干粉溶解于100%DMSO中得到6,400μg/ml的浓度来制备。
最小抑制浓度测试
MIC分析方法遵循临床和实验室标准协会或CLSI所述的程序(ClinicalandLaboratoryStandardsInstitute).MethodsforDilutionAntimicrobialSusceptibilityTestsforBacteriaThatGrowAerobically;ApprovedStandard—第八版CLSI文档M07-A8[ISBN1-56238-689-1]。ClinicalandLaboratoryStandardsInstitute,940WestValleyRoad,Suite1400,Wayne,Pennsylvania19087-19898USA,2009),并且利用自动移液器进行系列稀释和液体转移。用于MIC测定法的培养基为MuellerHintonII肉汤(MHBII-BectonDickinson,Sparks,MD;目录号212322;批次9044411)。金黄色葡萄球菌ATCC29213作为质量控制菌株,并且利奈唑胺作为质量控制抗生素来验证测定。在加入生长培养基之前NCL812和利奈唑胺均溶解于100%DMSO中。
大分子合成测定
细菌和生长条件
使用金黄色葡萄球菌ATCC29213研究NCL812对全细胞DNA、RNA、细胞壁、蛋白质和脂质合成的影响。细胞在TrypticaseSoy琼脂上35℃下生长过夜。平板的菌落用于接种10mlMuellerHinton肉汤II(MHBII),使培养物生长至指数生长早期(OD600=0.2至0.3),同时在35℃和200rpm的摇动器中温育。
DNA、RNA和蛋白质合成
当细胞达到指数生长早期时,将100μl培养物加入包含各种浓度测试化合物或20X终浓度溶解于100%DMSO的对照抗生素(5μl)的孔(三份)。5%DMSO处理培养物作为所有实验的“无药物”对照。细胞以105%加入MHBII以解释加入每个反应的药物量,或加入M9基本培养基以解释蛋白质合成反应。在室温下温育15min后,根据实验以0.5-1.0μCi/反应加入[3H]胸苷(DNA合成)、[3H]尿苷(RNA合成)或[3H]亮氨酸(蛋白质合成)。允许反应在室温下进行15-30min,然后通过加入12μl冷的5%三氯乙酸(TCA)或5%TCA/2%酪蛋白氨基酸(仅蛋白质合成)停止反应。反应在冰上温育30min,并且将TCA沉淀材料收集于25mmGF/A过滤器上。在用5ml冷的5%TCA洗涤三次后,用5ml100%乙醇漂洗过滤器两次,使其干燥,然后使用BeckmanLS3801液体闪烁计数器计数。
细胞壁合成
将指数生长早期的细菌细胞转移至M9基本培养基,并且如上所述加入包含各种浓度测试化合物或20X终浓度溶解于100%DMSO的对照抗生素(5μl)的1.5mleppendorf管中(100μl/管)。在37℃下温育5min后,每个管加入[14C]N-乙酰基葡糖胺(0.4μCi/反应),在37℃加热板中温育45min。通过向每个管加入100μl8%SDS来停止反应。然后反应在95℃加热板中加热30min,冷却,短暂离心,并点入预先润湿的HA过滤器(0.45μM)。在用5ml0.1%SDS洗涤三次后,用5ml去离子水漂洗过滤器两次,使其干燥,然后使用BeckmanLS3801液体闪烁计数器计数。
脂质合成
使细菌细胞在MHBII肉汤中生长至指数生长早期,并且如上所述加入包含各种浓度测试化合物或对照抗生素的1.5mleppendorf管中(一式三份)。在室温下温育5min后,以0.5μCi/反应加入[3H]甘油。
允许反应在室温下进行15min,然后通过加入375μl氯仿/甲醇(1:2)停止反应,然后每次加入后涡旋20秒。然后向每个反应加入氯仿(125μl),涡旋,然后加入125μldH2O并涡旋。以13,000rpm离心反应10min,然后将150μl有机相转移至闪烁小瓶,允许在通风柜中干燥至少1小时。样品计数通过液体闪烁计数进行。
结果
敏感性测试使用NCL812和金黄色葡萄球菌ATCC29213进行,以确定大分子合成测定所需的药物浓度。
图5显示出NCL812MIC为4μg/mL,而质量控制剂利奈唑胺在CLSI-建立的质量控制范围内(ClinicalandLaboratoryStandardsInstitute.PerformanceStandardsforAntimicrobialSusceptibilityTesting;NineteenthInformationalSupplement.CLSIdocumentM100-S20[ISBN1-56238-716-2].ClinicalandLaboratoryStandardsInstitute,940WestValleyRoad,Suite1400,Wayne,Pennsylvania19087-1898USA,2010)。在以相同方式制备但未接受金黄色葡萄球菌接种的平板中,≥8μg/mL观察到NCL812沉淀。对于金黄色葡萄球菌ATCC29213,大分子合成抑制研究使用等于MIC值(4μg/ml)0、0.25、0.5、1、2、4或8倍的NCL812浓度进行(图6-11)。
图6示出了NCL812对DNA合成的影响。NCL812显示在0.25倍MIC下无抑制,在0.5倍下40%抑制,并且在MIC下大约95%抑制。与对照组环丙沙星比较显示在8倍MIC(0.5μg/ml)下抑制大约51%。
NCL812抑制RNA合成的结果与DNA合成研究非常类似,利福平作为阳性对照(图7)。应当指出的是,在用于DNA和RNA合成测定的MuellerHinton肉汤II中,4至8倍MIC下观察到沉淀。
在0.25、0.5和1倍NCL812MIC值下蛋白质合成以剂量依赖性方式抑制,显示在MIC下最多97%抑制(图8)。利奈唑胺显示在8倍MIC(2μg/ml)下蛋白质合成大约61%抑制。在蛋白质合成测定中,NCL812的沉淀发生在4和8倍MIC。
在图9中,NCL812还显示出一定程度的细胞壁合成剂量依赖性抑制,但抑制从1倍大量增加至2倍MIC。然而,在4倍和8倍MIC下抑制分别降至大约68%和52%。在用于细胞壁合成测定的M9基本培养基中,NCL812沉淀发生在2、4和8倍MIC,这可能是导致抑制下降的因素。相比之下,阳性对照万古霉素显示在8倍MIC(2μg/ml)下96%抑制。
NCL812显示出与DNA和RNA合成所示类似的脂质合成抑制曲线,在MIC下达到大约90%抑制(图10)。阳性对照抑制剂浅蓝菌素显示出在8倍MIC(32μg/ml)下72%抑制。
图11展示了所有五个大分子合成反应的组合。可观察到每个途径的抑制曲线类似,暗示多个途径同时被NCL812整体抑制。NCL812靶向细胞膜,导致基本离子和/或代谢物渗漏,从而导致细胞合成途径整体关闭是可能的。
概括地说,在金黄色葡萄球菌生长培养物中,NCL812抑制DNA、RNA、蛋白质、细胞壁和脂质途径。虽然观察到途径剂量依赖性抑制的一些实例,但所有五个大分子合成反应对NCL812具有类似的敏感性。
实施例3:NCL812对ATP从金黄色葡萄球菌释放的影响
材料和方法
测试化合物
测试化合物NCL812在环境温度条件下运输,然后保存于2-8℃直到进行测试。原液通过将NCL812干粉溶解于100%DMSO中得到1,600μg/ml的浓度来制备。对照剂是多粘菌素B(Sigma,P-4932(批次044K11905))。
测试生物
金黄色葡萄球菌ATCC29213最初从美国典型培养物保藏中心(AmericanTypeCultureCollection(Manassas,VA))获得。
ATP释放测试
CellTiter-Glo发光法细胞活力测试(Promega)用于测定ATP从细菌的渗漏。培养物在Mueller-Hinton肉汤II中生长至指数生长早期(在600nm下0.2–0.3个光密度单位),然后用七种不同浓度的NCL812或多粘菌素B(阳性对照)处理,该处理利用每个化合物的MIC作为对照(0、0.25、0.5、1、2、3、4或8倍MIC)。阴性对照接受2%DMSO,其表示每个测试中的最终DMSO浓度。在暴露至药物30min后,离心沉淀细胞,分析上清液中ATP的存在。结果以释放至培养基的ATP浓度表示(μM)。
结果
NCL812MIC此前确定为4μg/ml。ATP释放测定通过以下方法进行:使金黄色葡萄球菌生长至指数生长期,然后以MIC的倍数加入抗生素,以检测剂量依赖性响应。
如图12所示,阳性对照多粘菌素B以剂量依赖性方式从金黄色葡萄球菌细胞释放ATP,在8倍MIC(256μg/ml)下最大释放量为大约0.34μMATP。在存在NCL812的情况下,ATP释放在0.5–1倍MIC下为剂量依赖性的,观察到在MIC(4μg/ml)下产生最大释放量(0.33μM)。然后在2至8倍MIC下,ATP释放实际上减少。应当指出的是,在先前的研究中,在MuellerHinton肉汤II中4至8倍MIC下观察到NCL812沉淀。
概括地说,NCL812显示出ATP从生长活跃的金黄色葡萄球菌细胞的剂量依赖性释放。ATP从细胞释放至生长培养基在MIC值下达到最大水平,然后是在高剂量下ATP释放量下降。数据显示NCL812可与金黄色葡萄球菌的细胞膜相互作用,导致重要代谢物例如ATP渗漏。
实施例4:NCL812对耐甲氧西林和甲氧西林敏感金黄色葡萄球菌的体外抗菌活性
材料和方法
抗微生物剂
NCL812原液(25.6-mg/ml)的等分试样在二甲基亚砜中制备,保存于-80℃,在使用前立即解冻。氨苄青霉素原液可购自Sigma-Aldrich(Australia)。抗微生物测试纸片可购自ThermoFisherScientific(Australia)。
微生物
获得二十九个MRSA临床分离株(图13)以及金黄色葡萄球菌对照生物ATCC49775。分离株鉴定通过传统表型方法确认,包括玻片凝固酶测试、Vogues-Proskauer测试、多粘菌素B敏感性测试(300个单位)和StaphytectPlus蛋白A乳胶玻片凝集测试(ThermoFisherScientificAustralia)。细菌在40%甘油液体中保存于-80℃,在37℃温育的绵羊血琼脂(SBA)上从原液例行生长。在后续的实验中,仅使用<24-h的新鲜培养物。
分离株分型
分离株收集物的抗生素敏感性表达谱使用Kirby-Bauer纸片扩散法进行,临床和实验室标准协会(CLSI)推荐在Mueller-Hinton琼脂上进行。分离株在37℃的SBA上过夜生长。菌落悬浮于生理盐水。浊度调整为0.5McFarland标准,并将悬浮液涂布于培养基上。将抗生素纸片转移到接种培养基,在37℃下温育24-h后进行分析。根据Kirby-Bauer测试标记为不耐β-内酰胺的MRSA的分离株在补充5-μg/ml氨苄青霉素的平板计数琼脂上从原液生长,并经受重复测试,因为青霉素结合蛋白质2a表达可由暴露于β-内酰胺抗微生物剂诱导。
蛋白A和mecA基因的分子检测确认MRSA状态
分离株种类使用靶向spa(蛋白A)和mecA(耐甲氧西林)基因的新型双重常规聚合酶链式反应(PCR)测试通过基因型确认。此外,分离株在mecA和spaSybr绿色实时PCR中测试。每个过夜细菌传代培养物的大约十个菌落悬浮于1×磷酸盐缓冲盐水(pH7.4)中并涡旋。分离株使用DNAMiniKit(Qiagen,Australia)根据制造商方案进行DNA提取。模板DNA在50-μl洗脱缓冲液中洗脱,并且直接用于PCR,或在DNA扩增前保存于-20℃,所述DNA扩增使用spa正向(5’-TGATACAGTAAATGACATTG-3’)和反向(5’-TTCTTATCAACAACAAGTTC-3’)引物以及mecA正向(5’-TTCGTGTCTTTTAATAAGTGAGG-3’)和反向(5’-ATGAAGTGGTAAATGGTAATATCG-3’)引物(Invitrogen,Australia)进行。常规PCR扩增在包含10-μlHotStarTaqPlusMasterMix(Qiagen,Australia)、0.5-μM每个spa引物、0.2-μM每个mecA引物和3-μl提取DNA的20-μl体积中进行。自动热循环仪(T100ThermalCycler,Bio-Rad)用于spa和mecA基因的PCR扩增。
表1:PCR和RT-PCR反应条件
分别为325-和120-bp的mecA和spa扩增产物通过GelRed染色然后是2%琼脂糖凝胶电泳检测。
最小抑制浓度测试
NCL812和氨苄青霉素阳性对照的体外活性在阳离子调节Mueller-HintonII肉汤中通过CLSI推荐的微量肉汤稀释确定。给包含两倍稀释的每种抗微生物剂的微量滴定板接种~105-CFU/ml每种分离株,最终体积为100-μl。平板在37℃下温育24-h。浊度(OD600下的吸光度)使用Bio-RadBenchmarkPlus微板分光光度计在Microplate软件5.2.1版(Bio-Rad)中测定。最小抑制浓度(MIC)端值定义为分光光度计评估的抑制细菌生长的最小抗微生物剂浓度。ATCC49775使用CLSI定义的断点包括于分离株收集物中,作为对照生物。计算MIC50、MIC90(分别抑制较低50%和90%的总生物生长的浓度)和MIC范围(最小值和最大值),以绘制分离株收集物的抗微生物剂敏感性曲线。
杀菌活性
NCL812的杀菌活性通过使用CLSI准则确定最小杀菌浓度(MBC)的和时间杀菌分析建立。MBC定义为其中消除99.95%的原始接种物的最低药物浓度。
ATCC49775的时间杀菌测定在微量滴定板中的阳离子调节Mueller-HintonII肉汤中,然后在抗微生物剂浓度等于1×和4×MIC的10-ml体积常量稀释测定中进行。常量稀释测定中的杀菌活性确定为从初始接种量减少3-log10。细菌在SBA上37℃下过夜培养。将菌落悬浮于肉汤中,并将浊度调整为0.5McFarland标准,以得到~105-CFU/ml的细菌悬浮液。细菌悬浮液在37℃下摇动温育。在加入抗微生物剂后0-、1-、2-、4-、8-、12-和24-h移除等分试样,稀释,接种于SBA上并且在37℃下温育48-h用于存活计数测定。使用Bio-RadBenchmarkPlus微板分光光度计监测600nm下光密度变化进行微量滴定板测定,获得金黄色葡萄球菌的比浊生长曲线。在加入抗微生物剂后0-、1-、2-、4-、8-、12和24-h测定光密度。
统计方法
微生物数据使用CLSI准则解释。数据使用司徒顿t-检验、费希尔精确检验、方差分析和适当的受试者之间影响测试的广义线性模型验证。在IBM19.0版(UniversityofAdelaide)中,0.05水平的差异被视为具有显著性。
结果
金黄色葡萄球菌种类和mecA状态确认
乳胶凝聚测试确认所有30个分离株均为蛋白A阳性。使用玻片凝聚进行分离株凝固酶活性阳性测试。Voges-Proskauer和耐多粘菌素B测试确认,除一个甲氧西林敏感分离株MSSADE-25之外,所有分离株均为金黄色葡萄球菌(图14)。根据spa基因PCR扩增,该分离株未鉴定为金黄色葡萄球菌分离株,尽管蛋白A乳胶凝聚和玻片凝固酶测试中为测试阳性。根据生物化学特性,该犬科动物来源葡萄球菌属物种鉴定为伪中间葡萄球菌。mecA常规和实时PCR结果确认,根据是否拥有mecA基因,将66.66%的分离株归类为耐甲氧西林。常规和实时PCR的mecA基因检测能力之间无显著性差异(P>0.05)。
金黄色葡萄球菌抗微生物剂敏感性曲线
抗微生物剂敏感性测定揭示,HA-MRSA分离株具有多抗微生物剂类别耐性的最高平均盛行率(P<0.000)。CA-MRSA分离株的耐性其次(P<0.007),然后是甲氧西林敏感葡萄球菌(P<0.037)(图15)。苯唑西林耐性在分别仅80.00%和10.00%的HA-MRSA和CA-MRSA分离株中表达。头孢替坦耐性在分别80.00%和20.00%的HA-MRSA和CA-MRSA分离株中表达。虽然苯唑西林和头孢替坦在检测MRSA的能力方面无显著性差异(P>0.05),但与纸片扩散法相比,使用mecAPCR时检测显著改善(P<0.013)。大部分HA-MRSA分离株表达阿莫西林-棒酸、头孢替坦、头孢氨苄、克林霉素、红霉素、苯唑西林和青霉素-G耐性,而大部分CA-MRSA分离株仅对克林霉素、红霉素和青霉素-G具有耐性。所有测试分离株均不具有万古霉素耐性。总体说来,最盛行耐性表型为青霉素-G(83.33%)、红霉素(73.33%)和克林霉素(43.33%),而仅单独一个分离株(3.33%)具有对三甲氧苄二氨嘧啶-磺胺甲噁唑和利福平耐性。
mec基因复合物相互作用
属于mec基因复合物A的所有MRSA分离株表达苯唑西林和头孢替坦二者耐性(图16)。然而,仅20%的mec基因复合物BMRSA分离株对这些抗微生物剂具有表型耐性。在属于mec基因复合物C2的MRSA分离株中,仅单独一个分离株表达甲氧西林和苯唑西林耐性,仅两个分离株表达头孢替坦耐性。未归类MRSA分离株表达完全的苯唑西林和头孢替坦耐性。
mecA实时PCR阴性的解链温度峰值导数图–dF/dT在mec基因复合物之间存在差异(P<0.003)(图17)。平均起来,mec基因复合物B和未归类的分离株显示出比其它SCCmec类型更高的解链温度峰值(P<0.012)。
NCL812的物理性质和MIC
NCL812的初始测试显示其可溶于DMSO,但当溶解于阳离子调节Mueller-HintonII肉汤(CAMHB)时生成浑浊溶液(图18)。在初始测试中,发现NCL812具有一致的MIC值(图18)。
体外抗菌活性:最小抑制浓度
化合物NCL812(4-和4-8-μg/ml)的MIC50和MIC90值示于图19中。MIC值随金黄色葡萄球菌分类而不同(敏感、HA或CA-MRSA)(P<0.005)。在很多情况下,与HA-MRSA相比,NCL812对CA-MRSA和甲氧西林敏感葡萄球菌的活性显著增加一个稀释度(分别地,P<0.002和P<0.020),然而甲氧西林敏感葡萄球菌和CA-MRSA的MIC值之间无显著性差异(P>0.05)。氨苄青霉素MIC值在根据CLSI准则预期的正常范围内。
体外抗菌活性:最小杀菌浓度
从NCL812确定的MBC分别等于93.33%和83.33%金黄色葡萄球菌分离株的MIC(图19)。在所有其余情况中,MBC高一个稀释度。对于NCL812,MBC分别在2-8-μg/ml和4-16-μg/ml的范围内。
时间杀菌研究
与ATCC49775的比浊生长曲线相比,在微量稀释测定中,当ATCC49775接种至补充NCL812的阳离子调节MuellerHintonII肉汤时,1×和4×MIC未观察到可见细菌生长(分别地,P<0.033和P<0.038)(图20)。
当用10-ml常量稀释测定法分析时,与生长对照相比,补充1×和4×MIC抗微生物剂并接种ATCC49775的肉汤显示出NCL812存活计数均显著减少(0.000<P<0.008)(图21)。另外,NCL812的时间杀菌曲线无显著差异(P>0.05)。两种抗微生物剂均保持杀菌性,直到抗微生物剂加入后大约8-12-h,此时观察到细菌再生长。观察到从8至24-hNCL812杀菌活性的显著变化。虽然NCL812在24-h后不再具有杀菌性,但1×MIC观察到的存活计数仍然显著低于得自未补充肉汤的那些(P<0.046)。
概括地说,上述实施例显示出NCL812对甲氧西林敏感葡萄球菌和MRSA二者的杀菌活性。MIC和MBC值在整个分离株的选择中一贯较小(MIC范围2-8-μg/ml)。NCL812对常见的多药物耐性MRSA分离株保持良好的体外抗微生物活性,包括传染性UKEMRSA-15、EMRSA-16和EMRSA-17、爱尔兰EMRSA-1、AUSEMRSA-3、NY/JAPANHA-MRSA和优势CA-MRSA克隆。NCL812还对一株最初鉴定为金黄色葡萄球菌菌株的伪中间葡萄球菌分离株具有活性。
初步测试暗示,NCL812靶向金黄色葡萄球菌细胞膜,导致重要代谢物例如腺苷-5’-三磷酸的剂量依赖性释放。细菌膜双层或构成细菌中膜功能的蛋白质的破裂是自然界中普遍存在的许多大型抗微生物剂的靶标,所述抗微生物剂包括糖脂、脂肽、脂蛋白、脂肪酸、中性脂质、磷脂和生物表面活性剂。虽然NCL812是低分子量(≤500-Da)合成化合物,但其看起来以靶向革兰氏阳性细胞膜的其它抗微生物剂,包括高分子量环状脂酯肽抗微生物剂达托霉素或化学结构目前未知的低分子量喹诺酮衍生HT61类似的方式发挥杀菌活性。由于存在外部脂双层膜,这些脂抗菌剂中的多者对革兰氏阴性微生物无效,该脂双层膜包含狭窄的孔蛋白通道,减少了一些化合物向细胞的净渗透。
即使在低浓度下NCL812也不溶于微生物介质可反映该抗微生物剂的两亲和低聚性质,并暗示实际MIC小于观测值,很可能其仅为溶解于溶液具有生物活性的NCL812。在时间杀菌研究中,NCL812发挥了快速ATCC49775体外杀菌活性。
重要的是,该抗微生物剂的表观体外半衰期短导致抗微生物剂加入后12-h观察到细菌再生长。这暗示如果活细菌群体在抗微生物剂失活之前的NCL812初始暴露存活,则出现细菌再生长。这些研究中排除了NCL812耐性发展,因为在收集、洗涤和MIC测试后,测试细菌保持NCL812敏感性。虽然NCL812的表观体外半衰期短可为未来体内应用所期望的特性,但这并非暗示在未来体内安全和功效实验中每8-h必须施用NCL812以保持足够的全身浓度,虽然从时间杀菌曲线可观察到NCL化合物系列是浓度依赖性而非时间依赖性抗微生物剂。
为克服甲氧西林敏感MRSA表型,将纸片扩散温育时间从24-h延长至48-h,以补偿mecR基因的去抑制减缓。虽然延长温育的效果未经检验,并且MRSA分离株的样品量较少妨碍了mec复合物相互作用的进一步研究;但在该研究中与确认分离株的mecA状态的表型方法相比,基因技术的灵敏度显著提高。虽然基因技术并非总是作为检测MRSA的日常方法,但实时PCR鉴定葡萄球菌属物种分离株中mecA基因的存在仍然是诊断的金标准。
实施例5:肺炎链球菌的新型抗微生物剂的体外药效动力学。
材料和方法
肺炎链球菌的抗微生物剂敏感性
肺炎链球菌菌株和生长条件
获得由八个特征性实验室菌株和12个临床分离株构成的二十个肺炎链球菌分离株。
表2:测试分离株的特性鉴定
国家典型培养物保藏中心(NationalCollectionofTypeCultures,NCTC)对照菌株D39用作所有MIC和MBC测定的生长对照。D39之后被指定用于杀菌动力学、耐性点测试和透射电子显微镜(TEM)研究,因为有大量证据证明其为具有确定的体内致病机理,显示出一致的NCL812MIC和MBC的实验室菌株。
对于所有体外测定,将新鲜的肺炎链球菌分离株在马血琼脂(HBA)平板(39g/LColumbia血琼脂基础[Oxoid]5%[v/v]脱纤维马血[Oxoid]上37℃下补充5%CO2生长过夜(O/N)。将含5%脱纤维绵羊血(MHSBARoseworthyMediaandBloodService)的Mueller-Hinton血琼脂用于圆盘扩散分析,如临床和实验室标准协会(CLSI)标准所述。肺炎链球菌在由4%裂解马血(LHB)和阳离子调节MuellerHinton肉汤(CAHMB,[Difco])组成的肉汤中37℃下补充5%CO2例行生长。马血清肉汤(HSB,10%(v/v)溶解于营养肉汤[10g/L蛋白胨、10g/LLabLemco(Oxiod)和5g/LNaCl]的供体马血清)还用于一些MIC测定。分离株溶解于HSB中保存于-80℃。
抗生素原液和试剂
NCL812由Neoculi以干粉形式提供。总共256mg分配于10ml100%二甲基亚砜(DMSO)中,制成25.6mg/ml的原液,然后在CAHMB中1:100稀释,制成256μg/ml的最终工作液。氨苄青霉素干粉得自SigmaA0166。将25.6mg/ml原液在盐水中1:100稀释,1:4、1:20以及最终1:16溶解于CAMHB中,制成0.18μg/ml的最终工作液。红霉素得自SigmaE077,以及氯化胆碱得自RocheDiagnostics。20微升0.05μg/ml红霉素在4.980mlCAMHB中1:25稀释,得到0.2μg/ml的最终工作液。将氯化胆碱(0.5%)加入4%LHB:CAMHB,用于特定的杀菌动力学测定。
确定肺炎链球菌分离株的抗微生物剂敏感性
分离株对12种不同抗微生物剂的敏感性(图22)通过CLSI和欧洲抗微生物剂敏感性测试委员会测试(EuropeanCommitteeonAntimicrobialSusceptibilityTesting,EUCAST)方法确定。抗微生物剂根据CLSI和EUCAST准则选择。除环丙沙星对肺炎链球菌之外抗微生物剂的抑菌圈直径通过CLSI标准确定;而环丙沙星抗微生物剂对肺炎链球菌敏感性的抑菌圈直径通过EUCAST确定。
表3:抑菌圈直径的解释标准
将标准化细菌悬浮液使用无菌棉拭子涂布到MHSBA。[肺炎链球菌的细菌悬浮液形式使用分光光度计标准化至OD600nm在0.08和0.1之间,然后1:20稀释。从O/N马血琼脂平板挑取细菌菌落。为确保1:20细菌悬浮液的纯度,将50μL涂布接种至马血琼脂并在37℃下5%CO2中温育O/N。计算CFU并与初始平板计数比较。]根据CLSI标准使用纸片分配器(Oxoid)放置抗生素圆纸片(Sigma)。MHSBA平板在37℃下5%CO2中温育16h–24h。使用尺子测量自然光反射生长的完整抑菌圈三次,近似到毫米,并以众数表示每个分离株的直径。肺炎链球菌分离株通过CLSI标准和质量控制(QC)范围归类为敏感、中敏(I)或耐性(R)(图22)。
NCL812MIC50、MIC90、MIC范围和MBC50、MBC90、MBC范围的确定
列于表2中的所有分离株的NCL812的MIC通过使用96-孔微量滴定板在37℃下5%CO2中温育24小时后,测量600nm光密度(OD600nm)(Spectramax分光光度计,MolecularDevicesCorporation)确定,作为细菌生长的指标。[微量肉汤稀释和96-孔板通过以下方法制备:使用多道移液管将90μL4%LHB:CAMHB等分至所有孔中。以1:2稀释比例向下非系列稀释滴定板的90μl抗微生物剂工作液。在规划96-孔板的准备时考虑阴性液体对照和稀释对照。]然后将10μL细菌悬浮液加入96孔板的适当孔中。每个测定包括适当的阳性(无抗微生物剂)、阴性(无抗微生物剂或细菌)和阴性稀释(抗微生物剂和液体的系列稀释对照)对照。杀菌动力学测定的MBC和平板计数通过从96-孔微量滴定板的每个孔将20μL等分至HBA,并在37℃下5%CO2中温育来确定。考虑稀释因素,MBC通过肺炎链球菌的99.95%抑制确定。MIC和MBC测定四次,以众数作为代表值。MIC50、MIC90和MIC范围以及MBC50、MBC90和MBC范围根据CLSI标准确定。MIC50和MIC90或MBC50和MBC90由最小浓度定义,当分离株的所有MIC和MBC从小到大排列时,其分别抑制分离株总量50%和90%。
使用菌株D39进行NCL812的微量肉汤稀释时间杀菌研究
细菌悬浮液一式三份加入包含NCL812的96-孔微量滴定板,起始浓度为128μg/ml,按顺序1:2系列稀释至0.25μg/ml的浓度。中值生长曲线减去阴性稀释对照得到合适的总细菌生成量指示。96-孔板在37℃下5%CO2中温育,前12小时每2小时读取一次600nm读数,然后在24和48小时读取最终读数。为进一步补充该数据,进行其中每半小时间隔使用分光光度计(Spectramax分光光度计,MolecularDevicesCorporation)从96-孔板自动读数14小时的单独实验,以确认原始微量肉汤稀释研究观察到的生长曲线趋势。
使用菌株D39进行NCL812的MBC时间杀菌研究
MBC杀菌动力学测定涉及三个96-孔微量滴定板的准备。在特定时间点,在HBA上37℃下5%CO2中温育后,提供来自这些板的等分试样的存活计数,并且在生长24小时后确定MBC。
NCL812的D39宏量肉汤稀释时间杀菌研究
细菌悬浮液和抗生素工作液如上所述制备。[对于制备宏量肉汤稀释,每支20ml试管加入9ml4%LHB:CAMHB。当加入一支试管时,1:2稀释9ml抗微生物剂工作液,然后从高浓度至低浓度抗微生物剂向下系列稀释。将1ml肺炎链球菌细菌悬浮液加入适当的试管,包括阳性对照。试管在37℃下5%CO2中温育,前12小时每10min用手轻轻摇晃NCL812处理的试管。在生长前12小时期间每2-3小时然后在24h和48h,将50μL每个细菌悬浮液涂布接种到HBA上,并在37℃下5%CO2中温育16-24h。]
表4:抗微生物剂的浓度
培养物在37℃下5%CO2中温育,前12小时每10min用手轻轻摇晃。在37℃下5%CO2中温育24小时后读取每个浓度的50μL等分试样的存活计数。在特定时间点使用pH试纸测定每个样品的pH。当计数超过1000个菌落/平板时,定义为汇合生长。杀菌效果定义为在24小时测定的每个浓度初始细胞悬浮液减少3-log10个单位(99.9%)。
NCL812的耐性测试点
宏量肉汤稀释如下所述制备。菌株D39的培养液(10ml)在存在2μg/ml和4μg/mlNCL812以及0.022μg/ml氨苄青霉素的情况下在37℃下5%CO2中温育6h。样品在101.45×g相对离心力(RCF)下离心10min,并在50ml磷酸盐缓冲盐水(PBS)中洗涤两次,移除所有残留抗微生物剂和/或细菌终产物和培养基。重悬洗涤的细菌并进行MIC测定。
NCL812对D39细胞膜超微结构的影响
透射电子显微镜法
细胞膜的外观形态和形态测定分析使用透射电子显微镜法(TEM)测定。D39的细菌悬浮液10ml培养物如上所述制备。样品在37℃下5%CO2中温育,每10min用手轻轻摇晃培养物。使培养物暴露至1μg/ml、4μg/ml或16μg/mlNCL812,并在6或12小时以101.45×g离心20min收集,在50mlPBS中洗涤两次。TEM工作的关键时间点通过分析杀菌动力学研究生成的生长曲线趋势确定。样品重悬于包含20%甘油的PBS中,并保存于-80℃直到使用。在固定前,通过离心移除20%甘油,在冰上用50mlPBS洗涤三次。
样品使用先前检验肺炎链球菌细胞壁超微结构的研究确定的修改方案固定(Hammerschmidt,S.等,2005.InfectImmun,第73卷,第4653-4667页)。赖氨酸乙酸盐基甲醛-戊二醛钌红-锇固定程序涉及用包含2%甲醛、2.5%戊二醛、0.075%钌红和0.075M赖氨酸乙酸盐的卡可酸盐缓冲溶液固定细菌沉淀1小时。在用包含0.075%钌红的卡可酸盐缓冲液洗涤三次后,用包含2%甲醛、2.5%戊二醛和0.075%钌红的卡可酸盐缓冲溶液进行第二次固定1.5小时。随后用包含0.075%钌红的卡可酸盐缓冲液洗涤细胞三次,并经历使用溶解于包含0.075%钌红的卡可酸盐的1%四氧化锇最终固定1小时。然后用仅包含0.075%钌红的卡可酸盐缓冲液洗涤样品三次。
样品使用等级系列乙醇(70%、90%、95%和100%)洗涤和脱水10-20min,每步两次。样品使用溶解于100%乙醇的50:50LR白树脂渗透1h,随后用100%LR白树脂洗涤1h,第三次更换100%LR白树脂保存O/N,以确保树脂足量渗透。然后将样品包埋至新鲜的LR白树脂中,在50℃下温育48小时。使用玻璃刀切成1μm切片,用甲苯胺蓝染色,在400x光学显微镜下查看,鉴定存在染色肺炎链球菌。然后使用金刚石刀将至少四块超薄切片切成90nm,并置于点阵网格上,每格一块切片。然后用乙酸双氧铀和柠檬酸铅对超薄切片间隔5min交替染色,然后在每次暴露期间用蒸馏水洗涤三次。然后将染色切片置于网格上,并在PhilipsCM100透射电子显微镜上25000x和130000x之间查看。获得130000x放大率的图像并使用analySIS[Olympus软件成像系统]分析。
统计分析
使用统计程序GraphPadPrism(第5版,GraphPadSoftwareInc.)forWindows进行统计分析。对于生长曲线,所示数据为每个数据的平均值和均值标准误差(SEM)(以误差条表示),不同的是对于宏量肉汤稀释研究,其中由于该测定涉及高成本不能得到多次平行测定结果。进行双尾、非配对t-检验。
结果
肺炎链球菌中NCL812的药效动力学
20个肺炎链球菌分离株的质量控制圆盘扩散分析
虽然用于圆盘扩散分析的12种抗微生物剂中的9种通过EUCAST建立了QC范围,但阿莫西林-克拉维酸盐、克拉霉素和克林霉素的QC范围未确定(表3)。
WCH16和WCH184均对至少两种抗微生物剂具有耐性,而EF3030和WCH137分别为三甲氧苄二氨嘧啶–磺胺甲噁唑中敏和耐性(图22)。其余16个分离株对所有12种抗微生物剂敏感。确认每个分离株的氨苄青霉素敏感性,使氨苄青霉素可在后续微量肉汤稀释测定中用作阳性对照(图22)。
NCL812在不同培养基中的溶解性和活性
将NCL812加入100%DMSO,但当用CAMHB或PBS进一步稀释时产生浑浊。
表5:NCL812的目视分析和氨苄青霉素溶解性
在NCL812MIC测定中,使用10%HSB(220ml马血清在180mlLemco营养肉汤中过滤成10%)进行肺炎链球菌菌株D39的生长,结果NCL812处理的D39MIC增加三倍,而阳性氨苄青霉素对照增加两倍。
表6:不同培养基中NCL812的活性差异。
在预先制备96-孔微量滴定板的不同保存条件下D39MIC无变化。
表7:宏量肉汤稀释的预制微量滴定板保存条件。
在宏量肉汤稀释期间,与适当的对照相比,培养基的pH不变(图23)。
肺炎链球菌的NCL812体外敏感性测定
NCL812MIC50、MIC90、MIC范围的测定
当对所有20个菌株进行测试时,NCL812表现出MIC50和MIC90为8μg/ml,MIC范围为4-8μg/ml。氨苄青霉素的MIC相当于最近公布的使用微量肉汤稀释作为肺炎链球菌分离株中抗微生物剂耐性端值的结果,从而确定所获得的NCL812MIC的准确性。
表8:NCL812和氨苄青霉素处理的分离株的MIC和MBC值
NCL812MBC50、MBC90、MBC范围的测定
测定NCL812和氨苄青霉素对所有二十个分离株的最小杀菌浓度(分别为MBC50、MBC90和MBC范围)。
表9:NCL812和氨苄青霉素对每个肺炎链球菌分离株的MIC和MBC值
NCL812处理的D39的微量肉汤稀释时间杀菌研究
在48小时时间期内暴露至亚抑制浓度(≤2μg/ml)NCL812的D39的生长类似于未暴露的对照(图24)。高浓度的NCL812(≥16μg/ml)使得48小时无细菌生长(图24)。这些生长特性使用Spectramax分光光度计通过微量液体杀菌动力学研究验证,所述分光光度计测量14小时内每半小时间隔NCL812和氨苄青霉素的生长(以OD600表示)(图25和26)。NCL812处理的D39的指数生长开始如图27所示。
将NCL812处理达48小时的D39的生长与氨苄青霉素或红霉素处理的D39比较(图28和29)。氨苄青霉素处理的D39表现出与暴露至NCL812达48小时的D39类似的生长(图28)。红霉素处理的D39产生与NCL812不同的生长曲线,其中观察到浓度之间的生长差异较大(图29)。在48小时时间期内将5%氯化胆碱加入培养基的结果是,与阳性和生长对照相比,NCL812的生长无显著性差异(图30和31)。
耐性点测试
用≤4μg/mlNCL812处理的D39在6小时进入对数生长期(图24),如四个独立实验所示。在5和6小时之间NLC812的抗微生物剂耐性可能性通过进一步确定暴露至2μg/mlNCL812、4μg/mlNCL812和0.0225μg/ml氨苄青霉素6小时的D39MIC研究。结果显示与生长对照和氨苄青霉素相比,暴露至NCL812的所有D39样品的MIC无显著增加。
表10:暴露至2ug/ml或4ug/mlNCL812达6小时的D39的MIC和MBC值
*D39生长对照:肺炎链球菌菌株D39在4%LHB:CAMHB中生长6小时。
**D39生长2对照:肺炎链球菌菌株D39在HBA上生长O/N,重悬于生理盐水中(0.1OD600)并在无菌生理盐水中1/20稀释。
通过测量特定时间点的相对MBC进行微量肉汤稀释
相对MBC在特定时间间隔使用液体稀释测定法测定,对于NCL812以及对照抗微生物剂氨苄青霉素和红霉素温育48小时(图32和33)。D39的氨苄青霉素(0.023μg/ml)和红霉素(0.00275μg/ml)MIC与其它肺炎链球菌分离株的公布结果的范围类似。观察NCL812、氨苄青霉素和红霉素之间的比较生长差异(图32和33)。氨苄青霉素和红霉素显示出细菌的时间依赖性减少。NCL812表现出快速杀菌作用,其证据是MBC在5小时内减少大约3倍。对于NCL812在整个48小时内杀菌浓度(8μg/ml)保持一致。
NCL812的D39宏量肉汤稀释时间杀菌研究
每个时间点的存活计数以NCL812(图34)和氨苄青霉素(图35)的log10CFU/ml减少表示。对于未暴露的对照和2μg/mlNCL812观察到一致的汇合生长(通过2x104CFU的限制确定)。128μg/mlNCL812的完整杀菌活性(定义为CFU减少3个数量级)以3小时内菌落形成单位(CFU)减少4个对数级观察,并且16μg/ml和64μg/mlNCL812之间的浓度对8小时内消除细菌生长有效(图34)。4μg/ml和8μg/mlNCL812看起来在暴露后11小时失活,因为观察到该时间点后菌株D39生长增加(图34)。
透射电子显微镜法
形态测定分析揭示,与生长对照相比,暴露至16μg/mLNCL8126小时后菌株D39的细胞膜显著改变。由于每个部分缺少可用的细菌细胞,4μg/ml处理的样品以及12小时培养物不考虑用于形态测定分析。与未处理的样品(4.35±0.24nm)相比,处理的样品具有显著更厚的细胞膜(6.43±0.29nm)(p<0.0001)(图36)。与未处理的样品(3.91±0.14nm)相比,16μg/mlNCL812处理的D39的周质空间(细胞膜和细胞壁之间的胞内空间)显著更大(4.54±0.096nm)(p<0.001)(图37)。
表11:平均细胞膜厚度和周质空间
概括地说,对于肺炎链球菌收集物,NCL812生成高度一致的MIC和等同的MBC,确认其对该生物具有杀菌性。在测量48小时时间期内相对MBC的杀菌动力学实验中,D39初始暴露至NCL8126小时后得到一致的杀菌效果。
杀菌活性的表现首先在肺炎链球菌中观察到。这表示NCL812在体外对肺炎链球菌有效。
在血液、血清或肉汤中,组分之间的竞争性结合减少了NCL812的抗微生物剂活性。这反映在不同肉汤类型和稀释剂之间观察到的MIC增加。在完成这些研究后,最近独立研究确认NCL812在PBS中沉淀,并且报道其在100%DMSO中初始稀释后,完全溶解于包含4%DMSO的水中。水溶性NCL812将大大提高体内生物利用率和血液或血清蛋白质之间的负相互作用。
根据该研究的发现,NCL812表现出对肺炎链球菌的作用机制不同于β-内酰胺或大环内酯类别,因为其看起来表现出浓度依赖性杀菌活性,而不是时间依赖性性质。确认体内NCL812的最大药代动力学血清浓度将有助于确认其浓度依赖性药效动力学活性。此外,氯化胆碱加入培养基确认NCL812的作用机制与细胞壁胆碱结合蛋白的亲和力无关,因此与细胞壁无关。
16μg/mlNCL812处理6小时的D39的细胞膜和周质空间的形态测定分析显示,与对照样品相比,处理样品中细胞膜和周质空间更大。膜表观尺寸增加是由于细胞膜下电子密集胞内材料的积聚。周质空间尺寸的增加是由于可能的去极化或ATP抑制导致的细胞膜破裂。NCL812的作用机制可能不是钙依赖性的,因为看起来电镜显微图中观察到NCL812和脂双层的钙通道抑制剂钌红之间的无竞争性结合。
最后,该体外研究显示NCL812具有多个所需特性,如看起来靶向肺炎链球菌的细胞膜的快速作用浓度依赖性杀菌抗微生物剂。这些特性是治疗急性肺炎链球菌感染所需的。由于NCL812可具有靶向细胞膜的作用机制,其作用比时间依赖性抗微生物剂例如β-内酰胺和大环内酯类更迅速,并且可能比具有胞内靶标的其它杀菌浓度依赖性抗微生物剂例如氟喹诺酮类更有效。
实施例6:澳大利亚甲氧西林敏感和耐甲氧西林伪中间葡萄球菌分离株的鉴定和新型抗葡萄球菌化合物的初步体外功效
材料和方法
甲氧西林敏感伪中间葡萄球菌(MSSP)和耐甲氧西林伪中间葡萄球菌(MRSP)的样品收集和鉴定
从狗获得总共23个伪中间葡萄球菌分离株(图38)。
该研究收集10个甲氧西林敏感和13个耐甲氧西林伪中间葡萄球菌。分离株根据体外苯唑西林耐性在表型上归类为耐甲氧西林,并且根据标准程序确定遗传学上存在mecA基因。
使用圆盘扩散技术进行苯唑西林和头孢西丁敏感性测试和Epsilometer测试。mecA基因鉴定使用聚合酶链式反应(PCR)进行
对23个伪中间葡萄球菌分离株进行以下抗微生物剂:青霉素、阿莫西林、红霉素、庆大霉素、克林霉素、环丙沙星、头孢氨苄、氯霉素、四环素、土霉素、万古霉素、头孢替坦、莫西沙星和利福平的CLSI圆盘扩散敏感性测试(图39)。
NCL812的最小抑制浓度(MIC)和最小杀菌浓度(MBC)测试使用CLSI方法进行,并且包括氨苄青霉素作为对照。然后对所有23个分离株测试抗葡萄球菌化合物,并根据标准方案测定最小抑制浓度(MIC)。在确定MIC后,进行最小杀菌浓度测试以确定这些化合物具有抑菌性还是杀菌性。
结果
mecA基因存在于13个MRSP分离株中,并且在10个MSSP中为阴性。根据纸片扩散(<=17mm)和E-测试MIC(>=0.5mg/L),所有MRSP分离株具有苯唑西林耐性。
当头孢西丁耐性断点设置为<=24mm时,3/13(23%)和5/13(38%)的测试MRSP是头孢西丁敏感的。当头孢西丁耐性断点设置为<=30mm时,在兽医诊断实验室仅1/13(7.7%)的测试MRSP是敏感的。
MRSP分离株对多个抗生素类别具有耐性。在13个MRSP分离株中,所有13个均为利福平敏感的。3/13(23%)为氯霉素敏感的;10/13(77%)为万古霉素敏感的。
有趣的是,3/13(23%)的MRSP分离株为阿莫西林敏感的;8/13(62%)为头孢噻吩敏感的;12/13(92%)为头孢替坦敏感的,并且12/13(92%)为莫西沙星敏感的,
根据MIC,所有23个分离株为NCL812敏感的。此外,根据最小杀菌浓度(MBC)测试,NCL812显示出具有杀菌性。
NCL812对伪中间葡萄球菌分离株的MIC范围被发现在1μg/mL和4μg/mL之间。
表12:NCL812对伪中间葡萄球菌分离株的MIC范围
分离株 | AMP | NCL812 |
S1P1 | 128 | 4 |
S2P2 | 128 | 2 |
S3P3 | 128 | 2 |
S4P4 | 128 | 1 |
S5P5 | 16 | 2 |
S6P6 | 64 | 2 |
S7P7 | 128 | 2 |
S8P8 | 128 | 2 |
S9P9 | 32 | 2 |
S10P10 | 64 | 2 |
S11P11 | 128 | 4 |
S12P12 | 32 | 2 |
S13P13 | 0.25 | 2 |
S14P14 | 1 | 2 |
S15P15 | 4 | 4 |
S16P16 | 0.25 | 2 |
S17P17 | 1 | 2 |
S18P18 | 4 | 4 |
S19P19 | 0.5 | 4 |
S20P20 | 4 | 4 |
S21P21 | 0.1 | 2 |
S22P22 | 8 | 4 |
S23P23 | 32 | 2 |
NCL812对伪中间葡萄球菌分离株的MIC50和MIC90被发现分别为2μg/mL和4μg/mL。NCL812对伪中间葡萄球菌分离株的MIC众数和MIC范围被发现分别为2μg/mL和1-4μg/mL。
表13:NCL812对伪中间葡萄球菌分离株的组合MIC值
耐甲氧西林伪中间葡萄球菌(MRSP)在狗、猫和马中造成的问题越来越多。据报道,在欧洲(ST71)和北美(ST68)从狗分离了两种主要MRSP克隆谱系。还有报道称,在日本MRSP对狗造成了影响,在香港报道了单个兽医工作者的MRSP病例。
在本研究中,使用mecA基因的存在和体外苯唑西林耐性的组合测定MRSP分离株。头孢西丁敏感性作为苯唑西林的替代品用于耐甲氧西林金黄色葡萄球菌。然而,使用耐甲氧西林金黄色葡萄球菌和凝固酶阴性葡萄球菌的人分离株推荐的说明准则进行头孢西丁圆盘扩散测试在MRSP鉴定中不可靠。Bemis等,2012提出了<=30mm=耐性和>=31=敏感的头孢西丁断点耐性。本研究同意该断点在预测耐甲氧西林伪中间葡萄球菌更可靠。MRSP分离株通常对多个抗生素类别具有耐性。因此,细菌培养物和抗生素敏感性被推荐至所有可疑MRSP感染,以允许适当选择抗生素。本研究需注意的限制是MRSP分离株对阿莫西林和先锋霉素(头孢噻吩和头孢替坦)的表观体外敏感性。NCL812对MSSP和MRSP二者的所有23个分离株有效。大量研究确认了NCL812对伪中间葡萄球菌的有效性,因为其为越来越多的家畜MRSP感染提供了安全的替代抗生素选择。
实施例7:NCL812对革兰氏阴性生物的活性。
本研究的目的是确定NCL812的抗菌活性靶标是否存在于革兰氏阴性细胞中。为确定NCL812靶标是否在革兰氏阴性细胞中,使用氨苄青霉素移除外膜和大多数细胞壁,然后用各种浓度的NCL812处理改性细胞(称为原生质球)。
原生质球状态诱导
大肠杆菌ATCC25922在琼脂上37℃下生长过夜。将过夜温育的两个菌落用于温育~20ml阳离子调节MuellerHinton肉汤。接种肉汤在37℃下温育18小时。将6ml过夜肉汤培养物加入20ml补充阳离子调节MuellerHinton肉汤(补充50mg/ml氨苄青霉素、0.4M蔗糖、8mMMgSO4),并在37℃下温育过夜。原生质球的形成使用相差显微镜确认。
NCL812的活性
将3ml原生质球培养物加入每支试管,并将50μl包含适当浓度NCL812的DMSO加入每支试管。对照试管仅加入50微升DMSO。用0、2、4、6、8和24小时采集的20μl样品温育原生质球24小时。20微升样品1:10系列稀释,将10ul适当稀释样品一式三份点入脑心浸出琼脂。脑心浸出琼脂在37℃下温育48小时,在24和48小时对菌落计数,以确定菌落形成单位的数量。
原生质球处理的NCL812的成像
在暴露至NCL812化合物24小时后采集样品,用台盼蓝染色并成像。
结果
持续观察到大于99%的原生质球诱导速率。NCL812的靶标被发现存在于大肠杆菌细胞中,随时间推移NCL812的浓度增加至≥32μg/ml,观察到菌落形成单位数显著减少(图40)。实验重复三次,图40中示出一个代表性实验。在暴露至NCL812化合物24小时后采集的原生质球图像显示,随着NCL812浓度的增加,多形细胞发育的频率增加(数据未示出)。这些结果显示NCL812作为抗菌剂对革兰氏阴性菌有效。
实施例8:NCL812的制剂。
使用本领域的标准方法制备以下制剂。
制剂A–局部用制剂-具有NCL812的基于PEG的凝胶
4.0gPEG4000;
3.5gPEG200;
0.6丙二醇;
1.9g水;和
0.204gNCL812。
将PEG4000、PEG200和丙二醇混合并加热到150℃,直到所有固体催化剂溶解。将NCL812加入水中,超声处理30分钟,直到完全悬浮。将NCL812溶液和凝胶溶液混合,并使之冷却和固化。制剂A展示出可接受的粘度、易于涂抹到皮肤上、均匀的悬浮液和一致且细腻的质地。
制剂B–局部用制剂-具有NCL812的基于PEG的凝胶
3.0gPEG4000;
1.0gPEG8000;
3.0gPEG200;
1.0g丙二醇;
1.9g水;和
0.202gNCL812。
将PEG4000、PEG8000、PEG200和丙二醇混合并加热到150℃,直到所有固体催化剂溶解。将NCL812加入水中,超声处理30分钟,直到完全悬浮。将NCL812溶液和凝胶溶液混合,并使之冷却和固化。制剂B展示出可接受的粘度、易于涂抹到皮肤上、均匀的悬浮液和一致且细腻的质地。
制剂C–局部用制剂-具有NCL812-Soluplus的基于PEG的凝胶
2.5gPEG4000;
4.0gPEG200;
2.5g丙二醇;
1.0g水;和
1.8gNCL812-SoluPlus固态分散体。
Soluplus购自BASF(www.soluplus.com)。使用本领域的标准方法制备NCL812-SoluPlus。
将PEG4000、PEG200、NCL812-SoluPlus和丙二醇混合并加热到150℃,直到所有固体催化剂溶解。添加水,然后将溶液超声处理。使溶液冷却和固化。制剂C展示出可接受的粘度、易于涂抹到皮肤上、均匀的悬浮液和一致且细腻的质地。
制剂D–片剂制剂
30mg脱水磷酸氢钙;
80mg微晶纤维素;
50mg乳糖;
8mg羟丙基甲基纤维素
1.5mg滑石
10mgNCL812
对赋形剂称重,并混合5分钟。将混合物送入压片机的喂料斗,使压片机根据本领域的标准程序运行。
制剂D展示出可接受的片剂硬度、崩解性和脆碎度。
制剂E–口服悬浮液
2.0ml甘油;
1.5ml无水乙醇;
600mgNCL812;和
至60ml媒介物(OraSweet和OraPlus,1:1)中
将NCL812粉末过75μm筛。将600mg过筛的NCL812与2.0ml甘油和1.5ml无水乙醇混合。将混合物置于研钵中,手动研磨直到NCL812均匀悬浮。将悬浮液超声处理30分钟。然后将媒介物(55mlOraSweet和OraPlus混合物)加到悬浮液中,再研磨10分钟。通过转移到量筒中将体积用Oraplus和Orasweet混合物补足到60ml
制剂E展示出可接受的悬浮液并暂时出可接受的短期稳定性。
制剂F–肌内注射
20mg/ml聚乙烯吡咯烷酮K30(PVPK30);
0.09mg/mlNCL812;和
50ml水。
通过向50mlMilliQ水中添加1.0gPVPK30制备了2%w/v的PVPK30溶液。然后将溶液置于超声仪中30分钟以进行平衡,将4.5mgNCL812加入PVP溶液中并置于10rpm最大速度下的振荡培养箱上24小时,温度控制在25±1℃。将溶液转移到5ml小瓶中并检查澄明度、外观、pH和短期稳定性。溶液的pH为7.25。
制剂F展示出可接受的透明度和短期稳定性。
实施例9:NCL812从制剂B中的释放。
本研究的目的是测量NCL812从实施例中制备的制剂B中的释放
将Franz扩散池用于对NCL812从其局部用制剂中的释放速率进行定量。将五毫升被选为理想的释放介质的无水乙醇装到接受室中。使用水夹套将接受液的温度保持恒定在32±1℃。选择孔径为0.45um(PallCorporation)的乙酰基纤维素膜,并置于供应室与接受室之间。之后,将多个试样(制剂B)装入供应室。通过采样口,在0.25、0.50、0.75、1、2、3、4、5、6、7、8和24小时的定期时间间隔收集一毫升接受液。将一毫升新鲜的无水乙醇立即返回到接受室中。将UV-HPLC用于分析所得的接受液的含量。
图41展示了NCL812随着时间的累积释放。该研究证实了制剂B提供可接受的NCL812释放谱。
实施例10:与其它类别的抗微生物剂的协同作用研究。
方法
将棋盘法(Gunics等,2000Int.J.Antimicrob.Agents.14:239-42)用于发现NCL812与四环素、氯霉素、红霉素(大环内酯)、氨苄青霉素(广谱β-内酰胺)、庆大霉素(氨基糖苷)、环丙沙星(氟喹诺酮)、磺胺甲噁唑(磺胺)或青霉素G(窄谱β-内酰胺)组合的相互作用(协同作用、拮抗作用、无作用)。对于初始实验,使用了金黄色葡萄球菌T3-129的实验室菌株,然而,该菌株对于一些抗微生物剂而言得到了不一致的结果,将对所有测试的抗微生物剂敏感的称为MK1的新葡萄球菌属物种菌株(当前正在鉴定中的确定种)用于后续测试。
首先,根据CLSI标准准则测定每种单独的抗生素的MIC。其次,一式两份地测试NCL812与每种上述抗生素的组合。为了评价该组合的作用,如下计算了每种抗生素的分级抑菌浓度(FIC):所测试的抗生素的FIC=所测试的抗生素的组合的MIC/单独的抗生素的MIC;NCL812的FIC=NCL812的组合的MIC/单独的NCL812的MIC;以及FICI=FIC指数=NCL812的FIC+每种测试的抗生素的FIC。
根据棋盘准则,将协同作用(S)定义为FICI<0.5。将无作用(NE)定义为0.5<FICI<4。将拮抗作用(A)定义为4<FICI。
结果
MIC、FIC、FICI和NCL812与八种抗生素之间的相互作用在表14中示出。代表不同类别的抗微生物剂的八种测试化合物均未显示出与NCL812的正(协同作用)或负(拮抗作用)相互作用,这与将抗微生物剂加到NCL812中时的相加作用一致。
表14:MIC、FIC、FICI和NCL812与八种抗生素之间的相互作用。
1金黄色葡萄球菌
菌株T3-29
2葡萄球菌属物种菌株MK1
FIC1=与NCL812组合的抗生素的MIC/单独的抗生素的MIC
FIC2=与抗生素组合的NCL812的MIC/单独的NCL812的
MIC
FICI=FIC
指数
实施例11:NCL812对金黄色葡萄球菌和粪肠球菌的抗微生物剂敏感性分离株的作用
材料和方法
菌株信息
将两个金黄色葡萄球菌分离株用于以下实验:金黄色葡萄球菌MK01人类皮肤菌株,和金黄色葡萄球菌KC01马皮肤菌株。这些分离株通过革兰氏染色和生物化学方法(包括RemelStaphaurex商业试剂盒)鉴定。一个粪肠球菌分离株(USA01)未鉴定为VRE菌株。因为该分离株之前已经形成物种,除了观察在血琼脂上的纯、特征性生长外,其未接受进一步的测试。
研究最小杀菌浓度(MBC)
CLSI方法
如在之前的实验中,将始于MIC的每个孔的10μL内容物接种到ColumbiaSBA板上并在37℃温育48h。在24和48h时对板进行检查,并将MBC记录为在板上未观察到细菌菌落(或与对照相比观察到明显的生长抑制)(CLSI2005)时的NCL812最低浓度。
金黄色葡萄球菌KC01和粪肠球菌USA01的杀菌动力学测定方法
使分别未被确定为MRSA或VRE的金黄色葡萄球菌KC01和粪肠球菌USA01在ColumbiaSBA上在37℃生长过夜。然后将一些细菌菌落悬浮在CAMHB(阳离子调节的MuellerHinton肉汤)中并调节到0.08至0.10的OD600。将细菌悬浮液按1:10稀释。将一毫升细菌加到9mL含各种(最高4×MIC)浓度的NCL的CAMHB中,以实现1至3×106CFU/mL的最终细菌浓度。将管子在37℃在恒定振荡下温育。为了确定在各个时间点存在的活细菌的数量,从每根管取出100μL等分试样并稀释。然后,将100μL各稀释样一式两份地涂到菌落计数琼脂上,并在37℃温育48h。在24h后,对存在于每个板上的菌落数计数,并因此对存在于原始悬浮液中的活细菌数计数。在48小时后对板再次检查。
结果
最小抑制浓度(MIC)
研究了分离株金黄色葡萄球菌MK01和KC01以及粪肠球菌USA01的NCL812MIC。结果是:金黄色葡萄球菌MK01=4-8μg/mL,金黄色葡萄球菌KC01=2μg/mL,粪肠球菌USA01=4μg/mL。
研究了金黃色葡萄球菌分离株MK01和KC01,未观察到生长或仅在低浓度NCL812(2μg/ml)下观察到生长,表明NCL812杀金黄色葡萄球菌。然而,对于所测试的粪肠球菌分离株(USA01),在所有测试的NCL812浓度下均观察到细菌生长。随着浓度的增加,存在细菌数的明显降低,但与金黃色葡萄球菌的无生长相比,存在生长。这些结果的汇总可见于表15。表15显示了对两个非MRSA金黄色葡萄球菌分离株和一个非VRE粪肠球菌分离株进行的NCL812MBC测试的结果。每个MBC测试一式两份地进行。在48h时未观察到结果的变化。表16显示了20个MRSA分离株的NCL812MBC值(μg/mL)。从NCL812MIC浓度开始到16倍MIC,一式两份地进行每个MBC测试。表17显示了10个VRE分离株的NCL812MBC值(μg/ml)从NCL812MIC浓度开始到32倍MIC,一式两份地进行每个MBC测试。
表15:对两个非MRSA金黄色葡萄球菌分离株和一个非VRE粪肠球菌分离株进行的NCL812MBC测试。
+=在绵羊血琼脂上生长;0=在绵羊血琼脂上无生长;N=未培养;括号中的数是每毫升样品在24小时后生长的细菌数(CFU/ml)
表16:20个MRSA分离株的NCL812MBC值(μg/ml)。
GB=在绵羊血琼脂上的细菌生长
N**=未在绵羊血琼脂上培养
表17:10个VRE分离株的NCL812MBC值。
*每毫升样品在24小时后生长的细菌数(CFU/ml);M=许多细菌在板上生长(太多而无法计数)
金黄色葡萄球菌KC01和粪肠球菌USA01的杀菌动力学测定方法
在t=0、120、240和360分钟,然后在24h时再次进行菌落计数。在2h时间点,金黃色葡萄球菌KC01显示出细菌数与初始数相比2.5log10的最小降低,并与相同时间点的对照相比大于3log10的降低。2log10的最小较低在6h温育时仍然明显,然而,在24h后,所存在的细菌数增加,并且这与对照无明显不同。
粪肠球菌USA01得到了类似的结果,然而,所观察到的细菌数降低低于金黄色葡萄球菌KC01。与生长对照相比,在2h时观察到了CFU/mL的2log10的降低。然而,与原始细菌数相比CFU/mL的降低刚好大于1log10。在4-16μg/mL的NCL812浓度下,该细菌数降低保持一致直到6h时间点。然而,在32和64μg/mL的浓度下,在相同的时间段内,存在约1log10的细菌数升高。在24h时,所有浓度下的数量均增加到几乎与生长对照相同的水平。
这些金黃色葡萄球菌和粪肠球菌菌株观察到的结果与所测试的所有MRSA和VRE分离株的杀菌动力学测定所观察到的结果一致。金黄色葡萄球菌KC01在不同NCL812浓度下温育最多24h的杀菌动力学测定在图42中示出。粪肠球菌USA01在不同NCL812浓度下温育最多24h的杀菌动力学测定在图43中示出。
实施例12:通过施用NCL812在体内治疗细菌感染的方法。
本研究的目的是确定含有NCL812的兽医试验用药在治疗小鼠皮肤感染中的功效
模型概述
有用的动物模型系统应为临床上相关、实验上稳健、伦理学上可接受、便于执行的并且应当提供可靠且可重现的结果。存在许多已描述的局部皮肤感染动物模型,包括巴豆油致炎皮肤模型(Akiyama,H.,H.Kanzaki,Y.Abe,J.Tada和J.Arata(1994)."Staphylococcusaureusinfectiononexperimentalcrotonoil-inflamedskininmice."JournalofDermatologicalScience8(1):1-10)、灼伤皮肤模型(Stieritz,D.D.,A.Bondi,D.McDermott和E.B.Michaels(1982)."Aburnedmousemodeltoevaluateanti-pseudomonasactivityoftopicalagents."JournalofAntimicrobialChemotherapy9(2):133-140)、皮肤缝合伤口模型(McRipley,R.J.和R.R.Whitney(1976)."CharacterizationandQuantitationofExperimentalSurgical-WoundInfectionsUsedtoEvaluateTopicalAntibacterialAgents."AntimicrobialAgentsandChemotherapy10(1):38-44)、皮肤胶带剥离模型(Kugelberg,E.,T.T.K.Petersen,T.Duvold,D.I.Andersson和D.Hughes(2005)."EstablishmentofaSuperficialSkinInfectionModelinMicebyUsingStaphylococcusaureusandStreptococcuspyogenes."AntimicrobialAgentsandChemotherapy49(8):3435-3441)以及线性全厚度手术刀切割方法(Guo,Y.,R.I.Ramos,J.S.Cho,N.P.Donegan,A.L.Cheung和L.S.Miller(2013)."InVivoBioluminescenceImagingToEvaluateSystemicandTopicalAntibioticsagainstCommunity-AcquiredMethicillin-ResistantStaphylococcusaureus-InfectedSkinWoundsinMice."AntimicrobialAgentsandChemotherapy57(2):855-863)。
在开展本研究之前的初步研究确立了源于上述模型的详细研究的新皮肤感染方法。简而言之,将研究小鼠麻醉,剪下一片背部皮肤以露出皮肤,并用手持式冲孔机取下一块圆形皮肤,在背部上留下具有中央腔的伤口。将伤口用已知数量的挑战性生物体感染。在感染后大约四至六小时,将伤口用媒介物制剂或活性制剂进行局部处理。每12小时对感染的皮肤伤口再次处理,总共处理14次。对小鼠实施人道安乐死,解剖原始感染伤口区域并取下,通过标准微生物学测试对细菌含量计数。以此方式,由于活性制剂处理而导致的细菌浓度变化可通过检查与媒介物对照相比的细菌载量降低而容易地确定。
材料和方法
感染种菌的制备
使细菌(金黄色葡萄球菌)的新鲜培养物在绵羊血琼脂上在37℃生长16-18小时。选择一些典型的菌落,悬浮在10ml胰酶大豆肉汤中并在振荡培养箱(240rpm)中在37℃温育过夜。将过夜悬浮液涡旋,并在新鲜胰酶大豆肉汤中稀释(1:100)(将100μl[0.1ml]溶于9.9ml肉汤)。将新鲜悬浮液在振荡培养箱(如上)中温育3小时以便得到对数中期细菌。将细菌通过在7,500rpm下离心10分钟而沉淀。移除肉汤上清液,将细菌悬浮在10ml磷酸盐缓冲生理盐水(PBS)中。将这些步骤再重复两次。使用分光光度计,以生理盐水作为空白,通过测量600nm处的吸光度而检查悬浮液的密度,以确认在约0.100的读数下的目标密度(与2.5x107CFU/ml的细菌密度一致)。将悬浮液置于放到具有冰砖的可上锁运输箱中的架子上,以在运输期间保持制冷,然后在到达后储存在小鼠皮肤感染实验室的冷房内。将最终悬浮液彻底混合,然后对小鼠中形成的皮肤伤口接种。
为了确保悬浮液的纯度和准确性,在置于上锁的箱子之前进行以下步骤。
通过将100μl最终悬浮液涂在SBA(绵羊血琼脂)板上,在37℃温育18小时并进行检查以确认一种菌落类型的均匀生长,而确保细菌悬浮液纯度。通过以下方式对最终悬浮液进行活菌计数:在Eppendorf管(每根管约900ul)中配制生理盐水,取出100μl样品并加到第一Eppendorf管中,将混合物涡旋,并使用含有生理盐水的第2根管重复。将该过程继续5至6根管。最后,将100μl第5次和第6次稀释样铺到平板计数琼脂上,在37℃温育18小时,并进行菌落计数以确认CFU/ml为约2.5x107。在接种伤口后,将该过程重复以确保在手术期间未发生污染或活菌计数的降低。
皮肤伤口手术操作
将每只小鼠置于诱导箱中并使用2%异氟烷诱导麻醉。将每只麻醉小鼠的眼用兽医眼科润滑剂覆盖以便防止角膜脱水。将每只小鼠从诱导室取出并置于手术区域上的各个麻醉鼻吸之前。在麻醉下,监控每只小鼠以评估麻醉深度(对疼痛、瞬目反射、骨骼肌张力的反应)和呼吸及心脏功能。用机械剪从手术区域剃去背部皮毛。将剃毛区域用沾到纸巾上的70%乙醇然后用10%w/v聚维酮碘溶液清洁。一旦碘溶液干燥后,施用非甾体抗炎剂美洛昔康的皮下注射。使用耳洞钳/活检穿孔器轻轻夹紧背部皮肤,以允许形成圆形全厚度伤口。媒介物对照和NCL812处理的小鼠将伤口用微量移液管通过10μl细菌悬浮液接种(2.5x105CFU/10μl)。一旦细菌悬浮液干燥后,将小鼠置于用小鼠编号标记的各个恢复箱中。记录接种时间。将每只小鼠的初始体重记录在适当的记录表上。小鼠在5分钟内恢复到全意识。将恢复的小鼠放回各个笼子,并每小时监控术后或麻醉并发症。
术后护理(术后4小时)
评估了小鼠的术后并发症,并将观察结果记录在临床记录表上。将每只小鼠小心从IVC取出并置于评估容器中,避免过多触摸或碰到手术部位。一旦小鼠在评估容器内后,即对其进行评估并将观察结果记录在术后临床记录表上。每当出现所建议的健康问题时,即施用术后止痛,并记录在临床记录表上。
动物监控和日常护理
抗生素施用(早上7点和下午6点)。媒介物或NCL812膏剂的第一次施用在术后4小时进行。在施用前对每个膏剂容器称重,并记录重量。小心地约束每只小鼠。将膏剂(媒介物或NCL812)涂到病变区域,并将经处理的小鼠返回到IVC,其中对每只小鼠进行观察以确保膏剂未因梳毛而被立即移除。记录膏剂容器的施用后重量。将媒介物和活性NCL产品在第一次施用后每12小时涂到皮肤伤口,总共连续处理14次。NCL812膏剂(制剂B,如实施例8中所示)以20mg/g的浓度含有氯苯胍。在每种情况下均涂抹约0.1-0.2g膏剂,从而向体重在18g与25g之间的小鼠递送28与56mg之间的NCL812总局部剂量。
每日监控。在大约中午12点每天一次对每只小鼠进行监控。将每只小鼠小心从IVC取出并置于观察容器中,避免过多触摸或碰到手术部位。仔细评估在容器中时的皮毛、姿势、眼睛、行为、发声和活动,并将观察结果记录在评估表上。检查小鼠粪便(笼底上或容器中)的稠度,并记录观察结果。当小鼠在容器中时测定每只小鼠的重量,并计算和记录体重变化。将观察容器用乙醇消毒,然后放在一边晾干,同时将新的容器用于下一只小鼠。每两天,将小鼠再次用2%异氟烷麻醉,并用尺子作为尺寸参考进行拍照。将这些照片用于评估试验期间的病变大小和感染进展。
组织分析和抗菌功效的评估
在7天皮肤伤口评估期结束时,对所有实验小鼠实施安乐死,然后收集伤口进行死后检查。从每只小鼠的背部解剖皮肤伤口。将样品置于样品管中,称重,然后添加1mlPBS和无菌组织均化微珠。将组织样品用组织匀化器(NextAdvanceBulletBlender)匀化10分钟,然后涡旋大约30秒。取出100μl上清液,置于含有900μlPBS的Eppendorf管中。使用系列稀释重复该程序,总共稀释8次。最后,将100μl各稀释样一式两份地移取到平板计数琼脂上,在37℃温育过夜。将十微升原始悬浮液置于绵羊血琼脂上以评估培养物纯度,并在37℃温育过夜。第二天,使用温育后的平板计数琼脂板进行活菌计数,并确认作为收获的菌株的金黄色葡萄球菌(挑战性生物体)的种类。
结果
在媒介物处理组中观察到的每克组织的平均菌落计数为5,888,436(6.77log10)。在NCL812组中观察到的每克组织的平均菌落计数为141,254(5.15log10)。每克组织的log10菌落形成单位数和降低%在下表中汇总。
表15:在局部施用媒介物和处理后每克组织的Log10菌落形成单位数和降低百分比
处理 | Log10(CFU/g) | 降低% |
媒介物 | 6.77 | |
NCL812 | 5.15 | 97.6 |
从该表中显而易见的是,用NCL812处理导致了感染性金黄色葡萄球菌的数量的高度降低。这些结果证实了在体内对细菌定植或感染的有效处理。
Claims (38)
1.一种治疗或预防受试者中细菌定植或感染的方法,所述方法包括以下步骤:将治疗有效量的氯苯胍或其治疗学上可接受的盐施用给所述受试者,其中所述细菌定植或感染因菌剂而导致。
2.根据权利要求1所述的方法,其中所述受试者选自:人、犬科动物、猫科动物、牛、绵羊、山羊、猪、鸟、鱼和马物种。
3.根据前述权利要求中任一项所述的方法,其中所述氯苯胍以0.1mg/kg至250mg/kg体重范围内的剂量施用给所述受试者。
4.根据前述权利要求中任一项所述的方法,其中所述菌剂为革兰氏阳性的。
5.根据权利要求4所述的方法,其中所述菌剂选自:软弱贫养菌、无胆甾原体属物种、猪放线杆菌、牛放线菌、欧洲放线菌、乔氏放线菌、戈氏放线菌、格雷费尼茨放线菌、大麦伤口放线菌、衣氏放线菌血清型II、衣氏放线菌、麦尔放线菌、内氏放线菌、纽氏放线菌、溶齿放线菌、罗氏放线菌、放线菌属物种、图列茨放线菌、粘性放线菌、异斯卡多维亚菌、产氢厌氧球菌、解乳厌氧球菌、莫道克厌氧球菌、八叠厌氧球菌、普氏厌氧球菌、四联厌氧球菌、阴道厌氧球菌、伯尔德隐秘杆菌(放线菌)、化脓隐秘杆菌(放线菌)、伯尔德隐秘杆菌、卡迪夫隐秘杆菌、芬氏隐秘杆菌、溶血隐秘杆菌、休斯顿隐秘杆菌、Arcanobacteriumlingnae、化脓隐秘杆菌(化脓放线菌)、节杆菌属、微小阿托波氏菌、极小阿托波氏菌、龈裂阿托波氏菌、阿托波氏菌属物种、阴道阿托波氏菌、炭疽芽孢杆菌、蜡状芽孢杆菌、环状芽胞杆菌、地衣芽孢杆菌、巨大芽胞杆菌、产黑色素芽孢杆菌、短小芽孢杆菌、球形芽孢杆菌、枯草芽孢杆菌、β溶血链球菌属物种、青春双歧杆菌、齿双歧杆菌、斯卡尔多维双歧杆菌、双歧杆菌、短短芽孢杆菌、侧孢短芽孢杆菌、短杆菌属、缓慢布雷德菌、Catabacterhongkongensis、CDC棒状杆菌群F-1和G、破伤风梭菌、巴氏梭菌、双酶梭菌、肉毒梭菌(A、B、C、D、E、F、G型)、肉毒梭菌、丁酸梭菌、气肿疽梭菌、鹌鹑梭菌、艰难梭菌、溶血梭菌、溶组织梭菌、诺氏梭菌A型、诺氏梭菌B型、诺氏梭菌、产气荚膜梭菌A型、产气荚膜梭菌A-E型、产气荚膜梭菌、毛状梭菌、多枝梭菌、败毒梭菌、索氏梭菌、楔形梭菌、螺状梭菌、梭菌属物种、第三梭菌、破伤风梭菌、产气柯林斯菌、邻居棒状杆菌、非发酵棒状杆菌非发酵亚种、非发酵棒状杆菌嗜脂亚种、无枝菌酸棒状杆菌、银色棒状杆菌、黏金色棒状杆菌、耳棒状杆菌、牛棒状杆菌、混乱棒状杆菌、膀胱炎棒状杆菌、白喉棒状杆菌、弗雷尼棒状杆菌、解葡萄糖苷棒状杆菌、杰氏棒状杆菌、克罗彭施泰特棒状杆菌、库彻氏棒状杆菌、黄色嗜脂棒状杆菌、麦利金氏棒状杆菌、马氏棒状杆菌、极小棒状杆菌、多毛棒状杆菌、丙酸棒状杆菌、假白喉棒状杆菌、假结核棒状杆菌、牛肾盂炎棒状杆菌、芮氏棒状杆菌、模仿棒状杆菌、纹带棒状杆菌、Corynebacteriumsundvallense、Corynebacteriumthomssensii、Corynebacteriumtuberculostearum、溃疡棒状杆菌、解脲棒状杆菌、结膜干燥棒状杆菌、马克洛斯氏菌、皮杆菌属、刚果嗜皮菌、刚果嗜皮菌、短埃格特菌、香港埃格特菌、娇弱埃格特菌、迟缓埃格特菌、微小埃格特菌、缠结埃格特菌、隐藏埃格特菌、中国埃格特菌、沟迹埃格特菌、纤细埃格特菌、埃格特菌属、鸟肠球菌、牛链球菌、铅黄/黄色肠球菌、盲肠肠球菌、殊异肠球菌、耐久肠球菌、粪肠球菌、屎肠球菌、鹑鸡肠球菌、浅黄肠球菌、海氏肠球菌、意大利肠球菌、病臭肠球菌、芒地肠球菌、苍白肠球菌、类鸟肠球菌、棉子糖肠球菌、血栖肠球菌、肠球菌属物种、猪丹毒丝菌、真细菌、龈沟产线菌、大芬戈尔德菌、巴尔涅斯孪生球菌、不解糖卟啉孪生球菌、博格孪生球菌、兔孪生球菌、溶血孪生球菌、麻疹孪生球菌、Gemellapalaticanis、血色孪生球菌、戈登氏菌属物种、毗邻颗粒链菌、细长颗粒链菌、副毗邻颗粒链菌、许氏盖球菌、嗜酸乳杆菌、干酪乳杆菌、发酵乳杆菌、戈氏乳杆菌、惰性乳杆菌、植物乳杆菌、鼠李糖乳杆菌、乳酸菌种、Lactobacillusultunensis、水生雷弗森菌、柠檬明串珠菌、乳明串珠菌、肠膜明串珠菌、类肠膜明串珠菌、假肠膜明串珠菌、格雷李斯特菌、无害李斯特菌、伊万诺夫李斯特菌、单核细胞增生李斯特菌、西尔李斯特菌、威尔逊李斯特菌、微杆菌属、柯氏动弯杆菌、羞怯动弯杆菌、动弯杆菌属物种、胆怯艰难杆菌、弱小艰难杆菌、Moryellaindoligenes、塞内加尔分枝杆菌、脓肿分枝杆菌、非洲分枝杆菌、Mycobacteriumarupense、亚洲分枝杆菌、欧巴涅分枝杆菌、鸟分枝杆菌群、鸟分枝杆菌类结核亚种、鸟分枝杆菌、博氏分枝杆菌、牛分枝杆菌、德氏分枝杆菌、卡内蒂分枝杆菌、山羊分枝杆菌、隐藏分枝杆菌、龟分枝杆菌、嵌合分枝杆菌、哥伦比亚分枝杆菌、康赛医院分枝杆菌、出众分枝杆菌、象分枝杆菌、鼻疽分枝杆菌、佛罗伦萨分枝杆菌、偶然分枝杆菌群、日内瓦分枝杆菌、古地分枝杆菌、嗜血分枝杆菌、半岛分枝杆菌、海德堡分枝杆菌、休斯顿分枝杆菌、免疫原分枝杆菌、居间分枝杆菌、胞内分枝杆菌、堪萨斯分枝杆菌、洛克司分枝杆菌、缓黄分枝杆菌、麻风分枝杆菌、鼠麻风分枝杆菌、马德里分枝杆菌、玛尔摩分枝杆菌、海洋分枝杆菌、马赛分枝杆菌、田鼠分枝杆菌、摩特弗分枝杆菌(鳗鱼)、莫娜分枝杆菌、产粘液分枝杆菌、内布拉斯加分枝杆菌、新金色分枝杆菌、新城分枝杆菌、沼泽分枝杆菌、副结核分枝杆菌(约内氏病)、帕尔门塞分枝杆菌、草分枝杆菌、弗卡分枝杆菌、鳍脚亚目动物分枝杆菌、猪分枝杆菌、假分枝杆菌(鱼)、假结核分枝杆菌、萨斯喀彻温分枝杆菌、瘰病分枝杆菌、Mycobacteriumsenuense、败血分枝杆菌、猴分枝杆菌、包皮垢分枝杆菌、分枝杆菌属物种、苏加分枝杆菌、土分枝杆菌/无色分枝杆菌群、三重分枝杆菌、肺结核分枝杆菌、托斯卡纳分枝杆菌、溃痕分枝杆菌、沃氏分枝杆菌、蟾分枝杆菌、分支杆菌属、星状诺卡氏菌、巴西诺卡氏菌、皮疽诺卡氏菌、新星诺卡氏菌、豚鼠耳炎诺卡氏菌、诺卡氏菌属物种、南非诺卡氏菌、厄氏菌属、口腔欧氏菌属物种、Olsenellaprofuse、齿龈欧氏菌、Oribacteriumsinus、蜂房类芽孢杆菌、微小微单胞菌、片球菌属、吲哚消化球菌、黑色消化球菌、不解糖嗜胨菌、戈巴赫嗜胨菌、兔嗜胨菌、吲哚嗜胨菌、艾弗嗜胨菌、泪腺嗜胨菌、奥尔森嗜胨菌、厌氧消化链球菌、口炎消化链球菌、疮疱丙酸杆菌、颗粒丙酸杆菌、丙酸丙酸杆菌、丙酸菌属、非解乳假枝杆菌、马红球菌、红串红球菌、束红球菌、玫瑰红红球菌、罗思氏菌属、产生瘤胃球菌、Slackiaexigua、还原天芥菜碱斯奈克氏菌、Solobacteriummoorei、阿尔莱特葡萄球菌、金黄色葡萄球菌厌氧亚种、金黄色葡萄球菌、耳葡萄球菌、头状葡萄球菌头状亚种、头状葡萄球菌解脲亚种、头状葡萄球菌、山羊葡萄球菌、肉葡萄球菌、溶酪葡萄球菌、产色葡萄球菌、科氏葡萄球菌科氏亚种、科氏葡萄球菌解脲亚种、科氏葡萄球菌、香料葡萄球菌、海豚葡萄球菌、表皮葡萄球菌、马胃葡萄球菌、猫葡萄球菌、福氏葡萄球菌、鸡葡萄球菌、溶血葡萄球菌、人葡萄球菌、猪葡萄球菌、中间葡萄球菌、克氏葡萄球菌、缓慢葡萄球菌、路邓葡萄球菌、水獭葡萄球菌、苍蝇葡萄球菌、尼泊尔葡萄球菌、巴氏葡萄球菌、佩氏葡萄球菌、鱼发酵葡萄球菌、伪中间葡萄球菌、普氏葡萄球菌、解糖葡萄球菌、腐生葡萄球菌、施氏葡萄球菌凝结亚种、施氏葡萄球菌、松鼠葡萄球菌、猿猴葡萄球菌、模仿葡萄球菌、葡萄球菌属物种、琥珀葡萄球菌、牛葡萄球菌、沃氏葡萄球菌、木糖葡萄球菌、牛葡萄球菌、粘滑口腔球菌(重新归类为粘滑罗斯菌)、无乳链球菌、咽峡炎链球菌种群(中间链球菌、星座链球菌和咽峡炎链球菌)、牛链球菌种群(解没食子酸链球菌解没食子酸亚种(过去称为牛链球菌生物型I)、牛链球菌、犬链球菌、停乳链球菌停乳亚种、马链球菌马亚种、马链球菌兽瘟亚种、豕链球菌、犬链球菌、猪链球菌、海豚链球菌)、停乳链球菌似马亚种、停乳链球菌、马链球菌(马链球菌马亚种)、马链球菌兽瘟亚种、马链球菌、马肠链球菌、似马链球菌(停乳链球菌似马亚种)、解没食子酸链球菌巴氏亚种(过去称为牛链球菌生物型II/2)、婴儿链球菌婴儿亚种、巴黎链球菌(过去称为牛链球菌生物型II/1)、缓症链球菌种群(嵴链球菌、婴儿链球菌、缓症链球菌、口腔链球菌、栖口腔链球菌、鼠口腔链球菌)和变异链球菌种群(大鼠链球菌、汗毛链球菌、野生链球菌、生殖道链球菌、猴链球菌、变异链球菌、大鼠链球菌、表兄链球菌、血链球菌群、格氏链球菌、副血链球菌、血链球菌)、肺炎链球菌、豕链球菌、化脓链球菌、唾液链球菌种群(非解乳糖链球菌、猪肠链球菌、婴儿链球菌、唾液链球菌、嗜热链球菌、前庭链球菌)、链球菌属物种、猪链球菌、乳房链球菌、兽瘟链球菌(马链球菌兽瘟亚种)、兽瘟链球菌、Trueperellaabortisuis、伯氏隐秘杆菌、Trueperellabialowiezensis、Trueperellabonasi、Trueperellapyogenes(化脓隐秘杆菌)、束村氏菌属物种、苏黎士菌属和Turicibactersanguine。
6.根据权利要求5所述的方法,其中所述菌剂选自:金黄色葡萄球菌、伪中间葡萄球菌、肺炎链球菌、化脓链球菌、无乳链球菌、屎肠球菌、粪肠球菌和艰难梭菌。
7.根据权利要求1至3中任一项所述的方法,其中所述菌剂为革兰氏阴性的。
8.根据权利要求7所述的方法,其中所述菌剂选自:醋酸杆菌科:-颈玫瑰单胞菌;费氏玫瑰单胞菌;吉氏玫瑰单胞菌--气单胞菌科:-异常嗜糖气单胞菌;嗜水气单胞菌;豚鼠气单胞菌;嗜水气单胞菌(和亚种);杀鲑气单胞菌;舒伯特气单胞菌;维罗纳气单胞菌温和生物变型(温和气单胞菌)--产碱杆菌科:-木糖氧化无色杆菌;粪产碱杆菌;Bordetellaansorpii;鸟博德特氏杆菌;支气管炎博德特氏杆菌;欣茨博德特氏杆菌;霍氏博德特氏杆菌;副百日咳博德特氏杆菌;百日咳博德特氏杆菌;彼得里博德特氏杆菌;创口博德特氏杆菌;解脲寡源杆菌;尿道寡源杆菌--无浆体科:-嗜吞噬细胞无浆体;扁平无浆体;牛无浆体;红血球内生无浆体;边缘无浆体;奥氏无浆体;羊无浆体;犬埃里希氏体;恰菲埃里希氏体;尤因埃里希氏体;小鼠埃里希氏体;羊埃里希氏体;反刍兽埃里希氏体;Neoehrlichialotoris;米库尔新埃里希氏体;蠕虫新立克次氏体;立氏新立克次氏体;腺热新立克次体;尖音库蚊沃尔巴克氏体--装甲菌科:-玫瑰装甲菌--拟杆菌科:-福赛斯拟杆菌;脆弱拟杆菌;产黑色素拟杆菌;赤拟杆菌;解脲拟杆菌--巴尔通氏体科:-阿尔萨斯巴尔通氏体;澳大利亚巴尔通氏体;杆状巴尔通氏体;Bartonellabirtlesii;牛巴尔通氏体;卡普里巴尔通氏体;Bartonellachomelii;克氏巴尔通氏体;多氏巴尔通氏体;伊丽莎白巴尔通氏体;格氏巴尔通氏体;汉氏巴尔通氏体;克勒巴尔通氏体;跳鼠巴尔通氏体;Bartonellaphoceensis;五日热巴尔通氏体;Bartonellarattimassiliensis;罗卡利巴尔通氏体;Bartonellaschoenbuchensis;鼹鼠巴尔通氏体;Bartonellatamiae;泰勒氏巴尔通氏体;特利波契巴尔通氏体;文森氏巴尔通氏体博格霍夫亚种;文森氏巴尔通氏体阿鲁潘亚种;文森氏巴尔通氏体文森氏亚种--蛭弧菌科:-蛭弧菌属物种--短螺旋体科:-短螺旋体属物种,包括汉普森短螺旋体、猪痢疾短螺旋体、莫道克短螺旋体、结肠菌毛样短螺旋体--布鲁氏菌科:-流产布鲁氏菌;犬布鲁氏菌;鲸型布鲁氏菌;马尔他布鲁氏菌;羊布鲁氏菌;鳍型布鲁氏菌;猪布鲁氏菌;人苍白杆菌;中间苍白杆菌--伯克氏菌科:-Burkholderiaaboris;须芒草伯克氏菌(基因组变型VII);Burkholderiaanthina(基因组变型VIII);新洋葱伯克氏菌(基因组变型III);洋葱伯克氏菌(基因组变型I);Burkholderiadiffusa;多罗萨伯克氏菌(基因组变型VI);Burkholderialatens;鼻疽伯克氏菌;Burkholderiametallica;多噬伯克氏菌(基因组变型II);类鼻疽伯克氏菌;吡咯伯克氏菌(基因组变型IX);Burkholderiaseminalis;稳定伯克氏菌(基因组变型IV);尤伯纳伯克氏菌(基因组变型X);越南伯克氏菌(基因组变型V);罕见贪铜菌;吉氏贪铜菌;皮氏罗尔斯顿菌;解甘露醇罗尔斯顿菌;Sphaerotilushippei;蒙大拿球衣菌;浮游球衣菌--弯曲菌科:-弓形菌属物种,包括斯氏弓形菌;大肠弯曲杆菌;简明弯曲杆菌;曲形弯曲杆菌;胚胎弯曲杆菌;纤细弯曲杆菌;瑞士弯曲杆菌;人弯曲杆菌;豚肠弯曲杆菌;海象弯曲杆菌;空肠弯曲杆菌;拉尼尔弯曲杆菌;红嘴鸥弯曲杆菌;海鸟弯曲杆菌;粘膜弯曲杆菌;直肠弯曲杆菌;昭和弯曲杆菌;痰液弯曲杆菌;乌普萨拉弯曲杆菌--Candidatus:-鲑鱼衣原体--心杆菌科:-人心杆菌;瓣膜心杆菌;节瘤偶蹄形菌--衣原体科:-衣原体属物种,包括鸟衣原体、家禽衣原体、鼠沙眼衣原体、猪衣原体、沙眼衣原体;嗜衣原体属物种,包括肺炎嗜衣原体、家畜嗜衣原体、鹦鹉热嗜衣原体、流产嗜衣原体、豚鼠嗜衣原体和猫嗜衣原体--Chthonomonadaceae:-Chthonomonascalidirosea.--丛毛单胞菌科:-睾丸酮丛毛单胞菌;Verminephrobacterspp.--柯克斯体科:-伯氏柯克斯体--噬纤维菌科:-柱状噬纤维菌;哈氏噬纤维菌;Flexibacterechinicida;华美屈挠杆菌;易挠屈挠杆菌;海滨屈挠杆菌;多形屈挠杆菌;玫瑰屈挠杆菌;红屈挠杆菌--脱硫弧菌科:-沃氏嗜胆菌;细胞内劳索尼亚氏菌--肠杆菌科:-戴氏西地西菌;拉氏西地西菌;奈氏西地西菌;无丙二酸柠檬酸杆菌;差异柠檬酸杆菌;弗氏柠檬酸杆菌;柯氏柠檬酸杆菌;康帝蒙提克罗诺杆菌;都柏林克罗诺杆菌;瑞士克罗诺杆菌;丙二酸克罗诺杆菌;穆汀斯克罗诺杆菌;Cronobacterpulveris;阪崎克罗诺杆菌;图列茨克罗诺杆菌;尤尼沃斯克罗诺杆菌;苏黎世克罗诺杆菌;鲶鱼爱德华氏菌;迟钝爱德华氏菌;产气肠杆菌;成团肠杆菌;阴沟肠杆菌;Enterobactercowanii;阿尔伯蒂埃希氏菌;大肠杆菌包括AIEC=粘附侵袭性大肠杆菌、EaggEC=肠凝集性大肠杆菌;EHEC=肠出血性大肠杆菌;EIEC=肠侵袭性大肠杆菌;EPEC=肠致病性大肠杆菌;ETEC=产肠毒素大肠杆菌;ExPEC=肠外致病性大肠杆菌、NMEC=新生儿脑膜炎大肠杆菌、NTEC=坏死性毒素大肠杆菌、UPEC=尿路致病性大肠杆菌;弗格森埃希氏菌;美洲爱文氏菌;蜂房哈夫尼菌;副蜂房哈夫尼菌;肉芽肿克雷伯氏菌;产酸克雷伯氏菌;肺炎克雷伯氏菌;抗坏血酸克吕沃尔氏菌;栖冷克吕沃尔氏菌;摩氏摩根氏菌;成团泛菌(过去称为成团肠杆菌);非共生发光杆菌;类志贺邻单胞菌;奇异变形杆菌;彭氏变形杆菌;普通变形杆菌;产碱普罗威登斯菌;雷氏普罗威登斯菌;斯氏普罗威登斯菌;Raoultellaelectrica;解鸟氨酸拉乌尔菌;植生拉乌尔菌;土生拉乌尔菌;邦戈尔沙门菌、肠沙门氏菌肠亚种(许多血清型);液化沙雷氏菌;粘质沙雷氏菌;鲍氏志贺氏菌;痢疾志贺氏菌;弗氏志贺氏菌;索氏志贺氏菌;小肠结肠炎耶尔森氏菌;鼠疫耶尔森氏菌;假结核耶尔森氏菌;鲁氏耶尔森氏菌--伞单胞菌科:-Fimbriimonasginsengisoli.--黄杆菌科:-动物溃疡伯杰氏;狗咬二氧化碳噬纤维菌;犬咬二氧化碳噬纤维菌;牙龈二氧化碳噬纤维菌;颗粒二氧化碳噬纤维菌;溶血二氧化碳噬纤维菌;Capnocytophagaleadbetteri;黄褐二氧化碳噬纤维菌;生痰二氧化碳噬纤维菌;产吲哚金黄杆菌;鱼金黄杆菌;脑膜炎败血伊丽莎白金菌;嗜鳃黄杆菌;柱状黄杆菌;小鳟鱼黄杆菌;杀鱼黄杆菌;嗜冷黄杆菌;气味类香菌;拟香味类香菌;鼻腔鸟杆菌;鸭瘟立默氏菌;哥伦比亚立默氏菌;鸽咽喉立默氏菌;Tenacibaculumdicentrarchi;脱色黏着杆菌;Tenacibaculumgallaicum;海洋黏着杆菌;鳎鱼黏着杆菌;有毒威克斯氏菌--弗朗西斯氏菌科:-土拉热弗朗西斯氏菌土拉热亚种;土拉热弗朗西斯氏菌全北区亚种;土拉热弗朗西斯氏菌新凶手亚种;蜃楼弗朗西斯氏菌;诺神弗朗西斯氏菌;诺神弗朗西斯氏菌东方亚种(也称为亚洲弗朗西斯氏菌)--梭杆菌科:-梭杆菌属物种,包括坏死梭杆菌、具核梭杆菌、多形梭杆菌--螺杆菌科:-同性恋螺杆菌;芬纳尔螺杆菌;幽门螺杆菌--军团菌科:-嗜肺军团和其它物种,包括;茴香军团菌;伯明瀚军团菌;博杰曼军团菌;辛辛那提军团菌;杜莫夫军团菌;费勒氏军团菌;戈曼军团菌;哈开里军团菌;约旦军团菌;兰斯格军团菌;长滩军团菌;马氏军团菌;麦氏军团菌;橡树岭军团菌;巴黎军团菌;赫伦荒原军团菌;图森军团菌;沃氏军团菌;沃斯利军团菌--钩端螺旋体科:-亚历山大钩端螺旋体(包括亚历山大钩端螺旋体七日热血清变型、亚历山大钩端螺旋体曼耗群3血清变型);Leptospiraalstoni(包括LeptospiraalstoniserovarPingchang、LeptospiraalstoniserovarSichuan);双曲钩端螺旋体(包括双曲钩端螺旋体安科纳血清变型、双曲钩端螺旋体卡内拉血清变型);博氏钩端螺旋体(包括博氏钩端螺旋体哈德乔血清变型、博氏钩端螺旋体哈德乔-牛血清变型、博氏钩端螺旋体波莫纳血清变型、博氏钩端螺旋体塔拉索夫血清变型);布鲁姆钩端螺旋体(包括布鲁姆钩端螺旋体赫斯布里治血清变型);弗氏钩端螺旋体(包括弗氏钩端螺旋体赫斯布里治血清变型);Leptospiraidonii;稻田氏钩端螺旋体(包括稻田氏钩端螺旋体莱姆血清变型、稻田氏钩端螺旋体马来亚血清变型);问号钩端螺旋体(包括问号钩端螺旋体澳大利亚血清变型、问号钩端螺旋体秋生血清变型、问号钩端螺旋体布拉迪斯拉发血清变型、问号钩端螺旋体犬血清变型、问号钩端螺旋体感冒伤寒血清变型、问号钩端螺旋体哈德乔血清变型、问号钩端螺旋体哈德乔-牛血清变型、问号钩端螺旋体出血性黄疸血清变型、问号钩端螺旋体波莫纳血清变型、问号钩端螺旋体致热血清变型、问号钩端螺旋体塔拉索夫血清变型);基施纳钩端螺旋体(包括基施纳钩端螺旋体保加利亚血清变型、基施纳钩端螺旋体蝙蝠血清变型、基施纳钩端螺旋体感冒伤寒血清变型);克氏钩端螺旋体;黎氏钩端螺旋体;迈氏钩端螺旋体(包括迈氏钩端螺旋体索非亚血清变型);野口氏钩端螺旋体(包括野口氏钩端螺旋体巴拿马血清变型、野口氏钩端螺旋体波莫纳血清变型);圣地罗斯钩端螺旋体;Leptospiraterpstrae;Leptospiravanthielii;韦氏钩端螺旋体(包括韦氏钩端螺旋体塞尔东尼血清变型、韦氏钩端螺旋体沙敏血清变型);沃尔氏钩端螺旋体;沃夫钩端螺旋体;柳川钩端螺旋体--纤毛菌科:-口腔纤毛菌;念珠状链杆菌--甲基杆菌科:-外链甲基杆菌群;藤泽氏甲基杆菌;嗜中温甲基杆菌;扎氏甲基杆菌--摩拉克氏菌科:-鲍氏不动杆菌(基因组种2);贝氏不动杆菌;布氏不动杆菌;乙酸钙不动杆菌(基因组种1);格尔纳不动杆菌;格氏不动杆菌;溶血不动杆菌(基因组种4);约氏不动杆菌(基因组种7);琼氏不动杆菌(基因组种5);鲁氏不动杆菌(基因组种8/9);帕氏不动杆菌;抗辐射不动杆菌(基因组种12);申氏不动杆菌;潭氏不动杆菌;特氏不动杆菌;汤氏不动杆菌;邬氏不动杆菌;威尼斯不动杆菌;亚特兰大摩拉克氏菌;鲍氏摩拉克氏菌;牛摩拉克氏菌;牛眼摩拉克氏菌;犬摩拉克氏菌;山羊摩拉克氏菌;卡他摩拉克氏菌;豚鼠摩拉克氏菌;兔摩拉克氏菌;马摩拉克氏菌;结膜炎摩拉克氏菌;林肯摩拉克氏菌;猴摩拉克氏菌;不液化摩拉克氏菌;Moraxellaoblonga;奥斯陆摩拉克氏菌;羊摩拉克氏菌;苯丙酮酸摩拉克氏菌;多动物摩拉克氏菌;猪摩拉克氏菌--摩替亚氏菌科:-深渊摩替亚氏菌;茶山站摩替亚氏菌;日本摩替亚氏菌;海洋摩替亚氏菌;深海摩替亚氏菌;粘摩替亚氏菌;雅氏摩替亚氏菌--奈瑟氏菌科:-紫色色杆菌;啮蚀艾肯氏菌;反硝化金氏菌、金氏金氏菌、口金氏菌、波塔新金氏杆菌;灰色奈瑟氏菌;长奈瑟氏菌;浅黄奈瑟氏菌;淋病奈瑟氏菌;乳糖奈瑟氏菌;脑膜炎奈瑟氏菌;粘液奈瑟氏菌;多糖奈瑟氏菌;干燥奈瑟氏菌;微黄奈瑟氏菌;Neisseriaweaver;透明颤菌属物种--亚硝化单胞菌科:-真养亚硝化单胞菌;嗜盐亚硝化单胞菌;寡养亚硝化单胞菌--巴斯德氏菌科:-伴放线放线杆菌;马驹放线杆菌;李氏放线杆菌;大叶性肺炎放线杆菌;精液放线杆菌;产琥珀酸放线杆菌;脲放线杆菌;伴放线凝聚杆菌、惰性凝聚杆菌、嗜沫凝聚杆菌;鸟禽杆菌;禽心内膜炎杆菌;鸡禽杆菌;副鸡禽杆菌;敏捷禽杆菌;Bibersteiniatrehalose;鸭源鸡杆菌;鸡杆菌基因组种1;鸡杆菌基因组种2;鸡杆菌基因组种3;鸡杆菌群V;虎皮鹦鹉鸡杆菌;输卵管炎鸡杆菌;海藻糖发酵鸡杆菌;埃及嗜血杆菌;鸟嗜血杆菌;杜克雷嗜血杆菌;溶血嗜血杆菌;流感嗜血杆菌;副溶血嗜血杆菌;副流感嗜血杆菌;副猪嗜血杆菌;睡眠嗜组织菌;豚鼠曼海姆菌;葡糖苷曼海姆菌;肉芽肿曼海姆菌;溶血曼海姆菌;反刍兽曼海姆菌;Mannheimiavarigena;Nicoletellasemolina;产气巴斯德氏菌;贝氏巴斯德氏菌;马巴斯德氏菌;犬巴斯德氏菌;达可马巴斯德氏菌;多杀巴斯德氏菌(多杀亚种、败毒亚种、败血亚种);侵肺巴斯德氏菌;口巴斯德氏菌;海藻巴斯德氏菌--鱼立克次氏体科:-鲑鱼立克次氏体--邻单胞菌科:-类志贺邻单胞菌--多囊菌科:-纤维堆囊菌--紫单胞菌科:-Dysgonomonascapnocytophagoides;Dysgonomonasgadei;Dysgonomonashofstadii;Dysgonomonasmossii;Dysgonomonasoryzarvi;Dysgonomonaswimpennyi;牙龈红棕色单胞菌--普雷沃氏菌科:-普雷沃氏菌属物种,包括中间普雷沃氏菌、产黑色素普雷沃氏菌--假单胞菌科:-浅黄金色单胞菌;铜绿假单胞菌;浅黄假单胞菌;荧光假单胞菌;恶臭假单胞菌;施氏假单胞菌;栖稻假单胞菌--根瘤菌科:-根癌农杆;放射根瘤菌--立克次氏体科:-Orientiachuto;恙虫病东方体;埃氏立克次氏体;非洲立克次氏体;小蛛立克次氏体;Rickettsiaargasii;亚洲立克次氏体;澳大利亚立克次氏体;贝利立克次氏体;加拿大立克次氏体;康氏立克次氏体;Rickettsiacooleyi;猫立克次氏体;黑龙江立克次氏体;瑞士立克次氏体;Rickettsiahonei;胡氏立克次体;虎林立克次氏体;Rickettsiahulinii;日本立克次氏体;Rickettsiamarmionii;Rickettsiamartinet;马赛立克次氏体;摩纳立克次氏体;蒙大拿立克次氏体;Rickettsiamonteiroi;Rickettsiamoreli;帕氏立克次氏体;皮氏立克次氏体;Rickettsiaphilipii;普氏立克次氏体;劳氏立克次氏体;扁头蜱立克次氏体;立氏立克次氏体;西伯利亚立克次氏体亚群;斯洛伐克立克次氏体;田村氏立克次氏体;斑疹伤寒立克次氏体--希瓦氏菌科:-腐败希瓦氏菌--鞘脂单胞菌科:-多食鞘氨醇杆菌;食神鞘氨醇杆菌;少动鞘氨醇单胞菌--螺菌科:-减少螺菌;迂回螺菌;维氏螺菌--螺旋体科:-阿氏疏螺旋体;鹅疏螺旋体;比塞蒂疏螺旋体;博氏疏螺旋体;质革疏螺旋体;达氏疏螺旋体;嘎氏疏螺旋体;赫氏疏螺旋体;西班牙疏螺旋体;日本疏螺旋体;Borrelialonestari;卢西塔尼亚疏螺旋体;宫本疏螺旋体;扁虱疏螺旋体;波斯疏螺旋体;回归热疏螺旋体;斯氏疏螺旋体;特里蜱疏螺旋体;特里蜱疏螺旋体;法雷斯疏螺旋体;斑点病密螺旋体;苍白密螺旋体地方亚种;苍白密螺旋体苍白亚种;苍白密螺旋体细长亚种--琥珀酸弧菌科:-厌氧螺菌属物种--萨特氏菌科:-萨特氏菌属物种包括华德萨特氏菌--栖热菌科:-亚栖热菌属物种--栖热袍菌科:-新阿波罗栖热袍菌--韦荣氏球菌科:-戴阿利斯特杆菌属物种;巨单胞菌属物种;巨球形菌属物种;梳状菌属物种;Pelosinusspp;Propionisporaspp;鼠孢菌属物种;韦荣氏球菌属物种;嗜酵母菌属物种--弧菌科:-美人鱼发光杆菌;适应弧菌;解藻酸弧菌;Vibrioazasii;坎氏弧菌;霍乱弧菌;美人鱼弧菌;河流弧菌;弗尼斯弧菌;霍利斯弧菌;麦奇尼科夫氏弧菌;最小弧菌;副溶血弧菌;创伤弧菌--沃尔巴克氏体科:-沃尔巴克氏体属物种--黄单胞菌科:-Luteimonasaestuarii;水生藤黄色单胞菌;食物堆肥藤黄色单胞菌;Luteimonaslutimaris;海洋藤黄色单胞菌;怪味藤黄色单胞菌;Luteimonasvadosa;Pseudoxanthomonasbroegbernensis;日本假黄单胞菌;嗜麦芽糖寡养单胞菌和亚硝酸盐还原寡养单胞菌。
9.根据权利要求8所述的方法,其中所述菌剂选自:不动杆菌属物种、嗜水气单胞菌、柠檬酸杆菌属物种、肠杆菌属物种、大肠杆菌、肺炎克雷伯氏菌、摩氏摩根氏菌、铜绿假单胞菌和嗜麦芽糖寡养单胞菌。
10.根据前述权利要求中任一项所述的方法,其中所述氯苯胍与降低所述菌剂的细胞壁完整性的化合物一起施用。
11.根据权利要求1至3中任一项所述的方法,其中所述菌剂无细胞壁。
12.根据权利要求11所述的方法,其中所述菌剂选自:支原体属物种、无乳支原体、产碱支原体、Mycoplasmaamphoriforme、精氨酸支原体、牛生殖道支原体、牛鼻支原体、牛支原体、牛眼支原体、颊支原体、加利福尼亚支原体、加拿大支原体、山羊支原体山羊亚种、山羊支原体山羊肺炎亚种、结膜支原体、犬支原体、殊异支原体、马生殖道支原体、咽喉支原体、狸猫支原体、发酵支原体(incognitus株)、鸡败血支原体(MG)、猫支原体、生殖道支原体、犬嗜血支原体、猫嗜血支原体、猪嗜血支原体(过去称为猪附红血球体)、人型支原体、猪肺炎支原体、猪鼻支原体、猪关节液支原体、衣阿华火鸡支原体(MM)、衣阿华支原体、利氏支原体、嗜脂支原体、火鸡支原体、霉状支原体山羊亚种、霉状支原体霉状亚种、霉状支原体霉状亚种(例如牛传染性胸膜肺炎(CBPP))、口腔支原体、羊肺炎支原体、牛支原体、穿透支原体、梨支原体、肺炎支原体、灵长类支原体、腐败支原体、唾液支原体、嗜精子支原体、猪支原体、关节液支原体(MS)、温氏支原体、支原体、尿原体属物种、微小尿原体、解脲尿原体、尿原体和差异尿原体。
13.根据权利要求1至12中任一项所述的方法,其中所述菌剂为革兰氏阳性的、革兰氏阴性的或不具有细胞壁并选自但不限于以下牲畜病原体:猪放线杆菌、牛放线菌、化脓隐秘杆菌、炭疽芽孢杆菌、蜡状芽孢杆菌、地衣芽孢杆菌、短小芽孢杆菌、产黑色素芽孢杆菌、枯草芽孢杆菌、肉毒梭菌、气肿疽梭菌、溶血梭菌、诺氏梭菌、产气荚膜梭菌、败毒梭菌、索氏梭菌、破伤风梭菌、鹌鹑梭菌、假结核棒状杆菌、牛肾盂炎棒状杆菌、刚果嗜皮菌、肠球菌属物种(例如粪肠球菌、屎肠球菌、耐久肠球菌、鸟肠球菌、海氏肠球菌)、猪丹毒丝菌、伊万诺夫李斯特菌、格雷李斯特菌、无害李斯特菌、西尔李斯特菌、威尔逊李斯特菌、单核细胞增生李斯特菌、鸟分枝杆菌、牛分枝杆菌、类结核分枝杆菌(副结核性肠炎)、支原体属(例如山羊支原体山羊肺炎亚种、山羊支原体山羊亚种、霉状支原体霉状亚种、无乳支原体、羊肺炎支原体、结膜支原体、精氨酸支原体、牛支原体和腐败支原体)牛支原体、殊异支原体、霉状支原体霉状亚种(例如牛传染性胸膜肺炎(CBPP))鸡败血支原体(MG)、衣阿华火鸡支原体(MM)、关节液支原体(MS)猪嗜血支原体(过去称为猪附红血球体)、产碱支原体、牛生殖道支原体、牛鼻支原体、牛眼支原体、加利福尼亚支原体、加拿大支原体、犬支原体、马生殖道支原体、猫支原体、犬嗜血支原体、猫嗜血支原体、猪肺炎支原体、猪鼻支原体、猪关节液支原体、衣阿华支原体、利氏支原体、火鸡支原体、霉状支原体山羊亚种、温氏支原体、猪支原体、马红球菌、表皮葡萄球菌、模仿葡萄球菌、猫葡萄球菌、木糖葡萄球菌、产色葡萄球菌、沃氏葡萄球菌、溶血葡萄球菌、松鼠葡萄球菌、腐生葡萄球菌、人葡萄球菌、山羊葡萄球菌、科氏葡萄球菌科氏亚种、科氏葡萄球菌解脲亚种、头状葡萄球菌头状亚种、头状葡萄球菌解脲亚种、猪葡萄球菌、金黄色葡萄球菌、伪中间葡萄球菌、海豚葡萄球菌、施氏葡萄球菌凝结亚种、金黄色葡萄球菌厌氧亚种、乳房链球菌、犬链球菌、无乳链球菌、停乳链球菌、化脓链球菌、牛链球菌、马链球菌兽瘟亚种、马肠链球菌、马链球菌(马链球菌马亚种)、似马链球菌(停乳链球菌似马亚种)、豕链球菌、猪链球菌、兽瘟链球菌、兽瘟链球菌(马链球菌兽瘟亚种)、停乳链球菌似马亚种、疮疱丙酸杆菌、颗粒丙酸杆菌、真杆菌属、吲哚消化球菌和厌氧消化链球菌;放线杆菌属、气单胞菌属、无形体属、弓形菌属、禽杆菌属、类杆菌属、巴尔通氏体属、博德特氏杆菌属、包柔氏螺旋体属、短螺旋体属、布氏杆菌属、弯曲杆菌属、二氧化碳噬纤维菌属、衣原体属、嗜衣原体属、金黄杆菌属、考克斯氏体属、噬纤维菌属、偶蹄形菌属、爱德华氏菌属、埃利希氏体属、埃希氏菌属、黄杆菌属、弗朗西斯氏菌属、梭杆菌属、鸡杆菌属、嗜血杆菌属、嗜组织菌属、克雷伯氏菌属、指甲花属、钩端螺旋体属、曼氏杆菌属、巨球形菌属、摩拉克氏菌属、新立克次氏体属、鸟杆菌属、巴斯德氏菌属、发光杆菌属、鱼衣原体属、鱼立克次氏体属、红棕色单胞菌属、普雷沃氏菌属、变形杆菌属、假单胞菌属、立克次氏体属、立默氏菌属、沙门氏菌属、链杆菌属、黏着杆菌属、弧菌属和耶尔森氏菌属。
14.根据前述权利要求中任一项所述的方法,其中所述感染或定植由选自以下的至少两种菌剂的混合物导致:革兰氏阳性菌、革兰氏阴性菌和无细胞壁的细菌。
15.根据权利要求1至14中任一项所述的方法,其中所述菌剂具有对选自以下的一种或多种化合物的耐性:青霉素类、先锋霉素类、碳青霉烯类、单环内酰胺类及其它β-内酰胺抗生素、夫西地烷类、氨基糖苷类、氟喹诺酮类、链阳性菌素类、四环素类、甘氨酰环素类、氯霉素及其它苯丙醇类、大环内酯类和酮内酯类、林可酰胺类、噁唑烷酮类、氨基环醇类、多粘菌素类、糖肽类、脂肽类、杆菌肽、莫匹罗星、截短侧耳素类、利福霉素类、磺胺类以及三甲氧苄二氨嘧啶。
16.根据权利要求15所述的方法,其中所述菌剂具有对选自以下的一种或多种化合物的耐性:β-内酰胺类、糖肽类、脂肽类、大环内酯类、噁唑烷酮类和四环素类。
17.根据权利要求15或16所述的方法,其中当所述化合物在选自0.001μg/mL-10,000μg/mL、0.01μg/mL-1000μg/mL、0.10μg/mL-100μg/mL和1μg/mL-50μg/mL的浓度范围内时,所述菌剂具有对所述化合物的耐性。
18.根据前述权利要求中任一项所述的方法,其中所述受试者中的所述细菌感染或定植基本上导致选自以下的适应症:由金黄色葡萄球菌或肺炎链球菌导致的医院获得性肺炎;由金黄色葡萄球菌、化脓链球菌或无乳链球菌导致的复杂性皮肤和皮肤结构感染;侵袭性肺炎球菌疾病,包括肺炎、支气管炎、急性窦炎、中耳炎、结膜炎、脑膜炎、菌血症、败血病、骨髓炎、脓毒性关节炎、心内膜炎、腹膜炎、心包炎、蜂窝织炎以及由肺炎链球菌导致的脑脓肿;由金黄色葡萄球菌或化脓链球菌导致的单纯性皮肤和皮肤结构感染;由肺炎链球菌、金黄色葡萄球菌或金黄色葡萄球菌导致的社区获得性肺炎;由甲氧西林敏感和耐甲氧西林分离株导致的血流感染(菌血症);耐万古霉素肠球菌感染;艰难梭菌相关腹泻(CDAD);和革兰氏阴性、革兰氏阳性或混合菌骨和关节感染;中枢神经系统感染;眼部感染;胃肠道感染;生殖道感染;腹腔内感染;呼吸道感染;外耳炎;中耳炎;败血病;全身性感染;腹腔内感染(IAI);泌尿道感染(UTI);以及菌血症。
19.根据前述权利要求中任一项所述的方法,其中通过经口施用将治疗有效量的氯苯胍或其治疗学上可接受的盐施用给所述受试者。
20.根据权利要求1至18中任一项所述的方法,其中通过肠胃外施用将治疗有效量的氯苯胍或其治疗学上可接受的盐施用给所述受试者。
21.根据权利要求1至18中任一项所述的方法,其中通过局部施用将治疗有效量的氯苯胍或其治疗学上可接受的盐施用给所述受试者。
22.一种抗菌药物组合物,其包含治疗有效量的氯苯胍或其治疗学上可接受的盐,并任选地包含药学上可接受的赋形剂或载体。
23.一种抗菌兽药组合物,其包含治疗有效量的氯苯胍或其治疗学上可接受的盐,并任选地包含兽药学上可接受的赋形剂或载体。
24.根据权利要求22或权利要求23所述的组合物,其中所述组合物包含选自抗菌剂和抗真菌剂的另一种抗微生物剂。
25.根据权利要求22或权利要求23所述的组合物,其中所述组合物适于经口施用。
26.根据权利要求22或权利要求23所述的组合物,其中所述组合物适于肠胃外施用。
27.根据权利要求22或权利要求23所述的组合物,其中所述组合物适于局部施用。
28.氯苯胍或其治疗学上可接受的盐在制造用于治疗受试者中的细菌定植或感染的药物中的用途。
29.根据权利要求28所述的用途,其中所述用途包括将治疗有效量的氯苯胍或其治疗学上可接受的盐施用给所述受试者。
30.根据权利要求29所述的用途,其中将所述氯苯胍以0.1mg/kg至250mg/kg体重范围内的剂量施用给所述受试者。
31.根据权利要求28-30中任一项所述的用途,其中通过经口施用将所述药物施用给所述受试者。
32.根据权利要求28-30中任一项所述的用途,其中通过肠胃外施用将所述药物施用给所述受试者。
33.根据权利要求28-30中任一项所述的用途,其中通过局部施用将所述药物施用给所述受试者。
34.一种用于治疗或预防受试者中的细菌定植或感染时的医疗装置,其中所述医疗装置包含根据权利要求24至27中任一项所述的组合物。
35.根据权利要求34所述的医疗装置,其中所述医疗装置为选自以下的形式:应用于受试者中的细菌或定植感染的膏药、绷带、敷料或植入物。
36.一种杀灭细菌的方法,所述方法包括用氯苯胍或其治疗学上可接受的盐接触所述细菌的步骤。
37.氯苯胍或其治疗学上可接受的盐杀灭细菌的用途,所述用途包括用氯苯胍或其治疗学上可接受的盐接触所述细菌的步骤。
38.一种方法、组合物、装置或用途,基本上如本文参照所附实施例和附图所述。
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NZ713615A (en) | 2020-05-29 |
RU2015150263A3 (zh) | 2018-03-19 |
RU2015150263A (ru) | 2017-06-06 |
AU2014262127A1 (en) | 2015-11-12 |
BR112015027699B1 (pt) | 2021-11-30 |
EP2991638B1 (en) | 2019-06-26 |
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WO2014176634A1 (en) | 2014-11-06 |
CA2910752C (en) | 2021-11-23 |
US9539223B2 (en) | 2017-01-10 |
US20160058717A1 (en) | 2016-03-03 |
US9775818B2 (en) | 2017-10-03 |
CN112656782A (zh) | 2021-04-16 |
BR112015027699A2 (pt) | 2017-08-29 |
US20170071884A1 (en) | 2017-03-16 |
AU2014262127B2 (en) | 2019-05-02 |
EP2991638A1 (en) | 2016-03-09 |
EP2991638A4 (en) | 2016-12-28 |
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JP6254257B2 (ja) | 2017-12-27 |
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