CN114774321A - 一种棒状杆菌及其在眼表抗真菌感染中的应用 - Google Patents
一种棒状杆菌及其在眼表抗真菌感染中的应用 Download PDFInfo
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Abstract
本发明提供一种棒状杆菌及其在眼表抗真菌感染中的应用,所述的棒状杆菌,为麦利金氏棒杆菌(Corynebacterium macginleyi)C.mac2株,于2021年11月16日保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23924。本发明再一个方面是提供所述的棒杆菌的一种新用途,是棒杆菌在制备用于治疗不同个体眼表疾病中的制品中的应用。本发明再一个方面还提供一种能够眼表定植的制品,所述的制品用于预防治疗真菌感染导致的眼表疾病;其中包含有所述的棒杆菌及其产物。本发明所提供的棒状杆菌正常情况下不会诱发眼部感染,将该棒状杆菌定植于眼表能够增强眼表免疫,保护角膜抵抗真菌感染。
Description
技术领域
本发明属于眼科治疗制品技术领域,具体涉及一种棒状杆菌及其在抗眼表真菌感染中的应用。
技术背景
眼表是介于内眼组织和外界环境之间的屏障。从解剖学上讲,眼表指起始于上、下睑缘灰线之间的全部黏膜上皮,包括角膜上皮和结膜上皮以及覆盖于眼球表面的泪膜。眼表组织易受到外界环境中的各种刺激而发生异常,眼表组织的稳定是维护眼内健康和清晰视觉的重要条件。角膜是眼表的重要组成部分,对维持正常眼表微环境的至关重要。
中国角膜失明和低视力的患病率为每万人中约21人,角膜失明约占致盲性眼病的四分之一,感染性角膜病又是角膜失明的主要原因。流行病学数据表明,微生物角膜炎在南亚、东南亚和东亚地区发病率更高,全世界每年病例数超过200万例。我国作为一个以农业人口众多的发展中国家,角膜植物外伤发生较多,同时由于抗生素、激素以及免疫抑制剂的不适当应用,导致真菌感染已成为感染性角膜炎的首位致病因素。
目前抗真菌药物被广泛应用于真菌性角膜炎的治疗中,但长期或不适当使用抗真菌药会导致免疫失调和组织损伤。另一方面真菌性角膜炎的误诊误治又提高了抗生素的滥用现象,导致耐药菌株出现。因此面对真菌性角膜炎疾病的治疗难题,需要需要寻找新的替代疗法。鉴于此,特提出本发明。
发明内容
本发明的目的是提供一种棒状杆菌及其在眼表抗真菌感染中的应用,从而克服现有的治疗方法中药物过量使用和产生耐药菌的缺陷。
本发明首先提供一种棒状杆菌,为麦利金氏棒杆菌(Corynebacteriummacginleyi)C.mac2株,于2021年11月16日保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23924。
本发明再一个方面是提供所述的棒杆菌的一种新用途,是棒杆菌在制备用于治疗不同个体眼表疾病中的制品中的应用;
所述的制品,为眼表定植制品。
所述的眼表疾病为真菌感染导致的疾病;
所述的真菌,作为实施例的具体记载,为镰刀菌;
本发明再一个方面还提供一种能够眼表定植的制品,所述的制品用于预防治疗真菌感染导致的眼表疾病;其中包含有所述的棒杆菌的活菌。
本发明所提供的棒状杆菌正常情况下不会诱发眼部感染,将该棒状杆菌定植于眼表能够增强眼表免疫,保护角膜抵抗真菌感染。
附图说明
图1:筛选的C.mac2株的菌落培养照片图;
图2:C.mac2株定植效果检测图,其中图2A为正常小鼠(WT)和同批次定植C.mac2正常小鼠(WT+C)结膜囊C.mac2含量检测图,结果表明定植组的菌落形成单位(CFU)比非定制组高66倍。
图2B为糖尿病小鼠(D)和同批次定植C.mac2糖尿病小鼠(D+C)结膜囊C.mac2浓度检测图,结果表明定植组的菌落形成单位(CFU)比非定制组高390倍。
图3:定植C.mac2后对上皮损伤修复的影响图,其中图3A为正常小鼠(WT)和同批次定植C.mac2正常小鼠(WT+C)角膜损伤修复的荧光素钠染色图,结果表明定植组上皮损伤面积明显小于非定植组。
图3B为糖尿病小鼠(D)和同批次定植C.mac2糖尿病小鼠(D+C)角膜损伤修复的荧光素钠染色图,结果表明定植组上皮损伤面积明显小于非定植组。
图4:小鼠角膜镰刀菌感染后的眼表情况照片图,其中图4A为正常小鼠(WT)和同批次定植C.mac2正常小鼠(WT+C)角膜的裂隙灯眼表照相,定植组镰刀菌感染情况明显轻于非定植组。
图4B为糖尿病小鼠(D)和同批次定植C.mac2糖尿病小鼠(D+C)角膜的裂隙灯眼表照相,定植组镰刀菌感染情况明显轻于非定植组。
图5:小鼠角膜镰刀菌感染后的角膜感染密度比较图,其中图5A为正常小鼠(WT)和同批次定植C.mac2正常小鼠(WT+C)角膜感染深度比较,定植组小鼠角膜感染镰刀菌的密度显著低于非定制组。
图5B为糖尿病小鼠(D)和同批次定植C.mac2糖尿病小鼠(D+C)角膜感染深度比较,定植组小鼠角膜感染镰刀菌的密度显著低于非定制组。
图6:小鼠角膜镰刀菌感染后的角膜感染面积比较图,其中图6A为正常小鼠(WT)和同批次定植C.mac2正常小鼠(WT+C)角膜感染面积比较图,结果显示定植组小鼠角膜感染镰刀菌的面积显著低于非定植组。
图6B:糖尿病小鼠(D)和同批次定植C.mac2糖尿病小鼠(D+C)角膜感染面积比较,定植组小鼠角膜感染镰刀菌的面积显著低于非定植组。
图7:C.mac2定植安全性评价图,野生型小鼠和糖尿病小鼠结膜囊定植C.mac2后追踪观察到60天眼部均未出现任何异常表现。
图8:棒状杆菌结膜囊定植抵抗镰刀菌感染的示意图。
具体实施方式
下面结合实施例和附图对本发明进行详细的描述。
实施例1:菌株分离及检测
2021年在山东第一医科大学附属眼科研究所,根据生物取样的伦理规定,从角膜屈光科就诊健康人结膜囊中分离获得了目的菌株。
具体的筛选分离步骤如下:
使用一次性无菌棉签轻轻擦拭健康人的结膜囊,立即对棉签进行无菌封存,在经紫外消毒过的微生物操作台中,将棉签放入LB肉汤中浸润搅拌,随后放入37℃恒温箱中培养,每日观察一次,待肉汤浑浊后,在无菌的条件下,用一次性接菌环蘸取肉汤菌液,均匀涂布到血琼脂平板,放入37℃恒温培养箱中进行培养。待有单克隆菌落形成后,用一次性接菌环挑取单克隆均匀涂布至滴有生理盐水的载玻片上,酒精灯烘干,进行革兰氏染色,结果呈现革兰氏染色阳性,镜下可观测到两端膨大着色较深的微生物。对菌株进行16S rDNA测序,菌株的鉴定结果如表1所示。
表1:筛选的微生物菌种鉴定结果报告表
对阳性菌株通过16SrRNA序列分析,具体步骤如下:
1)基因组DNA提取:使用擎科直扩试剂盒,裂解液裂解出DNA,具体方法参考使用说明书。
2)PCR扩增:PCR扩增采用50μL的体系,其中包括:1×T3 Mix 46μL,上、下游引物(10μmol/L)各1.0μL,DNA模板2.0μL。PCR反应扩增程序为:98℃预变性3min;98℃变性10S,55℃退火15S,72℃延伸20S,35个循环;72℃延伸2min,4℃保存。
3)PCR产物切胶回收后,进行测序,测得的序列如下:
GGGGGGGGCGTGCTTACCTGCAGTCGAACGGAAAGGCCCTGCTTGCGGGGTACTCGAGTGGCGAACGGGTGAGTAACACGTGGGTGATCTGCCCTGCACTTCGGGATAAGCTTGGGAAACTGGGTCTAATACCGGATAGGACCGCACTTTAGTGTGTGTGGTGGAAAGTTTTTTCGGTGTAGGATGAGCTCGCGGCCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGGCGACGGGTAGCCGGCCTGAGAGGGTGTACGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGGGGGATGAAGGCCTTCGGGTTGTAAACTCCTTTCGCTAGGGACGAAGCTTTTGTGACGGTACCTAGATAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGAATTACTGGGCGTAAAGGGCTCGTAGGTGGTTTGTCGCGTCGTCTGTGAAATTCTGGGGCTTAACTCCGGGCGTGCAGGCGATACGGGCATAACTTGAGTGCTGTAGGGGTAACTGGAATTCCTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGATGGCGAAGGCAGGTTACTGGGCAGTTACTGACGCTGAGGAGCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCTGTAAACGGTGGGCGCTAGGTGTGAGGGTTTTTCTACGATTCTCGTGCCGTAGCTAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGCTTGACATACACGAGATCGCTGCAGAGATGTAGTTTCCCTTTTGTGGTTGGTGTACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTATGTTGCCAGCATTTGGTTGGGGACTCATGAGAGACTGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGTCGGTACAACGCGCCTGCGACACTGTGAGGTGGAGCGAATCGCTGAAAGCCGGTCTTAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAATGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTTGGTAACACCCGAAGCCGGTGGCCTAAACGTGTTAGGGAGCCGTCGAAGGTGGGATTGGCGATGGGACGAAGTCGAACAAGATCCGGG(SEQ ID NO:1)。
经过BLAST比对,该序列与棒杆菌属(Corynebacterium sp.)亲缘关系最接近,命名为麦利金氏棒杆菌(Corynebacterium macginleyi)C.mac2株,于2021年11月16日保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23924。
菌株C.mac2在血琼脂平板中培养,菌落形状呈圆形、色灰白、光滑湿润的小菌落。
革兰染色阳性,菌体粗细不一,常一端或两端膨大呈棒状,排列不规则,呈栅栏状,无荚膜,无鞭毛。染色不均匀,两端有着色较深的异染颗粒。无芽孢,大多数菌株无动力,需氧或兼性厌氧。营养要求特殊,在普通培养基虽能生长,但菌体形态不典型,在血琼脂平板上生长良好。DNA中的G+C克分子含量为52-68%。胞壁多糖主要是阿拉伯糖和半乳糖。与分枝杆菌属、奴卡氏菌属和放线菌属相似,有交叉反应。
实施例2:C.mac2株在眼表定植的过程和效果
将棒状杆菌C.mac2株的活菌制剂、棒状杆菌灭活制剂、相关单一或混合细菌组成成分、以及细菌衍生产物进行眼表致病菌株的防治效果检测。
通过眼表定植的方式,将浓度为108cfu/mL的棒状杆菌C.mac2株活菌制剂滴注到野生型C57BL/C小鼠眼表,共计定植9天,定植完成后,对定植成功率进行评估。野生型C57BL/C小鼠定植C.mac2 2周后PCR检测定植效果如图2A所示,糖尿病C57BL/C小鼠定植C.mac2 2周后PCR检测定植效果如图2B所示。
实施例3:C.mac2株对小鼠眼表损伤修复的促进作用
在野生型C57BL/C小鼠(WT组)、定植C.mac2野生型小鼠(WT+C组)、糖尿病C57BL/C小鼠(D组)以及定植C.mac2糖尿病小鼠(D+C组)进行上皮愈合实验,小鼠在水合氯醛麻醉的状态下刮除角膜上皮中央2.5mm直径的上皮层,随后在0小时、12小时、24小时、36小时等时间点对小鼠眼表荧光素钠着色情况拍摄裂隙灯大体照,结果如图3所示,发现经过定植C.mac2野生型小鼠(WT+C组)和糖尿病小鼠(D+C组)相比各自的非定植组具有更快的上皮愈合速度。
实施例4:C.mac2株对眼表镰刀菌感染的防治效果
在将C.mac2株定植到小鼠眼表一个月后,对预使用活菌制剂糖尿病小鼠(D+C组)与对照组糖尿病小鼠(D组)一同进行眼表感染茄病镰刀杆菌操作,镰刀菌感染菌液的浓度为106CFU/mL。术后对四组小鼠进行临床病理学评分,具体包括病灶密度和病灶面积两个方面。在D组、D+C组、WT组、WT+C组中进行茄病镰刀菌感染实验,每组小鼠均在水合氯醛麻醉的状态下刮除角膜上皮中央2.5mm直径的上皮层,将直径3mm圆形浸有茄病镰刀菌悬液(106CFU/mL)的封口膜滤纸片覆盖在小鼠角膜表面,6-0缝合线缝合小鼠眼睑,24小时后拆除缝合线,每天裂隙灯拍照并从病灶密度和面积方面分别进行评分,以WT、D组为基准,绘制WT+C、D+C两组的相对倍数改变。从图4大体照可以看出,预先定植了C.mac2的WT+C组和D+C组的感染情况比未定植组更轻。病灶密度以大体照灰度值定量计算,如图5所示,WT+C、D+C两组比非定植组病灶密度显著降低。病灶面积以大体照中感染面积计算,如图6所示,WT+C、D+C两组比非定植组病灶面积显著减小。
上述结果表明预先在糖尿病小鼠眼滴注本发明筛选的棒状杆菌活菌制剂可以增强小鼠眼表免疫力,减轻茄病镰刀杆菌对角膜的浸润,一定程度上保护了小鼠角膜。同时获得更低的临床病理评分和更好的临床预后,棒状杆菌活菌制剂对糖尿病小鼠在应对角膜感染时起保护作用。
实施例5:定植安全性评价
将C57BL/C小鼠使用水合氯醛腹腔麻醉后,一次性干棉签擦拭小鼠泪膜,5μL106CFU/mL C.mac2悬液滴注到小鼠眼表,每3天一个周期,每周期内仅第一天第一次定植进行麻醉,连续共接种9天,在接种后的第1天、3天、5天、7天、14天、21天、2个月随机抽取各组中三只小鼠进行拍照,持续观察发现,角膜清亮透明,未见感染迹象(图7)。结果表明本发明筛选的菌株对眼表没有致病性。
序列表
<110> 山东第一医科大学附属眼科研究所(山东省眼科研究所、山东第一医科大学附属青岛眼科医院)
<120> 一种棒状杆菌及其在眼表抗真菌感染中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1448
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggggggggcg tgcttacctg cagtcgaacg gaaaggccct gcttgcgggg tactcgagtg 60
gcgaacgggt gagtaacacg tgggtgatct gccctgcact tcgggataag cttgggaaac 120
tgggtctaat accggatagg accgcacttt agtgtgtgtg gtggaaagtt ttttcggtgt 180
aggatgagct cgcggcctat cagcttgttg gtggggtaat ggcctaccaa ggcggcgacg 240
ggtagccggc ctgagagggt gtacggccac attgggactg agatacggcc cagactccta 300
cgggaggcag cagtggggaa tattgcacaa tgggcgcaag cctgatgcag cgacgccgcg 360
tgggggatga aggccttcgg gttgtaaact cctttcgcta gggacgaagc ttttgtgacg 420
gtacctagat aagaagcacc ggctaactac gtgccagcag ccgcggtaat acgtagggtg 480
cgagcgttgt ccggaattac tgggcgtaaa gggctcgtag gtggtttgtc gcgtcgtctg 540
tgaaattctg gggcttaact ccgggcgtgc aggcgatacg ggcataactt gagtgctgta 600
ggggtaactg gaattcctgg tgtagcggtg aaatgcgcag atatcaggag gaacaccgat 660
ggcgaaggca ggttactggg cagttactga cgctgaggag cgaaagcatg ggtagcgaac 720
aggattagat accctggtag tccatgctgt aaacggtggg cgctaggtgt gagggttttt 780
ctacgattct cgtgccgtag ctaacgcatt aagcgccccg cctggggagt acggccgcaa 840
ggctaaaact caaaggaatt gacgggggcc cgcacaagcg gcggagcatg tggattaatt 900
cgatgcaacg cgaagaacct tacctgggct tgacatacac gagatcgctg cagagatgta 960
gtttcccttt tgtggttggt gtacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1020
atgttgggtt aagtcccgca acgagcgcaa cccttgtctt atgttgccag catttggttg 1080
gggactcatg agagactgcc ggggttaact cggaggaagg tggggatgac gtcaaatcat 1140
catgcccctt atgtccaggg cttcacacat gctacaatgg tcggtacaac gcgcctgcga 1200
cactgtgagg tggagcgaat cgctgaaagc cggtcttagt tcggattggg gtctgcaact 1260
cgaccccatg aagtcggagt cgctagtaat cgcagatcag caatgctgcg gtgaatacgt 1320
tcccgggcct tgtacacacc gcccgtcacg tcatgaaagt tggtaacacc cgaagccggt 1380
ggcctaaacg tgttagggag ccgtcgaagg tgggattggc gatgggacga agtcgaacaa 1440
gatccggg 1448
Claims (7)
1.一种棒状杆菌,其特征在于,所述的棒状杆菌为麦利金氏棒杆菌,其保藏编号为CGMCC No.23924。
2.如权利要求1所述的棒杆菌,其特征在于,所述的棒状杆菌的16SrRNA序列为SEQ IDNO:1。
3.权利要求1所述的棒杆菌在制备用于治疗不同个体眼表疾病中的制品中的应用。
4.如权利要求3所述的应用,其特征在于,所述的制品为眼表定植微生物用制品。
5.如权利要求3所述的应用,其特征在于,所述的眼表疾病为真菌感染导致的疾病。
6.如权利要求5所述的应用,其特征在于,所述的真菌为镰刀菌。
7.一种用于眼表定植的制品,其特征在于,所述的制品用于预防治疗真菌感染导致的眼表疾病;其中包含有权利要求1所述的棒杆菌或其产物。
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Citations (4)
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WO2005054454A1 (fr) * | 2003-11-04 | 2005-06-16 | Universite De La Mediterranne (Aix-Marseille Ii) | Identification moleculaire des bacteries du genre corynebacterium |
CN111675635A (zh) * | 2013-05-01 | 2020-09-18 | 尼奥酷里私人有限公司 | 治疗感染的化合物和方法 |
CN112656782A (zh) * | 2013-05-01 | 2021-04-16 | 尼奥酷里私人有限公司 | 治疗细菌感染的方法 |
KR102334455B1 (ko) * | 2020-05-29 | 2021-12-03 | 코스맥스 주식회사 | 코리네박테리움 맥진레이 균주 및 그의 피부 상태 개선 용도 |
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WO2005054454A1 (fr) * | 2003-11-04 | 2005-06-16 | Universite De La Mediterranne (Aix-Marseille Ii) | Identification moleculaire des bacteries du genre corynebacterium |
CN111675635A (zh) * | 2013-05-01 | 2020-09-18 | 尼奥酷里私人有限公司 | 治疗感染的化合物和方法 |
CN112656782A (zh) * | 2013-05-01 | 2021-04-16 | 尼奥酷里私人有限公司 | 治疗细菌感染的方法 |
KR102334455B1 (ko) * | 2020-05-29 | 2021-12-03 | 코스맥스 주식회사 | 코리네박테리움 맥진레이 균주 및 그의 피부 상태 개선 용도 |
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