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颗粒链球菌属的研究进展

2011-08-15曹阳佩综述周学东施文元审校

华西口腔医学杂志 2011年6期
关键词:心内膜炎链球菌球菌

曹阳佩综述 周学东 施文元审校

颗粒链球菌属的研究进展

曹阳佩综述 周学东 施文元审校

(口腔疾病研究国家重点实验室,四川大学,成都 610041)

颗粒链球菌属是兼性厌氧、触酶阴性的革兰阳性球菌,口腔微生物组研究发现颗粒链球菌属是人口腔的优势菌,可引起牙周病、牙髓炎等机会感染。本文重点介绍颗粒链球菌属的最新研究进展。

颗粒链球菌属; 营养变异链球菌; 口腔; 生物学特征; 鉴定; 感染

口腔微生物组研究发现颗粒链球菌属(Granulicatella)是人口腔的优势菌,在口腔中多个部位检出,如唇前庭、软腭、硬腭、牙周袋[1]、颊上皮、舌侧面、扁桃体[2]、唾液[3-4]、舌背部[5-7]、龈下菌斑[8-11]等处。颗粒链球菌属可引起口腔、感染性心内膜炎等机会感染。目前国内相关介绍较少[12-15],本文对颗粒链球菌的研究现状介绍如下。

1 分类学

颗粒链球菌属是兼性厌氧、触酶阴性的革兰阳性球菌,Frenkel等[16]在1961年首次于亚急性细菌性心内膜炎和中耳炎患者的血培养分离得到。颗粒链球菌属细菌营养要求十分苛刻,需要补充一些巯基化合物以满足其生长要求或与其他细菌共生形成典型的卫星菌落现象。颗粒链球菌早期被称为营养变异链球菌(nutritionally variant streptococci,NVS)[17]。Bouvet等[18]通过DNA-DNA杂交证实了这些细菌与链球菌属的低基因相关度,并将NVS命名为缺陷链球菌(Streptococcus defectivus)和毗邻链球菌(Streptococcus adjacens)。Kawamura等[19]通过16S rRNA序列研究,认为NVS与链球菌属在系统发生学上无亲缘关系,提出其应为新的菌属,将其命名为乏养球菌属(Abiotrophia),并将缺陷链球菌和毗邻链球菌移入乏养球菌属,即缺陷乏养球菌(Abiotrophia defective)和毗邻乏养球菌(Abiotrophia adiacens)。此后,乏养球菌属又添加了3个新种,包括由心内膜炎患者血液中分离得到的苛求乏养球菌(Abiotrophia elegans)[20]、副毗邻乏养球菌(Abiotrophia para-adiacens)[21]、貂鲸乏养球菌(Abiotrophia balaenopterae)[22]。2000年Collins等[23]的16S rRNA测序结果表明:系统发生学上乏养球菌属由2个不同的谱系组成,一是模式株缺陷乏养球菌,另一个是由毗邻乏养球菌、苛求乏养球菌、副毗邻乏养球菌、貂鲸乏养球菌组成的相似菌群,因此,提出后4种应纳入新的菌属——颗粒链球菌属,包括毗邻颗粒链球菌(Granulicatella adiacens)、苛求颗粒链球菌(Granulicatella elegans)、副毗邻颗粒链球菌(Granulicatella para-adiacens)、貂鲸颗粒链球菌(Granulicatella balaenopterae)。

2 生物学特征

2.1 细胞形态和染色性

颗粒链球菌属细菌细胞是无动力、不形成芽胞的革兰阳性球菌,球状细胞可呈肿胀样,其革兰染色结果也往往呈现可变性。其排列呈多形性,成对、短链状或短杆状。细胞形态的多形性与其营养状态相关,在营养状况良好的条件下,可观察到革兰阳性的球菌,成对或短链状排列。因此,菌株形态上的不典型性使得根据形态鉴定十分困难[24]。

2.2 培养特性

颗粒链球菌属为兼性厌氧菌,10℃和45℃不生长,在6.5%NaCl肉汤中不生长。颗粒链球菌属对营养要求较高,常要在培养基中加入0.01%L-半胱氨酸或0.001%盐酸吡哆醛。对奥普托欣(Optochin)耐药,对万古霉素敏感。颗粒链球菌属在补充盐酸吡哆醛或L-半胱氨酸的5%或10%丹麦血平板(danish blood agar plate,DBA)或5%绵羊血平板(sheep blood agar plate,SBA)上生长。在10%DBA上培养48 h后,可观察到直径约1~3mm的灰白色菌落[25]。颗粒链球菌属细菌可在许多细菌周围呈现“卫星现象”生长,是一种典型的共生菌,这些细菌可以提供其生长需要的盐酸吡哆醛,被称为“助养细菌”。最早报道的“助养细菌”为嗜血杆菌属,在共同培育48 h后,在其接种划线区边缘可观察到菌落增大的现象,其他助养菌还包括葡萄球菌属、链球菌属、肠杆菌属及酵母菌[16,26-27]。

2.3 生化特性

颗粒链球菌属细菌触酶和氧化酶阴性,发酵葡萄糖的代谢产物为乳酸,不产气。毗咯芳氨酸酶和亮氨酸芳胺酶阳性,碱性磷酸酶、α-半乳糖苷酶、β-半乳糖苷酶阴性。

3 鉴定

3.1 表型鉴定

颗粒链球菌属菌株主要分离自血培养,其形态、染色不仅具有多形性,随着培养时间的延长,初代分离培养基的组成变化很大,故对其生长特性的描述也在不断变化。“卫星现象”和“吡哆醛依赖试验”可以辅助鉴定,但一些分离株还会出现“适应现象”,在未补充盐酸吡哆醛的培养基上可以生长良好,也不出现“卫星现象”,给分离鉴定造成更大困难。对鉴定颗粒链球菌属较为有价值的生物学特性包括是否产生α-半乳糖苷酶、β-半乳糖苷酶、β-葡糖苷酸酶,马尿酸盐水解,精氨酸水解,海藻糖、蔗糖、支链淀粉、塔格糖产酸等[25]。乏养球菌属和颗粒链球菌属的初步鉴定可采用“卫星现象”结合Rapid ID 32链球菌鉴定系统[28-29]。

3.2 分子生物学鉴定

分子生物学鉴定方法是最好的鉴定方法[30],以聚合酶链反应(polymerase chain reaction,PCR)为基础的细菌核糖体基因(rRNA)检测分析是金标准,通常进行16S rRNA基因序列分析[31]。除16S rRNA分析技术外,其他基于分子生物学技术的鉴定方法发展迅速。Drancourt等[32]采用了基于rpo B基因序列的鉴定方法以区分革兰阳性厌氧球菌中的链球菌属、肠球菌属、孪生球菌属、乏养球菌属和颗粒链球菌属。基于16S~23S核糖体DNA基因间隔区(intergenic spacer,ITS)的鉴定技术是新兴的菌种鉴定方法,由于ITS序列间具有较低的种群内差异性和较高的种群间差异性[33],鉴定结果准确可靠[34]。Tung等[35]采用ITS技术鉴定21种链球菌,1种乏养球菌,18种肠球菌,3种颗粒链球菌,正确率为98.2%(213/217),提示ITS可能成为受广泛认可的分子生物学鉴定技术。此外,ITS序列长度(189~601 bp)相比16S rRNA基因序列长度(约1.5 kb)更短,ITS序列测序会比目前普遍采用的16S rRNA测序更为准确有效,公共数据库中的ITS序列信息也在不断完善[36]。基于ITS发展的寡核苷酸微阵列(oligonucleotide microarray)技术鉴定菌种的结果也令人满意,Chen等[37]报道鉴定结果的敏感性和特异性分别为100%(312/312)和98.6%(72/73)。Tung等[29]报道结果的敏感性和特异性分别为100%(120/120)和95.6%(87/91)。以上优势提示ITS可能成为受广泛认可的分子生物学鉴定技术,甚至取代16S rRNA技术。

4 病原学

随着研究进一步深入,颗粒链球菌属所导致的疾病日益引起人们的重视。Christensen等[25]从97例患者中分离得到的乏养球菌和颗粒链球菌属主要来自血标本,但有3例来自眼部溃疡,58%患者诊断患有感染性心内膜炎,26%患者诊断患有菌血症或败血症,提示该类菌与感染关系密切。

4.1 口腔疾病

国外学者已有报道颗粒链球菌属与口腔疾病的密切关系,国内尚未见相关报道。

4.1.1 牙周炎 颗粒链球菌属被认为是可疑的牙周致病菌。16S rRNA基因序列技术已证实其在牙周病患者中的高检出率[8],在成人牙周炎患者[8-9,11,38]、坏死性溃疡性牙周炎患者[10]、复发性牙周炎患者所提取的样本中均检出了毗邻颗粒链球菌。在早期牙周炎患者的舌部样本中,毗邻颗粒链球菌的检出率更高,并具有相关性(P<0.05)[5]。最近,Colombo等[39]发现复发性牙周炎患者与单纯性牙周炎患者和牙周健康者相比,毗邻颗粒链球菌检出率更高(P<0.05)。以上报道提示颗粒链球菌属可能是牙周病的致病菌之一。

4.1.2 牙髓炎 在原发性牙髓感染和持续性牙髓感染的病例中均检出毗邻颗粒链球菌[38,40-41]。Siqueira等[42]发现毗邻颗粒链球菌在原发性牙髓感染样本中有14%的检出率。Rôças等[43]报道毗邻颗粒链球菌在有症状的牙髓感染中有较高的检出率(10%)。以上报道提示毗邻颗粒链球菌可能是牙髓感染的致病菌之一,其致病机制尚不清楚。

4.1.3 龋病 毗邻颗粒链球菌在儿童、成人龋病患者的样本中也有检出[44-45]。Ling等[46]报道颗粒链球菌属与龋病有明显的相关性(P<0.05)。Kanasi等[47]发现苛求颗粒链球菌与严重早期儿童龋有相关性(P<0.01)。以上报道提示颗粒链球菌属可能与龋病发病相关,尤其是早期儿童龋。

4.1.4 口臭 在大多数健康人和口臭患者的舌背部拭子中,毗邻颗粒链球菌都能被检测到[7]。Haraszthy等[6]检测了8个口臭患者和5个无口臭健康人的舌背部样本,毗邻颗粒链球菌在所有口臭患者中约有2%检出率,苛求颗粒链球菌仅能在口臭患者中检出(0.6%)。

4.2 感染性心内膜炎

关于颗粒链球菌引起感染性心内膜炎的机制尚不清楚。普遍认为心脏的病理情况是导致感染的发病因素之一,而口腔健康不佳、过去6个月内的牙科操作可能促进感染性心内膜炎的发生[48]。颗粒链球菌属所致的感染性心内膜炎占链球菌感染的4.3%~6%[49]。研究者们认为由于这类细菌对营养的特殊要求,可能导致在心内膜炎病例的病原学检测中常出现假阴性结果[17]。

分析30例NVS导致的感染性心内膜炎病例,颗粒链球菌感染的病情恶化常比肠球菌或草绿色链球菌更为严重,治疗更为困难。尽管给予了合适的抗生素治疗,但其复发率仍达41%[50]。Senn等[51]指出在老年患者中,颗粒链球菌属的致病性和死亡率更高,其生长特性使得临床分离鉴定较困难,应引起临床工作者的高度重视。

4.3 其他感染

近年来,颗粒链球菌属所引起的感染病例在国外多有报道,国内仅报道了1例[52]。除去上述提到的口腔疾病、感染性心内膜炎外,还涉及了多种感染,包括菌血症[25,53]、腹膜透析相关性腹膜炎[54]、感染性颅内动脉瘤[55]、化脓性关节炎[56]、脊椎骨髓炎[57-58]、关节盘炎[58]、乳房假体移植术后感染[59]、全膝关节成型术后感染[60]、脑脓肿[61]、中枢神经系统感染[62]、脑膜炎[63]、结膜炎[64]、上颌窦炎[25,28,65]、尿道感染[28]、股动脉血栓[48]。

5 治疗

临床发现颗粒链球菌属细菌所致感染治疗较为困难[49],可能是因为其对一些抗生素耐药。另外,传统的表型鉴定对此类细菌的低检出率也增加了诊断及治疗的难度。对于颗粒链球菌属所致的感染性心内膜炎,推荐的治疗方案是4~6周的青霉素G或氨苄青霉素联合庆大霉素治疗。仅当患者不能耐受青霉素G或氨苄青霉素时,才选用万古霉素。选用万古霉素时,可不联合庆大霉素进行治疗[66]。对此类细菌药物敏感性测试操作技术要求高,结果往往也不够准确,相应报道[67-70]往往也缺乏一致性。美国临床和实验室标准协会(Clinical and Laboratory Standards Institute,CLSI)推荐采用微量肉汤稀释法进行药敏试验:Mueller-Hinton肉汤添加2.5%~5%溶解的马血和0.001%盐酸吡哆醛[71]。CLSI指南提出,临床工作者首先应按照指南推荐方案治疗颗粒链球菌所致感染性心内膜炎病理,只有特殊病例需要时才进行药敏测试。

6 展望

目前有关颗粒链球菌属感染病例的报道日益增多,未来研究热点应着重于细菌的致病机制和其在生物膜中的作用,为临床的预防和治疗提供良好的依据。

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New progress in research of Granulicatella species

Cao Yangpei,Zhou Xuedong,Shi Wenyuan.(State Key Laboratory of Oral Diseases,Sichuan University,Chengdu610041,China)

Granulicatella species are facultative anaerobic,catalase-negative Gram-positive cocci,oral microbiome researches find out Granulicatella species are dominant bacteria in oral cavity which may cause opportunistic infection like periodontal disease,endodontic infection.This review summarized research progress of Granulicatella species.

Granulicatella species;nutritionally variant streptococci; oral cavity; biological property; identification;infection

R 780.2

A

10.3969/j.issn.1000-1182.2011.06.027

1000-1182(2011)06-0665-05

2011-07-29;

2011-09-23

曹阳佩(1988—),女,四川人,硕士

周学东,Tel:028-85501439

(本文编辑 胡兴戎)

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