草綠色鏈球菌之分子鑑定暨青黴素抗藥分析

草綠色鏈球菌viridans group streptococci(VGS) 是一群革蘭氏陽性兼性厭氧球菌(facultatively anaerobic gram-positive cocci)，在含血瓊脂培養基上呈現草綠色的不完全溶血而得名。草綠色鏈球菌為人體口腔內的一群正常菌叢(normal flora)，因此又稱之為口腔鏈球菌(oral streptococci)。此外，也存在於人體的上呼吸道、腸胃道以及泌尿生殖道等等。而在臨床上VGS所引起的重要感染則包含了感染性心內膜炎、腦膜炎、化膿性感染等疾病，在免疫不全的病人甚至會造成死亡，而且不同的 species常引起不同的感染，其抗藥性與治療方式也不盡相同，因此正確的將VGS鑑定到species level就顯得具有其意義與重要性。 VGS的命名與分類，目前是以16S rDNA的序列來當作為黃金準則(Gold standard)，並將VGS分為五個group，包括了anginosus group、mitis group、sanguinis group、mutans group以及salivarius group，另外並包含了二十幾個species。然而VGS菌種，是一群在演化上具有基因高度相似性特質的菌叢，其分類及命名在過去一直時常改變也十分紊亂，如何將其正確的鑑別出來，就成為一項重要而困難的工作。而目前一般臨床實驗室所使用的傳統及生化鑑定方法，在鑑定VGS時其結果也常常無法令人滿意，於是以分子鑑定為方向，來進一步的鑑定VGS的方法就成為一股研究的主流。此外，目前已有研究顯示16S rDNA因其高度的保守性而無法成為在VGS鑑定上十分良好的target，尤其是在VGS的anginosus、mitis group。因此我們選擇了groESL gene，這個普遍存在於細菌間，在演化上具有適度保守性卻兼具部份差異性特質的基因來作為VGS分類及鑑定的標的。 groESL operon包含了groES、groEL兩個基因，其所轉譯之蛋白質則包含了GroES與GroEL。GroESL是一種熱刺激蛋白，普通存在於原核及真核細胞，主要的功能為幫助細菌恢復在遭受外在環境壓力時所造成的蛋白質變性及參與細菌體中新合成的多胜肽的摺疊，自西元1989年以來，已有許多研究以GroESL為鑑定標的，成功的鑑定了分枝桿菌、葡萄球菌、腸球菌以及鏈球菌等等。而我們經由以往的研究，目前已知的是VGS參考菌株的groES 基因相似性在intraspecies、interspecies分別為94-99% 、62-95%，groEL 基因相似性在intraspecies、interspecies分別為92-99%、77-95%。因此接下來最想作的就是將在參考菌株所得到的資訊，再加以應用到臨床菌株的菌種鑑別，並希望能經由所發展的分子鑑定方法來對VGS的正確鑑定及臨床醫學有所貢獻。本實驗涵蓋了PCR and nucleotide sequencing、Group-specific PCR, PCR-RFLP、Group specific multiplex PCR快速鑑定等方法，應用在臨床菌株的anginosus、mitis、sanguinis group的 species identification，並成功完成鑑定VGS臨床菌株128株的鑑定，同時將結果與16S rDNA定序結果互相比較探討，並發現在鑑別基因高度相似性的VGS菌叢時，groESL基因能讓我們得到較優於16S rDNA的明確結果。此外，若是能克服目前在DataBank中資訊不足的限制，groESL基因將是未來鑑定VGS之十分具有潛力的新標的基因。另外，本實驗也嚐試將上列方法，直接應用於臨床檢體的快速鑑定，並成功的將一株S.intermedius自Brain abscess檢體中快速鑑定出來。此外，青黴素(penicillin)，是臨床上普遍用來治療VGS所引起的亞急性心內膜炎等感染的第一線用藥，其作用機制為與具有transpeptidase功能的青黴素結合蛋白Penicillin Binding Protein(PBP)結合，以阻斷peptidoglycan合成的最後步驟，影響細胞壁的合成，造成細菌脆弱與死亡。然而近年來，VGS對 penicillin 的抗藥比率明顯增加，造成治療上的困難，尤其是在mitis、sanguinis group。而在以往的研究顯示，VGS對penicillin的抗藥機轉，主要與PBP(如：PBP2B,2X,1A)的結構改變，所造成與藥物的親和性降低具關連性，尤其是發生在其三個重要的active motif附近。因此我們以PBP2B,2X,1A為標的，設計了PCR and nucleotide sequencing、TA cloning、PCR-RFLP等方法來分析mitis、sanguinis group的臨床菌株其PBP基因氨基酸的突變位置，並探討其與penicillin抗藥機制間的相關連性。結果顯示，在青黴素MIC分佈於intermediate、resistant中之臨床菌株其PBP2B,2X,1A都發現許多氨基酸的突變位置，尤其是發生在其active motif之中及其附近，因此可初步看出兩者間所具有的關連性。

關鍵字

草綠色鏈球菌； groESL基因；青黴素

並列摘要

Viridans group streptococci (VGS) are a group of heterogeneous gram positive facultative anaerobic cocci. They are usually considered to be commensals of the human oral cavity, but in recent years it has become clear that members of these species can be responsible for many infections, such as endocarditis, meningitis, purulent infections etc. VGS can also cause infection or death in immunocompromised patients. Furthermore, the clinical pathogenic and treatment may differ between species, thus, a correct identification for VGS to individual species level seems to be an important and meaningful work. Members of VGS can be divided into five major clusters according to their 16S rRNA sequences, which are highly conserved among bacteria and usually considered as gold standard. The five clusters include anginosus group, mitis group, sanguinis group, mutans group and salivarius group. Species identification of VGS has been difficult by conventional or automated systems. Differentiation between species by 16S rRNA gene is also not satisfactory due to their high similarity, therefore, some confusion may occur especially for anginosus group and mitis group. Our previous studies showed the groESL (cpn10/60 ) gene sequences were useful in differentiation of several bacterial species. The identities of the groES gene sequences among the VGS species for limited strains were about 62.1 to 95.1%, and the groEL gene sequences were 77.2 to 95.2%. Thus, we tested more clinical isolated using the same strategy to evaluate the clinical application for species identification. The tested isolates include anginosus, mitis, and sanguinis groups. We performed PCR and nucleotide sequencing, group-specific PCR, PCR- RFLP, multiplex PCR and compared the results with 16S rDNA identification. Our data revealed that the groESL genes can provide an additional parameter for species identification of VGS, particularly when 16S rRNA sequences share high degrees of similarity, otherwise, if more sufficient information can be available from Databank, the groESL gene is potentially useful in the differentiation of closely related species. Furthermore, the incidence of penicillin resistance in VGS has been increasing in recent years, and the major mechanism was known as modification of the PBPs. PBPs are made up of an N-terminal hydrophobic region, a central penicillin-binding domain, and a C-terminal domain, the active site of transpeptidase activity is formed by three conserved amino acid motifs, SXXK, SXN, and KTG. It has been reported that amino acid alternations in or flanking conserved motifs are associated with low-affinity variants of the PBPs, specifically decreased affinity of PBP1a, 2x, and 2b with β-lactams. These three PBPs are considered to be the key targets for β-lactams and were therefore chosen for examination in this study by using PCR-nucleotide sequencing, TA-cloning and PCR-RFLP methods. In this study, we found some possible significant amino acid alternations in penicillin-intermediate or resistant strains in both mitis and sanguinis groups. The amino acid alternations were in or near the active binding sites of PBP 1a, 2b and 2x, thus, possibly correlated with penicillin resistance.

並列關鍵字

viridans group streptococci ； groESL genes ； penicillin

參考文獻

Alam, S., Brailsford, S.R., Whiley, R.A., and Beighton, D. (1999) PCR-based methods for genotyping Viridans Group Streptococci. J. Clin. Microbiol. 37: 2772-2776.

Amoroso, A., Demares, D., Mollerach, M., Gutkind, G., and Coyette, J. (2001) All detectable high-molecular-mass penicillin-binding proteins are modified in a high-level beta-lactam-resistant clinical isolate of Streptococcus mitis. Antimicrob Agents Chemother 45: 2075-2081.

Bartie, K.L., Wilson, M.J., Williams, D.W., and Lewis, M.A.O. (2000) Macrorestriction fingerprinting of "Streptococcus milleri" group bacteria by pulsed-field gel electrophoresis. J. Clin. Microbiol. 38: 2141-2149.

Bosshard, P.P., Abels, S., Altwegg, M., Bottger, E.C., and Zbinden, R. (2004) Comparison of conventional and molecular methods for identification of aerobic catalase-negative gram-positive cocci in the clinical laboratory. J. Clin. Microbiol. 42: 2065-2073.

Caufield, P.W., Dasanayake, A.P., Li, Y., Pan, Y., Hsu, J., and Hardin, J.M. (2000) Natural history of Streptococcus sanguinis in the oral cavity of infants: Evidence for a discrete window of infectivity. Infect. Immun. 68: 4018-4023.

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草綠色鏈球菌之分子鑑定暨青黴素抗藥分析

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