轉醣鏈球菌群、NVS與Gemella屬之分子鑑定及Gemella morbillorum之groESL操縱子分析

Abstract

轉醣鏈球菌群屬於草綠色鏈球菌的其中一群，包含七種菌種及八個血清型：Streptococcus mutans (血清型c、e與f)、Streptococcus sobrinus (血清型d與g)、Streptococcus criceti (血清型a)、Streptococcus rattus (血清型b)、Streptococcus macacae (血清型c)、Streptococcus downei (血清型h)與Streptococcus ferus (血清型c)。在轉醣鏈球菌群當中，又以S. mutans與S. sobrinus這兩種細菌最常被分離出來，也因此最具代表性，其臨床上主要會造成蛀牙與心內膜炎。由於轉醣鏈球菌群的生化鑑定方法相當繁雜，因此近幾年來，有越來越多的分子鑑定方法被發表而為世人所知。本實驗利用groESL基因作為一個鑑定標的，除了可將轉醣鏈球菌群分為三大類之外，更可進一步將轉醣鏈球菌群與其他草綠色鏈球菌做一個劃分。 NVS (Abiotrophia defectiva、Granulicatella adiacens、Granulicatella elegans) 與Gemella屬(Gemella haemolysans與Gemella morbillorum)雖為人體常在菌，但多篇報導說明這些細菌因伺機感染而造成人類菌血症或心內膜炎的機會依然存在。此外，NVS與Gemella屬還共同具有無法生長於一般15%綿羊血片培養基、在巧克力培養基上雖可生長其菌落顆粒卻較細小的這個特性，因此本實驗嘗試設計一套multiplex PCR方法將這五種細菌做一個區分。此套multiplex PCR方法共使用了四個引子，可將五種細菌區分至Abiotrophia屬、Granulicatella屬與Gemella屬的層次。其中還發現Gemella屬的臨床菌株之groESL基因與ATCC參考菌株差異頗大，與16S rRNA基因的高保守性大不相同，其中是否還有subspecies的存在尚待確認。 因鑑定所需，本實驗得到Ge. morbillorum與Ge. haemolysans的groES上游序列，卻意外發現通常存在於革蘭氏陽性菌groESL操作子前方的第一型負向調控因子CIRCE並不存在，反倒發現了第三型調控因子CtsR所辨識之序列，其熱刺激反應並不明顯也頗令人疑惑。此外，實驗中還發現groESL操縱子之終止訊號似乎不夠強而造成mRNA有可能繼續轉錄，而與後方一條全長306 bp的open reading frame共同轉錄。Gemella屬的groESL操作子詳細調控機制、表現與組成情況尚待釐清。

Mutans group of streptococci is one of the five groups in viridans streptococci. Mutans streptococci consists of seven species which can be classified into eight serotypes a to h: Streptococcus mutans (serotypes c, e, and f), Streptococcus sobrinus (serotypes d and g), Streptococcus criceti (serotype a), Streptococcus downei (serotype h), Streptococcus ferus (serotype c), Streptococcus macacae (serotype c), and Streptococcus ratti (serotype b). According to previous reports, the most common species isolated from human sources are S. mutans and S. sobrinus. Species identification of mutans streptococci has been based on conventional methods including complicated biochemical tests, but it is time-consuming and sometimes unsatisfactory. In this study, we used groESL genes as a target for molecular identification. This PCR method could provide an alternative way for differentiation among members of mutans streptococci or from other viridans streptococci. NVS (Abiotrophia defectiva, Granulicatella adiacens, and Granulicatella elegans) and Gemella species (Gemella haemolysans and Gemella morbillorum) were commensal organisms in humans and they were sometimes responsible for opportunistic endocarditis. NVS and Gemella species had some common features in phenotypic characteristics. They could not grow on blood agar plates but could grow on chocolate agars, although their colonies were very small. It was easily misidentified in the microscopic examination because NVS were pleiomorphic in gram stain while Gemella species were easily decolorized. Because of these common characteristics, we designed a multiplex PCR assay based on groESL genes to identify NVS and Gemella species. Four primers were used in the multiplex PCR assay in which the five organisms could classify into Abiotrophia species, Granulicatella species and Gemella species. Besides, a clinical isolate of Gemella haemolysans displayed <90% identity in groEL gene with its reference strain but they shared the highest degree of similarities in 16S rRNA gene. Gemella haemolysans may be more heterogeneous than Gemella morbillorum. In our laboratory, we have successfully determined the full-length sequences of groESL operon in Gemella morbillorum. Unexpectedly, we could not find the CIRCE element, which was being found in groESL operons of gram positive bacteria, but found the CtsR binding site in the putative promoter region. This was an unusual event in gram positive bacteria. It was also confused that the heat shock response was not obvious in the data of northern blot and SDS-PAGE. Besides, it was seemed that termination of the groESL operon was not 100% efficient, and in some cases, the downstream gene could be cotranscribed as part of the operon. In this part of experiment, it relied on more data to confirm this observation.