Identification Of Streptococcus Mitis Species By Houseke

翠绿链球菌是一组革兰氏阳性菌，它构成了人类口腔和胃肠道中常驻菌群的一部分。它们偶尔会被发现是短暂菌血症的原因，特别是在牙科手术后。该组链球菌已成为亚急性细菌性心内膜炎的重要病原体之一，可导致严重的心脏瓣膜损伤。翠绿链球菌由至少22种组成，目前分为5个亚群：mitis、anginosus、mutans、salivarius和bovis。Mitis群链球菌是最常见的翠绿链球菌，可导致人类疾病。在过去的几十年里，随着基于分子的方法的出现，翠绿链球菌的重新分类一直在发展，有时令人困惑。歧义的原因之一是，用于鉴定临床实验室分离的大多数细菌的常规生化试验通常不能在物种水平上提供明确的鉴定，并且也可能错误地识别该组的成员。最近介绍了一种利用16S rDNA基因序列鉴定细菌病原体的新方法。然而，这种技术并没有帮助一些密切相关的生物，在他们的16S基因序列几乎相同。然而，用于鉴定临床重要细菌的16S rDNA测序方法具有真正的局限性。我们决定尝试多位点基因测序目标——管家基因——在物种水平上区分细菌，尽管这种淀粉凝胶蛋白电泳的分子版本尚未广泛用于物种区分。我们开始尝试这种方法的物种划分细菌分离属于mittis和sanguinis组的翠绿链球菌。虽然从逻辑上讲，为成功的菌株分型所需的更精细的区分表明，这种方法可能在物种水平上对生物体进行更清晰的区分，但尚不清楚是否可以在这两个群体的所有物种中找到用于PCR扩增目标基因区域的保守寡核苷酸；或者，在这些未经检验的物种中，在家养基因中检测到的任何序列差异是否能在物种水平上可靠地区分生物体，或者证明只是在短进化时间内稳定存在的随机突变，因此，只对在染色水平上区分细菌有用。我们能够证明，用于肺炎链球菌菌株分型的两个管家基因靶点也可以用于这两个临床重要的翠绿链球菌组的非常明确的物种区分。我们的研究成功地证明了所研究的两个基因gdh和gki可以应用于这些生物的物种鉴定。这种方法的鉴别能力比16S rDNA测序要好得多。这项工作已在一份提交的手稿中作了描述。

The viridans streptococci are a group of gram-positive bacteria that constitute part of the resident flora in the human oral cavity and gastrointestinal tract. They can occasionally be found as a cause of transient bacteremia, especially following dental procedures. This group of streptococci has become one of the important causative agents of subacute bacterial endocarditis that can result in serious damage of heart valves. Viridans streptococci are composed of at least 22 species, which are currently divided into 5 subgroups: mitis, anginosus, mutans, salivarius, and bovis. Mitis group streptococci are the most common viridans streptococci responsible for diseases in humans. In the past decades, with the emergence of molecular-based methods, re-classification of viridans streptococci has been evolving and is sometimes confusing. One of the reasons for the ambiguity is that conventional biochemical tests used to identify most of the bacteria isolated in the clinical laboratory often do not provide definitive identification at the species level, and may also misidentify members of this group. A new approach for the identification of bacterial pathogens using 16S rDNA gene sequences has been recently introduced. However, this technique has not been helpful in some closely related organisms that have nearly identical sequences in their 16S genes. The 16S rDNA sequencing approach for identification of clinically important bacteria, however, has real limitations. We decided to try the multilocus gene sequencing targets-- housekeeping genes --for differentiating bacteria at the species level, even though this molecular version of starch gel protein electrophoresis has not been widely used for species differentiation. We set out to try this approach for species delineation of bacterial isolates belonging to the mitis and sanguinis groups of viridans streptococci. Although logically the finer discriminations required for purposes of successful strain typing would suggest that this approach might give cleaner distinctions between organisms at the species level, it was unknown whether or not conserved oligonuceotides for PCR amplification of targeted gene regions could be found for all the species of these two groups; or whether any sequence differences detected in house keeping genes in these unexamined species would reliably differentiate organisms at the species level or prove to be just random mutations that are stably present for short evolutionary time periods and, thereby, only useful at distinguishing bacteria at the stain level. We were able to demonstrate that two of the housekeeping gene targets used for S. pneumoniae strain typing could also be used for very clear species discriminations in these two clinically important groups of viridans streptococci. Our studies successfully demonstrated that two of the genes studied, gdh and gki, could be applied to species identification in these organisms. The discriminatory power of this approach is far better that 16S rDNA sequencing. This work has been described in a submitted manuscript.