Harris J Kirk, De Groote Mary Ann, Sagel Scott D, Zemanick Edith T, Kapsner Robert, Penvari Churee, Kaess Heidi, Deterding Robin R, Accurso Frank J, Pace Norman R
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.
Proc Natl Acad Sci U S A. 2007 Dec 18;104(51):20529-33. doi: 10.1073/pnas.0709804104. Epub 2007 Dec 11.
Culture of bronchoalveolar lavage fluid (BALF) is the gold standard for detection of pathogens in the lower airways in cystic fibrosis (CF). However, current culture results do not explain all clinical observations in CF, including negative culture results during pulmonary exacerbation and inflammation in the absence of pathogens. We hypothesize that organisms not routinely identified by culture occur in the CF airway and may contribute to disease. To test this hypothesis we used a culture-independent molecular approach, based on use of rRNA sequence analysis, to assess the bacterial composition of BALF from children with CF and disease controls (DC). Specimens from 42 subjects (28 CF) were examined, and approximately 6,600 total clones were screened to identify 121 species of bacteria. In general, a single rRNA type dominated clone libraries from CF specimens, but not DC. Thirteen CF subjects contained bacteria that are not routinely assessed by culture. In four CF subjects, candidate pathogens were identified and include the anaerobe Prevotella denticola, a Lysobacter sp., and members of the Rickettsiales. The presumptive pathogens Tropheryma whipplei and Granulicatella elegans were identified in cases from the DC group. The presence of unexpected bacteria in CF may explain inflammation without documented pathogens and consequent failure to respond to standard treatment. These results show that molecular techniques provide a broader perspective on airway bacteria than do routine clinical cultures and thus can identify targets for further clinical evaluation.
支气管肺泡灌洗（BALF）培养是检测囊性纤维化（CF）患者下呼吸道病原体的金标准。然而，目前的培养结果并不能解释CF患者的所有临床观察结果，包括肺部病情加重期间培养结果为阴性以及在没有病原体的情况下出现炎症。我们推测，CF气道中存在未被常规培养方法鉴定出的微生物，它们可能与疾病的发生有关。为了验证这一假设，我们采用了一种基于rRNA序列分析的非培养分子方法，来评估CF患儿和疾病对照（DC）的BALF中的细菌组成。我们检测了42名受试者（28名CF患者）的样本，共筛选了约6600个克隆，以鉴定出121种细菌。总体而言，CF样本的克隆文库中通常以单一rRNA类型为主，而DC样本则不然。13名CF受试者体内含有常规培养方法未检测到的细菌。在4名CF受试者中，鉴定出了候选病原体，包括厌氧菌齿龈普雷沃菌、一种溶杆菌属细菌以及立克次氏体目成员。在DC组的病例中鉴定出了推测的病原体惠普尔嗜组织菌和优雅颗粒链菌。CF患者体内存在意外细菌可能解释了在没有记录到病原体的情况下出现的炎症以及对标准治疗无反应的情况。这些结果表明，与常规临床培养相比，分子技术能更全面地了解气道细菌，从而可以确定进一步临床评估的目标。
