Rogers Geraint B, Carroll Mary P, Serisier David J, Hockey Peter M, Kehagia Valia, Jones Graeme R, Bruce Kenneth D
Department of Life Sciences, King's College London, London, UK.
Respir Res. 2005 Jun 1;6(1):49. doi: 10.1186/1465-9921-6-49.
Chronic lung infections are the primary cause of morbidity and mortality in Cystic Fibrosis (CF) patients. Recent molecular biological based studies have identified a surprisingly wide range of hitherto unreported bacterial species in the lungs of CF patients. The aim of this study was to determine whether the species present were active and, as such, worthy of further investigation as potential pathogens.
Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiles were generated from PCR products amplified from 16S rDNA and Reverse Transcription Terminal Restriction Fragment Length Polymorphism (RT-T-RFLP) profiles, a marker of metabolic activity, were generated from PCR products amplified from 16S rRNA, both extracted from the same CF sputum sample. To test the level of activity of these bacteria, T-RFLP profiles were compared to RT-T-RFLP profiles.
Samples from 17 individuals were studied. Parallel analyses identified a total of 706 individual T-RF and RT-T-RF bands in this sample set. 323 bands were detected by T-RFLP and 383 bands were detected by RT-T-RFLP (statistically significant; P < or = 0.001). For the group as a whole, 145 bands were detected in a T-RFLP profile alone, suggesting metabolically inactive bacteria. 205 bands were detected in an RT-T-RFLP profile alone and 178 bands were detected in both, suggesting a significant degree of metabolic activity. Although Pseudomonas aeruginosa was present and active in many patients, a low occurrence of other species traditionally considered to be key CF pathogens was detected. T-RFLP profiles obtained for induced sputum samples provided by healthy individuals without CF formed a separate cluster indicating a low level of similarity to those from CF patients.
These results indicate that a high proportion of the bacterial species detected in the sputum from all of the CF patients in the study are active. The widespread activity of bacterial species in these samples emphasizes the potential importance of these previously unrecognized species within the CF lung.
慢性肺部感染是囊性纤维化（CF）患者发病和死亡的主要原因。最近基于分子生物学的研究在CF患者肺部发现了一系列迄今为止未报道的细菌种类，数量惊人。本研究的目的是确定这些存在的细菌种类是否具有活性，以及是否因此值得作为潜在病原体进行进一步研究。
从16S rDNA扩增的PCR产物生成末端限制性片段长度多态性（T-RFLP）图谱，从16S rRNA扩增的PCR产物生成代谢活性标志物逆转录末端限制性片段长度多态性（RT-T-RFLP）图谱，二者均从同一CF痰液样本中提取。为了检测这些细菌的活性水平，将T-RFLP图谱与RT-T-RFLP图谱进行比较。
对17名个体的样本进行了研究。平行分析在该样本集中共鉴定出706个单独的T-RF和RT-T-RF条带。T-RFLP检测到323条带，RT-T-RFLP检测到383条带（具有统计学意义；P≤0.001）。对于整个组，仅在T-RFLP图谱中检测到145条带，表明细菌代谢不活跃。仅在RT-T-RFLP图谱中检测到205条带，在二者中均检测到178条带，表明有显著程度的代谢活性。尽管许多患者体内存在并活跃着铜绿假单胞菌，但检测到传统上被认为是CF关键病原体的其他细菌种类的发生率较低。从无CF的健康个体提供的诱导痰液样本获得的T-RFLP图谱形成了一个单独的聚类，表明与CF患者的图谱相似度较低。
这些结果表明，在该研究中所有CF患者痰液中检测到的细菌种类中有很大一部分具有活性。这些样本中细菌种类的广泛活性强调了这些先前未被识别的细菌种类在CF肺部的潜在重要性。
