Ossewaarde Jacobus M, Meijer Adam
Microbiology (Reading). 1999 Feb;145 ( Pt 2):411-417. doi: 10.1099/13500872-145-2-411.
Respiratory tract infections in man may be caused by several members of the genus Chlamydia and also by two Chlamydia-like strains, 'Simkania negevensis' (Z-agent) and 'Parachlamydia acanthamoebae' (Bng). To facilitate diagnostic procedures a PCR assay able to detect all known Chlamydiaceae sequences in one reaction was developed. For this purpose, primers were selected to amplify a fragment of the 16S rRNA gene. Characterization of the amplified fragments was done by hybridization with specific probes and by sequencing. PCR assays were carried out using DNA isolated from nose/throat specimens or from peripheral blood mononuclear cells of patients with respiratory tract infections, and from vessel wall specimens of abdominal aneurysms. Six of the 42 nose/throat swab specimens analysed yielded strong bands and one yielded a faint band. Three of these bands were identified as Chlamydia pneumoniae and one as Chlamydia trachomatis by sequencing. Analysis of the three other bands yielded two different new sequences. DNA isolated from peripheral blood mononuclear cells of one patient yielded a third new sequence. DNA isolated from peripheral blood mononuclear cells of four healthy controls was negative. One of the abdominal aneurysm specimens also yielded a strong band. Sequencing revealed a fourth new sequence. All negative controls included during specimen processing and PCR analysis remained negative. The typical secondary structure of microbial 16S genes was present in all four new sequences indicating the validity of the sequence data. All four new sequences were distinct from other bacteria and clustered together with known Chlamydiaceae sequences. Phylogenetic analysis suggested a new lineage, separating the four new sequences, 'S. negevensis' and 'P. acanthamoebae' from the genus Chlamydia with the four known chlamydial species. In conclusion, this study provides evidence for the existence of several new members of the order Chlamydiales. Since the source of the Chlamydia-like strains has not been identified and serological and/or molecular cross-reactivities may be expected, results of identification of infecting recognized organisms should be interpreted cautiously.
人类呼吸道感染可能由衣原体属的几个成员引起，也可能由两种衣原体样菌株，即“内氏西曼卡菌”（Z病原体）和“棘阿米巴嗜衣原体”（Bng）引起。为了便于诊断，开发了一种能够在一次反应中检测所有已知衣原体科序列的聚合酶链反应（PCR）检测方法。为此，选择引物来扩增16S核糖体RNA（rRNA）基因的一个片段。通过与特异性探针杂交和测序对扩增片段进行表征。使用从呼吸道感染患者的鼻/咽标本或外周血单核细胞以及腹主动脉瘤血管壁标本中分离的DNA进行PCR检测。在分析的42份鼻/咽拭子标本中，有6份产生了强条带，1份产生了弱条带。通过测序，其中3条带被鉴定为肺炎衣原体，1条带被鉴定为沙眼衣原体。对另外3条带的分析产生了两个不同的新序列。从一名患者外周血单核细胞中分离的DNA产生了第三个新序列。从四名健康对照的外周血单核细胞中分离的DNA为阴性。其中一个腹主动脉瘤标本也产生了一条强条带。测序揭示了第四个新序列。在标本处理和PCR分析过程中纳入的所有阴性对照均保持阴性。所有四个新序列中均存在微生物16S基因的典型二级结构，表明序列数据的有效性。所有四个新序列均与其他细菌不同，并与已知的衣原体科序列聚集在一起。系统发育分析表明存在一个新的谱系，将这四个新序列、“内氏西曼卡菌”和“棘阿米巴嗜衣原体”与衣原体属以及四个已知衣原体物种区分开来。总之，本研究为衣原体目几个新成员的存在提供了证据。由于尚未确定衣原体样菌株的来源，并且可能存在血清学和/或分子交叉反应，因此对感染的已知病原体的鉴定结果应谨慎解释。
