A New Oligonucleotide Microarray for Detection of Pathogenic and Non-Pathogenic Legionella spp.

Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.

自军团菌病（legionellosis）这种致命疾病被发现以来，嗜肺军团菌（Legionella pneumophila）就被认定为该病的主要致病菌。后续研究发现，除嗜肺军团菌外的其他军团菌属物种，可引发多种非嗜肺军团菌感染。由于缺乏有效的诊断手段，非嗜肺军团菌感染的检出率很可能被低估。本研究对10种军团菌属物种及亚种的16S-23S rRNA基因内部转录间隔区（internal transcribed spacer, ITS）进行了测序，涵盖拟杆菌军团菌（L. anisa）、博兹曼军团菌（L. bozemanii）、杜莫夫军团菌（L. dumoffii）、费尔菲尔德军团菌（L. fairfieldensis）、戈曼军团菌（L. gormanii）、约旦军团菌（L. jordanis）、麦卡彻尼军团菌（L. maceachernii）、米克戴德军团菌（L. micdadei）、弗氏嗜肺军团菌亚种（L. pneumophila subspp. fraseri）以及帕斯库莱嗜肺军团菌亚种（L. pneumophila subspp. pasculleii）；并据此开发了一种快速寡核苷酸微阵列（oligonucleotide microarray）检测技术，可识别12种最常见的军团菌属物种，其中包括11种致病性物种：拟杆菌军团菌、博兹曼军团菌、杜莫夫军团菌、戈曼军团菌、约旦军团菌、长滩军团菌（L. longbeachae）、麦卡彻尼军团菌、米克戴德军团菌以及嗜肺军团菌（包含嗜肺亚种、弗氏亚种与帕斯库莱亚种），另有1种非致病性物种费尔菲尔德军团菌。该微阵列纳入了29条可重复性检测多种军团菌且特异性优异的探针。本研究共使用52株菌株（其中30株为目标致病菌，22株为非目标细菌）对该寡核苷酸微阵列检测方法进行验证。该检测方法的灵敏度为：基因组DNA检测下限为1.0 ng，军团菌培养物检测下限为13菌落形成单位（Colony-Forming Unit, CFU）/100 mL。该微阵列对7份空调冷凝水样本的检测准确率达100%，证实该技术在基础微生物学、临床诊断、食品安全以及流行病学监测等领域具备良好的应用前景。基于ITS的系统发育分析还显示，相较于其他9种致病性军团菌，非致病性的费尔菲尔德军团菌与嗜肺军团菌的亲缘关系最为接近。