A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp.

Boyang Cao,Xiangqian Liu,Lu Feng,Min Chen,Xiang Yu,Lei Wang,Yousef Abu Kwaik

doi:10.1371/journal.pone.0113863

Abstract

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.

Highlights

Legionella acquired its name after an outbreak of a -unknown ‘‘mystery disease’’ that affected 221 persons, and caused 34 deaths eventually, attending a convention of the American Legion in July 1976
We report the establishment of an oligonucleotide microarray method for the simultaneous detection of 11 pathogenic Legionella spp., L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila, and one non-pathogenic spp., L. fairfieldensis, based on the 16-23S rRNA gene internal transcribed spacer (ITS) regions
The following standard Legionella spp. strains were used for ITS sequencing: L. anisa (DSMZ 17627), L. bozemanii (ATCC 33217), L. dumoffii (ATCC 33279), L. fairfieldensis (ATCC 49588), L. gormanii (ATCC 43769), L. jordanis (DSMZ 19212), L. maceachernii (DSMZ 16642), L. micdadei (NCTC 11371), L. pneumophila subspp. fraseri (ATCC 35251), and L. pneumophila subspp. pascullei (ATCC 4585)

Summary

Introduction

Legionella acquired its name after an outbreak of a -unknown ‘‘mystery disease’’ that affected 221 persons, and caused 34 deaths eventually, attending a convention of the American Legion in July 1976. This epidemic, which occurred within days of the 200th anniversary of the signing of the Declaration of Independence, was widely publicized and raised great concern in the United States [1]. A few months later, the causative agent was identified as a previously unknown bacterium, which was subsequently named Legionella. L. pneumophila remains as the major cause of legionellosis, non-pneumophila infections have been reported to be caused by Legionella micdadei (60%), Legionella bozemanii (15%), Legionella dumoffii (10%), Legionella longbeachae (5%), and other species (10%) [6]

Methods

Results

Discussion

Conclusion