Development of a miniaturized DNA microarray for identification of 66 virulence genes of Legionella pneumophila.
BACKGROUND
For the last five years, Legionella sp. infections and legionnaire's disease in Poland have been receiving a lot of attention, because of the new regulations concerning microbiological quality of drinking water. This was the inspiration to search for and develop a new assay to identify many virulence genes of Legionella pneumophila to better understand their distribution in environmental and clinical strains. The method might be an invaluable help in infection risk assessment and in epidemiological investigations.


METHODS
The microarray is based on Array Tube technology. It contains 3 positive and 1 negative control. Target genes encode structural elements of T4SS, effector proteins and factors not related to T4SS. Probes were designed using OligoWiz software and data analyzed using IconoClust software. To isolate environmental and clinical strains, BAL samples and samples of hot water from different and independent hot water distribution systems of public utility buildings were collected.


RESULTS
We have developed a miniaturized DNA microarray for identification of 66 virulence genes of L. pneumophila. The assay is specific to L. pneumophila sg 1 with sensitivity sufficient to perform the assay using DNA isolated from a single L. pneumophila colony. Seven environmental strains were analyzed. Two exhibited a hybridization pattern distinct from the reference strain.


CONCLUSIONS
The method is time- and cost-effective. Initial studies have shown that genes encoding effector proteins may vary among environmental strains. Further studies might help to identify set of genes increasing the risk of clinical disease and to determine the pathogenic potential of environmental strains.
Department of Zoonoses and Tropical Diseases, Medical University of Warsaw, Żwirki i Wigury 61 Str., 02-091 Warsaw, Poland.
Mariusz
M
Żak
Piotr
P
Zaborowski
Milena
M
Baczewska-Rej
Aleksandra A
AA
Zasada
Renata
R
Matuszewska
Bożena
B
Krogulska
D004269:DNA, Bacterial / Q000235:genetics*; D006801:Humans; D016952:Legionella pneumophila / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D007877:Legionnaires' Disease / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011044:Poland; D014871:Water Microbiology; D014881:Water Supply / Q000032:analysis*
D004269:DNA, Bacterial