Currently, several PCR assays based on 16S rRNA and virulence-associated genes are available for detection of Legionella pneumophila. So far, no genotyping method has been published that can discriminate between serogroups and monoclonal subgroups of the most common L. pneumophila serogroup 1. Our first approach was to analyse LPS-associated genes of seven L. pneumophila serogroup 1 strains, and we developed two PCR-based methods specific for serogroup 1. Specific DNA fragments could be amplified from all the serogroup 1 strains (n=43) including the strains from the American Type Culture Collection. In contrast, none of the strains from serogroups 2–15 (n=41) contained these specific gene regions. In a second approach, primers specific for the lag-1 gene, encoding an O-acetyltransferase, which is responsible for the presence of the LPS epitope recognized by mAb 3/1, were designed and tested for their ability to differentiate between mAb 3/1-positive and -negative strains. All mAb 3/1-positive strains (n=30) contained the lag-1 gene, but in turn 4 of 13 tested mAb 3/1-negative strains were also positive in the PCR. Thus, the discrimination between mAb 3/1-positive and mAb 3/1-negative subgroups could not be achieved for all strains. In a third approach, two intergenic regions expected to be specific for monoclonal subgroup Knoxville and closely related subgroups Benidorm/Bellingham were identified and used for selective genotyping. These intergenic regions could not only be amplified in every tested strain belonging to the subgroups Knoxville, Benidorm and Bellingham, but also in some strains of other unrelated subgroups. The two PCR approaches with primers specific for serogroup 1 genes definitely represent a valuable tool in outbreak investigations and for risk assessment. They also might be used for culture-independent diagnosis of legionellosis caused by L. pneumophila serogroup 1.
CazaletC.,
RusniokC.,
BrüggemannH.,
ZidaneN.,
MagnierA.,
MaL.,
TichitM.,
JarraudS.,
BouchierC.other authors2004; Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat Genet 36:1165–1173[CrossRef]
CazaletC.,
JarraudS.,
Ghavi-HelmY.,
KunstF.,
GlaserP.,
EtienneJ.,
BuchrieserC.2008; Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species. Genome Res 18:431–441[CrossRef]
FryN. K.,
Alexiou-DanielS.,
BangsborgJ. M.,
BernanderS.,
Castellani PastorisM.,
EtienneJ.,
ForsblomB.,
GaiaV.,
HelbigJ. H.other authors1999; A multicenter evaluation of genotyping methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results from a pan-European study. Clin Microbiol Infect 5:462–477[CrossRef]
FryN. K.,
AfsharB.,
WewalkaG.,
HarrisonT. G.2006; Epidemiological typing of Legionella pneumophila in the absence of isolates. In Legionella: State of the Art 30 Years After its Recognition pp 152–155 Edited by
CianciottoN. P.,
Abu KwaikY.,
EdelsteinP. H.,
FieldsB. S.,
GearyD. F.,
HarrisonT. G.,
JosephC. A.,
RatcliffR. M.,
StoutJ. E.,
SwansonM. S.
Washington, DC: American Society for Microbiology;
GlöcknerG.,
Albert-WeissenbergerC.,
WeinmannE.,
JacobiS.,
SchunderE.,
SteinertM.,
HackerJ.,
HeunerK.2008; Identification and characterization of a new conjugation/type IVA secretion system ( trb / tra ) of Legionella pneumophila Corby localized on two mobile genomic islands. Int J Med Microbiol 298:411–428[CrossRef]
HelbigJ. H.,
KurtzJ. B.,
PastorisM. C.,
PelazC.,
LückP. C.1997; Antigenic lipopolysaccharide components of Legionella pneumophila recognized by monoclonal antibodies: possibilities and limitations for division of the species into serogroups. J Clin Microbiol 35:2841–2845
JolyJ. R.,
McKinneyR. M.,
TobinJ. O.,
BibbW. F.,
WatkinsI. D.,
RamsayD.1986; Development of a standardized subgrouping scheme for Legionella pneumophila serogroup 1 using monoclonal antibodies. J Clin Microbiol 23:768–771
KaplanJ. B.,
PerryM. B.,
MacLeanL. L.,
FurgangD.,
WilsonM. E.,
FineD. H.2001; Structural and genetic analyses of O polysaccharide from Actinobacillus actinomycetemcomitans serotype f. Infect Immun 69:5375–5384[CrossRef]
KozakN. A.,
BrownE.,
BensonR. F.,
SheltonB. G.,
FieldsB. S.2007; Presence of a lag-1 gene as a virulence marker for Legionella pneumophila serogroup 1 strains. In Abstracts of the 107th General Meeting of the American Society for Microbiology 2007. Toronto, Canada abstract D 144 Washington, DC: American Society for Microbiology;
KuchanaR.,
UldumS. A.2007; PCR method for detection of monoclonal subgroups of Legionella pneumophila serogroup 1. In 22nd Annual Meeting of the European Working Group on Legionella Infections, 2007. Stockholm, Sweden oral presentation
LückP. C.,
FreierT.,
SteudelC.,
KnirelY. A.,
LünebergE.,
ZähringerU.,
HelbigJ. H.2001; A point mutation in the active site of Legionella pneumophila O-acetyltransferase results in modified lipopolysaccharide but does not influence virulence. Int J Med Microbiol 291:345–352[CrossRef]
LückP. C.,
EckerC.,
ReischlU.,
LindeH. J.,
StempkaR.2007; Culture-independent identification of the source of an infection by direct amplification and sequencing of Legionella pneumophila DNA from a clinical specimen. J Clin Microbiol 45:3143–3144[CrossRef]
LünebergE.,
ZetzmannN.,
AlberD.,
KnirelY. A.,
KooistraO.,
ZahringerU.,
FroschM.2000; Cloning and functional characterization of a 30 kb gene locus required for lipopolysaccharide biosynthesis in Legionella pneumophila
. Int J Med Microbiol 290:37–49[CrossRef]
SatolaS. W.,
CollinsJ. T.,
NapierR.,
FarleyM. M.2007; Capsule gene analysis of invasive Haemophilus influenzae : accuracy of serotyping and prevalence of IS1016 among nontypeable isolates. J Clin Microbiol 45:3230–3238[CrossRef]
YuV. L.,
PlouffeJ. F.,
PastorisM. C.,
StoutJ. E.,
SchousboeM.,
WidmerA.,
SummersgillJ.,
FileT.,
HeathC. M.other authors2002; Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey. J Infect Dis 186:127–128[CrossRef]
YuJ.,
CarvalhoM. da G. S.,
BeallB.,
NahmM. H.2008; A rapid pneumococcal serotyping system based on monoclonal antibodies and PCR. J Med Microbiol 57:171–178[CrossRef]
ZouC. H.,
KnirelY. A.,
HelbigJ. H.,
ZähringerU.,
MintzC. S.1999; Molecular cloning and characterization of a locus responsible for O acetylation of the O polysaccharide of Legionella pneumophila serogroup 1 lipopolysaccharide. J Bacteriol 181:4137–4141