Abstract
Legionella bacteria are organisms of public health interest due to their ability to cause pneumonia (Legionnaires’ disease) in susceptible humans and their ubiquitous presence in water supply systems. Rapid diagnosis of Legionnaires’ disease allows the use of therapy specific for the disease. L. pneumophila serogroup 1 is the most common cause of infection acquired in community and hospital environments. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this work, simplex and duplex PCR assays with the use of new molecular markers pcs and pmtA involved in phosphatidylcholine synthesis were specified for rapid and cost-efficient identification and distinguishing Legionella species. The sets of primers developed were found to be sensitive and specific for reliable detection of Legionella belonging to the eight most clinically relevant species. Among these, four primer sets I, II, VI, and VII used for duplex-PCRs proved to have the highest identification power and reliability in the detection of the bacteria. Application of this PCR-based method should improve detection of Legionella spp. in both clinical and environmental settings and facilitate molecular typing of these organisms.
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References
Beaute J, Zucs P, de Jong B, European Legionnaires’ disease surveillance network (2013) Legionnaires’ disease in Europe, 2009–2010. Euro Surveill 18:20417
Behets J, Declerck P, Delaedt Y, Creemers B, Ollevier F (2007) Development and evaluation of a Taqman duplex real-time PCR quantification method for reliable enumeration of Legionella pneumophila in water samples. J Microbiol Methods 68:137–144
Blyth C, Adams N, Chen S (2009) Diagnostic and typing methods for investigating Legionella infection. NSW Pub Health Bull 20:157–161. doi:10.1071/NB08062
Cao B, Liu X, Chen M, Feng L, Wang L (2014) A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp. PLoS ONE 9(12):e113863. doi:10.1371/journal.pone.0113863.g002
Chang B, Sugiyama K, Taguri T, Amemura-Maekawa J, Kura F, Watanabe H (2009) Specific detection of viable Legionella cells by combined use of photoactivated ethidium monoazide and PCR/real-time PCR. Appl Environ Microbiol 75:147–153. doi:10.1128/AEM.00604-08
Cloud JL, Carroll KC, Pixton P, Erali M, Hillyard DR (2000) Detection of Legionella species in respiratory specimens using PCR with sequencing confirmation. J Clin Microbiol 38:1709–1712
Den Boer J, Yzerman E (2004) Diagnosis of Legionella infection in Legionnaires’ disease. Eur J Clin Microbiol Infect Dis 23:871–871. doi:10.1007/s10096-004-1248-8
Diederen BM (2008) Legionella spp and Legionnaires’ disease. J Infect 56:1–12. doi:10.1016/j.jinf.2007.09.010
Drożański WJ (1991) Sarcobium lyticum gen. nov. sp. nov. an obligate intracellular bacterial parasite of small free-living amoeba. Int J Syst Bacteriol 41:82–87. doi:10.1078/0723-2020-00068
Edelstein PH (2007) Urine antigen tests positive for Pontiac fever: implications for diagnosis and pathogenesis. Clin Infect Dis 44:229–231. doi:10.1086/510394
European Centre for Disease Prevention and Control (2014) Legionnaires’ disease in Europe, 2012. Stockholm: EDC; 2014. doi:10.2900/21087
Farnham A, Alleyne L, Cimini D, Balter S (2014) Legionnaires’ disease incidence and risk factors, New York, New York, USA 2002–2012. Emerg Infect Dis 20:1795–1802. doi:10.3201/eid2011.131872
Fields BS, Benson RF, Besser RE (2002) Legionella and legionnaires’ disease: 25 years of investigation. Clin Microbiol Rev 15:506–526. doi:10.1128/CMR.15.3.506-526.2002
Fittipaldi M, Codony F, Adrados B, Camper AK, Morato J (2011) Viable real-time PCR in environmental samples: can all data be interpreted directly? Microb Ecol 61:7–12. doi:10.1007/s00248-010-9719-1
Fujii T, Ohta M, Kono M, Hoshina S, Fukuhara K, Tsuruoka M (1999) Rapid detection of the gene of Legionella pneumophila using the fluorescence polarization with the asymmetric PCR. Nucleic Acids Symp Ser 42:59–60. doi:10.1093/nass/42.1.59
Fujinami Y, Kikkawa H, Kurosaki Y, Sakurada K, Yoahino M, Yasuda J (2011) Rapid discrimination of Legionella by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Microb Res 166:77–86. doi:10.1016/j.micres.2010.02.005
Geiger O, Lopez-Lara IM, Sohlenkamp C (2013) Phosphatidylcholine biosynthesis and function in bacteria. Biochim Biophys Acta 1831:503–513. doi:10.1016/j.bbalip.2012.08.009
Giglio S, Monis PT, Saint CP (2005) Legionella confirmation using real-time PCR and SYTO9 is an alternative to current methodology. Appl Environ Microbiol 71:8944–8948. doi:10.1128/AEM.71.12.8944-8948.2005
Haroon A, Koide M, Higa F, Tateyama M, Fujita J (2012) Identification of Legionella pneumophila serogroups and other Legionella species by mip gene sequencing. J Infect Chemother 18:276–281. doi:10.1007/s10156-011-0324-0
Jarraud S, Descours G, Ginevra C, Lina G, Etienne J (2013) Identification of Legionella in clinical samples. Methods Mol Biol 954:27–56. doi:10.1007/978-1-62703-161-5_2
Joly P, Falconnet PA, Andre J, Weill N, Reyrolle M, Vandenesch F, Maurin M, Etienne J, Jarraud S (2006) Quantitative real-time Legionella PCR for environmental water samples: data interpretation. Appl Environ Microbiol 72:2801–2808. doi:10.1128/AEM.72.4.2801-2808.2006
Kao PM, Tung MC, Hsu BM, Chiu YC, She CY, Shen SM, Huang YL, Huang WC (2013) Identification and quantitative detection of Legionella spp. in various aquatic environments by real-time PCR assay. Environ Sci Pollut Res Int 20:6128–6137. doi:10.1007/s11356-013-1534-z
Ko KS, Hong SK, Lee KH, Lee HK, Park MY, Miyamoto H, Kook YH (2003) Detection and identification of Legionella pneumophila by PCR-restriction fragment length polymorphism analysis of the RNA polymerase gene (rpoB). J Microbiol Methods 54:325–337. doi:10.1016/S0167-7012(03)00065-4
Lu X, Mo ZY, Zhao HB, Yan H, Shi L (2011) LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila. Appl Microbiol Biotechnol 92:179–187. doi:10.1007/s00253-011-3496-8
Maurin M, Hammer L, Gestin B, Timsit JF, Rogeaux O, Delavena E, Tous J, Epaulard O, Brion JP, Croize I (2010) Quantitative real-time PCR tests for diagnostic and prognostic purposes in cases of legionellosis. Clin Microbiol Infect 16:379–384. doi:10.1111/j.1469-0691.2009.02812.x
Merault N, Rusniok C, Jarraud S, Gomez-Valero L, Cazalet C, Marin M, Brachet E, Aegerter P, Gaillard JL, Etienne J, Herrmann JL, DELPH-I Study Group, Lawrence C, Buchrieser C (2011) Specific real-time PCR for simultaneous detection and identification of Legionella pneumophila serogroup 1 in water and clinical samples. Appl Environ Microbiol 77:1708–1717. doi:10.1128/AEM.02261-10
Moliner C, Ginevra C, Jarraud S, Flaudrops C, Bedotto M, Couderc C, Etienne J, Fournier P (2010) Rapid identification of Legionella species by mass spectrometry. J Med Microbiol 59:273–284. doi:10.1099/jmm.0.014100-0
Nazarian E, Bopp D, Saylors A, Limberger R, Musser K (2008) Design and implementation of a protocol for detection of Legionella in clinical and environmental samples. Diagn Microbiol Infect Dis 62:125–132. doi:10.1016/j.diagmicrobio.2008.05.004
O’Connor BA, Carman J, Eckert K, Tucker G, Givney R, Cameron S (2007) Does using potting mix make you sick? Results from a Legionella longbeachae case–control study in South Australia. Epidemiol Infect 135:34–39. doi:10.1017/S095026880600656X
Palusinska-Szysz M, Kalitynski R, Dawidowicz AL, Russa R, Drożański W (2008) Cellular envelope phospholipids from Legionella lytica. FEMS Microbiol Lett 283:239–246. doi:10.1111/j.1574-6968.2008.01177.x
Palusinska-Szysz M, Janczarek M, Kalitynski R, Dawidowicz AL, Russa R (2011) Legionella bozemanae synthesizes phosphatidylcholine from exogenous choline. Microbiol Res 166:87–98. doi:10.1016/j.micres.2010.02.004
Palusinska-Szysz M, Szuster-Ciesielska A, Kania M, Janczarek M, Chmiel E, Danikiewicz W (2014) Legionella dumoffii utilizes exogenous choline for phosphatidylcholine synthesis. Int J Mol Sci 15:8256–8279. doi:10.3390/ijms15058256
Phin N, Parry-Ford F, Harrison T, Stagg HR, Zhang N, Kumar K, Lortholary O, Zumla A, Abubakar I (2014) Epidemiology and clinical management of Legionnaires’ disease. Lancet Infect Dis 14:1011–1021. doi:10.1016/S1473-3099(14)70713-3
Pitcher DG, Saunders NA, Owen RJ (1989) Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol 8:151–156. doi:10.1111/j.1472-765X.1989.tb00262.x
Roig J, Rello J (2003) Legionnaires’ disease: a rational approach to therapy. J Antimicrob Chemother 51:1119–1129. doi:10.1093/jac/dkg191
Rowbotham TJ (1980) Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J Clin Pathol 33:1179–1183
Sabria M, Campins M (2003) Legionnaires’ disease: update on epidemiology and management options. Am J Respir Med 2:235–243
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Simonsen O, Wedege E, Kanestrom A, Bolstad K, Aaberge IS, Ragnhildstvient E, Ringstad J (2015) Characterization of the extent of a large outbreak of Legionnaires’ disease by serological assays. BMC Infect Dis 15:163. doi:10.1186/s12879-015-0903-2
Stolhaug A, Bergh K (2006) Identification and differentiation of Legionella pneumophila and Legionella spp. with real-time PCR targeting the 16S rRNA gene and species identification by mip sequencing. Appl Environ Microbiol 72:6394–6398. doi:10.1128/AEM.02839-05
Svarrer CW, Uldum SA (2012) The occurrence of Legionella species other than Legionella pneumophila in clinical and environmental samples in Denmark identified by mip gene sequencing and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Microbiol Infect 18:1004–1009. doi:10.1111/j.1469-0691.2011.03698.x
Wallis L, Robinson P (2005) Soil as a source of Legionella pneumophila serogroup 1 (Lp1). Aust N Z J Public Health 29:518–520. doi:10.1111/j.1467-842X.2005.tb00242.x
Waterer GW, Baselski VS, Wunderink RG (2001) Legionella and community-acquired pneumonia: a review of current diagnostic tests from clinician’s viewpoint. Am J Med 110:41–48. doi:10.1016/S0002-9343(00)00624-0
Wilderman PJ, Vasil AI, Martin WE, Murphy RC, Vasil ML (2002) Pseudomonas aeruginosa synthesizes phosphatidylcholine by use of the phosphatidycholine synthase pathway. J Bacteriol 184:4792–4799. doi:10.1128/JB.184.17.4792-4799.2002
Yanez MA, Carrasco-Serrano C, Barbera VM, Catalan V (2005) Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dot A gene. Appl Environ Microbiol 71:3433–3441. doi:10.1128/AEM.71.7.3433-3441.2005
Yang G, Benson R, Pelish T, Brown E, Winchell JM, Fields B (2010) Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region. Clin Microbiol Infect 16:255–261. doi:10.1111/j.1469-0691.2009.02766.x
Yaradou DF, Hallier-Soulier S, Moreau S, Poty F, Hillion Y, Reyrolle M, Andre J, Festoc G, Delabre K, Vandenesch F, Etienne J, Jarraud S (2007) Integrated real-time PCR for detection and monitoring of Legionella pneumophila in water systems. Appl Environ Microbiol 73:1452–1456. doi:10.1128/AEM.02399-06
Yong SF, Tan SH, Wee J, Tee JJ, Sansom FM, Newton HJ, Hartland EL (2010) Molecular detection of Legionella: moving on from mip. Front Microbiol 1(123):1–6. doi:10.3389/fmicb.2010.00123
Yu LV, Plouffe FJ, Pastoris MC, Stout JE, Schousboe M, Widmer A, Summersgill J, File T, Heath CM, Paterson DL, Chereshsky A (2002) 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
Zhou G, Cao B, Dou Y, Liu Y, Feng L, Wang L (2011) PCR methods for the rapid detection and identification of four pathogenic Legionella spp. and two Legionella pneumophila subspecies. Appl Microbiol Biotechnol 91:377–387. doi:10.1007/s00253-011-3283-6
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This work was supported by the grant from the Ministry of Science and Higher Education No. N N303 822640. The authors thank Maria Malek for excellent technical assistance.
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Communicated By: Agnieszka Szalewska-Palasz
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Janczarek, M., Palusińska-Szysz, M. PCR method for the rapid detection and discrimination of Legionella spp. based on the amplification of pcs, pmtA, and 16S rRNA genes. J Appl Genetics 57, 251–261 (2016). https://doi.org/10.1007/s13353-015-0317-2
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DOI: https://doi.org/10.1007/s13353-015-0317-2