Comparing methods of determining Legionella spp. in complex water matrices.
BACKGROUND
Legionella testing conducted at environmental laboratories plays an essential role in assessing the risk of disease transmission associated with water systems. However, drawbacks of culture-based methodology used for Legionella enumeration can have great impact on the results and interpretation which together can lead to underestimation of the actual risk. Up to 20% of the samples analysed by these laboratories produced inconclusive results, making effective risk management impossible. Overgrowth of competing microbiota was reported as an important factor for culture failure. For quantitative polymerase chain reaction (qPCR), the interpretation of the results from the environmental samples still remains a challenge. Inhibitors may cause up to 10% of inconclusive results. This study compared a quantitative method based on immunomagnetic separation (IMS method) with culture and qPCR, as a new approach to routine monitoring of Legionella.


RESULTS
First, pilot studies evaluated the recovery and detectability of Legionella spp using an IMS method, in the presence of microbiota and biocides. The IMS method results were not affected by microbiota while culture counts were significantly reduced (1.4 log) or negative in the same samples. Damage by biocides of viable Legionella was detected by the IMS method. Secondly, a total of 65 water samples were assayed by all three techniques (culture, qPCR and the IMS method). Of these, 27 (41.5%) were recorded as positive by at least one test. Legionella spp was detected by culture in 7 (25.9%) of the 27 samples. Eighteen (66.7%) of the 27 samples were positive by the IMS method, thirteen of them reporting counts below 10(3) colony forming units per liter (CFU l(-1)), six presented interfering microbiota and three presented PCR inhibition. Of the 65 water samples, 24 presented interfering microbiota by culture and 8 presented partial or complete inhibition of the PCR reaction. So the rate of inconclusive results of culture and PCR was 36.9 and 12.3%, respectively, without any inconclusive results reported for the IMS method.


CONCLUSIONS
The IMS method generally improved the recovery and detectability of Legionella in environmental matrices, suggesting the possibility to use IMS method as valuable indicator of risk. Thus, this method may significantly improve our knowledge about the exposure risk to these bacteria, allowing us to implement evidence-based monitoring and disinfection strategies.
Departamento de Microbiología General III, Facultad de Ciencias Biológicas, Universidad Complutense de Madrid, Campus Moncloa, 28040, Madrid, Spain. alvarodiazflores@pas.ucm.es.
Álvaro
Á
Díaz-Flores
Instituto de Ciencias de la Salud Ctra, de Extremadura Km. 114, 45600, Talavera de la Reina, Spain. jcmontero@jccm.es.
Juan Carlos
JC
Montero
Laboratorio Regional de Salud Pública Consejería de Sanidad y Consumo/Comunidad de Madrid, C/ Sierra del Alquife N 8, 2 Planta, 28053, Madrid, Spain. javier.castro@salud.madrid.org.
Francisco Javier
FJ
Castro
Laboratorio Regional de Salud Pública Consejería de Sanidad y Consumo/Comunidad de Madrid, C/ Sierra del Alquife N 8, 2 Planta, 28053, Madrid, Spain. eva.alejandres@salud.madrid.org.
Eva María
EM
Alejandres
Laboratorio Regional de Salud Pública Consejería de Sanidad y Consumo/Comunidad de Madrid, C/ Sierra del Alquife N 8, 2 Planta, 28053, Madrid, Spain. maria.bayon@salud.madrid.org.
Carmen
C
Bayón
Iproma, S.L, Cno.de la Raya 46, 12005, Castellón, Spain. isolis@iproma.com.
Inmaculada
I
Solís
Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, Consejo Superior de Investigaciones Científicas, Campus UAM-CSIC, 28049, Cantoblanco, Madrid, Spain. rfl@icp.csic.es.
Roberto
R
Fernández-Lafuente
Biótica, Bioquímica Analítica, S.L, Science and Technology Park of Jaume I University, Campus RiuSec - Espaitec 2, planta baja, E12071, Castellón de la Plana, Spain. guiller@biotica.es.
Guillermo
G
Rodríguez
D058491:Bacterial Load / Q000379:methods*; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D007875:Legionella / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D014871:Water Microbiology