Comparing methods of determining Legionella spp. in complex water matrices

BMC Microbiology

Guillermo Rodríguez²  Roberto Fernández-Lafuente⁶  Inmaculada Solís⁵  Carmen Bayón³  Eva María Alejandres³  Francisco Javier Castro³  Juan Carlos Montero⁴  Álvaro Díaz-Flores¹ 

[1] Departamento de Microbiología General III, Facultad de Ciencias Biológicas, Universidad Complutense de Madrid, Campus Moncloa, Madrid, 28040, Spain;Biótica, Bioquímica Analítica, S.L, Science and Technology Park of Jaume I University, Campus RiuSec - Espaitec 2, planta baja, Castellón de la Plana, E12071, Spain;Laboratorio Regional de Salud Pública Consejería de Sanidad y Consumo/Comunidad de Madrid, C/ Sierra del Alquife N 8, 2 Planta, Madrid, 28053, Spain;Instituto de Ciencias de la Salud Ctra, de Extremadura Km. 114, Talavera de la Reina, 45600, Spain;Iproma, S.L, Cno.de la Raya 46, Castellón, 12005, Spain;Departamento de Biocatálisis, Instituto de Catálisis y Petroleoquímica, Consejo Superior de Investigaciones Científicas, Campus UAM-CSIC, Madrid, 28049, Cantoblanco, Spain

关键词: Magnetic particles;    Environmental samples;    Detection;    Legionella;   

Others  :  1212055
DOI  :  10.1186/s12866-015-0423-7

 received in 2014-11-18, accepted in 2015-04-15,  发布年份 2015

【 摘 要 】

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 10³ 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.

Conclusion

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.

【 授权许可】

   
2015 Díaz-Flores et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

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