Development of Techniques on Environmental Monitoring for Legionellae
Date Issued
2011
Date
2011
Author(s)
Chen, Nai-Tzu
Abstract
Real-time quantitative PCR (qPCR) is popularly used to quantify legionellae; however, it may be interfered by environmental qPCR inhibitors and cannot differentiate viable and non-viable cells. This study tried to overcome such problems by optimizing DNA isolation procedure first. Evaluation was performed using samples spiked with humic acid (HA, ≤126.8 mg L-1), ferric ion alone (Fe, ≤3 mg L-1) or Fe/HA (≤3/100 mg L-1). QiAamp DNA Mini Kit (Q) is the appropriate DNA isolation method for quantifying legionellae by qPCR because it removed HA from 1.9-126.8 mg L-1 to an undetectable level (< 1 mg L-1), obtained the highest DNA yield, and relieved qPCR inhibition and acquired the highest cell recovery under 10- or 100-fold dilution. Further validation with cooling tower (CT) water shows that for detecting Legionella pneumophila and detecting/quantifying Legionella spp. by qPCR, Q with post 10-fold dilution is suggested to perform first. Afterward, L. pneumophila-positive samples should be re-analyzed a 100-fold dilution to acquire accurate cell concentrations.
Secondly, the capability of an internal inhibition control (IIC) to quantify the level of qPCR inhibition was assessed. Evaluation of L. pneumophila DNA-spiked samples, with or without the addition of HA or Fe, shows a significant and negative correlation between qPCR inhibition levels and qPCR-determined quantities of L. pneumophila DNA (r = -0.917). Such a dose-response relationship was also found in samples taken from CT and hot water systems (HWS) with detectable legionellae and > 5% partial qPCR inhibition levels (r = -0.635 and -0.826). These findings demonstrate that the IIC assay can reliably quantify > 5% partial qPCR inhibition levels. Moreover, for environmental samples, dilution treatment can significantly reduce qPCR inhibition levels and increase legionellae concentrations, highlighting the ability of the IIC assay to monitor the effectiveness of an intervention on the relief of inhibition. Thirdly, this study optimized the technique of combining ethidium monoazide (EMA, 0.9-45.5 μg mL-1) with qPCR (i.e. EMA-qPCR) and evaluated its environmental applicability on quantifying viable legionellae in water and biofilm of CT and HWS. qPCR with EMA at 2.3 μg mL-1 was determined as the optimal EMA-qPCR assay, which can accurately quantify viable L. pneumophila, L. anisa and legionellae-like amoebal pathogens 6 without interferences by heated legionellae, unheated non-legionellae cells, and CT water matrix (p > 0.05). For water and biofilm samples of CT and HWS, the viable legionellae counts determined by EMA-qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR. A nursing home survey conducted found a higher prevalence of legionellae in CT with a time interval between sampling and last cleaning > 50 days, dissolved organic carbon (DOC) > 20 mg L-1, and conductivity > 2,000 μs cm-1 (all p < 0.05), while legionellae concentrations were positively associated with non-operating CT and an increase in water hardness and total suspended solids (TSS) (all p < 0.05). Moreover, legionellae were more found in HWS with hardness > 50 mg L-1 as CaCO3, pH > 7.5, free chlorine ≤ 0.1 mg L-1, presence of Hartmannella vermfiormis, heterotrophic plate counts (HPC) in swab > 7 log CFU per cm2, building age > 24 years, and faucets without filters (all p < 0.05), while their abundance was increased with building age, pH, conductivity, and TSS (all p < 0.05). A higher level of HPC in HWS also increased legionellae concentration (p < 0.05). Furthermore, presence of legionellae in hot water was enhanced for DOC > 1 mg L-1 (p < 0.05), whereas legionellae prevalence and counts in swab were negatively correlated with water TSS (both p < 0.05). These risk factors should be considered in the control of legionellae contamination in CT and HWS to minimize the risk of legionellae infection. In conclusion, this study successfully optimizes DNA isolation procedure and develops the IIC assay for overcoming qPCR inhibition and demonstrates the applicability of EMA-qPCR for quantifying viable legionellae. Moreover, the risk factors for legionellae in CT and HWS are clearly identified, which would help to control legionellae and prevent legionellosis.
Secondly, the capability of an internal inhibition control (IIC) to quantify the level of qPCR inhibition was assessed. Evaluation of L. pneumophila DNA-spiked samples, with or without the addition of HA or Fe, shows a significant and negative correlation between qPCR inhibition levels and qPCR-determined quantities of L. pneumophila DNA (r = -0.917). Such a dose-response relationship was also found in samples taken from CT and hot water systems (HWS) with detectable legionellae and > 5% partial qPCR inhibition levels (r = -0.635 and -0.826). These findings demonstrate that the IIC assay can reliably quantify > 5% partial qPCR inhibition levels. Moreover, for environmental samples, dilution treatment can significantly reduce qPCR inhibition levels and increase legionellae concentrations, highlighting the ability of the IIC assay to monitor the effectiveness of an intervention on the relief of inhibition. Thirdly, this study optimized the technique of combining ethidium monoazide (EMA, 0.9-45.5 μg mL-1) with qPCR (i.e. EMA-qPCR) and evaluated its environmental applicability on quantifying viable legionellae in water and biofilm of CT and HWS. qPCR with EMA at 2.3 μg mL-1 was determined as the optimal EMA-qPCR assay, which can accurately quantify viable L. pneumophila, L. anisa and legionellae-like amoebal pathogens 6 without interferences by heated legionellae, unheated non-legionellae cells, and CT water matrix (p > 0.05). For water and biofilm samples of CT and HWS, the viable legionellae counts determined by EMA-qPCR were mostly greater than the culturable counts by culture assay but consistently lower than the total cell counts quantified by qPCR. A nursing home survey conducted found a higher prevalence of legionellae in CT with a time interval between sampling and last cleaning > 50 days, dissolved organic carbon (DOC) > 20 mg L-1, and conductivity > 2,000 μs cm-1 (all p < 0.05), while legionellae concentrations were positively associated with non-operating CT and an increase in water hardness and total suspended solids (TSS) (all p < 0.05). Moreover, legionellae were more found in HWS with hardness > 50 mg L-1 as CaCO3, pH > 7.5, free chlorine ≤ 0.1 mg L-1, presence of Hartmannella vermfiormis, heterotrophic plate counts (HPC) in swab > 7 log CFU per cm2, building age > 24 years, and faucets without filters (all p < 0.05), while their abundance was increased with building age, pH, conductivity, and TSS (all p < 0.05). A higher level of HPC in HWS also increased legionellae concentration (p < 0.05). Furthermore, presence of legionellae in hot water was enhanced for DOC > 1 mg L-1 (p < 0.05), whereas legionellae prevalence and counts in swab were negatively correlated with water TSS (both p < 0.05). These risk factors should be considered in the control of legionellae contamination in CT and HWS to minimize the risk of legionellae infection. In conclusion, this study successfully optimizes DNA isolation procedure and develops the IIC assay for overcoming qPCR inhibition and demonstrates the applicability of EMA-qPCR for quantifying viable legionellae. Moreover, the risk factors for legionellae in CT and HWS are clearly identified, which would help to control legionellae and prevent legionellosis.
Subjects
legionellae
real-time quantitative PCR
qPCR inhibition
internal inhibition control
nursing home
Type
thesis
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