Molecular and Cellular Probes xxx (2014) 1e6

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Legionella in water samples: How can you interpret the results obtained by quantitative PCR? Savina Ditommaso*, Elisa Ricciardi, Monica Giacomuzzi, Susan R. Arauco Rivera, Carla M. Zotti Department of Public Health and Pediatrics, University of Turin, P.zza Polonia, 94, 10126 Turin, Italy

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 June 2014 Accepted 9 September 2014 Available online xxx

Evaluation of the potential risk associated with Legionella has traditionally been determined from culture-based methods. Quantitative polymerase chain reaction (qPCR) is an alternative tool that offers rapid, sensitive and specific detection of Legionella in environmental water samples. In this study we compare the results obtained by conventional qPCR (iQ-Check™ Quanti Legionella spp.; Bio-Rad) and by culture method on artificial samples prepared in Page's saline by addiction of Legionella pneumophila serogroup 1 (ATCC 33152) and we analyse the selective quantification of viable Legionella cells by the qPCR-PMA method. The amount of Legionella DNA (GU) determined by qPCR was 28-fold higher than the load detected by culture (CFU). Applying the qPCR combined with PMA treatment we obtained a reduction of 98.5% of the qPCR signal from dead cells. We observed a dissimilarity in the ability of PMA to suppress the PCR signal in samples with different amounts of bacteria: the effective elimination of detection signals by PMA depended on the concentration of GU and increasing amounts of cells resulted in higher values of reduction. Using the results from this study we created an algorithm to facilitate the interpretation of viable cell level estimation with qPCR-PMA.

Keywords: Legionella spp PCR (polymerase chain reaction) Enumeration Environmental water

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction Evaluation of the risk associated with Legionella has traditionally been performed using culture-based methods. In fact, only culture method [1] and qPCR [2] are normalised methods for the quantitation of Legionella (as defined by standardisation of protocol, confirmation of its performance in different laboratories, applicability to different types of environmental samples and robustness). Bacterial culture is essential for identifying and typing Legionella strains, however, Legionella culture requires long incubation times (up to 10 days) before the results can be scored. Moreover, various factors can influence method accuracy: differences in the membrane, such as pore size, batches, fragility, crinkling, and electrostatic interactions, and differences in the procedures for washing the organisms from the membrane, such as using a shaker/vortex, ultrasound, or finger and thumb scraping. Quantitative polymerase chain reaction (qPCR) is an alternative tool that offers rapid, sensitive and specific detection of Legionella * Corresponding author. Tel.: þ39 0116705841; fax: þ39 0116705881. E-mail address: [email protected] (S. Ditommaso).

in environmental water samples [3e8]. This method may overcome many of the disadvantages associated with traditional culture, but it has two disadvantages: it can overestimate the amount of microorganisms that are present in the sample, and it cannot discriminate between viable culturable cells, viable but nonculturable (VBNC) cells, and non-viable cells or extraneous DNA persisting in the environment [9,10]. Therefore, the monitoring of Legionella contamination levels by conventional qPCR may result in an overestimation of the risk of infection because of false-positive results. However, the real risk from Legionella is limited to the live fraction of the total Legionella population. Only live or viable Legionella cells are able to replicate in pulmonary macrophages, which can result in severe pneumonia [11,12]. The development of more rapid, culture-independent methods that are capable of discriminating between live and dead cells is very important for the measurement of Legionella infection risks and prevention of legionellosis. Several commercial real-time kits designed for detection and quantification of Legionella species in water by the real-time PCR are currently available. These kits include the iQ-check real-time PCR kit (Bio-Rad France), the Aqua Screen Lp-qDual kit (Minerva

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Please cite this article in press as: Ditommaso S, et al., Legionella in water samples: How can you interpret the results obtained by quantitative PCR?, Molecular and Cellular Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.09.002

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Biolabs, Germany), GeneDisc legionella kit (GeneSystems, France), and the Mericon Quant Legionella spp. Kit (Qiagen). However, none of these kits provide a solution for discriminating between viable and nonviable Legionella bacteria. There are many publications that address this issue for various indicator organisms or environmental pathogens including for Legionella. The use of photoactivatable cell membrane impermeable nucleic acid intercalating dyes, such as ethidium monoazide (EMA) or propidium monoazide (PMA), followed by light exposure prior to DNA extraction and amplification has been one of the most successful approaches to detect viable cells. Light activation of DNA-bound dye molecules results in an irreversible DNA modification and subsequent inhibition of its amplification. Using nucleic acid-binding dyes (EMA or PMA) in combination with qPCR is an attractive alternative for selectively detecting and enumerating viable bacteria (leading to the term viability PCR, v-PCR) [13e15]. This technique is not limited to bacteria but has also successfully been applied to fungi, protozoa and viruses. Despite its advantages, there is evidence demonstrating that v-PCR has practical and theoretical limitations, especially when applied to environmental samples [16e18]. The greatest concern with using EMA lies in its capacity to penetrate bacterial cells with intact membranes and cause underestimation of live cell population and the greatest concern with using PMA is the generation of false-positive signals due to incomplete signal suppression. Several comparative studies have confirmed that PMA outperforms EMA in selective removal of dead cells when combined with real-time PCR [19e21]. For the successful application of v-PCR, several factors that can influence the outcome of the results must to be considered. These factors include the choice of dye, dye concentration, contact mode (cell suspension or cells impinged on filters), incubation conditions, light source, presence of a high number of dead cells, presence of high levels of suspended solids or biomass in the analysed samples, salt concentration in the reaction mix, pH of the reaction mix, length of the target gene and sequence of the target gene. Several published articles outline the various approaches that have been used to detect viable cells in combination with nucleic acid amplification methods which include the following: - PMA solution ranging from 6.25 mM [22] to 50 mM [23] to 400 mM [15], where the authors obtained signal reductions ranging from 3.85 to 5 log units. - Incubation periods in the dark ranging from 5 min [19,15,24] to 20 min [25] and light exposure from 2 min [23] to 20 min [26]. - Photoactivation carried out using high-power halogen lamps ranging from 500 to 750 W [23,27] and samples placed horizontally on ice a distance of 20e30 cm from the light source [19,24,28]. - Amplicon length affect qPCR: longer DNA sequences correlate with a higher probability that the DNA polymerase will encounter modifications in the stretch of DNA that is targeted by the primers which will result in an increased suppression of signals from membrane-compromised cells [24,29,30]. After applying PMA to heat-killed Legionella pneumophila, there was a significant difference in the number of dead cells that was measured when qPCR was based on the amplification of 16SrRNA (454 bp) or 5S rRNA (108 bp) [15]. - The ratio of live to dead cells can affect the efficiency of the method. Quantification of live cells by PMA-PCR in the presence of high numbers of dead cells was difficult when the concentration of live cells was lower than 105 cells ml1 [15]. The reason that dead cells influence vPCR signals is currently not clear. However, it appears plausible that dead cells have the ability to take up dye which lowers the concentration of available dye molecules per cell. Varma et al. [18] have suggested that

the effectiveness of PMA activity may be saturated by increasing cell numbers. - The treatment with the PMA can be carried out by applying the dye directly to the filter (used to concentrate the sample) [22,25] or into the tubes [23,26]. The aims of this study are to compare the results obtained by conventional qPCR and culture methods on artificial samples and to analyse the selective quantification of viable Legionella cells by the qPCR-PMA method. These comparisons will be used to define a simple algorithm for the interpretation of environmental Legionella monitoring results obtained using molecular methods. 2. Materials and methods 2.1. Comparison of Legionella quantification by culture and q-PCR The comparison of the quantitative results was made on artificial samples. L. pneumophila serogroup 1 (ATCC 33152) was inoculated into AYE with 0.0025% ferric pyrophosphate (Sigma) and 0.04% L-cysteine (Oxoid) and incubated with shaking at 170 r.p.m. until the culture broth reached an OD 600 of 0.5. The bacterial suspension was prepared in Page's saline and adjusted to a concentration of 1  106 cells ml1 which was confirmed by plate counting on BCYEa agar. For qPCR quantification, the cell cultures were serial diluted to obtain suspensions that contained 1  105, 1  104, 1  103, 1  102 and 1  101 cells ml1. Ten samples were prepared for each contamination level in order to evaluate the repeatability of the technique. The DNA extraction were performed following the Aquadien™ Bio-Rad protocol for the lysis, concentration and purification steps (without sample filtration) (Fig. 1). 2.2. Efficacy of PMA treatment The ability of qPCR-PMA to detect dead cells was tested either on artificial samples with different amounts of heat killed L. pneumophila cells and with different amounts of DNA extracted from Legionella. The last to assess the ability of PMA to bind to the DNA without the influence of the cellular membrane. 2.2.1. Heat killed cells A bacterial suspension was prepared in Page's saline and adjusted to a concentration of 108 cells ml1 as confirmed by plate counting. Seven serial dilutions of the bacterial suspension were prepared and each concentration was boiled for 15 min. The efficacy of heat treatment was confirmed by plating the suspension on BCYEa medium. For each concentration of the heat-killed Legionella suspension, two fractions were obtained and filtered through a 0.4 mm porosity polycarbonate membrane then the PMA solution (50 mM) was added directly to one filter. The data (not reported) obtained during optimisation of the PMA method (by modifying variables such as the concentration of PMA, incubation time, light source, distance from the light and light exposure time) indicated to us that the most efficient protocol is: PMA 50 mM, incubation in the dark for 10 min followed by a 10 min exposure to 500 W light at a distance of 20 cm from the light source on ice. After irradiation the bacterial DNA was extracted using the Aquadien™ extraction kit (Bio-Rad) according to the manufacturer's instructions (Fig. 1). 2.2.2. DNA Serial dilutions were made to prepare the samples which ranged from 1  107 cells ml1 to 1  101 cells ml1. We centrifuged 10 ml of each bacterial suspension at 3000 g at 4  C for 30 min. The supernatant was removed and the cells were resuspended in a lysis buffer for DNA extraction (Aquadien™ extraction kit Bio-Rad). For

Please cite this article in press as: Ditommaso S, et al., Legionella in water samples: How can you interpret the results obtained by quantitative PCR?, Molecular and Cellular Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.09.002

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Fig. 1. PMA treatment on Legionella cells and on Legionella DNA.

each concentration of Legionella DNA, two fractions were obtained and 50 mM. PMA was added to one aliquot of each concentration. The samples were then incubated in the dark for 10 min followed by a 10 min exposure to a 500 W halogen light, at a distance of 20 cm from the light source on ice (Fig. 1). 2.3. Quantification by qPCR The analysis was performed by iQ-Check™ Quanti Legionella spp., according to the manufacturer's instructions (Bio-Rad). The iQ-Check™ Quanti Legionella spp. is NF VALIDATION certified (certificate numbers BRD07/15-12/15), and it contains reagents to amplify and quantify about 100 bp fragment from the 5S rRNA gene of Legionella spp. Using this method Legionella can be quantified in less than 3 h following the water sample filtration and DNA extraction steps. The detection limit of this qPCR method is 5 Genomic Unit (GU) per well, corresponding to 80 GU L1. The quantification limit of the method is 608 GU L1. A genomic unit includes all gene copies (3 copies of 5S rRNA gene) contained in a cell. 2.4. Statistical analysis All qPCR data were analysed using Opticon Monitor Analysis Software version 3.4 (Bio-Rad). To compare the quantification by culture and by qPCR, the Dlog10 values were calculated (log10 value for qPCR  log10 values

for culture). Statistical analysis to calculate the mean values and the standard deviation were performed using Microsoft Excel. To compare the ability of PMA to reduce signal in heat killed cells or in extracted DNA, a simple linear regression line was generated for each data group (DNA and cells) and the two regression lines were compared to test the hypothesis of coincidence. To quantify the effect of PMA on heat killed cells, Dlog10 values were calculated (log10 value qPCR  log10 values qPCR-PMA). 3. Results 3.1. Comparison of Legionella quantification by culture and q-PCR We analysed 60 samples that contained different amounts of Legionella (106 e 101 CFU ml1). The results from the qPCR analysis were expressed in GU ml1 values, and they were compared to the culture results, which were expressed in CFU ml1. The amount of Legionella DNA (GU ml1) determined by qPCR was generally higher than the concentration of Legionella (CFU ml1) estimated using the culture method. The mean log difference was 1.45 (SD 0.24) equal to a load 28-fold higher than the load detected by culture (Table 1). 3.2. Efficacy of PMA treatment The experiments that used different amounts (101e107 CFU) of heat killed L. pneumophila cells and different amounts of extracted

Please cite this article in press as: Ditommaso S, et al., Legionella in water samples: How can you interpret the results obtained by quantitative PCR?, Molecular and Cellular Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.09.002

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Table 1 Comparison of results obtained by culture isolation and qPCR for enumeration of serially diluted live Legionella pneumophila cells. Culture log CFU ml1

qPCR log GU ml1(SD)

a

5 4 3 2 1 0

6.4 5.29 4.24 3.34 2.56 1.89

1.40 1.29 1.24 1.34 1.56 1.89

(±0.02) (±0.10) (±0.04) (±0.07) (±0.16) (±0.23)

Dlog

Standard deviation from ten independent experiments is given in brackets. Dlog mean ¼ 1.45 ± 0.24. a Dlog ¼ log GUlog CFU.

DNA demonstrated an elevated Dlog10 (log10 value qPCR  log10 values qPCR-PMA) for the higher concentration of Legionella (107), with values of 3.7 and 5.5. The signals from the samples that contained 106-103 heat killed cells or DNA were also inhibited by PMA even if the difference was less (Dlog102.91.7). However, in samples that contained low amounts of heat killed cells or DNA (102101), the signals remained detectable (Dlog10 0.6e0.8) (Table 2). Overall, increasing the Legionella cells concentrations increases the reduction of the signal starting from values of 0.63 log e 1.48 log 1.77 log 1.91 log e 2.17 log 2.93 log, up to a maximum of 3.67 log. The same trend was observed on extracted DNA. The signals reduction from the sample of heat killed cells and of the extracted DNA were compared and they were found to be similar. There were no significant differences between the values of the slope (1.964 vs 1.464; F ¼ 0.687; P ¼ 0.525) (Fig. 2). Taking into account that the bacteria load most frequently isolated from environmental samples ranged from 102 to 105 CFU/L [31e35], we calculated the mean of the delta values for 102, 103, 104 and 105 cell concentrations (Dlog mean ¼ 1.83). Therefore applying the described protocol (iQ-Check™ Quanti Legionella spp. combined with PMA treatment) it is possible to reduce 98.5 0% of the PCR signal from dead cells. 4. Discussion Legionella is found ubiquitously throughout aquatic habitats. From its natural reservoir (e.g., lakes, rivers, thermal springs) it can enter and colonise man-made water supply systems (e.g., water distribution plants, tanks, cooling towers) and from the water supplies it can spread to humans. Although no direct correlation has been demonstrated between Legionella load and risk of legionellosis, measurement of the microbial load underlies the analysis of the water samples taken from distribution systems.

Table 2 Efficacy of PMA treatment to eliminate detection signals from heat killed Legionella cells and extracted DNA in artificial samples. Cells Log concentration a heat-killed qPCR cell CFU log GU

Cells b

7 6 5 4 3 2 1

4.52 3.97 3.46 2.48 2.05 1.38 2.07

a

8.19 6.90 5.63 4.39 3.82 2.86 2.69

DNA c

DNA d

Cells aeb

DNA ced

qPCR PMA qPCR qPCR PMA Signal Signal log GU log GU log GU reductiona reductiona 9.20 8.26 6.80 4.99 4.11 2.41 1.96

3.73 5.54 4.93 3.23 2.27 1.54 1.15

3.67 2.93 2.17 1.91 1.77 1.48 0.63

5.47 2.72 1.87 1.76 1.84 0.87 0.81

Signal reduction ¼ Dlog10 values (log10 value qPCR  log10 values qPCR-PMA).

National [36] and international guidelines for Legionella prevention and control [37e39] set risk and type of intervention measures in water distribution systems based on the Legionella load detected in the samples. The most commonly used method for environmental surveillance of Legionella is the standard culture technique. Culture is essential for identifying and typing Legionella strains. However, culture methods require long incubation times (up to 10 days) before the results can be scored. This method can only be used by laboratories with consolidated experience in identifying various Legionella species from the many bacteria species that are found in an aquatic environment. To overcome the limitations of Legionella detection by culture, we utilised Bio-Rad kits to perform a qPCR quantitative assay that targeted the 5S rRNA of Legionella species (AquadienTM and iQCheckTM) and optimised the qPCR-PMA technique to quantify viable Legionella cells in environmental water samples. Comparing the quantification of live cells in artificial samples by culture to the values measured by qPCR showed us that Legionella counts calculated from qPCR were 28-fold higher (mean log difference ¼ 1.45) than those detected by culture. If we were to apply this method to environmental samples, then it must be considered that the potentially infectious fraction of L. pneumophila bacteria could be overestimated by conventional qPCR. Therefore, the high PCR signals should be critically interpreted and do not necessarily represent a health risk for exposed persons. This difference may be attributed to both the sensitivity difference between the two methods [8] and to the presence of Legionella doublets or chains, which are counted as only 1 CFU by the culture method but quantified as individual cells by qPCR. With regard to culture, some publications [14] suggest the hypothesis that several Legionella cells could be aggregated and only the origin of a colony, minimising the result. Moreover some legionella strains could have more 5S gene copies than the references strain used for the PCR standard curve [5]. The effects of PMA and its ability to quantify Legionella in environmental samples were comparatively analysed through the use of heat-killed cells and by using extracted Legionella DNA in order to assess the permeation of PMA through the membrane compromised cells. The experiments involving a Legionella pure culture that had been heat killed and treated with PMA demonstrated that there was an incomplete signal reduction in dead cells using the qPCRPMA method. These results have also been reported in other studies that targeted relatively short PCR amplicon sizes [20,40]. For both the EMA-PCR and PMA-PCR intercalating dye viability assays, complete dead cell signal suppression was observed when long-amplicon qPCR was amplified [29,30,41]. PMA treatment followed by short amplicon qPCR was previously shown to result in an incomplete signal reduction but, unlike the other methods, we observed a dissimilarity in the ability of PMA to suppress the PCR signal in samples with different amounts of bacteria. In our experiments, the effective elimination of detection signals by PMA depended on the concentration of GU and increasing amounts of cells resulted in higher values of reduction. This trend was also observed in DNA samples that were treated with PMA (Table 2). In contrast, when Slimani et al. [23] used the Bio-Rad kits (AquadienTM and iQ-CheckTM) and PMA at a concentration of 6.25 mM for the treatment of filtration membranes, a Dlog ¼ 3.87 for all of the bacterial load was obtained. The theory that links PMA treatment to the inhibition of DNA amplification in PCR must be considered with some caution. We demonstrated that by using heat-treated Legionella qPCR-PMA it is possible to reduce the PCR signal by 98.5% which is equal to a Dlog mean of 1.83.

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Fig. 2. Comparison of the effect of PMA þ light treatment on heat killed cells or extracted DNA. The linear regression model generated for each data group (DNA and cells) to test the hypothesis of coincidence showed no significant differences between the values of the slope (1.964 vs. 1.464; F ¼ 0.687; P ¼ 0.525).

Using the results of this study we created an algorithm, which is outlined in Fig. 3, to facilitate the interpretation of viable cell level estimation with qPCR-PMA. Taking into account that environmental samples of Legionella may include culturable cells, VBNC and dead cells, the final results of our method will include all viable (culturable and non culturable) and 1% of the dead Legionella cells (PMA was not able to eliminate all dead Legionella cells). To obtain a more accurate representation of the number of viable organisms in an environmental sample, we suggest to adjust qPCR-PMA results

by dividing the value expressed in GU 28 times. This number may pose a public health concern considering that the action levels for positive Legionella counts in national and international guidelines are based on culture values. These results present a basic tool for the application of our qPCR-PMA protocol in the field to assess the method for environmental monitoring of legionellae in water systems subjected to different disinfection procedures (continuous chlorination, thermal shock, etc).

Fig. 3. qPCR-PMA and quantification of Legionella in environmental samples:suggested algorithm for correlation of molecular and culture results.

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Acknowledgements We are grateful to Prof. Adriano Ceccarelli for good advice and expertise. The study was supported by a grant from the special Department fund ZOTC1PRE00.

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Please cite this article in press as: Ditommaso S, et al., Legionella in water samples: How can you interpret the results obtained by quantitative PCR?, Molecular and Cellular Probes (2014), http://dx.doi.org/10.1016/j.mcp.2014.09.002

Legionella in water samples: how can you interpret the results obtained by quantitative PCR?

Evaluation of the potential risk associated with Legionella has traditionally been determined from culture-based methods. Quantitative polymerase chai...
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