Roman Iakymchuk

Stef Graillat

Sylvain Collange

David Defour

ExBLAS: Reproducible and Accurate BLAS Library

We aim at providing algorithms and implementations for fundamental linear algebra operations -like the ones included in the Basic Linear Algebra Subprograms (BLAS) librarythat would deliver reproducible and accurate results with reasonable performance overhead compared to the standard non-reproducible implementations on modern parallel architectures such as Intel processors, Intel Xeon Phi co-processors, and GPU accelerators.

Introduction

In general, BLAS routines rely on the optimized version of parallel reduction and dot product involving floating-point additions and multiplications operations that are nonassociative. Due to non-associativity of these operations and dynamic scheduling on parallel architectures, getting a bitwise reproducible floating-point result for multiple executions of the same code on different or even similar parallel architectures is challenging. These discrepancies worsen on heterogeneous architectures -such as clusters composed of standard CPUs in conjunction with GPU accelerators and/or Intel Xeon Phi co-processors -, which combine together different programming environments that may obey various floating-point models and offer different intermediate precision or different operators. Such non-determinism and non-reproducibility of floating-point computations on parallel machines causes validation and debugging issues, and may even lead to deadlocks. Existing solutions to enhance reproducibility of BLAS routines are • Fixed reduction scheme such as Intel's "Conditional Numerical Reproducibility" (CNR) available as an option in the Math Kernel Library (MKL);

• Avoid rounding error by using the Kulisch accumulator [START_REF] Kulisch | Computer arithmetic and validity[END_REF];

• Mixed solution as the one proposed by Demmel and Nguyen for BLAS level-1.

Our Approach

We introduced in [START_REF] Sylvain Collange | Full-Speed Deterministic Bit-Accurate Parallel Floating-Point Summation on Multi-and Many-Core Architectures[END_REF] an approach to compute deterministic sums of floating-point numbers. This approach is based on a multi-level algorithm that combines efficiently floating-point expansion [START_REF] Hida | Algorithms for quad-double precision floating point arithmetic[END_REF], which are placed in registers, and Kulisch accumulator.

Performance Results

We provided implementations of the multi-level summation scheme on a range of parallel platforms: desktop and server CPUs, the Intel Xeon Phi many-core accelerator, and both NVIDIA and AMD GPUs. We relied on the parallel summation algorithm as well as exact multiplication to develop the fast, accurate, and reproducible implementations of fundamental linear algebra operations such as dot product, triangular solver, and matrixmatrix multiplication. We verified the accuracy of our implementations by comparing the computed results with the ones produced by the multiple precision MPFR library.

Conclusions and Future Work

We presented an approach to achieve correct rounding for the floating-point summation problem, along with implementations on multi-and many-core architectures. This yielded results that are both reproducible and accurate to the last bit at no performance lost. We applied this approach to the other BLAS routines. Even though the performance of triangular solver and matrix product can be argued, their output is consistently reproducible and accurate independently of threads scheduling and data partitioning. We plan to extend this approach to the rest of BLAS routines, derive specific implementations for compute-bound algorithms to be within 10 times performance overhead at most, and perform theoretical analysis of their accuracy.

Fig. 1 :

 1 Fig. 1: Hierarchical superaccumulation scheme.

Fig. 2 :Fig. 3 :Fig. 4 :

 234 Fig.2: The summation performance results on an 8-core Intel Xeon E5-4650L.

Acknowledgement

This work undertaken (partially) in the framework of CALSIMLAB is supported by the public grant ANR-11-LABX-0037-01 overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (reference: ANR-11-IDEX-0004-02). This work was also granted access to the HPC resources of ICS financed by Region Île-de-France and the project Equip@Meso (reference ANR-10-EQPX-29-01).


J Baudart
email: baudart@obs-banyuls.fr

A Mercier

P Lebaron

Rapid quantification of viable Legionella in nuclear cooling tower waters using filter cultivation, fluorescent in situ hybridization, and solid phase cytometry

Keywords: Legionella spp., microcolonies, FISH, cooling tower water, solid phase cytometry

Aims: to develop a rapid and sensitive method to quantify viable Legionella spp in cooling tower water samples.

Methods and Results:

A rapid, culture-based method capable of quantifying as few as 600

Legionella microcolonies per liter within 2 days in industrial waters was developed. The method combines a short cultivation step of microcolonies on GVPC agar plate, specific detection of Legionella cells by a fluorescent in situ hybridization (FISH) approach, and a sensitive enumeration using a solid phase cytometer. Following optimization of the cultivation conditions, the qualitative and quantitative performance of the method was assessed and the method was applied to 262 nuclear power plant cooling water samples.

Conclusions:

The performance of this method was in accordance with the culture method (NF-T 90-431) for Legionella enumeration.

Significance and Impact of Study:

The rapid detection of viable Legionella in water is a major concern to the effective monitoring of this pathogenic bacterium in the main water sources involved in the transmission of legionellosis infection (Legionnaires' disease). The new method proposed here appears to be a robust, efficient and innovative means for rapidly quantifing cultivable Legionella in cooling tower water samples within 48h.

Introduction

The Legionellaceae family comprises a wide variety of species. To date, about 50 species and more than 60 serogroups have been described. The most pathogenic of the Legionella species, L. pneumophila, is responsible for the majority of cases of legionellosis infection (Legionnaires' disease), and serogroup 1 has been found in 70% of the isolates from patients suffering from this disease [START_REF] Joseph | Legionnaire's disease in Europe 2000-2002[END_REF]. However, 19 other species have been involved in human infection [START_REF] Olsen | Alternative routes for dissemination of Legionella pneumophila causing three outbreaks in Norway[END_REF] or isolated from clinical cases [START_REF] Muder | Infection due to Legionella species other than L. pneumophila[END_REF].

The main non L. pneumophila species isolated from legionellosis infections are L. longbeachae, L. bozemanii, L. micdadei, L. feeleii, L. dumoffii, L. wadsworthii, and L. anisa. Human infection occurs through the inhalation of contaminated aerosols produced from aerosol-generating devices associated with various types of water system. Frequently, Legionella species have been isolated in cooling systems, and some cooling towers have also been shown to be implicated in outbreaks of legionellosis [START_REF] Morton | Outbreak of legionnaires' disease from a cooling water system in a power station[END_REF][START_REF] Bentham | Routing sampling and control of Legionella spp. in cooling tower water systems[END_REF][START_REF] Kirrage | Investigation of an outbreak of Legionnaires' disease: Hereford, UK[END_REF]. However, cooling tower associated outbreaks are mainly caused by L. pneumophila serogroup 1 strains. Although no cases of Legionnaires' disease have been linked to the presence of Legionella in nuclear power plant cooling circuits in France, an attention is also paid to monitor Legionella concentrations in the waters of these cooling towers by the authorities. Currently, the regulatory framework in France requires the monitoring of Legionella spp. in nuclear cooling tower water using culture-based methods (French guideline AFNOR T90-431, AFNOR, 2014). The results, which consider only cultivable cells, are expressed in colony forming units (CFUs) per liter. Legionella are fastidious bacteria with a slow in vitro growth rate, and as such they require some additional factors, such as L-cysteine and iron, to promote their growth on synthetic culture media. The visual macrocolonies that are counted on culture media contains about 10 6 cells, and usually appear after 2 to 7 cultivation days for Legionella cells isolated from an environmental water sample. The French normalized method (AFNOR T90-431), which is culture-based, stipulates the use of the GVPC medium for Legionella recovery in water samples [START_REF] Afnor | Water quality -Detection and enumeration of Legionella spp. and L. pneumophila. Method by direct inoculation and after concentration by membrane filtration or centrifugation[END_REF].

However, this method has some well-recognized limitations, especially their low sensitivity, which is caused by the overgrowth of non target microorganisms. This overgrowth occurs because of the long cultivation time, despite the presence of inhibitors in the culture media, and the large number of confirmation steps required to identify a few of the species using latex agglutination tests which are time-consuming (another 2 days). There is clearly a need for a faster culture-based method for Legionella monitoring in cooling water systems.

Fluorescent in situ hybridization (FISH) is a detection technique that uses specific fluorescent-labeled DNA probes which target the rRNA of the cells of selected microorganisms to rapidly identify bacterial cells [START_REF] Amann | Phylogenetic identification and in situ detection of individual microbial cells without cultivation[END_REF]. When the rRNA in the cell is targeted, only metabolically active bacteria are detected. However, starved environmental bacteria often show low metabolic activity, and, as a result, a low rRNA content may not be detected [START_REF] Amann | Phylogenetic identification and in situ detection of individual microbial cells without cultivation[END_REF]. To overcome this limitation, bacteria can be concentrated from samples and incubated on a solid medium to increase their metabolic activity and generate Legionella microcolonies that can be detected by FISH [START_REF] Wulling | The fluorescent in situ hybridization test in comparison with culture for detection of Legionella pneumophila in water samples[END_REF][START_REF] Satoh | Enumeration of Legionella CFU by colony hybridization using specific DNA probes[END_REF]. Reducing the growth time leading to microcolony formation will not only speed up the detection process, but will make it applicable to both the starved and active cells in water samples [START_REF] Wulling | The fluorescent in situ hybridization test in comparison with culture for detection of Legionella pneumophila in water samples[END_REF]. The Legionella cells that form microcolonies can be directly identified by FISH on a filter following filtration of the sample and a short time cultivation. The fluorescent microcolonies are then enumerated using a fluorescence detection device, such as digital imaging [START_REF] London | An automated system for rapid non-destructive enumeration of growing microbes[END_REF], an epifluorescence microscope [START_REF] Baba | Rapid enumeration of active Legionella pneumophila in freshwater environments by the microcolony method combined with direct fluorescent antibody staining[END_REF], Scan VIT [START_REF] Ditommaso | Evaluation of the usefulness of a new direct immunofluorescence assay (ScanVIT-Legionella TM ) for monitoring hospital water systems contaminated with Legionella spp[END_REF], or a solid phase cytometer. The methods that use FISH in combination with a brief filter cultivation method have been developed to specifically enumerate viable pathogenic bacteria, such as members of the Enterobacteriaceae family [START_REF] Ootsubo | Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample[END_REF], Listeria [START_REF] Fuchizawa | Specific detection and quantitative enumeration of Listeria spp. using fluorescent in situ hybridization in combination with filter cultivation (FISHFC)[END_REF], and Clostridium perfringens [START_REF] Shimizu | Fluorescent in situ hybridization in combination with filter cultivation (FISHFC) method for specific detection and enumeration of viable Clostridium perfringens[END_REF]. Recently, a combination of brief filter cultivation and FISH detection for Legionella enumeration in hospital water systems using a ScanVIT fluorescence detector was proposed by [START_REF] Ditommaso | Evaluation of the usefulness of a new direct immunofluorescence assay (ScanVIT-Legionella TM ) for monitoring hospital water systems contaminated with Legionella spp[END_REF]. However, no FISH method combined with the microcolonies enumeration for Legionella monitoring in industrial cooling tower systems as cooling tower of nuclear plants supplied with crude water has been proposed so far. This technique can constitute a favorable alternative for the monitoring of viable Legionella. Nevertheless, the quantitative performance of this method is mainly linked to the ability of the detector device to enumerate a large range of Legionella concentrations in a water sample. This ability is vital for Legionella monitoring in industrial cooling tower systems, because of the variability of the bacterial concentration, which is dependent on the quality of the water and the disinfection process used. Compared to other detectors, the main advantage of the solid phase cytometer is its very low detection limit and the large number of cells it can enumerate, as it scans the whole surface of the membrane filter. Moreover, it has a powerful ability to discriminate between fluorescent labeled cells and background fluorescence (Lemarchand et al., 2001, Baudart and[START_REF] Baudart | Rapid detection of Escherichia coli in waters using fluorescent in situ hybridization, direct viable counting and solid phase cytometry[END_REF].

The main objective of this study is to develop a filter method of cultivation for microcolonies of Legionella combined with a FISH method for the rapid enumeration of viable Legionella spp. by solid phase cytometry applicable to the cooling waters of nuclear power plants, and to compare the trial method with the normalized culture method (NF-T 90-431) for Legionella spp. enumeration on a total of 262 cooling tower water samples.

Materials and methods

Oligonucleotide probes and FISH assay. Four 16S rRNA oligonucleotide probes targeting specific sequences located at different positions on 16S rRNA for Legionella spp. [Leg705, CTGGTGTTCCTTCCGATC, and Leg226, TCGGACGCAGGCTAATCT [START_REF] Manz | In situ identification of Legionellaceae using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy[END_REF]; Legall11, CCTCCTCCCCACTGAAAGT, and Legall22, CACTGTATGTCAAGGGTAGG [START_REF] Leskelä | Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay[END_REF]] were selected from the literature.

The probes were selected on their specificity, which was tested in silico, and on their closest Tm values. Specificity was first tested for each oligonucleotide individually in silico, using the probe match function of the Ribosomal database project software package [START_REF] Maidak | The RDP-II (Ribosomal database project)[END_REF] and the BLAST function from the NCBI website [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]. Because of the use of multiple probes in the same mixture for the hybridization, the presence of secondary structures within a probe (self dimers) and between probes (cross dimers) was checked using the multiple primer analyzer module available online from the ThermoScientific website. No self dimers or cross dimers were detected in any of the four probes (http://www.thermoscientificbio.com/webtools/multipleprimer/). The oligonucleotides were synthesized and conjugated at their 5' end with 6-FAM (ThermoFisher Scientific, Ulm, Germany).

Bacterial strain and media for specificity test. The specificity of the fluorescent in situ hybridization (FISH) assay was tested on culture bacteria collections. A total of 39 Legionella strains and 21 non Legionella strains, provided from reference and environmental collections were used to check the specificity of the FISH assay (Table 1). The Legionella strains were grown on buffered charcoal-yeast extract supplemented with α-ketoglutarate (αBCYE) agar containing L-cysteine (Biomérieux) at 37°C for 48 to 72 h, and the non Legionella strains were grown at 30 or 37°C, depending on their optimum growth temperature, for 24 h on nutrient agar (Biomérieux). The tests were performed with freshly cultured cells in stationary growth phase. Cell suspensions were created in phosphate-buffered saline (PBS) adjusted to pH 6.9 (Sigma Aldrich), and then 100 µl of this cell suspension (diluted in PBS, several-fold if necessary) were filtered on a 0.40 µm black polyester membrane (CB04, Cycloblack, 25 mm, CHEMUNEX® -Biomérieux). Cells hybridization was performed directly on CB04 membrane as described in the fixation of cells and whole cells hybridization section. The cells were observed by epifluorescence microscopy.

Pre-treatment of environmental water samples before cultivation and hybridization. The environmental water samples were homogenized to disperse the bacterial cells and disrupt aggregates by means of an optimized mechanical treatment prior to the filter cultivation of the microcolonies. The homogenization treatment was performed as follows: 2 g of glass beads

(2-3 mm diameter, Dominique Dutscher, Brumath, France) were added to 35 ml of water sample. This sample was then mixed using a vortex and treated with ultrasonic energy for 1 min with a Branson sonicator operating at 42 kHz (Fischer Bioblock Scientific, Illkirch, France). This treatment was applied twice. The sample was mixed one final time using a vortex shaker for 1 min, prefiltered on a polycarbonate membrane (3.0 µm pore size, 47 mm diameter, Whatman, Dominique Dutscher), and then heat treated at 50°C for 30 min to slow the development of non Legionella microorganisms during the cultivation time. Five milliliters of the water sample were then filtered on a CB04 polyester membrane and acid treated by placing the CB04 membrane on 200 µl of acid solution (0.2 mol l -1 HCl-KCl buffer, pH 2.0) for 5 min at room temperature. Filter cultivation was then performed. The heat and acid treatments were performed as recommended by the standard method (NF T 90-431).

Optimization of cultivation conditions for microcolonies of Legionella. Cultivation time and temperature were tested on the selective GVPC agar plate to define the optimal conditions for growing microcolonies of Legionella on a filter from naturally contaminated cooling water samples. The GVPC selective agar medium [αBCYE, supplemented with 3 g of glycine, 100,000 U of polymyxin B, 80 mg of cychloheximide, and 1 mg of vancomycin per liter] (Biomérieux) is most often recommended for Legionella detection (ISO, 1998;CDC, 2005;[START_REF] Afnor | Water quality -Detection and enumeration of Legionella spp. and L. pneumophila. Method by direct inoculation and after concentration by membrane filtration or centrifugation[END_REF], as it inhibits non Legionella microorganisms, which can be responsible for the lack of recovery of Legionella spp. Two cultivation temperatures, 30 and 37°C, were compared for microcolonies recovery and various incubation times (ranging from 20 to 52 h) were tested. Tests were performed on a naturally contaminated 5 ml water sample following previous pre-treatment (homogenization, heat treatment, filtration of water sample on CB04 membrane and acid treatment), as described above. The microcolonies of were hybridized using the protocol described below, and then enumerated with solid phase cytometer.

Fixation of cells and whole cells hybridization.

The fixation of cells and hybridization were performed directly on the membrane as follows: the CB04 membrane was transferred onto a 25 mm cellulose pad (labeling pad, Millipore) soaked in 600 µl of 96.2% ethanol for fixation of cells. The membrane and pad were left in a Petri dish at room temperature for 4 ± 1 minutes, and then the membrane was dried at room temperature for 3 ± 1 minutes prior to being placed in 50 µl of hybridization buffer (900 mmol l -1 NaCl, 20 mmol l -1 Tris-HCl [pH 7.2], 0.1% sodium dodecyl sulfate, 20% deionized formamide, 10% dextran sulfate) containing the four probes (the final concentration of each probe being 5 ng µl -1 ) in hybridization chambers and incubated at 46°C for 120 min in a covered water bath. Following hybridization, the membranes were placed on a labeling pad soaked in 550 µl of washing buffer (40 mmol l -1 NaCl, 20 mmol l -1 Tris-HCl [pH 7.2], 0.01% sodium dodecyl sulfate, and 5 mmol l -1 EDTA).

Cells and microcolonies detection. For the specificity tests, the hybridized cells were analyzed by epifluorescence microscopy. The membrane containing cells was air dried and mounted on an antibleaching medium (Citifluor AF1; Citifluor Ltd., Houdon, United Kingdom) for observation using a WIBA filter block for FITC detection (Olympus model AX70, Hamburg, Germany). A negative control (without oligonucleotides) was set up to test the auto-fluorescence of the protocol for all the strains tested.

The microcolonies of Legionella spp. were counted using a solid phase cytometer, SPC (ScanRDI TM , CHEMUNEX® -Biomérieux) after: (i) a short cultivation time on a nutritive medium; and (ii) the staining of cells using the FISH assay described above. The membrane was then placed into the SPC's sample holder and on a 25 mm cellulose membrane (support pad, 0.45 µm pore size), which had previously been saturated with 100 µl of washing buffer.

The SPC system scans the sample on the support pad with an argon laser beam (488 nm emission wavelength), recording all the fluorescence events at 500-530 nm and 540-570 nm with two photomultiplier tubes. Fluorescence events are discriminated as targeted fluorescent bacterial signals or as false positives (autofluorescent particles) using a set of discriminant parameters [START_REF] Guillet | Solid phase cytometry for detection of rare events[END_REF]. The selected discriminant parameters were: peak intensity 100 to 20,000; secondary/primary ratio 0 to 0.9; number of lines 1 to 50; and number of samples 1 to 250. The analytical results were plotted in 2 dimensions, where all the discriminated fluorescence events are shown in terms of x and y coordinates on a schematic diagram of the membrane. One membrane can be scanned in 3 minutes, including the discrimination step. The validation step using epifluorescence microscopy was then performed, using a BH2 epifluorescence microscope (Olympus) equipped with an FITC filter block and a motorized stage driven by the ScanRDI TM system. The microcolonies of Legionella detected by the SPC were expressed as Microcolony Forming Unit (MicFU).

Quantitative performances of the assay. The sensitivity of the trial assay for enumerating

Legionella was tested on artificially contaminated cooling tower water samples. A different aliquot of a sterilized (121°C for 15 min) cooling tower water sample was spiked with various concentrations of L. pneumophila sg1 cells (ATCC 33152) and compared with theoretical counts of the cells added. The cell suspension was made from a fresh colony resuspended with milliQ water, and the cell concentrations were determined by SPC after staining of the viable cells using the ChemChrome V6 assay (CHEMUNEX ® -Biomérieux) [START_REF] Parthuisot | Evaluation of ChemChrome V6 for bacterial viability assessment in waters[END_REF]. Serial decimal dilutions were then made in water samples to obtain concentrations ranging from 10 2 to 10 7 cells l -1 . The repeatability of the method was then tested after 48 h of filter cultivation time by determining the coefficient of variation (CV) from 11 replicates performed by the same operator from the same cooling tower water sample. In this case, the test was performed on a naturally contaminated cooling tower water sample.

Naturally contaminated cooling tower water samples. A total of 262 cooling tower water samples were collected between 2009 and 2011 from 8 nuclear power plants in France. Fiftytwo water samples were collected during the process of disinfecting the cooling tower systems, and, for these samples, the residual free chlorine was neutralized by the addition of 20 mg l -1 of sodium thiosulfate. Two liters of water were collected in a sterile bottle. The filter cultivation was performed at 30°C for 24 and 48 h. The analysis was performed in triplicate.

Quantification of Legionella by the reference culture method. Legionella were enumerated and identified according to the AFNOR T90-431 standard (AFNOR, 2014) in compliance with the international standard ISO 11731 (ISO, 1998). Briefly, 200 µl of each cooling tower water sample was placed directly onto a selective GVPC plate (Biomérieux). In addition, water samples were concentrated by filtration through 0.45 μm pore size polycarbonate filters (Sartorius SAS, Palaiseau, France). The filtered volume was dependent on the sample's filterability, and ranged from 50 to 500 ml. The membrane was then transferred into 5 ml of sterile milliQ water and treated with ultrasonic energy for 2 x 1 min with a Branson sonicator (Fischer Bioblock Scientific, Illkirch, France) operating at 42 kHz. Volumes (100 µl) of the concentrate were also placed on GVPC plates following heat treatment (50°C for 30 min) and acid treatment (HCl 0.2 mol l -1 , KCl 0.2 mol l -1 , pH 2.0, for 5 min) to reduce the overgrowth of the non Legionella organisms during the long incubation period. The inoculated plates were incubated at 37 ± 2°C, and the colonies were counted after 3, 5, and 10 days. The colonies were then examined for fluorescence under a Wood lamp, and those exhibiting Legionella morphology were transferred to an αBCYE medium, an αBCYE medium without cysteine, and a blood agar medium (Biomérieux) as a control. At least five colonies per sample were identified by Legionella-specific immunolatex reagents (Oxoid). According to an AFNOR recommendation for dirty and non filterable waters, Legionella quantifications performed from 500 ml samples of cooling water leads to a limit of detection (LD) of the method of 100 CFU l -1 , and a limit of quantification of 500 CFU l -1 (AFNOR, 2014). All the statistics were calculated using XL stat software (Addinsoft, France).

Mathematical model of variation

The MicFU-FISH-SPC and reference culture methods were compared using the ISO 17994 criteria for establishing equivalence between microbiological methods (ISO, 2014). Briefly, the methods are considered as quantitatively equivalent (not different) if the mean relative difference of the paired confirmed counts does not vary significantly from zero.

Results

Oligonucleotide specificity for the FISH assay. The probe specificity was checked in silico using the probe matching function of the Ribosomal database project software package and the BLAST function from the NCBI website. According to the Blast results, all the probes are highly specific for the genus Legionella. In fact, of the 46 species of Legionella used, the probes matched with all 46 species for the Leg705 probe, and with 31 species, 33 species, and 39 species for the Leg226, Legall11, and Legall22 probes, respectively. Moreover, all the Legionella species tested were detected with at least 2 probes (Table S1). The specificity of the probe set (consisting of the four probes: Leg705, Leg226, Legall11, and Legall22) was then evaluated for a wide spectrum of bacterial strains obtained from reference collections or isolated from various environments. Thirty-nine Legionella strains and 21 non Legionella strains were used to assess the specificity of the FISH protocol. All the Legionella strains (100%) yielded a strong positive fluorescent signal with the probe set (Figure S1), and no fluorescent signal was generated with the non Legionella strains. No autofluorescent signal (without oligonucleotides) was detected for any of the bacterial strains tested (Table 1).

Influence of microcolonies cultivation conditions. The effect of cultivation temperature (30 and 37°C) performed on GVPC for the recovery of microcolonies by SPC was tested after 48 h of incubation. Analyses were performed on 11 cooling tower water samples collected from 3 different industrial sites. The microcolonies were counted by SPC from duplicate water samples, and the results were expressed as the number of Legionella spp. MicFUs per liter. The MicFU concentrations were significantly higher for cultivation at 30°C (signed Wilcoxon test, p =0.015), compared to those measured at 37°C (Figure 1). In addition, a few microcolonies were detected in two water samples (g and h, collected from site 2) at 30°C, while none were detected at 37°C. According to these results, the optimal temperature for microcolonies cultivation on GVPC agar is 30°C. A comparison of MicFUs measured at different cultivation times and CFUs enumerated using the standard method is presented in Figure 2. The standard plate count is 2.5 10 4 CFU l -1 , and MicFU counts measured in the same water sample increased gradually with cultivation time up to 48 h of incubation time (2.7 (±4.6) 10 2 l -1 for 20 h of incubation time up to 2.5 (±0.12) 10 4 l -1 for 48 h of incubation time).

After this incubation time, the MicFU counts remained constant and similar to the standard plate count. Consequently, the maximum number of recoveries on GVPC was obtained after 48 h at 30°C. In conclusion, for the cooling tower sample analyzed, the GVPC plate showed a better recovery rate for microcolonies of Legionella enumeration cultivated at 30°C. These cultivation conditions were applied to compare the MicFUs and the standard plate counts in other cooling tower water samples. However, because a shorter cultivation time could indicate the contamination level of the sample and be useful for faster circuit monitoring, we also enumerated the microcolonies after 24 of incubation time (Methods S1).

Quantitative performance of the MicFU-FISH-SPC method. The quantitative performance of the method for enumerating viable Legionella from cooling tower water samples was first evaluated on an artificially contaminated water sample with known log-decimal concentrations of L. pneumophila sg 1 to define the linearity domain, and the detection limit of the method. The water sample was analyzed prior to artificial contamination, and showed an absence of Legionella (equivalent to < 200 Legionella MicFU l -1 ). The quantification was performed on five replicates with a cell density level ranging from 10 2 to 10 7 viable cells added. The microcolonies were enumerated by SPC after 48 h, at 30°C. The theoretical counts and the MicFU counts were linearly correlated with the number of L. pneumophila cells added as the inoculums in a log-log plot (n = 30; r = 0.980, P < 0.0001; Spearman test correlation).

Consequently, we estimated the linearity domain of the trial method to range between 10 2 and 10 7 MicFU l -1 . The repeatability expressed as the precision under the same operating conditions was carried out on 11 analyses performed on the same day from the same natural water sample and analyzed by the same operator, as recommended by the French standard (ENV ISO 13843, Guidance on validation of microbiological methods) (AFNOR, 2001). The microcolonies were enumerated in the water sample after it was physically and chemically treated and filtered on a CB04 membrane of 5 ml of the sample. Enumerations were performed by SPC after 48 h of incubation at 30°C on GVPC. The concentration of Legionella spp. measured by both methods were similar and were 2.5 10 4 CFUs per liter for standard plate count and 2.1 10 4 MicFUs per liter. The repeatability of the trial method was investigated by determining the coefficient of variation (CV = 24.1%) measured from the number of microcolonies detected by SPC from each water sample (Table 2). The LD of the trial method was determined according to the French standardization procedure, set out in the document (ENV ISO 13843, AFNOR 2001). The theoretical LD of the method is 1 microcolony per membrane, corresponding to 200 MicFU l -1 when a 5 ml water sample is analyzed. Nevertheless, as expected in an environmental sample, the repeatability assay demonstrated that parallel determinations vary even more than is explained by the Poisson distribution. The samples are clearly overdispersed with an overdispersion factor of 0,049 (estimated with the repeatability assays). Because of this overdispersion, the Negative binomial model was applied to determine the LD of the method and was estimated to be 600 MicFU l -1 (3 microcolonies per analytical portion of 5 ml).

Legionella spp. quantification in naturally contaminated cooling tower water samples by

MicFU-FISH-SPC and comparison with standard plate counts. A total of 262 cooling tower water samples, collected between July 2009 and March 2011 from 8 different power plants located in France, were analyzed by both the trial and standard culture methods.

According to nuclear plant officials, the cooling circuits use freshwater water inputs from the larger rivers in France, such as the Seine, the Rhône, the Moselle, and the Loire. So, the physico-chemical qualities of the cooling tower water samples tested in this study varied, depending on the freshwater source and on temporal changes, such as the season. The microcolonies of Legionella from all the water samples were enumerated following application of the physical and chemical treatments described previously, and following cultivation performed 48 h at 30°C on GVPC agar. As reported in Table 3, 80.1% and 78.6% of the water samples were positive using the standard culture method and the trial method, respectively: Legionella CFU concentrations ranged between 5.10 2 and 8.5 10 5 CFU l -1 and the microcolonies concentrations ranged between 6.10 2 and 3.8 10 6 MicFU l -1 . The percentages of water samples for which the results of the two methods were in agreement (MicFU+/CFU+ and MicFU-/CFU-pair results) was 95% (n = 248) and the MicFU and CFU counts were linearly correlated (Pearson test, P<0.0001 for α=5%) (Figure 3).

The equivalence between the trial assay and the standardized culture method was tested according to ISO 17994 (ISO, 2014) to determine whether or not the trial method is 'at least as reliable' as the standardized culture method. Of the 262 water samples tested, 46 were found to be negative by both methods and excluded from the calculation, as recommended by ISO 17994. The 'two-sided' comparison performed on 216 water samples was considered appropriate for the acceptance of the trial method [START_REF] Bargellini | Inter-laboratory validation of a rapid assay for the detection and quantification of Legionella spp. in water samples[END_REF], and the results showed that the performance of the FISH method did not differ from that of the culture method (NF T 90-431), because the two values of the confidence interval (x L = -22.71, x H = 4.22) were on one side of zero and the second was on the other side of zero.

Discussion

The plate count methods for Legionella isolation and counting are currently standard ways to monitor Legionella in cooling tower water samples. Although they are widely used, these methods are time-consuming, because a long cultivation time is required to visualize the colonies, and because the procedure includes re-isolation of the strains and serological identification of microcolonies of Legionella. As a result of these limitations, the standard culture methods are not compatible with a rapid management of Legionella proliferation. However, they are still valuable detection methods for the information they provide on cell viability, because they are based on growth activity. Other methods have been proposed for the rapid detection of viable Legionella in water samples, such as the real-time PCR-based methods using ethidium monoazide or propidium monoazide [START_REF] Inoue | Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment[END_REF][START_REF] Chen | Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real-time quantitative PCR[END_REF], direct fluorescent antibody analysis (DFA), which includes a vital staining step to detect esterase activity [START_REF] Parthuisot | Occurrence of total and viable L. pneumophila cells in hot and natural waters measured by immunofluorescence-based assays and solid phase cytometry[END_REF] or FISH-based techniques [START_REF] Kirschner | Development of a new CARD-FISH protocol for quantification of Legionella pneumophila and its application in two hospital cooling towers[END_REF]. The real-time PCR-based methods have been successfully applied to Legionella monitoring in hot sanitary water (which can be described as 'clean water'). However, PCRbased methods have some drawbacks when applied to a dirty, turbid, and complex water, such as the water that characterizes industrial cooling tower systems. The occurrence of PCR inhibitor agents in water has been extensively documented and reported by several authors in cases where PCR was applied to environmental water samples, such as cooling tower waters [START_REF] Koide | Detection of Legionella spp. in cooling tower water by the polymerase chain reaction method[END_REF][START_REF] Yamamoto | Comparison of detection methods for Legionella species in environmental water by colony isolation, fluorescent antibody staining, and polymerase chain reaction[END_REF][START_REF] Catalan | Detection of Legionella pneumophila in wastewater by nested polymerase chain reaction[END_REF][START_REF] Villari | Comparison of conventional culture and PCR methods for the detection of Legionella pneumophila in water[END_REF][START_REF] Behets | Development and evaluation of a Taqman duplex real-time PCR quantification for reliable enumeration of Legionella pneumophila in water samples[END_REF][START_REF] Edagawa | Detection of culturable and nonculturable Legionella species from hot water systems of public building in Japan[END_REF][START_REF] Morio | Real-time PCR assay for the detection and quantification of Legionella pneumophila in environmental water samples: Utility for daily practice[END_REF]. These inhibitor agents are frequently present in turbid water, such as cooling tower water, as well as in untreated freshwater. In dirty water samples, even if purification DNA processes are applied during DNA extraction and before DNA amplification, they usually are too drastic to preserve all the DNA or too inefficient to remove PCR inhibitors. Currently, a tenfold serial dilution of DNA extract samples before amplification is proposed to reduce the inhibitor effect. In the case of industrial cooling tower systems, the presence of PCR inhibitors co-extracted with DNA can involve the application of a posteriori dilution factors of 5-100, which considerably decreases the sensitivity of the method, especially when the abundance of Legionella in the water is low, and leads to false-negative results [START_REF] Touron-Bodilis | Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems[END_REF]. Another concern is the diversified nature of the inhibitors. Their presence in water samples, lead to the success or failure of the PCR based methods applied for Legionella monitoring in these systems [START_REF] Chen | Rapid quantification of viable legionellae in water and biofilm using ethidium monoazide coupled with real-time quantitative PCR[END_REF][START_REF] Touron-Bodilis | Usefulness of real-time PCR as a complementary tool to the monitoring of Legionella spp. and Legionella pneumophila by culture in industrial cooling systems[END_REF]. Moreover, it has been demonstrated that viable PCR is not suitable for monitoring highly contaminated water samples, such as cooling tower water, especially following decontamination treatment. In the presence of high levels of suspended solids or biomass, the dye could undergo chemical adsorption onto other compounds, and the cross linking step by light could be inhibited, since the radiation probably would not be able to penetrate the liquid [START_REF] Fittipaldi | Viable real-time PCR in environmental samples: can all data be interpreted directly?[END_REF][START_REF] Slimani | Evaluation of propidium monoazide (PMA) treatment directly on membrane filter for the enumeration of viable but non cultivable Legionella by qPCR[END_REF].

Recently, a new DFA method combining a cellular test based on the detection of the esterase activity of bacteria cells and the specific detection of L. pneumophila cells by specific monoclonal antibodies has been proposed for the detection of active L. pneumophila in hot sanitary water and freshwaters [START_REF] Parthuisot | Occurrence of total and viable L. pneumophila cells in hot and natural waters measured by immunofluorescence-based assays and solid phase cytometry[END_REF]. This method offers an efficient means for detecting the Viable But Non Culturable (VBNC) L. pneumophila cells in water samples.

Nevertheless, its application to the detection of active Legionella spp. is currently still limited, because of the absence of highly specific antibodies able to directly target all Legionella species in a complex bacterial community, like those of cooling tower systems.

Finding a highly conserved nucleic acid region in Legionella species for its detection using the FISH technique can overcome this problem. The four probes that we selected target four distinct 16S rRNA sequences, which hybridize with targets in the same hybridization conditions. The specificity test performed on a large number of Legionella and non Legionella strains has revealed the high degree of specificity of the FISH technique, which has been extensively applied to the direct detection, identification, and enumeration of specific bacteria in an environmental water sample in combination with a fluorescence detection device. However, the majority of studies using these methods for cell detection in environmental samples have limited application because of the very low fluorescence signals given out following hybridization. These low fluorescence signals are mostly due to a low ribosomal content of individual and starved cells [START_REF] Amann | Phylogenetic identification and in situ detection of individual microbial cells without cultivation[END_REF]. To overcome the ribosomal content limitations, treatments like the catalyzed reporter deposition (CARD)-FISH technique have been proposed. This technique increases the fluorescent signal of hybridized cells using horseradish peroxydase-labeled probes [START_REF] Lebaron | A new sensitive, whole-cell hybridization technique for detection of bacteria involving a biotinylated oligonucleotide probe targeting rRNA and tyramide signal amplification[END_REF][START_REF] Baudart | Rapid and sensitive enumeration of viable diluted cells of members of the family Enterobacteriaceae cells in freshwater and drinking waters[END_REF]. A CARD-FISH technique was recently proposed by Kirchner et al. (2012) for the direct enumeration of L. pneumophila in two hospital cooling tower systems in combination with epifluorescence microscopy. With CARD-FISH, the authors measured concentrations of L. pneumophila that were higher than with standard plate counts by two to three orders, and up to six orders of magnitude, depending on the hospital investigated. The results obtained by Kirchner et al.

(2012) are closer to the counts usually measured by real-time PCR based on total DNA detection (included DNA extracted from dead cells, cultivable cells, and VBNC cells) than to the culture results. Some care should be taken in interpreting CARD-FISH results, particularly in the physiological interpretation of CARD-FISH-targeted cells. CARD does, in fact, efficiently increase the fluorescent signal of hybridized cells, leading to the detection of cells with a low ribosomal content, as is the case with starving or non viable cells [START_REF] Pernthaler | Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria[END_REF]. Consequently, and depending on the amplification signal emitted by the CARD process, viable cells, but also the non viable ones, can be detected by the CARD-FISH technique, providing counts that are similar to those in real-time PCR performed on the total DNA extracted. In addition, in assessing the performance of a rapid trial relative to that of a standard culture method, a major concern is that viability be measured using a widely accepted test in both methods which targets the same physiological function, such as the ability of a cell to grow

The microcolony-based method developed in this study offers the following advantages: cell viability, as determined by the cultivation, is based on the same physiological function, like cell growth ability, as in the standard method. The time of analysis can be reduced to 48 h, because microcolonies can be detected in their early stage of development when combined with solid phase cytometry. In addition, decreasing the cultivation time reduces the risk of the overgrowth of contaminating microorganisms, which can inhibit or mask Legionella growth.

The overgrowth of contaminating microorganisms is an important issue, as it leads to a decrease in the sensitivity of the culture method, especially for cooling tower water samples where microorganisms, other than Legionella, are abundant and diversified, which can make the results unusable.

For Legionella, the formation of the microcolonies depends on the cultivation conditions, as reported in our results. On the basis of ecological knowledge, Legionella growth occurs in an environment with temperatures in the 20 to 45°C range. An incubation temperature of 36°C ± 1 is advocated for Legionella isolation in the normalized culture method, but this temperature was selected because it corresponds to the optimal temperature of a human host, and allows better recovery of Legionella involved in human infections. Outside a human host, for example, Legionella species living in aquatic environments are able to grow over a large temperature range. A study performed on the isolation rate of Legionella species in groundwater have demonstrated that the rate of formation of Legionella isolates increases when the isolation process occurs at 30°C instead of 37°C [START_REF] Riffard | Occurrence of Legionella in Groundwater[END_REF]. Our study confirms that culture at 30°C improves the recovery rate of the microcolonies of Legionella from the cooling tower water samples analyzed.

Our objective was to determine a suitable cultivation time for naturally contaminated water samples for use with the trial method. Detectable microcolonies were counted by SPC after an incubation time as short as 20 h, but the maximum number was counted for an incubation time ranging between 44 h and 48 h, which is similar to the count yielded by the standard culture method. The increase in the recovery rate of the method is highly dependent on the cultivation time, as well as the cultivation temperature. This is explained by a hybridization signal intensity that may be affected by the ribosomal content of Legionella and the fact that the number of rRNA in cells is dependent on the incubation time [START_REF] Kerkhof | Small ribosomal RNA content in marine Proteobacteria during non-steady-state growth[END_REF]. In our study, the conditions of microcolonies cultivation were determined to be optimal at 30°C for 48 h on GVPC. Even though some studies have demonstrated that some non L. pneumophila grows poorly on GVPC medium [START_REF] Ta | Comparison of culture methods for monitoring Legionella species in hospital potable water systems and recommendations for standardization of such methods[END_REF], this medium remains currently used and suggested for the isolation of Legionella spp. from environmental water samples.

Under these conditions, the repeatability of the trial method measured by the standard deviation calculated from 11 replicates was acceptable (<25%) for the quantification of Legionella in cooling tower water and the detection limit was 600 MicFUs per liter.

In light of these results, MicFU-FISH-SCP appears to be an efficient method in accordance with the standard method to perform a rapid monitoring (within 2 days) of cultivable Legionella in industrial cooling tower systems, independently of the quality of the input freshwater and the presence of a biocide treatment. In addition, the analysis performed within 1 day (24 h test results shown in supplementary material) could be used as a rapid test to indicate whether or not a high level of Legionella is contaminating the cooling systems (semi quantitative test). The rapid analysis is performed on a small volume (5 ml vs 1 l), and a membrane sample can be stored for one month after cultivation, and ethanol treatment prior to hybridizing the microcolonies. These storage conditions can lead to wide-scale sampling, which is not always realizable with the standard method. The manipulation of a small volume of contaminated water could be another advantage, which would reduce the risk of exposure in laboratories and among healthcare employees. Some studies are needed before the industrialization of this method in order to make it more applicable in industrial surroundings and daily use.

Method S1 Quantitative performances of the assay performed for 24h of cultivation time

The quantitative performance of the method for enumerating microcolonies of Legionella from cooling tower water samples was evaluated on an artificially contaminated water sample with known log-decimal concentrations of L. pneumophila sg 1 to define the linearity domain, and the detection limit of the method. The water sample was analyzed prior to artificial contamination, and showed an absence of Legionella (equivalent to < 200 Legionella MicFU l -1 ). The quantification was performed on five replicates with a cell density level ranging from 10 2 to 10 7 viable cells added. The microcolonies were enumerated by SPC after 24 h at 30°C. The theoretical counts and the Legionella MicFU counts were linearly correlated with the number of L. pneumophila cells added as the inoculums in a loglog plot for both cultivation times (n = 20, r = 0.656, P = 0.002; Spearman test). Twenty out 30 water samples (66.7%) were positive using the approach for 24 h. For this shorter incubation time, no microcolony was detected for the theoretical cells added at 380 cells l -1 , and MicFUs were detected in 2 replicates of the 5 analyzed for theoretical cells added at 3800 cells l -1 . Consequently, we estimated the linearity domain of the trial method to range between 10 4 and 10 7 MicFU l -1 for the shorter incubation time (24 h). Enumerations were performed by SPC after 24 h of incubation at 30°C on GVPC. The concentration of Legionella spp. measured by the standard culture method was 2.5 10 4 CFUs per liter. The mean concentrations were 1.3 10 3 and 2.1 10 4 Legionella MicFUs per liter after 24 h of cultivation. The repeatability of the trial method was investigated by determining the coefficient of variation (CV) measured from the number of microcolonies detected by SPC from each water sample. The CV was 44.5% for enumeration performed after 24 h of cultivation. The microcolonies of Legionella were enumerated from all the water samples following application of the physical and chemical treatments described previously, and following cultivation performed for 24 h at 30°C on GVPC agar. About 80% of the water samples were positive using the standard culture method (Table A). Legionella CFU concentrations ranged between 5.10 2 and 8.5 10 5 CFU l -1 . In contrast, 62.7 % of the water samples were positive using the trial method for Legionella enumerated after 24 h. The MicFU concentrations ranged between 6.10 2 and 3.2 10 5 MicFU l -1 . The percentage of water samples for which the results of the two methods are in agreement (MicFU+/CFU+ and MicFU-/CFU-pair results) was 81% (n = 176) for cultivation of microcolonies performed for 24 h. If the results were not in agreement, this was mainly due to the MicFU-/CFU+ pair result for 24 h of cultivation.

  and limit of detection (LD) determination. The excess of Poisson randomness was detected qualitatively by the Poisson index of dispersion, and is called overdispersion. This additional random uncertainty was measured quantitatively by estimating variance, m = mean value) (FD T90- 2014). In overdispersed sample, a Negative Binomial model is appropriate to determine the LD. The LD is the particle number x (per analytical portion), where the probability p 0 of a negative result equals 5%. The particle number x is equal ² Normally distributed data were tested using the Shapiro-Wilk test, and the various cell concentrations were statistically compared using the Wilcoxon signed rank test when normality was not confirmed. The relation between SPC counts and standard plate counts was determined by regression models (Pearson test for the linear regression model).

Fig. 1

 1 Fig. 1 Legionella MicFU concentration measured by solid phase cytometry for 48 h at 37°C

Fig. 2

 2 Fig. 2 Legionella MicFU concentration measured on GVPC agar for cultivation times ranging

Fig. 3

 3 Fig. 3 Relationship between Legionella counts measured from naturally contaminated cooling

Legionella

  Figure1

Figure S1 .

 S1 Figure S1. Examples of epifluorescent micrographs of hybridized cultured Legionella cells obtained using the fluorescent in situ hybridization method. (A) L. longbeaches CIP 103881 matching with 2 probes; (B) L. jordanis CIP 105268 matching with 3 probes, and strains matching with the set of 4 probes; (C) L. anisa ATCC 35292; (D) L. pneumophila sg1 ATCC 4370; (E) L. pneumophila sg15 ATCC 32251; (F) L. sainthelensi CIP 103885.

  Legionella spp. quantification in naturally contaminated cooling tower water samples by MicFU-FISH-SPC after 24h of cultivation and comparison with standard plate counts.

Figure B .

 B Figure B. Relationship between MicFU counts of Legionella measured after 24 h and 48 h on GVPC at 30°C.

Figure C .

 C Figure C.Relationship between Legionella counts measured from naturally contaminated cooling tower water samples by the standard method and the trial method after: (A) 24 h; and (B) 48 h of cultivation on GVPC at 30°C.

Table 1

 1 Bacterial strains used for specificity control of the fluorescent in situ hybridization assay.

Table 2

 2 Repeatability of the MicFU counts measured from 11 replicates from the same cooling tower water sample.

Table 3

 3 Comparison of the number of sample (percentage in parentheses) positive or negative with MicFU-FISH-SPC (MicFU) assay performed for 48 h of incubation time and the standard culture method (CFU) for detecting Legionella spp. in 262 cooling tower water samples.

Table 2

 2

	Replicat	Numbre of MicFUs detected by SPC from 5ml of water sample	MicFu concentration ( l -1 )
	R1	75	15000
	R2	138	27600
	R3	119	23800
	R4	99	19800
	R5	126	25200
	R6	99	19800
	R7	75	15000
	R8	72	14400
	R9	145	29000
	R10	108	21600
	R11	128	25600
	Mean	107.64	21527.3
	Standard deviation	25.98	5195.6
	Coefficient of variation	24.1%

Table 3

 3

	MicFU
		Total
	+	-

Table S1 .

 S1 In silico specificity test of the four oligoprobes (Leg705, Leg226, Legall11, and Legall22) using the probe matching function of the Ribosomal database project software package[START_REF] Maidak | The RDP-II (Ribosomal database project)[END_REF] and the BLAST function on the NCBI website[START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF].

	Leg705	Leg 226	Legall11	Legall22	Number of matching
					probes
	L. dumoffii	L. dumoffii	L. dumoffii	L. dumoffii
	L wadsworthii	L wadsworthii	L wadsworthii	L wadsworthii
	L. gormanii	L. gormanii	L. gormanii	L. gormanii
	L. cherrii	L. cherrii		L. cherrii
	L. parisiensis	L. parisiensis	L. parisiensis	L. parisiensis
	L. anisa	L. anisa	L. anisa	L. anisa
	L. bozemanae	L. bozemanae	L. bozemanae	L. bozemanae
	L. cincinnatiensis	L. cincinnatiensis	L. cincinnatiensis	L. cincinnatiensis
	L. sainthelensi	L. sainthelensi	L. sainthelensi	L. sainthelensi
	L. gratiana	L. gratiana	L. gratiana	L. gratiana
	L. longbeaches		L. longbeaches
	L. santicrucis	L. santicrucis	L. santicrucis
	L. lytica	L. lytica	L. lytica	L. lytica
	L. steigerwaltii	L. steigerwaltii		L. steigerwaltii
	L. tucsonensis	L. tucsonensis	L. tucsonensis	L. tucsonensis
	L. lyticum		L. lyticum	L. lyticum
	L. shakespearei		L. shakespearei	L. shakespearei
	L. moravica		L. moravica	L. moravica
	L. worsleiensis		L. worsleiensis	L. worsleiensis
	L. quateirensis		L. quateirensis	L. quateirensis
	L. pneumophila	L. pneumophila	L. pneumophila	L. pneumophila
	L gresilensis	L gresilensis		L gresilensis
	L. waltersii		L. waltersii	L. waltersii
	L. londiniensis	L. londiniensis	L. londiniensis	L. londiniensis
	L. israelensis			L. israelensis
	L. fairfieldensis	L. fairfieldensis	L. fairfieldensis	L. fairfieldensis
	L. lansisgensis			L. lansisgensis
	L. birminghamensis		L. birminghamensis	L. birminghamensis
	L. quinlivanii		L. quinlivanii	L. quinlivanii
	L. geestiana		L. geestiana
	L. adelaidensis		L. adelaidensis	L. adelaidensis
	L. oakridgensis			L. oakridgensis
	L. nautarum			L. nautarum
	L. micdadei	L. micdadei	L. micdadei	L. micdadei
	L. maceacherni	L. maceacherni	L. maceacherni	L. maceacherni
	L. donaldsonii	L. donaldsonii	L. donaldsonii	L. donaldsonii
	L. feeleii	L. feeleii	L. feeleii	L. feeleii
	L. erythra	L. erythra	L. erythra	L. erythra
	L. spiritensis	L. spiritensis
	L. rubrilucens	L. rubrilucens	L. rubrilucens	L. rubrilucens
	L. taurisensis	L. taurisensis	L. taurisensis	L. taurisensis
	L. brunensis	L. brunensis
	L. beliardensis	L. beliardensis
	L. jamestowniensis	L. jamestowniensis		L. jamestowniensis
	L. hackeliae	L. hackeliae
	L. jordanis	L. jordanis		L. jordanis
	46 species	31 species	33 species	39 species

Table A .

 A Comparison of the number of sample (percentage in parentheses) positive or negative with MicFU-FISH-SPC (MicFU) assay performed for 24 h of incubation time and the standard culture method (CFU) for detecting Legionella spp. in 217 cooling tower water samples.In total, 217 water samples were analyzed with both methods. The MicFU counts were linearly correlated in a log-log plot, as reported in Figure B (P<0.0001; Pearson test). The counts enumerated after 48 h were significantly higher than those measured for the shorter cultivation time (Wilcoxon signed rank test, P<0.0001 for α=5%).

		MicFU
				Total
		+	-
	+	135 (62.2)	40 (18,4)	175
	CFU
	-	1 (0.5)	41 (18,9)	42
	total	136	81	217

Microbial Observatory of the Laboratoire ARAGO (MOLA) Strain Collection (Banyuls/mer, France).

Acknowledgments

We gratefully acknowledge the help of Marion Mottier from the Observatoire Océanologique of Banyuls-sur-Mer and of Gaëlle Le Meur from Euro Engineering with sample collection and Legionella culture.

Conflict of interest:

All authors declare that they do not have any conflict of interest.