Assessment of Viability of Legionella Pneumophila in Environmental Samples: On Filter Application of Ethidium Monoazide Bromide

Purpose: Culture method, Real-Time PCR (qPCR) and Ethidium Monoazide Bromide (EMA) qPCR have been compared in order to detect Legionella pneumophila (Lp) in water samples, to identify a method able to speed up the procedures, detect the “viable but not cultivable” bacteria (VBNC) and exclude dead bacteria using a commercial kit for extraction and amplication and modifying the protocol. Methods: Using these three methods, 34 environmental water samples and a series of samples articially spiked with alive, dead and VBNC Lp ATCC 33152 were analysed. ISO 11731-2-2004 culture method was applied, whereas a commercial kit was selected for both qPCR and EMA qPCR pretreatment. Results: only 35% (12/34) of the environmental samples were positive in both culture and qPCR methods. With regard to EMA qPCR, results showed the absence of dye toxicity on viable and VBNC strains and an incomplete effectiveness on the dead ones. In both viable and VBNC strains a decrease of bacterial DNA amplication was recorded as a function of sample dilution but not of EMA concentration. Conclusions: Discrepancies between culture method and EMA-qPCR were observed and could be due to different causes as membrane-dye interactions, presence of interfering compounds and the relatively low sensitivity of the kit used. Signicance Impact the Study:

incomplete effectiveness on the dead ones. In both viable and VBNC strains a decrease of bacterial DNA ampli cation was recorded as a function of sample dilution but not of EMA concentration.
Conclusions: Discrepancies between culture method and EMA-qPCR were observed and could be due to different causes as membrane-dye interactions, presence of interfering compounds and the relatively low sensitivity of the kit used.
Signi cance and Impact of the Study: In presence of one or more suspected cases of nosocomial legionellosis, the application of a rapid molecular method able to identify only the viable and VBNC Lp would be useful in order to quickly identify the source of infection and to intervene with sanitation treatments. However, because in our experience EMA pretreatment on lter membrane has not given the expected results, it would be necessary to proceed with other experiments and different dyes.
Key Points -Need for a rapid method able to identify both viable and VBNC Legionella pneumophila -Modi cation of the protocol and EMA pretreatment directly on lter -Results with discrepancies, no toxic effects even with increasing dye concentration Background Legionella is a Gram-negative, aerobic obligate bacterium, whose habitats are represented by both natural and arti cial aquatic environments, in a free-living form, as parasites of protozoa and within bio lms (Bianchi et al. 2016). Colonization by Legionella occurs more easily if the arti cial water network presents stagnation, bio lm, corrosion, limestone and temperatures between 25°C and 42°C.
Legionella, and particularly the species L.pneumophila (Lp), is the causative agent of Legionellosis, a generic term used to describe both pneumonic and non-pneumonic forms. Legionella is commonly transmitted by inhalation of contaminated aerosols, originating from air conditioning, cooling towers, hot and cold water systems, dental unit, humidi ers and whirlpool spas, but also by aspiration of contaminated water (Tesauro et al. 2018).
Legionellosis varies from a mild febrile illness (Pontiac fever) to a serious and sometimes fatal form of pneumonia (Legionnaires' disease) with an approximate mortality rate of 10-30% (Italian guidelines).
Some authors (Dusserre et al. 2008, Ducret et al. 2014, Li et al. 2014, Mansi et al. 2014, Casini et al. 2018 reports that Lp, if submitted to prolonged stress, such as continuous disinfection treatments, can enter in a state of low metabolic activity that makes it "viable but not cultivable" (VBNC). VBNC bacteria are not able to replicate in culture but regain virulence when environmental conditions return to be optimal, representing a risk for human health.
Laboratory methods currently used for both research and quanti cation of Lp in water are culture method and Real-Time PCR, both with strengths and limitations.
Culture method allows to detect and isolate all Legionella species and permits to correlate clinical strains with the environmental ones. Using this method, however, the results, expressed in Colony Forming Units Furthermore, in case of high concentration of dead bacteria (>10 5 CFU/ml), the amount of EMA may not be enough to inactivate their DNA, with risk of having false positives (Fittipaldi et al. 2011, Qin et al. 2012). Subsequently, it would be necessary to optimize EMA concentration according to chlorine concentration possibly present in examined water samples. Finally, it is important to consider the rather signi cant cost of the method.
Aims of this work were i) to compare the culture and the qPCR methods on the same water samples and ii) to applicate and develop the EMA qPCR, using a commercial kit and modifying its protocol, in order to identify a rapid method able to speed up the procedures, detect the viable and "viable but not cultivable" bacteria (VBNC) and exclude dead bacteria, especially in situations where discriminating is particularly relevant, as in presence of clusters or outbreaks of Legionellosis.

Methods
Study design has included several phases: 1) Applicate both culture and molecular analysis on the same water sample. Thirty-four water samples of 2 liters were collected using sterile bottles containing Sodium Thiosulfate (LP italiana SpA, Milan); refrigerated samples were transported to the laboratory where they were analyzed both by culture method and qPCR, within 24 hours from the sampling. The samples were collected from Healthcare Facilities distributed in Lombardy, Piedmont and Liguria (North Italy). Some of these water systems were treated in continuous with disinfectants as chlorine dioxide and monochloramine. 2) Applicate and develop EMA qPCR method. Water samples arti cially spiked with viable, dead and VBNC Lp ATCC 33152 in serial dilutions were tested. Initially, 3 different concentrations of the dye (1,25-2,5-5mg/ml) were used, and higher concentrations (2,5-10-20 mg/ml) were subsequently Culture method in environmental samples ISO 11731-2-2004 "Water quality-detection and enumeration of Legionella" Part 2: "Direct membrane ltration method for waters with low bacterial counts" was followed to test viable cells through culture method. Brie y, after ltration, the cellulose acetate membrane (0,22µm) was placed directly on GVPN Agar Base medium and Petri dishes were incubated in thermostat with CO 2 at 2.5%, at 37°C for 10 days.
Subsequently, suspicious colonies were tested for identi cation of genus both on BCYE Agar and CYE Agar (ThermoFisher Scienti c, Oxoid, Rodano, Italy) and only those BYCE positive were veri ed through latex agglutination tests (Legionella Latex Test, ThermoFisher Scienti c, Oxoid, Rodano, Italy) to identify species and serogroups. For the ampli cation reaction, 1 cycle was performed at 95°C for 5 minutes, followed by 45 cycles so composed: "denaturation" (95°C for 30 seconds), "annealing" (55°C for 30 seconds) and "elongation and data collection" (60°C for 45 seconds).

EMA treatment
Each arti cially spiked sample was ltered using membranes supplied by the AquaScreen FastExtract (Fx) extraction kit and EMA treatment was carried out directly on membranes, to integrate dye treatment with the kit protocol. Each membrane was placed in a Petri dish and covered with 500ml of EMA at different concentrations. Petri dishes were incubated in dark in a thermostat at 21°C for 10 minutes, then placed in a tray with ice and exposed for 5 minutes to light (500W) at 15/20 cm from the light source. Finally, each membrane was taken and placed in a new Petri dish for bacterial DNA extraction and ampli cation procedures, as provided by kit instructions.

Data interpretation
Values of Threshold cycle (Ct) indicate presence or absence of Lp and the ampli cation reaction is to be considered positive with Ct <40 and negative with Ct ≥ 40.
The GU determined refer to the total number of bacterial cells, alive and cultivable, alive but not cultivable and death. It should be noted that the kit has a "Limit of Detection" (LOD) of 20 GU and a "Limit of Quanti cation" (LOQ) of 50 GU.

Results
Data regarding the comparison between culture method and qPCR, carried out on 34 environmental samples and collected in different 13 Healthcare facilities in North Italy are synthetically reported in Table   1. There is correspondence between samples positive and negative with both methods in 12/34 (35%) and 10/34 (29%) cases, respectively. In the other cases there were discrepancies between the two methods. In 9/12 water samples chlorine dioxide concentration was reported, with mean values of 0.32 mg/L and a range of 0.18-0.58 mg/L. Lp was present in the water network with variable concentrations ranging from "not detected" to 3.3Log and 50% of the isolated strains belonged to Lp serogroup 1. GUs range from 2.48Log to 5Log, only in rare circumstances high values of CFU/L correspond to high values of GU. As shown in Figure 1, we observed a decrease of DNA ampli cation as a function of sample dilution, both in viable and VBNC strains at all three dye concentrations, almost overlapping. EMA inhibited the ampli cation of DNA of dead bacterial cells only at the concentration of 2.5µg/ml (4th dilution) and of 10µg/ml (3rd and 4th dilution); in the other cases, qPCR detected genetic material. Con rmation that these bacteria were died was given by culture method, which always gave results equal to 0 CFU/L.

Discussion And Conclusion
Legionnaires' disease represents an emerging problem for public health, especially if it occurs in healthcare facilities, causing not only isolated cases but also clusters or outbreaks. Hence, environmental surveillance in health facilities plays an important role in preventing these infections, in order to establish usual or extraordinary treatment interventions to reduce and control the risk of acquiring the infection.
Currently, with the aim to detect and quantify the contamination degree in water networks, culture method is considered the gold standard, but qPCR is still possible on water samples, even without international validation.
Many studies con rmed qPCR as a fast and sensitive method to detect and amplify the mip gene of Lp, and useful to highlight also VBNC bacteria. Nevertheless, the problem of DNA detection from dead bacteria remains, along with other economic and methodological limitations. Other studies highlight the possibility of overcoming the problem of overestimating the presence of dead bacterial cells, using Ethidium bromide monoazide (EMA) to bind to DNA of dead cells and prevent its ampli cation by PCR (EMA-qPCR) (Reyneke et al. 2017).
Culture method, qPCR and EMA-qPCR, with a variation to the protocol applying the dye directly on the lter, were compared in the environmental water samples and arti cially spiked ones with serial dilutions of alive, dead and VBNC Legionella pneumophila ATCC 33152.
The comparison between culture method and qPCR gave overlapping results in most of the cases, showing that the AquaScreen® FastExtract kit and AquaScreen® Legionella pneumophila kit, respectively for extraction and ampli cation, have acceptable and satisfactory characteristics. We can hypothesize that discrepancies regarding samples with positive cultures and negative qPCR are due to presence of ferruginous deposits or other substances, such as residues of disinfection treatments, that may play a role, interfering with DNA extraction and blocking DNA ampli cation. We cannot exclude that there was also a problem regarding the analytical sensitivity of the q-PRC (20UG/L), being not able to detect very low charges of the microorganism, a condition that often occurs in structures with continuous treatment with biocide. In this case, the likelihood of acquiring the infection is very small, as demonstrated by the absence of cases of Legionnaire's disease among the patients.
Otherwise, in the case of samples with negative cultures and qPCR positive, we can hypothesize that the presence of dead or VBNC bacterial cells has been highlighted, with the ampli cation of bacterial DNA. Thereby, we consider essential to try to solve or contain this possibility.
Unfortunately, our experiments with EMA-qPCR were not satisfactory at low concentrations of dye (1.25-2.5-5 µg/ml) and even at higher concentrations of 2.5-10-20 µg/ml. Despite toxicity data to bacteria reported in literature, in our series of tests the dye has not proven to be toxic on live bacteria or even on VBNC ones, observing ampli cations at all dilutions. At the same time, it has not been shown to be effective in inhibiting the ampli cation of the DNA of dead bacterial cells.
Our idea to associate qPCR to the use of dye directly on the lter to evidence VBNC cells and exclude the dead ones derived from the necessity to best represent the real contamination from Lp in the water network in a short time, as emerged also in other studies. This membrane lter is used to retain the bacteria using a nitrocellulose disc inside a home-made cartridge. Subsequently the immunodetection of the bacteria retained in the nitrocellulose (blocking, antibody incubation, washings and developing) was performed.
We assumed the same idea to use and optimize the ltration on membrane to the following phase of selection of dead cells from viable and VBNC ones in our experiments. We choose not to carry out the treatment on pellets but directly on membranes in order to use the commercial qPCR kit and to speed up the duration of the analyses. Unfortunately, from our results, we can hypothesize that interactions have occurred between lter and the dye, making its effect less effective on bacterial cells.
In conclusion, while cultural method remains the Gold standard for the research of Lp in waters, it is necessary to develop alternative methods that allow to shorten the analyses time and therefore to be able to intervene more quickly in case of positivity. At the same time, there is still a need to have a laboratory method that can detect VBNC bacteria and exclude dead ones. For this purpose, the qPCR could be conducted in parallel with culture method, even better when combined with a pre-treatment that allows to exclude dead bacteria. We therefore plan to continue our experience, modifying conditions of isolation, reagents, or dye.

Declarations
The data analyzed during the current study are available from the corresponding author.
Ethics approval and consent to participate not applicable.
Consent for publication not applicable.

competing interests
The authors declare that they have no competing interests.

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