Sample collection
Thirty-four samples of cold and hot water were collected using sterile bottles containing Sodium Thiosulfate (LP italiana SpA, Milan) after flushing water for 5 min; refrigerated samples were transported to the laboratory where they were analyzed both by culture method and qPCR, within 24 h of the sampling. The samples were collected from Healthcare Facilities distributed in Lombardy, Piedmont and Liguria (north Italy). Some of these water systems were treated continuously with disinfectants such as chlorine dioxide and monochloramine.
Study design included several phases: (1) applying both culture and molecular analysis to the same water sample; (2) applying and developing EMA qPCR method. Pure water samples, which were artificially spiked with viable, non-viable and VBNC Lp ATCC 33152 in serial dilutions, were tested. Initially, 3 different concentrations of the dye (1.25:2.5 and 5 μg/ml) were used, and higher concentrations (2.5, 10 and 20 μg/ml) were subsequently tested. Non-viable cells were obtained by heat treating, in an incubator, at 95 °C for 15 min (Qin et al. 2012, Ditommaso et al. 2015, Slimani et al. 2012) while VBNC cells were treated at 59 °C for 30 min (Slimani et al. 2012).
Culture method in environmental samples
ISO 11731-2-2004 “Water quality—detection and enumeration of Legionella” Part 2: “Direct membrane filtration method for waters with low bacterial counts” was followed to test viable cells through culture method (ISO 2014). Briefly, after filtration, the cellulose acetate membrane (0.22 μm) was placed directly on GVPN Agar Base medium and Petri dishes were incubated in an incubator with CO2 at 2.5%, at 37 °C for 10 days. Subsequently, suspicious colonies were tested for genus identification both on BCYE Agar and CYE Agar (ThermoFisher Scientific, Oxoid, Rodano, Italy) and only those which were BYCE positive were verified through latex agglutination tests (Legionella Latex Test, ThermoFisher Scientific, Oxoid, Rodano, Italy) to identify species and serogroups.
DNA extraction
The AquaScreen® FastExtract, validated according to ISO/TS 12869: 2012, was used to extract DNA from water samples, following the instructions provided by the manufacturer (see details in Fig. 1). Water samples were filtered through the polycarbonate membranes (size pores of 0.45 μm), provided by the kit, and any bacterium present was subjected to lysis directly onto the membrane, as required by the supplier’s protocol. Then, binding, removal of contaminants and elution of DNA were all enacted.
Quantitative PCR
Bacterial DNA was amplified by qPCR using the AquaScreen® Legionella pneumophila kit, validated according to AFNOR T90-471 and ISO/TS 12869: 2012, following the instructions provided by the manufacturer and using the StepOnePlus Thermocycler (Applied Biosystems, Foster City, CA, USA)
The amplification target is the Lp mip gene, which encodes for a protein implicated in the bacterium virulence mechanisms. The related primers are the following: Forward Legmip_f: 5′-GGG (AG)ATT(ACG)T TTATGA AGA TGA (AG)A(CT) TGG; Reverse Legmip_r: 5′-TC(AG) TT(ATCG) GG(ATG) CC(ATG) AT(ATCG)GG(ATCG) CC(ATG) CC.
A Reaction Mix, provided by the kit, contained, in addition to an internal control, all required primers, probes, dNTPs and Taq polymerase and was used for the amplification of the extracted DNA.
For each group of samples subjected to amplification, a negative control (PCR grade water), a positive control (provided by the kit) and a series of 6 standard dilutions of Lp were also analyzed.
Standard dilutions (PCR Quantification Standard), made from a stock solution of 108 GU provided by the kit, were necessary for the construction of a standard reference curve and the quantification of the amplified DNA (GU).
For the amplification reaction, 1 cycle was performed at 95 °C for 5 min, followed by 45 cycles composed as so: “denaturation” (95 °C for 30 s), “annealing" (55 °C for 30 s) and “elongation and data collection” (60 °C for 45 s).
EMA treatment
Each pure water sample, artificially spiked for the tests, was filtered using membranes supplied by the AquaScreen FastExtract (Fx) extraction kit and EMA treatment was carried out directly on membranes, to integrate dye treatment to the kit protocol (Fig. 1).
Two different experimental tests were carried out: one with EMA at concentrations of 1.25; 2.5 and 5 μg/ml and a second with EMA at concentrations of 2.5, 10 and 20 μg/ml (Mansi et al. 2014, Qin et al. 2012, Chang et al. 2009, Chen and Chang, 2010, Delgado et al. 2009). Each membrane was placed in a Petri dish and covered with 500μl of EMA at different concentrations. Petri dishes were incubated in an incubator at 21 °C for 10 min, then placed in a tray with ice and exposed to light for 5 min (500 W) at 15/20 cm from the light source (Qin et al. 2012). Lastly, each membrane was taken and placed in a new Petri dish for bacterial DNA extraction and amplification procedures, as provided by kit instructions.
Data interpretation
Values of threshold cycle (Ct) indicate the presence or absence of Lp and the amplification reaction is to be considered positive with Ct < 40 and negative with Ct ≥ 40 (from AquaScreen Legionella pneumophila Instructions for use).
The detected GUs refer to the total number of bacterial cells, viable and cultivable, viable but not cultivable and non-viable. It should be noted that the AquaScreen® Legionella pneumophila kit has a “limit of detection” (LOD) of 20 GU and a “limit of quantification” (LOQ) of 50 GU.
The Fisher’s exact test, along with sensitivity, specificity and predictive values, was calculated for cultural and qPCR results on water samples from healthcare facilities. The non-parametric linear by linear trend test was applied to the results deriving from qPCR and EMA-qPCR experiments for viable, VBNC and non-viable strains, using Stata17.0, Stata Corp., College Station, TX, USA 2021.