A comparative pre-post ecological study was conducted from September 2006 to January 2011.
Water sampling and microbiological analysis
Plumbing and toilet water tanks on passenger trains were examined.
Two samples were collected from every sampling site at a single time point: one sample was harvested immediately after the tap was switched on and without flaming for qualitative evaluation of the Legionella species and serogroup and the second sample was collected for quantitative determination (Legionella CFU/L of water) after the water ran for at least 5–10 minutes, which is more representative of the water flowing in the system. At least 6 to 12 water samples were collected weekly.
One litre of water was collected in sterile bottles with 0.01% sodium thiosulphate to neutralise any residual chlorine; water samples were transported in a suitable cool box, protected from direct light and processed within four hours after collection.
Microbiological detection of Legionella spp. was conducted following the methods described in the “Italian Guidelines for Legionellosis prevention and control” [19, 20].
Ten millilitres of the sample were first put into a sterile screw-capped Falcon tube, and then, a one-litre sample of water was concentrated by filtration on a 0.20 μm pore-diameter cellulose membrane filter.
The membranes were aseptically removed and placed into the sterile screw-capped Falcon tube previously filled with 10 ml of water sample collected; the material collected onto the membrane filter was vortex-mixed continuously for 2 min.
Ten millilitres of the sample was divided into two aliquots of 5 ml each; one aliquot was incubated at 50°C in a water bath for 30 minutes (treated sample) and the second aliquot remained at room temperature (non-treated sample).
Next, 0.1 ml of the treated and non-treated samples was spread on a 90 mm Petri dish containing GVPC-selective medium (glycine, polymyxin B, vancomycin and cycloheximide). These plates were incubated at 37°C in a humidified atmosphere with 2.5% CO2. Plates were evaluated for a maximum of 14 days before reporting them negative.
Suspected colonies that displayed a distinctive surface with an iridescent and faceted cut glass appearance were counted from each sampling. Gram staining was performed on the suspected colonies; weakly staining, gram-negative bacilli were observable. Suspicious colonies were sub-cultured both onto buffered charcoal yeast extract (BCYE) agar with cysteine and charcoal yeast extract agar (CYE Agar Base – Oxoid) for verification.
These plates were incubated at 37°C in a humidified atmosphere with 2.5% CO2 for ≥2 days.
The isolated colonies growing only on BCYE, but that did not grow on CYE, were determined to be Legionella colonies. Subsequently, these colonies were serotyped by the agglutination Legionella Latex Test (Oxoid) that provides separate identification of L. pneumophila serogroup 1, L. pneumophila serogroups 2–14 and other species of Legionella spp. that have been implicated in human disease: L. longbeachae 1 and 2, L. bozemanii 1 and 2, L. dumoffii, L. gormanii, L. jordanis, L. micdadei and L. anisa.
According to the Italian Guidelines , quantitative evaluation (CFU/L) was performed considering the following items: the count of colonies growth in each plate, the concentration of the original sample and the dilutions successively made.
Water supply on board trains and environmental decontamination procedures
The water distributed in the toilets of passenger cars, both for the washbasin and for sanitary services, must have, at the time of supply, the characteristics required for human consumption. Because of the characteristics of hydraulic systems on board, these requirements cannot be fully insured, so water is labelled "no drinking".
Passenger railway cars are fitted with steel or resin tanks in which the total volume of water is variable depending on the type of car (ranging from 200 to 1,800 litres).
The water tanks are located under the car’s roof and the water flows into the toilets by gravity.
Tanks are refilled at stations or through installation maintenance by jet spouts placed on side rails and discharge takes place through a special tap below the wagon.
When Legionella bacterial load was greater than 103 CFU/L, decontamination procedures were performed [19, 20]. Decontamination was completed in September 2008 (pre-decontamination step = September 2006-September 2008), so October 2008 is considered the beginning of the post-decontamination period.
The measures of supply reservoir and pipeline decontamination involve chemical de-scaling with 20% acetic acid and hyper-chlorination with 20 mg/l for 2 hours or 50 mg/l for 1 hour [19, 20], followed by washing to reach 0.2 mg/l of free residual chlorine concentration, or even replacement in limited cases of obvious damage.
The prevalence proportion of all positive samples was calculated; the following bacterial load values were adopted to perform a statistical analysis: <100 CFU/L, 100–999 CFU/L, 1,000-9,999 CFU/L, >10,000 CFU/L.
The unpaired t-test was performed to evaluate statistically significant differences between the mean load values before and after the decontamination procedures; statistical significance was set at p ≤ 0.05.