LEGIONELLA SPP SURVIVAL AFTER DIFFERENT DISINFECTION PROCEDURES: COMPARISON BETWEEN CONVENTIONAL CULTURE AND EMA-QPCR

Quantitative real-time-PCR (q-PCR) is largely used to evaluate different pathogens in both environmental and clinical samples, including Legionella spp Advantages on traditional culture include the high sensitivity, accuracy and rapid evaluation of germ contamination. The main disadvantage is that this method does not distinguish dead/viable cells, a relevant aspect in case of continuous disinfection like in swimming pools. This study presents a new quantitative PCR method using ethidium monoazide (EMA) to detect only viable cells. The comparison with conventional culture can further inform on culturable and VBNC (viable but not culturable) Legionella spp. To develop the new test, experiments have been conducted with L. pneumophila ATCC 33152. We compared EMA addition at different concentrations on both viable cells during exponential growth period and dead cells. After DNA extraction, q-PCR reaction was made using LightMix kit Legionella spp with amplification of the fragment 386 bp of 16S gene. Water samples from swimming pools and hot water distribution systems treated with both chlorine and other disinfection procedures have been collected for analysis by EMA-qPCR. All samples have been also analysed with conventional culture (ISO-11731) to isolate Legionella spp. EMA in the range 100-6 μM was able to completely inhibit DNA amplification in samples containing dead legionellae at concentration of 3x105 UFC/L. In contrast, EMA at concentrations of 6 μM did not reduce the q-PCR results on viable Legionella. However, higher EMA levels were associated with partial inhibition of DNA amplification on viable cells due to dye toxicity. The analyses on real water samples are still in course. Our results confirm that the Ema-qPCR can represent a new procedure able to distinguish dead and viable legionellae. We underline the need for a balance between EMA toxicity and efficacy, depending on Legionella spp concentration. The results of this method on water samples collected from both swimming pools and hot water distribution systems will be useful to compare EMA-qPCR and culture in evaluating differences in dead/viable/culturable germ cells according to various disinfection procedures.