Occurrence of Legionella spp. in thermal environments: Virulence factors and biofilm formation in isolates from a spa

Abstract

The aim of the study was to evaluate the occurrence of Legionella spp. in the water system of a spa in the city of Naples by analyzing water, air and surface samples. On the whole, 312 samples were collected and analyzed in the course of 10months. Legionella CYE Agar Base and Legionella Latex Test (Oxoid©) were used to identify and serotype presumptive Legionella pneumophila strains. A further identification was carried out by rDNA16S and ITS region amplification followed by a sequence analysis by DNA Sequencing Analysis software (Applied Biosystems). Similarity search was performed using BLAST algorithm against the GenBank database (NCBI GenBank). Specific in-vitro tests aimed to evaluate the production of esoenzymes (hemolysins, collagenases, mucinases, lipases, proteinases, DNAses, elastases) on GC-FC Agar were also carried out. Finally, a crystal violet staining method (absorbance at 570nm) was used to evaluate the ability of the strains to produce biofilm in a 96-multiwell polyethylene plate. All samples were negative for L. pneumophila. Six different Legionella strains were isolated from water samples and identified as Legionella londiniensis and Legionella spp. A significant (from 1000 to 10,000CFU/L) and a low to moderate (from 100 to 1000CFU/L) contamination were detected respectively in the 5% and 4% of samples; 91% of water samples showed a Legionella spp. amount less than 100CFU/L. Two Legionella londiniensis isolates showed collagenases, caseinases, proteinases and gelatinases activities, being classified as potentially pathogenic bacteria. None of the isolates were classified as strong biofilm producer but they showed a moderate to weak ability to form biofilm on polyethylene. This result is significant because large part of the spa pipelines is plastic-coated.The highest frequency of isolation of Legionella spp. was detected in the unit for Thermal Mud Therapy, which showed a relative risk value equal to 1.69 (CI 95% 0.60–4.70).Although our results proved a moderate contamination in different water samples, the presence of potentially pathogenic environmental strains of Legionella spp. should not be underestimated because most part of costumers attending the spa are old and sick people, and Legionella strains can represent a real risk.