Serological and molecular identification of Legionella spp. isolated from water and surrounding air samples in Italian healthcare facilities.

BACKGROUND: Legionella is an intracellular microorganism living in natural and artificial aquatic environments. Although its transmission to humans is linked to the inhalation of contaminated aerosols, there is no validated air sampling method for the control and prevention of the disease. The aim of the present study was to provide more information on the distribution of Legionella spp. in indoor environments and to determine whether the same Legionella strains are isolated from air and water samples. METHODS: Ten healthcare facilities located in seven regions of Italy were enrolled. The serological typing of Legionella spp. from water samples and the surrounding air by active and passive sampling was assessed using polyvalent and monovalent antisera. Subsequently, the strains identified as Legionella pneumophila (Lpn) underwent molecular typing by sequence-based typing (SBT) using seven genes (flaA, pilE, asd, mip, mompS, proA, and neuA). The allelic profile number was assigned using the European Working Group for Legionella Infections-SBT database. RESULTS: Lpn serogroup 6 was the most prevalent serogroup; it was found simultaneously in the air and water samples of three different healthcare facilities. In the remaining seven hospitals, Lpn serogroups 1, 6, 7, 9, and 12 were isolated exclusively from water samples. The molecular investigation showed that Lpn strains in the water and air samples of each positive healthcare facility had the same allelic profile. Strains, identified as sequence types (STs) 728 and ST 1638+ST 1324, were isolated in two respective healthcare facilities, and a new strain, identified as ST 1989, was obtained in one healthcare facility. CONCLUSION: The application of the SBT method allowed to verify the homology among Legionella strains from water samples and the surrounding air. The results showed that the same Lpn strains were present in the air and water samples, and a new Legionella strain was identified.

[Legionella spp. contamination in indoor air: preliminary results of an Italian multicenter study]. / La contaminazione indoor da Legionella spp: risultati preliminari di una indagine multicentrica italiana.

OBJECTIVE: To propose a standardized protocol for the evaluation of Legionella contamination in air. DESIGN: A bathroom having a Legionella contamination in water >1,000 cfu/l was selected in 10 different healthcare facilities. Air contamination was assessed by active (Surface Air System, SAS) and passive (Index of Microbial Air, IMA) sampling for 8 hours, about 1 m away from the floor and 50 cm from the tap water. Two hundred liters of air were sampled by SAS every 12 min, after flushing water for 2 min. The IMA value was calculated as the mean value of colony forming units/16 plates exposed during sampling (2 plates/hour). Water contamination was evaluated at T0, after 4 and 8 hours, according to the standard methods. RESULTS: Air contamination by Legionella was found in three healthcare facilities (one with active and two with passive sampling), showing a concomitant tap water contamination (median=40,000; range 1,100-43,000 cfu/l). The remaining seven hospitals isolated Legionella spp. exclusively from water samples (median=8,000; range 1,200-70,000 cfu/l). CONCLUSIONS: Our data suggest that environmental Legionella contamination cannot be assessed only through the air sampling, even in the presence of an important water contamination.

The Presence of Legionella in Water Used for Car Washing: Implications for Public Health.

Although today all of the aspects of Legionella are better understood than in the past, in many countries the interest is still mainly focused on healthcare and tourism facilities. Other at-risk areas are less explored, such as those where workers are often in contact with water during their activities. In reality, any water system capable of producing aerosols can be considered a potential source of Legionella transmission, including car washes, where a large number of users work and flow through annually. From January to May 2022, 120 samples were carried out in 30 car washes located in Messina (Italy): 60 samples of water and 60 of aerosols. The aim of this investigation was to evaluate the risk of legionellosis in car washing workers exposed to potentially contaminated aerosols. To increase the probability of finding Legionella, the sample collections were organized on different days of the week. Of the total samples taken, 10 (8.3%) were positive for Legionella: seven (11.7%) water (range 100-1000 CFU) and three (5%) aerosol (range 10-150 CFU) samples. Detected serogroups were L. pneumophila sgr 1, 7, 10 and Legionella gormanii. Given the results obtained, preventative measures should be implemented in such facilities in order to protect the health of users and car wash operators.

The contribution of professors of Hygiene to the progress and development of public health in Italy: one hundred years of history.

In 1917 Achille Sclavo, a distinguished researcher and founder of the Italian Society of Hygiene, ended up the first term as Rector of the University of Siena. Since then, the contribution of professors of hygiene in Italian universities has ranged over several relevant topics including vaccinations, environmental hygiene, hospital hygiene, healthcare organization and management, with an important contribution to the health reform of 1978 by Augusto Giovanardi and Alessandro Seppilli. Several Academic Schools (Roman, Neapolitan, Genoese, Sicilian, Venetian, Lombard, etc.) have produced excellent researchers, teachers and mentors who have also occupied important positions in the panorama of the Italian health system. This note analyzes the main research topics, the most famous institutes and departments of hygiene and public health and the contributions of the most famous professors for the development of the discipline, the management of the post- graduated Schools in hygiene and preventive medicine, the commitment in scientific associations and the role of some of them in important institutional positions. Even through its contribution and constant commitment to the institutions, Italian public health has achieved the reputation of being one of the best known and appreciated in the international scientific community.

Innate Immune Surveillance in the Central Nervous System Following Legionella pneumophila Infection.

BACKGROUND & OBJECTIVE: The innate immune response is a common occurrence in many neuroinflammatory diseases. Central Nervous System (CNS) resident immune cells are able to detect and react to infections and sterile trauma. Peripheral immune cell migration into CNS is regulated by the blood-brain barrier, although peripheral immune cells can invade CNS through meninges, choroid plexus, perivascular spaces, and cerebrospinal fluid. Consequently, in the brain, immune reactions can be mediated by both resident and peripheral immune cells. Both in the periphery and within the CNS, innate immune response is regulated by a wide array of pattern recognition receptors, including Tolllike, scavenger, Retinoic Acid-inducible Gene-1 like, and nucleotide-binding oligomerization domainslike responsible for inflammasome formation. Inflammasome pathway activation induces pyroptosis, a highly inflammatory cell death pattern that occurs to remove intracellular pathogens. Legionella pneumophila is an intracellular microorganism responsible for Legionnaires' disease, a lung infection always associated to neurological dysfunctions. Recent studies have been shown that Toll-like receptors, nucleotide-binding oligomerization domains-like receptors, and RIG-1 like, are activated by L. pneumophila. This flagellated bacterium is able to replicate in phagocytic cells, including macrophages and microglia, responding by activating inflammasome pathways that may be the cause of CNS dysfunction detected in several infected patients. CONCLUSION: The aim of this review is to bring together the latest findings concerning L. pneumophila infection and innate immune host cell responses. A deeper knowledge of these processes could allow the use of immunomodulatory compounds able to counteract CNS involvement following L. pneumophila infection.

Evaluation of Legionella Air Contamination in Healthcare Facilities by Different Sampling Methods: An Italian Multicenter Study.

Healthcare facilities (HF) represent an at-risk environment for legionellosis transmission occurring after inhalation of contaminated aerosols. In general, the control of water is preferred to that of air because, to date, there are no standardized sampling protocols. Legionella air contamination was investigated in the bathrooms of 11 HF by active sampling (Surface Air System and Coriolis®µ) and passive sampling using settling plates. During the 8-hour sampling, hot tap water was sampled three times. All air samples were evaluated using culture-based methods, whereas liquid samples collected using the Coriolis®µ were also analyzed by real-time PCR. Legionella presence in the air and water was then compared by sequence-based typing (SBT) methods. Air contamination was found in four HF (36.4%) by at least one of the culturable methods. The culturable investigation by Coriolis®µ did not yield Legionella in any enrolled HF. However, molecular investigation using Coriolis®µ resulted in eight HF testing positive for Legionella in the air. Comparison of Legionella air and water contamination indicated that Legionella water concentration could be predictive of its presence in the air. Furthermore, a molecular study of 12 L. pneumophila strains confirmed a match between the Legionella strains from air and water samples by SBT for three out of four HF that tested positive for Legionella by at least one of the culturable methods. Overall, our study shows that Legionella air detection cannot replace water sampling because the absence of microorganisms from the air does not necessarily represent their absence from water; nevertheless, air sampling may provide useful information for risk assessment. The liquid impingement technique appears to have the greatest capacity for collecting airborne Legionella if combined with molecular investigations.