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Legionella species are Gram negative, facultative, intracellular bacteria found in natural and engineered water systems. Understanding the bacterial interactions underlying the success of Legionella in aquatic environments could be beneficial for control. We aimed to profile, by 16S rRNA amplicon sequencing, the bacterial communities in premise plumbing systems of buildings in four European cities (Copenhagen, Warsaw, Rome, Athens), and identify positive and negative associations of specific community members to culturable Legionella. The coarse taxonomic composition was similar across the four cities, but Copenhagen and Warsaw had richer, more diverse communities than Athens and Rome, with a greater number of city-specific amplicon sequence variants (ASVs). The cities had statistically significant differences in bacterial communities at the ASV level, with relatively few shared ASVs. Out of 5,128 ASVs, 73 were classified as Legionella, and one or more of these were detected in most samples from each city (88.1% overall). Interestingly, the relative abundance of Legionella ASVs did not correlate with Legionella culture status. Overall, 44.2% of samples were Legionella culture positive: 71.4% in Warsaw, 62.2% in Athens, 22.2% in Rome, and 15.2% in Copenhagen. 54 specific ASVs and 42 genera had significant positive or negative associations with culturable Legionella. Negative associations included Staphylococcus, Pseudomonas, and Acinetobacter. Positive associations included several Nitrospira ASVs and one classified as Nitrosomodaceae oc32, ASVs in the amoeba-associated genera Craurococcus-Caldovatus and Reyranella, and the predatory genus Bdellovibrio. Some of these associations are well supported by laboratory studies, but others are the opposite of what was expected. This highlights the difficulties in translating pure culture results to into complex real-life scenarios. However, these positive and negative associations held across the four cities, across multiple buildings and plumbing compartments. This is important because developing better control measures, including probiotic approaches, will require an understanding of ecological relationships that can be generalised across different engineered water systems.ImportanceThis study provides a snapshot of the diversity of microbial communities among premise plumbing systems in four European cities, providing new information on bacterial ASVs and genera that have positive or negative associations with culturable Legionella across a broad geographical and climatic range. This could inform studies aimed at confirming both in vitro and real-life scenarios around the role of other microbial community members in modulating Legionella proliferation. It could also help in the development of probiotic approaches to controlling this opportunistic pathogen.

© 2022 Ricci et al. This article is available under a CreativeCommons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/)., Legionella pneumophila serogroup 1 (Lp1) sequence type (ST) 23 is one of the most commonly detected STs in Italy where it currently causes all investigated outbreaks. ST23 has caused both epidemic and sporadic cases between 1995 and 2018 and was analysed at genomic level and compared with ST23 isolated in other countries to determine possible similarities and differences. A core genome multi-locus sequence typing (cgMLST), based on a previously described set of 1,521 core genes, and single-nucleotide polymorphisms (SNPs) approaches were applied to an ST23 collection including genomes from Italy, France, Denmark and Scotland. DNAs were automatically extracted, libraries prepared using NextEra library kit and MiSeq sequencing performed. Overall, 63 among clinical and environmental Italian Lp1 isolates and a further seven and 11 ST23 from Denmark and Scotland, respectively, were sequenced, and pangenome analysed. Both cgMLST and SNPs analyses showed very few loci and SNP variations in ST23 genomes. All the ST23 causing outbreaks and sporadic cases in Italy and elsewhere, were phylogenetically related independent of year, town or country of isolation. Distances among the ST23s were further shortened when SNPs due to horizontal gene transfers were removed. The Lp1 ST23 isolated in Italy have kept their monophyletic origin, but they are phylogenetically close also to ST23 from other countries. The ST23 are quite widespread in Italy, and a thorough epidemiological investigation is compelled to determine sources of infection when this ST is identified in both LD sporadic cases and outbreaks.

Legionella pneumophila is ubiquitous in aquatic environments and responsible for severe pneumonia in humans through inhalation of aerosol containing Legionella spp. Macrolides and fluoroquinolones are frequently used antimicrobials, but treatment failures are increasingly being reported. As susceptibility testing is not routinely performed, this study aimed to determine the minimum inhibitory concentrations (MICs) on 58 environmental Legionella pneumophila strains (24 of serogroup 1 and 34 of non-serogroup 1) isolated in Northern Italy. MICs of azithromycin, erythromycin, ciprofloxacin, levofloxacin, and rifampicin were determined by the microdilution method using buffered yeast extract broth supplemented with α-ketoglutarate (BYEα). Seventy-five percent of Legionella pneumophila isolates showed MIC values below the tentative highest MICs indicated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), rifampicin was the most active agent with MIC90 values below 0.008 mg/L. Interestingly, one isolate was tested and found to be PCR-positive for the azithromycin LpeAB active efflux system, further confirmed by the reserpine/resazurin microtiter assay. In conclusion, this study has provided additional susceptibility data for environmental Legionella pneumophila isolates from Northern Italy demonstrating, in general, low MICs values for the tested antimicrobials, although one strain tested was shown to possess the LpeAB resistance determinant, indicating that future surveillance studies are warranted.

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids’ stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 104, 103 and 102 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2–8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2–8 °C was 83–100% and 75–95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella.

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids’ stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 104, 103 and 102 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2–8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2–8 °C was 83–100% and 75–95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella.

Legionella pneumophila is ubiquitous in aquatic environments and responsible for severe pneumonia in humans through inhalation of aerosol containing Legionella spp. Macrolides and fluoroquinolones are frequently used antimicrobials, but treatment failures are increasingly being reported. As susceptibility testing is not routinely performed, this study aimed to determine the minimum inhibitory concentrations (MICs) on 58 environmental Legionella pneumophila strains (24 of serogroup 1 and 34 of non-serogroup 1) isolated in Northern Italy. MICs of azithromycin, erythromycin, ciprofloxacin, levofloxacin, and rifampicin were determined by the microdilution method using buffered yeast extract broth supplemented with α-ketoglutarate (BYEα). Seventy-five percent of Legionella pneumophila isolates showed MIC values below the tentative highest MICs indicated by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); rifampicin was the most active agent with MIC90 values below 0.008 mg/L. Interestingly, one isolate was tested and found to be PCR-positive for the azithromycin LpeAB active efflux system, further confirmed by the reserpine/resazurin microtiter assay. In conclusion, this study has provided additional susceptibility data for environmental Legionella pneumophila isolates from Northern Italy demonstrating, in general, low MICs values for the tested antimicrobials, although one strain tested was shown to possess the LpeAB resistance determinant, indicating that future surveillance studies are warranted.

In September 2018 in Brescia province, northern Italy, an outbreak of Legionnaires' disease (LD) caused by Legionella pneumophila serogroup 2 (Lp2) occurred. The 33 cases (two fatal) resided in seven municipalities along the Chiese river. All cases were negative by urinary antigen test (UAT) and most were diagnosed by real-time PCR and serology. In only three cases, respiratory sample cultures were positive, and Lp2 was identified and typed as sequence type (ST)1455. In another three cases, nested sequence-based typing was directly applied to respiratory samples, which provided allelic profiles highly similar to ST1455. An environmental investigation was undertaken immediately and water samples were collected from private homes, municipal water systems, cooling towers and the river. Overall, 533 environmental water samples were analysed and 34 were positive for Lp. Of these, only three samples, all collected from the Chiese river, were Lp2 ST1455. If and how the river water could have been aerosolised causing the LD cases remains unexplained. This outbreak, the first to our knowledge caused by Lp2, highlights the limits of UAT for LD diagnosis, underlining the importance of adopting multiple tests to ensure that serogroups other than serogroup 1, as well as other Legionella species, are identified.

The COVID-19 pandemic started in China in early December 2019, and quickly spread around the world. The epidemic gradually started in Italy at the end of February 2020, and by May 31, 2020, 232,664 cases and 33,340 deaths were confirmed. As a result of this pandemic, the Italian Ministerial Decree issued on March 11, 2020, enforced lockdown; therefore, many social, recreational, and cultural centers remained closed for months. In Apulia (southern Italy), all non-urgent hospital activities were suspended, and some wards were closed, with a consequent reduction in the use of the water network and the formation of stagnant water. This situation could enhance the risk of exposure of people to waterborne diseases, including legionellosis. The purpose of this study was to monitor the microbiological quality of the water network (coliforms, E. coli, Enterococci, P. aeruginosa, and Legionella) in three wards (A, B and C) of a large COVID-19 regional hospital, closed for three months due to the COVID-19 emergency. Our study revealed that all three wards' water network showed higher contamination by Legionella pneumophila sg 1 and sg 6 at T1 (after lockdown) compared to the period before the lockdown (T0). In particular, ward A at T1 showed a median value = 5600 CFU/L (range 0–91,000 CFU/L) vs T0, median value = 75 CFU/L (range 0–5000 CFU/L) (p-value = 0.014); ward B at T1 showed a median value = 200 CFU/L (range 0–4200 CFU/L) vs T0, median value = 0 CFU/L (range 0–300 CFU/L) (p-value = 0.016) and ward C at T1 showed a median value = 175 CFU/L (range 0–22,000 CFU/L) vs T0, median value = 0 CFU/L (range 0–340 CFU/L) (p-value, Highlights • Microbiological quality of the water network in a COVID-19 hospital was monitored. • The water network showed high contamination by L. pneumophila. • Coliforms and P.aeruginosa were detected in temporarily closed units.

Legionella spp are the causative agents of Legionnaires&rsquo, diseases, which is a pneumonia of important public health concern. Ubiquitous freshwater and soil inhabitants can reach man-made water systems and cause illness. Legionella enumeration and quantification in water systems is crucial for risk assessment and culture examination is the gold standard method. In this study, Legionella recovery from potable water samples, at presumably a low concentration of interfering microorganisms, was compared by plating on buffered charcoal yeast extract (BCYE) and glycine, vancomycin, polymyxin B, cycloheximide (GVPC) Legionella agar media, according to the International Standard Organization (ISO) 11731: 2017. Overall, 556 potable water samples were analyzed and 151 (27.1%) were positive for Legionella. Legionella grew on both BCYE and GVPC agar plates in 85/151 (56.3%) water samples, in 65/151 (43%) on only GVPC agar plates, and in 1/151 (0.7%) on only BCYE agar plates. In addition, GVPC medium identified Legionella species other than pneumophila in six more samples as compared with the culture on BCYE. Although the medians of colony forming units per liter (CFU/L) detected on the BCYE and GVPC agar plates were 2500 and 1350, respectively (p-value &lt, 0.0001), the difference did not exceed one logarithm, and therefore is not relevant for Legionella risk assessment. These results make questionable the need to utilize BCYE agar plates to analyze potable water samples.

In July 2018, a large outbreak of Legionnaires’ disease (LD) caused by Legionella pneumophila serogroup 1 (Lp1) occurred in Bresso, Italy. Fifty-two cases were diagnosed, including five deaths. We performed an epidemiological investigation and prepared a map of the places cases visited during the incubation period. All sites identified as potential sources were investigated and sampled. Association between heavy rainfall and LD cases was evaluated in a case-crossover study. We also performed a case–control study and an aerosol dispersion investigation model. Lp1 was isolated from 22 of 598 analysed water samples; four clinical isolates were typed using monoclonal antibodies and sequence-based typing. Four Lp1 human strains were ST23, of which two were Philadelphia and two were France-Allentown subgroup. Lp1 ST23 France-Allentown was isolated only from a public fountain. In the case-crossover study, extreme precipitation 5–6 days before symptom onset was associated with increased LD risk. The aerosol dispersion model showed that the fountain matched the case distribution best. The case–control study demonstrated a significant eightfold increase in risk for cases residing near the public fountain. The three studies and the matching of clinical and environmental Lp1 strains identified the fountain as the source responsible for the epidemic.

In July 2018, a large outbreak of Legionnaires’ disease (LD) caused by Legionella pneumophila serogroup 1 (Lp1) occurred in Bresso, Italy. Fifty-two cases were diagnosed, including five deaths. We performed an epidemiological investigation and prepared a map of the places cases visited during the incubation period. All sites identified as potential sources were investigated and sampled. Association between heavy rainfall and LD cases was evaluated in a case-crossover study. We also performed a case–control study and an aerosol dispersion investigation model. Lp1 was isolated from 22 of 598 analysed water samples; four clinical isolates were typed using monoclonal antibodies and sequence-based typing. Four Lp1 human strains were ST23, of which two were Philadelphia and two were France-Allentown subgroup. Lp1 ST23 France-Allentown was isolated only from a public fountain. In the case-crossover study, extreme precipitation 5–6 days before symptom onset was associated with increased LD risk. The aerosol dispersion model showed that the fountain matched the case distribution best. The case–control study demonstrated a significant eightfold increase in risk for cases residing near the public fountain. The three studies and the matching of clinical and environmental Lp1 strains identified the fountain as the source responsible for the epidemic.

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Legionnaires’ disease (LD) is a severe pneumonia caused by bacteria belonging to the genus Legionella. This is a major public health concern and infections are steadily increasing worldwide. Several sources of infection have been identified, but they have not always been linked to human isolates by molecular match. The well-known Legionella contamination of private homes has rarely been associated with the acquisition of the disease, although some patients never left their homes during the incubation period. This study demonstrated by genomic matching between clinical and environmental Legionella isolates that the source of an LD cluster was a private building. Monoclonal antibodies and sequence-based typing were used to type the isolates, and the results clearly demonstrated the molecular relationship between the strains highlighting the risk of contracting LD at home. To contain this risk, the new European directive on the quality of water intended for human consumption has introduced for the first time Legionella as a microbiological parameter to be investigated in domestic water systems. This should lead to a greater attention to prevention and control measures for domestic Legionella contamination and, consequently, to a possible reduction in community acquired LD cases.

According to European Guidelines for Legionnaires’ Disease prevention and control, travel-associated Legionnaires’ disease (TALD) cases are managed differently if classified as sporadic or as part of a cluster and more stringent control measures are deployed after clusters are identified. In this study, we propose to modify the current cluster definition: ‘two or more cases of Legionnaires’ disease (LD) who stayed at, or visited, the same commercial accommodation site 2–10 days before onset of illness and whose onset is within the same 2-year period’ with a new cluster definition, i.e. accommodation sites associated with multiple cases regardless of the time elapsed between them. TALD cases occurred in Italy and in the Balearic Islands between 2005 and 2015 were analysed applying the current European Legionnaires’ Disease Surveillance Network (ELDSNet) cluster definition. In a sample of selected accommodation sites with multiple cases, a microbiological study was also conducted. Using the new definition, 63 additional sites (16.4% increase) and 225 additional linked cases (19.5% increase) were identified.Legionella pneumophilasg1 was isolated from 90.7% of the selected accommodation sites. The use of the here proposed TALD cluster definition would warrant a full investigation for each new identified case. This approach should therefore increase the number of sites that will require a risk assessment and, in the presence of an increased risk, the adoption of LD control measures to hopefully prevent additional cases.

Aim To evaluate real-time PCR as a diagnostic method for Legionnaires’ disease (LD). Detection of Legionella DNA is among the laboratory criteria of a probable LD case, according to the European Centre for Disease Prevention and Control, although the utility and advantages, as compared to culture, are widely recognised. Methods Two independent laboratories, one using an in-house and the other a commercial real-time PCR assay, analysed 354 respiratory samples from 311 patients hospitalised with pneumonia between 2010–15. The real-time PCR reliability was compared with that of culture and urinary antigen tests (UAT). Concordance, specificity, sensitivity and positive and negative predictive values (PPV and NPV, respectively) were calculated. Results Overall PCR detected eight additional LD cases, six of which were due to Legionella pneumophila (Lp) non-serogroup 1. The two real-time PCR assays were concordant in 99.4% of the samples. Considering in-house real-time PCR as the reference method, specificity of culture and UAT was 100% and 97.9% (95% CI: 96.2–99.6), while the sensitivity was 63.6% (95%CI: 58.6–68.6) and 77.8% (95% CI: 72.9–82.7). PPV and NPV for culture were 100% and 93.7% (95% CI: 91.2-96.3). PPV and NPV for UAT were 87.5% (95% CI: 83.6-91.4) and 95.8% (95% CI: 93.5-98.2). Conclusion Regardless of the real-time PCR assay used, it was possible to diagnose LD cases with higher sensitivity than using culture or UAT. These data encourage the adoption of PCR as routine laboratory testing to diagnose LD and such methods should be eligible to define a confirmed LD case.

SUMMARYAn unusually long-lasting community-acquired outbreak of Legionnaires’ disease (LD) occurred in the inhabitants of a town in northern Italy from 2005 to 2008. Overall, 43 cases were diagnosed including five deaths. Hundreds of water samples were collected forLegionellaisolation but only two clinical samples were obtained. Clinical strains were ST23 as were environmental isolates detected in mostLegionella-positive patients' homes and those from a public fountain. Although noLegionellawas found in the municipal water mains, a continuous chlorination was applied in 2008. This action resulted in a halving of cases, although incidence remained tenfold higher than the Italian average incidence until the end of 2013, when it dropped to the expected rate. Retrospective analyses of prevalent wind direction suggested that a hidden cooling tower could have been the main cause of this uncommon outbreak, highlighting the importance of implementation of cooling tower registers in supporting LD investigations.

Molecular typing methods for discriminating different bacterial isolates are essential epidemiological tools in prevention and control of Legionella infections and outbreaks. A selection of 56 out of 184 Legionella pneumophila serogroup 1 (Lp1) clinical isolates, collected from different Italian regions between 1987 and 2012, and stored at the National Reference Laboratory for Legionella , were typed by monoclonal antibody (MAb) subgrouping, amplified fragment length polymorphism (AFLP) and sequence based typing (SBT). These strains were isolated from 39 community (69.6%), 14 nosocomial (25%) and 3 travel associated (5.4%) Legionnaires’disease cases. MAb typing results showed a prevalence of MAb 3/1 positive isolates (75%) with the Philadelphia subgroup representing 35.7%, followed by Knoxville (23.2%), Benidorm (12.5%), Allentown/France (1.8%), Allentown/France-Philadelphia (1.8%). The remaining 25% were MAb 3/1 negative, namely 11 Olda (19.6%), 2 Oxford (3.6%) and 1 Bellingham (1.8%) subgroups. AFLP analysis detected 20 different genomic profiles. SBT analysis revealed 32 different sequence types (STs) with high diversity of STs (IOD STs = 0.952) 12 of which were never described before. ST1 and ST23 were most frequently isolated as observed worldwide. A helpful analysis of data from SBT, MAb subgrouping and AFLP is provided, as well as a comparison to the Lp1 types investigated from other countries. This study describes the first Italian Lp1 strains database, providing molecular epidemiology data useful for future epidemiological investigations, especially of travel associated Legionnaires’ diseases (TALD) cases, Italy being the country associated with the highest number of clusters.

Legionella quantification in environmental samples is overestimated by qPCR. Combination with a viable dye, such as Propidium monoazide (PMA), could make qPCR (named then vPCR) very reliable. In this multicentre study 717 artificial water samples, spiked with fixed concentrations of Legionella and interfering bacterial flora, were analysed by qPCR, vPCR and culture and data were compared by statistical analysis. A heat-treatment at 55 °C for 10 minutes was also performed to obtain viable and not-viable bacteria. When data of vPCR were compared with those of culture and qPCR, statistical analysis showed significant differences (P0.001). However, although the heat-treatment caused an abatement of CFU/mL ≤1 to 1 log10 unit, the comparison between untreated and heat-treated samples analysed by vPCR highlighted non-significant differences (P0.05). Overall this study provided a good experimental reproducibility of vPCR but also highlighted limits of PMA in the discriminating capability of dead and live bacteria, making vPCR not completely reliable.

Aims: To perform an international trial to derive alert and action levels for the use of quantitative PCR (qPCR) in the monitoring of Legionella to determine the effectiveness of control measures against legionellae. Methods and Results: Laboratories (7) participated from six countries. Legionellae were determined by culture and qPCR methods with comparable detection limits. Systems were monitored over ‡10 weeks. For cooling towers (232 samples), there was a significant difference between the log mean difference between qPCR (GU l )1 ) and culture (CFU l )1 ) for Legionella pneumophila (0AE71) and for Legionella spp. (2AE03). In hot and cold water (506 samples), the differences were less, 0AE62 for Leg. pneumophila and 1AE05 for Legionella spp. Results for individual systems depended on the nature of the system and its treatment. In cooling towers, Legionella spp. GU l )1 always exceeded CFU l )1 , and usually Legionella spp. were detected by qPCR when absent by culture. The pattern of results by qPCR for Leg. pneumophila followed the culture trend. In hot and cold water, culture and qPCR gave similar results, particularly for Leg. pneumophila. There were some marked exceptions with temperatures ‡50� C, or in the presence of supplementary biocides. Action and alert levels for qPCR were derived that gave results comparable to the application of the European Guidelines based on culture. Algorithms are proposed for the use of qPCR for routine monitoring. Conclusions: Action and alert levels for qPCR can be adjusted to ensure public health is protected with the benefit that remedial actions can be validated earlier with only a small increase in the frequency of action being required. Significance and Impact of the Study: This study confirms it is possible to derive guidelines on the use of qPCR for monitoring the control of legionellae with consequent improvement to response and public health protection.

The aim of this study was to determine the in vitro activity of Melaleuca alternifolia Cheel (tea tree) oil (TTO) against 22 strains of Legionella pneumophila of different serogroup and source of isolation. Both a standard broth micro-dilution method, with slight modifications, and a micro-atmosphere diffusion method were used. Furthermore, we have established a simple sealing procedure in the micro-dilution method to determine the antibacterial activity of TTO against Legionella in aqueous phase. The results showed that L. pneumophila, quite irrespective of serogroup and source of isolation, is exquisitely sensitive to TTO, with minimal inhibitory concentration (MIC) ranging from 0.125 to 0.5% v/v, and a bactericidal activity at 0.5% v/v. In addition, we show here that TTO vapours exert critical activity, that must be controlled for reproducible MIC determinations. Overall, our data suggest that TTO could be active as anti-Legionella disinfectant, for control of water system contamination, especially in spas, in small waterlines or in particular respiratory medical devices.

In 2004, an outbreak of legionnaires disease occurred in a hospital in northern Italy with a water system that had been disinfected multiple times since 1990 and equipped with a continuous disinfecting system. Molecular typing linked the outbreak to contamination of the hospital water system and demonstrated the persistence of a predominant strain ofLegionella pneumophilafor 15 years.

Between August and October 2003, 15 cases of Legionnaires' disease were detected in the 9th district of Rome. To identify possible sources of Legionella exposure, a matched case-control study was conducted and environmental samples were collected. Hospital discharge records were also retrospectively analysed for the period July–November 2003, and results were compared with the same period during the previous 3 years. The case-control study revealed a significantly increased risk of disease among those frequenting a specific department store in the district (OR 9·8, 95% CI 2·1–46·0), and Legionella pneumophila was isolated from the store's cooling tower. Genotypic and phenotypic analysis of human and environmental isolates demonstrated that the cluster was caused by a single strain of L. pneumophila serogroup 1, and that the cooling tower of the store was the source of infection. The increased number of hospital admissions for microbiologically undiagnosed pneumonia during the study period may indicate that some legionellosis cases were not identified.

A middle-aged immunocompetent woman was diagnosed and treated for a severe pulmonary human herpesvirus 3 infection. During the treatment, an infection from Legionella pneumophila serogroup 1 was also diagnosed. This coinfection threatened the life of the patient and led to serious permanent sequelae. This report highlights the importance of preventing Legionella environmental contamination, suspecting Legionella coinfection in patients with viral pneumonia, and vaccinating susceptible adults against chickenpox.

Legionella pneumophila is a pathogen widespread in aquatic environment, able to multiply both within amoebae and human macrophages. The aim of this study was to identify genes differently expressed in a spontaneous avirulent Legionella pneumophila serogroup 6 mutant, named Vir-, respect the parental strain (Vir+), and to determine their role in the loss of virulence. Protein profiles revealed some differences in Vir- proteomic maps, and among the identified proteins the undetectable 3-hydroxybutyrate dehydrogenase (BdhA) and a down-produced lipase. Both Legionella enzymes were studied before and were here further characterized at genetic level. A significant down-regulation of both genes was observed in Vir- at the transcriptional level, but the use of defined mutants demonstrated that they did not affect the intracellular multiplication. A mutant (MS1) showed an accumulation of poly-3-hydroxybutyrate (PHB) granules suggesting a role of bdhA gene in its degradation process. The lipase deduced amino acid sequence revealed a catalytic triad, typical of the 'lipase box' characteristic of PHB de-polymerase enzymes, that let us suppose a possible involvement of lipase in the PHB granule degradation process. Our results revealed unexpected alterations in secondary metabolic pathways possibly linking the loss of virulence to Legionella lack of energy sources.

This chapter talks about the role of the flagellum in the observed protection from legionella infection that was investigated by the authors. First, the minimal lethal dose (MLD) of two different strains of Legionella pneumophila was determined by infecting intraperitoneally (i.p.) female A/J mice, aged 4 to 6 weeks, with different doses of an L. pneumophila serogroup 6 strain of human origin (VIR+), or from the L. pneumophila serogroup 1 Corby strain. A flagellar preparation was performed from a 6-day culture of the flagellated VIR+ strain on buffered charcoal-yeast extract (BCYE; Oxoid, Italy) agar plates at 36№C, in humidified atmosphere and 2.5% CO2. Mice were observed for signs of illness and for survival for 15 days. In flagella-immunized mice, no mortality was observed, compared with 100% mortality of similarly challenged mice immunized with the sham-flagellar preparation or unimmunized control mice. Involvement of humoral immune responses in the protective effect due to flagellar immunization was investigated, also considering that flagella are present in most Legionella species and serogroups, and their composition is common in all of them.

Legionella pneumophila causes a severe atypical pneumonia in humans, the so-called Legionnaires' disease. Although the disease mostly occurs in immunocompromised subjects, several virulence factors of Legionella have been described that confer to the microorganism an intrinsic aggressive potential. The ability of this pathogen to replicate in host macrophages is considered a major virulence trait. For this reason, the susceptibility to the experimental infection with L. pneumophila observed in guinea pigs and in selected mouse strains has been generally explained by the permissiveness of macrophages of these animals for Legionella invasion and intracellular replication in vitro, as reported, for elicited peritoneal macrophages from susceptible A/J mice. Researchers found that the i.p. inoculation of virulent Legionella cells from the VIR+ strain was rapidly lethal for A/J mice, since 100 and 60% of the animals acutely died within 2 days following the challenge with 2 X 108 or 2 X 107 bacterial cells. Unchecked TNF-α production upon i.v. Legionella inoculation has recently been reported as the causative factor in the death of Legionella replication-unpermissive BALB/c mice. Accordingly, the protection afforded by IL-10 treatment or TNF-α neutralization in the mice model also indicates that the release of TNF-α, a well-known toxic shock-related cytokine, plays a major role in mediating the lethal effects of the virulent legionellae. Therefore induction of proinflammatory cytokines, in particular TNF-α, besides being an important factor facilitating the resolution of L. pneumophila infections, might also be regarded as a possible contributory factor to the pathogenic action of virulent L. pneumophila strains.

Different methods have been applied to control Legionella in water systems such as copper-silver ionization, chlorine dioxide, thermal treatments, hyperchlorination, UV radiation. In this chapter, authors evaluated the efficacy of a continuous system based on a silver-hydrogen peroxide mixture in two nursing home water systems. Recent studies showed a decline of Legionnaire's disease (LD) in health care facility-acquired cases where environmental monitoring of the water system and installation of a disinfection method were performed. Therefore, when the plumbing system is heavily contaminated by Legionella, it is important to apply control measures to reduce Legionella colonization and infection risk. Studies on the efficacy of the silver-hydrogen peroxide disinfection system are currently lacking, and to our knowledge, the present study is the first reported for Legionella control in hot water systems. In vitro studies demonstrated that hydrogen peroxide is a weak disinfectant in the absence of a catalyst. The combined action of hydrogen peroxide with silver as the catalyst characterize the disinfection power of the mixture used in this study. Results of this study, although preliminary, show that such disinfection systems can effectively control Legionella water system colonization even if some increase of Legionella levels at distal sites is observed.

MICs of clarithromycin, amikacin, isoniazid, rifabutin, ciprofloxacin, sparfloxacin, ethambutol, and clofazimine were determined for six isolates of Mycobacterium avium complex (MAC) from AIDS patients both by the radiometric method and by an ex vivo model of infection in human macrophages. The median MICs in macrophages were similar or slightly lower than values found in broth, except for amikacin, which had slightly higher MICs inside the cells. Combinations of clarithromycin with other antimicrobial agents showed that clarithromycin-clofazimine and clarithromycin-rifabutin were synergistic on five of six strains while clarithromycin-amikacin and clarithromycin-isoniazid were antagonistic on one and two strains, respectively. The addition of amikacin made the combinations of clarithromycin-clofazimine and clarithromycin-ethambutol synergistic against all the MAC strains. In the macrophage model, the combination of clarithromycin-clofazimine (mean survival, 21%) and clarithromycin-rifabutin (mean survival, 29%) showed a strong reduction in viable counts compared with single drugs, while clarithromycin-amikacin was less active than single drugs alone. In general, the addition of amikacin did not improve the activity of the combinations, except for clarithromycin-isoniazid-amikacin (mean survival, 19%), which was significantly more active than either clarithromycin-isoniazid or clarithromycin-amikacin. The use of the macrophage model can suggest new combinations of antimicrobial agents with anti-MAC activity which, on the basis of their in vitro effectiveness, would probably be disregarded for assay in animal models.