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Legionnaires' disease (LD) is a serious type of pneumonia, typically contracted by susceptible people through the inhalation of aerosols contaminated with Legionella pneumophila (Lp). In this report, the first case of coinfection with Lp–Bordetella bronchiseptica (Bb) is described. A possible source of the Lp infection may be the hotel in Paris (France) where the patient had stayed before developing the symptoms. The Bb infection may have been transmitted by the dog with which he had constant contact, although this has not been proven.

IntroductionLegionella 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.Materials and methodsWe aimed to profile, by 16S rRNA gene amplicon sequencing (V3-V4), 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.ResultsThe 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.DiscussionSome 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 in 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.

A very rare case of pulmonary Klebsiella pneumoniae-Legionella pneumophila coinfection in a double kidney transplanted man affected by the chronic renal disease is described. Cases of Legionnaires' disease with an incubation period of 14 days have rarely been documented. Despite the long period of hospitalization, typing of clinical and environmental L. pneumophila strains demonstrated that the patient's home water distribution system was the source of infection, highlighting that Legionella house contamination can be a hidden risk, especially for immune-compromised people.

Legionella pneumophila (Lp), responsible for a severe pneumonia called Legionnaires’ disease, represents an important health burden in Europe. Prevention and control of Lp contamination in warm water systems is still a great challenge often due to the failure in disinfection procedures. The aim of this study was to evaluate the in vitro activity of Terpinen-4-ol (T-4-ol) as potential agent for Lp control, in comparison with the essential oil of Melaleuca alternifolia (tea tree) (TTO. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of T-4-ol were determined by broth micro-dilution and a micro-atmosphere diffusion method to investigate the anti-Lp effects of T-4-ol and TTO vapors. Scanning Electron Microscopy (SEM) was adopted to highlight the morphological changes and Lp damage following T-4-ol and TTO treatments. The greatest antimicrobial activity against Lp was shown by T-4-ol with a MIC range of 0.06–0.125% v/v and MBC range of 0.25–0.5% v/v. The TTO and T-4-ol MIC and MBC decreased with increasing temperature (36 °C to 45 ± 1 °C), and temperature also significantly influenced the efficacy of TTO and T-4-ol vapors. The time-killing assay showed an exponential trend of T-4-ol bactericidal activity at 0.5% v/v against Lp. SEM observations revealed a concentration- and temperature- dependent effect of T-4-ol and TTO on cell surface morphology with alterations. These findings suggest that T-4-ol is active against Lp and further studies may address the potential effectiveness of T-4-ol for control of water systems.

Legionella spp are the causative agents of Legionnaires’ 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 < 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.

Detection and enumeration of Legionella in water samples is of great importance for risk assessment analysis. The plate culture method is the gold standard, but has received several well-known criticisms, which have induced researchers to develop alternative methods. The purpose of this study was to compare Legionella counts obtained by the analysis of potable water samples through the plate culture method and through the IDEXX liquid culture Legiolert method. Legionella plate culture, according to ISO 11731:1998, was performed using 1 L of water. Legiolert was performed using both the 10 mL and 100 mL Legiolert protocols. Overall, 123 potable water samples were analyzed. Thirty-seven (30%) of them, positive for L. pneumophila, serogroups 1 or 2–14 by plate culture, were used for comparison with the Legiolert results. The Legiolert 10 mL test detected 34 positive samples (27.6%) and the Legiolert 100 mL test detected 37 positive samples, 27.6% and 30% respectively, out of the total samples analyzed. No significant difference was found between either the Legiolert 10 mL and Legiolert 100 mL vs. the plate culture (p = 0.9 and p = 0.3, respectively) or between the Legiolert 10 mL and Legiolert 100 mL tests (p = 0.83). This study confirms the reliability of the IDEXX Legiolert test for Legionella pneumophila detection and enumeration, as already shown in similar studies. Like the plate culture method, the Legiolert assay is also suitable for obtaining isolates for typing purposes, relevant for epidemiological investigations.