Occurrence of Legionella spp. in Man-Made Water Sources: Isolates Distribution and Phylogenetic Characterization in the Emilia-Romagna Region

Legionella species distribution in the Emilia-Romagna region, involving hospital (H) and community (C) environments, was conducted. Legionella culture, agglutination test, and mip-gene sequencing were applied on 240 isolates. The analysis showed a higher prevalence of non-Legionellapneumophila (n-Lp) species (84.1%) compared with L. pneumophila (Lp) (15.9%), with a higher frequency of n-Lp with respect to Lp species in both environments (77.6% and 96.4%, in H and C, respectively). The Shannon index showed a significant difference in Legionella distribution (p = 0.00017), with a significant abundance of Lp in the H compared with C environment (p = 0.00028). The continuous disinfection treatment in H could contribute to adaptive survival of the Lp species. Phylogenetic analysis revealed a conservative clade distribution between H and C: L. feeleii clade with three subclades in C and the Lp clade with five subclades in H and two in C, respectively. Our findings suggest the importance of Legionella surveillance both in H and C, with a focus on n-Lp species less connected to human disease. The Legionella prevalence and diversity found here indicate that geographical and temporal isolate evolution should be considered during surveillance, particularly in the light of global warming and changes in population risk factors

Draft Genome Sequence of Legionella Species Isolated from Drinking Water in an Italian Industry

We report the draft genome sequences of an environmentalLegionellastrainisolated from an industrial water distribution system in Italy. Macrophage infectivity poten-tiator (mip)andb-subunit of RNA polymerase (rpoB) genes were used to perform the spe-cies identification. Whole-genome sequencing (WGS) and average nucleotide identity (ANI)identified the isolate as belonging to a presumptive novelLegionellaspecies, with a ge-nome length of 3,281,851 bp

The Role of Sensor-Activated Faucets in Surgical Handwashing Environment as a Reservoir of Legionella

Surgical handwashing is a mandatory practice to protect both surgeons and patients in order to control Healthcare-Associated Infections (HAIs). The study is focused on Legionella and Pseudomonas aeruginosa contamination in Surgical Handwashing Outlets (SHWOs) provided by sensor-activated faucets with Thermostatic Mixer Valves (TMVs), as correlated to temperature, technologies, and disinfection used. Samples were analyzed by standard culture techniques, comparing hot- and cold-water samples. Legionella isolates were typed by an agglutination test and by mip sequencing. Legionella contamination showed the same distribution between hot and cold samples concerning positive samples and mean concentration: 44.5% and 1.94 Log10 cfu/L vs. 42.6% and 1.81 Log10 cfu/L, respectively. Regarding the distribution of isolates (Legionella pneumophila vs. Legionella non-pneumophila species), significant di_erences were found between hot- and cold-positive samples. The contamination found in relation to ranges of temperature showed the main positive samples (47.1%) between 45.1–49.6 _C, corresponding to high Legionella concentrations (2.17 Log10 cfu/L). In contrast, an increase of temperature (>49.6 _C) led to a decrease in positive samples (23.2%) and mean concentration (1.64 Log10 cfu/L). A low level of Pseudomonas aeruginosa was found. For SHWOs located in critical areas, lack of consideration of technologies used and uncorrected disinfection protocols may lead to the development of a high-risk environment for both patients and surgeons

New Insight regarding Legionella Non- Pneumophila Species Identification: Comparison between the Traditional mip Gene Classification Scheme and a Newly Proposed Scheme Targeting the rpoB Gene

The identification of Legionella non-pneumophila species (non-Lp) in clinical and environmental samples is based on the mip gene, although several studies suggest its limitations and the need to expand the classification scheme to include other genes. In this study, the development of a new classification scheme targeting the rpoB gene is proposed to obtain a more reliable identification of 135 Legionella environmental isolates. All isolates were sequenced for the mip and rpoB genes, and the results were compared to study the discriminatory power of the proposed rpoB scheme. Complete concordance between the mip and rpoB results based on genomic percent identity was found for 121/135 (89.6%) isolates; in contrast, discordance was found for 14/135 (10.4%) isolates. Additionally, due to the lack of reference values for the rpoB gene, inter- and intraspecies variation intervals were calculated based on a pairwise identity matrix that was built using the entire rpoB gene (∼4,107 bp) and a partial region (329 bp) to better evaluate the genomic identity obtained. The interspecies variation interval found here (4.9% to 26.7%) was then proposed as a useful sequence-based classification scheme for the identification of unknown non-Lp isolates. The results suggest that using both the mip and rpoB genes makes it possible to correctly discriminate between several species, allowing possible new species to be identified, as confirmed by preliminary whole-genome sequencing analyses performed on our isolates. Therefore, starting from a valid and reliable identification approach, the simultaneous use of mip and rpoB associated with other genes, as it occurs with the sequence-based typing (SBT) scheme developed for Legionella pneumophila, could support the development of multilocus sequence typing to improve the knowledge and discovery of Legionella species subtypes

Legionella bononiensis sp. nov., isolated from a hotel water distribution system in northern Italy

Legionella-like isolates, strains 27fs60, 30fs61 and 30cs62T, were isolated from a hotel water distribution system in the Emilia-Romagna region, Italy. Isolates were Gram- and Ziehl Neelsen-stain-negative, rod-shaped, with transitory flagella presence and able to grow at 32–37 °C (with an optimum at 32 °C) on buffered charcoal–yeast extract agar with l-cysteine, glycine–vancomycin–polymyxin B–cycloheximide agar and Wadowsky–Yee medium agar. The strains showed positive reactions for oxidase, hippurate and gelatinase and a weakly positive reaction for catalase. Based on the EUCAST cut-off, strain 30cs62T was resistant to ciprofloxacin (5 mg l−1). The mip and rpoB gene sequences of the three strains showed close matches to those of Legionella quateirensis ATCC 49507T with similarity values of 98.2 and 94.5 %, respectively. Whole genome sequencing of the three strains was performed, resulting in G+C contents of 39.0, 39.1 and 39.0 mol%, respectively. The identity percentage measured by average nucleotide identity between the three strains and their respective closest strains were: 91.32 % L. quateirensis NCTC 12376T, 91.45 % L. quateirensis ATCC 49507T and 91.45 % L. quateirensis ATCC 49507T, respectively. The digital DNA–DNA hybridization analysis demonstrated how the isolates were separated from the most related phylogenetic Legionella species (L. quateirensis ATCC 49507T, ≤40.10 % DNA–DNA relatedness). The concatenated phylogenetic tree based on 16S rRNA, mip, rpoB and rnpB genes, shows a close relationship with L. quateirensis ATCC 49507T. The results obtained confirm the status of an independent species. The name proposed for this species is Legionella bononiensis sp. nov. with 30cs62T (=ATCC TSD-262T= DSM 112526T) as the type strain

Dynamics of Legionella Community Interactions in Response to Temperature and Disinfection Treatment: 7 Years of Investigation

none5noIn man-made water distribution systems, Legionella community interactions remain unknown, due to their ability to change from sessile to planktonic states or live in viable but non-culturable forms, in response to anthropic and environmental stress. During 7 years of hospital Legionella surveillance, in 191 hot water positive samples, the interactions among the Legionella species, temperature, and disinfection treatment were evaluated. Legionella was isolated following ISO 11731:2017, and identification was performed by mip gene sequencing and sequence-based typing (SBT) for L. anisa or L. rubrilucens and L. pneumophila, respectively. The species with the higher frequency of isolation was L. pneumophila serogroup 1 (78.53%; 4865.36 ± 25,479.11 cfu/L), followed by L. anisa (54.45%; 558.79 ± 2637.41 cfu/L) and L. rubrilucens (21.99%; 307.73 ± 1574.95 cfu/L), which were sometimes present together. Spearman’s rho correlation test was conducted among the species with respect to temperature and disinfectant (H2O2/Ag+). The results showed a generally positive interaction among these species sharing the same environment, except for competition between L. anisa and L. rubrilucens. High temperature (48.83 ± 2.59 °C) and disinfection treatment (11.58 ± 4.99 mg/L) affected the presence of these species. An exception was observed with L. anisa, which showed disinfection treatment resistance. For the purposes of environmental surveillance, it is fundamental to better understand the interactions and dynamic of the Legionella community in man-made water systems in order to choose the proper physical or chemical treatments. The simultaneous presence of different Legionella species could result in an increased resistance to high temperature and disinfectant treatment, leading to changes in contamination level and species diversity.openLuna Girolamini, Silvano Salaris, Maria Rosaria Pascale, Marta Mazzotta, Sandra CristinoLuna Girolamini, Silvano Salaris, Maria Rosaria Pascale, Marta Mazzotta, Sandra Cristin

Study of Long-Term Effectiveness of Water Safety Plan Applied to Microfiltered Water Dispensers to Improve Water Quality

none6noThe use of Microfiltered Water Dispensers (MWDs) is increasing in offices, companies, or commercial facilities, as a response to plastic pollution. Despite their widespread use, poor data are available about the water quality and pathogens developed. Starting from a high contamination found in MWDs, a Water Safety Plan (WSP) was implemented on 57 MWDs to improve the water quality. To assess the effectiveness of WSP during the period 2017–2021, the environmental monitoring of heterotrophic plate counts (HPCs) at 36 ◦C and 22 ◦C, Enterococcus spp., Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, and Clostridium perfringens, as prescribed by Italian regulation for drinking water, was conducted. A high level of HPCs at both temperatures was observed, over the regulation limit; contrarily, the pathogenic bacteria were absent. The contamination found was studied with respect to the temperature threshold of 25 ◦C, suggested by directives. No significant differences were found between samples above and below the threshold, while a significant decrease over the years was observed for HPCs at 36 ◦C (p = 0.0000000001) and 22 ◦C (p = 0.000006). The WSP implementation resulted in a 43.09% decrease for HPCs at 36 ◦C and a 24.26% decrease for HPCs at 22 ◦C. Moreover, during the SARS-CoV-2 pandemic, the WSP contributed to limit the microbial contamination, preserving the MWDs’ functionality and hygienic conditions and the drinking water quality.openMarta Mazzotta, Silvano Salaris, Maria Rosaria Pascale, Luna Girolamini, Graziella Ciani, Sandra CristinoMarta Mazzotta, Silvano Salaris, Maria Rosaria Pascale, Luna Girolamini, Graziella Ciani, Sandra Cristin

How Molecular Typing Can Support Legionella Environmental Surveillance in Hot Water Distribution Systems: A Hospital Experience

In this study, we aimed to associate the molecular typing of Legionella isolates with a culture technique during routine Legionella hospital environmental surveillance in hot water distribution systems (HWDSs) to develop a risk map able to be used to prevent nosocomial infections and formulate appropriate preventive measures. Hot water samples were cultured according to ISO 11731:2017. The isolates were serotyped using an agglutination test and genotyped by sequence-based typing (SBT) for Legionella pneumophila or macrophage infectivity potentiator (mip) gene sequencing for non-pneumophila Legionella species. The isolates\u2019 relationship was phylogenetically analyzed. The Legionella distribution and level of contamination were studied in relation to temperature and disinfectant residues. The culture technique detected 62.21% of Legionella positive samples,characterized by L. pneumophila serogroup 1, Legionella non-pneumophila, or both simultaneously. The SBT assigned two sequence types (STs): ST1, the most prevalent in Italy, and ST104, which had never been isolated before. The mip gene sequencing detected L. anisa and L. rubrilucens. The phylogenetic analysis showed distinct clusters for each species. The distribution of Legionella isolates showed significant dierences between buildings, with a negative correlation between the measured level of contamination, disinfectant, and temperature. The Legionella molecular approach introduced in HWDSs environmental surveillance permits (i) a risk map to be outlined that can help formulate appropriate disinfection strategies and (ii) rapid epidemiological investigations to quickly identify the source of Legionella infections

Draft Genome Sequences of Legionella Presumptive Novel Species Isolated during Environmental Surveillance in Artificial Water Systems

none7noWe present the draft genome sequences of three Legionella strains that were isolated from a hotel water distribution system. Legionella species identification was performed by macrophage infectivity potentiator (mip) and RNA polymerase β subunit (rpoB) gene sequencing. Whole-genome sequencing and average nucleotide identity results supported the hypothesis of new Legionella species isolationopenLuna Girolamini, Silvano Salaris, Massimiliano Orsini, Maria Rosaria Pascale, Marta Mazzotta, Antonella Grottola, Sandra CristinoLuna Girolamini, Silvano Salaris, Massimiliano Orsini, Maria Rosaria Pascale, Marta Mazzotta, Antonella Grottola, Sandra Cristin

Combining Traditional and Molecular Techniques Supports the Discovery of a Novel Legionella Species During Environmental Surveillance in a Healthcare Facility

none9siLegionella surveillance plays a significant role not only to prevent the risk of infection but also to study the ecology of isolates, their characteristics, and how their prevalence changes in the environment. The difficulty in Legionella isolation, identification, and typing results in a low notification rate; therefore, human infection is still underestimated. In addition, during Legionella surveillance, the special attention given to Legionella pneumophila leads to an underestimation of the prevalence and risk of infection for other species. This study describes the workflow performed during environmental Legionella surveillance that resulted in the isolation of two strains, named 8cVS16 and 9fVS26, associated with the genus Legionella. Traditional and novel approaches such as standard culture technique, MALDI-TOF MS, gene sequencing, and whole-genome sequencing (WGS) analysis were combined to demonstrate that isolates belong to a novel species. The strain characteristics, the differences between macrophage infectivity potential (mip), RNA polymerase b subunit (rpoB), and reference gene sequences, the average nucleotide identity (ANI) of 90.4%, and the DNA–DNA digital hybridization (dDDH) analysis of 43% demonstrate that these isolates belong to a new Legionella species. The finding suggests that, during the culture technique, special attention should be paid to the characteristics of the isolates that are less associated with the Legionella genus in order to investigate the differences found using more sensitive methods. The characterization of the two newly discovered isolates based on morphological, biochemical, and microscopic characteristics is currently underway and will be described in another future study.openLuna Girolamini, Maria Rosaria Pascale, Marta Mazzotta, Simona Spiteri, Federica Marino, Silvano Salaris, Antonella Grottola, Massimiliano Orsini, Sandra CristinoLuna Girolamini, Maria Rosaria Pascale, Marta Mazzotta, Simona Spiteri, Federica Marino, Silvano Salaris, Antonella Grottola, Massimiliano Orsini, Sandra Cristin