Evaluating Fourier-transform infrared spectroscopy with IR Biotyper as a faster and simpler method for investigating the sources of an outbreak of legionellosis.

Fourier-transform infrared (FTIR) spectroscopy using the IR Biotyper and core genome single nucleotide polymorphism (cgSNP) analysis were performed on 12 Legionella isolates associated with an outbreak at a spa house in Kanagawa Prefecture, Japan, and 3 non-outbreak isolates. The discriminative power of FTIR spectroscopy for 48-h incubation conditions of L. pneumophila in this outbreak was lower than cgSNP-based typing but higher than serogroup typing. FTIR spectroscopy could screen outbreak isolates from a group of genetically related isolates and may be useful as an initial typing method in Legionella outbreak investigations.

Legionella Serositis: A Rare Presentation.

BACKGROUND: Legionella has a higher prevalence in India than in the world. Legionaries' disease most commonly involves the lungs but because of increased awareness, extrapulmonary manifestations are also being diagnosed more frequently. CASE DESCRIPTION: We present a case of a young female with acute onset of fever and chest pain. On initial investigation, an electrocardiogram (ECG) reported widespread pulse rate (PR) depression suggestive of pericarditis which was confirmed by ECG. High-resolution computed tomography (HRCT) thorax suggested mild bilateral pleural effusion with normal lung parenchyma. elevated erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) added to the diagnosis of serositis. Serological study for atypical organisms was remarkable for positive immunoglobulin M (IgM) for Legionella. She was treated with a high dose of steroids and azithromycin successfully. CONCLUSION: Isolated extrapulmonary presentation of legionaries disease is often overlooked and is common. So it should be always included in the diagnostic armamentarium as treatment is highly efficacious if started early.

Outbreak of Legionnaires' disease in Rzeszów in 2023. / Ognisko choroby legionistów w Rzeszowie w 2023 roku.

BACKGROUND: Legionnaires' disease is a type of severe pneumonia caused by Legionella bacteria. The case fatality rate in this disease is 5-10%. People with various comorbidities, smokers and the elderly are at greater risk of developing the disease. OBJECTIVE: The aim of the work is to present the results of an epidemiological investigation into the outbreak of Legionnaires' disease that occurred in the city of Rzeszów and the surrounding area in August and September 2023 and to present the threat related to the presence of Legionella bacteria in water supply installations and networks. MATERIAL AND METHODS: The material for this publication was data from an epidemiological investigation conducted in the outbreak of Legionnaires disease in Rzeszów in 2023. RESULTS: Epidemiological investigation revealed 165 cases of Legionnaires' disease in the outbreak, including 152 confirmed cases and 13 probable cases. The case fatality rate in a legionellosis outbreak was 15%. Environmental tests were carried out in residential and public buildings and industrial installations during the investigation. As part of environmental tests, 187 water samples were collected, including 87 warm water samples. CONCLUSIONS: The outbreak of Legionnaires' disease in the city of Rzeszów draws attention to the potential threat from the Legionella bacteria to the health and life of especially elderly people suffering from chronic diseases. The environmental tests carried out confirmed the highest number of Legionella bacteria at medium and high levels in water samples taken in the private apartments of sick people. Despite the lack of strict legal regulations clearly specifying the obligations regarding periodic disinfection of internal hot water supply installations, cooperation with their owners should be undertaken to enforce plans and actions in this area.

Legionella anisa or Legionella bozemanii? Traditional and molecular techniques as support in the environmental surveillance of a hospital water network.

Understanding the actual distribution of different Legionella species in water networks would help prevent outbreaks. Culture investigations followed by serological agglutination tests, with poly/monovalent antisera, still represent the gold standard for isolation and identification of Legionella strains. However, also MALDI-TOF and mip-gene sequencing are currently used. This study was conducted to genetically correlate strains of Legionella non pneumophila (L-np) isolated during environmental surveillance comparing different molecular techniques. Overall, 346 water samples were collected from the water system of four pavilions located in a hospital of the Apulia Region of Italy. Strains isolated from the samples were then identified by serological tests, MALDI-TOF, and mip-gene sequencing. Overall, 24.9% of water samples were positive for Legionella, among which the majority were Legionella pneumophila (Lpn) 1 (52.3%), followed by Lpn2-15 (20.9%), L-np (17.4%), Lpn1 + Lpn2-15 (7.1%), and L-np + Lpn1 (2.3%). Initially, L-np strains were identified as L. bozemanii by monovalent antiserum, while MALDI-TOF and mip-gene sequencing assigned them to L. anisa. More cold water than hot water samples were contaminated by L. anisa (p < 0.001). PFGE, RAPD, Rep-PCR, and SAU-PCR were performed to correlate L. anisa strains. Eleven out of 14 strains identified in all four pavilions showed 100% of similarity upon PFGE analysis. RAPD, Rep-PCR, and SAU-PCR showed greater discriminative power than PFGE.

Chasing Waterborne Pathogens in Antarctic Human-Made and Natural Environments, with Special Reference to Legionella spp.

Waterborne pathogenic diseases are public health issues, especially for people staying in remote environments, such as Antarctica. After repeated detection of Legionella by PCR from the shower room of Syowa Station, the Japanese Antarctic research station, we wanted to understand the occurrence of waterborne pathogens, especially Legionella, in the station and their potential sources. In this study, we analyzed water and biofilm samples collected from the water facilities of Syowa Station, as well as water samples from surrounding glacier lakes, by 16S rRNA gene-based amplicon sequencing. For Legionella spp., we further attempted to obtain a detailed community structure by using genus-specific primers. The results showed that potentially pathogenic genera were mostly localized in the station, while Legionella spp., Pseudomonas spp., and Mycobacterium spp. were also widely distributed in lakes. Genus-specific analysis of Legionella spp. within the lake environments confirmed the presence of diverse Legionella amplicon sequence variants (ASVs) that were distinctly different from the Legionella ASVs detected in the station. The majority of the Legionella ASVs inhabiting Antarctic lake habitats were phylogenetically distinct from known Legionella species, whereas the ASVs detected in the human-made station tended to contain ASVs highly similar to well-described mesophilic species with human pathogenicity. These data suggest that unexpected Legionella diversity exists in remote Antarctic cold environments and that environmental differences (e.g., temperature) in and around the station affect the community structure.IMPORTANCE We comprehensively examined the localization of potential waterborne pathogens in the Antarctic human-made and natural aquatic environment with special focus on Legionella spp. Some potential pathogenic genera were detected with low relative abundance in the natural environment, but most detections of these genera occurred in the station. Through detailed community analysis of Legionella spp., we revealed that a variety of Legionella spp. was widely distributed in the Antarctic environment and that they were phylogenetically distinct from the described species. This fact indicates that there are still diverse unknown Legionella spp. in Antarctica, and this genus encompasses a greater variety of species in low-temperature environments than is currently known. In contrast, amplicon sequence variants closely related to known Legionella spp. with reported pathogenicity were almost solely localized in the station, suggesting that human-made environments alter the Legionella community.

Improvements to the Success of Outbreak Investigations of Legionnaires' Disease: 40 Years of Testing and Investigation in New York State.

Since 1978, the New York State Department of Health's public health laboratory, Wadsworth Center (WC), in collaboration with epidemiology and environmental partners, has been committed to providing comprehensive public health testing for Legionella in New York. Statewide, clinical case counts have been increasing over time, with the highest numbers identified in 2017 and 2018 (1,022 and 1,426, respectively). Over the course of more than 40 years, the WC Legionella testing program has continuously implemented improved testing methods. The methods utilized have transitioned from solely culture-based methods for organism recovery to development of a suite of reference testing services, including identification and characterization by PCR and pulsed-field gel electrophoresis (PFGE). In the last decade, whole-genome sequencing (WGS) has further refined the ability to link outbreak strains between clinical specimens and environmental samples. Here, we review Legionnaires' disease outbreak investigations during this time period, including comprehensive testing of both clinical and environmental samples. Between 1978 and 2017, 60 outbreaks involving clinical and environmental isolates with matching PFGE patterns were detected in 49 facilities from the 157 investigations at 146 facilities. However, 97 investigations were not solved due to the lack of clinical or environmental isolates or PFGE matches. We found 69% of patient specimens from New York State (NYS) were outbreak associated, a much higher rate than observed in other published reports. The consistent application of new cutting-edge technologies and environmental regulations has resulted in successful investigations resulting in remediation efforts. IMPORTANCE Legionella, the causative agent of Legionnaires' disease (LD), can cause severe respiratory illness. In 2018, there were nearly 10,000 cases of LD reported in the United States (https://www.cdc.gov/legionella/fastfacts.html; https://wonder.cdc.gov/nndss/static/2018/annual/2018-table2h.html), with actual incidence believed to be much higher. About 10% of patients with LD will die, and as high as 90% of patients diagnosed will be hospitalized. As Legionella is spread predominantly through engineered building water systems, identifying sources of outbreaks by assessing environmental sources is key to preventing further cases LD.

Comparison of BCYEα+AB agar and MWY agar for detection and enumeration of Legionella spp. in hospital water samples.

BACKGROUND: This study illustrates for the first time the performance (sensitivity and selectivity) of the selective medium BCYEα +AB suggested by the new edition of ISO 11731 for legionella isolation and enumeration. We compared the efficacy of the selective BCYEα +AB medium with that of the highly selective MWY medium. RESULTS: Legionella spp. was detected in 48.2 and 47.1% of the samples by BCYEα +AB and MWY agar, respectively. For optimal detection of Legionella spp., most protocols recommend using selective media to reduce the number of non-Legionella bacteria. Agreement between the two media was 86.7%. CONCLUSIONS: According to the results, both media have a very similar performance and they both have advantages and disadvantages over each other. In AB medium there is the risk of being less selective so more interfering microbiota may grow but in MWY medium there is the risk of being too selective. The low selectivity of the AB medium could be resolved if other treatments are applied after filtration, e.g. acid and/or heat treatment, but it must be taken into account that these treatments still reduce the number of viable Legionella. In conclusion, we recommend using MWY as a selective medium for the detection of Legionella spp. as it is easier discern suspected colonies and facilitate the final Legionella spp.

Outbreaks Associated with Treated Recreational Water - United States, 2015-2019.

Outbreaks associated with treated recreational water can be caused by pathogens or chemicals in aquatic venues such as pools, hot tubs, water playgrounds, or other artificially constructed structures that are intended for recreational or therapeutic purposes. For the pseriod 2015-2019, public health officials from 36 states and the District of Columbia (DC) voluntarily reported 208 outbreaks associated with treated recreational water. Almost all (199; 96%) of the outbreaks were associated with public (nonbackyard) pools, hot tubs, or water playgrounds. These outbreaks resulted in at least 3,646 cases of illness, 286 hospitalizations, and 13 deaths. Among the 155 (75%) outbreaks with a confirmed infectious etiology, 76 (49%) were caused by Cryptosporidium (which causes cryptosporidiosis, a gastrointestinal illness) and 65 (42%) by Legionella (which causes Legionnaires' disease, a severe pneumonia, and Pontiac fever, a milder illness with flu-like symptoms). Cryptosporidium accounted for 2,492 (84%) of 2,953 cases resulting from the 155 outbreaks with a confirmed etiology. All 13 deaths occurred in persons affected by a Legionnaires' disease outbreak. Among the 208 outbreaks, 71 (34%) were associated with a hotel (i.e., hotel, motel, lodge, or inn) or a resort, and 107 (51%) started during June-August. Implementing recommendations in CDC's Model Aquatic Health Code (MAHC) (1) can help prevent outbreaks associated with treated recreational water in public aquatic venues.

Legionella septentrionalis sp. nov., isolated from aquatic environments in the northern PR China.

Four strains (km711T, km714, km542 and km524), representing a novel Legionella species, were isolated from aquatic environments in northern PR China. Cells were Gram-stain-negative, rod-shaped, microaerobic, motile and growth depended on l-cysteine. They grew at 25‒42 °C (optimum, 35‒37 °C) and could tolerate up to 1.5 % (w/v) NaCl (optimum, 0.5 %). The major fatty acids (>5 %) of the type strain km711T were C17 : 0 anteiso, C15 : 0 anteiso, iso-C16 : 0 and C16 : 1 ω7c and/or iso-C15 : 0 2OH. The pairwise comparison values were <96.1 % for 16S rRNA gene sequences, 23.3‒28.7 % interspecies variation for mip gene sequences, <93.6 % average nucleotide identity and <72.8 % average amino acid identity between these four strains and related type strains within the genus Legionella. The phylogenetic tree based on the four concatenated genes (16S rRNA, mip, rpoB and rnpB) and protein-concatamer tree based on concatenation of 21 protein markers both revealed that these four strains formed a separate phylogenetic branch cluster within the genus Legionella. The results of phenotypic and genotypic features suggest that these four strains represent a novel species of the genus Legionella, for which the name Legionella septentrionalis sp. nov. is proposed (type strain km711T=KCTC 15655T=NBRC 113219T).

Occurrence of the Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran; first detection of Legionella cherrii.

BACKGROUND: This study aimed to investigate the occurrence of Legionella species in the respiratory samples of patients with pneumonia symptoms from Ahvaz, Iran by culture and the real-time PCR of 23S-5S rRNA gene spacer region. METHODS AND RESULTS: A total of 123 clinical respiratory samples including 63 pleural aspirates, 57 bronchoalveolar lavage (BAL), and 3 sputum were collected from 65 males and 58 females with pneumonia symptoms. All samples were cultured on the Modified Wadowsky-Yee (MWY) agar. The Legionella species was identified by routine bacteriological tests. The presence of the 16S-23S rRNA spacer region gene was investigated by real-time PCR. The Legionella species were differentiated by sequencing of 16S-23S rRNA gene. A total of 2 (1.6%) BAL specimens were positive for Legionella species by culture method. No Legionella spp. were identified in pleural aspirates and sputum samples by the culture method. Using real-time PCR, 9 (7.3%) samples including 6 BAL, 1 sputum, and 2 pleural aspirates were positive for legionella species. These species were detected in 3 (5.2%) females and 6 males (9.2%). The results of sequencing showed that eight species were L. pneumophila while one was L. cherrii. Also, the 2 isolates that were identified by culture method, were confirmed as L. pneumophila by sequencing. CONCLUSIONS: The results showed that using the real-time PCR has a more efficacy for detecting of Legionella species in respiratory samples. Also, L. pneumophila was the most prevalent species circulating in the southwest region of Iran. So, periodic monitoring programs is recommended to prevent epidemics due to this bacterium.

Isolation and identification of Legionella spp. from hot spring water in Algeria by culture and molecular methods.

AIMS: Due to infectious risk associated with the presence of Legionella in warm water, we determined the prevalence of living Legionella spp. in hot spring water in Algeria. METHODS AND RESULTS: Detection of Legionella by culture was done by using two methods, direct culture on agar plates and co-culture with amoeba. Fifty samples were taken from different hot springs in northern Algeria, including swimming pools, showers and thermal sources. Legionella pneumophila serotypes were predominant, accounting for 60% of positive samples. Direct method allowed the isolation of 13 L. pneumophila only of 50 samples (26%), whereas co-culture using a panel of three free living amoeba allowed the isolation of 119 Legionella species from the same samples (80%) CONCLUSIONS: Amoeba co-culture allowed the isolation of several Legionella sp., while direct culture allowed the isolation of L. pneumophila only. Remarkably, Legionella longbeachae, usually isolated from soil and compost, was isolated for the first time in thermal water in three samples using Vermamoeba vermiformis co-culture. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of Legionella in the water of hot springs in Algeria, which are mainly frequented by individuals at risk of Legionellosis, requires urgent control measures.

Loop-mediated amplification as promising on-site detection approach for Legionella pneumophila and Legionella spp.

Recently Legionella pneumophila is the main causative waterborne organism of severe respiratory infections. Additionally, other Legionella species are documented as human pathogens. In our work, we describe a rapid detection method which combines two advantages for sensitive and specific detection of the genus Legionella: the fast isothermal amplification method "Loop-mediated isothermal AMPlification" (LAMP), and a colorimetric detection method using the metal indicator hydroxynaphtol blue (HBN) which allows to determine an optical signal with a simple readout (with the naked eye). Moreover, we present two approaches for minimizing the assay volume using a stationary microchip LAMP and droplet digital-based LAMP (ddLAMP) as promising highly sensitive setups.

Distribution of Legionella species and serogroups in patients with culture-confirmed Legionella pneumonia.

Legionella species are consistently identified as some of the most common causative agents of severe community-acquired pneumonia (CAP) or nosocomial pneumonia. Although the number of reported Legionella infection cases is gradually increasing in Japan, most cases are diagnosed by a urinary antigen test, which identifies only L. pneumophila serogroup 1. Therefore, assessment of pneumonia-causing Legionella species and serogroups would be important. The Japan Society for Chemotherapy Legionella committee has collected the isolates and clinical information on cases of sporadic community-acquired Legionella pneumonia throughout Japan. Between December 2006 and March 2019, totally 140 sporadic cases were identified, in which L. pneumophila was the most frequently isolated species (90.7%) followed by L. bozemanae (3.6%), L. dumofii (3.6%), L. micdadei (1.4%), and L. longbeachae (0.7%). Among 127 isolates of L. pneumophila, 111 isolates were of serogroup 1, two of serogroup 2, four of serogroup 3, one of serogroup 4, one of serogroup 5, seven of serogroup 6, and one was of serogroup 10. We also assessed in vitro activity of antibiotics against these isolates and showed that quinolones and macrolides have potent anti-Legionella activity. Our study showed that pneumonia-causing Legionella species and serogroup distribution was comparable to that reported in former surveillances. L. pneumophila was the most common etiologic agent in patients with community-acquired Legionella pneumonia, and L. pneumophila serogroup 1 was the predominant serogroup.

Effects of Acanthamoeba castellanii on the dissolved oxygen and the microbial community under the experimental aquatic model.

Acanthamoeba castellanii is a protist that has a high predation efficiency for bacteria in a number of monoxenic culture experiments. However, the role of A. castellanii in the microbial community is still unknown because of the lack of studies on multiple-species interactions. The aim of this study was to investigate the change of bacterial composition after A. castellanii emerges in a water environment. We added A. castellanii to an environmental water sample and incubated it for two days. Then, we performed 16S ribosomal RNA sequencing techniques to analyze the changes in bacterial composition. In this study, A. castellanii slightly increased the relative abundance of a few opportunistic pathogens, such as Legionella, Roseomonas, and Haemophilus. This result may be related to the training ground hypothesis. On the other hand, the growth of some bacteria was inhibited, such as Cyanobacteria and Firmicutes. Although A. castellanii did not drastically change the whole bacterial community, we surprisingly found the dissolved oxygen concentration was increased after incubation with A. castellanii. We applied environmental water at the laboratory scale to investigate the interactions among A. castellanii, complex microbial communities and the environment. We identified the bacteria that are sensitive to A. castellanii and further found the novel relationship between dissolved oxygen and microbial interaction. Our results helped to clarify the role of A. castellanii in microbial communities.

Ten-Year Retrospective Analysis of Legionella Diffusion in Hospital Water Systems and Its Serogroup Seasonal Variation.

INTRODUCTION: Legionella spp. are ubiquitous aquatic organisms found to be associated with community-acquired pneumoniae (CAP) as well as hospital-acquired pneumonia (HAP). Direct inhalation of aerosols from environmental colonisation is typically the source of infection. The aim of this study was to determine the level of colonisation in hospital water supply systems in order to assess the criticality of the water distribution network and strengthen preventive measures. METHODS: From 2009 to 2018, 769 water samples were collected and then analysed according to the standard methods indicated in ISO11731-2:2004 and ISO11731:2017 for Legionella detection. RESULTS: The samples were positive in 37.1% cases (n. 285) and negative in 62.9% cases (n. 484). The threshold of 10,000 CFU/L was exceeded in 15.1% cases and led to decolonisation as indicated by Italian and European ECDC guidelines. In the autumn-winter period SG1 showed a positivity of 41.2% (n. 40) with a decrease in the spring-summer period with 9.6% (n. 18) of positivity. In contrast, SG2-15 showed a positivity of 30.9% (n. 30) in autumn-winter, which tends to increase to 56.9% (n. 112) in spring-summer (p < 0.001). CONCLUSION: Surprisingly, besides showing a seasonal trend already described previously in the literature, the positivity of our sample was not balanced even for serogroups in the two periods. This could be due to genetic differences and ecological niches to be further investigated that could also have links with the greater pathogenicity of SG1. Environmental microbiological surveillance and risk assessment should be performed more frequently and disinfection must be carried out, especially in health facilities where people are more susceptible to infections.

Legionella pneumonia: increased risk after COVID-19 lockdown? Italy, May to June 2020.

We report a case of Legionella pneumonia in a dishwasher of a restaurant in Rome, Italy, just after the end of the lockdown that was in place to control the SARS-CoV-2 epidemic. The case highlights the importance of strict monitoring of water and air systems immediately before reopening business or public sector buildings, and the need to consider Legionella infections among the differential diagnosis of respiratory infections after lockdown due to the ongoing COVID-19 pandemic.

Cutaneous Legionella infections in allogeneic hematopoietic cell transplantation recipients.

To date, only twenty cases of cutaneous legionellosis have been reported. Cutaneous legionellosis has heterogeneous manifestations including abscesses, nodules, and cellulitis. The detection of most cutaneous Legionella species requires specific diagnostic cultures and assays. Herein, we report a case of cutaneous legionella in a hematopoietic cell transplantation recipient with culture-negative nodules unresponsive to empiric antibiotics. We also discuss the varied morphology of cutaneous legionellosis and important diagnostic considerations.

Rapid detection of Legionella spp in water samples by ScanVIT method: comparison of acid vs heat treatment.

BACKGROUND: A rapid detection of Legionella bacteria in water samples is crucial to minimize the risk of acquiring infections, especially in health care facilities. Different detection methods and different decontamination procedures have been reported to affect the recovery of Legionella spp. Our goal was to test the recovery of Legionella pneumophila and Legionella non-pneumophila species using a kit based on non-specific and species-specific probes to treat water samples after two different decontamination procedures. METHODS: The study was conducted with samples collected in the teaching hospital "Le Scotte" of Siena (Italy). Waters samples were analyzed by: i) ScanVIT method after treatment with acids; ii) ScanVIT method after heating; and iii) cultural standard method after heating. The results of the decontamination procedures and the detection methods were evaluated by comparing the number of Legionella-positive and -negative samples, and the recovery rates (CFU l-1) obtained by ScanVIT and the standard method. RESULTS: We find that ScanVIT method is highly sensitive with both decontamination treatments, yielding a higher recovery of L. pneumophila compared to the standard method. Conversely, ScanVIT associated with the acid-treatment yielded the highest recovery of L. non-pneumophila. CONCLUSIONS: The acid-treatment combined to the ScanVIT method increases the recovery of L. non-pneumophila in water samples compared to both ScanVIT associated with heat-treatment and standard culture method. Thus, this method may represent the best choice to detect L. non-pneumophila in water samples and reduce the risk of infection due to underestimation of Legionella loads.

Removal Efficacy of Opportunistic Pathogens and Bacterial Community Dynamics in Two Drinking Water Treatment Trains.

Drinking water treatment processes (DWTPs) impact pathogen colonization and microbial communities in finished water; however, their efficacies against opportunistic pathogens are not fully understood. In this study, the effects of treatment steps on the removal of Legionella spp., Legionella pneumophila, nontuberculous mycobacteria, Mycobacterium avium, and two amoeba hosts (Vermamoeba vermiformis, Acanthamoeba) are evaluated in two parallel trains of DWTPs equipped with different pretreatment units. Quantitative polymerase chain reaction analysis demonstrates significantly reduced numbers of total bacteria, Legionella, and mycobacteria during ozonation, followed by a rebound in granular activated carbon (GAC) filtration, whereas sand filtration exerts an overarching effect in removing microorganisms in both treatment trains. V. vermiformis is more prevalent in biofilm (34%) than water samples (7.7%), while Acanthamoeba is not found in the two trains of DWTPs. Illumina sequencing of bacterial 16S rRNA genes reveals significant community shifts at different treatment steps, as well as distinct bacterial community structures in water and biofilm samples in parallel units (e.g., ozonation, GAC, sand filtration) between the two trains (analysis of similarities (ANOSIM), p < 0.05), implying the potential influence of different pretreatment steps in shaping the downstream microbiome. Overall, the results provide insights to mitigation of opportunistic pathogens and engineer approaches for managing bacterial communities in DWTPs.

Legionella qingyii sp. nov., isolated from water samples in China.

Three Legionella-like strains, designed km488T, km489 and km521, were isolated from freshwater samples in China. Cells were Gram-stain-negative, rod-shaped and non-spore-forming. Growth was observed on BCYEα agar, but not on BCYEα agar without l-cysteine, chocolate agar with PolyViteX or Columbia blood agar. The major fatty acids (>5 %) of strains km488T, km489 and km521 were C16 : 0, anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The mip gene sequences (574 nt) showed the isolates were almost identical with more than 99.7 % sequence similarities, and closely matched to L. gormanii ATCC 33297T with 95.4-95.6 % sequence similarities. Phylogenetic analyses based on concatenated gene (16S rRNA, mip, rpoB and rnpB) sequences indicated that the isolates formed a distinct cluster along with L. gormanii within the genus Legionella. Matrix-assisted laser desorption ionization time-of-flight analyses also demonstrated a clear separation between the isolates and other closely and distantly related Legionella species. DNA-DNA hybridization studies demonstrated that the isolates were closely related (92.0 -95.0 % DNA-DNA relatedness) but differentiated from their phylogenetic neighbours (<70 % DNA-DNA relatedness). The whole genome of km488T was sequenced, and showed a G+C content of 37.8 mol%. Based on the findings from this polyphasic taxonomic study, the isolates are considered to represent a single novel species, for which the name Legionella qingyii sp. nov. is proposed. The type strain is km488T (KCTC 15636T=CCTCC AB 2018025T=NRBC 113223T).