Correlation between bacterial microbiome and Legionella species in water from public bath facilities by 16S rRNA gene amplicon sequencing.

Public bath facilities are a major source of Legionella infections in Japan. In this study, we performed 16S rRNA gene amplicon sequencing to characterize the bacterial community in bath and shower water from public bath facilities, along with chemical parameters, and investigated the effect of the bacterial microbiome on the presence of Legionella species. Although no significant difference in bacterial community richness was observed between bath and shower water samples, there was a remarkable difference in the bacterial community structure between them. Distance-based redundancy analysis revealed that several factors (free residual chlorine, pH, and conductivity) were correlated with the bacterial community in bath water. The most abundant bacterial genera in the samples were Pseudomonas (13.7%) in bath water and Phreatobacter (13.6%) in shower water, as indicated by the taxonomic composition, and the dominant bacteria differed between these environmental samples. Legionella pneumophila was the most frequently detected Legionella species, with additional 15 other Legionella species detected in water samples. In Legionella-positive water samples, several unassigned and uncultured bacteria were enriched together. In addition, the co-occurrence network showed that Legionella was strongly interconnected with two uncultured bacteria. Corynebacterium and Sphingomonas negatively correlated with Legionella species. The present study reveals the ecology of Legionella species, especially their interactions with other bacteria that are poorly understood to date. IMPORTANCE: Public bath facilities are major sources of sporadic cases and outbreaks of Legionella infections. Recently, 16S rRNA gene amplicon sequencing has been used to analyze bacterial characteristics in various water samples from both artificial and natural environments, with a particular focus on Legionella bacterial species. However, the relationship between the bacterial community and Legionella species in the water from public bath facilities remains unclear. In terms of hygiene management, it is important to reduce the growth of Legionella species by disinfecting the water in public bath facilities. Our findings contribute to the establishment of appropriate hygiene management practices and provide a basis for understanding the potential health effects of using bath and shower water available in public bath facilities.

Prevention of legionella infections from toilet flushing cisterns.

INTRODUCTION: Immunocompromised patients are at an increased risk of severe legionella infections. We present the results of an outbreak investigation initiated following a fatal case of hospital-acquired legionellosis linked to contaminated water from a toilet-flushing cistern. Additionally, we provide experimental data on the growth of Legionella spp. in flushing cisterns and propose a straightforward protocol for prevention. METHODS: We monitored the growth of Legionella spp. in the building's hot- and cold-water systems using quantitative bacterial culture on selective agar. Molecular typing of Legionella pneumophila isolates from the infected patient and the water system was conducted through core-genome multi-locus sequence typing (cgMLST). RESULTS: Legionella contamination in the hospital building's cold-water system was significantly higher than in the hot-water system and significantly higher in toilet flushing cistern's water compared with cold water from bathroom sinks and showers. Isolates from the patient and from the flushing cistern of the patient's bathroom were identical by cgMLST. In an experimental setting, daily toilet flushing for a period of 21 days resulted in a 67% reduction in the growth of Legionella spp. in the water of toilet flushing cisterns. Moreover, a one-time disinfection of cisterns with peracetic acid, followed by daily flushing, decreased legionella growth to less than 1% over a period of at least seven weeks in these setting. CONCLUSIONS: One-time disinfection of highly contaminated cisterns with peracetic acid and daily toilet flushing as short-term measure can significantly reduce legionella contamination in flushing cisterns. These measures may aid in preventing legionella infection among immunocompromised patients.

Estimating the burden of illness caused by domestic waterborne Legionnaires' disease in Canada: 2015-2019.

Legionellosis is a disease caused by the bacterium Legionella that most commonly presents as Legionnaires' disease (LD), a severe form of pneumonia. From 2015 to 2019, an average of 438 LD cases per year were reported in Canada. However, it is believed that the actual number of cases is much higher, since LD may be underdiagnosed and underreported. The purpose of this study was to develop an estimate of the true incidence of illnesses, hospitalizations, and deaths associated with LD in Canada. Values were derived using a stochastic model, based on Canadian surveillance data from 2015 to 2019, which were scaled up to account for underdiagnosis and underreporting. Overall, there were an estimated 1,113 (90% CrI: 737-1,730) illnesses, 1,008 (90% CrI: 271-2,244) hospitalizations, and 34 (90% CrI: 4-86) deaths due to domestically acquired waterborne LD annually in Canada from 2015 to 2019. It was further estimated that only 36% of illnesses and 39% of hospitalizations and deaths were captured in surveillance, and that 22% of illnesses were caused by Legionella serogroups and species other than Legionella pneumophila serogroup 1 (non-Lp1). This study highlights the true burden and areas for improvement in Canada's surveillance and detection of LD.

Legionnaires' Disease Outbreak Associated With a Hot Tub Display at the North Carolina Mountain State Fair, September 2019.

OBJECTIVES: On September 23, 2019, the North Carolina Division of Public Health identified a legionellosis increase in western North Carolina; most patients had recently attended the North Carolina Mountain State Fair. We conducted a source investigation. METHODS: Cases were fair attendees with laboratory-confirmed legionellosis and symptom onset within 2 to 14 days (Legionnaires' disease) or ≤3 days (Pontiac fever). We conducted a case-control study matching cases to non-ill fair attendees as control participants and an environmental investigation, and we performed laboratory testing (Legionella bacteria culture and polymerase chain reaction) of 27 environmental samples from fairgrounds and hot tubs and 14 specimens from case patients. We used multivariable unconditional logistic regression models to calculate adjusted odds ratios for potential Legionella exposure sources and risk factors. RESULTS: Of 136 people identified with fair-associated legionellosis, 98 (72%) were hospitalized and 4 (3%) died. Case patients were more likely than control participants to report walking by hot tub displays (adjusted odds ratio = 10.0; 95% CI, 4.2-24.1). Complete hot tub water treatment records were not kept, precluding evaluation of water maintenance conducted on display hot tubs. Legionella pneumophila sequence types (STs) were consistent among 10 typed clinical specimens (ST224) but distinct from the only positive environmental sample from the fair (ST7 and ST8). CONCLUSIONS: Hot tub displays were identified as the most likely outbreak source, making this the largest hot tub-associated Legionnaires' disease outbreak worldwide. Following the investigation, the North Carolina Division of Public Health and the Centers for Disease Control and Prevention released guidance on mitigating risk of Legionella exposure from hot tub displays. Results highlight the importance of properly maintaining equipment that aerosolizes water, including hot tubs intended for display purposes only.

The potential risks of Legionella arising from ligature-resistant fixtures.

To provide a safe environment, behavioral health settings must adhere to "ligature-resistant" protocols for patients at risk of harm to themselves or others. However, certain bathroom ligature-resistant fixtures alter environmental controls, such as sinks and showerheads, and increase the risk of water-borne pathogens due to low water output settings, highlighting the importance of an interdisciplinary water management program. We describe how ligature-resistant water fixtures may have been associated with a possible case of hospital-associated Legionellosis.

Prevalence and genetic distribution of Legionella spp. in public bath facilities in Kobe City, Japan.

Legionella is an important waterborne pathogen that causes legionellosis. Public baths are considered the primary cause of legionellosis infection in Japan. We investigated the prevalence and genetic distribution of 338 Legionella spp. isolates from 81 public bath facilities, including 35 hot springs and 46 other facilities, through annual periodic surveillance in Kobe, Japan, from 2016 to 2021. In addition, the genotypes of nine clinical strains of unknown infectious source from the same period were compared to those of bathwater isolates. We elucidated the differences in the distribution of Legionella species, serogroups, and genotypes between hot springs and other public baths. Legionella israelensis, L. londiniensis, and L. micdadei colonized hot springs along with L. pneumophila. The minimum spanning tree analysis based on multiple-locus variable number tandem repeat analysis (MLVA) also identified four major clonal complexes (CCs) in L. pneumophila SG1 and found that CC1 of the four CCs is a specific novel genotype with the lag-1 gene in hot springs. The same MLVA genotypes and sequence types as those of the clinical strains were not present among the strains isolated from bath water. Thus, our surveillance is useful for estimating the sources of legionellosis infection in Japan and developing prevention strategies.

Legionellosis in Poland in 2018-2021.

Objectives: The aim of this study is to compare the epidemiological situation of legionellosis in Poland in 2018-2021 to prior years, taking into account the impact of the COVID-19 pandemic in 2020-2021. Material and methods: The assessment is based on national surveillance data published in the annual bulletin "Infectious Diseases and Poisons in Poland" from 2013 to 2021, as well as data from Legionnaires' disease case reports collected and sent to the Department of Epidemiology of Infectious Diseases and Surveillance NIPH NIH - NRI by sanitary and epidemiological stations or submitted to EpiBase. Results: In Poland, both Legionnaires' disease (an acute infection that progresses to pneumonia) and Pontiac fever (a mild, flu-like sickness) are reported. In 2018-2021, a total of 255 cases of legionellosis were registered, including 236 cases of Legionnaires' disease and 19 cases of Pontiac fever. Each year, there was an increase in the number of notifications compared to the annual median number of cases from 2013-2017. The annual incidence rate in 2019 (0.23 per 100,000 population) was the highest since the start of legionellosis case registration in Poland. It declined again during the years of the COVID-19 pandemic. The notifications occurred throughout the country, but the highest notification rate was observed in the western belt of voivodeships. Pomorskie reported the highest incidence, accounting for more than 20% of all registered cases. The median incidence of Legionnaires' disease in men (0.23 per 100,000) was more than twice that of women (0.10), with the highest incidence (0.58) recorded in men 65 years of age or older. All indigenous cases of Legionnaires' disease were sporadic; all but three patients were hospitalized. State Sanitary Inspection reported 26 fatal cases of Legionnaires' disease (mortality = 11%). Twenty-four cases were linked to contaminated water systems in health-care settings, and 21 cases were likely associated with travel abroad. Summary and conclusions: Although the number of notifications has increased in recent years, Legionnaires' disease is still an infrequently diagnosed respiratory infection in Poland, and the reported incidence remains one of the lowest in the entire EU. The most affected demographic group is men aged 65 and older. Improving the early diagnosis of Legionnaires' disease in healthcare settings remains a priority.

Bacterial persistence in Legionella pneumophila clinical isolates from patients with recurring legionellosis.

Bacterial persisters are a transient subpopulation of non-growing, antibiotic-tolerant cells. There is increasing evidence that bacterial persisters play an important role in treatment failure leading to recurring infections and promoting the development of antibiotic resistance. Current research reveals that recurring legionellosis is often the result of relapse rather than reinfection and suggests that the mechanism of bacterial persistence may play a role. The development of single-cell techniques such as the Timerbac system allows us to identify potential persister cells and investigate their physiology. Here, we tested the persister forming capacity of 7 pairs of Legionella pneumophila (Lp) clinical isolates, with isolate pairs corresponding to two episodes of legionellosis in the same patient. We distinguished non-growing subpopulations from their replicating counterparts during infection in an amoeba model. Imaging flow cytometry allowed us to identify single non-growing bacteria within amoeba cells 17 h post-infection, thus corresponding to this subpopulation of potential persister cells. Interestingly the magnitude of this subpopulation varies between the 7 pairs of Lp clinical isolates. Biphasic killing kinetics using ofloxacin stress confirmed the persister development capacity of ST1 clinical isolates, highlighting enhanced persister formation during the host cell infection. Thus, persister formation appears to be strain or ST (sequence type) dependent. Genome sequence analysis was carried out between ST1 clinical isolates and ST1 Paris. No genetic microevolution (SNP) linked to possible increase of persistence capacity was revealed among all the clones tested, even in clones issued from two persistence cycle experiments, confirming the transient reversible phenotypic status of persistence. Treatment failure in legionellosis is a serious issue as infections have a 5-10% mortality rate, and investigations into persistence in a clinical context and the mechanisms involved may allow us to combat this issue.

Legionellosis on the rise: A scoping review of sporadic, community-acquired incidence in the United States.

Over the past two decades, the incidence of legionellosis has been steadily increasing in the United States though there is noclear explanation for the main factors driving the increase. While legionellosis is the leading cause of waterborne outbreaks in the US, most cases are sporadic and acquired in community settings where the environmental source is never identified. This scoping review aimed to summarise the drivers of infections in the USA and determine the magnitude of impact each potential driver may have. A total of 1,738 titles were screened, and 18 articles were identified that met the inclusion criteria. Strong evidence was found for precipitation as a major driver, and both temperature and relative humidity were found to be moderate drivers of incidence. Increased testing and improved diagnostic methods were classified as moderate drivers, and the ageing U.S. population was a minor driver of increasing incidence. Racial and socioeconomic inequities and water and housing infrastructure were found to be potential factors explaining the increasing incidence though they were largely understudied in the context of non-outbreak cases. Understanding the complex relationships between environmental, infrastructure, and population factors driving legionellosis incidence is important to optimise mitigation strategies and public policy.

Water quality influences Legionella pneumophila determination.

Legionellosis is a respiratory disease of public health concern. The bacterium Legionella pneumophila is the etiologic agent responsible for >90% of legionellosis cases in the United States. Legionellosis transmission primarily occurs through the inhalation or aspiration of contaminated water aerosols or droplets. Therefore, a thorough understanding of L. pneumophila detection methods and their performance in various water quality conditions is needed to develop preventive measures. Two hundred and nine potable water samples were collected from taps in buildings across the United States. L. pneumophila was determined using three culture methods: Buffered Charcoal Yeast Extract (BCYE) culture with Matrix-assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) identification, Legiolert® 10- and 100-mL tests, and one molecular method: quantitative Polymerase Chain Reaction (qPCR) assay. Culture and molecular positive results were confirmed by secondary testing including MALDI-MS. Eight water quality variables were studied, including source water type, secondary disinfectant, total chlorine residual, heterotrophic bacteria, total organic carbon (TOC), pH, water hardness, cold- and hot-water lines. The eight water quality variables were segmented into 28 categories, based on scale and ranges, and method performance was evaluated in each of these categories. Additionally, a Legionella genus qPCR assay was used to determine the water quality variables that promote or hinder Legionella spp. occurrence. L. pneumophila detection frequency ranged from 2 to 22% across the methods tested. Method performance parameters of sensitivity, specificity, positive and negative predictive values, and accuracy were >94% for the qPCR method but ranged from 9 to 100% for the culture methods. Water quality influenced L. pneumophila determination by culture and qPCR methods. L. pneumophila qPCR detection frequencies positively correlated with TOC and heterotrophic bacterial counts. The source water-disinfectant combination influenced the proportion of Legionella spp. that is L. pneumophila. Water quality influences L. pneumophila determination. To accurately detect L. pneumophila, method selection should consider the water quality in addition to the purpose of testing (general environmental monitoring versus disease-associated investigations).

Legionella longbeachae wound infection: case report and review of reported Legionella wound infections.

Extrapulmonary manifestations of infection with Legionella species, of which 24 may cause disease in humans, are very rare. Here, we describe a case of a 61-year-old woman with no history of immunosuppression presenting with pain and swelling of her index finger after a prick by rose thorns during gardening. Clinical examination showed fusiform swelling of the finger with mild redness, warmth, and fever. The blood sample revealed a normal white blood cell count and a slight increase in C-reactive protein. Intraoperative observation showed extensive infectious destruction of the tendon sheath, while the flexor tendons were spared. Conventional cultures were negative, while 16S rRNA PCR analysis identified Legionella longbeachae that also could be isolated on buffered charcoal yeast extract media. The patient was treated with oral levofloxacin for 13 days, and the infection healed quickly. The present case report, with a review of the literature, indicates that Legionella species wound infections may be underdiagnosed due to the requirement for specific media and diagnostic methods. It emphasizes the need for heightened awareness of these infections during history taking and clinical examination of patients presenting with cutaneous infections.

Increased Incidence of Legionellosis after Improved Diagnostic Methods, New Zealand, 2000-2020.

Legionellosis, notably Legionnaires' disease, is recognized globally and in New Zealand (Aotearoa) as a major cause of community-acquired pneumonia. We analyzed the temporal, geographic, and demographic epidemiology and microbiology of Legionnaires' disease in New Zealand by using notification and laboratory-based surveillance data for 2000‒2020. We used Poisson regression models to estimate incidence rate ratios and 95% CIs to compare demographic and organism trends over 2 time periods (2000-2009 and 2010-2020). The mean annual incidence rate increased from 1.6 cases/100,000 population for 2000-2009 to 3.9 cases/100,000 population for 2010-2020. This increase corresponded with a change in diagnostic testing from predominantly serology with some culture to almost entirely molecular methods using PCR. There was also a marked shift in the identified dominant causative organism, from Legionella pneumophila to L. longbeachae. Surveillance for legionellosis could be further enhanced by greater use of molecular typing of isolates.

Legionelosis: Recomendaciones para la prevención, la vigilancia epidemiológica y el control de casos y brotes / Legionellosis: Recommendations for prevention, epidemiological surveillance and control of cases and outbreaks

La enfermedad de los legionarios es un importante problema de salud pública particularmente por su frecuente presentación en forma de brotes, tanto comunitarios como nosocomiales, y por su letalidad, especialmente en personas de edad avanzada o con otras enfermedades. La notificación oportuna de casos y/o brotes de enfermedad y la investigación epidemiológica permiten la identificación de la/s fuentes de exposición y la adopción de medidas de prevención y control adecuadas. Las infecciones por Legionella son más frecuentes entre adultos mayores de 50 años, hombres, fumadores y huéspedes inmunocomprometidos o con ciertas enfermedades crónicas subyacentes. La infección en niñas/os es rara, con ≤ 1% de los casos de neumonía causada por Legionella, y puede ser asintomática o leve y no detectada. La Legionella puede multiplicarse si el agua no es tratada de manera adecuada o si los sistemas de agua no son mantenidos adecuadamente.

Environmental Investigation and Surveillance for Legionella in Aotearoa New Zealand, 2000-2020.

The reported rate of legionellosis is increasing in Aotearoa New Zealand (NZ) with most cases community-acquired, sporadic (non-outbreak) and without an identifiable source. This analysis used two datasets to describe the environmental sources that contribute to Legionella in NZ, based on linkages with outbreaks and sporadic clinical cases, and analysis of environmental testing data. These findings highlight the need for enhanced environmental investigation of clinical cases and outbreaks. There is also a need for systematic surveillance testing of high-risk source environments to support more rigorous controls to prevent legionellosis.

Investigation of a legionellosis outbreak in Sydney CBD - a brief report.

OBJECTIVE: To identify and control a source of Legionella in Sydney CBD. METHODS: Clinical, epidemiological, environmental and genomic techniques were employed to identify cases and the source of Legionella. RESULTS: Eleven legionellosis cases were linked to Sydney CBD with a median age of 69 years. All were hospitalised and had risk factors for Legionella infection. Eight of 11 cases identified as male. Genomic analysis linked three cases to a contaminated cooling water source in Sydney CBD, with a further case infected with a similar strain to that found in Sydney CBD. Another case, although epidemiologically linked to Sydney CBD, was infected with a genomically different strain to that found in Sydney CBD. Six other cases had no viable sample for genomic analysis. CONCLUSION/IMPLICATIONS FOR PUBLIC HEALTH: An outbreak of legionellosis is a serious public health threat that requires rapid investigation and environmental control. We were able to identify a source in Sydney CBD through the application of clinical, epidemiological, environmental and genomic techniques. Genomic analysis is a powerful tool that can be used to confirm the source location but requires close collaboration between clinicians, public health units and microbiologists to recover viable sputum cultures from cases diagnosed with legionellosis.

Temporal trends in legionellosis national notification data and the effect of COVID-19, Switzerland, 2000-2020.

The notification rate of legionellosis in Switzerland and other European countries has markedly increased over the last 20 years. Here, we investigated the Swiss notification data on legionellosis from 2000 to 2020 in regards of overall time trend, content and data quality. We further explored the impact of the COVID-19 pandemic on the reported case numbers using an interrupted time series approach. Between 2000 and 2020, 5980 cases were included in our analysis. The annual crude notification rate for legionellosis cases increased from 1.1/100,000 population (CI: 0.9-1.4) in 2000 to 5.6/100,000 population (CI: 5.1-6.1) in 2020. In recent years, the summer peaks have been more pronounced and some shifted earlier in the year. The highest notification rate was recorded in 2018 with 6.7/100,000 population (CI: 6.2-7.3). The hospitalisation rate for notified cases remained high across all study years (89.9%), while the case fatality rate slightly decreased (from 7.7% to 3.6%). COVID-19 containment measures, such as travel restrictions and/or related behavioural changes, are associated with a temporary decline in cases of 35%. Overall, the quality of the notification data was good. Clinical data were more susceptible to interferences than data from laboratory reporting, which could be observed most clearly in the decline of clinical reports by 4.3 percentage points in 2020. As the case classification for Legionnaires' disease includes pneumonia symptoms, this decline could lead to an underestimation of Legionnaires' disease cases, yet the continuous reporting though the diagnostic laboratories suggested a robust surveillance system for legionellosis in Switzerland.

Risk of Legionellosis in residential areas around farms irrigating with municipal wastewater.

The conservation of freshwater is of both global and national importance, and in the United States, agriculture is one of the largest consumers of this resource. Reduction of the strain farming puts on local surface or groundwater is vital for ensuring resilience in the face of climate change, and one possible option is to irrigate with a combination of freshwater and reclaimed water from municipal wastewater treatment facilities. However, this wastewater can contain pathogens that are harmful to human health, such as Legionella pneumophila, which is a bacterium that can survive aerosolization and airborne transportation and cause severe pneumonia when inhaled. To assess an individual adult's risk of infection with L. pneumophila from a single exposure to agricultural spray irrigation, a quantitative microbial risk assessment was conducted for a scenario of spray irrigation in central Illinois, for the growing seasons in 2017, 2018, and 2019. The assessment found that the mean risk of infection for a single exposure exceeded the safety threshold of 10-6 infections/exposure up to 1 km from a low-pressure irrigator and up to 2 km from a high-pressure irrigator, although no median risk exceeded the threshold for any distance or irrigator pressure. These findings suggest that spray irrigation with treated municipal wastewater could be a viable option for reducing freshwater consumption in Midwest farming, as long as irrigation on windy days is avoided and close proximity to the active irrigator is limited.