Legionella pneumophila serogroup 1 sequence types (ST) 213 and 222, a single-locus variant of ST213, were first detected in the early 1990s in the Midwest United States (U.S.) and the late 1990s in the Northeast U.S. and Canada. Since 1992, these STs have increasingly been implicated in community-acquired sporadic and outbreak-associated Legionnaires' disease (LD) cases. We were interested in understanding the change in LD frequency due to these STs and identifying genetic features that differentiate these STs from one another. For the geographic area examined here (Mountain West to Northeast) and over the study period (1992-2020), ST213/222-associated LD cases identified by the Centers for Disease Control and Prevention increased by 0.15 cases per year, with ST213/222-associated LD cases concentrated in four states: Michigan (26%), New York (18%), Minnesota (16%), and Ohio (10%). Additionally, between 2002 and 2021, ST222 caused at least five LD outbreaks in the U.S.; no known outbreaks due to ST213 occurred in the U.S. during this time. We compared the genomes of 230 ST213/222 isolates and found that the mean of the average nucleotide identity (ANI) within each ST was high (99.92% for ST222 and 99.92% for ST213), with a minimum between ST ANI of 99.50% and a maximum of 99.87%, indicating low genetic diversity within and between these STs. While genomic features were identified (e.g., plasmids and CRISPR-Cas systems), no association explained the increasing geographic distribution and prevalence of ST213 and ST222. Yet, we provide evidence of the expanded geographical distribution of ST213 and ST222 in the U.S.IMPORTANCESince the 1990s, cases of Legionnaires' disease (LD) attributed to a pair of closely related Legionella pneumophila variants, ST213 and ST222, have increased in the U.S. Furthermore, between 2002 and 2021, ST222 caused at least five outbreaks of LD in the U.S., while ST213 has not been linked to any U.S. outbreak. We wanted to understand how the rate of LD cases attributed to these variants has changed over time and compare the genetic features of the two variants. Between 1992 and 2020, we determined an increase of 0.15 LD cases ascribed to ST213/222 per year in the geographic region studied. Our research shows that these STs are spreading within the U.S., yet most of the cases occurred in four states: Michigan, New York, Minnesota, and Ohio. Additionally, we found little genetic diversity within and between these STs nor could specific genetic features explain their geographic spread.

As part of the response to the highly pathogenic avian influenza A(H5N1) virus outbreak in U.S. cattle and poultry and the associated human cases, CDC and partners are monitoring influenza A virus levels and detection of the H5 subtype in wastewater. Among 48 states and the District of Columbia that performed influenza A testing of wastewater during May 12-July 13, 2024, a weekly average of 309 sites in 38 states had sufficient data for analysis, and 11 sites in four states reported high levels of influenza A virus. H5 subtype testing was conducted at 203 sites in 41 states, with H5 detections at 24 sites in nine states. For each detection or high level, CDC and state and local health departments evaluated data from other influenza surveillance systems and partnered with wastewater utilities and agriculture departments to investigate potential sources. Among the four states with high influenza A virus levels detected in wastewater, three states had corresponding evidence of human influenza activity from other influenza surveillance systems. Among the 24 sites with H5 detections, 15 identified animal sources within the sewershed or adjacent county, including eight milk-processing inputs. Data from these early investigations can help health officials optimize the use of wastewater surveillance during the upcoming respiratory illness season.

In October 2022, CDC's National Wastewater Surveillance System began routine testing of U.S. wastewater for Monkeypox virus. Wastewater surveillance sensitivity, positive predictive value (PPV), and negative predictive value (NPV) for Monkeypox virus were evaluated by comparing wastewater detections (Monkeypox virus detected versus not detected) to numbers of persons with mpox in a county who were shedding virus. Case ascertainment was assumed to be complete, and persons with mpox were assumed to shed virus for 25 days after symptom onset. A total of 281 cases and 3,492 wastewater samples from 89 sites in 26 counties were included in the analysis. Wastewater surveillance in a single week, from samples representing thousands to millions of persons, had a sensitivity of 32% for detecting one or more persons shedding Monkeypox virus, 49% for detecting five or more persons shedding virus, and 77% for detecting 15 or more persons shedding virus. Weekly PPV and NPV for detecting persons shedding Monkeypox virus in a county were 62% and 80%, respectively. An absence of detections in counties with wastewater surveillance signified a high probability that a large number of cases were not present. Results can help to guide the public health response to Monkeypox virus wastewater detections. A single, isolated detection likely warrants a limited public health response. An absence of detections, in combination with no reported cases, can give public health officials greater confidence that no cases are present. Wastewater surveillance can serve as a useful complement to case surveillance for guiding the public health response to an mpox outbreak.

Wastewater surveillance, the measurement of pathogen levels in wastewater, is used to evaluate community-level infection trends, augment traditional surveillance that leverages clinical tests and services (e.g., case reporting), and monitor public health interventions (1). Approximately 40% of persons infected with SARS-CoV-2, the virus that causes COVID-19, shed virus RNA in their stool (2); therefore, community-level trends in SARS-CoV-2 infections, both symptomatic and asymptomatic (2) can be tracked through wastewater testing (3-6). CDC launched the National Wastewater Surveillance System (NWSS) in September 2020 to coordinate wastewater surveillance programs implemented by state, tribal, local, and territorial health departments to support the COVID-19 pandemic response. In the United States, wastewater surveillance was not previously implemented at the national level. As of August 2021, NWSS includes 37 states, four cities, and two territories. This report summarizes NWSS activities and describes innovative applications of wastewater surveillance data by two states, which have included generating alerts to local jurisdictions, allocating mobile testing resources, evaluating irregularities in traditional surveillance, refining health messaging, and forecasting clinical resource needs. NWSS complements traditional surveillance and enables health departments to intervene earlier with focused support in communities experiencing increasing concentrations of SARS-CoV-2 in wastewater. The ability to conduct wastewater surveillance is not affected by access to health care or the clinical testing capacity in the community. Robust, sustainable implementation of wastewater surveillance requires public health capacity for wastewater testing, analysis, and interpretation. Partnerships between wastewater utilities and public health departments are needed to leverage wastewater surveillance data for the COVID-19 response for rapid assessment of emerging threats and preparedness for future pandemics.

In October 2018, the Centers for Disease Control and Prevention was notified of a cluster of Legionnaires' disease cases in workers at a racetrack facility. The objective of the resulting investigation was to determine the extent of the outbreak and identify potential sources of exposure to halt transmission. Case-finding and interviews were conducted among symptomatic racetrack workers who were known to be at the facility within 14 days prior to symptom onset. An environmental assessment of the facility and surrounding area was conducted for sources of potential Legionella exposure. In total, 17 legionellosis cases were identified. The environmental assessment revealed a poorly maintained hot tub in the jockey locker room as the most likely source. Further investigation identified deficiencies in the facility's ventilation systems, which suggested a transmission mechanism for workers who never entered the locker room floor. Considering indirect exposure routes via air handling systems can be useful for source identification and case-finding in legionellosis outbreaks.

A recent report described a sharp increase in calls to poison centers related to exposures to cleaners and disinfectants since the onset of the coronavirus disease 2019 (COVID-19) pandemic (1). However, data describing cleaning and disinfection practices within household settings in the United States are limited, particularly concerning those practices intended to prevent transmission of SARS-CoV-2, the virus that causes COVID-19. To provide contextual and behavioral insight into the reported increase in poison center calls and to inform timely and relevant prevention strategies, an opt-in Internet panel survey of 502 U.S. adults was conducted in May 2020 to characterize knowledge and practices regarding household cleaning and disinfection during the COVID-19 pandemic. Knowledge gaps were identified in several areas, including safe preparation of cleaning and disinfectant solutions, use of recommended personal protective equipment when using cleaners and disinfectants, and safe storage of hand sanitizers, cleaners, and disinfectants. Thirty-nine percent of respondents reported engaging in nonrecommended high-risk practices with the intent of preventing SARS-CoV-2 transmission, such as washing food products with bleach, applying household cleaning or disinfectant products to bare skin, and intentionally inhaling or ingesting these products. Respondents who engaged in high-risk practices more frequently reported an adverse health effect that they believed was a result of using cleaners or disinfectants than did those who did not report engaging in these practices. Public messaging should continue to emphasize evidence-based, safe practices such as hand hygiene and recommended cleaning and disinfection of high-touch surfaces to prevent transmission of SARS-CoV-2 in household settings (2). Messaging should also emphasize avoidance of high-risk practices such as unsafe preparation of cleaning and disinfectant solutions, use of bleach on food products, application of household cleaning and disinfectant products to skin, and inhalation or ingestion of cleaners and disinfectants.

During a Legionnaires disease outbreak at a Missouri hotel in 2015, the Centers for Disease Control and Prevention assisted state and local health departments to identify possible sources and transmission factors and to recommend improvements to water management. We performed an environmental assessment to understand the hotels water systems and identify areas of risk for Legionella amplifi cation and transmission. We obtained samples from the pool, spa, and potable water systems for Legionella culture. In the potable water system, we noted temperatures ideal for Legionella amplifi cation and areas of water stagnation. Additionally, we found inadequate documentation of pool and spa disinfection and maintenance. Of 40 water samples, Legionella pneumophila serogroup 1 that matched the sequence type of one available clinical isolate was recovered from five sink and shower fixtures. A comprehensive environmental assessment proved crucial to identifying maintenance issues in the hotels water systems and underscored the need for a water management program to reduce Legionnaires disease risk.

Legionella spp. are the cause of a severe bacterial pneumonia known as Legionnaires' disease (LD). In some cases, current genetic subtyping methods cannot resolve LD outbreaks caused by common, potentially endemic L. pneumophila (Lp) sequence types (ST), which complicates laboratory investigations and environmental source attribution. In the United States (US), ST1 is the most prevalent clinical and environmental Lp sequence type. In order to characterize the ST1 population, we sequenced 289 outbreak and non-outbreak associated clinical and environmental ST1 and ST1-variant Lp strains from the US and, together with international isolate sequences, explored their genetic and geographic diversity. The ST1 population was highly conserved at the nucleotide level; 98% of core nucleotide positions were invariant and environmental isolates unassociated with human disease (n = 99) contained ~65% more nucleotide diversity compared to clinical-sporadic (n = 139) or outbreak-associated (n = 28) ST1 subgroups. The accessory pangenome of environmental isolates was also ~30-60% larger than other subgroups and was enriched for transposition and conjugative transfer-associated elements. Up to ~10% of US ST1 genetic variation could be explained by geographic origin, but considerable genetic conservation existed among strains isolated from geographically distant states and from different decades. These findings provide new insight into the ST1 population structure and establish a foundation for interpreting genetic relationships among ST1 strains; these data may also inform future analyses for improved outbreak investigations.

The majority of Legionnaires' disease (LD) cases are caused by Legionella pneumophila, a genetically heterogeneous species composed of at least 17 serogroups. Previously, it was demonstrated that L. pneumophila consists of three subspecies: pneumophila, fraseri and pascullei. During an LD outbreak investigation in 2012, we detected that representatives of both subspecies fraseri and pascullei colonized the same water system and that the outbreak-causing strain was a new member of the least represented subspecies pascullei. We used partial sequence based typing consensus patterns to mine an international database for additional representatives of fraseri and pascullei subspecies. As a result, we identified 46 sequence types (STs) belonging to subspecies fraseri and two STs belonging to subspecies pascullei. Moreover, a recent retrospective whole genome sequencing analysis of isolates from New York State LD clusters revealed the presence of a fourth L. pneumophila subspecies that we have termed raphaeli. This subspecies consists of 15 STs. Comparative analysis was conducted using the genomes of multiple members of all four L. pneumophila subspecies. Whereas each subspecies forms a distinct phylogenetic clade within the L. pneumophila species, they share more average nucleotide identity with each other than with other Legionella species. Unique genes for each subspecies were identified and could be used for rapid subspecies detection. Improved taxonomic classification of L. pneumophila strains may help identify environmental niches and virulence attributes associated with these genetically distinct subspecies.

During the summer of 2015, New York, New York, USA, had one of the largest and deadliest outbreaks of Legionnaires' disease in the history of the United States. A total of 138 cases and 16 deaths were linked to a single cooling tower in the South Bronx. Analysis of environmental samples and clinical isolates showed that sporadic cases of legionellosis before, during, and after the outbreak could be traced to a slowly evolving, single-ancestor strain. Detection of an ostensibly virulent Legionella strain endemic to the Bronx community suggests potential risk for future cases of legionellosis in the area. The genetic homogeneity of the Legionella population in this area might complicate investigations and interpretations of future outbreaks of Legionnaires' disease.

Legionella spp. present in some human-made water systems can cause Legionnaires' disease in susceptible individuals. Although legionellae have been isolated from the natural environment, variations in the organism's abundance over time and its relationship to aquatic microbiota are poorly understood. Here, we investigated the presence and diversity of legionellae through 16S rRNA gene amplicon and metagenomic sequencing of DNA from isolates collected from seven sites in three watersheds with varied land uses over a period of 1 year. Legionella spp. were found in all watersheds and sampling sites, comprising up to 2.1% of the bacterial community composition. The relative abundance of Legionella tended to be higher in pristine sites than in sites affected by agricultural activity. The relative abundance levels of Amoebozoa, some of which are natural hosts of legionellae, were similarly higher in pristine sites. Compared to other bacterial genera detected, Legionella had both the highest richness and highest alpha diversity. Our findings indicate that a highly diverse population of legionellae may be found in a variety of natural aquatic sources. Further characterization of these diverse natural populations of Legionella will help inform prevention and control efforts aimed at reducing the risk of Legionella colonization of built environments, which could ultimately decrease the risk of human disease. IMPORTANCE Many species of Legionella can cause Legionnaires' disease, a significant cause of bacterial pneumonia. Legionella in human-made water systems such as cooling towers and building plumbing systems are the primary sources of Legionnaires' disease outbreaks. In this temporal study of natural aquatic environments, Legionella relative abundance was shown to vary in watersheds associated with different land uses. Analysis of the Legionella sequences detected at these sites revealed highly diverse populations that included potentially novel Legionella species. These findings have important implications for understanding the ecology of Legionella and control measures for this pathogen that are aimed at reducing human disease.

We report here the complete genome sequences of two of the earliest known strains of Legionella pneumophila subsp. fraseri Detroit-1 is serogroup 1 and was isolated from a lung biopsy specimen in 1977. Dallas 1E is serogroup 5 and was isolated in 1978 from a cooling tower.

Legionella pneumophila was first recognized as a cause of severe and potentially fatal pneumonia during a large-scale outbreak of Legionnaires' disease (LD) at a Pennsylvania veterans' convention in Philadelphia, 1976. The ensuing investigation and recovery of four clinical isolates launched the fields of Legionella epidemiology and scientific research. Only one of the original isolates, "Philadelphia-1", has been widely distributed or extensively studied. Here we describe the whole-genome sequencing (WGS), complete assembly, and comparative analysis of all Philadelphia LD strains recovered from that investigation, along with L. pneumophila isolates sharing the Philadelphia sequence type (ST36). Analyses revealed that the 1976 outbreak was due to multiple serogroup 1 strains within the same genetic lineage, differentiated by an actively mobilized, self-replicating episome that is shared with L. pneumophila str. Paris, and two large, horizontally-transferred genomic loci, among other polymorphisms. We also found a completely unassociated ST36 strain that displayed remarkable genetic similarity to the historical Philadelphia isolates. This similar strain implies the presence of a potential clonal population, and suggests important implications may exist for considering epidemiological context when interpreting phylogenetic relationships among outbreak-associated isolates. Additional extensive archival research identified the Philadelphia isolate associated with a non-Legionnaire case of "Broad Street pneumonia", and provided new historical and genetic insights into the 1976 epidemic. This retrospective analysis has underscored the utility of fully-assembled WGS data for Legionella outbreak investigations, highlighting the increased resolution that comes from long-read sequencing and a sequence type-matched genomic data set.

Here, we report the complete genome sequences of Legionella pneumophila serogroup 1 strains OLDA and Pontiac, which predate the 1976 Philadelphia Legionnaires' disease outbreak. Strain OLDA was isolated in 1947 from an apparent sporadic case, and strain Pontiac caused an explosive outbreak at a Michigan health department in 1968.

We report here the complete genome sequences of three Legionella pneumophila isolates that are associated with a Legionnaires' disease outbreak in New York in 2012. Two clinical isolates (D7630 and D7632) and one environmental isolate (D7631) were recovered from this outbreak. A single isolate-specific virulence gene was found in D7632. These isolates were included in a large study evaluating the genomic resolution of various bioinformatics approaches for L. pneumophila serogroup 1 isolates.

A total of 30 Legionella pneumophila serogroup 1 isolates representing 10 separate legionellosis laboratory investigations ("outbreaks") that occurred in New York State between 2004 and 2012 were selected for evaluation of whole-genome sequencing (WGS) approaches for molecular subtyping of this organism. Clinical and environmental isolates were available for each outbreak and were initially examined by pulsed-field gel electrophoresis (PFGE). Sequence-based typing alleles were extracted from WGS data yielding complete sequence types (ST) for isolates representing 8 out of the 10 outbreaks evaluated in this study. Isolates from separate outbreaks sharing the same ST also contained the fewest differences in core genome single nucleotide polymorphisms (SNPs) and the greatest proportion of identical allele sequences in a whole-genome multilocus sequence typing (wgMLST) scheme. Both core SNP and wgMLST analyses distinguished isolates from separate outbreaks, including those from two outbreaks sharing indistinguishable PFGE profiles. Isolates from a hospital-associated outbreak spanning multiple years shared indistinguishable PFGE profiles but displayed differences in their genome sequences, suggesting the presence of multiple environmental sources. Finally, the rtx gene demonstrated differences in the repeat region sequence among ST1 isolates from different outbreaks, suggesting that variation in this gene may be useful for targeted molecular subtyping approaches for L. pneumophila This study demonstrates the utility of various genome sequence analysis approaches for L. pneumophila for environmental source attribution studies while furthering the understanding of Legionella ecology. IMPORTANCE: We demonstrate that whole-genome sequencing helps to improve resolution of Legionella pneumophila isolated during laboratory investigations of legionellosis compared to traditional subtyping methods. These data can be important in confirming the environmental sources of legionellosis outbreaks. Moreover, we evaluated various methods to analyze genome sequence data to help resolve outbreak-related isolates.

Legionnaires' disease is a severe respiratory disease that is estimated to cause between 8,000 and 18,000 hospitalizations each year, though the exact burden is unknown due to under-utilization of diagnostic testing. Although Legionella pneumophila is the most common species detected in clinical cases (80-90%), other species have also been reported to cause disease. However, little is known about Legionnaires' disease caused by these non-pneumophila species. We designed a multiplex real-time PCR assay for detection of all Legionella spp. and simultaneous specific identification of four clinically-relevant Legionella species, L. anisa, L. bozemanii, L. longbeachae, and L. micdadei, using 5'-hydrolysis probe real-time PCR. The analytical sensitivity for detection of nucleic acid from each target species was ≤50fg per reaction. We demonstrated the utility of this assay in spiked human sputum specimens. This assay could serve as a tool for understanding the scope and impact of non-pneumophila Legionella species in human disease.

Legionnaires' disease (LD) is an often severe and potentially fatal form of bacterial pneumonia caused by an extensive list of Legionella species. These ubiquitous freshwater and soil inhabitants cause human respiratory disease when amplified in man-made water or cooling systems and their aerosols expose a susceptible population. Treatment of sporadic cases and rapid control of LD outbreaks benefit from swift diagnosis in concert with discriminatory bacterial typing for immediate epidemiological responses. Traditional culture and serology were instrumental in describing disease incidence early in its history; currently, diagnosis of LD relies almost solely on the urinary antigen test, which captures only the dominant species and serogroup, Legionella pneumophila serogroup 1 (Lp1). This has created a diagnostic "blind spot" for LD caused by non-Lp1 strains. This review focuses on historic, current, and emerging technologies that hold promise for increasing LD diagnostic efficiency and detection rates as part of a coherent testing regimen. The importance of cooperation between epidemiologists and laboratorians for a rapid outbreak response is also illustrated in field investigations conducted by the CDC with state and local authorities. Finally, challenges facing health care professionals, building managers, and the public health community in combating LD are highlighted, and potential solutions are discussed.