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.

On February 1, 2022, the U.S. Virgin Islands (USVI) Department of Health (VIDOH) was notified of a confirmed case of Legionnaires disease in an adult U.S. resident (Figure). The patient, a man aged 55 years, returned to his U.S. state of residence from leisure travel in USVI on January 22 and developed a cough, shortness of breath, and fatigue on January 23. On January 29, he was hospitalized for shortness of breath and received a positive SARS-CoV-2 test result at admission. The combination of the patient’s symptoms and recent travel history prompted administration of a urinary antigen test (UAT) for Legionnaires disease specific to Legionella pneumophila serogroup 1 (Lp1); a positive result was returned on January 31. Inpatient treatment administered for COVID-19 pneumonia and Legionnaires disease included remdesivir, oral levofloxacin, oral and intravenous steroid therapy, and as-needed use of a bronchodilator inhaler and an expectorant. Remdesivir was discontinued during inpatient treatment because of elevated liver enzymes. The patient recovered and was discharged on February 2.

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.

Taxonomic classification of Clostridium botulinum is based on the production of botulinum neurotoxin (BoNT) while closely-related, non-toxic organisms are classified as Clostridium sporogenes. However, this taxonomic organization does not accurately mirror phylogenetic relationships between these species. A phylogenetic reconstruction using 2,016 orthologous genes shared among strains of C. botulinum Group I and C. sporogenes clearly separated these two species into discrete clades which showed approximately 93% average nucleotide identity (ANI) between them. Clustering of strains based on the presence of variable orthologs revealed 143 C. sporogenes clade-specific genetic signatures, a subset of which was further evaluated for their ability to correctly classify a panel of presumptive C. sporogenes strains by PCR. Genome sequencing of several C. sporogenes strains lacking these signatures confirmed that they clustered with C. botulinum strains in a core genome phylogenetic tree. Our analysis also identified C. botulinum strains that contained C. sporogenes clade-specific signatures and phylogenetically clustered with C. sporogenes strains. The genome sequences of two bontB2-containing strains belonging to the C. sporogenes clade contained regions with similarity to a bont-encoding plasmid (pCLD) while two different strains belonging to the C. botulinum clade encoded bontB2 on the chromosome. These results indicate that bont/B2 was likely acquired by C. sporogenes strains through horizontal gene transfer. The genome-based classification of these species used to identify candidate genes for the development of rapid assays for molecular identification may be applicable to additional bacterial species that are challenging with respect to their classification.