Detecting Mpox Cases Through Wastewater Surveillance - United States, August 2022-May 2023.

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.

Using Wastewater Surveillance Data to Support the COVID-19 Response - United States, 2020-2021.

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.

Legionellosis Cluster Associated With Working at a Racetrack Facility in West Virginia, 2018.

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.

Knowledge and Practices Regarding Safe Household Cleaning and Disinfection for COVID-19 Prevention - United States, May 2020.

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.

Genomic heterogeneity differentiates clinical and environmental subgroups of Legionella pneumophila sequence type 1.

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.

Comparative genome analysis reveals a complex population structure of Legionella pneumophila subspecies.

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.

Legionnaires' Disease Outbreak Caused by Endemic Strain of Legionella pneumophila, New York, New York, USA, 2015.

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.

Characterization of Legionella Species from Watersheds in British Columbia, Canada.

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.

Complete Genome Sequences of Legionella pneumophila subsp. fraseri Strains Detroit-1 and Dallas 1E.

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.

Genomic Analysis Reveals Novel Diversity among the 1976 Philadelphia Legionnaires' Disease Outbreak Isolates and Additional ST36 Strains.

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.

Complete Genome Sequences of the Historical Legionella pneumophila Strains OLDA and Pontiac.

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.

Complete Genome Sequences of Three Outbreak-Associated Legionella pneumophila Isolates.

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.

Genomic Resolution of Outbreak-Associated Legionella pneumophila Serogroup 1 Isolates from New York State.

UNLABELLED: 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.

Simultaneous detection of Legionella species and L. anisa, L. bozemanii, L. longbeachae and L. micdadei using conserved primers and multiple probes in a multiplex real-time PCR assay.

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.

Current and emerging Legionella diagnostics for laboratory and outbreak investigations.

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.

Epithelial adhesion mediated by pilin SpaC is required for Lactobacillus rhamnosus GG-induced cellular responses.

Lactobacillus rhamnosus GG is a widely used probiotic, and the strain's salutary effects on the intestine have been extensively documented. We previously reported that strain GG can modulate inflammatory signaling, as well as epithelial migration and proliferation, by activating NADPH oxidase 1-catalyzed generation of reactive oxygen species (ROS). However, how strain GG induces these responses is unknown. Here, we report that strain GG's probiotic benefits are dependent on the bacterial-epithelial interaction mediated by the SpaC pilin subunit. By comparing strain GG to an isogenic mutant that lacks SpaC (strain GGΩspaC), we establish that SpaC is necessary for strain GG to adhere to gut mucosa, that SpaC contributes to strain GG-induced epithelial generation of ROS, and that SpaC plays a role in strain GG's capacity to stimulate extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) signaling in enterocytes. In addition, we show that SpaC is required for strain GG-mediated stimulation of cell proliferation and protection against radiologically inflicted intestinal injury. The identification of a critical surface protein required for strain GG to mediate its probiotic influence advances our understanding of the molecular basis for the symbiotic relationship between some commensal bacteria of the gut lumen and enterocytes. Further insights into this relationship are critical for the development of novel approaches to treat intestinal diseases.

Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species.

The resident prokaryotic microbiota of the metazoan gut elicits profound effects on the growth and development of the intestine. However, the molecular mechanisms of symbiotic prokaryotic-eukaryotic cross-talk in the gut are largely unknown. It is increasingly recognized that physiologically generated reactive oxygen species (ROS) function as signalling secondary messengers that influence cellular proliferation and differentiation in a variety of biological systems. Here, we report that commensal bacteria, particularly members of the genus Lactobacillus, can stimulate NADPH oxidase 1 (Nox1)-dependent ROS generation and consequent cellular proliferation in intestinal stem cells upon initial ingestion into the murine or Drosophila intestine. Our data identify and highlight a highly conserved mechanism that symbiotic microorganisms utilize in eukaryotic growth and development. Additionally, the work suggests that specific redox-mediated functions may be assigned to specific bacterial taxa and may contribute to the identification of microbes with probiotic potential.

Circuitry linking the Csr and stringent response global regulatory systems.

CsrA protein regulates important cellular processes by binding to target mRNAs and altering their translation and/or stability. In Escherichia coli, CsrA binds to sRNAs, CsrB and CsrC, which sequester CsrA and antagonize its activity. Here, mRNAs for relA, spoT and dksA of the stringent response system were found among 721 different transcripts that copurified with CsrA. Many of the transcripts that copurified with CsrA were previously determined to respond to ppGpp and/or DksA. We examined multiple regulatory interactions between the Csr and stringent response systems. Most importantly, DksA and ppGpp robustly activated csrB/C transcription (10-fold), while they modestly activated csrA expression. We propose that CsrA-mediated regulation is relieved during the stringent response. Gel shift assays confirmed high affinity binding of CsrA to relA mRNA leader and weaker interactions with dksA and spoT. Reporter fusions, qRT-PCR and immunoblotting showed that CsrA repressed relA expression, and (p)ppGpp accumulation during stringent response was enhanced in a csrA mutant. CsrA had modest to negligible effects on dksA and spoT expression. Transcription of dksA was negatively autoregulated via a feedback loop that tended to mask CsrA effects. We propose that the Csr system fine-tunes the stringent response and discuss biological implications of the composite circuitry.

Molecular geometry of CsrA (RsmA) binding to RNA and its implications for regulated expression.

The global regulatory protein CsrA binds to the 5'-untranslated leader of target transcripts and alters their translation and/or stability. CsrA is a symmetrical homodimer containing two identical RNA-binding surfaces. Gel shift assays with model RNA substrates now show that CsrA can bind simultaneously at two target sites within a transcript (bridging or dual-site binding). An intersite distance of approximately 18 nucleotides (nt) was optimal, although bridging occurred with an intersite distance of 10 to >or=63 nt. The close 10-nt spacing reduced the stability of dual-site binding, as competition for one site by a second CsrA dimer readily occurred. Both RNA-binding surfaces of a single CsrA protein were essential for efficient in vitro repression of a glgC'-'lacZ translational fusion that contains four CsrA target sites within the untranslated leader. Heterodimeric CsrA (HD-CsrA) containing a single R44A replacement, which was defective for binding at its mutant surface but bound RNA normally at its wild-type (WT) surface, was approximately 14-fold less effective at repression than homodimeric WT-CsrA. Furthermore, deletion of a CsrA target site of glgC that lies upstream from the Shine-Dalgarno sequence did not affect regulation by HD-CsrA but decreased regulation by WT-CsrA, confirming a regulatory role of dual-site binding. Finally, we propose a mechanism whereby a globular ribonucleoprotein complex is formed between CsrA and its noncoding RNA antagonist, CsrB. Because many target sites of CsrB are located closer together than is optimal for bridging, binding to nonadjacent sites should be energetically favored, causing multiple CsrA dimers to tether CsrB into the observed globular form rather than an extended CsrA-CsrB complex.

CsrA inhibits translation initiation of Escherichia coli hfq by binding to a single site overlapping the Shine-Dalgarno sequence.

Csr (carbon storage regulation) of Escherichia coli is a global regulatory system that consists of CsrA, a homodimeric RNA binding protein, two noncoding small RNAs (sRNAs; CsrB and CsrC) that function as CsrA antagonists by sequestering this protein, and CsrD, a specificity factor that targets CsrB and CsrC for degradation by RNase E. CsrA inhibits translation initiation of glgC, cstA, and pgaA by binding to their leader transcripts and preventing ribosome binding. Translation inhibition is thought to contribute to the observed mRNA destabilization. Each of the previously known target transcripts contains multiple CsrA binding sites. A position-specific weight matrix search program was developed using known CsrA binding sites in mRNA. This search tool identified a potential CsrA binding site that overlaps the Shine-Dalgarno sequence of hfq, a gene that encodes an RNA chaperone that mediates sRNA-mRNA interactions. This putative CsrA binding site matched the SELEX-derived binding site consensus sequence in 8 out of 12 positions. Results from gel mobility shift and footprint assays demonstrated that CsrA binds specifically to this site in the hfq leader transcript. Toeprint and cell-free translation results indicated that bound CsrA inhibits Hfq synthesis by competitively blocking ribosome binding. Disruption of csrA caused elevated expression of an hfq'-'lacZ translational fusion, while overexpression of csrA inhibited expression of this fusion. We also found that hfq mRNA is stabilized upon entry into stationary-phase growth by a CsrA-independent mechanism. The interaction of CsrA with hfq mRNA is the first example of a CsrA-regulated gene that contains only one CsrA binding site.