Multistate nontyphoidal Salmonella and Shiga toxin-producing Escherichia coli outbreaks linked to international travel-United States, 2017-2020.

Enteric bacterial infections are common among people who travel internationally. During 2017-2020, the Centers for Disease Control and Prevention investigated 41 multistate outbreaks of nontyphoidal Salmonella and Shiga toxin-producing Escherichia coli linked to international travel. Resistance to one or more antimicrobial agents was detected in at least 10% of isolates in 16 of 30 (53%) nontyphoidal Salmonella outbreaks and 8 of 11 (73%) Shiga toxin-producing E. coli outbreaks evaluated by the National Antimicrobial Resistance Monitoring System. At least 10% of the isolates in 14 nontyphoidal Salmonella outbreaks conferred resistance to one or more of the clinically significant antimicrobials used in human medicine. This report describes the epidemiology and antimicrobial resistance patterns of these travel-associated multistate outbreaks. Investigating illnesses among returned travellers and collaboration with international partners could result in the implementation of public health interventions to improve hygiene practices and food safety standards and to prevent illness and spread of multidrug-resistant organisms domestically and internationally.

Reoccurring Escherichia coli O157:H7 Strain Linked to Leafy Greens-Associated Outbreaks, 2016-2019.

Genomic characterization of an Escherichia coli O157:H7 strain linked to leafy greens-associated outbreaks dates its emergence to late 2015. One clade has notable accessory genomic content and a previously described mutation putatively associated with increased arsenic tolerance. This strain is a reoccurring, emerging, or persistent strain causing illness over an extended period.

Tracking early lung cancer metastatic dissemination in TRACERx using ctDNA.

Circulating tumour DNA (ctDNA) can be used to detect and profile residual tumour cells persisting after curative intent therapy1. The study of large patient cohorts incorporating longitudinal plasma sampling and extended follow-up is required to determine the role of ctDNA as a phylogenetic biomarker of relapse in early-stage non-small-cell lung cancer (NSCLC). Here we developed ctDNA methods tracking a median of 200 mutations identified in resected NSCLC tissue across 1,069 plasma samples collected from 197 patients enrolled in the TRACERx study2. A lack of preoperative ctDNA detection distinguished biologically indolent lung adenocarcinoma with good clinical outcome. Postoperative plasma analyses were interpreted within the context of standard-of-care radiological surveillance and administration of cytotoxic adjuvant therapy. Landmark analyses of plasma samples collected within 120 days after surgery revealed ctDNA detection in 25% of patients, including 49% of all patients who experienced clinical relapse; 3 to 6 monthly ctDNA surveillance identified impending disease relapse in an additional 20% of landmark-negative patients. We developed a bioinformatic tool (ECLIPSE) for non-invasive tracking of subclonal architecture at low ctDNA levels. ECLIPSE identified patients with polyclonal metastatic dissemination, which was associated with a poor clinical outcome. By measuring subclone cancer cell fractions in preoperative plasma, we found that subclones seeding future metastases were significantly more expanded compared with non-metastatic subclones. Our findings will support (neo)adjuvant trial advances and provide insights into the process of metastatic dissemination using low-ctDNA-level liquid biopsy.

PulseNet International Survey on the Implementation of Whole Genome Sequencing in Low and Middle-Income Countries for Foodborne Disease Surveillance.

PulseNet International (PNI) is a global network of 88 countries who work together through their regional and national public health laboratories to track foodborne disease around the world. The vision of PNI is to implement globally standardized surveillance using whole genome sequencing (WGS) for real-time identification and subtyping of foodborne pathogens to strengthen preparedness and response and lower the burden of disease. Several countries in North America and Europe have experienced significant benefits in disease mitigation after implementing WGS. To broaden the routine use of WGS around the world, challenges and barriers must be overcome. We conducted this study to determine the challenges and barriers countries are encountering in their attempts to implement WGS and to identify how PNI can provide support to improve and become a better integrated system overall. A survey was designed with a set of qualitative questions to capture the status, challenges, barriers, and successes of countries in the implementation of WGS and was administered to laboratories in Africa, Asia-Pacific, Latin America and the Caribbean, and Middle East. One-third of respondents do not use WGS, and only 8% reported using WGS for routine, real-time surveillance. The main barriers for implementation of WGS were lack of funding, gaps in expertise, and training, especially for data analysis and interpretation. Features of an ideal system to facilitate implementation and global surveillance were identified as an all-in-one software that is free, accessible, standardized and validated. This survey highlights the minimal use of WGS for foodborne disease surveillance outside the United States, Canada, and Europe to date. Although funding remains a major barrier to WGS-based surveillance, critical gaps in expertise and availability of tools must be overcome. Opportunities to seek sustainable funding, provide training, and identify solutions for a globally standardized surveillance platform will accelerate implementation of WGS worldwide.

Azithromycin and Ciprofloxacin Treatment Outcomes During an Outbreak of Multidrug-Resistant Shigella sonnei Infections in a Retirement Community-Vermont, 2018.

BACKGROUND: In 2018, the Centers for Disease Control and Prevention and the Vermont Department of Health investigated an outbreak of multidrug-resistant Shigella sonnei infections in a retirement community that offered a continuum of care from independent living through skilled nursing care. The investigation identified 24 culture-confirmed cases. Isolates were resistant to trimethoprim-sulfamethoxazole, ampicillin, and ceftriaxone, and had decreased susceptibility to azithromycin and ciprofloxacin. METHODS: To evaluate clinical and microbiologic response, we reviewed inpatient and outpatient medical records for treatment outcomes among the 24 patients with culture-confirmed S. sonnei infection. We defined clinical failure as diarrhea (≥3 loose stools per day) for ≥1 day after treatment finished, and microbiologic failure as a stool culture that yielded S. sonnei after treatment finished. We used broth microdilution to perform antimicrobial susceptibility testing, and whole genome sequencing to identify resistance mechanisms. RESULTS: Isolates contained macrolide resistance genes mph(A) and erm(B) and had azithromycin minimum inhibitory concentrations above the Clinical and Laboratory Standards Institute epidemiological cutoff value of ≤16 µg/mL. Among 24 patients with culture-confirmed Shigella infection, 4 were treated with azithromycin; all had clinical treatment failure and 2 also had microbiologic treatment failure. Isolates were susceptible to ciprofloxacin but contained a gyrA mutation; 2 patients failed treatment with ciprofloxacin. CONCLUSIONS: These azithromycin treatment failures demonstrate the importance of clinical breakpoints to aid clinicians in identifying alternative treatment options for resistant strains. Additionally, these treatment failures highlight a need for comprehensive susceptibility testing and systematic outcome studies, particularly given the emergence of multidrug-resistant Shigella among an expanding range of patient populations.

Agritourism and Kidding Season: A Large Outbreak of Human Shiga Toxin-Producing Escherichia coli O157 (STEC O157) Infections Linked to a Goat Dairy Farm-Connecticut, 2016.

The objective of this study was to determine sources of Shiga toxin-producing Escherichia coli O157 (STEC O157) infection among visitors to Farm X and develop public health recommendations. A case-control study was conducted. Case-patients were defined as the first ill child (aged <18 years) in the household with laboratory-confirmed STEC O157, or physician-diagnosed hemolytic uremic syndrome with laboratory confirmation by serology, who visited Farm X in the 10 days prior to illness. Controls were selected from Farm X visitors aged <18 years, without symptoms during the same time period as case-patients. Environment and animal fecal samples collected from Farm X were cultured; isolates from Farm X were compared with patient isolates using whole genome sequencing (WGS). Case-patients were more likely than controls to have sat on hay bales at the doe barn (adjusted odds ratio: 4.55; 95% confidence interval: 1.41-16.13). No handwashing stations were available; limited hand sanitizer was provided. Overall, 37% (29 of 78) of animal and environmental samples collected were positive for STEC; of these, 62% (18 of 29) yielded STEC O157 highly related by WGS to patient isolates. STEC O157 environmental contamination and fecal shedding by goats at Farm X was extensive. Farms should provide handwashing stations with soap, running water, and disposable towels. Access to animal areas, including animal pens and enclosures, should be limited for young children who are at risk for severe outcomes from STEC O157 infection. National recommendations should be adopted to reduce disease transmission.

Shiga Toxin-Producing Escherichia coli Infections Associated With Romaine Lettuce-United States, 2018.

BACKGROUND: Produce-associated outbreaks of Shiga toxin-producing Escherichia coli (STEC) were first identified in 1991. In April 2018, New Jersey and Pennsylvania officials reported a cluster of STEC O157 infections associated with multiple locations of a restaurant chain. The Centers for Disease Control and Prevention (CDC) queried PulseNet, the national laboratory network for foodborne disease surveillance, for additional cases and began a national investigation. METHODS: A case was defined as an infection between 13 March and 22 August 2018 with 1 of the 22 identified outbreak-associated E. coli O157:H7 or E. coli O61 pulsed-field gel electrophoresis pattern combinations, or with a strain STEC O157 that was closely related to the main outbreak strain by whole-genome sequencing. We conducted epidemiologic and traceback investigations to identify illness subclusters and common sources. A US Food and Drug Administration-led environmental assessment, which tested water, soil, manure, compost, and scat samples, was conducted to evaluate potential sources of STEC contamination. RESULTS: We identified 240 case-patients from 37 states; 104 were hospitalized, 28 developed hemolytic uremic syndrome, and 5 died. Of 179 people who were interviewed, 152 (85%) reported consuming romaine lettuce in the week before illness onset. Twenty subclusters were identified. Product traceback from subcluster restaurants identified numerous romaine lettuce distributors and growers; all lettuce originated from the Yuma growing region. Water samples collected from an irrigation canal in the region yielded the outbreak strain of STEC O157. CONCLUSIONS: We report on the largest multistate leafy greens-linked STEC O157 outbreak in several decades. The investigation highlights the complexities associated with investigating outbreaks involving widespread environmental contamination.

An Overview of PulseNet USA Databases.

PulseNet USA is the molecular surveillance network for foodborne disease in the United States. The network consists of state and local public health laboratories, as well as food regulatory agencies, that follow PulseNet's standardized protocols to perform pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) and analyze the results using standardized software. The raw sequences are uploaded to the GenomeTrakr or PulseNet bioprojects at the National Center for Biotechnology Information. The PFGE patterns and analyzed sequence data are uploaded in real time with associated demographic data to the PulseNet national databases managed at the Centers for Disease Control and Prevention. The PulseNet databases are organism specific and provide a central storage location for molecular and demographic data related to an isolate. Sequences are compared in the databases, thereby facilitating the rapid detection of clusters of foodborne diseases that may represent widespread outbreaks. WGS genotyping data, for example, antibiotic resistance and virulence profiles, are also uploaded in real time to the PulseNet databases to improve food safety surveillance activities.

The H/ACA complex disrupts triplex in hTR precursor to permit processing by RRP6 and PARN.

Human telomerase RNA (hTR) is transcribed as a precursor that is then posttranscriptionally modified and processed. A fraction of the transcripts is oligoadenylated by TRAMP and either processed into the mature hTR or degraded by the exosome. Here, we characterize the processing of 3' extended forms of varying length by PARN and RRP6. We show that tertiary RNA interactions unique to the longer transcripts favor RNA degradation, whereas H/ACA RNP assembly stimulates productive processing. Interestingly, the H/ACA complex actively promotes processing in addition to protecting the mature 3' end. Processing occurs in two steps with longer forms first being trimmed by RRP6 and shorter forms then being processed by PARN. These results reveal how RNA structure and RNP assembly affect the kinetics of processing and degradation and ultimately determine the amount of functional telomerase produced in cells.

Outbreak of E. coli O157:H7 Infections Associated with Exposure to Animal Manure in a Rural Community - Arizona and Utah, June-July 2017.

On June 26, 2017, a hospital in southern Utah notified the Utah Department of Health of Shiga toxin-producing Escherichia coli (STEC) O157:H7 infections in two children from a small community on the Arizona-Utah border. Both children developed hemolytic uremic syndrome, characterized by hemolytic anemia, acute kidney failure, and thrombocytopenia and died within a few days of illness onset. Over the next few days, several more STEC-associated illnesses were reported in residents of the community. A joint investigation by local and state health agencies from Arizona and Utah and CDC was initiated to identify the outbreak source and prevent additional cases; a total of 12 cases were identified, including the two children who died. Investigators initially explored multiple potential sources of illness; epidemiologic and environmental information revealed cow manure contact as the likely initial cause of the outbreak, which was followed by subsequent person-to-person transmission. One of the outbreak strains was isolated from bull and horse manure collected from a yard near a community household with two ill children. Local health agencies made recommendations to the public related to both animal contact and hand hygiene to reduce the risk for STEC transmission. Animal or animal manure contact should be considered a potential source of STEC O157:H7 during outbreaks in communities where ruminants are kept near the home.

Pof8 is a La-related protein and a constitutive component of telomerase in fission yeast.

Telomerase reverse transcriptase (TERT) and the non-coding telomerase RNA subunit (TR) constitute the core of telomerase. Here we now report that the putative F-box protein Pof8 is also a constitutive component of active telomerase in fission yeast. Pof8 functions in a hierarchical assembly pathway by promoting the binding of the Lsm2-8 complex to telomerase RNA, which in turn promotes binding of the catalytic subunit. Loss of Pof8 reduces TER1 stability, causes a severe assembly defect, and results in critically short telomeres. Structure profile searches identified similarities between Pof8 and telomerase subunits from ciliated protozoa, making Pof8 next to TERT the most widely conserved telomerase subunits identified to date.

Shiga Toxin-Producing E. coli Infections Associated with Flour.

BACKGROUND: In 2016, a multijurisdictional team investigated an outbreak of Shiga toxin-producing Escherichia coli (STEC) serogroup O121 and O26 infections linked to contaminated flour from a large domestic producer. METHODS: A case was defined as infection with an outbreak strain in which illness onset was between December 21, 2015, and September 5, 2016. To identify exposures associated with the outbreak, outbreak cases were compared with non-STEC enteric illness cases, matched according to age group, sex, and state of residence. Products suspected to be related to the outbreak were collected for STEC testing, and a common point of contamination was sought. Whole-genome sequencing was performed on isolates from clinical and food samples. RESULTS: A total of 56 cases were identified in 24 states. Univariable exact conditional logistic-regression models of 22 matched sets showed that infection was significantly associated with the use of one brand of flour (odds ratio, 21.04; 95% confidence interval [CI], 4.69 to 94.37) and with tasting unbaked homemade dough or batter (odds ratio, 36.02; 95% CI, 4.63 to 280.17). Laboratory testing isolated the outbreak strains from flour samples, and whole-genome sequencing revealed that the isolates from clinical and food samples were closely related to one another genetically. Trace-back investigation identified a common flour-production facility. CONCLUSIONS: This investigation implicated raw flour as the source of an outbreak of STEC infections. Although it is a low-moisture food, raw flour can be a vehicle for foodborne pathogens.

Laboratory Investigation of Salmonella enterica serovar Poona Outbreak in California: Comparison of Pulsed-Field Gel Electrophoresis (PFGE) and Whole Genome Sequencing (WGS) Results.

INTRODUCTION: Recently, Salmonella enterica serovar Poona caused a multistate outbreak, with 245 out of 907 cases occurring in California. We report a comparison of pulsed-field gel electrophoresis (PFGE) results with whole genome sequencing (WGS) for genotyping of Salmonella Poona isolates. METHODS: CA Salmonella Poona isolates, collected from July to August 2015, were genotyped by PFGE using XbaI restriction enzyme. WGS was done using Nextera XT library kit with 2x300 bp or 2x250 bp sequencing chemistry on the Illumina MiSeq Sequencer.  Reads were mapped to the de novo assembled serovar Poona draft genome (48 contigs, N50= 223,917) from the outbreak using CLCbio GW 8.0.2. The phylogenetic tree was generated based on hqSNPs calling. Genomes were annotated with CGE and PHAST online tools. In silico MLST was performed using the CGE online tool. RESULTS: Human (14) and cucumber (2) Salmonella Poona isolates exhibited 3 possibly related PFGE patterns (JL6X01.0018 [predominant], JL6X01.0375, JL6X01.0778).  All isolates that were related by PFGE also clustered together according to the WGS. One isolate with a divergent PFGE pattern (JL6X01.0776) served as an outlier in the phylogenetic analysis and substantially differed from the outbreak clade by WGS. All outbreak isolates were assigned to MLST sequence type 447. The majority of the outbreak-related isolates possessed the same set of Salmonella Pathogenicity Islands with few variations. One outbreak isolate was sequenced and analyzed independently by CDC and CDPH laboratories; there was 0 SNP difference in results. Additional two isolates were sequenced by CDC and the raw data was processed through CDPH and CDC analysis pipelines. Both data analysis pipelines also generated concordant results.  Discussion: PFGE and WGS results for the recent CA Salmonella enterica serovar Poona outbreak provided concordant assignment of the isolates to the outbreak cluster. WGS allowed more robust determination of genetic relatedness, provided information regarding MLST-type, pathogenicity genes, and bacteriophage content. WGS data obtained independently at two laboratories showed complete agreement.

Signaled drug delivery and transport across the blood-brain barrier.

We use a mathematical model to describe the delivery of a drug to a specific region of the brain. The drug is carried by liposomes that can release their cargo by application of focused ultrasound (US). Thereupon, the drug is absorbed through the endothelial cells that line the brain capillaries and form the physiologically important blood-brain barrier (BBB). We present a compartmental model of a capillary that is able to capture the complex binding and transport processes the drug undergoes in the blood plasma and at the BBB. We apply this model to the delivery of levodopa (L-dopa, used to treat Parkinson's disease) and doxorubicin (an anticancer agent). The goal is to optimize the delivery of drug while at the same time minimizing possible side effects of the US.

Human Telomerase RNA Processing and Quality Control.

The non-coding RNA subunit of telomerase provides the template for telomerase activity. In diverse fungi, 3' end processing of telomerase RNA involves a single cleavage by the spliceosome. Here, we examine how human telomerase RNA (hTR) primary transcripts are processed into the mature form of precisely 451 nt. We find that the splicing inhibitor isoginkgetin mimics the effects of RNA exosome inhibition and causes accumulation of long hTR transcripts. Depletion of exosome components and accessory factors reveals functions for the cap binding complex (CBC) and the nuclear exosome targeting (NEXT) complex in hTR turnover. Whereas longer transcripts are predominantly degraded, shorter precursor RNAs are oligo-adenylated by TRF4-2 and either processed by poly(A)-specific ribonuclease (PARN) or degraded by the exosome. Our results reveal that hTR biogenesis involves a kinetic competition between RNA processing and degradation and suggest treatment options for telomerase insufficiency disorders.