Multidrug-Resistant Campylobacter jejuni Outbreak Linked to Puppy Exposure - United States, 2016-2018.

Campylobacter causes an estimated 1.3 million diarrheal illnesses in the United States annually (1). In August 2017, the Florida Department of Health notified CDC of six Campylobacter jejuni infections linked to company A, a national pet store chain based in Ohio. CDC examined whole-genome sequencing (WGS) data and identified six isolates from company A puppies in Florida that were highly related to an isolate from a company A customer in Ohio. This information prompted a multistate investigation by local and state health and agriculture departments and CDC to identify the outbreak source and prevent additional illness. Health officials from six states visited pet stores to collect puppy fecal samples, antibiotic records, and traceback information. Nationally, 118 persons, including 29 pet store employees, in 18 states were identified with illness onset during January 5, 2016-February 4, 2018. In total, six pet store companies were linked to the outbreak. Outbreak isolates were resistant by antibiotic susceptibility testing to all antibiotics commonly used to treat Campylobacter infections, including macrolides and quinolones. Store record reviews revealed that among 149 investigated puppies, 142 (95%) received one or more courses of antibiotics, raising concern that antibiotic use might have led to development of resistance. Public health authorities issued infection prevention recommendations to affected pet stores and recommendations for testing puppies to veterinarians. This outbreak demonstrates that puppies can be a source of multidrug-resistant Campylobacter infections in humans, warranting a closer look at antimicrobial use in the commercial dog industry.

Heavy Rainfall, Sewer Overflows, and Salmonellosis in Black Skimmers (Rynchops niger).

Extreme weather events, particularly heavy rainfall, are occurring at greater frequency with climate change. Although adverse human health effects from heavy rainfall are often publicized, impacts to free-ranging wildlife populations are less well known. We first summarize documented associations of heavy rainfall on wildlife health. We then report a novel investigation of a salmonellosis outbreak in a colony of black skimmers (Rynchops niger) in Florida, USA. During June-September 2016, heavy rainfall resulted in the discharge of millions of gallons of untreated wastewater into the Tampa Bay system, contaminating the water body, where adult skimmers foraged. At least 48 fledglings died, comprising 39% of the colony's nesting season's offspring. Of eight examined deceased birds from the colony, six had a systemic salmonellosis infection. Isolates were identified as Salmonella enterica serotype Typhimurium. Their pulsed-field gel electrophoresis patterns were identical to each other and matched those from several human Salmonella sp. infections. Differences among whole-genome sequences were negligible. These findings and the outbreak's epidemic curve suggest propagated transmission occurred within the colony. A multidisciplinary and One Health approach is recommended to mitigate any adverse effects of climate change-driven stochastic events, especially when they place already imperiled wildlife at further risk.

Early Emergence Phase of SARS-CoV-2 Delta Variant in Florida, US.

SARS-CoV-2, the causative agent of COVID-19, emerged in late 2019. The highly contagious B.1.617.2 (Delta) variant of concern (VOC) was first identified in October 2020 in India and subsequently disseminated worldwide, later becoming the dominant lineage in the US. Understanding the local transmission dynamics of early SARS-CoV-2 introductions may inform actionable mitigation efforts during subsequent pandemic waves. Yet, despite considerable genomic analysis of SARS-CoV-2 in the US, several gaps remain. Here, we explore the early emergence of the Delta variant in Florida, US using phylogenetic analysis of representative Florida and globally sampled genomes. We find multiple independent introductions into Florida primarily from North America and Europe, with a minority originating from Asia. These introductions led to three distinct clades that demonstrated varying relative rates of transmission and possessed five distinct substitutions that were 3-21 times more prevalent in the Florida sample as compared to the global sample. Our results underscore the benefits of routine viral genomic surveillance to monitor epidemic spread and support the need for more comprehensive genomic epidemiology studies of emerging variants. In addition, we provide a model of epidemic spread of newly emerging VOCs that can inform future public health responses.

Molecular Epidemiology of Salmonellosis in Florida, USA, 2017-2018.

The state of Florida reports a high burden of non-typhoidal Salmonella enterica with approximately two times higher than the national incidence. We retrospectively analyzed the population structure and molecular epidemiology of 1,709 clinical isolates from 2017 and 2018. We found 115 different serotypes. Rarefaction suggested that the serotype richness did not differ between children under 2 years of age and older children and adults and, there are ~22 well-characterized dominant serotypes. There were distinct differences in dominant serotypes between Florida and the USA as a whole, even though S. Enteritidis and S. Newport were the dominant serotypes in Florida and nationally. S. Javiana, S. Sandiego, and S. IV 50:z4, z23:- occurred more frequently in Florida than nationally. Legacy Multi Locus Sequence Typing (MLST) was of limited use for differentiating clinical Salmonella isolates beyond the serotype level. We utilized core genome MLST (cgMLST) hierarchical clusters (HC) to identify potential outbreaks and compared them to outbreaks detected by Pulse Field Gel Electrophoresis (PFGE) surveillance for five dominant serotypes (Enteritidis, Newport, Javiana, Typhimurium, and Bareilly). Single nucleotide polymorphism (SNP) phylogenetic-analysis of cgMLST HC at allelic distance 5 or less (HC5) corroborated PFGE detected outbreaks and generated well-segregated SNP distance-based clades for all studied serotypes. We propose "combination approach" comprising "HC5 clustering," as efficient tool to trigger Salmonella outbreak investigations, and "SNP-based analysis," for higher resolution phylogeny to confirm an outbreak. We also applied this approach to identify case clusters, more distant in time and place than traditional outbreaks but may have been infected from a common source, comparing 176 Florida clinical isolates and 1,341 non-clinical isolates across USA, of most prevalent serotype Enteritidis collected during 2017-2018. Several clusters of closely related isolates (0-4 SNP apart) within HC5 clusters were detected and some included isolates from poultry from different states in the US, spanning time periods over 1 year. Two SNP-clusters within the same HC5 cluster included isolates with the same multidrug-resistant profile from both humans and poultry, supporting the epidemiological link. These clusters likely reflect the vertical transmission of Salmonella clones from higher levels in the breeding pyramid to production flocks.

Spatial Epidemiology of Salmonellosis in Florida, 2009-2018.

Non-typhoidal Salmonella enterica infections cause a high disease burden in the United States with an estimated 1.2 million illnesses annually. The state of Florida consistently has a relatively high incidence compared to other states in the United States. Nevertheless, studies regarding the epidemiology of nontyphoidal salmonellosis and its spatial and temporal patterns in Florida were rarely reported. We examined the spatial and temporal patterns of 62,947 salmonellosis cases reported to FL Health Charts between 2009 and 2018. Dominant serotypes circulating in Florida were also explored using whole genome sequencing (WGS) based serotype-prediction for 2,507 Salmonella isolates sequenced by the Florida Department of Health during 2017 and 2018. The representativeness of laboratory-sequenced isolates for reported cases was determined by regression modeling. The annual incidence rate of salmonellosis decreased from 36.0 per 100,000 population in 2009 to 27.8 per 100,000 in 2016, and gradually increased in 2017 and 2018. Increased use of culture-independent testing did not fully explain this increase. The highest incidence rate was observed in children, contributing 40.9% of total reported cases during this period. A seasonal pattern was observed with the incidence peaking in September and October, later than the national average pattern. Over these 10 years, the Northeast and Northwest regions of the state had higher reported incidence rates, while reported rates in the Southeast and South were gradually increasing over time. Serotypes were predicted based on WGS data in the EnteroBase platform. The top-five most prevalent serotypes in Florida during 2017-2018 were Enteritidis, Newport, Javiana, Sandiego and Braenderup. The highest percentage of isolates was from children under 5 years of age (41.4%), and stool (84.7%) was the major source of samples. A zero-inflated negative binomial regression model showed that the reported case number was a strong predictor for the number of lab-sequenced isolates in individual counties, and the geospatial distribution of sequenced isolates was not biased by other factors such as age group. The spatial and temporal patterns identified in this study along with the prevalence of different serotypes will be helpful for the development of efficient prevention and control strategies for salmonellosis in Florida.

Aureimonas altamirensis masquerading as Brucella spp. in a blood culture from a 68-year-old male with cancer.

Staphylococcus epidermidis and a Gram negative bacillus (GNB) were isolated in blood cultures from a 68-year-old male with cancer. The GNB was suspicious for Brucella spp., but was identified using 16S rDNA sequencing as Aureimonas altamirensis. The complexity of the identification is described in this case study.

Employing Molecular Phylodynamic Methods to Identify and Forecast HIV Transmission Clusters in Public Health Settings: A Qualitative Study.

Molecular HIV surveillance is a promising public health strategy for curbing the HIV epidemic. Clustering technologies used by health departments to date are limited in their ability to infer/forecast cluster growth trajectories. Resolution of the spatiotemporal dynamics of clusters, through phylodynamic and phylogeographic modelling, is one potential strategy to develop a forecasting tool; however, the projected utility of this approach needs assessment. Prior to incorporating novel phylodynamic-based molecular surveillance tools, we sought to identify possible issues related to their feasibility, acceptability, interpretation, and utility. Qualitative data were collected via focus groups among field experts (n = 17, 52.9% female) using semi-structured, open-ended questions. Data were coded using an iterative process, first through the development of provisional themes and subthemes, followed by independent line-by-line coding by two coders. Most participants routinely used molecular methods for HIV surveillance. All agreed that linking molecular sequences to epidemiological data is important for improving HIV surveillance. We found that, in addition to methodological challenges, a variety of implementation barriers are expected in relation to the uptake of phylodynamic methods for HIV surveillance. The participants identified several opportunities to enhance current methods, as well as increase the usability and utility of promising works-in-progress.

Characterization of the grappa gene, the Drosophila histone H3 lysine 79 methyltransferase.

We have identified a novel gene named grappa (gpp) that is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development.

Mechanism of chromosomal boundary action: roadblock, sink, or loop?

Boundary elements or insulators subdivide eukaryotic chromosomes into a series of structurally and functionally autonomous domains. They ensure that the action of enhancers and silencers is restricted to the domain in which these regulatory elements reside. Three models, the roadblock, sink/decoy, and topological loop, have been proposed to explain the insulating activity of boundary elements. Strong predictions about how boundaries will function in different experimental contexts can be drawn from these models. In the studies reported here, we have designed assays that test these predictions. The results of our assays are inconsistent with the expectations of the roadblock and sink models. Instead, they support the topological loop model.

Protein:protein interactions and the pairing of boundary elements in vivo.

Although it is now well-established that boundary elements/insulators function to subdivide eukaryotic chromosomes into autonomous regulatory domains, the underlying mechanisms remain elusive. One idea is that boundaries act as barriers, preventing the processive spreading of "active" or "silenced" chromatin between domains. Another is that the partitioning into autonomous functional units is a consequence of an underlying structural subdivision of the chromosome into higher order "looped" domains. In this view, boundaries are thought to delimit structural domains by interacting with each other or with some other nuclear structure. The studies reported here provide support for the looped domain model. We show that the Drosophila scs and scs' boundary proteins, Zw5 and BEAF, respectively, interact with each other in vitro and in vivo. Moreover, consistent with idea that this protein:protein interaction might facilitate pairing of boundary elements, we find that that scs and scs' are in close proximity to each other in Drosophila nuclei.

Global changes in the expression patterns of RNA isolated from the hippocampus and cortex of VX exposed mice.

One of the established activities of the nerve agent VX is inhibition of the enzyme acetylcholinesterase (AChE). This inhibition affects the cholinergic nervous system by decreasing the activity of the neurotransmitter-hydrolyzing enzyme cholinesterase (ChE). In an effort to gain a more comprehensive understanding of the molecular pathways affected by low-level exposure to VX, an expression profiling approach was used to identify genes with altered RNA expression patterns after exposure.Specifically, mice were exposed to 0.1, 0.2, 0.4, and 0.6 LD50 VX for a period of 2 weeks. At 2 h, 72 h, and 2 weeks after the final exposure, RNA was isolated from both the hippocampus and the cortex. Changes in gene expression levels were assessed by DNA microarray technology and grouped according to their expression patterns. Data presented here demonstrate that 2 weeks postexposure all up-regulated gene expression has returned to pre-exposure levels, including genes related to the central nervous system. Additionally, this investigation has revealed non-AChE pathway genes involved in other neuronal functions that display altered expression profiles after VX exposure.