Limited Metabolomic Overlap between Commensal Bacteria and Marine Sponge Holobionts Revealed by Large Scale Culturing and Mass Spectrometry-Based Metabolomics: An Undergraduate Laboratory Pedagogical Effort at Georgia Tech.

Sponges are the richest source of bioactive organic small molecules, referred to as natural products, in the marine environment. It is well established that laboratory culturing-resistant symbiotic bacteria residing within the eukaryotic sponge host matrix often synthesize the natural products that are detected in the sponge tissue extracts. However, the contributions of the culturing-amenable commensal bacteria that are also associated with the sponge host to the overall metabolome of the sponge holobiont are not well defined. In this study, we cultured a large library of bacteria from three marine sponges commonly found in the Florida Keys. Metabolomes of isolated bacterial strains and that of the sponge holobiont were compared using mass spectrometry to reveal minimal metabolomic overlap between commensal bacteria and the sponge hosts. We also find that the phylogenetic overlap between cultured commensal bacteria and that of the sponge microbiome is minimal. Despite these observations, the commensal bacteria were found to be a rich resource for novel natural product discovery. Mass spectrometry-based metabolomics provided structural insights into these cryptic natural products. Pedagogic innovation in the form of laboratory curricula development is described which provided undergraduate students with hands-on instruction in microbiology and natural product discovery using metabolomic data mining strategies.

Multistate Outbreak of Burkholderia cepacia Complex Bloodstream Infections After Exposure to Contaminated Saline Flush Syringes: United States, 2016-2017.

BACKGROUND: Burkholderia cepacia complex (Bcc) has caused healthcare-associated outbreaks, often in association with contaminated products. The identification of 4 Bcc bloodstream infections in patients residing at a single skilled nursing facility (SNF) within 1 week led to an epidemiological investigation to identify additional cases and the outbreak source. METHODS: A case was initially defined via a blood culture yielding Bcc in a SNF resident receiving intravenous therapy after 1 August 2016. Multistate notifications were issued to identify additional cases. Public health authorities performed site visits at facilities with cases to conduct chart reviews and identify possible sources. Pulsed-field gel electrophoresis (PFGE) was performed on isolates from cases and suspect products. Facilities involved in manufacturing suspect products were inspected to assess possible root causes. RESULTS: An outbreak of 162 Bcc bloodstream infections across 59 nursing facilities in 5 states occurred during September 2016-January 2017. Isolates from patients and pre-filled saline flush syringes were closely related by PFGE, identifying contaminated flushes as the outbreak source and prompting a nationwide recall. Inspections of facilities at the saline flush manufacturer identified deficiencies that might have led to the failure to sterilize a specific case containing a partial lot of the product. CONCLUSIONS: Communication and coordination among key stakeholders, including healthcare facilities, public health authorities, and state and federal agencies, led to the rapid identification of an outbreak source and likely prevented many additional infections. Effective processes to ensure the sterilization of injectable products are essential to prevent similar outbreaks in the future.

Complete Genome Sequence of Mycobacteriumchimaera Strain CDC2015-22-71.

Mycobacterium chimaera is a nontuberculous mycobacterium species commonly found in the environment. Here, we report the first complete genome sequence of a strain from the investigation of invasive infections following open-heart surgeries that used contaminated LivaNova Sorin Stockert 3T heater-cooler devices.

Invasive Nontuberculous Mycobacterial Infections among Cardiothoracic Surgical Patients Exposed to Heater-Cooler Devices1.

Invasive nontuberculous mycobacteria (NTM) infections may result from a previously unrecognized source of transmission, heater-cooler devices (HCDs) used during cardiac surgery. In July 2015, the Pennsylvania Department of Health notified the Centers for Disease Control and Prevention (CDC) about a cluster of NTM infections among cardiothoracic surgical patients at 1 hospital. We conducted a case-control study to identify exposures causing infection, examining 11 case-patients and 48 control-patients. Eight (73%) case-patients had a clinical specimen identified as Mycobacterium avium complex (MAC). HCD exposure was associated with increased odds of invasive NTM infection; laboratory testing identified patient isolates and HCD samples as closely related strains of M. chimaera, a MAC species. This investigation confirmed a large US outbreak of invasive MAC infections in a previously unaffected patient population and suggested transmission occurred by aerosolization from HCDs. Recommendations have been issued for enhanced surveillance to identify potential infections associated with HCDs and measures to mitigate transmission risk.

Investigation of First Identified mcr-1 Gene in an Isolate from a U.S. Patient - Pennsylvania, 2016.

In 2015, scientists reported the emergence of the plasmid-encoded mcr-1 gene conferring bacterial resistance to the antibiotic colistin (1), signaling potential emergence of a pandrug-resistant bacterium. In May 2016, mcr-1-positive Escherichia coli was first isolated from a specimen from a U.S. patient (2) when a Pennsylvania woman was evaluated for a urinary tract infection. The urine culture and subsequent testing identified the gene in an extended-spectrum beta-lactamase (ESBL)-producing E. coli with reduced susceptibility to colistin. The patient had no international travel for approximately 1 year, no livestock exposure, and a limited role in meal preparation with store-bought groceries; however, she had multiple and repeated admissions to four medical facilities during 2016.

Clinical Correlates of Surveillance Events Detected by National Healthcare Safety Network Pneumonia and Lower Respiratory Infection Definitions-Pennsylvania, 2011-2012.

OBJECTIVE To determine the clinical diagnoses associated with the National Healthcare Safety Network (NHSN) pneumonia (PNEU) or lower respiratory infection (LRI) surveillance events DESIGN Retrospective chart review SETTING A convenience sample of 8 acute-care hospitals in Pennsylvania PATIENTS All patients hospitalized during 2011-2012 METHODS Medical records were reviewed from a random sample of patients reported to the NHSN to have PNEU or LRI, excluding adults with ventilator-associated PNEU. Documented clinical diagnoses corresponding temporally to the PNEU and LRI events were recorded. RESULTS We reviewed 250 (30%) of 838 eligible PNEU and LRI events reported to the NHSN; 29 reported events (12%) fulfilled neither PNEU nor LRI case criteria. Differences interpreting radiology reports accounted for most misclassifications. Of 81 PNEU events in adults not on mechanical ventilation, 84% had clinician-diagnosed pneumonia; of these, 25% were attributed to aspiration. Of 43 adult LRI, 88% were in mechanically ventilated patients and 35% had no corresponding clinical diagnosis (infectious or noninfectious) documented at the time of LRI. Of 36 pediatric PNEU events, 72% were ventilator associated, and 70% corresponded to a clinical pneumonia diagnosis. Of 61 pediatric LRI patients, 84% were mechanically ventilated and 21% had no corresponding clinical diagnosis documented. CONCLUSIONS In adults not on mechanical ventilation and in children, most NHSN-defined PNEU events corresponded with compatible clinical conditions documented in the medical record. In contrast, NHSN LRI events often did not. As a result, substantial modifications to the LRI definitions were implemented in 2015. Infect Control Hosp Epidemiol 2016;37:818-824.

Use of nonpharmaceutical interventions to reduce transmission of 2009 pandemic influenza A (pH1N1) in Pennsylvania public schools.

BACKGROUND: School-based recommendations for nonpharmaceutical interventions (NPIs) were issued in response to the threat of 2009 pandemic influenza A (pH1N1). The implementation and effectiveness of these recommendations has not been assessed. METHODS: In November 2009, a Web-based survey of all Pennsylvania public schools was conducted to assess the use of recommended NPIs. RESULTS: Overall, 1040 (31%) of 3351 schools participated in the survey. By fall 2009, 820 (84%) of 979 respondents reported that their school had an influenza plan in place, a 44% higher proportion than in the spring 2009 (p < .01). Most schools communicated health messages (eg, staying home when sick), implemented return to school requirements, and made hand sanitizer available. Schools with a spring influenza plan (N = 568) were less likely to report substantial influenza-like illness (ILI) during the fall wave of influenza than the 299 schools without a spring influenza plan (63% vs 71%, p = .02). This association persisted after controlling for schools with substantial ILI in the spring. CONCLUSION: The reported use of NPIs in participating Pennsylvania public schools improved substantially from spring to fall and was generally consistent with issued recommendations. The proactive implementation of a number of NPIs and the early implementation of communication and education initiatives might have cumulatively reduced the impact of pH1N1 in some schools.

Outbreak of influenza A (H3N2) variant virus infection among attendees of an agricultural fair, Pennsylvania, USA, 2011.

During August 2011, influenza A (H3N2) variant [A(H3N2)v] virus infection developed in a child who attended an agricultural fair in Pennsylvania, USA; the virus resulted from reassortment of a swine influenza virus with influenza A(H1N1)pdm09. We interviewed fair attendees and conducted a retrospective cohort study among members of an agricultural club who attended the fair. Probable and confirmed cases of A(H3N2)v virus infection were defined by serology and genomic sequencing results, respectively. We identified 82 suspected, 4 probable, and 3 confirmed case-patients who attended the fair. Among 127 cohort study members, the risk for suspected case status increased as swine exposure increased from none (4%; referent) to visiting swine exhibits (8%; relative risk 2.1; 95% CI 0.2-53.4) to touching swine (16%; relative risk 4.4; 95% CI 0.8-116.3). Fairs may be venues for zoonotic transmission of viruses with epidemic potential; thus, health officials should investigate respiratory illness outbreaks associated with agricultural events.

Epidemiological and laboratory characterization of a yellow fever outbreak in northern Uganda, October 2010-January 2011.

BACKGROUND: In November 2010, following reports of an outbreak of a fatal, febrile, hemorrhagic illness in northern Uganda, the Uganda Ministry of Health established multisector teams to respond to the outbreak. METHODS: This was a case-series investigation in which the response teams conducted epidemiological and laboratory investigations on suspect cases. The cases identified were line-listed and a data analysis was undertaken regularly to guide the outbreak response. RESULTS: Overall, 181 cases met the yellow fever (YF) suspected case definition; there were 45 deaths (case fatality rate 24.9%). Only 13 (7.5%) of the suspected YF cases were laboratory confirmed, and molecular sequencing revealed 92% homology to the YF virus strain Couma (Ethiopia), East African genotype. Suspected YF cases had fever (100%) and unexplained bleeding (97.8%), but jaundice was rare (11.6%). The overall attack rate was 13 cases/100000 population, and the attack rate was higher for males than females and increased with age. The index clusters were linked to economic activities undertaken by males around forests. CONCLUSIONS: This was the largest YF outbreak ever reported in Uganda. The wide geographical case dispersion as well as the male and older age preponderance suggests transmission during the outbreak was largely sylvatic and related to occupational activities around forests.

Response to 2009 pandemic influenza A H1N1 among public schools of Georgia, United States--fall 2009.

BACKGROUND: Little is known about the extent of implementation or the effectiveness of the Centers for Disease Control and Prevention's (CDC) recommended non-pharmaceutical interventions (NPIs) in schools to control the spread of 2009 pandemic influenza A H1N1 (pH1N1). METHODS: A web-based, cross-sectional survey of all public K-12 schools in Georgia, USA was conducted about preparedness and response to pH1N1, and absenteeism and respiratory illness. Schools that reported ≥10% absenteeism and at least two times the normal level of respiratory illness in the same week were designated as having experienced significant respiratory illness and absenteeism (SRIA) during that week. RESULTS: Of 2248 schools surveyed, 704 (31.3%) provided sufficient data to include in our analysis. Participating schools were spread throughout Georgia, USA and were similar to non-participating schools. Of 704 schools, 160 (22.7%) reported at least 1 week of SRIA. Most schools reported implementing the CDC recommendations for the control of pH1N1, and only two schools reported canceling or postponing activities. Schools that communicated with parents about influenza in the summer, had shorter school days, and were located in urban areas were less likely to experience SRIA. CONCLUSIONS: Most Georgia schools in the United States adopted the CDC recommendations for pH1N1 mitigation and few disruptions of school activities were reported. Early and timely communication with parents, as well as shorter school days, may have been effective in limiting the effect of pH1N1 on schools.

Enabled (Xena) regulates neural plate morphogenesis, apical constriction, and cellular adhesion required for neural tube closure in Xenopus.

Regulation of cellular adhesion and cytoskeletal dynamics is essential for neurulation, though it remains unclear how these two processes are coordinated. Members of the Ena/VASP family of proteins are localized to sites of cellular adhesion and actin dynamics and lack of two family members, Mena and VASP, in mice results in failure of neural tube closure. The precise mechanism by which Ena/VASP proteins regulate this process, however, is not understood. In this report, we show that Xenopus Ena (Xena) is localized to apical adhesive junctions of neuroepithelial cells during neurulation and that Xena knockdown disrupts cell behaviors integral to neural tube closure. Changes in the shape of the neural plate as well as apical constriction within the neural plate are perturbed in Xena knockdown embryos. Additionally, we demonstrate that Xena is essential for cell-cell adhesion. These results demonstrate that Xena plays an integral role in coordinating the regulation of cytoskeletal dynamics and cellular adhesion during neurulation in Xenopus.

Integrin alpha5 is required for somite rotation and boundary formation in Xenopus.

The morphogenesis of somites in Xenopus laevis is characterized by a complex process of cell turning that requires coordinated regulation of cell shape, adhesion, and motility. The integrin alpha5 subunit has been implicated in the formation of somite boundaries in organisms utilizing epithelialization to create morphologically distinct somites, but its function has not been examined in Xenopus. We used a splice-blocking morpholino to knock down expression of integrin alpha5 during somite formation. Loss of integrin alpha5 delayed somite turning and accumulation of integrin beta1 at somite boundaries, and disrupted the fibronectin matrix surrounding developing somites. Irregular somite boundaries with a sparse and discontinuous fibronectin matrix formed upon eventual completion of somite turning. Recovery of somite morphology was improved, but still incomplete in far posterior somites. These data demonstrate that the role of integrin alpha5 in somite boundary formation is conserved in a species using a unique mechanism of somitogenesis.

Regulation of otic vesicle and hair cell stereocilia morphogenesis by Ena/VASP-like (Evl) in Xenopus.

The inner ear is derived from a thickening in the embryonic ectoderm, called the otic placode. This structure undergoes extensive morphogenetic movements throughout its development and gives rise to all components of the inner ear. Ena/VASP-like (Evl) is an actin binding protein involved in the regulation of cytoskeletal dynamics and organization. We have examined the role of Evl during the morphogenesis of the Xenopus inner ear. Evl (hereafter referred to as Xevl) is expressed throughout otic vesicle formation and is enriched in the neuroblasts that delaminate to form the vestibulocochlear ganglion and in hair cells that possess mechanosensory stereocilia. Knockdown of Xevl perturbs epithelial morphology and intercellular adhesion in the otic vesicle and disrupts formation of the vestibulocochlear ganglion, evidenced by reduction of ganglion size, disorganization of the ganglion, and defects in neurite outgrowth. Later in embryogenesis, Xevl is required for development of mechanosensory hair cells. In Xevl knockdown embryos, hair cells of the ventromedial sensory epithelium display multiple abnormalities including disruption of the cuticular plate at the base of stereocilia and disorganization of the normal staircase appearance of stereocilia. Based on these data, we propose that Xevl plays an integral role in regulating morphogenesis of the inner ear epithelium and the subsequent development of the vestibulocochlear ganglion and mechanosensory hair cells.

Regulation of somitogenesis by Ena/VASP proteins and FAK during Xenopus development.

The metameric organization of the vertebrate body plan is established during somitogenesis as somite pairs sequentially form along the anteroposterior axis. Coordinated regulation of cell shape, motility and adhesion are crucial for directing the morphological segmentation of somites. We show that members of the Ena/VASP family of actin regulatory proteins are required for somitogenesis in Xenopus. Xenopus Ena (Xena) localizes to the cell periphery in the presomitic mesoderm (PSM), and is enriched at intersomitic junctions and at myotendinous junctions in somites and the myotome, where it co-localizes with beta1-integrin, vinculin and FAK. Inhibition of Ena/VASP function with dominant-negative mutants results in abnormal somite formation that correlates with later defects in intermyotomal junctions. Neutralization of Ena/VASP activity disrupts cell rearrangements during somite rotation and leads to defects in the fibronectin (FN) matrix surrounding somites. Furthermore, inhibition of Ena/VASP function impairs FN matrix assembly, spreading of somitic cells on FN and autophosphorylation of FAK, suggesting a role for Ena/VASP proteins in the modulation of integrin-mediated processes. We also show that inhibition of FAK results in defects in somite formation, blocks FN matrix deposition and alters Xena localization. Together, these results provide evidence that Ena/VASP proteins and FAK are required for somite formation in Xenopus and support the idea that Ena/VASP and FAK function in a common pathway to regulate integrin-dependent migration and adhesion during somitogenesis.

Regulation of actin cytoskeleton architecture by Eps8 and Abi1.

BACKGROUND: The actin cytoskeleton participates in many fundamental processes including the regulation of cell shape, motility, and adhesion. The remodeling of the actin cytoskeleton is dependent on actin binding proteins, which organize actin filaments into specific structures that allow them to perform various specialized functions. The Eps8 family of proteins is implicated in the regulation of actin cytoskeleton remodeling during cell migration, yet the precise mechanism by which Eps8 regulates actin organization and remodeling remains elusive. RESULTS: Here, we show that Eps8 promotes the assembly of actin rich filopodia-like structures and actin cables in cultured mammalian cells and Xenopus embryos, respectively. The morphology of actin structures induced by Eps8 was modulated by interactions with Abi1, which stimulated formation of actin cables in cultured cells and star-like structures in Xenopus. The actin stars observed in Xenopus animal cap cells assembled at the apical surface of epithelial cells in a Rac-independent manner and their formation was accompanied by recruitment of N-WASP, suggesting that the Eps8/Abi1 complex is capable of regulating the localization and/or activity of actin nucleators. We also found that Eps8 recruits Dishevelled to the plasma membrane and actin filaments suggesting that Eps8 might participate in non-canonical Wnt/Polarity signaling. Consistent with this idea, mis-expression of Eps8 in dorsal regions of Xenopus embryos resulted in gastrulation defects. CONCLUSION: Together, these results suggest that Eps8 plays multiple roles in modulating actin filament organization, possibly through its interaction with distinct sets of actin regulatory complexes. Furthermore, the finding that Eps8 interacts with Dsh and induced gastrulation defects provides evidence that Eps8 might participate in non-canonical Wnt signaling to control cell movements during vertebrate development.

Non-traditional roles for the Adenomatous Polyposis Coli (APC) tumor suppressor protein.

The Adenomatous Polyposis Coli (APC) tumor suppressor is a multifunctional protein that is mutated in a majority of colon cancers. The role of APC as an antagonist of the Wnt signaling pathway is well known and it is widely accepted that inappropriate activation of this pathway through loss of APC function contributes to the progression of colon cancers. However, a body of evidence is growing to support the idea that APC plays non-traditional functions outside of the Wnt pathway with roles in cell migration, adhesion, chromosome segregation, spindle assembly, apoptosis, and neuronal differentiation. This review highlights the research into alternate functions for APC beyond its role in Wnt signaling and discusses the possible contributions for these non-traditional functions of APC in tumor formation.

Income inequality and risk of suicide in New York City neighborhoods: a multilevel case-control study.

Evidence on the relationship between income inequality and suicide is inconsistent. Data from the New York City Office of the Chief Medical Examiner for all fatal injuries was collected to conduct a multilevel case-control study. In multilevel models, suicide decedents (n=374) were more likely than accident controls (n=453) to reside in neighborhoods with greater income inequality even after controlling for individual characteristics; this relation was modified by age with an effect overall and among decedents aged 15-34 but not among decedents 35-64. These data suggest that income inequality may contribute to the risk of suicide in younger adults.

Cloning and developmental expression of Xenopus Enabled (Xena).

Regulation of actin dynamics, organization, and interaction with cell surface adhesion proteins is critical for tissue morphogenesis during development. The Ena/VASP family of actin-binding proteins function in several cellular processes that involve dynamic regulation of the actin cytoskeleton, including axon guidance, platelet aggregation, cell migration, and cell adhesion. The vertebrate Ena/VASP family is composed of three genes: Ena (Enabled), VASP (Vasodilator Stimulated Phosphoprotein), and Evl (Ena/VASP-Like). To better understand the role of Ena/VASP proteins during vertebrate development, we have cloned and characterized the developmental expression of Ena in Xenopus laevis. Analysis of the temporal expression of Xenopus Ena (Xena) demonstrates that multiple isoforms of Xena are detected throughout embryogenesis and that the presence of different isoforms is developmentally regulated. In situ hybridization analyses reveal that Xena is broadly expressed throughout development. During gastrulation and neurulation, Xena is detected in the neuroepithelium, notochord, and somites. In tadpoles, Xena expression is restricted to dorsal regions of the brain, whereas it is expressed at lower levels throughout the spinal cord. Xena expression is also detected in the notochord, myotome, heart, pronephros, and cranial placodes, including the olfactory and otic placodes. Analysis of the subcellular localization of Xena using a GFP fusion protein revealed that Xena localizes to adherens junctions and focal adhesions in Xenopus animal caps and NIH3T3 fibroblasts, respectively. These results define spatiotemporal windows in which Xena may function during early Xenopus development to modulate actin-dependent processes such as cell adhesion and migration.

Molecular cloning and expression of Ena/Vasp-like (Evl) during Xenopus development.

Ena/VASP proteins are actin-binding proteins implicated in the regulation of axon guidance, platelet aggregation, cell motility, and cell adhesion. The vertebrate Ena/VASP family is comprised of three genes: Ena (Enabled), VASP (Vasodilator Stimulated Phosphoprotein), and Evl (Ena/VASP-Like). We have cloned and characterized cDNAs encoding three alternatively spliced isoforms of Xenopus laevis Evl, designated Xevl, Xevl-I and Xevl-H. Analysis of the temporal expression of Xevl, Xevl-I and Xevl-H demonstrates that transcripts for each isoform are first detectable at low levels at stage 18, show increased abundance by stage 23, and persist throughout the remainder of embryogenesis. In situ hybridization analyses using a probe that detects all three Xevl isoforms or a probe that specifically detects the Xevl-H isoform revealed expression in the cement gland, brain, neural tube, myotome, and neural placodes, including the otic, lateral line, and olfactory placodes. These results suggest roles for Xevl in regulating actin dynamics and cell adhesion in neural and mesodermal tissues during later stages of Xenopus development.

Dishevelled activates Ca2+ flux, PKC, and CamKII in vertebrate embryos.

Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.