Dana Marshall

Multidrug resistant Acinetobacter baumannii: a descriptive study in a city hospital

BackgroundMultidrug resistant Acinetobacter baumannii, (MRAB) is an important cause of hospital acquired infection. The purpose of this study is to determine the risk factors for MRAB in a city hospital patient population.MethodsThis study is a retrospective review of a city hospital epidemiology data base and includes 247 isolates of Acinetobacter baumannii (AB) from 164 patients. Multidrug resistant Acinetobacter baumannii was defined as resistance to more than three classes of antibiotics. Using the non-MRAB isolates as the control group, the risk factors for the acquisition of MRAB were determined.ResultsOf the 247 AB isolates 72% (177) were multidrug resistant. Fifty-eight percent (143/247) of isolates were highly resistant (resistant to imipenem, amikacin, and ampicillin-sulbactam). Of the 37 patients who died with Acinetobacter colonization/infection, 32 (86%) patients had the organism recovered from the respiratory tract. The factors which were found to be significantly associated (p ≤ 0.05) with multidrug resistance include the recovery of AB from multiple sites, mechanical ventilation, previous antibiotic exposure, and the presence of neurologic impairment. Multidrug resistant Acinetobacter was associated with significant mortality when compared with sensitive strains (p ≤ 0.01). When surgical patients (N = 75) were considered separately, mechanical ventilation and multiple isolates remained the factors significantly associated with the development of multidrug resistant Acinetobacter. Among surgical patients 46/75 (61%) grew a multidrug resistant strain of AB and 37/75 (40%) were resistant to all commonly used antibiotics including aminoglycosides, cephalosporins, carbepenems, extended spectrum penicillins, and quinolones. Thirty-five percent of the surgical patients had AB cultured from multiple sites and 57% of the Acinetobacter isolates were associated with a co-infecting organism, usually a Staphylococcus or Pseudomonas. As in medical patients, the isolation of Acinetobacter from multiple sites and the need for mechanical ventilation were significantly associated with the development of MRAB.ConclusionsThe factors significantly associated with MRAB in both the general patient population and surgical patients were mechanical ventilation and the recovery of Acinetobacter from multiple anatomic sites. Previous antibiotic use and neurologic impairment were significant factors in medical patients. Colonization or infection with MRAB is associated with increased mortality.

Bridging the gap between systems biology and medicine

Systems biology has matured considerably as a discipline over the last decade, yet some of the key challenges separating current research efforts in systems biology and clinically useful results are only now becoming apparent. As these gaps are better defined, the new discipline of systems medicine is emerging as a translational extension of systems biology. How is systems medicine defined? What are relevant ontologies for systems medicine? What are the key theoretic and methodologic challenges facing computational disease modeling? How are inaccurate and incomplete data, and uncertain biologic knowledge best synthesized in useful computational models? Does network analysis provide clinically useful insight? We discuss the outstanding difficulties in translating a rapidly growing body of data into knowledge usable at the bedside. Although core-specific challenges are best met by specialized groups, it appears fundamental that such efforts should be guided by a roadmap for systems medicine drafted by a coalition of scientists from the clinical, experimental, computational, and theoretic domains.

New Data Analysis and Mining Approaches Identify Unique Proteome and Transcriptome Markers of Susceptibility to Autoimmune Diabetes

Non-obese diabetic (NOD) mice spontaneously develop autoimmunity to the insulin producing beta cells leading to insulin-dependent diabetes. In this study we developed and used new data analysis and mining approaches on combined proteome and transcriptome (molecular phenotype) data to define pathways affected by abnormalities in peripheral leukocytes of young NOD female mice. Cells were collected before mice show signs of autoimmunity (age, 2–4 weeks). We extracted both protein and RNA from NOD and C57BL/6 control mice to conduct both proteome analysis by two-dimensional gel electrophoresis and transcriptome analysis on Affymetrix expression arrays. We developed a new approach to analyze the two-dimensional gel proteome data that included two-way analysis of variance, cluster analysis, and principal component analysis. Lists of differentially expressed proteins and transcripts were subjected to pathway analysis using a commercial service. From the list of 24 proteins differentially expressed between strains we identified two highly significant and interconnected networks centered around oncogenes (Myc and Mycn) and apoptosis-related genes (Bcl2 and Casp3). The 273 genes with significant strain differences in RNA expression levels created six interconnected networks with a significant over-representation of genes related to cancer, cell cycle, and cell death. They contained many of the same genes found in the proteome networks (including Myc and Mycn). The combination of the eight, highly significant networks created one large network of 272 genes of which 82 had differential expression between strains either at the protein or the RNA level. We conclude that new proteome data analysis strategies and combined information from proteome and transcriptome can enhance the insights gained from either type of data alone. The overall systems biology of prediabetic NOD mice points toward abnormalities in regulation of the opposing processes of cell renewal and cell death even before there are any clear signatures of immune system activation.

Fetuin-A associates with histones intracellularly and shuttles them to exosomes to promote focal adhesion assembly resulting in rapid adhesion and spreading in breast carcinoma cells

The present analyses were undertaken to define the mechanisms by which fetuin-A modulates cellular adhesion. FLAG-tagged fetuin-A was expressed in breast carcinoma and HEK-293T cells. We demonstrated by confocal microscopy that fetuin-A co-localizes with histone H2A in the cell nucleus, forms stable complexes with histones such as H2A and H3 in solution, and shuttles histones to exosomes. The rate of cellular adhesion and spreading to either fibronectin or laminin coated wells was accelerated significantly in the presence of either endogenous fetuin-A or serum derived protein. More importantly, the formation of focal adhesion complexes on surfaces coated by laminin or fibronectin was accelerated in the presence of fetuin-A or histone coated exosomes. Cellular adhesion mediated by histone coated exosomes was abrogated by heparin and heparinase III. Heparinase III cleaves heparan sulfate from cell surface heparan sulfate proteoglycans. Lastly, the uptake of histone coated exosomes and subsequent cellular adhesion, was abrogated by heparin. Taken together, the data suggest a mechanism where fetuin-A, either endogenously synthesized or supplied extracellularly can extract histones from the nucleus or elsewhere in the cytosol/membrane and load them on cellular exosomes which then mediate adhesion by interacting with cell surface heparan sulfate proteoglycans via bound histones.

Molecular Phenotyping of Immune Cells from Young NOD Mice Reveals Abnormal Metabolic Pathways in the Early Induction Phase of Autoimmune Diabetes

Islet leukocytic infiltration (insulitis) is first obvious at around 4 weeks of age in the NOD mouse – a model for human type 1 diabetes (T1D). The molecular events that lead to insulitis and initiate autoimmune diabetes are poorly understood. Since TID is caused by numerous genes, we hypothesized that multiple molecular pathways are altered and interact to initiate this disease. We evaluated the molecular phenotype (mRNA and protein expression) and molecular networks of ex vivo unfractionated spleen leukocytes from 2 and 4 week-old NOD mice in comparison to two control strains. Analysis of the global gene expression profiles and hierarchical clustering revealed that the majority (∼90%) of the differentially expressed genes in NOD mice were repressed. Furthermore, analysis using a modern suite of multiple bioinformatics approaches identified abnormal molecular pathways that can be divided broadly into 2 categories: metabolic pathways, which were predominant at 2 weeks, and immune response pathways, which were predominant at 4 weeks. Network analysis by Ingenuity pathway analysis identified key genes/molecules that may play a role in regulating these pathways. These included five that were common to both ages (TNF, HNF4A, IL15, Progesterone, and YWHAZ), and others that were unique to 2 weeks (e.g. MYC/MYCN, TGFB1, and IL2) and to 4 weeks (e.g. IFNG, beta-estradiol, p53, NFKB, AKT, PRKCA, IL12, and HLA-C). Based on the literature, genes that may play a role in regulating metabolic pathways at 2 weeks include Myc and HNF4A, and at 4 weeks, beta-estradiol, p53, Akt, HNF4A and AR. Our data suggest that abnormalities in regulation of metabolic pathways in the immune cells of young NOD mice lead to abnormalities in the immune response pathways and as such may play a role in the initiation of autoimmune diabetes. Thus, targeting metabolism may provide novel approaches to preventing and/or treating autoimmune diabetes.

Alpha-2 Heremans Schmid Glycoprotein (AHSG) Modulates Signaling Pathways in Head and Neck Squamous Cell Carcinoma Cell Line SQ20B

This study was performed to identify the potential role of Alpha-2 Heremans Schmid Glycoprotein (AHSG) in Head and Neck Squamous Cell Carcinoma (HNSCC) tumorigenesis using an HNSCC cell line model. HNSCC cell lines are unique among cancer cell lines, in that they produce endogenous AHSG and do not rely, solely, on AHSG derived from serum. To produce our model, we performed a stable transfection to down-regulate AHSG in the HNSCC cell line SQ20B, resulting in three SQ20B sublines, AH50 with 50% AHSG production, AH20 with 20% AHSG production and EV which is the empty vector control expressing wild-type levels of AHSG. Utilizing these sublines, we examined the effect of AHSG depletion on cellular adhesion, proliferation, migration and invasion in a serum-free environment. We demonstrated that sublines EV and AH50 adhered to plastic and laminin significantly faster than the AH20 cell line, supporting the previously reported role of exogenous AHSG in cell adhesion. As for proliferative potential, EV had the greatest amount of proliferation with AH50 proliferation significantly diminished. AH20 cells did not proliferate at all. Depletion of AHSG also diminished cellular migration and invasion. TGF-β was examined to determine whether levels of the TGF-β binding AHSG influenced the effect of TGF-β on cell signaling and proliferation. Whereas higher levels of AHSG blunted TGF-β influenced SMAD and ERK signaling, it did not clearly affect proliferation, suggesting that AHSG influences on adhesion, proliferation, invasion and migration are primarily due to its role in adhesion and cell spreading. The previously reported role of AHSG in potentiating metastasis via protecting MMP-9 from autolysis was also supported in this cell line based model system of endogenous AHSG production in HNSCC. Together, these data show that endogenously produced AHSG in an HNSCC cell line, promotes in vitro cellular properties identified as having a role in tumorigenesis.

QTL mapping arthritis traits in CXB mice.

B6 mice are of intermediate susceptibility to collagen-induced arthritis (CIA) while Balb/c mice are resistant, but are highly susceptible to proteoglycan-induced arthritis. Antigen presentation is H-2 directed but the disease that results is thought to be driven by regions outside of the MHC therefore, CXB mouse strains afford the opportunity to look at the influence of these regions on CIA. H2-b CXB strains are predicted to show variation in disease parameters relative to C57Bl/6, depending on which Balb/c chromosome regions is present. Nine of thirteen CXB strains are H-2b while one has a recombined H-2 region (CXB9). The four H-2d strains were crossed with C57Bl/6ByJ to generate F1 mice that could present collagen via the B6-contributed H-2b locus, while possibly identifying Balb/c loci that would have a dominant effect on disease progression. A number of disease and immunological parameters were collected and gene expression analysis was done on resting spleens. A range of incidence and severity of disease was seen and mice were assigned to susceptibility groups based on collected parameters. Preliminary QTL analysis has identified regions on chromosomes 13, 15 and 19 that correlate with susceptibility to CIA.

Genomic and proteomic characterization of multi-drug resistant Acinetobacter baumannii

Background Multidrug resistant Acinetobacter baumanii (MRAB) is an emerging pathogen that is an important cause of hospital acquired infection and has been shown to increase mortality and length of hospital stay. MRAB is the predominant multidrug resistant bacteria at Nashville General Hospital at Meharry Medical College (NGHM). The goal of this study is to determine the major genomic and proteomics patterns of MRAB at NGHM.

Next-gen sequencing of multi-drug resistant Acinetobacter baumannii to determine antibiotic resistance genotypes

Background Multi-drug resistant (MDR) Acinetobacter baumannii is an important cause of hospital acquired infection and often increases mortality and length of stay[1-3]. The mechanisms of resistance include: (1) antimicrobialinactivating enzymes such as -lactamases, (2) alteration of membrane porin channels, and (3) mutations that change cellular functions [4]. Accurate genotyping and correlation to antimicrobial susceptibility will help prevent and treat outbreaks of Acinetobacter. The genome of A. baumannii ranges from 3.2 Megabases (Mb) in the drug sensitive SDF strain up to 3.9 Mb in the MDR AYE strain. A surprisingly high proportion of baumannii ORFs, (15%-20%), are located in resistance islands or “alien islands” long stretches of DNA acquired from a foreign source. The MDR AYE strain has an 86Kb island containing 45-50 drug resistance genes located in an insertion hotspot [5]. Our study aims to sequence several A. baumannii isolates from Metro Nashville General (NGH) Hospital and conduct a strain-to-reference genomic characterization of clinical virulence factors.

Proceedings of the 16th Annual UT-KBRIN Bioinformatics Summit 2016: proceedings

Fig. 1 (abstract P1). RAST server annotations for A. baumanii clinical isolate. Genes associated with virulence are highly represented. P1 Proteogenomic characterization of a clinical isolate of Acinetobacter baumanii from a case of fulminant sepsis: What does the data mean clinically? L Leon Dent, Sammed N Mandape, Siddharth Pratap, Jianan Dong, Jamaine Davis, Jennifer A Gaddy, Kofi Amoah, Steve Damo, Dana R Marshall Department of Surgery, Meharry Medical College, Nashville, TN 37208, USA; Bioinformatics Core, Meharry Medical College, Nashville, TN 37208, USA; Department of Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN 37208, USA; Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA; Department of Life and Physical Sciences, Fisk University, Nashville, TN 37208, USA; Department of Pathology, Anatomy and Cell Biology, Meharry Medical College, Nashville, TN 37208, USA Correspondence: Dana R Marshall (dmarshall@mmc.edu) BMC Proceedings 2017, 11(Suppl 9):P1