Microbiology Commons^™

High-Temperature Fluorescent In Situ Hybridization For Detecting Escherichia Coli In Seawater Samples, Using Rrna-Targeted Oligonucleotide Probes And Flow Cytometry, Ying Zhong Tang, Karina Yew Hoong Gin, Tok Hoon Lim

OES Faculty Publications

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E. coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with …

Ridding Ships' Ballast Water Of Microorganisms, Fred C. Dobbs, Andrew Rogerson

OES Faculty Publications

( First paragraph) A complex assemblage of microorganisms exists in nearly every aquatic system on earth. In lakes and oceans, every milliliter of water contains about 10² protists (single-celled eukaryotes), 10⁶ bacteria, and 10⁷–10⁹ viruses. Therefore, billions of microorganisms inevitably enter ships’ ballast tanks during normal operations. It has been argued that microorganisms must certainly be frequent invaders of coastal ecosystems, given the high densities of bacteria and viruses in ballast water—10⁸ and 10⁹ organisms per liter, respectively (1)—their potentially high reproductive rates, broad tolerances to physical conditions, and ability to form resting …

Transport Of The Harmful Bloom Alga Aureococcus Anophagefferens By Oceangoing Ships And Coastal Boats, Martina A. Doblin, Linda C. Popels, Kathryn J. Coyne, David A. Hutchins, S. Craig Cary, Fred C. Dobbs

OES Faculty Publications

It is well established that cyst-forming phytoplankton species are transported in ships' ballast tanks. However, there is increasing evidence that other phytoplankton species which do not encyst are also capable of surviving ballast transit. These species have alternative modes of nutrition (hetero- or mixotrophy) and/or are able to survive long-term darkness. In our studies of no-ballast-on-board vessels arriving in the Great Lakes, we tested for the presence of the harmful algal bloom species Aureococcus anophagefferens (brown tide) in residual (i.e., unpumpable) ballast water using methods based on the PCR. During 2001, the brown tide organism was detected in 7 of …

Ultrastructure Of Mycobacterium Marinum Granuloma In Striped Bass Morone Saxatilis, D. T. Gauthier, W. K. Vogelbein, C. A. Ottinger

Biological Sciences Faculty Publications

An emerging epizootic of mycobacteriosis currently threatens striped bass Morone saxatilis populations in Chesapeake Bay, USA. Several species of mycobacteria, including Mycobacterium marinum, species resembling M. avium, M. gordonae, M. peregrinum, M. scrofulaceum and M. terrae, and the new species M. shottsii have been isolated from diseased and healthy bass. In this study, we describe the ultrastructure of developing M. marinum granulomas in experimentally infected bass over a period of 45 wk. The primary host response to injected mycobacteria was formation of large macrophage aggregations containing phagocytosed bacilli. M. marinum were always contained within phagosomes. Close …

Virulence Factors Of The Group A Streptococci And Genes That Regulate Their Expression, Wayne Hines

Biological Sciences Faculty Publications

Group A streptococci produce a wide variety of extracellular (cell-associated and released) virulence factors. The function of these factors varies and includes roles in adhesion, spreading, tissue destruction, immune system evasion, and cell toxicity. How these factors are regulated with regard to one another is important for this organism's ability to bring about the variety of diseases this microbe is capable of causing. Therefore, along with the multitude of virulence factors, there are a number of regulatory systems that regulate, either directly or indirectly, the production of these factors and therefore influence the pathogenesis of group A streptococcal infections.

Dissecting The Loci Of Low-Level Quinine Resistance In Malaria Parasites, Michael T. Ferdig, Roland A. Cooper, Jianbing Mu, Bingbing Deng, Deirdre A. Joy, Xin-Zhuan Su, Thomas E. Wellems

Biological Sciences Faculty Publications

Quinine (QN) remains effective against Plasmodium falciparum, but its decreasing efficacy is documented from different continents. Multiple genes are likely to contribute to the evolution of QN resistance. To locate genes contributing to QN response variation, we have searched a P. falciparum genetic cross for quantitative trait loci (QTL). Results identify additive QTL in segments of chromosomes (Chrs) 13, 7 and 5, and pairwise effects from two additional loci of Chrs 9 and 6 that interact, respectively, with the QTL of Chrs 13 and 7. The mapped segments of Chrs 7 and 5 contain pfcrt, the determinant of …

Identification And Characterization Of A Nontypeable Haemophilus Influenzae Putative Toxin-Antitoxin Locus, Dayle A. Daines, Justin Jarisch, Arnold L. Smith

Biological Sciences Faculty Publications

Background: Certain strains of an obligate parasite of the human upper respiratory tract, nontypeable Haemophilus influenzae (NTHi), can cause invasive diseases such as septicemia and meningitis, as well as chronic mucosal infections such as otitis media. To do this, the organism must invade and survive within both epithelial and endothelial cells. We have identified a facilitator of NT(Hi) survival inside human cells, virulence-associated protein D (vapD_Hi, encoded by gene H10450). Both vapD_Hi and a flanking gene, H10451, exhibit the genetic and physical characteristics of a toxin/antitoxin ( TA) locus, with VapD_Hi serving as the …

Isolation And Characterization Of Mycobacteria From Striped Bass Morone Saxatilis From The Chesapeake Bay, Martha W. Rhodes, Howard Kator, Ilsa Kaattari, David Gauthier, Wolfgang Vogelbein, Christopher A. Ottinger

Biological Sciences Faculty Publications

Mycobacteriosis in striped bass Morone saxatilisof Chesapeake Bay, USA, was first diagnosed in 1997 based on the presence of granulomatous inflammation and acid-fast bacteria in skin and spleen. To confirm histopathology, bacteriological detection and identification of mycobacteria were begun using splenic tissue from fish with and without skin ulcerations. On the basis of initial studies using a variety of selective and nonselective media, decontamination, homogenization and incubation conditions, a simple and quantitative recovery method using aseptic necropsy of splenic tissue was developed. Optimal recovery was obtained by spread-plating homogenates on Middlebrook 7H10 agar with incubation for 3 mo at …

Dinitrogen Fixation And Release Of Ammonium And Dissolved Organic Nitrogen By Trichodesmium Ims101, Margaret R. Mulholland, Deborah A. Bronk, Douglas G. Capone

OES Faculty Publications

Two methods used to measure dinitrogen (N₂) fixation (acetylene reduction and ¹⁵N₂ uptake) often result in different N₂ fixation rates. Part of the discrepancy may arise from the observation that Trichodesmium can release a fraction of their recently fixed N₂ as dissolved organic nitrogen (DON) and/or ammonium (NH4 ⁺). To resolve outstanding issues regarding N₂ fixation and the production of dissolved combined nitrogen (N) by Trichodesmium, we conducted a comprehensive analysis of N₂ fixation and the production of DON and NH₄⁺ in cultures of Trichodesmium IMS101. We performed …

An Investigation Of Dissolved Organic Matter In A Shallow Coastal Bay Subject To Aureococcus Anophagefferens Blooms, Jean-Paul Simjouw

OES Theses and Dissertations

Aureococcus anophagefferens, the pelagophyte responsible for brown tide blooms, was identified in Chincoteague Bay in 1997 and has “bloomed” there since at least 1998. Aureococcus anophagefferens is capable of using dissolved organic nitrogen (DON) and dissolved organic carbon (DOC) substrates to support growth, and this utilization is hypothesized to give the organism a competitive advantage relative to other phytoplankton when inorganic nutrient concentrations are low or depleted. Because previous studies suggest dissolved organic matter (DOM) is important in initiating and sustaining brown tide blooms, a field study of the variations in DOC concentration and DOM composition was performed at …

Characterization Of Spy1600 A Putative Hyaluronidase Gene In Group A Streptococci, Karin M. Berling

Biological Sciences Theses & Dissertations

Group A Streptococci (GAS), also known as Streptococcus pyogenes, can cause a variety of human diseases ranging from asymptomatic to life threatening. Exactly how a single type of organism is able to inflict such a multitude of diseases remains to be fully understood. One possibility includes the large number of secreted virulence factors expressed by the organism. The recent sequencing of three streptococcal genomes has indicated the existence of several previously unknown genes, some of which may encode possible virulence factors. Among these is Spy1600, which based on its sequence similarities has been proposed to encode a hyaluronidase, a …

Hyaluronate Lyase Gene Of Streptococcus Pyogenes: Molecular Characterization And Regulation, Sheryl Lynne Walton

Theses and Dissertations in Biomedical Sciences

Streptococcus pyogenes causes an assortment of diseases ranging from pharyngitis to necrotizing fasciitis. This bacterium has the ability to elaborate several extracellular products capable of causing tissue damage; one of which is a hyaluronate lyase. Little information is available regarding the regulation of streptococcal hyaluronidase. As such, the expression of hylA in the hyaluronate lyase-producing strain ATCC 10403 and in strains that do not produce detectable enzyme was analyzed by RT-PCR and quantitative PCR. Hyaluronate lyase transcript was detected throughout growth for all strains; however, expression of hylA in the enzymatically inactive strains, 71698 and SF370, was determined …

Real-Time Study Of Multidrug Resistance Mechanism In Pseudomonas Aeruginosa Using Nanoparticle Optics And Single Live Cell Imaging, Sophia Vasou Kyriacou

Chemistry & Biochemistry Theses & Dissertations

This thesis centers on the study of the xenobiotic efflux system in Pseudomonas aeruginosa, which is a ubiquitous bacterium. It resists many structurally and functionally diverse substrates due to expression of Mex-extrusion pumps, including MexAB-OprM, MexCD-OprJ, MexEF-OprN and MexXY-OprM systems. Despite extensive research, the structure and mechanism of multidrug resistance is unclear (1-9). For example, (i) how do MexA, MexB and OprM proteins assemble to extrude antibiotics? (ii) What is the antibiotic susceptibility of MexA, MexB, and OprM proteins? (iii) How do substrates cross the outer membrane of P. aeruginosa? (iv) Where are antibiotics accumulated inside the cell? This thesis …

Comparison Of Methods For Dna Isolation From Food Samples For Detection Of Shiga Toxin-Producing Escherichia Coli By Real-Time Pcr, Loree C. Heller, Carisa R. Davis, K. Kealy Peak, David Wingfield, Andrew C. Cannons, Philip T. Amuso, Jacqueline Cattani

Bioelectrics Publications

In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

Experimental Mycobacteriosis In Striped Bass Morone Saxatilis, D. T. Gauthier, M. W. Rhodes, W. K. Vogelbein, H. Kator, C. A. Ottinger

Biological Sciences Faculty Publications

Striped bass Morone saxatilis were infected intraperitoneally with approximately 10⁵ Mycobacterium marinum, M. shottsii sp. nov., or M. gordonae. Infected fish were maintained in a flow-through freshwater system at 18 to 21°C, and were examined histologically and bacteriologically at 2, 4, 6, 8, 17, 26, 36 and 45 wk post-infection (p.i.). M. marinum caused acute peritonitis, followed by extensive granuloma development in the mesenteries, spleen and anterior kidney. Granulomas in these tissues underwent a temporal progression of distinct morphological stages, culminating in well-circumscribed lesions surrounded by normal or healing tissue. Mycobacteria were cultured in high numbers from …

The Role Of Physical, Chemical, And Microbial Heterogeneity On The Field-Scale Transport And Attachment Of Bacteria, Brian J. Mailloux, Mark E. Fuller, Tullis C. Onstott, James Hall, Hailiang Dong, Mary F. Deflaun, Sheryl H. Streger, Randi K. Rothmel, Maria Green, Donald J. P. Swift, Jon Radke

OES Faculty Publications

A field-scale bacterial transport experiment was conducted at the Narrow Channel Focus Area of the South Oyster field site located in Oyster, Virginia. The goal of the field experiment was to determine the relative influence of subsurface heterogeneity and microbial population parameters on flow direction, velocity, and attachment of bacteria at the field scale. The field results were compared with results from laboratory-scale column experiments to develop a method for predicting field-scale bacterial transport. The field site is a shallow, sandy, unconfined, aerobic aquifer that has been characterized by geophysical, sedimentological, and hydrogeological methods. Comamonas sp. strain DA001 and a …

Multiple Transporters Associated With Malaria Parasite Responses To Chloroquine And Quinine, Jianbing Mu, Michael T. Ferdig, Xiaorong Feng, Deirdre A. Joy, Junhui Duan, Tetsuya Furuya, G. Subramanian, L. Aravind, Roland A. Cooper, John C. Wootton, Momia Xiong, Xin-Zhuan Su

Biological Sciences Faculty Publications

Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CO), quinine (ON) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CO resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CO and ON. Such molecules may contribute to increasing incidences of ON treatment failure, the molecular basis …

Il-12 Plasmid Delivery By In Vivo Electroporation For The Successful Treatment Of Established Subcutaneous B16.F10 Melanoma, M. Lee Lucus, Loree Heller, Domenico Coppola, Richard Heller

Bioelectrics Publications

Interleukin-12 (IL-12) has been used in numerous immunotherapy protocols against melanoma. However, delivery of IL-12 in the form of recombinant protein can result in severe toxicity, and gene therapy has had limited success against B16.F10 murine melanoma. The purpose of this study was to examine the effectiveness of in vivo electroporation for the delivery of plasmid DNA encoding IL-12 as an antitumor agent against B16.F10 melanoma. We treated mice bearing established B16.F10 melanoma tumors with intratumoral (i.t.) or intramuscular (i.m.) injections of a plasmid encoding IL-12, followed by in vivo electroporation. For i.t. treatments, we used an applicator containing six …

Rickettsial Infection In Dermacentor Variabilis (Acari : Ixodidae) Inhibits Transovarial Transmission Of A Second Rickettsia, Kevin R. Macaluso, Daniel E. Sonenshine, Shane M. Ceraul, Abdu F. Azad

Biological Sciences Faculty Publications

This study examined the ability of ticks to maintain multiple species of spotted fever group rickettsiae via transovarial transmission. Using a capillary feeding method, previously established Rickettsia montana- and Rickettsia rhipicephali-infected cohorts of Dermacentor variabilis (Say) were exposed to R. rhipicephali and R. montana, respectively, in two reciprocal challenge experiments. Eggs collected from individual females, for two successive generations, of each cohort were assessed for rickettsial infection by polymerase chain reaction for each challenge experiment. Assessment of the eggs from challenged ticks identified that both R. montana- and R. rhipicephali-infected ticks were refractory to their …

Effects Of Nonequilibrium Atmospheric Pressure Plasmas On The Heterotrophic Pathways Of Bacteria And On Their Cell Morphology, Mounir Laroussi, J. Paul Richardson, Fred C. Dobbs

OES Faculty Publications

To date, most research on the interaction of nonequilibrium, atmospheric pressure plasma discharges with bacteria has concentrated on the germicidal effects. Therefore, published results deal mainly with killing efficacy and little attention is given to physical mechanisms and biochemical pathways and their potential alterations when cells of microorganisms are exposed to the plasma. In this letter, an attempt to investigate the effects of plasma exposure on the biochemical pathways of bacteria is presented. In addition, using electron microscopy, we investigate if any gross morphological changes take place when cells are exposed to a lethal dose of plasma. We are testing …

Extended Tailing Of Bacteria Following Breakthrough At The Narrow Channel Focus Area, Oyster, Virginia, Pengfei Zhang, William P. Johnson, Timothy D. Scheibe, Keun-Hyung Choi, Fred C. Dobbs, Brian J. Mailloux

OES Faculty Publications

Extended tailing of low bacterial concentrations following breakthrough at the Narrow Channel focus area was observed for 4 months. Bacterial attachment and detachment kinetics associated with breakthrough and extended tailing were determined by fitting a one-dimensional transport model to the field breakthrough-tailing data. Spatial variations in attachment rate coefficient (k(f)) were observed under forced gradient conditions (i.e., k(f) decreased as travel, distance increased), possibly because of decreased bacterial adhesion with increased transport distance. When pore water velocity decreased by an order of magnitude at 9 days following injection, apparent bacterial attachment rate coefficients did not decrease with velocity as expected …

Development Of An Electrotransformation Technique For Streptococcus Iniae And Preliminary Characterization Of The Hemolysin Associated With This Bacterium, Kimberly Ann Hahn

Biological Sciences Theses & Dissertations

Streptococcus iniae is a gram-positive organism responsible for causing disease in both freshwater as well as saltwater fish. Every year fisheries lose fish by the tons due to diseases caused by this organism. In 1991, the first reported human case of disease associated with this organism was described. Since this initial case, there have been numerous other reported cases of S. iniae infections. This organism is a catalase negative, facultatively anaerobic organism that produces a capsule and when plated onto blood agar demonstrates β-hemolytic activity.

In this study, the relationship between the hemolysin produced by S. iniae and the lactate …

Interactions Among Murine Cytomegalovirus Us22 Family Gene Products That Influence Viral Pathogenesis, Zaruhi Karabekian

Theses and Dissertations in Biomedical Sciences

Cytomegalovirus (CMV) is a complex, ubiquitous herpesvirus that is characterized by acute, chronic, and latent infections. Monocytes-macrophages are the key target cell type involved in pathogenesis, which is most effectively studied using the murine model of CMV infection. Previously three murine CMV (MCMV) genes (M139, M140, and M141) were identified to regulate viral expression in cultured macrophages and in mice. These genes are members of the US22 gene family with respect to HCMV homology. There is no function assigned to the proteins encoded by these genes. However, deletion of M139, M140, and M141 significantly curtails growth of MCMV in macrophages …

Effect Of Magnesium On The Production Of Scna And Scnr By Streptococcus Pyogenes, Strain Ff22, Laura E. Quinn

Biological Sciences Theses & Dissertations

The lantibiotic SA-FF22, produced by Streptococcus pyogenes strain FF22, was the first described antimicrobial peptide produced by a Streptococcus species. S. pyogenes is a group A Streptococcus responsible for such human illness as strep-throat, rheumatic fever, necrotizing fasciitis, endocarditis, and meningitis. Previous studies of SA-FF22 have shown that antimicrobial activity is lost in the presence of lmM magnesium. We hypothesize that the lack of SA-FF22 activity in the presence of magnesium is due to an absence of transcription of scnA, the gene encoding SA-FF22. The lack of transcriptional activation of scnA may be due to an absence of ScnR, a …

Characterization Of Murine Cytomegalovirus M142 And M143 Essential Gene Products, Bridget Linette Dalton

Theses and Dissertations in Biomedical Sciences

Cytomegalovirus (CMV) is a species-specific virus belonging to the Herpesviridae family. This DNA virus causes severe disease or even death in newborns and immunosuppressed patients. Murine CMV (MCMV) provides an opportunity to study the role of various viral products in replication and pathology in the natural host. Successful replication of CMV in the host depends upon the expression of a cascade of viral genes: immediate early (IE), early (E), and late (L). To date only three MCMV IE proteins have been characterized (IE1, IE2 and IE3). Our laboratory recently identified a novel IE gene region within the Hind III I …

Investigations Into The Innate Resistance Of The Tick, Dermacentor Variabilis, When Challenged With The Bacterium, Escherichia Coli, Shane Michael Ceraul

Biological Sciences Theses & Dissertations

In addition to the soluble response, many invertebrates control bacterial infections by means of phagocytosis or melanotic encapsulation. In some insects, Escherichia coli growth is reported to be inhibited by aggregation/encapsulation. Soluble and phagocytic responses to bacterial challenge have been reported in ticks, but evidence of an aggregation/encapsulation response was reported only for inanimate (araldite) implants. This study was done to determine how ticks control infection by E. coli. Ticks were challenged by direct inoculation of bacteria into the hemocoel cavity. Using plate counts, no viable E. coli were detected I hour post-inoculation. A direct fluorescence assay (DF A) …

Short-Term Temporal And Spatial Dynamics Of Bacterioplankton Near Barbados In The Caribbean Sea, Keun-Hyung Choi, Fred C. Dobbs, Robert K. Cowen

OES Faculty Publications

To investigate temporal (ca 1 mo) and spatial dynamics of bacterioplankton in the Caribbean Sea, time-series measurements of chlorophyll a concentration, bacterial abundance, and thymidine incorporation rate were performed at 6 stations near Barbados, In addition, a series of incubation experiments was carried out with seawater samples collected from various depths and stations to determine bacterial growth rates and removal rates by bacterivory. Vertically integrated phytoplankton and bacterioplankton biomasses averaged 1290 and 1200 mg C m(-2), respectively, and heterotrophic bacterial production was 106 mg C m(-2), with in situ bacterial growth of 0.1 d(-1), Approximately halfway through the sampling period, …

Characterization Of The Human Cytomegalovirus Ul75 Late Gene Promoter, Bernard J. P. Mcwatters

Theses and Dissertations in Biomedical Sciences

Gene expression during productive infection by the human cytomegalovirus (HCMV) occurs in an ordered and sequential manner, beginning with immediate early (IE), then early (E) and finally late (L) gene expression. Significant work has addressed the regulation of IE and E gene expression while relatively little work has addressed the control of late gene expression. In order to further address HCMV late gene expression, the promoter of the HCMV UL75 (glycoprotein H, gH) late gene was characterized. The data obtained in this study were combined with observations made in two other studies that have addressed HCMV late gene expression to …

Kinetic Characterization Of Tetracycline Binding And Release By Demineralized Bone - Dfdba, Orner Kabil

Biological Sciences Theses & Dissertations

The microbial etiology of periodontal diseases has led to widespread research in the development of methods and local delivery systems to increase the efficacy of antibiotic therapy. Several drug delivery systems employing biodegradable and nonbiodegradable carriers have been shown to release antibiotics directly into periodontal pockets. The purpose of this study was to determine the binding and release kinetics of tetracycline by demineralized bone. Further aspects of the study include in vitro evaluation of DFDBA (demineralized freeze-dried bone allografts) as a tetracycline carrier system for periodontal therapy. Experiments were performed which employed different tetracycline concentrations with constant amounts of bone …

Hyaluronidases Of Gram-Positive Bacteria, Wayne L. Hynes, Sheryl Lynne Walton

Biological Sciences Faculty Publications

Bacterial hyaluronidases, enzymes capable of breaking down hyaluronate, are produced by a number of pathogenic Gram-positive bacteria that initiate infections at the skin or mucosal surfaces. Since reports of the hyaluronidases first appeared, there have been numerous suggestions as to the role of the enzyme in the disease process. Unlike some of the other more well studied virulence factors, much of the information on the role of hyaluronidase is speculative, with little or no data to substantiate proposed roles. Over the last 5 years, a number of these enzymes from Gram-positive organisms have been cloned, and the nucleotide sequence determined. …