Academic Degrees and Honors

• B.S.   Microbiology, University of Arizona (1968)

• Ph.D. Microbiology, Georgetown University (1973)

• National Research Council (Canada) Postdoctoral Fellowship (1973-1974)

• Academy of Sciences Exchange Scientist, Romania (1982)

• First Citizens Bank Scholar Award for Excellence in Research (1988)

• Fellow, American Academy of Microbiology (1990-)

• Member, Phi Kappa Phi (Academic Excellence), Phi Beta Delta (Honor Society for International Scholars)

• Visiting Professor, University of Göteborg, Sweden (1990)

• Visiting Professor, North Carolina State University (1994,1995,1996)

• Cone Distinguished Professor for Teaching (1998)

• Visiting Professor, Royal Veterinary and Agricultural University, Copenhagen, Denmark (1998)

• Burroughs Wellcome Fund Visiting Professor in the Microbiological Sciences (1999)

• Member, Standard Methods Committee, Standard Council, American Water Works Association (2000-)

• Member, Working Party on Culture Media, International Committee on Food Microbiology and Hygiene, International Union of Microbiological Sciences (2000-)

• Harshini V. de Silva Graduate Student Mentoring Award (2002)

• Senior Faculty Fellow, Global Institute for Energy and Environmental Systems (2001-)

• Joint Task Group for Section 9213 Recreational Waters for Standard Methods for the Examination of Water and Wastewater. American Water Works Association (2004-)

• Member, Editorial Board, FEMS Microbiology Ecology, 2005-

   

Courses Taught

• BIOL 4250 Microbiology
• BIOL 4257 Microbial Physiology and Metabolism
• BIOL 8000 Microbiology and Immunology (Ph.D. core course)

Areas of Research

The major areas of study in my laboratory (Vibrio vulnificus and other pathogenic marine Vibrio spp., Helicobacter pylori, the “viable but nonculturable” state, and bacterial stress responses and their relationship to survival and virulence) are described briefly here, followed by a description of the individual students and projects underway.

Vibrio vulnificus

First described in 1976, Vibrio vulnificus is a halophilic bacterial species that causes primary septicemia and wound infections.  Disease in humans results from contamination of a skin lesion or ingestion of contaminated seafood.  The striking feature of both the septicemia and wound infections is the speed with which they develop and the high mortality rate (65% and 25%, respectively) associated with them.  Deaths typically occur within 4-5 days from onset.  The infectious dose of V. vulnificus is believed to be low and fatal infections have been reported after the ingestion of a single oyster.  Fatal wound infections acquired by contamination of ant bite lesions have also been reported. Regional V. vulnificus surveillance has been conducted by all the

Bacterial Stress Responses

Many of the so-called heat shock proteins, such as chaperones (e.g. DnaK and GroEL) and proteases (e.g. Clp, Lon), are also induced by other environmental changes, such as the addition of ethanol, heavy metals, oxidative agents, high osmolarity, pollutants, starvation, exposure to low temperature, or interaction with eukaryotic hosts.  Therefore, the heat shock response can more accurately be considered a general stress response.  Through a process termed cross protection, this response improves thermotolerance, salt tolerance, tolerance to heavy metals and UV exposure, starvation survival, and plays an important role in pathogenesis.  These stress responses are critical for bacterial adaptation to changes in the environment, and are therefore a major links between microbial ecology and microbial pathogenesis.  One example is the htrA system, the product of which is essential for bacterial growth only at elevated (e.g. human body) temperatures.  This system is activated by the alternate sigma factor, σ^E, encoded by the rpoE gene.  This gene has been shown to control mucoidy in cystic fibrosis isolates of Pseudomonas aeruginosa.  In E. coli, transcriptional activation of the heat shock genes is induced by the alternate sigma factor, σ³² (product of the rpoH gene).  Our lab is interested in the bacterial response to a variety of environmental stresses, especially starvation, cold, osmotic, and oxidative stress.  The bacteria under active investigation are Vibrio vulnificus (the cause of 95% of all seafood-related deaths in this country) and Helicobacter pylori (the cause of virtually all gastric and duodenal ulcers).  In both cases, we study the mechanisms by which these bacteria survive in the natural environment, the molecular regulator mechanisms and protein products which occur during these survival responses, and the role of these proteins in pathogenesis.

The Viable but Nonculturable State

            Microbial ecologists have long recognized that large proportions of the microbial populations inhabiting natural habitats appear to be nonculturable.  Indeed, plate counts of bacteria in soil, rivers and oceans typically indicate that far less than 1% of the total bacteria observed by direct microscopic examination can be grown on culture media.  It has also long been known that certain portions of bacterial populations in natural environments seem to disappear during certain seasons, only to reappear at other times.  We now understand that at least part of the explanation for these observations is not due to seasonal die-off of the cells, but to their entry into what is most commonly called the “viable but nonculturable” state.

        A bacterial cell in the viable but nonculturable (VBNC) state may be defined as one which fails to grow on the routine bacteriological media on which it would normally grow and develop into a colony, but which is in fact alive and metabolically active.  Bacteria enter into this dormant state in response to one or more environmental stresses which might otherwise be ultimately lethal to the cell.  Thus, the VBNC state should be considered a means of cell survival.  Eventually, when the inducing stress is removed, these cells are able to emerge from the VBNC state, and again become culturable on routine media.

        The typical VBNC response is seen in Fig. 1, which shows the response of the human pathogen, Vibrio vulnificus, to exposure to low temperature (5^oC).  Such a temperature is below that at which this aquatic bacterium can grow and, if it were not for the VBNC response, is a temperature which would eventually lead to death of the population.

            As is evident from Fig. 1, cells lose their ability to be cultured (open squares) in a rather linear manner, eventually reaching a point where platings suggest a total lack of any living cells.  However, whereas death of a bacterial population generally leads to lysis of the cells and loss of cell structure, direct examination of cells entering the VBNC state indicates that the cells remain intact (closed symbols).  Such cells could, of course, have died, but simply not undergone lysis.  The primary evidence that such cells are alive, even if nonculturable, is from data obtained when one of the “direct viability” assays is applied to such cultures, or continues production of  mRNA is detected.  These assays allow the direct determination of the viability of individual cells in a population, without the need for culture.  As seen in Fig. 1 (open circles), such assays often indicate that a large proportion of the apparently dead population is indeed alive.

Cells entering the VBNC state generally undergo a reduction in size, and during this time, significant changes in membrane structure, protein composition, ribosomal content, and possibly even DNA arrangement are experienced.  However, decreases macromolecular synthesis (Fig. 2) do not mean that all synthesis has ceased.  Indeed, protein synthesis appears to be essential for entry into this state, and under these conditions V. vulnificus produces some 40 new proteins not seen during growth at “normal” temperatures.   At the same time, dramatic decreases in membrane fatty acid composition, and in nutrient transport and respiration rates have generally been reported to occur as cells enter this dormant state.  Cell wall synthesis, or at least metabolism of the constituents of these structures, also apparently continues.

Helicobacter pylori

The gram-negative, microaerophilic bacterium, Helicobacter pylori, is now realized to be the main etiological agent of human chronic gastritis and peptic ulcer disease, and is a significant risk factor for gastric adenocarcinoma and non-Hodgkins lymphoma of the stomach.  It has been estimated that more than half the world’s population are infected by this organism.   Despite this, its reservoirs and mode of transmission have yet to be established. While strong epidemiological evidence has been presented that transmission by water and/or the fecal-oral route occurs, investigators around the world have found it enigmatic that its culture from the environment has proved unsuccessful.  Indeed, its presence in environmental samples such as water has only been shown using such sophisticated methods as the polymerase chain reaction (PCR) and hybridization with specific gene probes.

        Complicating the epidemiology of H. pylori infections is the fact that the organism exists in two forms, an actively dividing spiral form, and a coccoid form. When cultured under favorable conditions in vitro, the majority of H. pylori bacteria have the spiral or bacillary appearance, while aging or exposure to a variety of unfavorable conditions (including antibiotics) results in conversion to the coccoid form.  There is no question that the spiral form is involved in infection, but what role the coccoid form, which is not culturable by standard laboratory methods, may play in pathogenesis is unclear.  Although not culturable, there are indications that these forms are viable and infectious.  

        Another concern is that H. pylori infections are often difficult to eliminate, with recurrences appearing in persons who are culture-negative for H. pylori following antibiotic treatment.  It has been proposed that the cause of both the antibiotic resistance often apparent in vivo, and of the recurrences of gastritis and peptic ulcer disease, is a consequence of the nonculturable coccoid form of this bacterium.  

        Failure to detect this organism in the environment by culture methods, despite epidemiological evidence that waterborne transmission is likely, leads to the notion that H. pylori cells are capable of transition to a viable but nonculturable state (the coccoid form), and that this is the state in which the bacterium survives in the environment. 

Studies on the Two Genotypes of Vibrio vulnificus,

and on the Role of Alternate Sigma Factors in their Survival

Thomas M. Rosche, Ph.D.

       My work is focused on the marine pathogen, Vibrio vulnificus, mainly using molecular techniques.  Specifically, I have been involved in studies to show the role that the alternate sigma factor, RpoS, plays in the ability of the bacterium to adapt and respond to changes in environmental conditions.  We have shown that RpoS is necessary for survival of V. vulnificus after exposure to many adverse environmental stresses, including oxidative, osmotic, and acidic conditions, as well as for motility and expression of several exo-enzymes potentially involved in virulence. 

     Additionally, I am using genomic techniques, such as RAPD-PCR, in order to classify V. vulnificus strains according to potential virulence.  We have recently developed a PCR-based screen which is able to sort V. vulnificus into two groups, which are highly associated with clinical or environmental origin.  We plan to use this screen to study the ecology of these two strain types in the natural environment in the hopes of understanding why some bacteria present in oysters cause disease and others do not.  We also intend to study and additional differences between the two strain types in the hopes of identifying previously unknown virulence factors. 

     Finally, I am studying the mechanism of capsule expression in V. vulnificus.  Expression of the polysaccharide capsule is necessary for virulence, but under laboratory conditions acapsular variants arise at fairly high frequency (~1/100), with certain environmental stresses dramatically increasing this switch rate.  Once such acapsular (translucent) colonies arise, they do not appear able to revert back to the capsule-expressing (opaque) morphology.  We aim to uncover the mechanism of this capsule switching, with the intention of developing a protocol to treat freshly harvested oysters to generate switching to the acapsular, and therefore avirulent form.

In Situ and In vitro Studies on the VBNC State

of Estuarine Vibrio spp. 

Karen Dyer (M.S. Candidate)

            The VBNC state allows the survival of non-spore forming bacteria, such as V. vulnificus, V. cholerae, and V. parahaemolyticus, when they are exposed to stressful environmental conditions. While the exact mechanism remains unknown, it has been determined that (1) many bacterial species become nonculturable while remaining viable, (2) the factors which induce this state vary considerably, but typically represent various environmental stresses (3) entrance in to the VBNC state involves a variety of morphological, physiological, biochemical, and genetic changes in the cells, and (4) culture of cells in the “nonculturable” state may be possible, if the appropriate conditions are provided.

            The objectives of my project involve various aspects of the VBNC state in V. vulnificus, V. cholerae, and V. parahaemolyticus.  First, I am conducting a multi-year study on the distribution and ecology of these three pathogens, correlating their presence with some 10 physical and chemical parameters, and an additional 4 microbiological parameters. I am examining especially the role of temperature and nutrient on the in situ entrance of these three bacteria into the VBNC state at sites on the Neuse and Pamlico rivers, as well as in vitro studies which will be conducted in our laboratory. I have also developed and optimized PCR technology to detect these pathogens while in the VBNC state. Thirdly, I am determininh the conditions required for resuscitation of these species. 

Virulence Determinants in Vibrio vulnificus

Tamara Hilton (M.S. candidate)

            Vibrio vulnificus is an estuarine bacterium that can cause extreme septicemia and wound infections that may lead to death. In the course of lab passage, a virulent strain of V. vulnificus C7184° mutated to the point of highly attenuated virulence.  In previous studies, this strain was shown to have a significantly increased LD₅₀ compared to the wild type strain. Several virulence factors are known for V. vulnificus, but so far none have been found to be deficient in this mutant strain.  Studying this mutant is important in understanding the virulence of these bacteria.  In my research, I am looking at several putative virulence factors that could be lacking in this strain. These include attachment ability, production of alternate (stress) sigma factors, SSR repeats, motility, capsule switching, and quorum sensing. I am also comparing the proteomics and RAPD-PCR patterns between the two strains.

In situ and in vitro gene expression in Vibrio vulnificus

Ben Smith (M.S. candidate)

            Vibrio vulnificus is an estuarine bacterium capable of causing fatal septicemia and wound infections.  These infections occur following consumption of raw or undercooked seafood, or after exposure of wounds to water containing this organism.  V. vulnificus strains can be classified as either environmental (isolated from the environment) or clinical (isolated from a patient suffering infection) and these strains have been shown to vary in genetic composition.  Several researchers have previously investigated in vitro hemolysin gene expression in V. vulnificus clinical and environmental strains, but no studies of in situ gene expression have been reported.  I plan to study in vitro and in situ gene expression in environmental and clinical strains of V. vulnificus by RT-PCR.  vvhA (hemolysin), rpoS (stress sigma factor), and tufA (elongation factor) are three genes of interest and will be studied.  I will examine similarities and differences in expression between clinical and environmental strains during entry and survival in the VBNC state, osmotic and cold-shock responses, starvation, and logarithmic growth.  Such findings could uncover survival strategies of this bacterium, as well as differences in expression of distinct genes which may contribute to virulence of this pathogen.  Moreover, my research will potentially show differences in gene expression between in vitro lab experiments and what occurs in the natural environment of this organism.

Identification of Virulence Proteins in Vibrio vulnificus

Liza Warner

Vibrio vulnificus is an estuarine bacterium that can cause septicemia and wound infections that may lead to death.   One of the strains of V. vulnificus studied in our Lab, C7184^o, has mutated over time.  LD₅₀ studies conducted previously show that the virulence of C7184^o is attenuated when compared with its parent C7184^oK.   One of my projects is to study phenotypic expression in both strains to identify changes that may have contributed to the loss of virulence.   Previous studies have identified differences in protein expression and I plan on also continuing that work.  I will conduct both 1 and 2-D SDS-PAGE protein gels as well as phenotypic microarray analysis. 

         The second project I am working on relates to a proposed addiction module system for programmed cell death (PCD) in E. coli.  Various studies have identified an addiction module that consists of two adjacent genes mazE and mazF in E. coli that codes for a labile anti-toxin MazE and a stable toxin MazF that interact to form a complex.  During periods of stress, the labile anti-toxin is degraded and the toxin causes a type of PCD.   One of the questions raised in the current research is if this is actual cell death (or apoptosis) or if it leads to a type of bacteriostasis.   I plan on growing the E. coli strains under conditions of amino acid starvation to activate the module and to conduct cell counts until they are no longer culturable, then attempt to resuscitate the cells.  I will also use special stains to determine if the cells are actually dead, or have entered a “viable but non-culturable” state.

Survival of Helicobacter pylori on Foods

Alan Buck

Helicobacter pylori is the bacterium recognized as the major cause of gastritis and peptic ulcers. Currently, half of the world’s population is infected with this bacterium, yet its mode of transmission from person to person is not understood. Many researchers believe transmission is through contaminated water (due to the very high rate of infection - 80% - of individuals residing in developing countries), but this potential reservoir has not yielded culturable H. pylori cells. Others have looked to the survival of H. pylori on vegetables that were irrigated with contaminated water. Previous studies from our lab have shown that H. pylori can remain culturable for up to 5 days on spinach and tomatoes. However, it is believed that H. pylori  enters a viable but nonculturable (VBNC) state and that it may actually survive for extended periods of time in waters or on fruits and vegetables while remaining completely undetectable by conventional culture means.  This may provide a reservoir for the organism in the environment. Critics, however, have questioned the VBNC evidence due to unsuccessful attempts to resuscitate this bacterium back to the culturable state. If H. pylori does indeed enter the VBNC state, then there should exist some evidence of activity – most likely in the production of mRNA.

My research involves inoculating various vegetables with H. pylori, where the cells will enter the VBNC state. Once in that state, I will test for the presence of mRNA using a technique known as RT-PCR. Given the extremely short half-life of RNA (2-4 minutes), any RNA detected from the VBNC cells would be a positive indication of life in these nonculturable cells, and provide more evidence for the VBNC phenomenon.

The Role of Acanthamoeba and Quorum Sensing in Survival of Helicobacter pylori

Emily Davis (Honors Undergraduate Student)

The inability to culture H. pylori directly from the environment may be a result of its entrance into the viable but nonculturable (VBNC) state. Bacteria in the VBNC state do not grow on routine biological media, yet are alive and capable of being resuscitated into a culturable state.  Bacteria entering the VBNC state do so in response to environmental stresses such as temperature downshifts or nutrient depletion.  If a method to resuscitate H. pylori from its VBNC state could be found, this could affirm the mode of transmission of the organism.  Studies in our lab have shown that H. pylori exposed to freshwater environments for 10 days decline in culturability to <10 CFU/ml, yet a large population of viable cells still exist (i.e. are in the VBNC state).  Using RT-PCR, it has also been found that H. pylori in the VBNC state continue to transcribe several genes, some of which have been determined to be virulence factors (Adams, B.L., T.C. Bates, and J. D. Oliver. 2003. Survival of Helicobacter pylori in a natural freshwater environment. Appl. Environ. Microbiol. 69(12):7462-7466.

            Legionella pneumophila, the causative agent of Legionnaire’s Disease, is also known to enter the VBNC state when incubated in a low nutrient environment. Studies have shown that co-incubation with Acanthamoeba castellanii, a natural amoeba host for this bacterial pathogen, resulted in a return to the fully culturable state of L. pneumophila.  

Similar to the studies described above with L. pneumophila, some researchers have been able to demonstrate successful co-incubation of H. pylori with A. castellanii.  In contrast to the few days that H. pylori is usually culturable when removed from rich laboratory media, it was able to remain fully culturable for several weeks when incubated with A. castellanii.  The bacterium was shown to survive in vacuoles within the amoeba, demonstrating continued metabolic activity. Co-incubation of H. pylori with A. castellanii could prove to be instrumental in the survival of this pathogen in the environment and in its transmission.  A type II secretion system does not exist in H. pylori, yet a similar type IV secretion system is present.  One of the genes encoding for the type IV secretion system of H. pylori is vacA.  vacA and other homologues of the genes required for L. pneumophila survival in A. castellanii may also be expressed by H. pylori during co-incubation with A. castellanii.  I wish to study the role which A. castellanii may have in the resuscitation of H. pylori from the VBNC into a fully culturable state.

Of additional interest is the role quorum sensing may play within A. castellanii.  Quorum sensing is a signaling mechanism between bacteria which greatly influences gene expression.  Quorum sensing involves the production and detection of signaling molecules that function to communicate information to a population of cells about their external environment and cell density. When the concentration of signaling molecules, specifically AI-2 in H. pylori, is high enough there can be either activation or suppression of target genes.  Incubation within A. castellanii would provide an enclosed environment to facilitate quorum sensing in H. pylori. I plan to study the role quorum sensing may also play in the resuscitation of H. pylori from the VBNC state.  RT-PCR techniques will be used to determine the effect quorum sensing may play in gene expression.  I also intend to integrate the studies involving A. castellanii with the quorum sensing studies to determine any relationship between these two factors.  It has been shown that genes required for the invasion of A. castellanii by L. pneumophila are in the LuxR family.  LuxR proteins are integral to quorum sensing.  Within A. castellanii, AI-2 signaling molecules remain bound inside the amoeba membrane increasing their concentration.  The increased concentration of AI-2 molecules may prolong culturability or influence the VBNC state of H. pylori.  I also hope to determine how the presence of AI-2 signaling with or without the presence of A. castellanii affects gene expression in H. pylori. 

In conclusion, our understanding of the epidemiology of H. pylori has been limited by its lack of culturability due its entrance into the viable but nonculturable state.  It is known that H. pylori continues to exhibit gene expression when the cells enter this state.  Furthermore, studies show that co-incubation with A. castellanii prolongs the culturability of H. pylori significantly. The quorum sensing mechanism allows bacteria to evaluate their surroundings and the cell densities in that environment.  This mechanism may allow H. pylori to recognize its presence within its natural amoeba host.  It is hoped that the studies described here will provide a better understanding of the role incubation within A. castellanii and quorum sensing play in the gene expression of this organism.  A better understanding of which genes are expressed under these conditions may allow resuscitation of this pathogen into a fully culturable state and prove instrumental in determining its epidemiology and mode of transmission.

Physiological Differences Between Clinical and Environmental Genotypes of Vibrio vulnificus

Ryan Bogard (M.S. Candidate) 

     Vibrio vulnificus is a gram-negative, marine pathogen that is of great concern to the shellfish industry. In fact, 95% of deaths resulting from seafood consumption are caused by this bacterium (Oliver and Kaper, 2001). V. vulnificus is observed to have two morphotypes, opaque and translucent, which correspond to the presence or absence of capsule. The presence of this complex polysaccharide capsule is a major virulence factor, having antiphagocytic properties. Recent studies involving analysis of a 200 bp RAPD-PCR amplicon led to a novel PCR based method of distinguishing between clinical (C) and environmental (E) isolates (Rosche et al. 2005), thus concluding that two distinct genotypes of V. vulnificus exist. Of those strains examined, 93% (28/30) of the environmental isolates had the E-genotype. This finding suggests that C-genotype strains of V. vulnificus are relatively rare in the environment, which is supported by the lower than expected frequency of infection. I hypothesize that bactericidal resistance differences exist between both the colony (capsular) morphotypes and the two genotypes of V. vulnificus. Genotypic differences may have arisen due to selective pressures of a stressful marine environment, which in turn allowed the successful transition of V. vulnificus from a generalist marine microbe to a human pathogen.  These genotypic differences may involve a suite of stress proteins that protect against oxidative, temperature, and nutrient deficiency stresses. Preliminary studies in the Oliver lab lend support to the hypothesis of differential stress toleration between genotypes. In one study clinical strains of V. vulnificus were observed to survive significantly better than environmental strains when incubated at 40° C. Another study confirmed that stress proteins play a critical role in survivability of V. vulnificus when exposed to stresses such as oxidation, osmotic changes, pH and temperature-shock. Comparative survivability analyses among both morphotypes of environmentally and clinically isolated E-type and C-type strains are necessary to distinguish physiological differences that may exist between the two strains. Survivability, measured in CFU’s, will be represented by the strains ability to resist the bactericidal effects of exposure to human sera. The ability of each strain to withstand opsonization effects will be measured by comparing bacterial survivability in heat-inactivated serum to survivability in normal human serum (NHS). Included in the strains to be examined are several well studied clinical and environmental strains, two clinically isolated E-type strains, two environmentally isolated E-type strains, as well as an RpoS stress protein mutant. These strains will be exposed to various temperature, oxidative, and nutritional conditions, incubated in NHS and analyzed for differences in survivability.

Physiological Differences Between Clinical and Environmental Genotypes of Vibrio vulnificus

Cristina Price (MS Candidate)

     My research efforts are based on recent findings on genomic differences between clinical (C) and environmental (E) strains of Vibrio vulnificus.  Specifically, I will be working on identifying physiological characteristics that may contribute to different virulence factors between    C and E strains. I am also examining differences in growth characteristics of the two genotypes, and their responses to varying salinities.

Environmental Conditions Leading to Loss of Capsule in Vibrio vulnificus

Brett Froelich (Lab Assistant)

I am currently engaged in two projects, both involving the bacterium Vibrio vulnificus, a potentially fatal food-borne pathogen.  The first project involves determining the conditions that lead to the down-regulation of capsule by this bacterium.  V. vulnificus is the leading cause of sea-food borne death and capsule expression is the only definitive virulence factor.  The second project includes examining isolates of the two genotypes (clinical and environmental) of V. vulnificus to observe any differences in production of extracellular enzymes.  Discerning a difference may provide evidence of additional virulence factors. 

Recent Publications (2000-2005)

Effect of starvation and the viable but nonculturable state on green fluorescent protein (GFP) in GFP-tagged Pseudomonas fluorescens. 2000. Lowder, M.A., A. Unge, N. Maraha, J.K. Jansson, J. Swiggertt, and J.D. Oliver. Appl. Environ. Microbiol. 66:3160-3165.

The viable but nonculturable state and cellular resuscitation. 2000. Oliver, J.D. Intern. Symp. Microb. Ecol. 723-730.

The public health significance of viable but nonculturable bacteria.  2000. Oliver, J.D.  In: "Nonculturable Microorganisms in the Environment", R.R. Colwell and D.J. Grimes (ed.).  Amer. Soc. Microbiol. Press, Washington, D.C.

Problems in detecting dormant (VBNC) cells and the role of DNA elements in this response. 2000. Oliver, J.D. pp. 1-15, In: Marker and Reporter Genes, J.K. Jansson, J.D. van Elsas, and M.J. Bailey (eds.). Landes Biosciences, Georgetown, TX.

Culture media for the isolation and enumeration of pathogenic Vibrio species in foods and environmental samples. 2001. Oliver, J.D. In: Culture Media for Food Microbiology, 2nd Rev. Ed. J.E.L. Corry, G.D.W. Curtis, and R.M. Baird (eds.), Elsevier Science, Netherlands.

Essential role for estrogen in protection against Vibrio vulnificus induced endotoxic shock. 2001. Merkel, S.M., S. Alexander, J.D. Oliver, and Y.M. Huet-Hudson. Infect. Immun. 69:6119-6122. 

The use of GFP as a reporter for metabolic activity in Pseudomonas putida. 2001. Lowder, M. and J.D. Oliver. Microbiol. Ecol. 41:310-313.

Vibrio species. Oliver, J.D. and J. Kaper. 2001. pp. 263-300 In: Food Microbiology:  Fundamentals and Frontiers, 2^nd ed. M.P. Doyle, L.R. Beuchat, T.J. Montville (ed.).  Amer. Soc. Microbiol.

Effects of refrigeration and alcohol on the load of Aeromonas hydrophila in oysters.  2002.  Birkenhauer, J.B. and J.D. Oliver. J. Food Protect. 65:560–562.

Use of diacetyl to reduce the load of Vibrio vulnificus in the Eastern oyster, Crassostrea virginica. 2003. Birkenhauer, J.B. and J.D. Oliver. J. Food Protect. 66:38-43.

 A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments.  2003. Pfeffer, C. and J.D. Oliver.  Lett. Appl. Microbiol. 36:150-151.

Analysis of Vibrio vulnificus from market oysters and septicemia cases for virulence markers.  2003. DePaola, A. et al. (11 authors).  Appl. Environ. Microbiol. 69: 4006-4011.

The ecology of Vibrio vulnificus in estuarine waters of eastern North Carolina.  2003. Pfeffer, C.S. and J.D. Oliver.  Appl. Environ. Microbiol. 69:3526-3531.

Culture media for the isolation and enumeration of pathogenic Vibrio species in foods and environmental samples.  2003. Oliver, J.D. pp. 249-269 In: Handbook of Culture media for Food Microbiology, 2^nd ed.  J.E.L. Corry, G.D.W. Curtis, and R.M. Baird (eds.).  Vol. 37 of Progress in Industrial Microbiology. Elsevier.  Amsterdam.

RpoS-dependent stress response and exoenzyme production in Vibrio vulnificus.   Huelsmann, A., T.M. Rosche, I.-S. Kong, H.M. Hassan, D.M Beam, and J.D. Oliver. Appl. Environ. Microbiol. 69:6114-6120.

Survival of Helicobacter pylori in a natural freshwater environment.  2003. Adams, B.L., T.C. Bates, and J.D. Oliver. Appl. Environ. Microbiol. 69:7462-7466.

Effects of temperature on detection of plasmid or chromosomally encoded gfp- and lux­-labeled Pseudomonas fluorescens in soil.  2004. Bunker, S.T., T.C. Bates, and J.D. Oliver.  Environ. Biosaf. Res. 3:83-90.

Biochemical and virulence characterization of viable but nonculturable cells of Vibrio parahaemolyticus.  2004. Wong, H.-C. and J.D. Oliver.  J. Food Prot. 67:2430-2435.

The viable but nonculturable state of Vibrio parahaemolyticus.  2004. Bates, T.C. and J.D. Oliver.  J. Microbiol. 42:74-79.

Role of catalase and oxyR in the viable but nonculturable state of Vibrio vulnificus.  2004. Kong, I.-S., T.C. Bates, A. Hülsmann, , H. Hassan, and J.D. Oliver.  FEMS Microbiol. Ecol. 50:133-142.

Pulsed-field electrophoresis analysis of Vibrio vulnificus strains isolated from Taiwan and United States.  2004. Wong, H.-c., S.Y. Chen, M.-Y. Chen, J.D. Oliver, L.-I. Hor, and W.-Ch. Tsai. Appl. Environ. Microbiol. 70:5153-5158.

Changes in membrane fatty acid composition during entry of Vibrio vulnificus in the viable but nonculturable state.  2004. Day, A.P. and J.D. Oliver.  J. Microbiol. 42:69-73.

Induction of Escherichia coli and Salmonella typhimurium into the viable but nonculturable state following chlorination of wastewater.  2005. Oliver, J.D., M. Dagher, and K. Linden. J. Water and Health 3.3:249-257.

Wound infections caused by Vibrio vulnificus and other marine bacteria.  “Special Article”.  Oliver, J.D.  2005.  Epidemiol. Infect. 133:383-391.

A rapid and simple PCR analysis indicates there are two subgroups of Vibrio vulnificus which correlate with clinical or environmental isolation.  2005. Rosche, T.M., Y. Yano, and J.D. Oliver. Microbiol. Immunol. 49:381-389.

The viable but nonculturable state in bacteria.  2005.  Oliver, J.D. J. Microbiol. 43:93-100.

Cloning, sequencing and expression of a GroEL-like protein gene of Vibrio vulnificus.  2005. Wong, H.-c., K.H. Lu, and J.D. Oliver.  Taiw. J. Agric. Chem. Food Sci. 43:1-7.

RpoS involvement in osmotically-induced cross protection in Vibrio vulnificus.  Rosche, T.M., T.C. Bates, D.J. Smith, E.E. Parker, and J.D. Oliver.  FEMS. Microbiol. Ecol. 53:455-462.

Intrapopulational variation in Vibrio vulnificus levels in Crassostrea virginica is associated with the host size but not with disease status or developmental stability.  2005. Sokolova, I.M., L. Leamy, M. Harrison, and J.D. Oliver.  J. Shellfish Res. 24:503-508.

Vibrio vulnificus. 2005.  Oliver, J.D. In: Biology of Vibrios.  F.L. Thompson, B. Austin, and J. Swing. (eds.).  Amer. Soc. Microbiol. Press, Washington, D.C. (in press).

In situ and in vitro gene expression by Vibrio vulnificus during entry into, persistence within, and resuscitation from the viable but nonculturable state.  Smith, B.E. and J.D. Oliver. Appl. Environ. Microbiol. (submitted).

In situ and in vitro gene expression by Vibrio vulnificus during the starvation-survival state.  Smith, B.E. and J.D. Oliver.  Appl. Environ. Microbiol. (submitted).

An AFLP approach to identify genetic markers associated with resistance to Vibrio vulnificus and Perkinsus marinus in eastern oysters.  Sokolova, I.M., J.D. Oliver, and L.J. Leamy. J. Shellfish Res. (submitted).

Viable but nonculturable bacteria in food environments. 2005. Oliver, J.D. In:  Food-borne pathogens: Microbiology and Molecular Biology.  P.M. Fratamico, A.K. Bhunia, and J.L. Smith (eds.). Caister Academic Press, Norfolk, UK.

Vibrio vulnificus.  2005. Oliver, J.D. In: Bacterial Pathogens in Sea Water. Belkin, S. (ed.). Plenum Publishing. (In press).

Gene expression by Helicobacter pylori in a natural freshwater environment.  Adams, B. L. and J. D. Oliver.  Appl. Environ. Microbiol. (submitted).

 The role of cross protection in survival of Helicobacter pylori during stress.  Adams, B.L. and J.D. Oliver. Helicobacter  (submitted).

Recent Graduate Students  

Bunker, Stephen.  2000.  Effects of environmental stresses on culturability of genetically modified Pseudomonas fluorescens in soil.

Bates, Tonya.  2001. The VBNC state in Vibrio parahaemolyticus.

Birkenhauer, Jennifer. 2001. Use of GRAS compounds to reduce loads of Vibrio vulnificus in oysters.

Day, Ashley. 2002.  Membrane fatty acid changes in Vibrio vulnificus induced by low temperature and osmotic shock.

Courtney Pfeffer. 2002.  Involvement of Vibrio vulnificus in wound infections in the Neuse River area of North Carolina.

Daren Beam.  2004. Quorum sensing and pathogenesis in Vibrio vulnificus.

Ben Smith. 2005.  In situ and in vitro gene expression by Vibrio vulnificus during the VBNC and starvation-survival states.

Tamara Hilton.  2005. Capsular switching among the clinical- and environmental-genotypes of Vibrio vulnificus.  2005.

Karen Dyer Blackwell. 2005. The viable but nonculturable states of Vibrio vulnificus, Vibrio cholerae, and Vibrio parahaemolyticus in the natural environment.  2005.