Tiffany C. Williams

Pyrosequencing-Based Comparative Genome Analysis of Vibrio vulnificus Environmental Isolates

Between 1996 and 2006, the US Centers for Disease Control reported that the only category of food-borne infections increasing in frequency were those caused by members of the genus Vibrio. The gram-negative bacterium Vibrio vulnificus is a ubiquitous inhabitant of estuarine waters, and is the number one cause of seafood-related deaths in the US. Many V. vulnificus isolates have been studied, and it has been shown that two genetically distinct subtypes, distinguished by 16S rDNA and other gene polymorphisms, are associated predominantly with either environmental or clinical isolation. While local genetic differences between the subtypes have been probed, only the genomes of clinical isolates have so far been completely sequenced. In order to better understand V. vulnificus as an agent of disease and to identify the molecular components of its virulence mechanisms, we have completed whole genome shotgun sequencing of three diverse environmental genotypes using a pyrosequencing approach. V. vulnificus strain JY1305 was sequenced to a depth of 33×, and strains E64MW and JY1701 were sequenced to lesser depth, covering approximately 99.9% of each genome. We have performed a comparative analysis of these sequences against the previously published sequences of three V. vulnificus clinical isolates. We find that the genome of V. vulnificus is dynamic, with 1.27% of genes in the C-genotype genomes not found in the E- genotype genomes. We identified key genes that differentiate between the genomes of the clinical and environmental genotypes. 167 genes were found to be specifically associated with environmental genotypes and 278 genes with clinical genotypes. Genes specific to the clinical strains include components of sialic acid catabolism, mannitol fermentation, and a component of a Type IV secretory pathway VirB4, as well as several other genes with potential significance for human virulence. Genes specific to environmental strains included several that may have implications for the balance between self-preservation under stress and nutritional competence.

Interactive effects of cadmium and hypoxia on metabolic responses and bacterial loads of eastern oysters Crassostrea virginica Gmelin

Pollution by toxic metals including cadmium (Cd) and hypoxia are important stressors in estuaries and coastal waters which may interactively affect sessile benthic organisms, such as oysters. We studied metabolic responses to prolonged hypoxic acclimation (2 weeks at 5% O2) in control and Cd-exposed (30 d at 50 μg L(-1) Cd) oysters Crassostrea virginica, and analyzed the effects of these stressors on abundance of Vibrio spp. in oysters. Hypoxia-acclimated oysters retained normal standard metabolic rates (SMR) at 5% O2, in contrast to a decline of SMR observed during acute hypoxia. However, oysters spent more time actively ventilating in hypoxia than normoxia resulting in enhanced Cd uptake and 2.7-fold higher tissue Cd burdens in hypoxia. Cd exposure led to a significant decrease in tissue glycogen stores, increase in free glucose levels and elevated activity of glycolytic enzymes (hexokinase and aldolase) indicating a greater dependence on carbohydrate catabolism. A compensatory increase in activities of two key mitochondrial enzymes (citrate synthase and cytochrome c oxidase) was found during prolonged hypoxia in control oysters but suppressed in Cd-exposed ones. Cd exposure also resulted in a significant increase in abundance of Vibrio parahaemolyticus and Vibrio vulnificus levels during normoxia and hypoxia, respectively. Overall, Cd- and hypoxia-induced changes in metabolic profile, Cd accumulation and bacterial flora of oysters indicate that these stressors can synergistically impact energy homeostasis, performance and survival of oysters in polluted estuaries and have significant consequences for transfer of Cd and bacterial pathogens to the higher levels of the food chain.

Apparent loss of Vibrio vulnificus from North Carolina oysters coincides with a drought-induced increase in salinity.

ABSTRACT Despite years of successful isolation of Vibrio vulnificus from estuarine waters, beginning in 2007, it was extremely difficult to culture V. vulnificus from either North Carolina estuarine water or oyster samples. After employing culture-based methods as well as PCR and quantitative PCR for the detection of V. vulnificus, always with negative results, we concluded that this pathogen had become nearly undetectable in the North Carolina estuarine ecosystem. We ensured that the techniques were sound by seeding North Carolina oysters with V. vulnificus and performing the same tests as those previously conducted on unadulterated oysters. V. vulnificus was readily detected in the seeded oysters using both classes of methods. Furthermore, oysters were obtained from the Gulf of Mexico, and V. vulnificus was easily isolated, confirming that the methodology was sound but that the oysters and waters of North Carolina were lacking the V. vulnificus population studied for decades. Strikingly, the apparent loss of detectable V. vulnificus coincided with the most severe drought in the history of North Carolina. The drought continued until the end of 2009, with an elevated water column salinity being observed throughout this period and with V. vulnificus being nearly nonexistent. When salinities returned to normal after the drought abated in 2010, we were again able to routinely isolate V. vulnificus from the water column, although we were still unable to culture it from oysters. We suggest that the oysters were colonized with a more salt-tolerant bacterium during the drought, which displaced V. vulnificus and may be preventing recolonization.

Interspecific Quorum Sensing Mediates the Resuscitation of Viable but Nonculturable Vibrios

ABSTRACT Entry and exit from dormancy are essential survival mechanisms utilized by microorganisms to cope with harsh environments. Many bacteria, including the opportunistic human pathogen Vibrio vulnificus, enter a form of dormancy known as the viable but nonculturable (VBNC) state. VBNC cells can resuscitate when suitable conditions arise, yet the molecular mechanisms facilitating resuscitation in most bacteria are not well understood. We discovered that bacterial cell-free supernatants (CFS) can awaken preexisting dormant vibrio populations within oysters and seawater, while CFS from a quorum sensing mutant was unable to produce the same resuscitative effect. Furthermore, the quorum sensing autoinducer AI-2 could induce resuscitation of VBNC V. vulnificus in vitro, and VBNC cells of a mutant unable to produce AI-2 were unable to resuscitate unless the cultures were supplemented with exogenous AI-2. The quorum sensing inhibitor cinnamaldehyde delayed the resuscitation of wild-type VBNC cells, confirming the importance of quorum sensing in resuscitation. By monitoring AI-2 production by VBNC cultures over time, we found quorum sensing signaling to be critical for the natural resuscitation process. This study provides new insights into the molecular mechanisms stimulating VBNC cell exit from dormancy, which has significant implications for microbial ecology and public health.

Viable but nonculturable and persister cells coexist stochastically and are induced by human serum

ABSTRACT Dormancy holds a vital role in the ecological dynamics of microorganisms. Specifically, entry into dormancy allows cells to withstand times of stress while maintaining the potential for reentry into an active existence. The viable but nonculturable (VBNC) state and antibiotic persistence are two well-recognized conditions of dormancy demonstrated to contribute to bacterial stress tolerance and, as a consequence, yield populations that are tolerant to high-dose antibiotics. Aside from this commonality, more evidence is being presented that indicates the relatedness of these two states. Here, we demonstrate that VBNC cells are present during persister isolation experiments, further indicating that these cells coexist and are induced by the same conditions. Interestingly, we reveal that VBNC cells can exist stochastically in unstressed growing cultures, a finding that is characteristic of persisters. Furthermore, human serum induces the formation of both VBNC cells and persisters, a finding not previously described for either dormancy state. Lastly, we describe the role of toxin-antitoxin systems (TAS) in the induction of the VBNC state and report that these TAS, which are classically implicated in persister cell formation, are also induced during incubation in human serum. This study provides evidence for the recently proposed “dormancy continuum hypothesis” and substantiates the physical and molecular relatedness of VBNC and persister cells in a standardized model organism. Notably, these results provide new evidence for the clinical significance of VBNC and persister cells.

Implications of Chitin Attachment for the Environmental Persistence and Clinical Nature of the Human Pathogen Vibrio vulnificus

ABSTRACT Vibrio vulnificus naturally inhabits a variety of aquatic organisms, including oysters, and is the leading cause of seafood-related death in the United States. Strains of this bacterium are genetically classified into environmental (E) and clinical (C) genotypes, which correlate with source of isolation. E-genotype strains integrate into marine aggregates more efficiently than do C-genotype strains, leading to a greater uptake of strains of this genotype by oysters feeding on these aggregates. The causes of this increased integration of E-type strains into marine “snow” have not been demonstrated. Here, we further investigate the physiological and genetic causalities for this genotypic heterogeneity by examining the ability of strains of each genotype to attach to chitin, a major constituent of marine snow. We found that E-genotype strains attach to chitin with significantly greater efficiency than do C-genotype strains when incubated at 20°C. Type IV pili were implicated in chitin adherence, and even in the absence of chitin, the expression level of type IV pilin genes (pilA, pilD, and mshA) was found to be inherently higher by E genotypes than by C genotypes. In contrast, the level of expression of N-acetylglucosamine binding protein A (gbpA) was significantly higher in C-genotype strains. Interestingly, incubation at a clinically relevant temperature (37°C) resulted in a significant increase in C-genotype attachment to chitin, which subsequently provided a protective effect against exposure to acid or bile, thus offering a clue into their increased incidence in human infections. This study suggests that C- and E-genotype strains have intrinsically divergent physiological programs, which may help explain the observed differences in the ecology and pathogenic potential between these two genotypes.

Transcriptome Sequencing Reveals the Virulence and Environmental Genetic Programs of Vibrio vulnificus Exposed to Host and Estuarine Conditions

Vibrio vulnificus is a natural inhabitant of estuarine waters worldwide and is of medical relevance due to its ability to cause grievous wound infections and/or fatal septicemia. Genetic polymorphisms within the virulence-correlated gene (vcg) serve as a primary feature to distinguish clinical (C-) genotypes from environmental (E-) genotypes. C-genotypes demonstrate superior survival in human serum relative to E-genotypes, and genome comparisons have allowed for the identification of several putative virulence factors that could potentially aid C-genotypes in disease progression. We used RNA sequencing to analyze the transcriptome of C-genotypes exposed to human serum relative to seawater, which revealed two divergent genetic programs under these two conditions. In human serum, cells displayed a distinct “virulence profile” in which a number of putative virulence factors were upregulated, including genes involved in intracellular signaling, substrate binding and transport, toxin and exoenzyme production, and the heat shock response. Conversely, the “environmental profile” exhibited by cells in seawater revealed upregulation of transcription factors such as rpoS, rpoN, and iscR, as well as genes involved in intracellular signaling, chemotaxis, adherence, and biofilm formation. This dichotomous genetic switch appears to be largely governed by cyclic-di-GMP signaling, and remarkably resembles the dual life-style of V. cholerae as it transitions from host to environment. Furthermore, we found a “general stress response” module, known as the stressosome, to be upregulated in seawater. This signaling system has been well characterized in Gram-positive bacteria, however its role in V. vulnificus is not clear. We examined temporal gene expression patterns of the stressosome and found it to be upregulated in natural estuarine waters indicating that this system plays a role in sensing and responding to the environment. This study advances our understanding of gene regulation in V. vulnificus, and brings to the forefront a number of previously overlooked genetic networks.

A new culture-based method for the improved identification of Vibrio vulnificus from environmental samples, reducing the need for molecular confirmation

Vibrio vulnificus is an opportunistic human pathogen responsible for 95% of seafood related deaths in the US. Monitoring the presence of this bacterium in estuarine waters and shellfish is of medical and economic importance due to its ability to cause severe wound infections and fulminant septicemia. Current methods for isolating V. vulnificus from environmental samples typically employ an initial selective medium which requires subsequent molecular confirmation of presumptive V. vulnificus isolates. Although culture-based methods are accessible and inexpensive, they lack the specificity needed to definitively identify V. vulnificus. The goal of this study was to develop a more accurate, culture-based method for the initial detection of V. vulnificus, thereby decreasing or eliminating the requirement for confirmatory molecular tests. Colony color characteristics of a variety of Vibrio species were determined on three commonly employed media to identify those which present as false-positive isolates for V. vulnificus. We subsequently developed a triple-plating method which utilizes three media in combination to greatly decrease the number of false positive isolates. The number of isolates positively identified as V. vulnificus using the triple-plating method were compared to a typical single-plating method and revealed over a 2-fold increase in ability to accurately predict V. vulnificus isolates. We suggest that this new method will enhance the predictive power of culture-based methods, reduce the cost and time spent on additional detection methods, and may be a valuable alternative when molecular methods are not available or unaffordable.

Serum Survival of Vibrio vulnificus: Role of Genotype, Capsule, Complement, Clinical Origin, and in Situ Incubation

Virulence of the human pathogen, V. vulnificus, is associated with encapsulation, serum complement resistance, and genotype. The C-genotype of this bacterium is correlated (>90%) with virulence and with isolation source (clinical settings). E-genotype strains are highly correlated with environmental isolation (93%) but appear less virulent. In this study, we characterized the importance of genotype, encapsulation, serum complement, and in situ exposure to estuarine water on the survival of the two genotypes in human serum. Results confirmed the superior ability of C-genotype strains to survive exposure to human serum, as well as the significance of complement, and revealed that lack of capsule allowed serum killing of both C- and E-genotypes. Cells incubated in situ responded similarly to cells incubated in vitro with the exception of E-environmental strains. Interestingly, our studies found that those cells of the E-genotype, typically considered non-pathogenic, which were isolated from wound infections demonstrated serum survival similar to that of virulent, C-genotype, strains.

Molecular and Physical Factors That Influence Attachment of Vibrio vulnificus to Chitin.

ABSTRACT The human pathogen Vibrio vulnificus is the leading cause of seafood-related deaths in the United States. Strains are genotyped on the basis of alleles that correlate with isolation source, with clinical (C)-genotype strains being more often implicated in disease and environmental (E)-genotype strains being more frequently isolated from oysters and estuarine waters. Previously, we have shown that the ecologically distinct C- and E-genotype strains of V. vulnificus display different degrees of chitin attachment, with C-genotype strains exhibiting reduced attachment relative to their E-genotype strain counterparts. We identified type IV pili to be part of the molecular basis for this observed genotypic variance, as E-genotype strains exhibit higher levels of expression of these genes than C-genotype strains. Here, we used a C-genotype quorum-sensing (QS) mutant to demonstrate that quorum sensing is a negative regulator of type IV pilus expression, which results in decreased chitin attachment. Furthermore, calcium depletion reduced E-genotype strain attachment to chitin, which suggests that calcium is necessary for proper functioning of the type IV pili in E-genotype strains. We also found that starvation or dormancy can alter the efficiency of chitin attachment, which has significant implications for the environmental persistence of V. vulnificus. With the increasing incidence of wound infections caused by V. vulnificus, we investigated a subset of E-genotype strains isolated from human wound infections and discovered that they attached to chitin in a manner more similar to that of C-genotype strains. This study enhances our understanding of the molecular and physical factors that mediate chitin attachment in V. vulnificus, providing insight into the mechanisms that facilitate the persistence of this pathogen in its native environment.