Mesrop Ayrapetyan

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.

Integration of Vibrio vulnificus into marine aggregates and its subsequent uptake by Crassostrea virginica oysters.

ABSTRACT Marine aggregates are naturally forming conglomerations of larvacean houses, phytoplankton, microbes, and inorganics adhered together by exocellular polymers. In this study, we show in vitro that the bacterial pathogen Vibrio vulnificus can be concentrated into laboratory-generated aggregates from surrounding water. We further show that environmental (E-genotype) strains exhibit significantly more integration into these aggregates than clinical (C-genotype) strains. Experiments where marine aggregates with attached V. vulnificus cells were fed to oysters (Crassostrea virginica) resulted in greater uptake of both C and E types than nonaggregated controls. When C- and E-genotype strains were cocultured in competitive experiments, the aggregated E-genotype strains exhibited significantly greater uptake by oyster than the C-genotype strains.

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.

Development of a Matrix Tool for the Prediction of Vibrio Species in Oysters Harvested from North Carolina

ABSTRACT The United States has federal regulations in place to reduce the risk of seafood-related infection caused by the estuarine bacteria Vibrio vulnificus and Vibrio parahaemolyticus. However, data to support the development of regulations have been generated in a very few specific regions of the nation. More regionally specific data are needed to further understand the dynamics of human infection relating to shellfish-harvesting conditions in other areas. In this study, oysters and water were collected from four oyster harvest sites in North Carolina over an 11-month period. Samples were analyzed for the abundances of total Vibrio spp., V. vulnificus, and V. parahaemolyticus; environmental parameters, including salinity, water temperature, wind velocity, and precipitation, were also measured simultaneously. By utilizing these data, preliminary predictive management tools for estimating the abundance of V. vulnificus bacteria in shellfish were developed. This work highlights the need for further research to elucidate the full suite of factors that drive V. parahaemolyticus abundance.

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.

Survival of Vibrio vulnificus Genotypes in Male and Female Serum, and Production of Siderophores in Human Serum and Seawater

Vibrio vulnificus is an estuarine bacterium responsible for 95% of all seafood-related deaths in the United States. Several studies have demonstrated that V. vulnificus infections are enhanced when host iron availability is increased, such as occurs with chronic liver disease. Because of the gender difference seen in these infections, we examined whether there was a difference in the survival in both male and female serum by both the C (clinical) and E (environmental) genotypes of V. vulnificus. We further determined the significance of the catecholate and hydroxamate siderophores produced by this pathogen during both human infections and while in its natural estuarine environment. We found that only C-genotype strains were capable of growth in human serum, regardless of inoculum size, with growth in male and female serum being equal. We found the catecholate outer membrane receptor gene (vuuA) to be expressed significantly more than that for the hydroxamate siderophore (fhuA) when the cells were exposed to human serum, regardless of the genotype. When cells were exposed to natural seawater, fhuA showed increased expression over time, while vuuA showed decreased expression. Our data suggest that the catecholate siderophore is important during human infections, whereas the hydroxamate siderophore may be more important in the estuarine environment this pathogen inhabits.

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.