D. Jay Grimes

Ecology of Vibrio parahaemolyticus and Vibrio vulnificus in the Coastal and Estuarine Waters of Louisiana, Maryland, Mississippi, and Washington (United States)

ABSTRACT Vibrio parahaemolyticus and Vibrio vulnificus, which are native to estuaries globally, are agents of seafood-borne or wound infections, both potentially fatal. Like all vibrios autochthonous to coastal regions, their abundance varies with changes in environmental parameters. Sea surface temperature (SST), sea surface height (SSH), and chlorophyll have been shown to be predictors of zooplankton and thus factors linked to vibrio populations. The contribution of salinity, conductivity, turbidity, and dissolved organic carbon to the incidence and distribution of Vibrio spp. has also been reported. Here, a multicoastal, 21-month study was conducted to determine relationships between environmental parameters and V. parahaemolyticus and V. vulnificus populations in water, oysters, and sediment in three coastal areas of the United States. Because ecologically unique sites were included in the study, it was possible to analyze individual parameters over wide ranges. Molecular methods were used to detect genes for thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and tdh-related hemolysin (trh) as indicators of V. parahaemolyticus and the hemolysin gene vvhA for V. vulnificus. SST and suspended particulate matter were found to be strong predictors of total and potentially pathogenic V. parahaemolyticus and V. vulnificus. Other predictors included chlorophyll a, salinity, and dissolved organic carbon. For the ecologically unique sites included in the study, SST was confirmed as an effective predictor of annual variation in vibrio abundance, with other parameters explaining a portion of the variation not attributable to SST.

Semantics and Strategies

The term “nonculturable” was invoked by Xu et al. in 1982 (47) to describe starved but viable cells in a survival or dormant state of existence, observed to occur after an actively growing culture of Vibrio cholerae O1 or Escherichia coli was placed in a nutrient-free microcosm, e.g., a saline solution free of nutrient and incubated at low temperature. The cells were grown in a nutrient broth under optimal conditions, harvested by centrifugation, washed, and placed in sterile artificial seawater (15‰ salinity). The cells were enumerated directly, using both acridine orange and fluorescent-antibody staining, and viewed by epifluorescent microscopy. They were also plated using media optimized for their growth. It was determined by direct microscopic observation that the total number of cells did not decrease with time. However, as incubation continued over several days, it was found that colonies no longer formed when the same samples were plated on media that had been optimized for growth of the culture. To determine whether such cells were viable or dead, a microscopic technique developed by Kogure et al. (25) was employed to examine the cultures that yielded no colonies on transfer to solid media and no growth in liquid media. The Kogure et al. (25) method, a direct-viable-count procedure, when applied to these cultures revealed that nearly all of the cells enumerated by acridine orange direct counting (AODC) were metabolically active, even though they could not be recovered on any plating medium or in broth. Hence, the phrase “viable but nonculturable” (VBNC) was coined to describe these bacteria that were apparently in a dormant state.

Enumeration of Vibrio parahaemolyticus in the viable but nonculturable state using direct plate counts and recognition of individual gene fluorescence in situ hybridization

Vibrio parahaemolyticus is a gram-negative, halophilic bacterium indigenous to marine and estuarine environments and it is capable of causing food and water-borne illness in humans. It can also cause disease in marine animals, including cultured species. Currently, culture-based techniques are used for quantification of V. parahaemolyticus in environmental samples; however, these can be misleading as they fail to detect V. parahaemolyticus in a viable but nonculturable (VBNC) state which leads to an underestimation of the population density. In this study, we used a novel fluorescence visualization technique, called recognition of individual gene fluorescence in situ hybridization (RING-FISH), which targets chromosomal DNA for enumeration. A polynucleotide probe labeled with Cyanine 3 (Cy3) was created corresponding to the ubiquitous V. parahaemolyticus gene that codes for thermolabile hemolysin (tlh). When coupled with the Kogure method to distinguish viable from dead cells, RING-FISH probes reliably enumerated total, viable V. parahaemolyticus. The probe was tested for sensitivity and specificity against a pure culture of tlh(+), tdh(-), trh(-)V. parahaemolyticus, pure cultures of Vibrio vulnificus, Vibrio harveyi, Vibrio alginolyticus and Vibrio fischeri, and a mixed environmental sample. This research will provide additional tools for a better understanding of the risk these environmental organisms pose to human health.

The Importance of Viable but Nonculturable Bacteria in Biogeochemistry

Biogeochemical cycles are critical parts of life itself—if elements do not cycle, life will grind to a halt, with essential elements being buried. Recycling of the elements allows for their reappearance in the food web to be used again and again, thereby allowing life to replenish itself. A simple example of this is shown in Fig. 1 for the carbon cycle, in which energy flow on Earth is diagrammatically linked to the carbon cycle. In this diagram we see the two major sources of energy on the planet (photic energy and geothermal energy) linked to life directly (through photosynthesis) and indirectly (through conversion of geothermal energy to reduced substrates that are used for lithotrophic metabolism). Both processes lead to the “fixation” or reduction of CO2 to organic carbon—e.g., the conversion of carbon dioxide into biomass. The organic carbon is then recycled to the atmosphere via respiration by carbon-oxidizing bacteria and eukaryotes.

A novel agar formulation for isolation and direct enumeration of Vibrio vulnificus from oyster tissue

A new selective and differential medium, Vibrio vulnificus X-Gal (VVX), was developed for direct enumeration of V. vulnificus (Vv) from oyster samples. This agar utilizes cellobiose and lactose as carbon sources, and the antibiotics colistin and polymyxin B as selective agents. Hydrolysis of 5-bromo-4-chloro-3-indolyl- beta-d-galactopyranoside (x-gal), used in the agar as a lactose analog, produces an insoluble blue dye that makes lactose positive colonies easily distinguishable from any non-lactose fermenting bacteria. Various bacterial species were spot plated onto thiosulfate-citrate-bile salts-sucrose agar (TCBS), and CHROMagar Vibrio, two vibrio-specific selective agars, non-selective agar, and VVX to compare selectivity of VVX to other widely used media. A V. vulnificus pure culture was serially diluted on VVX and non-selective agar to determine the VVX percent recovery. Water and oyster samples were spread plated on VVX agar and allowed to incubate for 16-18 h at 33 °C. Blue and white colonies from VVX agar were picked and screened by end point PCR for the Vv hemolysin vvhA. VVX agar showed a significant improvement over TCBS and CHROMagar at preventing non-target growth. There was an 87.5% recovery compared to non-selective plating and a 98% positivity rate of blue colonies picked from oyster tissue plating. The findings suggest that this new agar is a fast, distinctive, and accurate method for enumeration of V. vulnificus from the environment.

Aquaculture and Animal Pathogens in the Marine Environment with Emphasis on Marine Shrimp Viruses

The Oxford English Dictionary defines agriculture as “The science and art of cultivating the soil; including the allied pursuits of gathering in the crops and rearing live stock”. Modifying the OED definition by replacing “soil” with “Earth’s surface” makes aquatic agriculture—or in shorthand “aquaculture”, the fastest growing sector of agriculture (FAO, 2002).A characteristic that sets the aquaculture industry apart from terrestrial livestock production is the rearing of species that are also natural resources. Although the spread of a terrestrial livestock pathogen serves as a primary concern to the livestock industry, dissemination of an aquaculture pathogen concerns commercial fisheries, recreational fisheries, and a wider environmental community. A converse concern is also felt by the aquaculture community; pathogens in natural populations may spread more easily into the aquaculture industry because of the shared host species. As human population size and wealth have grown, so has demand for seafood. Expansion of marine aquaculture meets much of the new demand. According to the data in the FAO database FishStat, the capture fishery sector of seafood production has been level since the 1980s; however,mariculture has continued an 8%per annumexponential growth rate that began in the 1950s (Fig. 19.1). Further, the percentage of marine seafood coming from mariculture has increased steadily over that time and now approaches 25%. Given that a population (either wild or cultured) carries a pathogen, the probability of transferring that pathogen to the other population increases as the number of effective contacts between those two populations increases. An effective contact is one that carries with it some non-zero probability of transmission-of-infection. One correlate to the growth of marine aquaculture is increased contacts between wild and cultured host populations. More contacts presently occur because of an increase in cultured animals inhabiting the environment and therefore more opportunities for contact between wild and cultured animals. Effective contact can occur between cultured animals and local populations during fish rearing or more distant populations after harvest.

Nontoxigenic Vibrio cholerae Non-O1/O139 Isolate from a Case of Human Gastroenteritis in the U.S. Gulf Coast

ABSTRACT An occurrence of Vibrio cholerae non-O1/O139 gastroenteritis in the U.S. Gulf Coast is reported here. Genomic analysis revealed that the isolate lacked known virulence factors associated with the clinical outcome of a V. cholerae infection but did contain putative genomic islands and other accessory virulence factors. Many of these factors are widespread among environmental strains of V. cholerae, suggesting that there might be additional virulence factors in non-O1/O139 V. cholerae yet to be determined. Phylogenetic analysis revealed that the isolate belonged to a phyletic lineage of environmental V. cholerae isolates associated with sporadic cases of gastroenteritis in the Western Hemisphere, suggesting a need to monitor non-O1/O139 V. cholerae in the interest of public health.

Bacterial Species Identified on the Skin of Bottlenose Dolphins Off Southern California via Next Generation Sequencing Techniques

The dermis of cetaceans is in constant contact with microbial species. Although the skin of the bottlenose dolphin provides adequate defense against most disease-causing microbes, it also provides an environment for microbial community development. Microbial community uniqueness and richness associated with bottlenose dolphin skin is a function of varying habitats and changing environmental conditions. The current study uses ribosomal DNA as a marker to identify bacteria found on the skin of coastal and offshore bottlenose dolphins off of Southern California. The unique microbial communities recovered from these dolphins suggest a greater microbial diversity on the skin of offshore ecotype bottlenose dolphins, while microbial populations associated with the coastal ecotype include species that are more closely related to each other and that suggest exposure to communities that are likely to be associated with terrestrial runoff.