Systematic Analysis of White Pox Disease in Acropora palmata of the Florida Keys and Role of Serratia marcescens

White pox disease (WPD) affects the threatened elkhorn coral, Acropora palmata. Owing in part to the lack of a rapid and simple diagnostic test, there have been few systematic assessments of the prevalence of acroporid serratiosis (caused specifically by Serratia marcescens) versus general WPD signs. Six reefs in the Florida Keys were surveyed between 2011 and 2013 to determine the disease status of A. palmata and the prevalence of S. marcescens. WPD was noted at four of the six reefs, with WPD lesions found on 8 to 40% of the colonies surveyed. S. marcescens was detected in 26.9% (7/26) of the WPD lesions and in mucus from apparently healthy colonies both during and outside of disease events (9%; 18/201). S. marcescens was detected with greater frequency in A. palmata than in the overlying water column, regardless of disease status (P = 0.0177). S. marcescens could not be cultured from A. palmata but was isolated from healthy colonies of other coral species and was identified as pathogenic pulsed-field gel electrophoresis type PDR60. WPD lesions were frequently observed on the reef, but unlike in prior outbreaks, no whole-colony death was observed. Pathogenic S. marcescens was circulating on the reef but did not appear to be the primary pathogen in these recent WPD episodes, suggesting that other pathogens or stressors may contribute to signs of WPD. Results highlight the critical importance of diagnostics in coral disease investigations, especially given that field manifestation of disease may be similar, regardless of the etiological agent.     

Enzyme-Linked Immunosorbent Assays for the Specific and Sensitive Quantification of Methanosarcina mazei and Methanobacterium bryantii

Three microtitration plate enzyme-linked immunosorbent assays (ELISAs) have been developed: a competitive ELISA and a two-site (or indirect sandwich) ELISA for Methanosarcina mazei S6 and a two-site ELISA for Methanobacterium bryantii FR-2. The assays were sensitive, with limits of cell protein detection of 3 ng ml⁻¹, 5 ng ml⁻¹, and 50 ng ml⁻¹, respectively, and showed good precision. The M. mazei assays used monoclonal antibodies and were entirely species specific, showing no cross-reaction with methanogens of other genera or with other species of the same genus. The Methanobacterium bryantii assay, which used two polyclonal antisera, showed only a slight cross-reaction with one other Methanobacterium species but no cross-reaction with methanogens of other genera. The use of the ELISAs for quantitative analysis of mixed cultures and of sewage sludge samples was investigated. Sludge diluted at 1:10³ or more caused no significant interference in any of the three ELISAs. Various cultures of bacteria, methanogens, and nonmethanogens at a protein concentration of 50 μg ml⁻¹ showed no significant interference in the M. mazei competitive assay and the Methanobacterium bryantii two-site assay, although they did cause falsely low results in the M. mazei two-site assay.     

Shifting white pox aetiologies affecting Acropora palmata in the Florida Keys, 1994–2014

We propose ‘the moving target hypothesis’ to describe the aetiology of a contemporary coral disease that differs from that of its historical disease state. Hitting the target with coral disease aetiology is a complex pursuit that requires understanding of host and environment, and may lack a single pathogen solution. White pox disease (WPX) affects the Caribbean coral Acropora palmata. Acroporid serratiosis is a form of WPX for which the bacterial pathogen (Serratia marcescens) has been established. We used long-term (1994–2014) photographic monitoring to evaluate historical and contemporary epizootiology and aetiology of WPX affecting A. palmata at eight reefs in the Florida Keys. Ranges of WPX prevalence over time (0–71.4%) were comparable for the duration of the 20-year study. Whole colony mortality and disease severity were high in historical (1994–2004), and low in contemporary (2008–2014), outbreaks of WPX. Acroporid serratiosis was diagnosed for some historical (1999, 2003) and contemporary (2012, 2013) outbreaks, but this form of WPX was not confirmed for all WPX cases. Our results serve as a context for considering aetiology as a moving target for WPX and other coral diseases for which pathogens are established and/or candidate pathogens are identified. Coral aetiology investigations completed to date suggest that changes in pathogen, host and/or environment alter the disease state and complicate diagnosis.     

Signaling-mediated cross-talk modulates swarming and biofilm formation in a coral pathogen Serratia marcescens

Interactions within microbial communities associated with marine holobionts contribute importantly to the health of these symbiotic organisms formed by invertebrates, dinoflagellates and bacteria. However, mechanisms that control invertebrate-associated microbiota are not yet fully understood. Hydrophobic compounds that were isolated from surfaces of asymptomatic corals inhibited biofilm formation by the white pox pathogen Serratia marcescens PDL100, indicating that signals capable of affecting the associated microbiota are produced in situ. However, neither the origin nor structures of these signals are currently known. A functional survey of bacteria recovered from coral mucus and from cultures of the dinoflagellate Symbiodinium spp. revealed that they could alter swarming and biofilm formation in S. marcescens. As swarming and biofilm formation are inversely regulated, the ability of some native α-proteobacteria to affect both behaviors suggests that the α-proteobacterial signal(s) target a global regulatory switch controlling the behaviors in the pathogen. Isolates of Marinobacter sp. inhibited both biofilm formation and swarming in S. marcescens PDL100, without affecting growth of the coral pathogen, indicative of the production of multiple inhibitors, likely targeting lower level regulatory genes or functions. A multi-species cocktail containing these strains inhibited progression of a disease caused by S. marcescens in a model polyp Aiptasia pallida. An α-proteobacterial isolate 44B9 had a similar effect. Even though ∼4% of native holobiont-associated bacteria produced compounds capable of triggering responses in well-characterized N-acyl homoserine lactone (AHL) biosensors, there was no strong correlation between the production of AHL-like signals and disruption of biofilms or swarming in S. marcescens.     

Quantitation of the diastereoisomers of l-buthionine-(R,S)-sulfoximine in human plasma: a validated assay by capillary electrophoresis

An assay for the diastereoisomers of the biochemical modifier $\text{l}\text{-buthionine}\text{-(R,S)-}\text{sulfoximine}$ (BSO) in human plasma has been developed using capillary electrophoresis (CE). Separation of the diastereoisomers is achieved by the micellar electrokinetic chromatography (MEKC) mode of CE. Plasma is injected directly onto the separation capillary without any extraction step, and BSO is detected directly by ultraviolet absorbance measurements at 190 nm without prior derivatization. The whole assay, including capillary conditioning, takes approximately 30 min. Intra- and inter-day R.S.D. values are approximately 7% at sample concentrations around 25 μg ml⁻¹, and approximately 3% at sample concentrations around 500 μg ml⁻¹. The limit of detection in plasma is 3.9 μg ml⁻¹ (S/N = 2). The assay has been used to quantitate the diastereoisomers of BSO in patient samples in a pharmacokinetic study.     

Anthropogenic nutrient enrichment of seagrass and coral reef communities in the Lower Florida Keys: discrimination of local versus regional nitrogen sources

Land-based nutrient pollution represents a significant human threat to coral reefs globally. We examined this phenomenon in shallow seagrass and coral reef communities between the Content Keys (southern Florida Bay) and Looe Key (south of Big Pine Key) in the Lower Florida Keys by quantifying the role of physical forcing (rainfall, wind, tides) and water management on mainland South Florida to nutrient enrichment and blooms of phytoplankton, macroalgae, and seagrass epiphytes. Initial studies (Phase I) in 1996 involved daily water quality sampling (prior to, during, and following physical forcing events) at three stations (AJ, an inshore area directly impacted by sewage discharges; PR, a nearshore patch reef located inshore of Hawk Channel; and LK, an offshore bank reef at Looe Key) to assess the spatial and temporal patterns in advection of land-based nutrients to the offshore reefs. Concentrations of dissolved inorganic nitrogen (DIN=NH₄⁺+NO₃⁻+NO₂⁻), soluble reactive phosphorus (SRP), and chlorophyll a increased at PR and LK following a wind event (∼15 knots, northeast) in mid-February. The highest DIN (mostly NH₄⁺) and SRP concentrations of the entire study occurred at the inshore AJ during an extreme low tide in March. Following the onset of the wet season in May, mean NH₄⁺ and chlorophyll a concentrations increased significantly to maximum seasonal values at PR and LK during summer; relatively low concentrations of NO₃⁻ and a low f-ratio (NO₃⁻/NH₄⁺+NO₃⁻) at all stations during summer do not support the hypothesis that the seasonal phytoplankton blooms resulted from upwelling of NO₃⁻. A bloom of the seagrass epiphyte Cladosiphon occidentalis (phaeophyta) followed the onset of the rainy season and increased NH₄⁺ concentrations at LK, resulting in very high epiphyte:blade ratios (∼3:1) on Thalassia testudinum. Biomass of macroalgae increased at all three stations from relatively low values (<50 g dry wt m⁻²) in winter and early spring to higher values (∼100-300 g dry wt m⁻²) typical of eutrophic seagrass meadows and coral reefs following the onset of the rainy season. The mean δ¹⁵N value of Laurencia intricata (rhodophyta) during 1996 at AJ (+4.7‰) was within the range reported for macroalgae growing on sewage nitrogen; lower values at the more offshore PR (+3.1‰) and LK (+2.9‰) were at the low end of the sewage range, indicating an offshore dilution of the sewage signal during the 1996 study. However, transient increases in δ¹⁵N of Cladophora catanata (chlorophtyta) from ~+2% to +5% at LK concurrent with elevated NH₄⁺ concentrations following rain and/or wind events in May and July suggest episodic advection of sewage nitrogen to the offshore LK station. The Phase II study involved sampling of macroalgae for δ¹⁵N along a gradient from the Content Keys through Big Pine Key and offshore to LK in the summer wet season of 2000 and again in the drought of spring 2001. During the July 2000 sampling, macroalgae in nearshore waters around Big Pine Key had elevated δ¹⁵N values (~+4‰) characteristic of sewage enrichment; lower values (~+2‰) at LK were similar to values reported for macroalgae in upstream waters of western Florida Bay influenced by nitrogen-rich Everglades runoff. That pattern contrasted with the drought sampling in March 2001, when δ¹⁵N values of macroalgae were elevated (+6‰) to levels characteristic of sewage enrichment over a broad spatial scale from the Content Keys to LK. These results suggest that regional-scale agricultural runoff from the mainland Everglades watersheds as well as local sewage discharges from the Florida Keys are both significant nitrogen sources supporting eutrophication and algal blooms in seagrass and coral reef communities in the Lower Florida Keys. Hydrological and physical forcing mechanisms, including rainfall, water management on the South Florida mainland, wind, and tides, regulate the relative importance and variability of these anthropogenic nitrogen inputs over gradients extending to the offshore waters of the Florida Reef Tract.     

Immunoquantitative Real-Time PCR for Detection and Quantification of Staphylococcus aureus Enterotoxin B in Foods

A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of immunocapture of SEB and real-time PCR amplification of the DNA probe linked to the detection antibody. iqPCR was compared to an in-house enzyme-linked immunosorbent assay (ELISA) using the same couple of capture-detection antibodies and to commercial kits for detection of S. aureus enterotoxins (SE). The iqPCR was approximately 1,000 times more sensitive (<10 pg ml⁻¹) than the in-house ELISA and had a dynamic range of approximately 10 pg ml⁻¹ to approximately 30,000 pg ml⁻¹. iqPCR was not inhibited by any of the foods tested and was able to detect SEB present in these foods. No cross-reactivity with SE other than SEB was observed. Application of iqPCR for detection of SEB in cultures of S. aureus revealed the onset of SEB production after 4 h of incubation at 22, 37, and 42°C, which was in the first half of the exponential growth phase. The total amounts of SEB produced by the two strains tested were larger at 42°C than at 37°C and were strain dependent.     

Comparison of the Seasonal Variations of Synechococcus Assemblage Structures in Estuarine Waters and Coastal Waters of Hong Kong

Seasonal variation in the phylogenetic composition of Synechococcus assemblages in estuarine and coastal waters of Hong Kong was examined through pyrosequencing of the rpoC1 gene. Sixteen samples were collected in 2009 from two stations representing estuarine and ocean-influenced coastal waters, respectively. Synechococcus abundance in coastal waters gradually increased from 3.6 × 10³ cells ml⁻¹ in March, reaching a peak value of 5.7 × 10⁵ cells ml⁻¹ in July, and then gradually decreased to 9.3 × 10³ cells ml⁻¹ in December. The changes in Synechococcus abundance in estuarine waters followed a pattern similar to that in coastal waters, whereas its composition shifted from being dominated by phycoerythrin-rich (PE-type) strains in winter to phycocyanin-only (PC-type) strains in summer owing to the increase in freshwater discharge from the Pearl River and higher water temperature. The high abundance of PC-type Synechococcus was composed of subcluster 5.2 marine Synechococcus, freshwater Synechococcus (F-PC), and Cyanobium. The Synechococcus assemblage in the coastal waters, on the other hand, was dominated by marine PE-type Synechococcus, with subcluster 5.1 clades II and VI as the major lineages from April to September, when the summer monsoon prevailed. Besides these two clades, clade III cooccurred with clade V at relatively high abundance in summer. During winter, the Synechococcus assemblage compositions at the two sites were similar and were dominated by subcluster 5.1 clades II and IX and an undescribed clade (represented by Synechococcus sp. strain miyav). Clade IX Synechococcus was a relatively ubiquitous PE-type Synechococcus found at both sites, and our study demonstrates that some strains of the clade have the ability to deal with large variation of salinity in subtropical estuarine environments. Our study suggests that changes in seawater temperature and salinity caused by the seasonal variation of monsoonal forcing are two major determinants of the community composition and abundance of Synechococcus assemblages in Hong Kong waters.     

Bitten down to size: Fish predation determines growth form of the Caribbean coral reef sponge Mycale laevis

Interactions between organisms add complexity to ecosystem function, particularly on coral reefs. The Caribbean orange icing sponge Mycale laevis is semi-cryptic, often growing under coral colonies or between coral branches. This association is reportedly a mutualism, with the sponge deterring boring sponges from invading the coral skeleton and the coral providing an expanding surface for sponge growth. But is there an alternative explanation for the proximity of sponge and coral? We examined the importance of fish predation on the growth of the sponge. While the semi-cryptic growth form of M. laevis predominates on reefs off the Florida Keys and the Bahamas Islands, M. laevis grows with a non-cryptic, erect morphology off Bocas del Toro, Panama. Surveys revealed that sponge-eating fishes were rare or absent at Bocas del Toro compared to sites in the Florida Keys. Because past studies were inconsistent about the palatability of M. laevis to fish predators, we conducted feeding experiments with sponges from all three sites. Crude organic extracts of M. laevis from all three sites were palatable to generalist fish predators in aquarium assays, and field feeding assays and caging experiments conducted in the Florida Keys confirmed that spongivorous fishes readily ate exposed fragments of M. laevis. Our results suggest that M. laevis is restricted to its semi-cryptic growth form by spongivorous predators, with corals providing a physical refuge from predation. This alternative explanation supports the broader hypothesis that Caribbean reef sponges can be categorized on the basis of chemical defense into defended, palatable, and preferred species, the last of which are restricted to refugia.     

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C_T) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C_T and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10⁻⁵ pg of E. coli O157:H7 DNA ml⁻¹ equivalent to approximately 6.4 × 10³ CFU of E. coli O157:H7 ml⁻¹ based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 10⁴ CFU g⁻¹. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g⁻¹ with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

Abundance and Distribution of Ostreococcus sp. in the San Pedro Channel, California, as Revealed by Quantitative PCR

Ostreococcus is a genus of widely distributed marine phytoplankton which are picoplanktonic in size (<2 μm) and capable of rapid growth. Although Ostreococcus has been detected around the world, little quantitative information exists on its contribution to planktonic communities. We designed and implemented a genus-specific TaqMan-based quantitative PCR (qPCR) assay to investigate the dynamics and ecology of Ostreococcus at the USC Microbial Observatory (eastern North Pacific). Samples were collected from 5 m and the deep chlorophyll maximum (DCM) between September 2000 and August 2002. Ostreococcus abundance at 5 m was generally <5.0 × 10³ cells ml⁻¹, with a maximum of 8.2 × 10⁴ cells ml⁻¹. Ostreococcus abundance was typically higher at the DCM, with a maximum of 3.2 × 10⁵ cells ml⁻¹. The vertical distribution of Ostreococcus was examined in March 2005 and compared to the distribution of phototrophic picoeukaryotes (PPE) measured by flow cytometry. The largest contribution to PPE abundance by Ostreococcus was ~70% and occurred at 30 m, near the DCM. Despite its relatively low abundance, the depth-integrated standing stock of Ostreococcus in March 2005 was ~30 mg C m⁻². Our work provides a new technique for quantifying the abundance of Ostreococcus and demonstrates the seasonal dynamics of this genus and its contribution to picoeukaryote biomass at our coastal sampling station.     

Ubiquitous associations and a peak fall prevalence between apicomplexan symbionts and reef corals in Florida and the Bahamas

Although apicomplexans are a widely recognized and important parasitic group, little is known about those associated with invertebrates, such as reef-building scleractinian corals. To resolve the potential impact of apicomplexans on coral health, it is first necessary to further describe this group of putative parasites and determine their prevalence among host species. Here, it was hypothesized that apicomplexan prevalence would vary seasonally, similar to what occurs in other marine apicomplexans as well as some coral symbionts. To test this, Caribbean scleractinian species Porites astreoides, Montastraea (=Orbicella) annularis, M. (=O.) faveolata, and Siderastrea siderea were sampled seasonally from two reefs each in the Florida Keys and the Bahamas for 9- and 5.5-year periods, respectively. Utilizing a PCR-based screening assay, apicomplexan DNA was detected from most Floridian (80.1 %: n = 555/693) and Bahamian (90.7 %: n = 311/343) coral tissue samples collected over these multi-year periods. Furthermore, apicomplexan DNA was detected from nearly all (98.7 %: n = 78/79) single polyps sampled at multiple locations within six M. faveolata colonies, indicating little to no intracolonial variation in the screening assay. Mixed-model logistic regression was utilized to determine the effects of season, host species, and reef on apicomplexan prevalence. The model identified a significant seasonal effect, with the highest apicomplexan prevalence occurring during fall. There also was a large effect of host species, with apicomplexan prevalence significantly lower among S. siderea colonies relative to the other species. While reef did not have a significant effect in the full model, there was a significant difference in apicomplexan prevalence between Floridian and Bahamian reefs for S. siderea, implying regional differences in this host species. Despite seasonal and species-specific differences in prevalence, apicomplexans are ubiquitous constituents of these particular scleractinian coral species from Florida and the Bahamas.     

Australian Dust Storm Associated with Extensive Aspergillus sydowii Fungal “Bloom” in Coastal Waters

A massive central Australian dust storm in September 2009 was associated with abundant fungal spores (150,000/m³) and hyphae in coastal waters between Brisbane (27°S) and Sydney (34°S). These spores were successfully germinated from formalin-preserved samples, and using molecular sequencing of three different genes (the large subunit rRNA gene [LSU], internal transcribed spacer [ITS[, and beta-tubulin gene), they were conclusively identified as Aspergillus sydowii, an organism circumstantially associated with gorgonian coral fan disease in the Caribbean. Surprisingly, no human health or marine ecosystem impacts were associated with this Australian dust storm event. Australian fungal cultures were nontoxic to fish gills and caused a minor reduction in the motility of Alexandrium or Chattonella algal cultures but had their greatest impacts on Symbiodinium dinoflagellate coral symbiont motility, with hyphae being more detrimental than spores. While we have not yet seen any soft coral disease outbreaks on the Australian Great Barrier Reef similar to those observed in the Caribbean and while this particular fungal population was non- or weakly pathogenic, our observations raise the possibility of future marine ecosystem pathogen impacts from similar dust storms harboring more pathogenic strains.     

An Improved Protocol for Quantification of Freshwater Actinobacteria by Fluorescence In Situ Hybridization

We tested a previously described protocol for fluorescence in situ hybridization of marine bacterioplankton with horseradish peroxidase-labeled rRNA-targeted oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) in plankton samples from different lakes. The fraction of Bacteria detected by CARD-FISH was significantly lower than after FISH with fluorescently monolabeled probes. In particular, the abundances of aquatic Actinobacteria were significantly underestimated. We thus developed a combined fixation and permeabilization protocol for CARD-FISH of freshwater samples. Enzymatic pretreatment of fixed cells was optimized for the controlled digestion of gram-positive cell walls without causing overall cell loss. Incubations with high concentrations of lysozyme (10 mg ml⁻¹) followed by achromopeptidase (60 U ml⁻¹) successfully permeabilized cell walls of Actinobacteria for subsequent CARD-FISH both in enrichment cultures and environmental samples. Between 72 and >99% (mean, 86%) of all Bacteria could be visualized with the improved assay in surface waters of four lakes. For freshwater samples, our method is thus superior to the CARD-FISH protocol for marine Bacteria (mean, 55%) and to FISH with directly fluorochrome labeled probes (mean, 67%). Actinobacterial abundances in the studied systems, as detected by the optimized protocol, ranged from 32 to >55% (mean, 45%). Our findings confirm that members of this lineage are among the numerically most important Bacteria of freshwater picoplankton.     

Utilization of Mucus from the Coral Acropora palmata by the Pathogen Serratia marcescens and by Environmental and Coral Commensal Bacteria

In recent years, diseases of corals caused by opportunistic pathogens have become widespread. How opportunistic pathogens establish on coral surfaces, interact with native microbiota, and cause disease is not yet clear. This study compared the utilization of coral mucus by coral-associated commensal bacteria (“Photobacterium mandapamensis” and Halomonas meridiana) and by opportunistic Serratia marcescens pathogens. S. marcescens PDL100 (a pathogen associated with white pox disease of Acroporid corals) grew to higher population densities on components of mucus from the host coral. In an in vitro coculture on mucus from Acropora palmata, S. marcescens PDL100 isolates outgrew coral isolates. The white pox pathogen did not differ from other bacteria in growth on mucus from a nonhost coral, Montastraea faveolata. The ability of S. marcescens to cause disease in acroporid corals may be due, at least in part, to the ability of strain PDL100 to build to higher population numbers within the mucus surface layer of its acroporid host. During growth on mucus from A. palmata, similar glycosidase activities were present in coral commensal bacteria, in S. marcescens PDL100, and in environmental and human isolates of S. marcescens. The temporal regulation of these activities during growth on mucus, however, was distinct in the isolates. During early stages of growth on mucus, enzymatic activities in S. marcescens PDL100 were most similar to those in coral commensals. After overnight incubation on mucus, enzymatic activities in a white pox pathogen were most similar to those in pathogenic Serratia strains isolated from human mucosal surfaces.Serratia is a gammaproteobacterium frequently isolated from waters, plants, and animals (7). Some isolates of Serratia are well-characterized symbionts of invertebrates. Serratia marcescens and Serratia liquefaciens have been identified as vertically transmitted symbionts of the sugar beet maggot (9). Serratia colonizes male and female reproductive tracts of the maggots, eggs, and pharyngeal filter. There, the bacteria are hypothesized to aid in metamorphosis by digesting chitinous puparial walls (9). In the gut of another insect, the diamondback moth, strains of S. marcescens appear to live as commensals capable of modestly (5 to 8%) increasing growth rates of the host (8). Serratia strains have also been isolated from feces and cloacal swabs from clinically normal captive birds, but not from organs or carcasses of sick or diseased animals housed within the same facility (3, 20). Serratia spp. have also been linked to diseases of invertebrate animals and their larvae (for reviews, see references 7, 15, and 21). To cause diseases in nematodes and flies, S. marcescens first colonizes the intestines, degrades cells of the alimentary tract and then spreads to other organs (14, 21). There are, however, exceptions to this mode of infection. Serratia entomophila, the causal agent of amber disease in grubs, grows within the alimentary tract of the animal to >10⁶ CFU. However, bacteria neither attach to nor colonize surfaces of the gut; rather, they adhere to gut contents (10) and cause the appearance of signs by producing the Sep toxin that inhibits accumulation of the insect''s digestive serine proteases and disrupts the cytoskeletal network (6). It appears, therefore, that various isolates of Serratia are capable of entering into a full range of interactions (from mutualistic to commensal to pathogenic) with their animal hosts (for reviews, see references 7, 15, and 21).A strain of S. marcescens, PDL100, was shown to be associated with white pox disease of the threatened Caribbean coral Acropora palmata (22, 27). White pox disease results in coral tissue necrosis, exposing carbonate skeleton at a rate of 2.5 cm² day⁻¹ (22). It is not yet clear how S. marcescens PDL100 colonizes and infects corals. It is likely that to cause disease, the pathogen first needs to colonize and establish within the coral surface mucus layer.The coral surface mucus layer contains polymers of mixed origin. Coral mucus is made in the mucocytes of the polyp, where the photosynthate produced by the coral symbiotic dinoflagellate Symbiodinium spp. is converted into polymers that are excreted onto the coral surface (for a review, see reference 2). A glycoprotein is the major component of coral mucus from both hard and soft corals (16, 17, 19). The composition of the glycoprotein differs among coral species (4, 17). The mucus polymer of Acropora formosa, for example, contains 36 to 38% of neutral sugars, 18 to 22% of amino sugars, and 19 to 30% of amino acids; lipids make up 4.2% of the polymer (17). In the mucus of A. formosa, the oligosaccharide decorations (two to four sugar residues long) are attached to the polypeptide backbone by an O-glycosidic link to serine or threonine through the carbon 1 of mannose (16). The glycoproteins from A. formosa and Pseudopterogorgia americana corals contain terminal arabinose residues linked by a β1→2 or β1→3 bond. In the mucus of acroporid corals, arabinose, N-acetyl-glucosamine, mannose, glucose, galactose, N-acetyl-galactosamine, and fucose were the major sugars; serine and threonine were the major amino acids (4, 17). The elucidation of the chemical structure of coral mucus is complicated by the fact that the mucus contains excretions of coral mucocytes, extracellular substances produced by the associated microbiota as well as oligomers that may result from the degradation of these polymers (for reviews, see references 2 and 24).In this study, we tested the hypothesis that S. marcescens PDL100 is capable of a more extensive utilization of A. palmata mucus than other environmental or pathogenic isolates of S. marcescens. This hypothesis is based on the recent discoveries that pathogenic and commensal host-associated bacteria differ in their patterns of carbon source utilization during growth on components of the mucus that lines host surfaces (5, 26). These different strategies of mucus utilization may allow pathogenic bacteria to outcompete native residents and establish within the host''s mucosa (5, 13, 26). To test this hypothesis, growth of the strain PDL100 on coral mucus and enzymatic activities induced during growth on mucus were assayed and compared to those of pathogenic and environmental isolates of S. marcescens and three native coral-associated bacteria.     

Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus, Produced under Various Conditions

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 μg ml⁻¹. The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 μg of cereulide g⁻¹ (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 μg of cereulide ml⁻¹ in liquid medium at room temperature (21 ± 1°C) in 1 to 3 days, during the stationary growth phase when cell density was 2 × 10⁸ to 6 × 10⁸ CFU ml⁻¹. Cereulide production at temperatures at and below 8°C or at 40°C was minimal.     

Quantification of virus genes provides evidence for seed-bank populations of phycodnaviruses in Lake Ontario,Canada

Using quantitative PCR, the abundances of six phytoplankton viruses DNA polymerase (polB) gene fragments were estimated in water samples collected from Lake Ontario, Canada over 26 months. Four of the polB fragments were most related to marine prasinoviruses, while the other two were most closely related to cultivated chloroviruses. Two Prasinovirus-related genes reached peak abundances of >1000 copies ml⁻¹ and were considered ‘high abundance'', whereas the other two Prasinovirus-related genes peaked at abundances <1000 copies ml⁻¹ and were considered ‘low abundance''. Of the genes related to chloroviruses, one peaked at ca 1600 copies ml⁻¹, whereas the other reached only ca 300 copies ml⁻¹. Despite these differences in peak abundance, the abundances of all genes monitored were lowest during the late fall, winter and early spring; during these months the high abundance genes persisted at 100–1000 copies ml⁻¹ while the low abundance Prasinovirus- and Chlorovirus-related genes persisted at fewer than ca 100 copies ml⁻¹. Clone libraries of psbA genes from Lake Ontario revealed numerous Chlorella-like algae and two prasinophytes demonstrating the presence of candidate hosts for all types of viruses monitored. Our results corroborate recent metagenomic analyses that suggest that aquatic virus communities are composed of only a few abundant populations and many low abundance populations. Thus, we speculate that an ecologically important characteristic of phycodnavirus communities is seed-bank populations with members that can become numerically dominant when their host abundances reach appropriate levels.     

Real-Time PCR Assays for Quantification and Differentiation of Vibrio vulnificus Strains in Oysters and Water

Vibrio vulnificus is an autochthonous estuarine bacterium and a pathogen that is frequently transmitted via raw shellfish. Septicemia can occur within 24 h; however, isolation and confirmation from water and oysters require days. Real-time PCR assays were developed to detect and differentiate two 16S rRNA variants, types A and B, which were previously associated with environmental sources and clinical fatalities, respectively. Both assays could detect 10² to 10³ V. vulnificus total cells in seeded estuarine water and in oyster homogenates. PCR assays on 11 reference V. vulnificus strains and 22 nontarget species gave expected results (type A or B for V. vulnificus and negative for nontarget species). The relationship between cell number and cycle threshold for the assays was linear (R² = >0.93). The type A/B ratio of Florida clinical isolates was compared to that of isolates from oysters harvested in Florida waters. This ratio was 19:17 in clinical isolates and 5:8 (n = 26) in oysters harvested from restricted sites with poor water quality but was 10:1 (n = 22) in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB (both primer sets amplified), but no isolates from overlying waters were type AB. The real-time PCR assays were sensitive, specific, and quantitative in water samples and could also differentiate the strains in oysters without requiring isolation of V. vulnificus and may therefore be useful for rapid detection of the pathogen in shellfish and water, as well as further investigation of its population dynamics.     

A propidium monoazide–quantitative PCR method for the detection and quantification of viable Enterococcus faecalis in large-volume samples of marine waters

The development of rapid detection assays of cell viability is essential for monitoring the microbiological quality of water systems. Coupling propidium monoazide with quantitative PCR (PMA-qPCR) has been successfully applied in different studies for the detection and quantification of viable cells in small-volume samples (0.25–1.00 mL), but it has not been evaluated sufficiently in marine environments or in large-volume samples. In this study, we successfully integrated blue light-emitting diodes for photoactivating PMA and membrane filtration into the PMA-qPCR assay for the rapid detection and quantification of viable Enterococcus faecalis cells in 10-mL samples of marine waters. The assay was optimized in phosphate-buffered saline and seawater, reducing the qPCR signal of heat-killed E. faecalis cells by 4 log₁₀ and 3 log₁₀ units, respectively. Results suggest that high total dissolved solid concentration (32 g/L) in seawater can reduce PMA activity. Optimal PMA-qPCR standard curves with a 6-log dynamic range and detection limit of 10² cells/mL were generated for quantifying viable E. faecalis cells in marine waters. The developed assay was compared with the standard membrane filter (MF) method by quantifying viable E. faecalis cells in seawater samples exposed to solar radiation. The results of the developed PMA-qPCR assay did not match that of the standard MF method. This difference in the results reflects the different physiological states of E. faecalis cells in seawater. In conclusion, the developed assay is a rapid (～5 h) method for the quantification of viable E. faecalis cells in marine recreational waters, which should be further improved and tested in different seawater settings.