Human sewage identified as likely source of white pox disease of the threatened Caribbean elkhorn coral,Acropora palmata

Caribbean elkhorn coral, Acropora palmata, has been decimated in recent years, resulting in the listing of this species as threatened under the United States Endangered Species Act. A major contributing factor in the decline of this iconic species is white pox disease. In 2002, we identified the faecal enterobacterium, Serratia marcescens, as an etiological agent for white pox. During outbreaks in 2003 a unique strain of S. marcescens was identified in both human sewage and white pox lesions. This strain (PDR60) was also identified from corallivorious snails (Coralliophila abbreviata), reef water, and two non‐acroporid coral species, Siderastrea siderea and Solenastrea bournoni. Identification of PDR60 in sewage, diseased Acropora palmata and other reef invertebrates within a discrete time frame suggests a causal link between white pox and sewage contamination on reefs and supports the conclusion that humans are a likely source of this disease.     

Bacterial community associated with black band disease in corals

Black band disease (BBD) is a virulent polymicrobial disease primarily affecting massive-framework-building species of scleractinian corals. While it has been well established that the BBD bacterial mat is dominated by a cyanobacterium, the quantitative composition of the BBD bacterial mat community has not described previously. Terminal-restriction fragment length polymorphism (T-RFLP) analysis was used to characterize the infectious bacterial community of the bacterial mat causing BBD. These analyses revealed that the bacterial composition of the BBD mat does not vary between different coral species but does vary when different species of cyanobacteria are dominant within the mat. On the basis of the results of a new method developed to identify organisms detected by T-RFLP analysis, our data show that besides the cyanobacterium, five species of the division Firmicutes, two species of the Cytophaga-Flexibacter-Bacteroides (CFB) group, and one species of delta-proteobacteria are also consistently abundant within the infectious mat. Of these dominant taxa, six were consistently detected in healthy corals. However, four of the six were found in much higher numbers in BBD mats than in healthy corals. One species of the CFB group and one species of Firmicutes were not always associated with the bacterial communities present in healthy corals. Of the eight dominant bacteria identified, two species were previously found in clone libraries obtained from BBD samples; however, these were not previously recognized as important. Furthermore, despite having been described as an important component of the pathogenetic mat, a Beggiatoa species was not detected in any of the samples analyzed. These results will permit the dominant BBD bacteria to be targeted for isolation and culturing experiments aimed at deciphering the disease etiology.     

Human pathogen shown to cause disease in the threatened eklhorn coral Acropora palmata

Coral reefs are in severe decline. Infections by the human pathogen Serratia marcescens have contributed to precipitous losses in the common Caribbean elkhorn coral, Acropora palmata, culminating in its listing under the United States Endangered Species Act. During a 2003 outbreak of this coral disease, called acroporid serratiosis (APS), a unique strain of the pathogen, Serratia marcescens strain PDR60, was identified from diseased A. palmata, human wastewater, the non-host coral Siderastrea siderea and the corallivorous snail Coralliophila abbreviata. In order to examine humans as a source and other marine invertebrates as vectors and/or reservoirs of the APS pathogen, challenge experiments were conducted with A. palmata maintained in closed aquaria to determine infectivity of strain PDR60 from reef and wastewater sources. Strain PDR60 from wastewater and diseased A. palmata caused disease signs in elkhorn coral in as little as four and five days, respectively, demonstrating that wastewater is a definitive source of APS and identifying human strain PDR60 as a coral pathogen through fulfillment of Koch's postulates. A. palmata inoculated with strain PDR60 from C. abbreviata showed limited virulence, with one of three inoculated fragments developing APS signs within 13 days. Strain PDR60 from non-host coral S. siderea showed a delayed pathogenic effect, with disease signs developing within an average of 20 days. These results suggest that C. abbreviata and non-host corals may function as reservoirs or vectors of the APS pathogen. Our results provide the first example of a marine "reverse zoonosis" involving the transmission of a human pathogen (S. marcescens) to a marine invertebrate (A. palmata). These findings underscore the interaction between public health practices and environmental health indices such as coral reef survival.     

Characterization of the bacterial consortium associated with black band disease in coral using molecular microbiological techniques

The bacterial community associated with black band disease (BBD) of the scleractinian corals Diploria strigosa, Montastrea annularis and Colpophyllia natans was examined using culture-independent techniques. Two complementary molecular screening techniques of 16S rDNA genes [amplified 16S ribosomal DNA restriction analysis (ARDRA) of clone libraries and denaturing gradient gel electrophoresis (DGGE)] were used to give a comprehensive characterization of the community. Findings support previous studies indicating low bacterial abundance and diversity associated with healthy corals. A single cyanobacterial ribotype was present in all the diseased samples, but this was not the same as that identified from Phormidium corallyticum culture isolated from BBD. The study confirms the presence of Desulfovibrio spp. and sulphate-reducing bacteria that have previously been associated with the BBD consortium. However, the species varied between diseased coral samples. We found no evidence of bacteria from terrestrial, freshwater or human sources in any of the samples. We report the presence of previously unrecognized potential pathogens [a Cytophaga sp. and an alpha-proteobacterium identified as the aetiological agent of juvenile oyster disease (JOD)] that were consistently present in all the diseased coral samples. The molecular biological approach described here gives an increasingly comprehensive and more precise picture of the bacterial population associated with BBD. To understand the pathogenesis of BBD, our attention should be focused on the pervasive ribotypes identified in this study (the Cyanobacterium sp., the Cytophaga sp. and the JOD pathogen).     

Bacterial community structure associated with the Antarctic soft coral, Alcyonium antarcticum

The structure and composition of microbial communities inhabiting the soft coral Alcyonium antarcticum were investigated across three differentially contaminated sites within McMurdo Sound, Antarctica. Diverse microbial communities were revealed at all sites using culture-based analysis, denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene clone-library analysis, and FISH. Phylogenetic analysis of isolates and retrieved sequences demonstrated close affiliation with known psychrophiles from the Antarctic environment and high similarity to Gammaproteobacteria clades of sponge-associated microorganisms. The majority of bacteria detected with all techniques reside within the Gammaproteobacteria, although other phylogenetic groups including Alpha- and Betaproteobacteria, Bacteroidetes, Firmicutes, Actinomycetales, Planctomycetes, and Chlorobi and bacteria from the functional group of sulfate-reducing bacteria were also present. Multivariate (nMDS) analysis of DGGE banding patterns and principal component analysis of quantitative FISH data revealed no distinct differences in community composition between differentially contaminated sites. Rather, conserved coral-associated bacterial groups were observed within and between sites, providing evidence to support specific coral-microbial interactions. This is the first investigation of microbial communities associated with Antarctic soft corals, and the results suggest that spatially stable microbial associations exist across an environmental impact gradient.     

Experimental antibiotic treatment identifies potential pathogens of white band disease in the endangered Caribbean coral Acropora cervicornis

Coral diseases have been increasingly reported over the past few decades and are a major contributor to coral decline worldwide. The Caribbean, in particular, has been noted as a hotspot for coral disease, and the aptly named white syndromes have caused the decline of the dominant reef building corals throughout their range. White band disease (WBD) has been implicated in the dramatic loss of Acropora cervicornis and Acropora palmata since the 1970s, resulting in both species being listed as critically endangered on the International Union for Conservation of Nature Red list. The causal agent of WBD remains unknown, although recent studies based on challenge experiments with filtrate from infected hosts concluded that the disease is probably caused by bacteria. Here, we report an experiment using four different antibiotic treatments, targeting different members of the disease-associated microbial community. Two antibiotics, ampicillin and paromomycin, arrested the disease completely, and by comparing with community shifts brought about by treatments that did not arrest the disease, we have identified the likely candidate causal agent or agents of WBD. Our interpretation of the experimental treatments is that one or a combination of up to three specific bacterial types, detected consistently in diseased corals but not detectable in healthy corals, are likely causal agents of WBD. In addition, a histophagous ciliate (Philaster lucinda) identical to that found consistently in association with white syndrome in Indo-Pacific acroporas was also consistently detected in all WBD samples and absent in healthy coral. Treatment with metronidazole reduced it to below detection limits, but did not arrest the disease. However, the microscopic disease signs changed, suggesting a secondary role in disease causation for this ciliate. In future studies to identify a causal agent of WBD via tests of Henle–Koch''s postulates, it will be vital to experimentally control for populations of the other potential pathogens identified in this study.     

Gene expression associated with white syndromes in a reef building coral,Acropora hyacinthus

Background

Corals are capable of launching diverse immune defenses at the site of direct contact with pathogens, but the molecular mechanisms of this activity and the colony-wide effects of such stressors remain poorly understood. Here we compared gene expression profiles in eight healthy Acropora hyacinthus colonies against eight colonies exhibiting tissue loss commonly associated with white syndromes, all collected from a natural reef environment near Palau. Two types of tissues were sampled from diseased corals: visibly affected and apparently healthy.

Results

Tag-based RNA-Seq followed by weighted gene co-expression network analysis identified groups of co-regulated differentially expressed genes between all health states (disease lesion, apparently healthy tissues of diseased colonies, and fully healthy). Differences between healthy and diseased tissues indicate activation of several innate immunity and tissue repair pathways accompanied by reduced calcification and the switch towards metabolic reliance on stored lipids. Unaffected parts of diseased colonies, although displaying a trend towards these changes, were not significantly different from fully healthy samples. Still, network analysis identified a group of genes, suggestive of altered immunity state, that were specifically up-regulated in unaffected parts of diseased colonies.

Conclusions

Similarity of fully healthy samples to apparently healthy parts of diseased colonies indicates that systemic effects of white syndromes on A. hyacinthus are weak, which implies that the coral colony is largely able to sustain its physiological performance despite disease. The genes specifically up-regulated in unaffected parts of diseased colonies, instead of being the consequence of disease, might be related to the originally higher susceptibility of these colonies to naturally occurring white syndromes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1540-2) contains supplementary material, which is available to authorized users.     

Bacterial community structure in Apis florea larvae analyzed by denaturing gradient gel electrophoresis and 16S rRNA gene sequencing

This study characterizes the colonization and composition of bacterial flora in dwarf Asian honeybee (Apis florea) larvae and compares bacterial diversity and distribution among different sampling locations. A. florea larvae were collected from 3 locations in Chiang Mai province, Thailand. Bacterial DNA was extracted from each larva using the phenol–chloroform method. Denaturing gradient gel electrophoresis was performed, and the dominant bands were excised from the gels, cloned, and sequenced for bacterial species identification. The result revealed similarities of bacterial community profiles in each individual colony, but differences between colonies from the same and different locations. A. florea larvae harbor bacteria belonging to 2 phyla (Firmicutes and Proteobacteria), 5 classes (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Bacilli, and Clostridia), 6 genera (Clostridium, Gilliamella, Melissococcus, Lactobacillus, Saccharibacter, and Snodgrassella), and an unknown genus from uncultured bacterial species. The classes with the highest abundance of bacteria were Alphaproteobacteria (34%), Bacilli (25%), Betaproteobacteria (11%), Gammaproteobacteria (10%), and Clostridia (8%), respectively. Similarly, uncultured bacterial species were identified (12%). Environmental bacterial species, such as Saccharibacter floricola, were also found. This is the first study in which sequences closely related to Melissococcus plutonius, the causal pathogen responsible for European foulbrood, have been identified in Thai A. florea larvae.     

Composition of the oil-slime microbial community as determined by analysis of the 16S rRNA gene

Analysis of the 16S rRNA genes of the cultured microorganisms of industrial oil-slime revealed predominance (～85–90%) of the Gammaproteobacteria in the community of aerobic heterotrophs and specific oil-slime degraders. Relation of the isolated strains with members of the genera Pseudomonas, Stenotrophomonas, and Enterobacter was established. Analysis of the same gene in the total DNA from the oil-slime revealed greater microbial diversity (～20 operative taxonomic units determined by T-RFLP) than in the cultured part of the community, which included ～12 different colony types. Three major restriction fragments were found, with their total area ～50%. These results demonstrated the low morphological and phylogenetic diversity of the oil-slime bacterial community.     

Bacterioplankton community structure in the Arctic waters as revealed by pyrosequencing of 16S rRNA genes

Fjords and open oceans are two typical marine ecosystems in the Arctic region, where glacial meltwater and sea ice meltwater have great effects on the bacterioplankton community structure during the summer season. This study aimed to determine the differences in bacterioplankton communities between these two ecosystems in the Arctic region. We conducted a detailed census of microbial communities in Kongsfjorden (Spitsbergen) and the Chukchi Borderland using high-throughput pyrosequencing of the 16S rRNA gene. Gammaproteobacteria and Bacteroidetes were the dominant members of the bacterioplankton community in Kongsfjorden. By contrast, the most abundant bacterial groups in the surface seawater samples from the Chukchi Borderland were Alphaproteobacteria and Actinobacteria. Differences in bacterial communities were found between the surface and subsurface waters in the investigation area of the Chukchi Borderland, and significant differences in bacterial community structure were also observed in the subsurface water between the shelf and deep basin areas. These results suggest the effect of hydrogeographic conditions on bacterial communities. Ubiquitous phylotypes found in all the investigated samples belonged to a few bacterial groups that dominate marine bacterioplankton communities. The sequence data suggested that changes in environmental conditions result in abundant rare phylotypes and reduced amounts of other phylotypes.     

Cyanobacteria associated with coral black band disease in Caribbean and Indo-Pacific Reefs

For 30 years it has been assumed that a single species of cyanobacteria, Phormidium corallyticum, is the volumetrically dominant component of all cases of black band disease (BBD) in coral. Cyanobacterium-specific 16S rRNA gene primers and terminal restriction fragment length polymorphism analyses were used to determine the phylogenetic diversity of these BBD cyanobacteria on coral reefs in the Caribbean and Indo-Pacific Seas. These analyses indicate that the cyanobacteria that inhabit BBD bacterial mats collected from the Caribbean and Indo-Pacific Seas belong to at least three different taxa, despite the fact that the corals in each case exhibit similar signs and patterns of BBD mat development.     

Analysis of bacterial community structure in the natural circulation system wastewater bioreactor by using a 16S rRNA gene clone library

A variety of physical and chemical parameters are routinely monitored during operation of the Natural Circulation System, a wastewater purification bioreactor in which only natural materials and no synthetic chemicals are used. However, the microbial community structures existing in the Natural Circulation System have not been well characterized. Thus, bacterial community structure and composition in this system were studied using clone library analysis of 16S ribosomal RNA genes amplified using PCR with universal bacterial primer sets. The PCR products were then subcloned into the pGEM-T vector. Each unique restriction fragment length polymorphism pattern, created by using two pairs of restriction endonucleases, was designated as an operational taxonomic unit (OTU). The Natural Circulation System comprises five tanks, the second and third of which play a major role in the bioreactor. Clone library pro-files and principal component analysis revealed differences in the bacterial community structures of the second (anaerobic chamber) and the third (aerobic chamber) tanks. However, the beta-proteobacteria, Bacteroidetes/ Chlorobi and gamma-proteobacteria groups were dominant in both tanks. Bacterial composition was more complex in the second tank (107 OTUs) than in the third tank (68 OTUs). Of a total of 154 OTUs in the clone libraries, only 21 were common to the two tanks. The results obtained in this study should provide important information for future research into and management of the Natural Circulation System wastewater bioreactor.     

A meta-analysis of 16S rRNA gene clone libraries from the polymicrobial black band disease of corals

Black band disease (BBD) is a polymicrobial disease affecting corals on reefs worldwide. Since 2002, researchers have constructed clone libraries from the BBD consortium using 16S rRNA gene primers targeting a variety of phyla. In the present study, a meta-analysis was conducted of published libraries from 2002 through the present that contain bacterial sequence data associated with individual clones and BBD samples. The libraries analyzed were from 87 BBD samples, collected from 16 species of scleractinian corals in 10 different geographic locations, and included 327 unique operational taxonomic units (OTUs). One OTU (cyanobacterial) was present in 62 (71%) samples, and three (one Cytophaga-Flavobacter-Bacteriodetes and two alphaproteobacterial) were present in 11 (13%) of the samples. The remaining 323 unique OTUs were present in <10% of the samples. The Alphaproteobacteria was the most diversely represented group. Analysis of clone libraries using nonmetric multidimensional scaling indicated strong regional specificity of BBD microbial populations, but limited host coral specificity. The results of this analysis support the hypotheses that: (1) a specific cyanobacterium may be the primary pathogen of BBD; (2) additional functional groups, required for BBD pathobiology, are represented by variable opportunistic species; and (3) opportunistic BBD microorganisms are primarily derived from the environment.     

Characterization of Cytophaga-Flavobacteria community structure in the Bering Sea by cluster-specific 16S rRNA gene amplification analysis

A newly designed Cytophaga-Flavobacteria-specific 16S rRNA gene primer pair was employed to investigate the CF community structure in the Bering Sea, revealing a previously unknown and unexpected high CF diversity in this high latitude cold sea. In total, 56 clones were sequenced and 50 unique CF 16 rRNA gene fragments were obtained, clustering into 16 CF subgroups, including nine cosmopolitan subgroups, five psychrophilic subgroups, and two putatively autochthonous subgroups. The majority of sequences (82%) were closely related to uncultured CF species and could not be classified into known CF genera, indicating the presence of a large number of so-far uncultivated CF species in the Bering Sea.