Carnobacterium maltaromaticum associated with meningoencephalitis and otitis in stranded common thresher sharks (Alopias vulpinus).

Juvenile common thresher sharks (Alopias vulpinus) have been recently stranding along the California coastline. Using Illumina sequencing of the bacterial 16S rRNA gene along with necropsy, cytological, bacteriological, and histological techniques, we screened microbial communities and described lesions characterizing affected sharks with the purpose of identifying potential pathogen sources and pathologic processes. Histopathological assessment of moribund sharks revealed severe meningoencephalitis, as previously described in stranded salmon sharks (Lamna ditropis), along with inflammation of the inner ear and subcutaneous tissues surrounding the endolymphatic ducts. Furthermore, inflamed areas were characterized by the prevalence of Carnobacterium maltaromaticum, suggesting this bacterium as a potential pathogen that gains access to the inner ear through the endolymphatic ducts, with subsequent spread into the brain. The absence or low abundance of this bacterium in the spiral valve in both healthy and infected sharks suggests that Carnobacterium is not a commensal member of their digestive communities and the spiral valve is unlikely to be the source of the pathogen. Furthermore, phylogenetic analysis suggests that C. maltaromaticum strains isolated from diseased sharks have minimal genetic variation and differ from other strains originating from food or diseased teleosts. While a C. maltaromaticum-like organism has previously been associated with meningoencephalitis in salmon shark strandings, this is the first study to report common thresher shark strandings associated with C. maltaromaticum, involving the endolymphatic ducts as portals of entry to the brain.

Impact of wave action and rainfall on incidence and antibiotic resistance of total coliforms in Southern California beaches.

Coliforms are important bacterial contamination indicators in recreational waters. Little is known about the antibiotic resistance of coliforms from Southern California beaches. This study examined the numbers of coliforms as well as the incidence of antibiotic-resistant coliforms in beaches with restricted and non-restricted wave action by sampling from the shores of both types of beaches following dry and wet weather. Total coliforms were selected by membrane filtration onto mEndo agar and then enumerated. Randomly selected isolates from each location were screened for resistance to nine classes of antibiotics by disk diffusion, and the multiple antibiotic resistance (MAR) index was calculated. Numbers of total coliforms were significantly higher following rain compared to dry weather. Total coliform numbers were not significantly elevated at non-restricted wave action sites. Restricted wave action sites had a 78.5% increase in MAR index following wet weather compared to dry weather. Resistance to ampicillin was observed in almost 50% of isolates and was not significantly impacted by wave action or weather. Minimum inhibitory concentration testing revealed that many isolates were highly resistant to ampicillin. This study is the first to report on the antibiotic resistance of coliforms found in Southern California beaches and highlights the prevalence of ampicillin resistance.

Schizasterid Heart Urchins Host Microorganisms in a Digestive Symbiosis of Mesozoic Origin.

Because of their lifestyles, abundance, and feeding habits, infaunal marine deposit feeders have a significant impact on the ocean floor. As these animals also ingest microorganisms associated with their sediment and seawater diet, their digestive tract usually contains a diverse array of bacteria. However, while most of these microorganisms are transients, some may become part of a resident gut microbiome, in particular when sheltered from the main flow of digesta in specialized gut compartments. Here, we provide an in-depth analysis of the structure and contents of the intestinal caecum (IC), a hindgut diverticulum found exclusively in schizasterid heart urchins (Echinoidea: Spatangoida: Schizasteridae). Based on specimens of Brisaster townsendi, in addition to various other schizasterid taxa, our structural characterization of the IC shows that the organ is a highly specialized gut compartment with unique structural properties. Next generation sequencing shows that the IC contains a microbial population composed predominantly of Bacteroidales, Desulfobacterales, and Spirochaetales. The microbiome of this gut compartment is significantly different in composition and lower in diversity than the microbial population in the sediment-filled main digestive tract. Inferences on the function and evolution of the IC and its microbiome suggest that this symbiosis plays a distinct role in host nutrition and that it evolved at least 66 million years ago during the final phase of the Mesozoic.

Broad Environmental Tolerance for a Salicola Host-Phage Pair Isolated from the Cargill Solar Saltworks, Newark, CA, USA.

Phages greatly influence the ecology and evolution of their bacterial hosts; however, compared to hosts, a relatively low number of phages, especially halophilic phages, have been studied. This study describes a comparative investigation of physicochemical tolerance between a strain of the halophilic bacterium, Salicola, isolated from the Cargill Saltworks (Newark, CA, USA) and its associated phage. The host grew in media between pH 6-8.5, had a salinity growth optimum of 20% total salts (ranging from 10%-30%) and an upper temperature growth limit of 48 °C. The host utilized 61 of 190 substrates tested using BIOLOG Phenotype MicroArrays. The CGφ29 phage, one of only four reported Salicola phages, is a DNA virus of the Siphoviridae family. Overall, the phage tolerated a broader range of environmental conditions than its host (salinity 0-30% total salts; pH 3-9; upper thermal limit 80 °C) and is the most thermotolerant halophilic phage ever reported. This study is the most comprehensive investigation to date of a Salicola host-phage pair and provides novel insights into extreme environmental tolerances among bacteriophages.

Characterization of Chemosynthetic Microbial Mats Associated with Intertidal Hydrothermal Sulfur Vents in White Point, San Pedro, CA, USA.

The shallow-sea hydrothermal vents at White Point (WP) in Palos Verdes on the southern California coast support microbial mats and provide easily accessed settings in which to study chemolithoautotrophic sulfur cycling. Previous studies have cultured sulfur-oxidizing bacteria from the WP mats; however, almost nothing is known about the in situ diversity and activity of the microorganisms in these habitats. We studied the diversity, micron-scale spatial associations and metabolic activity of the mat community via sequence analysis of 16S rRNA and aprA genes, fluorescence in situ hybridization (FISH) microscopy and sulfate reduction rate (SRR) measurements. Sequence analysis revealed a diverse group of bacteria, dominated by sulfur cycling gamma-, epsilon-, and deltaproteobacterial lineages such as Marithrix, Sulfurovum, and Desulfuromusa. FISH microscopy suggests a close physical association between sulfur-oxidizing and sulfur-reducing genotypes, while radiotracer studies showed low, but detectable, SRR. Comparative 16S rRNA gene sequence analyses indicate the WP sulfur vent microbial mat community is similar, but distinct from other hydrothermal vent communities representing a range of biotopes and lithologic settings. These findings suggest a complete biological sulfur cycle is operating in the WP mat ecosystem mediated by diverse bacterial lineages, with some similarity with deep-sea hydrothermal vent communities.

Stable Isotope Phenotyping via Cluster Analysis of NanoSIMS Data As a Method for Characterizing Distinct Microbial Ecophysiologies and Sulfur-Cycling in the Environment.

Stable isotope probing (SIP) is a valuable tool for gaining insights into ecophysiology and biogeochemical cycling of environmental microbial communities by tracking isotopically labeled compounds into cellular macromolecules as well as into byproducts of respiration. SIP, in conjunction with nanoscale secondary ion mass spectrometry (NanoSIMS), allows for the visualization of isotope incorporation at the single cell level. In this manner, both active cells within a diverse population as well as heterogeneity in metabolism within a homogeneous population can be observed. The ecophysiological implications of these single cell stable isotope measurements are often limited to the taxonomic resolution of paired fluorescence in situ hybridization (FISH) microscopy. Here we introduce a taxonomy-independent method using multi-isotope SIP and NanoSIMS for identifying and grouping phenotypically similar microbial cells by their chemical and isotopic fingerprint. This method was applied to SIP experiments in a sulfur-cycling biofilm collected from sulfidic intertidal vents amended with (13)C-acetate, (15)N-ammonium, and (33)S-sulfate. Using a cluster analysis technique based on fuzzy c-means to group cells according to their isotope ((13)C/(12)C, (15)N/(14)N, and (33)S/(32)S) and elemental ratio (C/CN and S/CN) profiles, our analysis partitioned ~2200 cellular regions of interest (ROIs) into five distinct groups. These isotope phenotype groupings are reflective of the variation in labeled substrate uptake by cells in a multispecies metabolic network dominated by Gamma- and Deltaproteobacteria. Populations independently grouped by isotope phenotype were subsequently compared with paired FISH data, demonstrating a single coherent deltaproteobacterial cluster and multiple gammaproteobacterial groups, highlighting the distinct ecophysiologies of spatially-associated microbes within the sulfur-cycling biofilm from White Point Beach, CA.

Lignocellulose-responsive bacteria in a southern California salt marsh identified by stable isotope probing.

Carbon cycling by microbes has been recognized as the main mechanism of organic matter decomposition and export in coastal wetlands, yet very little is known about the functional diversity of specific groups of decomposers (e.g., bacteria) in salt marsh benthic trophic structure. Indeed, salt marsh sediment bacteria remain largely in a black box in terms of their diversity and functional roles within salt marsh benthic food web pathways. We used DNA stable isotope probing (SIP) utilizing (13)C-labeled lignocellulose as a proxy to evaluate the fate of macrophyte-derived carbon in benthic salt marsh bacterial communities. Overall, 146 bacterial species were detected using SIP, of which only 12 lineages were shared between enriched and non-enriched communities. Abundant groups from the (13)C-labeled community included Desulfosarcina, Spirochaeta, and Kangiella. This study is the first to use heavy-labeled lignocellulose to identify bacteria responsible for macrophyte carbon utilization in salt marsh sediments and will allow future studies to target specific lineages to elucidate their role in salt marsh carbon cycling and ultimately aid our understanding of the potential of salt marshes to store carbon.

Patterns of microbial diversity along a salinity gradient in the Guerrero Negro solar saltern, Baja CA Sur, Mexico.

The goal of this study was to use environmental sequencing of 16S rRNA and bop genes to compare the diversity of planktonic bacteria and archaea across ponds with increasing salinity in the Exportadora de Sal (ESSA) evaporative saltern in Guerrero Negro, Baja CA S., Mexico. We hypothesized that diverse communities of heterotrophic bacteria and archaea would be found in the ESSA ponds, but that bacterial diversity would decrease relative to archaea at the highest salinities. Archaeal 16S rRNA diversity was higher in Ponds 11 and 12 (370 and 380 g l(-1) total salts, respectively) compared to Pond 9 (180 g l(-1) total salts). Both Pond 11 and 12 communities had high representation (47 and 45% of clones, respectively) by Haloquadratum walsbyi-like (99% similarity) lineages. The archaeal community in Pond 9 was dominated (79%) by a single uncultured phylotype with 99% similarity to sequences recovered from the Sfax saltern in Tunisia. This pattern was mirrored in bop gene diversity with greater numbers of highly supported phylotypes including many Haloquadratum-like sequences from the two highest salinity ponds. In Pond 9, most bop sequences, were not closely related to sequences in databases. Bacterial 16S rRNA diversity was higher than archaeal in both Pond 9 and Pond 12 samples, but not Pond 11, where a non-Salinibacter lineage within the Bacteroidetes >98% similar to environmental clones recovered from Lake Tuz in Turkey and a saltern in Chula Vista, CA was most abundant (69% of community). This OTU was also the most abundant in Pond 12, but only represented 14% of clones in the more diverse pond. The most abundant OTU in Pond 9 (33% of community) was 99% similar to an uncultured gammaproteobacterial clone from the Salton Sea. Results suggest that the communities of saltern bacteria and archaea vary even in ponds with similar salinity and further investigation into the ecology of diverse, uncultured halophile communities is warranted.

Characterization of halophiles isolated from solar salterns in Baja California, Mexico.

Solar salterns are extreme hypersaline environments that are five to ten times saltier than seawater (150-300 g L(-1) salt concentration) and typically contain high numbers of halophiles adapted to tolerate such extreme hypersalinity. Thirty-five halophile cultures of both Bacteria and Archaea were isolated from the Exportadora de Sal saltworks in Guerrero Negro, Baja California, Mexico. 16S rRNA sequence analysis showed that these cultured isolates included members belonging to the Halorubrum, Haloarcula, Halomonas, Halovibrio, Salicola, and Salinibacter genera and what may represent a new archaeal genus. For the first time, metabolic substrate usage of halophile isolates was evaluated using the non-colorimetric BIOLOG Phenotype MicroArray plates. Unique carbon substrate usage profiles were observed, even for closely related Halorubrum species, with bacterial isolates using more substrates than archaeal cultures. Characterization of these isolates also included morphology and pigmentation analyses, as well as salinity tolerance over a range of 50-300 g L(-1) salt concentration. Salinity optima varied between 50 and 250 g L(-1) and doubling times varied between 1 and 12 h.

Spatial and temporal variability in a stratified hypersaline microbial mat community.

Hypersaline microbial mat communities have recently been shown to be more diverse than once thought. The variability in community composition of hypersaline mats, both in terms of spatial and temporal dimensions, is still poorly understood. Because this information is essential to understanding the complex biotic and abiotic interactions within these communities, terminal restriction fragment analysis and 16S rRNA gene sequencing were used to characterize the near-surface community of a hypersaline microbial mat in Guerrero Negro, Mexico. Core samples were analyzed to assay community variability over large regional scales (centimeter to kilometer) and to track depth-related changes in population distribution at 250-microm intervals over a diel period. Significant changes in total species diversity were observed at increasing distances across the mat surface; however, key species (e.g. Microcoleus sp.) were identified throughout the mat. The vertical position and abundance of >50% of the 60 peaks detected varied dramatically over a diel cycle, including Beggiatoa sp., cyanobacteria, Chloroflexus sp., Halochromatium sp., Bacteroidetes sp. and several as-yet-identified bacteria. Many of these migrations correlated strongly with diel changes in redox conditions within the mat, contributing to strong day-night community structure differences.

High rates of sulfate reduction in a low-sulfate hot spring microbial mat are driven by a low level of diversity of sulfate-respiring microorganisms.

The importance of sulfate respiration in the microbial mat found in the low-sulfate thermal outflow of Mushroom Spring in Yellowstone National Park was evaluated using a combination of molecular, microelectrode, and radiotracer studies. Despite very low sulfate concentrations, this mat community was shown to sustain a highly active sulfur cycle. The highest rates of sulfate respiration were measured close to the surface of the mat late in the day when photosynthetic oxygen production ceased and were associated with a Thermodesulfovibrio-like population. Reduced activity at greater depths was correlated with novel populations of sulfate-reducing microorganisms, unrelated to characterized species, and most likely due to both sulfate and carbon limitation.

Linkage of high rates of sulfate reduction in Yellowstone hot springs to unique sequence types in the dissimilatory sulfate respiration pathway.

Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89 degrees C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.

UV-acclimation responses in natural populations of cyanobacteria (Calothrix sp.).

Phenotypic acclimation to changing conditions is typically thought to be beneficial to organisms in the environment. UV radiation is an important parameter affecting photosynthetic organisms in natural environments. We measured the response of photosynthetic carbon fixation in populations of cyanobacteria inhabiting a hot spring following acclimation to different UV treatments. These two very closely related populations of cyanobacteria, differing in their content of the extracellular UV-screening pigment scytonemin, were acclimated in situ under natural solar irradiance modified by filters that excluded both UVA/B, only UVB or transmitted both UVA/B. Cells from each preacclimation treatment were subsequently assayed for photosynthetic performance under all UV conditions (incubation treatment) giving a two-factor experimental design for each population. No acclimation filter treatment effects were observed even after two months under different acclimation treatments. This suggests that UV photoacclimation does not occur in either of these populations, regardless of the presence of scytonemin. By contrast, cells showed significant UV-inhibition during 1 h incubations under full sun. The population with high levels of scytonemin usually had lower rates of photosynthetic carbon fixation than the scytonemin-lacking population. However, the degree of UV inhibition, especially UVA inhibition, was higher for the cells without scytonemin pigment. These results suggest that closely related natural cyanobacterial populations respond differently to natural irradiance conditions and may be adopting different strategies of UV tolerance.

The synthesis of the UV-screening pigment, scytonemin, and photosynthetic performance in isolates from closely related natural populations of cyanobacteria (Calothrix sp.).

Two populations of the cyanobacterium Calothrix sp. found in Yellowstone thermal spring outflows differ greatly in their contents of scytonemin, a UV-screening pigment, and in their photosynthetic carbon assimilation rates. Clonal isolates from both populations were used to investigate these phenotypic differences. Identical partial 16S rDNA sequences ( approximately 900 bp) suggest a very close relationship between the two Calothrix populations and indicate that environmental differences may, in part, explain the field observations. The effects of native spring water on scytonemin synthesis and photosynthesis were tested during experiments using plated cells. Results show differences in the spring water environment were at least partly responsible for the differences in scytonemin content observed in the field. Furthermore, spring water effects on photosynthetic performance suggest adaptation in these strains to their spring of origin. Controlled experiments performed using cultures grown in artificial liquid medium showed no significant difference in photosynthetic carbon uptake between strains. However, significant differences were detected in their ability to synthesize scytonemin indicating genetic differences between populations. These findings suggest that both genetic and environmental differences are responsible for the naturally occurring variation in scytonemin content and photosynthetic ability in these two closely related populations.

Effect of environmental factors on the synthesis of scytonemin, a UV-screening pigment, in a cyanobacterium (Chroococcidiopsis sp.).

Abstract. The UV-screening pigment scytonemin is found in many species of ensheathed cyanobacteria. Past work has shown that the pigment is synthesized in response to exposure to UV-A irradiance. This study investigated the effect of other correlated stress factors including heat, osmotic and oxidative stress on the synthesis of scytonemin in a clonal cyanobacterial isolate ( Chroococcidiopsis sp.) from an epilithic desert crust. Stress experiments were carried out both in conjunction with UV-A irradiance and in isolation. Increases in both temperature and photooxidative conditions in conjunction with UV-A caused a synergistic increase in the rate of scytonemin production. In contrast, increased salt concentration under UV-A irradiance inhibited scytonemin synthesis. However, unlike the responses to temperature and oxidative stress, cells synthesized low levels of scytonemin under osmotic stress in the absence of scytonemin-inducing irradiance. These results suggest that scytonemin induction may be regulated as a part of a complex stress response pathway in which multiple environmental signals affect its synthesis.