第885回千葉医学会例会・千葉大学第二外科例会

<p><b>A</b>. NMDS ordination of 16S rRNA gene-derived microbial community structure. Similarity profile analysis, an <i>a priori</i> statistical approach that uses permutation to identify groups of communities that are more dissimilar than expected by chance, identified two distinct clusters of communities. Ellipses represent the 95% confidence intervals around the centroid for each cluster (the spatial mean in NMDS space of the communities belonging to each cluster). Lines emanating from the centroids indicate to which cluster each community belongs. Bacterial families well-correlated with the ordination (r² > 0.40) are displayed; vector length is proportional to the Pearson correlation coefficient for each family and vector direction corresponds to the direction of increasing abundance relative to the ordinated communities. Legend indicates the dune from which each ordinated community originated. Final 2-dimensional stress of the ordination is 0.12. <b>B</b>. Linear discriminant analysis (LDA) of bacterial classes indicates that the two clusters of microbial communities identified by similarity profile analysis are driven by the disparity between a high abundance of <i>Gammaproteobacteria</i> in one set of communities and more diverse population in the other set of communities. Only classes with effect size > 2.0 are displayed. <b>C</b>. NMDS ordination is based only on samples for which environmental parameters were measured. Parameters with r² > 0.1 are displayed. Final 2-dimensional stress of the ordination is 0.07.</p

Anr absences decrease attachment and aggregation in <i>P. extremaustralis</i>.

<p>(A) Attachment to polystyrene plates in 0.5 NE2 medium supplemented with glucose, KNO₃ and casaminoacids. Values represent media ± SD of 5 independent experiments with 12 wells per strain. Different letters showed significant differences among strains (P<0.05) using ANOVA (B). Autoaggregation experiment. The cells were incubated during 2 h without agitation and a culture sample was stained with DAPI. I) wild type strain. II) <i>anr</i> mutant strain. III) <i>anr</i> complemented strain. All observations were performed at 1000X. (C). Slide culture assay to investigate twitching motility. Cells were incubated for 15 h. I) wild type strain. II) <i>anr</i> mutant strain. III) <i>anr</i> complemented strain. Arrows showed rafts in the edge of the culture. All observations were performed using contrast phase microscopy at 400× magnification.</p

Effect of <i>anr</i> mutation on expression of <i>pilG</i> and <i>morA</i> genes in microaerobic cultures.

<p>(A) qPCR Real Time experiments were carried out in microaerobic cultures. Values were expressed as the ratio between the raw level of expression of each target gene and the 16S rRNA gene, and represent the mean ± SD of three independent experiments. The asterisk (*) denotes significant differences (P<0.05) using the Student’s t test.</p

Organization of the P. <i>extremaustralis</i> intergenic <i>pilG</i> and <i>morA</i> region showing putative Anr boxes.

<p>(A) The sequences −35 and −10 of a probable σ⁷⁰ promoter are underlined. The <i>pilG</i> and <i>morA</i> start codons and the start or the stop codon of the neighbors genes are shown by boldface type and arrows indicate the direction of the transcription. Anr boxes are boxed. (B) Sequence logo of 5 Anr boxes located in genes influenced by Anr in <i>P. extremaustralis</i> were used to generate the Anr position weight matrix sequence.</p

Biofilm parameters of wild type and <i>anr</i> mutant <i>of P. extremaustralis</i>. Values represent the mean ± SD of three independent experiments.

<p>The asterisk (*) denotes significant differences among strains (P<0.05) using the Student’s t test.</p

Swimming motility is increased in <i>anr</i> mutant strain.

<p>(A) Swimming motility. The asterisk (*) denotes significant differences (P<0.05) using the Student’s t test. (B) Transmission electron microscopy. I) wild type strain. II) <i>anr</i> mutant strain. Observations were performed at 46000X magnification.</p

Limited impact of weathered residues from the Deepwater Horizon oil spill on the gut-microbiome and foraging behavior of sheepshead minnows (<i>Cyprinodon variegatus</i>)

The Deepwater Horizon disaster of April 2010 was the largest oil spill in U.S. history and exerted catastrophic effects on several ecologically important fish species in the Gulf of Mexico (GoM). Within fish, the microbiome plays a key symbiotic role in maintaining host health and aids in acquiring nutrients, supporting immune function, and modulating behavior. The aim of this study was to examine if exposure to weathered oil might produce significant shifts in fish gut-associated microbial communities as determined from taxa and genes known for hydrocarbon degradation, and whether foraging behavior was affected. The gut microbiome (16S rRNA and shotgun metagenomics) of sheepshead minnow (Cyprinodon variegatus) was characterized after fish were exposed to oil in High Energy Water Accommodated Fractions (HEWAF; tPAH = 81.1 ± 12.4 µg/L) for 7 days. A foraging behavioral assay was used to determine feeding efficiency before and after oil exposure. The fish gut microbiome was not significantly altered in alpha or beta diversity. None of the most abundant taxa produced any significant shifts as a result of oil exposure, with only rare taxa showing significant shifts in abundance between treatments. However, several bioindicator taxa known for hydrocarbon degradation were detected in the oil treatment, primarily Sphingomonas and Acinetobacter. Notably, the genus Stenotrophomonas was detected in high abundance in 16S data, which previously was not described as a core member of fish gut microbiomes. Data also demonstrated that behavior was not significantly affected by oil exposure. Potential low bioavailability of the oil may have been a factor in our observation of minor shifts in taxa and no behavioral effects. This study lays a foundation for understanding the microbiome of captive sheepshead minnows and indicates the need for further research to elucidate the responses of the fish gut-microbiome under oil spill conditions.</p

Results of ANOVA and PERMANOVA analyses.

<p>P values less than 0.05 are bolded.</p

Comparison of the relative abundance of phyla between cultivation, deep 16S rRNA gene amplicon sequencing and metagenomics.

<p>Comparison of the relative abundance of phyla between cultivation, deep 16S rRNA gene amplicon sequencing and metagenomics.</p

Heatmap showing the most abundant OTUs in all samples.

<p>OTU names are given at the lowest available taxonomic level; OTUs that could not be classified below the level of order are termed Unclassified. Dendrograms were generated using hierarchical clustering with complete linkage. Plot indicates the relative fraction of sequences in each sample that were classified as <i>Gammaproteobacteria</i> at the level of class. Sample key: dune name; location on dune face (C = crest, M = middle, B = base); sample number.</p