Culture Enrichment Combined With Long-Read Sequencing Facilitates Genomic Understanding of Hadal Sediment Microbes

Culture enrichment was developed to discover the uncharted microbial species in the environmental microbiota. Yet this strategy has not been widely used to study microbes of deep-sea environments. Here, we report the cultivation and metagenomic analysis of oceanic sediment microbiota collected from 6,477 m deep in the Mariana Trench. The sediment samples were cultured anaerobically in the laboratory for 4 months, before being subjected to full-length 16S rRNA gene sequencing using the PacBio technique and metagenome sequencing using both the Illumina and Oxford Nanopore techniques. The 16S rRNA gene analyses revealed 437 operational taxonomic units specific to the cultured microbes, despite the lower diversity of the cultured microbiota in comparison with the original. Metagenome analyses revealed the prevalence of functions related to respiration, energy production, and stress response in the cultured microbes, suggesting these functions may contribute to microbial proliferation under laboratory conditions. Binning of the assembled metagenome contigs of the cultured microbiota generated four nearly complete genomes affiliated to yet unclassified species under the genera Alcanivorax, Idiomarina, Sulfitobacter, and Erythrobacter. Excepting Alcanivorax, the other three genera were almost undetectable in the original samples and largely enriched in the cultured samples. The four genomes possessed a variety of genes for carbohydrate utilization and nitrite reduction, pointing to an ability to respire diverse carbon sources using nitrite as the final electron acceptor. Taken together, the findings suggest that a combination of culture enrichment and long-read sequencing is an ideal way to mine novel microbial species in the hadal environment, particularly species that are rare in their native environmental niches, and thus expand our understanding of the hadal microbial diversity and function.

Microbiome population dynamics of cold smoked sockeye salmon during refrigerated storage and after culture enrichment

Cold smoked salmon is a ready-to-eat seafood product of high commercial importance. The processing and storage steps facilitate the introduction, growth and persistence of foodborne pathogens and spoilage bacteria. The growth of commensal bacteria during storage and once the product is opened also influence the quality and safety of cold smoked salmon. Here we investigated the microbial community through targeted 16s rRNA gene and shotgun metagenomic sequencing, as means to better understand the interactions among bacteria in cold smoked salmon. Cold smoked salmon samples were tested over 30 days of aerobic storage at 4℃ and cultured at each timepoint in buffered Listeria enrichment broth (BLEB) commonly used to detect Listeria in foods. The microbiomes were comprised of Firmicutes and Proteobacteria namely, Carnobacterium , Brochothrix , Pseudomonas , Serratia , and Psychrobacter . Pseudomonas species were the most diverse species with 181 taxa identified. Additionally, we identified potential homologs to 10 classes of bacteriocins in microbiomes of cold smoked salmon stored at 4°C and corresponding BLEB culture enrichments. The findings presented here contribute to our understanding of microbiome population dynamics in cold smoked salmon, including changes in bacterial taxa during aerobic cold storage and after culture enrichment. This may facilitate improvements to pathogen detection and quality preservation of this food.

Flow-Through Electrochemical Biosensor for the Detection of Listeria monocytogenes Using Oligonucleotides

Consumption of food contaminated by Listeria monocytogenes can result in Listeriosis, an illness with hospitalization rates of 94% and mortality rates up to 30%. As a result, U.S. regulatory agencies governing food safety retain zero-tolerance policies for L. monocytogenes. However, detection at such low concentrations often requires strategies such as increasing sample size or culture enrichment. A novel flow-through immunoelectrochemical biosensor has been developed for Escherichia coli O157:H7 detection in 1 L volumes without enrichment. The current work further augments this biosensor’s capabilities to (1) include detection of L. monocytogenes and (2) accommodate genetic detection to help overcome limitations based upon antibody availability and address specificity errors in phenotypic assays. Herein, the conjugation scheme for oligo attachment and the conditions necessary for genetic detection are laid forth while results of the present study demonstrate the sensor’s ability to distinguish L. monocytogenes DNA from L. innocua with a limit of detection of ~2 × 104 cells/mL, which agrees with prior studies. Total time for this assay can be constrained to <2.5 h because a timely culture enrichment period is not necessary. Furthermore, the electrochemical detection assay can be performed with hand-held electronics, allowing this platform to be adopted for near-line monitoring systems.

INDIVIDUALIZATION AND SOCIALIZATION OF PRESCHOOL CHILDREN

Changes in the political, social and economic spheres of modern Kazakhstan society dictate the need to increase attention to the socialization of preschool children in the family and preschool organizations. The integrity of the pedagogical process is understood as the integrity of the processes of socialization and individualization of the preschool child, preservation of the child's nature and its development in culture, enrichment of individual cultural experience in the process of inclusion in the socio-cultural experience, unity of development and education. The modern pedagogical process is designed as a system of conditions that allow each child to realize individual needs and at the same time interact with the children's community. The organization of children's activities initiates the creation of children's associations in which each child performs a favorite function and simultaneously cooperates with other children. In such an educational space, the processes of socialization and individualization leading to preschool age harmoniously complement each other.

Colony-Forming Unit Spreadplate Assay versus Liquid Culture Enrichment-Polymerase Chain Reaction Assay for the Detection of Bacillus Endospores in Soils

A liquid culture enrichment-polymerase chain reaction (E-PCR) assay was investigated as a potential tool to overcome inhibition by chemical component, debris, and background biological impurities in soil that were affecting detection assay performance for soil samples containing Bacillus atrophaeus subsp. globigii (a surrogate for B. anthracis). To evaluate this assay, 9 g of matched sets of three different soil types (loamy sand [sand], sandy loam [loam] and clay) was spiked with 0, ~4.5, 45, 225, 675 and 1350 endospores. One matched set was evaluated using a previously published endospore concentration and colony-forming unit spreadplate (CFU-S) assay and the other matched set was evaluated using an E-PCR assay to investigate differences in limits of detection between the two assays. Data illustrated that detection using the CFU-S assay at the 45-endospore spike level started to become sporadic whereas the E-PCR assay produced repeatable detection at the ~4.5-endospore spike concentration. The E-PCR produced an ~2-log increase in sensitivity and required slightly less time to complete than the CFU-S assay. This study also investigated differences in recovery among pure and blended sand and clay soils and found potential activation of B. anthracis in predominately clay-based soils.