A Laboratory Manual for the Preparation of Chemical Reagents, Solutions and Special Indicators

A Laboratory Manual for the Preparation of Chemical Reagents, Solutions and Special Indicators. ETHIOPIAN BIODIVERSITY INSTITUTE, Microbial Biodiversity Directorate. This manual includes detailed view of the following: Laboratory Safety Precautions, Chemical Reagent Preparation, Buffer and Chemical Solution Preparation, Staining Solution Preparation, Indicators and Dye Solution Preparation, Preparation of Special Reagents, Known and Suspected List of Carcinogens and References.

Characterization of Biological Aerosols Over Northeast India: A Metagenomic Approach

Northeast India is considered as one of the major biodiversity hotspots in the world but the region is underexplored for their microbial biodiversity. Extensive characterization of biological aerosol (bioaerosol) samples collected from various locations of Northeast India was carried out for all the four seasons in a year. These were characterized in terms of particulate matters (inhalable, thoracic, and alveolic), their constituents (pollens, fungal spores, animal debris, and non-biological components), and finally the bacterial diversity was determined by DNA based metagenomic approach. The non-biological (non-viable) component of aerosols is found to vary from 30- 89% in pre-monsoon season which coexists with pollens (4-20%), animal debris (1-24%) and fungal spores (1-17%). The highest number of culturable microbial population in terms of CFU count was observed in the samples collected in pre-monsoon season (i.e., 125.24-632.45 CFU/m3) and the lowest CFU was observed in monsoon season (i.e., 20.83- 319.0 CFU/m3). The metagenomic approach with the samples collected during pre-monsoon season showed a total of bacterial 184 OTUs (operational taxonomic units) with 28,028 abundance count comprising with 7 major phylum, 6 classes, 10 orders, 15 families, 13 genus, and 8 species of bacteria. The species level distribution clearly shows the presence of Gammaproteobacteria (52%) most abundantly followed by Bacilli (21%), Alphaproteobacteria (14%), Betaproteobacteria (5%, Flavobacteria (5%), and Sphingobacteria (3%). It is the first report from entire Northeast India to uncover bacterial diversity in aerosol samples through DNA based metagenomic approach.

Extracellular DNA in environmental samples: Occurrence, extraction, quantification, and impact on microbial biodiversity assessment

Environmental DNA, i.e., DNA directly extracted from environmental samples, has been applied to understand microbial communities in the environments and to monitor contemporary biodiversity in the conservation context. Environmental DNA often contains both intracellular DNA (iDNA) and extracellular DNA (eDNA). eDNA can persist in the environment and complicate environmental DNA sequencing-based analyses of microbial communities and biodiversity. Although several studies acknowledged the impact of eDNA on DNA-based profiling of environmental communities, eDNA is still being neglected or ignored in most studies dealing with environmental samples. In this article, we summarize key findings on eDNA in environmental samples and discuss the methods used to extract and quantify eDNA as well as the importance of eDNA on the interpretation of experimental results. We then suggest several factors to consider when designing experiments and analyzing data to negate or determine the contribution of eDNA to environmental DNA-based community analyses. This field of research will be driven forward by: (i) carefully designing environmental DNA extraction pipelines by taking into consideration technical details in methods for eDNA extraction/removal and membrane-based filtration and concentration; (ii) quantifying eDNA in extracted environmental DNA using multiple methods including qPCR and fluorescent DNA binding dyes; (iii) carefully interpretating effect of eDNA on DNA-based community analyses at different taxonomic levels; and (iv) when possible, removing eDNA from environmental samples for DNA-based community analyses.

Universal activity-based labeling method for ammonia- and alkane-oxidizing bacteria

The advance of metagenomics in combination with intricate cultivation approaches has facilitated the discovery of novel ammonia-, methane-, and other short-chain alkane-oxidizing microorganisms, indicating that our understanding of the microbial biodiversity within the biogeochemical nitrogen and carbon cycles still is incomplete. The in situ detection and phylogenetic identification of novel ammonia- and alkane-oxidizing bacteria remain challenging due to their naturally low abundances and difficulties in obtaining new isolates from complex samples. Here, we describe an activity-based protein profiling protocol allowing cultivation-independent unveiling of ammonia- and alkane-oxidizing bacteria. In this protocol, 1,7-octadiyne is used as a bifunctional enzyme probe that, in combination with a highly specific alkyne-azide cycloaddition reaction, enables the fluorescent or biotin labeling of cells harboring active ammonia and alkane monooxygenases. Biotinylation of these enzymes in combination with immunogold labeling revealed the subcellular localization of the tagged proteins, which corroborated expected enzyme targets in model strains. In addition, fluorescent labeling of cells harboring active ammonia or alkane monooxygenases provided a direct link of these functional lifestyles to phylogenetic identification when combined with fluorescence in situ hybridization. Furthermore, we show that this activity-based labeling protocol can be successfully coupled with fluorescence-activated cell sorting for the enrichment of nitrifiers and alkane-oxidizing bacteria from complex environmental samples, enabling the recovery of high-quality metagenome-assembled genomes. In conclusion, this study demonstrates a novel, functional tagging technique for the reliable detection, identification, and enrichment of ammonia- and alkane-oxidizing bacteria present in complex microbial communities.

Microbiomes of natural and abandoned agricultural soils of the Central part of Yamal region

Soil cover of the northern most regions of Eurasia are considered as underestimated in terms of their possible role in expansions of current agriculture to the cryolithozone. In this context, abandoned agricultural soils of Yamal region were investigated in terms of morphology, chemistry and taxonomy microbiome compositions and compared in these terms with mature tundra and taiga soils of pristine environments. The level of soil fertility was low in all cases – former agricultural soils and pristine ones. The level of microorganism’s biodiversity was higher in soils of agricultural lands. This fact indicates that the agricultural soil treatment in polar terrestrial ecosystem results in increasing of soil microbial biodiversity due to diversification of ecological niches. Also the is an essential lack of nitrogen sources in all permafrost affected soils studied.

Influence of pneumococcal conjugate vaccine 13 on upper respiratory tract microbial biodiversity in infants

Kozhakhmetov S, Aitenov Y, Ramazanova B, Koloskova Y, Burkutbayeva T, Yeraliyeva L, Mustafina K, Beglarova G, Yergazina M, Kushugulova A. 2021. Influence of pneumococcal conjugate vaccine 13 on upper respiratory tract microbial biodiversity in infants. Biodiversitas 22: 5055-5060. Pneumococcal disease and its associated mortality are burdens on the healthcare system. The intensive introduction of pneumococcal vaccines has provided robust control and infection management worldwide. The Pneumococcal Conjugate Vaccine (PCV) has been successfully employed in the national programs of many countries. However, no studies have yet analyzed the effect of pneumococcal vaccines on Central Asian populations. We investigated the effect of the pneumococcal vaccine on the nasopharyngeal microbiome of infants under the age of two years. Samples were collected from healthy patients as part of routine hospital check-ups, then subjected to high-throughput sequencing of the V1–V3 region of 16S rRNA for bioinformatics analysis. The obtained data were combined with the results of a previously published study (with matching criteria) to increase statistical power. No significant differences were found in vaccination status, sex or age. Nevertheless, the results demonstrated structural changes in the microbiome of the upper respiratory tract under the influence of the PCV13 vaccine. The results of beta-diversity unweighted UniFrac distance measuring showed that the experimental groups differed in their qualitative (taxonomic) structure (p < 0.1). In the vaccinated group, the abundance of several symbiotic taxa was significantly decreased, including Streptococcus pneumonia.

Broad-Spectrum Inhibitor of Bacterial Polyphosphate Homeostasis Attenuates Virulence Factors and Helps Reveal Novel Physiology of Klebsiella pneumoniae and Acinetobacter baumannii

Acinetobacter baumannii and Klebsiella pneumoniae currently rank amongst the most antibiotic-resistant pathogens, responsible for millions of infections each year. In the wake of this crisis, anti-virulence therapeutics targeting bacterial polyphosphate (polyP) homeostasis have been lauded as an attractive alternative to traditional antibiotics. In this work, we show that the small molecule gallein, a known G-protein βγ subunit modulator, also recently proven to have dual-specificity polyphosphate kinase (PPK) inhibition in Pseudomonas aeruginosa, in turn exhibits broad-spectrum PPK inhibition in other priority pathogens. Gallein treatment successfully attenuated virulence factors of K. pneumoniae and A. baumannii including biofilm formation, surface associated motility, and offered protection against A. baumannii challenge in a Caenorhabditis elegans model of infection. This was highlighted most importantly in the critically understudied A. baumannii, where gallein treatment phenocopied a ppk1 knockout strain of a previously uncharacterized PPK1. Subsequent analysis revealed a unique instance of two functionally and phenotypically distinct PPK1 isoforms encoded by a single bacterium. Finally, gallein was administered to a defined microbial community comprising over 30 commensal species of the human gut microbiome, demonstrating the non-disruptive properties characteristic of anti-virulence treatments as microbial biodiversity was not adversely influenced. Together, these results emphasize that gallein is a promising avenue for the development of broad-spectrum anti-virulence therapeutics.