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Capillary Zone Electrophoresis with Fraction Collection for Separation, Culturing, and Identification of Bacteria from an Environmental Microbiome

Author:
Huge, Bonnie Jaskowski, Champion, Matthew M., Dovichi, Norman J.
Source:
Analytical chemistry 2019 v.91 no.7 pp. 4649-4655
ISSN:
1520-6882
Subject:
Escherichia coli, automation, bacteria, capillary zone electrophoresis, detectors, fractionation, genes, microbiome, models, phylogeny, ribosomal DNA, ribosomal RNA, sequence analysis, sewage effluent
Abstract:
Capillary zone electrophoresis (CZE) can produce high-resolution separations of biological samples, including microbial mixtures. The study of complex populations of microorganisms using CZE is limited because most detectors have limited sensitivity, are destructive, and provide limited information for microbial identification. To address these issues, we developed an integrated capillary zone electrophoresis apparatus to fractionate bacteria from complex mixtures. We deposited fractions onto nutrient agar in a Petri dish for microbial culturing, and we subjected the observed colonies to Sanger sequencing of a phylogenetic marker, the 16S rRNA gene, for microbial identification. We separated and cultured both a single bacteria species, the model Gram-negative organism Escherichia coli, and a complex environmental isolate of primary sewage effluent. Sequence analysis of the 16S rRNA genes from this mixture identified 15 ± 5 distinct bacterial species per run. This approach requires minimal manipulation of microbial populations and combines electrophoretic fractionation of bacterial cells with automated collection for accurate identification of species. This approach should be applicable to microorganisms in general and may enable discrimination of physiologically different cells in complex assemblages, such as in microbiome samples.
Agid:
6355717