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Bias problems in culture-independent analysis of environmental bacterial communities: a representative study on hydrocarbonoclastic bacteria.

ABSTRACT: Culture-dependent methods for bacterial community analysis are currently considered obsolete; therefore, molecular techniques are usually used instead. The results of the current study on hydrocarbonoclastic bacteria in various oily habitats in Kuwait showed however, that the bacterial identities varied dramatically according to the analytical approach used. For six desert and six seawater samples used in this study, the culture-independent and culture-dependent techniques each led to a unique bacterial composition. Problems related to the culture-dependent technique are well known. The results of the current study highlighted bias problems other than those already recorded in the literature for the molecular approaches. Thus, for example, in contrast to the culture-dependent technique, the primers used in the molecular approach preferentially amplified the 16S rDNAs of hydrocarbonoclastic bacteria in total genomic DNAs of all the studied environmental samples, and in addition, failed to reveal in any environmental sample members of the Actinobacteria. The primers used in the molecular approach also amplified certain "pure" 16S rDNAs, but failed to do so when these DNAs were in mixture. In view of these results, it is recommended that the two analytical approaches should be used simultaneously because their combined results would reflect the bacterial community composition more precisely than either of them can do alone.

SUBMITTER: Al-Awadhi H

PROVIDER: S-EPMC3769543 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Bias problems in culture-independent analysis of environmental bacterial communities: a representative study on hydrocarbonoclastic bacteria.

Al-Awadhi Husain H Dashti Narjis N Khanafer Majida M Al-Mailem Dina D Ali Nidaa N Radwan Samir S

SpringerPlus 20130801

Culture-dependent methods for bacterial community analysis are currently considered obsolete; therefore, molecular techniques are usually used instead. The results of the current study on hydrocarbonoclastic bacteria in various oily habitats in Kuwait showed however, that the bacterial identities varied dramatically according to the analytical approach used. For six desert and six seawater samples used in this study, the culture-independent and culture-dependent techniques each led to a unique b ...[more]

PMID: 24040582

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Project description:BACKGROUND: Plants harbor a diverse bacterial community, both as epiphytes on the plant surface and as endophytes within plant tissue. While some plant-associated bacteria act as plant pathogens or promote plant growth, others may be human pathogens. The aim of the current study was to determine the bacterial community composition of organic and conventionally grown leafy salad vegetables at the point of consumption using both culture-dependent and culture-independent methods. RESULTS: Total culturable bacteria on salad vegetables ranged from 8.0 × 10(3) to 5.5 × 10(8) CFU g(-1). The number of culturable endophytic bacteria from surface sterilized plants was significantly lower, ranging from 2.2 × 10(3) to 5.8 × 10(5) CFU g(-1). Cultured isolates belonged to six major bacterial phyla, and included representatives of Pseudomonas, Pantoea, Chryseobacterium, and Flavobacterium. Eleven different phyla and subphyla were identified by culture-independent pyrosequencing, with Gammaproteobacteria, Betaproteobacteria, and Bacteroidetes being the most dominant lineages. Other bacterial lineages identified (e.g. Firmicutes, Alphaproteobacteria, Acidobacteria, and Actinobacteria) typically represented less than 1% of sequences obtained. At the genus level, sequences classified as Pseudomonas were identified in all samples and this was often the most prevalent genus. Ralstonia sequences made up a greater portion of the community in surface sterilized than non-surface sterilized samples, indicating that it was largely endophytic, while Acinetobacter sequences appeared to be primarily associated with the leaf surface. Analysis of molecular variance indicated there were no significant differences in bacterial community composition between organic versus conventionally grown, or surface-sterilized versus non-sterilized leaf vegetables. While culture-independent pyrosequencing identified significantly more bacterial taxa, the dominant taxa from pyrosequence data were also detected by traditional culture-dependent methods. CONCLUSIONS: The use of pyrosequencing allowed for the identification of low abundance bacteria in leaf salad vegetables not detected by culture-dependent methods. The presence of a range of bacterial populations as endophytes presents an interesting phenomenon as these microorganisms cannot be removed by washing and are thus ingested during salad consumption.

Project description:The recently proposed candidatus order Altiarchaeales remains an uncultured archaeal lineage composed of genetically diverse, globally widespread organisms frequently observed in anoxic subsurface environments. In spite of 15 years of studies on the psychrophilic biofilm-producing Candidatus Altiarchaeum hamiconexum and its close relatives, very little is known about the phylogenetic and functional diversity of the widespread free-living marine members of this taxon. From methanogenic sediments in the White Oak River Estuary, NC, USA, we sequenced a single cell amplified genome (SAG), WOR_SM1_SCG, and used it to identify and refine two high-quality genomes from metagenomes, WOR_SM1_79 and WOR_SM1_86-2, from the same site. These three genomic reconstructions form a monophyletic group, which also includes three previously published genomes from metagenomes from terrestrial springs and a SAG from Sakinaw Lake in a group previously designated as pMC2A384. A synapomorphic mutation in the Altiarchaeales tRNA synthetase ? subunit, pheT, caused the protein to be encoded as two subunits at non-adjacent loci. Consistent with the terrestrial spring clades, our estuarine genomes contained a near-complete autotrophic metabolism, H2 or CO as potential electron donors, a reductive acetyl-CoA pathway for carbon fixation, and methylotroph-like NADP(H)-dependent dehydrogenase. Phylogenies based on 16S rRNA genes and concatenated conserved proteins identified two distinct sub-clades of Altiarchaeales, Alti-1 populated by organisms from actively flowing springs, and Alti-2 which was more widespread, diverse, and not associated with visible mats. The core Alti-1 genome suggested Alti-1 is adapted for the stream environment with lipopolysaccharide production capacity and extracellular hami structures. The core Alti-2 genome suggested members of this clade are free-living with distinct mechanisms for energy maintenance, motility, osmoregulation, and sulfur redox reactions. These data suggested that the hamus structures found in Candidatus Altiarchaeum hamiconexum are not present outside of stream-adapted Altiarchaeales. Homologs to a Na(+) transporter and membrane bound coenzyme A disulfide reductase that were unique to the brackish sediment Alti-2 genomes, could indicate adaptations to the estuarine, sulfur-rich environment.

Dataset Information

Bias problems in culture-independent analysis of environmental bacterial communities: a representative study on hydrocarbonoclastic bacteria.

Publications

Bias problems in culture-independent analysis of environmental bacterial communities: a representative study on hydrocarbonoclastic bacteria.

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