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      Characterization of the Bacterial Community Naturally Present on Commercially Grown Basil Leaves: Evaluation of Sample Preparation Prior to Culture-Independent Techniques

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          Abstract

          Fresh herbs such as basil constitute an important food commodity worldwide. Basil provides considerable culinary and health benefits, but has also been implicated in foodborne illnesses. The naturally occurring bacterial community on basil leaves is currently unknown, so the epiphytic bacterial community was investigated using the culture-independent techniques denaturing gradient gel electrophoresis (DGGE) and next-generation sequencing (NGS). Sample preparation had a major influence on the results from DGGE and NGS: Novosphingobium was the dominant genus for three different basil batches obtained by maceration of basil leaves, while washing of the leaves yielded lower numbers but more variable dominant bacterial genera including Klebsiella, Pantoea, Flavobacterium, Sphingobacterium and Pseudomonas. During storage of basil, bacterial growth and shifts in the bacterial community were observed with DGGE and NGS. Spoilage was not associated with specific bacterial groups and presumably caused by physiological tissue deterioration and visual defects, rather than by bacterial growth.

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          16S ribosomal DNA amplification for phylogenetic study.

          D J R Lane, D A Pelletier, S M Barns … (1991)
          A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.
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            Metagenomics: application of genomics to uncultured microorganisms.

            Jo Handelsman (2004)
            Metagenomics (also referred to as environmental and community genomics) is the genomic analysis of microorganisms by direct extraction and cloning of DNA from an assemblage of microorganisms. The development of metagenomics stemmed from the ineluctable evidence that as-yet-uncultured microorganisms represent the vast majority of organisms in most environments on earth. This evidence was derived from analyses of 16S rRNA gene sequences amplified directly from the environment, an approach that avoided the bias imposed by culturing and led to the discovery of vast new lineages of microbial life. Although the portrait of the microbial world was revolutionized by analysis of 16S rRNA genes, such studies yielded only a phylogenetic description of community membership, providing little insight into the genetics, physiology, and biochemistry of the members. Metagenomics provides a second tier of technical innovation that facilitates study of the physiology and ecology of environmental microorganisms. Novel genes and gene products discovered through metagenomics include the first bacteriorhodopsin of bacterial origin; novel small molecules with antimicrobial activity; and new members of families of known proteins, such as an Na(+)(Li(+))/H(+) antiporter, RecA, DNA polymerase, and antibiotic resistance determinants. Reassembly of multiple genomes has provided insight into energy and nutrient cycling within the community, genome structure, gene function, population genetics and microheterogeneity, and lateral gene transfer among members of an uncultured community. The application of metagenomic sequence information will facilitate the design of better culturing strategies to link genomic analysis with pure culture studies.
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              The Diversity of Archaea and Bacteria in Association with the Roots of Zea mays L.

              E.W. Triplett, M.K. Chelius (2001)
              The diversity of bacteria and archaea associating on the surface and interior of maize roots (Zea mays L.) was investigated. A bacterial 16S rDNA primer was designed to amplify bacterial sequences directly from maize roots by PCR to the exclusion of eukaryotic and chloroplast DNA. The mitochondrial sequence from maize was easily separated from the PCR-amplified bacterial sequences by size fractionation. The culturable component of the bacterial community was also assessed, reflecting a community composition different from that of the clone library. The phylogenetic overlap between organisms obtained by cultivation and those identified by direct PCR amplification of 16S rDNA was 48%. Only 4 bacterial divisions were found in the culture collection, which represented 27 phylotypes, whereas 6 divisions were identified in the clonal analysis, comprising 74 phylotypes, including a member of the OP10 candidate division originally described as a novel division level lineage in a Yellowstone hot spring. The predominant group in the culture collection was the actinobacteria and within the clone library, the a-proteobacteria predominated. The population of maize-associated proteobacteria resembled the proteobacterial population of a typical soil community within which resided a subset of specific plant-associated bacteria, such as Rhizobium- and Herbaspirillum-related phylotypes. The representation of phylotypes within other divisions (OP10 and Acidobacterium) suggests that maize roots support a distinct bacterial community. The diversity within the archaeal domain was low. Of the 50 clones screened, 6 unique sequence types were identified, and 5 of these were highly related to each other (sharing 98% sequence identity). The archaeal sequences clustered with good bootstrap support near Marine group I (crenarchaea) and with Marine group II (euryarchaea) uncultured archaea. The results suggest that maize supports a diverse root-associated microbial community composed of species that for the first time have been described as inhabitants of a plant-root environment.
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                Author and article information

                Contributors
                Paul B. Tchounwou: Role: Academic Editor
                Journal
                Journal ID (nlm-ta): Int J Environ Res Public Health
                Journal ID (iso-abbrev): Int J Environ Res Public Health
                Journal ID (publisher-id): ijerph
                Title: International Journal of Environmental Research and Public Health
                Publisher: MDPI
                ISSN (Print): 1661-7827
                ISSN (Electronic): 1660-4601
                Publication date (Electronic): 21 August 2015
                Publication date (Print): August 2015
                Volume: 12
                Issue: 8
                Pages: 10171-10197
                Affiliations
                [1 ]Faculty of Bioscience Engineering, Department of Food Safety and Food Quality, Laboratory of Food Microbiology and Food Preservation (LFMFP), Ghent University, Ghent 9000, Belgium; E-Mails: siele.ceuppens@ 123456ugent.be (S.C.); stefanie_dlbk@ 123456hotmail.com (S.D.); jolien_boussemaere@ 123456hotmail.com (J.B.)
                [2 ]Faculty of Pharmaceutical Sciences, Department of Pharmaceutics, Laboratory of Pharmaceutical Biotechnology (LabFBT), Ghent University, Ghent 9000, Belgium; E-Mail: dieter.deconinck@ 123456ugent.be
                [3 ]Faculty of Bioscience Engineering, Department of Biochemical and Microbial Technology, Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Ghent 9000, Belgium; E-Mail: nico.boon@ 123456ugent.be
                Author notes
                [* ]Author to whom correspondence should be addressed; E-Mail: mieke.uyttendaele@ 123456ugent.be ; Tel.: +32-92-646-178; Fax: +32-92-255-510.
                Article
                Publisher ID: ijerph-12-10171
                DOI: 10.3390/ijerph120810171
                PMC ID: 4555336
                PubMed ID: 26308033
                SO-VID: 9bccd83b-321d-4da9-8e5f-edd83fbc440f
                Copyright © © 2015 by the authors; licensee MDPI, Basel, Switzerland.
                License:

                This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 07 July 2015
                Date accepted : 19 August 2015
                Categories
                Subject: Article

                ScienceOpen disciplines: Public health
                Keywords: novosphingobium,fresh herbs,16s rrna,dgge,next-generation sequencing ngs
                Data availability:
                ScienceOpen disciplines: Public health
                Keywords: novosphingobium, fresh herbs, 16s rrna, dgge, next-generation sequencing ngs

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