title: Validation of the (GTG)(5)-rep-PCR fingerprinting technique for rapid classification and identification of acetic acid bacteria, with a focus on isolates from Ghanaian fermented cocoa beans.
abstract: Amplification of repetitive bacterial DNA elements through the polymerase chain reaction (rep-PCR fingerprinting) using the (GTG)(5) primer, referred to as (GTG)(5)-PCR fingerprinting, was found a promising genotypic tool for rapid and reliable speciation of acetic acid bacteria (AAB). The method was evaluated with 64 AAB reference strains, including 31 type strains, and 132 isolates from Ghanaian, fermented cocoa beans, and was validated with DNA:DNA hybridization data. Most reference strains, except for example all Acetobacter indonesiensis strains and Gluconacetobacter liquefaciens LMG 1509, grouped according to their species designation, indicating the usefulness of this technique for identification to the species level. Moreover, exclusive patterns were obtained for most strains, suggesting that the technique can also be used for characterization below species level or typing of AAB strains. The (GTG)(5)-PCR fingerprinting allowed us to differentiate four major clusters among the fermented cocoa bean isolates, namely A. pasteurianus (cluster I, 100 isolates), A. syzygii- or A. lovaniensis-like (cluster II, 23 isolates), and A. tropicalis-like (clusters III and IV containing 4 and 5 isolates, respectively). A. syzygii-like and A. tropicalis-like strains from cocoa bean fermentations were reported for the first time. Validation of the method and indications for reclassifications of AAB species and existence of new Acetobacter species were obtained through 16S rRNA sequencing analyses and DNA:DNA hybridizations. Reclassifications refer to A. aceti LMG 1531, Ga. xylinus LMG 1518, and Ga. xylinus subsp. sucrofermentans LMG 18788(T).
authors.affiliation: Research Group of Industrial Microbiology and Food Biotechnology, Department of Applied Biological Sciences and Engineering, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium. ldvuyst@vub.ac.be
authors.forename: Luc
authors.initials: L
authors.lastname: De Vuyst
authors.forename: Nicholas
authors.initials: N
authors.lastname: Camu
authors.forename: Tom
authors.initials: T
authors.lastname: De Winter
authors.forename: Katrien
authors.initials: K
authors.lastname: Vandemeulebroecke
authors.forename: Vincent
authors.initials: V
authors.lastname: Van de Perre
authors.forename: Marc
authors.initials: M
authors.lastname: Vancanneyt
authors.forename: Paul
authors.initials: P
authors.lastname: De Vos
authors.forename: Ilse
authors.initials: I
authors.lastname: Cleenwerck
mesh_terms: D019342:Acetic Acid / Q000378:metabolism*; D000091:Acetobacter / Q000145:classification* / Q000302:isolation & purification* / Q000378:metabolism; D015373:Bacterial Typing Techniques; D002099:Cacao / Q000382:microbiology*; D016172:DNA Fingerprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D005516:Food Microbiology; D005784:Gene Amplification; D008969:Molecular Sequence Data; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D000431:Ethanol; D019342:Acetic Acid