Identification of Dekkera bruxellensis (Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes

Abstract

A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification ofBrettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results
obtained from testing 127 different yeast strains, including 78 Brettanomycesisolates from wine, show that the spoilage organismBrettanomyces belongs to the species D. bruxellensis and that the new method is able to identifyBrettanomyces (D. bruxellensis) with 100% sensitivity and 100% specificity.

Full text

APPLIED AND ENVIRONMENTAL MICROBIOLOGY,
0099-2240/01/$04.00⫹0 DOI: 10.1128/AEM.67.2.938–941.2001
February 2001, p. 938–941 Vol. 67, No. 2
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Identification of Dekkera bruxellensis (Brettanomyces) from Wine
by Fluorescence In Situ Hybridization Using
Peptide Nucleic Acid Probes
HENRIK STENDER,
1
* CLETUS KURTZMAN,
2
JENS J. HYLDIG-NIELSEN,
1
DITTE SØRENSEN,
1
ADAM BROOMER,
1
KENNETH OLIVEIRA,
1
HEATHER PERRY-O’KEEFE,
1
ANDREW SAGE,
3
BARBARA YOUNG,
3
AND JAMES COULL
1
Boston Probes, Inc., Bedford, Massachusetts 01730
1
; Microbial Properties Research Unit, National Center for
Agricultural Utilization Research, USDA Agricultural Research Service, Peoria, Illinois 61604
2
; and Millipore
Corporation, Bedford, Massachusetts 01730
3
Received 28 August 2000/Accepted 2 November 2000
A new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification
of Brettanomyces is described. The test is based on fluorescein-labeled PNA probes targeting a species-specific
sequence of the rRNA of Dekkera bruxellensis. The PNA probes were applied to smears of colonies, and results
were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains,
including 78 Brettanomyces isolates from wine, show that the spoilage organism Brettanomyces belongs to the
species D. bruxellensis and that the new method is able to identify Brettanomyces (D. bruxellensis) with 100%
sensitivity and 100% specificity.
Brettanomyces is a well-recognized wine spoilage yeast that
causes an undesirable flavor. The sensory character of this
“Bretty” flavor is often described as mousiness, barnyard,
horse sweat, or Band-Aid (5, 9). Current methods for identi-
fication and enumeration of Brettanomyces contamination take
1 to 2 weeks and rely on growth on a semiselective culture
medium, followed by final identification by biochemical and
physiological analysis and morphology as determined by mi-
croscopic examination (3). Morphological characterization of
Brettanomyces is somewhat subjective, and there have been
various morphological descriptions, such as bud scars, bullet
shape, and Mickey Mouse-like. Newer techniques for rapid
detection and identification of Brettanomyces, such as an en-
zyme-linked immunosorbent assay (7) and, more recently,
PCR (6), have also been described.
The nomenclature of Brettanomyces used in the wine indus-
try differs from that of the recently revised taxonomy of yeasts
(11, 12). Enologists refer to the spoilage organism as Bretta-
nomyces or “Brett” or, in some publications, by the species
names Dekkera intermedia and Brettanomyces intermedius (3),
Brettanomyces lambicus (3), Brettanomyces custersii,orDekkera
bruxellensis (6). Today, only D. bruxellensis is an accepted spe-
cies name, and the other names are considered synonyms.
Peptide nucleic acid (PNA) molecules are pseudopeptides
which are able to hybridize to complementary nucleic acid
targets (RNA and DNA) obeying Watson-Crick base pairing
rules (2, 10). Due to their uncharged, neutral backbone, PNA
probes exhibit favorable hybridization characteristics, such as
high specificity, strong affinity, and rapid kinetics resulting in
improved hybridization to highly structured targets, such as
rRNA (13). In addition, the relatively hydrophobic character
of PNAs compared to DNA oligonucleotides makes PNA
probes capable of penetrating the hydrophobic cell wall fol-
lowing mild fixation conditions that do not lead to disruption of
cell morphology (14). These unique characteristics of PNA
have opened new possibilities for molecular diagnostic assays.
The D1-D2 region of 26S ribosomal DNA (rDNA) of eu-
caryotic organisms shows a high degree of species variation
and has been used for identification and taxonomy of yeast
species (1, 8). In this study, 26S rDNA sequence information
was used to design species-specific probes targeting the rRNA
of D. bruxellensis. These probes were used to develop a new
fluorescence in situ hybridization (FISH) method for identifi-
cation of Brettanomyces.
MATERIALS AND METHODS
Yeast strains. Five type strains representing the five Dekkera and Brettanomy-
ces species, 10 reference strains representing synonyms of D. bruxellensis, and 26
yeast species potentially found in wine were obtained from the Agricultural
Research Service Culture Collection (Peoria, Ill.) and the American Type Cul-
ture Collection (Manassas, Va.). Seventy-eight wine isolates of Brettanomyces
were kindly provided by E&J Gallo (Modesto, Calif.), California State University
at Fresno (Fresno, Calif.), Sutter Home (St. Helena, Calif.), Robert Mondavi
Winery (Oakville, Calif.), and Boston Probes, Inc. (Bedford, Mass.). Eight wine
isolates of cycloheximide-resistant spheroidal yeasts were kindly provided by
Beringer (St. Helena, Calif.), Vinquiry, Inc. (Windsor, Calif.), Columbia Winery
(Woodinville, Wash.), and Robert Mondavi Winery. The spheroid yeasts were
included because they grow relatively slowly on cycloheximide containing media,
like Brettanomyces, and may therefore be misidentified as Brettanomyces.
Wine samples. Three wine samples confirmed to be positive for Brettanomyces
by microscopy were kindly provided by Vinquiry, Inc.
Culture media and growth conditions. A nonselective yeast and mold medium
(YM) (Difco Laboratories, Detroit, Mich.) and a Brettanomyces-selective me-
dium (BSM) (Millipore Corp., Bedford, Mass.) were used. BSM contains cyclo-
heximide as well as antibiotics that inhibit bacterial growth. Yeast strains were
propagated in YM at 25°C.
For FISH analysis, strains were spread onto YM agar and incubated at 30°C,
whereas wine samples were filtered through 47-mm-diameter, 0.45-␮m-pore-size
HVLP filter membranes (Millipore) and then incubated at 30°C on a pad soaked
with 2 ml of BSM in a small petri dish.
* Corresponding author. Mailing address: Boston Probes, Inc., 75E
Wiggins Ave., Bedford, MA 01730. Phone: (781) 271-1100. Fax: (781)
276-4931. E-mail: HStender@BostonProbes.com.
938

Preparation of smears. For each smear, 1 drop of phosphate-buffered saline
was placed in the well of a Teflon-coated microscope slide (Erie Scientific,
Portsmouth, N.H.). A small portion of a colony was picked with a clean, sterile
toothpick and suspended in the phosphate-buffered saline by gentle mixing in the
microscope well. The slide was then placed on a 50°C slide warmer for 30 min,
after which the smears were dry.
Selection of probe sequence. Sequence processing was performed by using
computer software from DNASTAR (Madison, Wis.). Alignment of closely
related yeast D1-D2 26S rDNA sequences (1, 8) was performed by using the
Megalign (version 4.03) program. From the alignments, species-specific se-
quences of D. bruxellensis were identified and subsequently checked for signifi-
cant sequence similarity with the whole GenBank database by using the Gene-
Man (version 3.30) software and an Advanced BLAST search of the GenBank
nr-database (www.ncbi.nlm.nih.govlast). Complementary 15-mer probe se-
quences were checked for significant levels of secondary structure by using the
PrimerSelect program (version 4.03).
Synthesis of fluorescein-labeled PNA probes. PNAs were synthesized by using
an Expedite 8909 nucleic acid synthesis system with the PNA option and reagents
from PE Biosystems, Foster City, Calif. The aqueous solubility of the PNAs was
enhanced by flanking the nucleobase sequence with solubility enhancers (4). The
N terminus of each PNA was extended by using an 8-amino-3,6-dioxaoctanoic
acid spacer (PE Biosystems). Following removal of the terminal Fmoc protecting
group, the N terminus of the resin-bound PNA was labeled with 5(6)-carboxy-
fluorescein. Specifically, the resin was treated with 250 ␮l of a solution containing
0.5 M 5(6)-carboxyfluorescein (Aldrich, Milwaukee, Wis.), 0.5 M N,N⬘-diisopro-
pylcarbodiimide (Aldrich), and 0.5 M 1-hydroxy-7-azabenzotriazole (PE Biosys-
tems) in dimethylformamide (Burdick & Jackson, Muskegon, Mich.) (15). The
synthesis support was then washed and dried under a high vacuum. After re-
moval from the synthesis cartridge, the resin was transferred to an Ultrafree spin
cartridge (Millipore Corp.) for cleavage and deprotection (User’s Guide. PNA
Chemistry for the Expedite Nucleic Acid Synthesis System, Perspective Biosys-
tems, Inc., Framingham, Mass.). The product was analyzed by high-performance
liquid chromatography and matrix-assisted laser desorption ionization-time of
flight mass spectrometry to confirm its purity and identity. The fluorescein-
labeled PNA probe was finally purified by using standard reversed-phase C
18
chromatographic methods.
FISH. Smears were covered with approximately 20 ␮l of a hybridization solu-
tion containing 10% (wt/vol) dextran sulfate (Sigma Chemical Co., St. Louis,
Mo.), 10 mM NaCl (J. T. Baker), 30% (vol/vol) formamide (Sigma), 0.1%
(wt/vol) sodium pyrophosphate (Sigma), 0.2% (wt/vol) polyvinylpyrrolidone (Sig-
ma), 0.2% (wt/vol) Ficoll (Sigma), 5 mM Na
2
EDTA (Sigma), 0.1% (vol/vol)
Triton X-100 (Aldrich), 50 mM Tris-HCl (pH 7.5), and 100 nM fluorescein-
labeled PNA probe. Coverslips were put on the smears to ensure even coverage
with hybridization solution, and the slides were subsequently placed on a slide
warmer with a humidity chamber (Slidemoat, Boeckel, Germany) and incubated
for 30 min at 50°C. Following hybridization, the coverslips were removed by
submerging the slides in approximately 20 ml of prewarmed 5 mM Tris–15 mM
NaCl–0.1% (vol/vol) Triton X-100 (pH 10) per slide in a water bath at 50°C and
washed for 30 min. The slides were then cooled to room temperature by brief
immersion in H
2
O and air dried following brief immersion in ethanol. Each
smear was finally mounted by using 1 drop of IMAGEN mounting fluid (DAKO,
Ely, United Kingdom) and covered with a coverslip. Microscopic examination
was conducted with a fluorescence microscope (Optiphot; Nikon Corporation,
Tokyo, Japan) equipped with a 60⫻/1.4 oil objective (Nikon), an HBO 100-W
mercury lamp, and a fluorescein isothiocyanate-Texas Red dual-band filter set
(Chroma Technology Corp., Brattleboro, Vt.). Images were obtained by using a
color charge-coupled device camera (Diagnostic Instruments, Inc., Sterling
Heights, Mich.) connected to a computer system.
RESULTS
Sequences of D1-D2 26S rDNA from yeast species poten-
tially found in wine were aligned in order to identify species-
specific target regions of D. bruxellensis rRNA. The optimal
target sequence was found in all synonyms of D. bruxellensis
and differed by at least four bases from the sequences of other
yeast species (Fig. 1). In addition, a BLAST search did not
reveal other eucaryotic or bacterial rDNA sequences with the
exact same target sequence.
Initially, the specificity of BRE26S14 labeled with fluores-
cein (BRE26S14/Flu) was tested by FISH by using the type
strains of the five species of Dekkera and Brettanomyces (Table
1), as well as 10 reference strains representing different syn-
onyms of D. bruxellensis (Table 2). Twenty-six other yeast spe-
cies potentially found in wine were also examined for reactivity
with the probe (Table 3). As predicted from the alignment of
sequences in the target area, BRE26S14/Flu hybridized only to
the type strain of D. bruxellensis and synonyms thereof,
whereas it did not detect any of the other 26 yeast species. In
addition, BRE26S14/Flu did not react with any of eight isolates
of spheroid yeasts capable of growing on BSM. These uniden-
tified spheroid yeasts grow relatively slowly on cycloheximide-
containing media, like Brettanomyces, and are therefore among
FIG. 1. Alignment of partial yeast D1-D2 26S rDNA sequences for probe selection. The anti-parallel hybridization sequence of the BRE26S14
PNA probe is shown above the alignment. Base differences between the target sequences and other sequences are highlighted.
VOL. 67, 2001 D. BRUXELLENSIS IN WINE 939

the species most likely to be misidentified as Brettanomyces by
persons without experience with identification of Brettanomy-
ces.
The sensitivity of BRE26S14/Flu for detection of the actual
spoilage organism, Brettanomyces, was then assessed by ana-
lyzing 78 wine isolates of Brettanomyces. All isolates were iden-
tified by the probe; thus, there was 100% correlation with the
results of methods used by wine makers to identify Brettano-
myces isolated from wine. This result provided further proof
that the spoilage organism named Brettanomyces belongs to the
species D. bruxellensis.
Finally, the routine applicability of the method for identifi-
cation of colonies of Brettanomyces obtained directly from wine
samples was also evaluated with three Brettanomyces-contam-
inated wines. Colonies from all three wine samples were iden-
tified by BRE26S14/Flu.
Figure 2 shows images obtained by the FISH method with
smears of colonies grown for 1 to 2 weeks on BSM following
membrane filtration. Individual cells of Brettanomyces were
identified by their bright green fluorescence, whereas undetec-
ted cells were reddish brown. Often mixtures of cells exhibiting
high, medium, low, and no green fluorescence were observed
in smears of cells from a Brettanomyces colony. This was not
due to a mixed population as all cells originated from the same
colony. Instead, it was most likely a result of variable amounts
of target rRNA in the individual cells due to different meta-
bolic stages of the cells in a colony, so that some cells were
growing and multiplying while others may have been resting or
even dead. Alternatively, the variability in intensity may have
been due to variable permeability of the cell wall. The images
also demonstrate that the morphology of the cells was not
affected by the FISH procedure. However, some of the mor-
phological characteristics were not as pronounced when this
method was used as they were when bright-field microscopy
was used because the cell membrane was not fluorescent since
the rRNA molecules were located in the cell cytoplasm.
DISCUSSION
We showed that using fluorescently labeled PNA oligomers
is a powerful method for identifying colonies of the spoilage
organism Brettanomyces (D. bruxellensis). The FISH method
described here provides a combination of the high specificity
offered by molecular techniques with the simplicity of micros-
copy. In contrast to the previous subjective method of identi-
fication based on morphology, this new method provides 100%
definitive identification of Brettanomyces irrespective of the
experience and skill of the wine technologist.
This study also shows that Brettanomyces, the spoilage or-
ganism in wine, belongs to the species D. bruxellensis. Probes
designed by using sequence data from taxonomic studies have
been shown to detect all 78 confirmed isolates of Brettanomy-
ces. To our knowledge, this is the first study that provides a link
between the recently revised taxonomy of yeasts and the spoil-
age organism Brettanomyces. The various descriptions of the
flavors caused by Brettanomyces, as well as the many somewhat
dubious morphological descriptions and the many synonyms,
can all be ascribed to D. bruxellensis. Although D. anomala, the
other species of the genus Dekkera, may spoil wine, it is not
associated with the wine spoilage organism Brettanomyces.
In summary, our new method for identification of Brettano-
TABLE 1. Detection of type strains of Dekkera and Brettanomyces
accepted species with BRE26S14/Flu
Organism Strain
a
Result
Dekkera anomala NRRL Y-17522 ⫺
Dekkera bruxellensis NRRL Y-12961 ⫹
Brettanomyces naardenensis NRRL Y-17526 ⫺
Brettanomyces custersianus NRRL Y-6653 ⫺
Brettanomyces nanus NRRL Y-17527 ⫺
a
NRRL, Agricultural Research Service Culture Collection, Peoria, Ill.
TABLE 2. Detection of D. bruxellensis reference strains (synonyms)
with BRE26S14/Flu
Organism Strain
a
Result
Brettanomyces bruxellensis NRRL Y-1411 ⫹
Brettanomyces lambicus NRRL Y-1413 ⫹
Mycotorula intermedia NRRL Y-17534 ⫹
Brettanomyces bruxellensis NRRL Y-1412 ⫹
Brettanomyces schanderlii NRRL Y-17523 ⫹
Brettanomyces abstinens NRRL Y-17525 ⫹
Dekkera intermedia ATCC 52904
b
⫹
Dekkera intermedia ATCC 56869 ⫹
Dekkera intermedia ATCC 64276 ⫹
Dekkera lambica ATCC 10563
c
⫹
a
NRRL, Agricultural Research Service Culture Collection, Peoria, Ill.;
ATCC, American Type Culture Collection, Manassas, Va.
b
Equivalent to strain NRRL Y-17523.
c
Equivalent to strain NRRL Y-1413.
TABLE 3. Reactions of other yeast species potentially found in
wine with BRE26S14/FLU
Organism Strain
a
Result
Hanseniaspora uvarum NRRL Y-1614 ⫺
Hanseniaspora guilliermondii NRRL Y-1625 ⫺
Hanseniaspora occidentalis NRRL Y-7946 ⫺
Hanseniaspora osmophila NRRL Y-1613 ⫺
Hanseniaspora valbyensis NRRL Y-1626 ⫺
Hanseniaspora vineae NRRL Y-17529 ⫺
Kloeckera lindneri NRRL Y-17531 ⫺
Torulaspora delbrueckii NRRL Y-866 ⫺
Debaryomyces hansenii NRRL Y-7426 ⫺
Debarymyces carsonii NRRL YB-4275 ⫺
Candida stellata NRRL Y-1446 ⫺
Metschnikowia pulcherrima NRRL Y-7111 ⫺
Rhodotorula fujisanensis NRRL YB-4824 ⫺
Rhodotorula glutinis NRRL Y-2502 ⫺
Rhodotorula graminis NRRL Y-2474 ⫺
Schizosaccharomyces pombe NRRL Y-12796 ⫺
Pichia anomala NRRL Y-366 ⫺
Pichia membranifaciens NRRL Y-2026 ⫺
Pichia farinosa NRRL Y-7553 ⫺
Saccharomyces cerevisiae ATCC 4098 ⫺
Saccharomyces kluyveri NRRL Y-12651 ⫺
Saccharomycodes ludwigii NRRL Y-12793 ⫺
Zygosaccharomyces bailii ATCC 66825 ⫺
Zygosaccharomyces bisporus NRRL Y-12626 ⫺
Zygosaccharomyces rouxii NRRL Y-229 ⫺
Zygosaccharomyces florentinus NRRL Y-1560 ⫺
a
NRRL, Agricultural Research Service Culture Collection, Peoria, Ill.;
ATCC, American Type Culture Collection, Manassas, Va.
940 STENDER ET AL. APPL.ENVIRON.MICROBIOL.

myces is easily adapted to microscopic techniques currently
used in wine laboratories, except that a fluorescence micro-
scope is required. Furthermore, the uncertainty and subjectiv-
ity associated with the currently used methods are eliminated
by the specificity of the PNA probe, which provides definitive
identification of the spoilage organism.
ACKNOWLEDGMENTS
We thank Rich Morenzoni (E&J Gallo), Kenneth Fugelsang (Cal-
ifornia State University at Fresno), Glenn Andrade (Sutter Home),
Judy Miles (Beringer), Pat Paris (Robert Mondavi Winery), Neil
Brown (Vinquiry, Inc.), and Bruce Watson (Columbia Winery) for
providing many yeast isolates. S. Casey, J. MacNeill, and S. Voetsch
are acknowledged for synthesis of the PNA probes.
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