DNA Probe For Lactobacillus Delbrueckii Subsp. Lactis

Journal of Dairy Research (1999) 66 303311
Printed in the United Kingdom
303
DNA probe for Lactobacillus delbrueckii subsp. lactis

B GIORGIO GIRAFFA* DIEGO MORA
* Istituto Sperimentale Lattiero-Caseario, via Lombardo 11, I-26900 Lodi, Italia
Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, UniversitaZ degli
Studi di Milano, via Celoria 2, I-20133 Milano, Italia
(Received 21 November 1997 and accepted for publication 6 October 1998)
Lactobacilli are of great commercial value because they are widely used as
starters in food fermentation. The species Lactobacillus delbrueckii, which comprises
the three subspecies bulgaricus, lactis and delbrueckii, is important in dairy products
and vegetables. The subspecies bulgaricus is present in yogurt, and subspecies lactis
is recovered from whey, starter cultures and cheeses (Stiles & Holzapfel, 1997). It is
unusual to recover Lb. delbrueckii subsp. delbrueckii (Lb. delbrueckii) from dairy
products, from which Lb. delbrueckii subsp. lactis (Lb. lactis) and subsp. bulgaricus
(Lb. bulgaricus) are typically isolated. However, given the similarity of these two
latter subspecies, a clear identification of isolates on the basis of phenotype criteria
alone is often problematic (Dellaglio, 1989 ; Millie' re et al. 1996).
The use of DNA probes and methods based on polymerase chain reaction (PCR)
have greatly facilitated diagnostic identification of bacteria. For the lactobacilli,
DNA probes have been described for Lb. helveticus, Lb. acidophilus, Lb. fermentum,
Lb. plantarum and most of the other Lactobacillus species (Pot et al. 1994 ; Tailliez et
al. 1994 ; Quere et al. 1997). For Lb. delbrueckii, an EcoRI DNA fragment of the
plasmid pY85 was used as a probe for this species, although it was not able to
discriminate its three subspecies (Delley et al. 1990).
In our laboratory, a specific amplification of a DNA fragment of " 1n7 kbp using
universal primers for the amplification of ribosomal RNA (rRNA) genes was found
only for dairy isolates of Lb. lactis and not for Lb. bulgaricus, Lb. delbrueckii, Lb.
helveticus and Lb. acidophilus (G. Giraffa, P. de Vecchi and L. Rossetti, unpublished
results). This prompted us to test the possible use of this fragment as a specific DNA
probe for Lb. lactis. To this end, Southern and dot blot hybridization experiments
were carried out with total DNA of several strains belonging to different lactic acid
bacteria (LAB) species.

Strains used
The bacterial strains examined in this study and belonging to different LAB
species are listed in Table 1. Strains were identified by means of physiological tests,
which included fermentation of carbohydrates, growth at different temperatures (10
and 45 mC), production of CO from glucose and production of (k)- or -lactic acid.
#
Fermentation profiles were also obtained with the API 50 CHL system (API
Biome! rieux, F-69280 Marcy lEtoile, France), according to the manufacturers
instructions. Interpretation of the fermentation profiles was facilitated by sys For correspondence.
304
Table 1. Bacterial strains tested by Southern and dot blot hybridization.

Species
ATCC 15808
ATCC 10697
ATCC 12315T
ATCC 11842T
ATCC 1961
ATCC 9649T
ATCC 15009T
ATCC 10386
ATCC 4356T
ATCC 14931T
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Strain
L3
L48
Ll1
Ll3
Ll5
Ll7
Ll9
Ll12
Ll13
Ll15
Ll17
10T
13T
18T
7E
9E
20E
21E
1G
4G
9G
3H
18H
6I
15I
18I
19I
1G24
Signal with the

1n7 kbp amplicon
j
j
j
k
k
k
k
k
k
k
j
j
j
j
j
k
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
G. G D. M
Lactobacillus delbrueckii subsp. lactis

Lb. delbrueckii subsp. lactis
Lb. delbrueckii subsp. bulgaricus
Lb. delbrueckii subsp. delbrueckii
Lb. helveticus
Lb. helveticus
Lb. acidophilus
Lb. fermentum
Source
3G24
10A48
3D48
7D48
9D48
3F48
6F48
1I48
Lb2
Lb4
Lb5
Lb6
Lb8
Lb9
Lb14
10I
7D48
18D48
L8
4D48
Lh10
Lh17
Lh20
Lh37
Lh47
Lh73
La2
La5
La6
Lc1
Lp1
Lp3
Sd1
Sd124
Lcn8
j
j
j
j
j
j
j
j
k
j
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
305
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Cheese isolate
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
Our collection
CNRZ 318
Our collection
ATCC 14917T
Our collection
Our collection
ATCC 19435T
CNRZ 141
CNRZ 144
ATCC 19257T
CNRZ 105
Our collection
Our collection
Our collection
DNA probe for lactobacilli

Lb. helveticus
Lb. helveticus
Lb. helveticus
Lb. helveticus
Lb. helveticus
Lb. helveticus
Lb. acidophilus
Lb. acidophilus
Lb. acidophilus
Lb. casei
Lb. casei
Lb. plantarum
Lb. plantarum
Lb. plantarum
Lactococcus lactis subsp. lactis
Lc. lactis subsp. lactis
Lc. lactis subsp. lactis
Lc. lactis subsp. cremoris
Lc. lactis subsp. cremoris
Lc. lactis subsp. lactis biovar diacetilactis
Lc. lactis subsp. lactis biovar diacetilactis
Leuconostoc mesenteroides subsp. dextranicum
306
Table 1. continued.
Source
Streptococcus thermophilus
Str. thermophilus
Str. thermophilus
Str. thermophilus
Pediococcus pentosaceous
Pc. acidilactici
Enterococcus faecium
Ec. faecium
Ec. faecalis
Ec. faecalis
Ec. faecalis
ATCC 19258T
Our collection
Our collection
Our collection
NCSU
NCSU
ATCC 19434T
Our collection
ATCC 19433T
Our collection
Our collection
Strain
St6
St8
St20
FBB- 611
NCK 137
Fs3
Fs7
Signal with the

1n7 kbp amplicon
k
k
k
k
k
k
k
k
k
k
k
CNRZ, Centre National de Recherche Zootechnique (F-78352 Jouy-en-Josas, France) ; ATCC, American Type Culture Collection (Rockville, MD 20852, USA ; a superscript
T after the strain number indicates that the strain is the type strain of the species or subspecies) ; NCSU, North Carolina State University (Raleigh, NC 27695, USA ; strains
kindly provided by Dr Todd Klaenhammer).
Code used by the Istituto Sperimentale Lattiero-Caseario.
Probe used for dot blot hybridization : j, hybridization ; k, no hybridization.
G. G D. M
Species
307
tematically comparing all the results obtained from the bacterium studied with the
computer-aided data base API-LAB Plus (Api Biome! rieux).
Maintenance of cultures
Bacterial strains were maintained as frozen stocks at k80 mC with 150 ml
glycerol\l as cryoprotective agent. Working cultures were prepared through three
transfers in MRS medium (Biokar, F-60000 Beauvais, France) for lactobacilli and
pediococci, and in M17 medium (Oxoid, Basingstoke RG24 0PW, UK) for lactococci,
enterococci, leuconostocs, and Streptococcus thermophilus. Strains were cultivated at
30 mC (for lactococci) or 37 mC for 1618 h. Purity was checked by plating on
corresponding agar media and microscopic examination.
DNA extraction and polymerase chain reaction amplifications
Total DNA from different LAB was extracted from broth cultures by the method
of De los Reyes-Gavila! n et al. (1992) or, for the enterococci alone, by guanidium
thiocyanate (Pitcher et al. 1989). DNA quantification and purity were obtained by
measuring A and A as described by Sambrook et al. (1989).
#'!
#)!
PCR experiments were performed using the primers described by Vaneechoutte
et al. (1992). The primer sequences, which were derived from conserved regions
present in the 16S and 23S rDNA, were : 5h-TGGCTCAGATTGAACGCTGGCGGC-3h
and 5h-CCTTTCCCTCACGGTACTGGT-3h. These primers have been used previously
to amplify rRNA genes from Str. thermophilus and other bacteria (Vaneechoutte et
al. 1992 ; Salzano et al. 1994). The sequences of the primers were analysed by MailFASTA (Internet address, www.ebi.ac.uk\htbin\fasta.py?request), based on the
FASTA program developed by Pearson & Lipman (1988) as implemented in the GCG
package (Devereux et al. 1984). This allowed us to perform fast and sensitive
comparisons of our primer sequences against various databases including the
European Molecular Biology Laboratories (EMBL ; http :\\srs.ebi.ac.uk) entries.
Amplifications were performed in volumes of 100 l with 1 mol\l of each primer,
2n5 U Amplitaq polymerase (Perkin Elmer Italia, I-20052 Monza, Italy), 2n5 mMgCl , 200 ng template DNA from the type strain Lb. lactis ATCC 12315T, and
#
200 mol\l each of dNTP. Reactions were carried out in a model 2400 Perkin Elmer
thermal cycler under the following conditions. After one cycle at 94 mC for 2 min, 40
cycles were performed of 94 mC for 30 s, 55 mC for 30 s and 72 mC for 1 min. The final
extension was carried out at 72 mC for 10 min. The amplified products were run on
agarose gel (12 g\l) and a fragment of " 1n7 kbp as the main PCR product was
excised from the gel and purified with the Geneclean spin kit (Bio 101, Vista, CA
92083, USA) according to the suppliers instructions. The purified DNA was used as
a probe in Southern and dot blot hybridization. A positive PCR control of 500 bp was
amplified from bacteriophage DNA using the GeneAmp Lambda Control kit
(Perkin Elmer Italia), following the manufacturers instructions.
DNADNA hybridization
Total DNA from different LAB strains was digested with EcoRI (Life
Technologies Italia, I-20098 Milano, Italy), according to the manufacturers
instructions. The DNA restriction fragments were separated by electrophoresis in
agarose gels (20 g\l) and Southern blotted under alkaline conditions (0n4 -NaOH)
on a Hybond N+ nylon membrane (Amersham Pharmacia Biotech Italia, I-20093
Milano, Italy). Hybridization was carried out using the enhanced chemiluminescence
direct nucleic acid labelling and detection systems (Amersham), according to the
308
G. G D. M
suppliers instructions. In other experiments, total DNA samples were dot blotted
using a Bio-Rad (Bio-Rad Laboratories, I-20090 Milano, Italy) apparatus. Southern
and dot blot hybridizations were carried out under stringent conditions (6 -urea,
42 mC) using the gene-cleaned 1n7 kbp fragment as a Lb. lactis DNA probe.
Amplification of total DNA from Lb. lactis ATCC 12315T using the primers and
conditions described above gave as the main PCR product the expected fragment of
" 1n7 kbp, which served as a potential DNA probe for Lb. lactis. This fragment was
amplified by DNA from all the other Lb. lactis strains, but not from Lb. bulgaricus or
Lb. delbrueckii. Some strains are shown in Fig. 1 (a) as examples. In addition, a
2n4 kbp PCR product was the main PCR product from the DNA of Lb. delbrueckii
and Lb. bulgaricus, but not from Lb. lactis (Fig. 1 b, lanes 3, 5 and 4 respectively ; type
strains are shown as examples).
Southern and dot blot hybridization experiments indicated that 38 out of 39 Lb.
lactis strains were recognized by the probe ; only Lb. lactis Ll7 gave a false negative
signal. No hybridization took place with the total DNA of 52 strains belonging to
other LAB species, except for Lb. bulgaricus Lb4, which gave a false positive signal
(Table 1).
The alignment of nucleotide sequences of the two primers used for amplification
with the library sequences showed 90 % homology of the primer 5h-TGGCTCAGATTGAACGCTGGCGGC-3h to region 5679 of the 16S rRNA sequence of Lb. lactis
ATCC 12315T (accession no. M58823). Similarly, the primer 5h-CCTTTCCCTCACGGTACTGGT-3h was aligned with region 489510 of the 23S rRNA gene of Lb.
delbrueckii 11Lb1 (accession no. X68426), because the sequence of the 23S rRNA of
Lb. lactis is not reported in the Genebank.
It is widely accepted that correct classification and identification of LAB is

difficult without the support of genotype techniques. Although fermentation
reactions (mostly examined with commercially available test systems) remain
important as tools for strain identification of lactobacilli, the large number of species
and strains and the overall phenotype similarity among closely related species
prevent a reliable method of species determination. This is particularly true for the
biovars Lb. bulgaricus and Lb. lactis of the species Lb. delbrueckii, which can be
confused if only the phenotype characteristics are known (Dellaglio, 1989). This
phenotype variation is marked in wild strains of Lb. bulgaricus isolated from natural
habitats, which often display unusual carbohydrate fermentation patterns and
chemotaxonomic characteristics (Dellaglio & Torriani, 1985 ; Bosi et al. 1986 ; Millie' re
et al. 1996).
A DNA fragment of " 1n7 kbp was amplified from the total DNA of Lb. lactis
under stringent conditions and by using universal primers for the amplification of
ribosomal RNA genes. With these primers, a 2n4 kbp fragment encompassing the
16S, the spacer region and part of the 23S rDNA is amplified in Str. thermophilus and
the Comamonadaceae (Salzano et al. 1994 ; Vaneechoutte et al. 1992). The specificity
of the amplification of the 1n7 kbp fragment in Lb. lactis suggested that the presence
(or absence) of this specific amplicon after amplification under the protocol described
may be sufficient on its own to distinguish Lb. lactis, which belongs to a phylogenetic
group (the Lb. delbrueckii group ) with homologies of 16S rRNA from 90n8 to 99n3 %
309

Molecular
size, kb (a )
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17181920 21 22
30
16
17
10
Molecular
size, kb (b)
30
16
1 2 3 4 5 6
24
17
10
Fig. 1. Agarose gel electrophoresis of different amplified fragments from Lactobacillus delbrueckii
strains listed in Table 1. (a) Amplification of the 1n7 kbp fragment : lanes 13, Lb. lactis ATCC 15808,
ATCC 10697 and ATCC 12315 ; lane 4, Lb. bulgaricus ATCC 11842 ; lanes 510, Lb. lactis Ll1, Ll3, Ll5,
Ll9, Ll12 and Ll13 ; lane 11, Lb. delbrueckii ATCC 9649 ; lane 12, Lb. bulgaricus Lb2 ; lane 13, Lb. lactis
Ll15 ; lane 14, Lb. bulgaricus ATCC 1961 ; lane 15, empty ; lane 16, Lb. bulgaricus Lb5 ; lane 17, empty ;
lane 18, Lb. lactis Ll17 ; lane 19, Lb. bulgaricus Lb6 ; lane 20, 500 bp fragment polymerase chain
reaction (PCR) positive control after amplification of bacteriophage DNA (GeneAmp Lambda
Control ; Perkin Elmer Italia, I-20052 Monza, Italy) ; lane 21, negative control, no target DNA added ;
lane 22, DNA molecular size marker 1 kbp ladder (Life Technologies Italia, I-20098 Milano, Italy). (b)
Amplification of the 2n4 kbp fragment : lane 3, Lb. delbrueckii ATCC 9649 ; lane 4, Lb. lactis ATCC
15808 ; lane 5, Lb. bulgaricus ATCC 11842 ; lane 7, 500 bp fragment PCR positive control after
amplification of bacteriophage DNA (GeneAmp Lambda Control) ; lane 2, negative control, no
target DNA added ; lane 6, DNA molecular size marker 100 bp ladder (Life Technologies) ; lanes 1 and
8, DNA molecular size marker 1 kbp ladder (Life Technologies).
(Collins et al. 1991), from other thermophilic lactobacilli. The three subspecies of Lb.
delbrueckii cannot be distinguished even by rRNA sequence analysis (Hertel et al.
1993 ; Vandamme et al. 1996). This prompted us to use the 1n7 kbp amplicon as the
DNA probe for Lb. lactis.
Hybridization showed that the 1n7 kbp amplicon specifically probed to DNA from
Lb. lactis strains alone. All other strains tested from different species of LAB were
negative. It should be noted that the hybridization conditions adopted were
stringent, and that false positive (or negative) results were obtained in only two out
of 91 strains. Thus the 1n7 kb amplicon has been shown to be a genetic probe with
good specificity for Lb. lactis.
310
G. G D. M
The alignment of the primers used with the nucleotide sequences of the 16S and
23S genes of Lb. lactis and Lb. delbrueckii published in data banks suggested that the
amplicon of 1n7 kbp originated from rRNA regions. Since the size of the 16S23S
rRNA spacer region of Lb. lactis is 220221 bp (Tilsala-Timisjarvi & Alatossava,
1997), the size of the amplicon obtained with the primers used should have been
2n4 kbp, instead of the " 1n7 kbp obtained experimentally. In previous reports,
moreover, differently sized PCR products were reported for Lb. delbrueckii using
primers specific for the 16S rRNA gene (Drake et al. 1996) or the 16S23S rRNA
intergenic region (Moschetti et al. 1997 ; Tilsala-Timisjarvi & Alatossava, 1997). DNA
heterogeneity of the number, length, and composition of the ribosomal rRNA
operons in Lb. delbrueckii and its subspecies (Gurtler & Stanisich, 1996 ; Moschetti et
al. 1997) provides a reasonable explanation for this discrepancy.
The authors are grateful to Lia Rossetti and Patrizia De Vecchi for their excellent
technical assistance.
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DNA Probe For Lactobacillus Delbrueckii Subsp. Lactis

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Journal of Dairy Research (1999) 66 303311

Printed in the United Kingdom

DNA probe for Lactobacillus delbrueckii subsp. lactis

Table 1. Bacterial strains tested by Southern and dot blot hybridization.

Signal with the

Lactobacillus delbrueckii subsp. lactis

DNA probe for lactobacilli

Lb. delbrueckii subsp. lactis

Signal with the

DNA probe for lactobacilli

It is widely accepted that correct classification and identification of LAB is

DNA probe for lactobacilli

B, F., M, L. & B, V. 1986 Physiological and genetic characteristics of variant strains of

DNA probe for lactobacilli

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