ARTICLE IN PRESS
International Dairy Journal 17 (2007) 1115–1122
www.elsevier.com/locate/idairyj
Characterization of spontaneous phage-resistant variants of
Streptococcus thermophilus by randomly amplified polymorphic DNA
analysis and identification of phage-resistance mechanisms
A.G. Binettia, V.B. Suáreza, P. Tailliezb,1, J.A. Reinheimera,
a
Instituto de Lactologı´a Industrial (INLAIN), Facultad de Ingenierı´a Quı´mica (Universidad Nacional del Litoral),
Santiago del Estero 2829, 3000 Santa Fe, Argentina
b
Unité de Recherches Laitières et Génétique Apliquée, URLGA, INRA, Jouy-en-Josas, France
Received 26 June 2006; accepted 25 January 2007
Abstract
A total of 100 spontaneous phage-resistant mutants isolated from nine commercial Streptococcus thermophilus strains were
characterized preliminarily by randomly amplified polymorphic DNA (RAPD) and the nature of their phage-resistance mechanisms was
investigated. Only for mutants isolated from one strain, free phages were detected in their culture supernatants when these were titrated
on the sensitive strain, suggesting that the mutants could have acquired the resistance phenotype by integrating the phage in their
genomes (lysogeny). Adsorption interference was observed in the derivatives isolated from two strains. For mutants isolated from two
other strains, restriction–modification (R–M) type systems were detected. In one of these cases, R–M was probably combined with
another intracellular anti-phage system. In most cases, the molecular profiles (RAPD fingerprints) obtained with four arbitrary primers
showed a high similarity among parent strains and their respective phage-resistant mutants. Some of these mutants were identified as
potentially improved strains for industrial use.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Streptococcus thermophilus; Phages; RAPD-PCR
1. Introduction
Despite the development of a variety of countermeasures
(culture rotation, improved sanitation strategies and use of
bacteriophage-resistant starter strains) phage infection
during product manufacture continues to be the leading
cause of failed or retarded dairy fermentations (Brüssow &
Desière, 2001; Coffey, Coakley, Mc Garry, Fitzgerald, &
Ross, 1998; Forde & Fitzgerald, 1999; Klaenhammer
& Fitzgerald, 1994; Neve, 1996; Vadeboncoeur & Moineau, 2004). Several factors, such as lysogenic lactic acid
Corresponding author. Tel.: +54 342 4530302; fax: +54 342 4571162.
E-mail address: anabinetti@fiqus.unl.edu.ar (A.G. Binetti).
Present address: Unité d’Ecologie Microbienne des Insectes
et Interactions, Hôte-Pathogène, UMR INRA—Université Montpellier
II, France.
1
0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.idairyj.2007.01.007
bacteria (LAB) present in raw milk processed daily and the
use of non-sterile fermentation media (pasteurized milk)
determine the entrance and dynamics of phage populations
in dairy plant environments. Thus, the success of commercial lactic starter cultures depends, primarily, on the
selection of phage-unrelated strains, which are able to
withstand viral infections. The isolation of phage-resistant
mutants with satisfactory technological performance from
sensitive-strains represents a very interesting approach for
obtaining improved strains for industrial purposes (Coffey
et al., 1998; Klaenhammer, 1984; Guglielmotti et al., 2006;
Quiberoni, Reinheimer, & Tailliez, 1998). Although this
technique has the advantage of simplicity and rapidity, it is
rarely used for commercial Lactococcus strains since
bacteriophage insensitive mutants often exhibit a variety
of negative qualities that may exclude them for being used
in industrial dairy fermentations (Coffey et al., 1998; Forde
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& Fitzgerald, 1999; Klaenhammer, 1984; Moineau, 1999;
Sturino & Klaenhammer, 2004). However, it was successfully employed to isolate phage-resistant variants with
good technological abilities from Lactobacillus helveticus,
allowing their use in cheese making (Quiberoni, Reinheimer, & Suárez, 1999).
Streptococcus thermophilus is one of the most important
thermophilic LAB because of its worldwide use in the dairy
industry. In Argentina, besides its relevance as a starter in
the yoghurt industry, this species is used in the production
of other fermented milks and a large variety of hard and
semi-hard cheeses (Reinheimer et al., 1997). From this kind
of processes many specific phages have been isolated over
the past years (Suárez, Quiberoni, Binetti, & Reinheimer,
2002). Low frequencies at which S. thermophilus spontaneous phage-resistant mutants occur were previously
reported (Moineau, 1999; Viscardi, Capparelli, & Iannelli,
2003; Viscardi, Capparelli, Di Mateo et al., 2003).
Notwithstanding, we have recently isolated (Binetti, Bailo,
& Reinheimer, 2007) such variants from sensitive commercial strains used in Argentinean dairy plants by the
secondary culture method with a frequency that was
strain-dependent. Since some of these mutants showed
excellent levels of phage resistance and stability, as well as
acidifying and proteolytic activities, they could be used as
improved strains for industrial purposes. However, the
mechanisms involved in their resistance have not been
elucidated yet.
The aim of this work was to investigate, by means of a
primary identification, the resistance mechanisms present
in phage-resistant mutants of S. thermophilus and characterize these derivatives based on randomly amplified
polymorphic DNA (RAPD) fingerprints.
2. Materials and methods
2.1. Bacterial strains, bacteriophages and culture conditions
Spontaneous phage-resistant variants were obtained
from nine S. thermophilus strains (identified as 4-C, 5-C,
YDS10-C, Jo1-C, M1-C, M8-C, M11-C, MiC1 and MiC7),
isolated from commercial starters used in Argentinean
dairy industries (INLAIN Culture Collection), and three
Italian S. thermophilus strains (identified as I49, I53 and
I54) belonging to the Istituto Sperimentale Lattiero Caseario (ISLC, Lodi, Italy) Culture Collection. Bacteriophages
used were nine autochthonal S. thermophilus phages
(f021-4, f031, fCYM, fCYS1, fQP2, fQPL2, fQPL10 ,
fMi2 and fMi1) isolated from Argentinean dairy plants
(INLAIN Phage Collection) and three Italian S. thermophilus phages (f49, f53 and f54) (ISLC Phage Collection)
(Table 1). To isolate spontaneous phage-resistant mutants,
the secondary culture method was used (Carminati,
Zennaro, Neviani, & Giraffa, 1993). Strains and phageresistant derivatives were grown in M17 broth or M17 agar
(Biokar, Beaubois, France) at 42 1C and stored ( 80 1C) in
M17 broth supplemented with 15% (v/v) glycerol and in
Table 1
Phage-resistance mechanisms present in phage-resistant mutants isolated
from commercial S. thermophilus strains
Sensitive
strain
Phage
nRa
4-C
5-C
YSD10
M1-C
M8-C
M11-C
Jo1-C
MiC1
MiC7
I49
I53
I54
f021-4
f031
fCYM
fQP2
fQLP2
fQLP10
fCYS1
fMi2
fMi1
f49
f53
f54
3
3
2
6
10
1
1
4
8
26
17
9
Lysogenyb
+
Adsorption
ratec
85.5
93.3
89.9
82.1718.1
80.673.4
99.9
95.7
96.173.6
48.573.2
96.276.6
83.177.1
49.279.0
a
nR: Number of phage-resistant mutants isolated; : absence; +:
presence.
b
Spontaneous induction of free phages, detected in the supernatants of
mutants cultures.
c
% (mean value of each group) of adsorbed phages in M17-Ca broth
after 30 min at 45 1C. Standard deviation was calculated when nR43.
non-fat dry skim milk (Merck, Darmstadt, Germany).
Phage enumerations were carried out by double-layer
plaque titration method from IDF Standards (1991) using
M17 soft agar on M17 agar supplemented with 10 mM
CaCl2 (M17-Ca) and 100 mM glycine (Lillehaug, 1997).
2.2. Characterization of strains
For S. thermophilus strains and their phage-resistant
variants, cell (phase contrast, 1000 , Microscope Jenamed
2 Carl Zeiss, Jena, Germany) and colony (on M17 agar)
morphologies were observed. To evaluate sugar fermentation patterns, API 50 CHS (Bio Merieux, Marcy l’Etoile,
France) galleries were used, according to the manufacturer’s instructions.
2.3. Phage-resistance mechanisms
Lysogeny and adsorption rates were determined for all
phage-resistant mutants as previously described (Quiberoni
et al., 1998). All assays were performed in triplicate.
In the case of mutants that exhibited a relatively high
adsorption rate of phage particles and a late lysis in broth,
the presence of restriction–modification (R–M) type
resistance mechanisms was investigated according to de
los Reyes-Gavilán, Limsowtin, Tailliez, Séchaud, and
Accolas (1992) modified as follows: a phage suspension
was titrated on the sensitive strain and on the phageresistant mutant, and Efficiency of Plaquing (EOP) (first
value) was calculated. One or two lysis plaques obtained
from the titration on the phage-resistant variant were
picked up and suspended in 5 mL of M17-Ca broth. Phage
suspensions were kept 24 h at 4 1C and then inoculated with
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0.2 mL of phage-resistant mutant overnight culture
and incubated at 42 1C until total lysis, to allow the
propagation of potentially modified phages. The lysate
obtained was filtered and titrated on both strains (sensitive and resistant) for determining the second EOP
value (in the presence of a R–M system, this value must
be approximately equal to the unit). From the titre plate
of the sensitive strain, one or two lysis plaques were
picked up and suspended in 5 mL of M17-Ca broth
and, after 24 h at 4 1C, inoculated with 0.2 mL of sensitive
strain culture and incubated at 42 1C until total lysis.
The resulting lysate was filtered and titrated on both
strains (sensitive and resistant). The respective EOP-value
was also determined for this stage (if it is similar to
that obtained in the first stage, it indicates the presence
of an active R–M type phage-resistance mechanism
in the mutant studied). All assays were performed in
triplicate.
2.4. RAPD analysis
Total DNA of strains was obtained by phenol-chloroform extraction as was described previously (de los ReyesGavilán et al., 1992) and quantified by electrophoresis on
0.8% (w/v) agarose gels (Seakem, Tebu, France). Optimized polymerase chain reaction (PCR) amplification
reactions were performed in a total volume of 100 mL
(10 mmol L 1 Tris-HCl buffer pH 9.0, containing
1.5 mmol L 1 MgCl2) with 1 mL of template DNA (20–
100 mg), 0.5 mmol L 1 primer (Bioprobe, Montreuil-sousBois, France), 2.5 U Taq Polymerase (Qbiogène, Illkirch
Cedex, France) and 200 mmol L 1 of each dNTP (Boehringer Mannheim, Mannheim, Germany). Four single
arbitrary primers, P1 (50 TGCTCTGCCC 30 ), P2
(50 GGTGACGCAG 30 ), P3 (50 GTCCACACGG 30 ) and
P4 (50 CTGCTGGGAC 30 ) were used in separate PCR
reactions. A Perkin-Elmer (Courtaboeuf, France) thermo
cycler (model 9600) was used to submit DNA samples to 30
cycles of amplification (94 1C for 1 min, 36 1C for 2 min and
72 1C for 2 min). Amplification products were analysed by
electrophoresis in 1% (w/v) GTG agarose gels (Seakem,
Tebu, Le Perray-en-Yvelines, France) containing
200 mg L 1 of ethidium bromide (Sigma, Saint Quentin
Fallavier, France) and viewed by ultraviolet (UV) transillumination at 254 nm. A DNA molecular weight marker,
123 bp DNA Ladder (Gibco BRL, Cergy Pontoise, France)
was used as a standard. Photographs of gels under UV
light were taken using Polaroid film type 665 and negative
pictures were digitalized using a Hewlett Packard (Issy les
Moulineaux, France) SCanJet IIcx/T. Digitalized pictures
were analysed using the Gel Compare software (AppliedMaths, Sint-Martens-Latem, Belgium). Band profiles
obtained with the four primers were normalized and
subsequently combined. Densitometric traces were
grouped in clusters using the Unweighted Pair Group
Method with Arithmetic Average (UPGMA; Romersburg,
1984).
1117
3. Results
3.1. Characterization of strains
All phage-resistant variants were identical to their parent
strains in cell- and colony-morphologies and sugar
fermentation patterns.
3.2. Phage-resistance mechanisms
3.2.1. Lysogeny
Only for phage-resistant mutants isolated from the
sensitive strain S. thermophilus I49, free phages able to
infect the parent strain, but not themselves, were detected
in their broth culture supernatants. This fact suggested that
the resistance phenotype could be linked to integration of
f49 in the streptococcal genomes, leading to a possible
phage-resistance mechanism associated to lysogeny (by
lysogenic immunity; Table 1).
3.2.2. Adsorption rates
In general, a significant inhibition of phage adsorption
was not detected for resistant variants since their mean
adsorption rates were higher than 82%, except for those
isolated from S. thermophilus MiC7 and I54 that were
partially unable to bind phage particles (mean adsorption
rates of 48.5% and 49.2%, respectively, Table 1).
3.2.3. R–M mechanisms
The variants isolated from strains S. thermophilus 5-C
and I53 with phages f031 and f53, respectively, showed
high adsorption rates (93.3% and 83.1%, respectively, as
mean value, Table 1) and a late lysis in broth. Additionally,
they were the only group of phage-resistant mutants which
exhibited the ability to form visible lysis plaques when they
were infected with phages (EOP mean values of 4.6 10 7
and 2.2 10 6, respectively, Binetti et al., 2007). Therefore,
they were investigated for the presence of R–M systems.
Based on the results obtained (Table 2), it was possible to
detect the presence of active R–M type mechanisms in
mutant R5-C031-10 (derived from S. thermophilus 5-C). At
first, it exhibited a high resistance level against f031 (EOP
value: 5.0 10 7). After titrating the potentially modified
phage (f031m) on strains S. thermophilus 5-C and R5C031-10, the resulting EOP was near the unit. Finally,
when f031m was replicated on the parent strain and then
titrated again on both strains, the EOP decreased to a value
(3.5 10 6) near to that obtained at a first stage.
Also, four mutants isolated from S. thermophilus I53
showed high resistance levels against f53 (EOP values
from 1.6 10 7 to 1.2 10 6, Table 2). At the second
stage, when the potentially modified phages were propagated on the resistant variants and then titrated on both
strain types, EOP values slightly lower than 1 (from
2.2 10 1 to 8.6 10 1) were obtained. Finally, at the
third stage of the assay, the EOPs appeared not near the
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Table 2
Evidence of restriction–modification mechanisms in phage-resistant mutants isolated from commercial S. thermophilus strains
1a
EOPb
Sensitive strain/phageresistant mutant
Phage
Assay stage
PFU mL
5-C
R5031-10
f031
1st
1.2 10871.1 108
6.0 10174.0 101
5.0 10
5-C
R5031-10
f031c
5.1 10771.3 107
5.6 10773.5 107
1.1
5-C
R5031-10
f031
I53
RI53-9
RI53-10
RI53-12
RI53-21
fI53
I53
RI53-9
I53
RI53-10
I53
RI53-12
I53
RI53-21
fI53(RI53-9)c
I53
RI53-9
I53
RI53-10
I53
RI53-12
I53
RI53-21
fI53(RI53-9)
2nd
7
3rd
1st
6
1.0 10 78.7 10
3.5 10172.2 101
3.5 10
6
2.7 10871.4 108
9.0 10176.7 101
3.3 10271.4 101
4.3 10172.1 101
1.5 10271.9 102
3.3 10
1.2 10
1.6 10
5.6 10
7
2.2 10
1
8.6 10
1
2.3 10
1
6.6 10
1
5.5 10
2
1.3 10
1
6.5 10
2
6.5 10
2
9.0 10875.4 107
2.0 10873.0 108
2.2 10974.1 108
1.9 10971.1 109
1.9 10971.7 109
4.5 10873.9 109
8.7 10772.9 107
5.7 10772.8 107
2nd
c
fI53(RI53-10)
c
fI53(RI53-12)
c
fI53 (RI53-21)
3.3 10873.5 108
1.8 10771.0 107
1.3 10979.1 108
1.7 10871.1 108
1.1 10979.9 108
7.3 10777.2 107
1.5 10971.2 109
1.0 10871.3 108
3rd
fI53(RI53-10)
fI53(RI53-12)
fI53(RI53-21)
7
6
7
7
a
Mean value of three determinations.
Efficiency of plaquing.
c
Modified phage, obtained by replication of the lytic phage on the resistant variant.
b
unit but higher (approximately 5 log orders) than those
obtained at the first stage (from 5.5 10 2 to 1.3 10 1).
3.3. RAPD analysis
Individual dendrograms of RAPD profiles were obtained
for S. thermophilus phage-sensitive strains and their
respective phage-resistant variants (Fig. 1 shows five
examples). For variants isolated from S. thermophilus
YDS10, one of them (RYDS10-3) appeared almost
identical (93% similarity) to the parent strain, while the
other one (RYDS10-17) showed higher molecular diversity
(83% similarity), mainly in the genome region amplified
with primer P2 (Fig. 1A). For S. thermophilus M8-C and its
derivatives, two clusters (82% similarity) were obtained
from their RAPD profiles (Fig. 1B), principally based on
the differences detected with primer P4. Within both
groups, a high (490%) similarity coefficient was observed
among the respective strains. For S. thermophilus Jo1-C
and its single mutant, the similarity coefficient was 84%
and the molecular diversity was also detected by primer P4
(Fig. 1C). In the case of the S. thermophilus I53-variants
group, the similarity with the parent strain was 84%; the
molecular diversity was most evident in the RAPD profiles
obtained with the P2 primer (Fig. 1D). Finally, the
dendrogram of the S. thermophilus I54-variants group
enabled to distinguish two clusters, linked by a similarity
coefficient of 79%. The strains belonging to each cluster
showed a molecular diversity lower than 15%, which was
most significant in the RAPD profiles obtained with primer
P2 (Fig. 1E). For the other strains (S. thermophilus 4-C,
5-C, M1-C, M11-C, MiC1, MiC7 and I49) and their
respective phage-resistant mutants, the similarity coefficients were also higher than 70% (data not shown).
4. Discussion
Over the last decade, and due to the evolution of the
processes and the extensive use of commercial thermophilic
cultures, the dairy industry has faced increasing phage
problems with S. thermophilus strains (Brüssow, Bruttin,
Desière, Lucchini, & Foley, 1998; Coffey et al., 1998; Forde
& Fitzgerald, 1999; Klaenhammer & Fitzgerald, 1994;
Moineau, 1999; Neve, 1996; Suárez, Quiberoni, Binetti, &
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1119
Fig. 1. Dendrograms obtained by comparison (Program Gel Compar, Applied Maths, Sint-Martens-Latem, Belgium) and clustering (UPGMA method:
Unweighted Pair Group Method using Arithmetic Averages) of RAPD profiles of S. thermophilus strains (A: YDS-10, B: M8-C, C: Jo1-C, D: I53 and
E: I54) and their respective spontaneous phage-resistant mutants, using the primers P1, P2, P3 and P4. Parent strains are indicated in bold.
Reinheimer, 2002). In an effort to find strains with an
improved phage resistance, 100 spontaneous phage-resistant mutants were isolated from commercial S. thermophilus strains under selective pressure of specific phages
(Binetti et al., 2007). In the present work, it was possible to
establish that different mechanisms of phage resistance are
present among these mutants. Spontaneous phage induction was detected only for the variants derived from
S. thermophilus I49, which was probably due to the
integration of the f49 genome in their chromosomes, a
phage-resistance mechanism linked to super-infection
immunity or lysogeny. This resistance mechanism occurs
very frequently in lactococci and lactobacilli, but is almost
inexistent in S. thermophilus (Carminati & Giraffa, 1992;
Josephsen & Neve, 1998; Mercenier, 1990; Neve, 1996).
The failure of the challenging phage to adsorb to the
cell, presumably due to mutations in the receptor gene,
holds very often for bacteriophage-insensitive mutants of
lactococci (Moineau, 1999; Sturino & Klaenhammer, 2004;
Vadeboncoeur & Moineau, 2004). In case of streptococcal
resistant variants, instead, a normal adsorption of phage
particles was observed, except for those derived from
S. thermophilus MiC7 and I54 that were only partially
unable to adsorb viruses. Recently, Viscardi, Capparelli,
and Iannelli (2003) and Viscardi, Capparelli, Di Mateo
et al. (2003) developed two methods for selection of phageresistant S. thermophilus strains by means of flow
cytometry that allowed the isolation of variants in which
phage adsorption had been blocked. Interference with
phage adsorption as frequently observed in other LAB,
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such as different species of Lactobacillus (Neviani, Carminati, & Giraffa, 1992; Quiberoni et al., 1998; Reinheimer,
Morelli, Callegari, & Bottazzi, 1993; Reinheimer, Quiberoni, Tailliez, Binetti, & Suárez, 1996; Ventura, Callegari, &
Morelli, 1999) and Lactococcus (for review, see Forde &
Fitzgerald, 1999) is in most cases plasmid-encoded. In
general, the adsorption inhibition shown by spontaneous
phage-resistant mutants presents some drawbacks since it
is highly specific and reversion to phage sensitivity can
occur at a high frequency (Forde & Fitzgerald, 1999;
Limsowtin & Terzaghi, 1976; Moineau, 1999). Consequently, the inherent instability of this system and the
persistence of phage particles in the environment may limit
the significance of adsorption inhibition as a potent defence
mechanism (Forde & Fitzgerald, 1999). Therefore, phageresistant mutants from S. thermophilus MiC7 and I54 may
be of restricted value as industrial strains.
In contrast, R–M systems are powerful defence mechanisms since they interrupt the infection process prior to the
initiation of phage-directed cell death and they remove
phage particles from the environment. The combination of
R–M and Abi systems can be even more effective by
countering the genetic flexibility of phages (usually evolving to escape the restriction) increasing the degree of
insensitivity and expanding the range of phage types
against which the host is resistant (Forde & Fitzgerald,
1999; Sturino & Klaenhammer, 2004). In this work, one
derivative from S. thermophilus 5-C (resistant to f031)
could have acquired the phage-resistance phenotype by
means of a tentative R–M type system that could be silent
or absent in the parent strain. The R–M phenotype in this
case was clearly evidenced by the classical methodology
proposed by de los Reyes Gavilán, Limsowtin, Séchaud,
Veaux, and Accolas (1990). Furthermore, in some mutants
derived from S. thermophilus I53 (resistant to f53) the
nature of the phage-resistance mechanism was not clear,
but our results suggested the presence of R–M type
mechanisms that would be combined with other anti-phage
systems. The phenotype characteristic typical of R–M
systems was verified but, an increase in phage particles
following the third stage of the R–M screen was indicative
for an extra mechanism (probably another intracellular
system like Abi) being also present, which could increase
the phage-resistance power of these mutants. This hypothesis is based on the fact that any other mechanism
(lysogeny and adsorption interference) would have been
detected by means of our preliminary study. Additional
studies are being made to elucidate this hypothesis. Most of
known R–M and Abi systems have been identified in
lactococci and they have mainly a plasmid location (Forde
& Fitzgerald, 1999; Klaenhammer, 1984; Klaenhammer &
Fitzgerald, 1994; Moineau, 1999). R–M systems have
also been detected in other members of LAB, such as
L. helveticus (de los Reyes-Gavilán et al., 1990) (combined
with Abi systems), L. delbrueckii subsp. lactis and
L. fermentum (Moineau, 1999). In S. thermophilus, for
which very few phage-resistance mechanism have been
reported, the situation may be different as a consequence of
the relative absence of plasmids in this species (Moineau,
1997).
The use of a molecular technique such as RAPD
fingerprinting enables the identification of many organisms
at the species level and the study of the diversity among
strains belonging to a particular species. The classical
method is based on the use of a single oligonucleotide to
generate a fingerprint of PCR products for the direct
comparison of the genomes (Welsh & McClelland, 1990;
Williams, Kubelik, Livak, Rafaliski, & Tingey, 1990). For
LAB, it has allowed the identification and characterization
of lactobacilli species (L. acidophilus, L. casei, L. delbrueckii
and L. helveticus) from dairy products (Cocconcelli, Parisi,
Senini, & Bottazzi, 1997; Drake, Small, Spence, & Swanson,
1996; Giraffa et al., 2004; Guglielmotti et al., 2006; Mora
et al., 2002; Moschetti et al., 1998). Several authors
(Quiberoni et al., 1998; Tailliez, Quénée, & Chopin, 1996;
Tailliez, Tremblay, Ehrlich, & Chopin, 1998) have used this
technique with three arbitrary primers to study the molecular
diversity among L. plantarum, L. pentosus, L. helveticus and
L. lactis strains. In this study, RAPD was utilized to detect
molecular diversity and relationship among the phageresistant mutants and their respective parent strains. We
modified the conventional method used for S. thermophilus
(Giraffa & Rossetti, 2004; Mora et al., 2002; Moschetti et al.,
1998) to increase sensitivity and reproducibility by means of
four primers according to Tailliez et al. (1998). The
comparison of profiles obtained for the different groups of
phage-resistant strains clearly showed that all of them had
molecular profiles highly similar to that of the corresponding
phage-sensitive strain. In most cases, the phage-resistance
phenotype could arise from changes in the genome that were
not amplified by the primers used in this work. However, the
differences detected for some phage-resistant mutants
(similarity coefficients o85%) could correspond to genetic
diversity related to changes in phage receptors (derivatives
from S. thermophilus MiC7 and I54), R–M type mechanisms
(derivatives from S. thermophilus 5-C) or another possible genetic modification responsible to phage-resistance
phenotype (derivatives from S. thermophilus I53 and other
mutants for which the phage-resistance mechanism was not
identified).
5. Conclusions
Even though additional studies are required to clarify the
tentative phage-resistance mechanisms identified in this
work, some spontaneous phage-resistant strains would be
potentially suitable for the formulation of industrial starter
cultures with enhanced features. The most favourable
representative would be S. thermophilus RI53-9, derivative
from S. thermophilus I53 that, based in our results,
exhibited the combination of strong phage-resistance
mechanisms. Additionally, from a previous report (Binetti
et al., 2007), this strain showed an excellent industrial
performance and a high stability of its phage-resistance
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phenotype. Also, certain variants of S. thermophilus M1-C
and Jo1-C, in spite of the undetermined nature of their
phage-resistance mechanisms, exhibited high levels and
stability of their phenotypes and good technological
characteristics (Binetti et al., 2007). Probably they have
acquired these phenotypes by means of a combination of
strong intracellular systems like Abi since spontaneous
lysogeny, adsorption interference and R–M systems were
not detected.
The RAPD technique have been shown to be a useful
tool to rapidly confirm the parental relationships between
phage-sensitive strains and their phage-resistant derivatives
and indicated that contaminations did not occur in the
isolation of mutants.
Acknowledgements
This work was supported by the Consejo Nacional de
Investigaciones Cientı́ficas y Técnicas (CONICET) of
Argentina (Proyecto PIP 2000 No 02035), the Universidad
Nacional del Litoral (Santa Fe, Argentina)—Programación CAI+D 2002 (Proyecto No 155) and the Agencia
Nacional de Promoción Cientı́fica y Tecnológica de
Argentina (Proyecto PICT 2000, No 09-08200). We would
like to thank Dr. Domenico Carminati (ISLC, Lodi, Italy)
who provides us with phages f49, f53 and f54 and their
S. thermophilus host-strains.
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