Characterization of Lactococcus lactis isolates from bovine

mastitis
Carme Plumed-Ferrer *, Kaisa Uusikyla , Jenni Korhonen, Atte von Wright
Food Biotechnology, Institute of Public Health and Clinical Nutrition, University of Eastern Finland, P.O. Box 1627, FI-70210 Kuopio, Finland
1. Introduction
L. lactis and its subspecies lactis and cremoris are widely
used mesophilic dairy starters in the production of several
types of cheeses, fermented milks and plant materials
(see von Wright, 2012 for a review), and have been
included in the Qualied Presumption of Safety (QPS) list
of the European Food Safety Authority (Leuschner et al.,
2010).
In the past, only the reported opportunistic infections
associated with the genus Lactococcus were rare (Collins
et al., 1983; Teixeira et al., 1996). Nowadays, Lactococcus
garvieae, another species of Lactococcus and closely related
to L. lactis, is classied as an emerging pathogen (Morita
et al., 2011 causing infections in sh (Vendrell et al., 2006),
bovine mastitis (Collins et al., 1983; Teixeira et al., 1996)
and has been associated with human clinical cases
(Reimundo et al., 2011). Recently, also L. lactis has been
isolated from diseased sh (Wang et al., 2008; Chen et al.,
2012; Perez et al., 2011), bovine mastitis cases (Devriese
et al., 1999; Fortin et al., 2003; Haguingan et al., 2010;
Wyder et al., 2011; Romero et al., 2011), bird infections
(Goyache et al., 2001) and numerous human clinical
infections (for review, Mofredj et al., 2007; Uchida et al.,
2011). Moreover, the difculties to distinguish lactococci
from either streptococci or enterococci may have caused a
misidentication of many earlier clinical isolates, and the
number of infections caused by L. lactis can be considerably
higher than the published reports indicate (Uchida et al.,
2011).
Veterinary Microbiology 167 (2013) 592599
A R T I C L E I N F O
Article history:
Received 21 May 2013
Received in revised form 4 September 2013
Accepted 6 September 2013
Keywords:
Lactococcus lactis
Bovine mastitis
Starter strains
A B S T R A C T
Lactococcus lactis is a widely used mesophilic dairy starter and has been included in the
Qualied Presumption of Safety (QPS) list of the European Food Safety Authority. However,
it is increasingly found as the cause of human or animal infections, such as bovine mastitis,
probably due to the improvement of the identication of the infective microorganisms.
Since there are some grounds to suspect that at least certain variants of L. lactis may cause
animal or human diseases, it is important to properly identify the differences between the
strains associated with infections and the safe starter strains. Bovine mastitis isolates and
dairy starter strains were genotypically characterized and clustered by the 16S rRNA gene
sequence and RAPD-PCR ngerprint patterns, and phenotypically characterized by their
tolerance to different stress conditions typically found in the intestinal tract of mammals,
the carbohydrate- and antibiotic resistance prole, as well as the in vitro adhesion capacity
to udder epithelial cells. Genotypically, there were no differences between the mastitis
isolates and the dairy starter strains. However, there were clear phenotypic distinctions
between mastitis isolates and typical starter strains, the former showing an increased
tolerance to temperature, lysozyme, bile salts, pH and antibiotics, as well as improved
carbohydrate fermentation capacity, and in vitro adhesion to udder epithelial cells.
Although these differences might not be considered as actual virulence factors, they
improve the ability of the strain to survive in the body of homeothermic animals and,
interestingly, are also typical properties associated with potential probiotic strains.
2013 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +358 403553573; fax: +358 16173322.
E-mail address: Carme.Plumed@uef. (C. Plumed-Ferrer).
Contents lists available at ScienceDirect
Veterinary Microbiology
j ou r nal h o mepag e: w ww . el sevi er . co m/ l oc at e/ vet m i c
0378-1135/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.vetmic.2013.09.011
Mastitis is considered as the most frequent and costly
disease in dairy cows worldwide (Wyder et al., 2011). The
standard mastitis control practices have successfully
reduced the prevalence of contagious pathogens such as
Streptococcus agalactiae and Staphylococcus aureus, but also
opened a window of opportunity for the environmental
and opportunistic bacteria to become dominant, even in
well-managed dairy herds (Nam et al., 2010; Petrovski
et al., 2011). Mastitis bacterial isolates are mainly
identied using protocols based on conventional culturing
and biochemical characteristics (NMC, 1999), and the
differentiation between some groups of bacteria (espe-
cially gram-positive, catalase-negative cocci), such as
Lactococcus, Streptococcus and Enterococcus, might have
not been accurate (Fortin et al., 2003).
Since there are some grounds to suspect that at least
certain variants of L. lactis may cause animal or human
diseases, it is especially important to properly study the
differences between the strains associated with infections
and the safe starter strains and to differentiate pathogenic
from non-pathogenic strains. The aim of this work is to
characterize L. lactis strains of different origins and
specically to compare mastitis isolates and dairy starter
strains for both, genotypic and phenotypic characteristics.
2. Materials and methods
2.1. Strains, media and culture conditions
The strains/isolates used in this study are listed in the
supplementary material 1. Of the altogether 27 L. lactis
isolates from clinical/subclinical mastitis cases originated
in different herds, 23 were isolated and characterized by
the Finnish Food Safety Authority (Evira) and the rest by
Valio Oy (Helsinki, Finland). The isolates were gram-
positive, catalase-negative, esculin-positive, penicillin-
susceptible, originated from the milk of quarters with
high somatic cell counts (SCC > 3 10
6
cells/ml), were
monocultures or the main isolate, and were identied as
Lactococcus lactis/Enterococcus faecium using API Strep
(bioMe rieux, Marcy lEtoile, France) (Pitkala et al., 2004, for
the origin of the isolates). For the comparison, seven bulk
starter strains from Valio Oy and one commercial Direct
Vat Set (DVS) starter strain were also included in the study,
as well as a L. garvieae 20684 strain isolated from a mastitis
case (Collins et al., 1983). All strains were routinely grown
in M17 broth (Oxoid Ltd., Hampshire, United Kingdom) at
30 8C.
2.2. Phenotypic characterization and antibiotic resistance
The isolates and starter strains were examined for
phenotypic properties including colony morphology and
pigmentation, production of oxidase and catalase, Gram
staining, and cellular morphology. The temperature
tolerance at 37 8C was also monitored. Moreover, the
carbohydrate fermentation prole of the isolates and
starter strains was studied using API 50 CHL assay
(bioMe rieux) according to the manufacturers instructions.
The minimum inhibitory concentrations (MICs) of nine
antibiotics (gentamicin, kanamycin, streptomycin, ery-
thromycin, chloramphenicol, tetracycline, ampicillin, neo-
mycin and penicillin) were determined according to ISO
10932:2010 standard except that the agar dilution
method, as described by Korhonen et al. (2007), was used
instead of broth microdilution, and aerobic conditions
were applied. Epidemiological cut-off values were dened
according to the committee on Antimicrobial Suscept-
ibility Testing (EUCAST, http://www.eucast.org).
2.3. Molecular identication of isolates and phylogenetic
analysis
The identities of the mastitis isolates and starter strains
were veried by molecular methods, including partial
amplication of 16S rRNA gene, sequencing and sequence
comparison. For this, total genomic DNA from overnight
cultures was isolated using a DNA extraction kit (NucleoS-
pin Tissue, Macherey-Nagel, Du ren, Germany). Genomic
DNA quality and concentrations were determined with a
nanodrop 1000 Spectrophotometer (Thermo Fisher Scien-
tic Inc., Wilmington, DE, USA) and 1% agarose gels. A
fragment of approx. 650 bp of the 16S rRNA gene was
amplied by polymerase chain reaction (PCR) using the
primers 685r (5
0
-TCTACGCATTTCACCGCTAC-3
0
) and H121
(5
0
-GAGTTTGATCCTGGCTCAGGA-3
0
) or 27F (5
0
-AGAGTTT-
GATCCTGGCTCAG-3
0
). PCR reaction was performed in a 50-
ml total volume containing 1 unit of GoTaq DNA
polymerase (Promega, Mannheim, Germany), 10 pmol of
each primer, 200 mM of each dNTP, 3 mM MgCl
2
and 50 ng
of genomic DNA. PCR amplications were performed using
Biometria T3 thermocycler (Thistle Scientic Ltd., Glasgow,
UK). The PCR parameters were following: initial denatura-
tion at 94 8C for 6 min; 30 cycles of 94 8C for 1 min, 54 8C for
1 min and 72 8C for 1 min; nal extension at 72 8C for
10 min. Amplicons were puried using a PCR clean-up gel
extraction kit (NucleoSpin Extract II, Macherey-Nagel) and
sent for sequencing (LGC Genomics GmbH, Berlin, Ger-
many). Homology searches were performed using Gen-
Bank and Ribosomal Database Project II databases.
Phylogenetic and molecular evolutionary analyses were
conducted by MEGA 5 software (Tamura et al., 2011) using
the neighbor-joining method. Bootstraps values were
obtained from 1000 replicates.
2.4. RAPD-PCR analysis
In order to further characterize the mastitis isolates,
randomly amplied polymorphic DNA (RAPD-PCR) analy-
sis was carried out using three different primers: P16 (5
0
-
TCGCCAGCCA-3
0
), P17 (5-CAGACAAGCC-3
0
) and P2 (5
0
-
GATCGGACGG-3
0
). PCR reaction was performed in 25-ml
total volume containing 1 unit of GoTaq DNA polymerase
(Promega), 10 pmol of primer, 200 mM of each dNTP, 3 mM
MgCl
2
and 50 ng of genomic DNA. PCR amplications were
performed as follows: initial denaturation at 94 8C for
5 min; 30 cycles of 94 8C for 30 s, 24 8C for 2 min and 72 8C
for 2 min; nal extension at 72 8C for 5 min. Amplication
products were electrophoretically separated in 1% agarose
gels containing SYBR safe DNA gel staining (Life Technol-
ogies Ltd., Paisley, UK). Gels were visualized and photo-
graphed using the Gel Doc UV transilluminator 2000
C. Plumed-Ferrer et al. / Veterinary Microbiology 167 (2013) 592599 593
(Bio-Rad Laboratories Inc., Hercules, CA, USA). Band
patterns were analyzed using GelCompar II (Applied
Maths NV, Sint-Martens-Latem, Belgium).
2.5. The potential to survive in mammalian host
The sensitivity of the strains to some host defense
mechanisms, namely to lysozyme, bile and low pH were
determined for all the isolates and control strains. The
growth of bacteria exposed to a range of lysozyme
concentrations (0.053 mg/ml) was determined using a
Thermo Bioscreen C automatic turbidometer (Labsystems
Oy, Helsinki, Finland) as described by Plumed-Ferrer and
von wright (2011). The growth performance was analyzed
using M17 broth at 30 8C. The tolerances to bile and low pH
were determined by standard plating of broth cultures on
M17 plates after different exposure times. The pH values
tested were 2.5 and 6.7, while the bile acid resistance was
tested in the presence of 0.3% ox bile (SigmaAldrich,
Steinheim, Germany). For all the tests, at least 3 replicates
were performed.
2.6. Surface properties
Surface hydrophobicity was assessed using the bacter-
ial adherence to hydrocarbons method described by
Rosenberg (1984) with some modications. Bacteria were
grown overnight in M17 broth at 30 8C. Cells were
harvested by centrifugation, washed three times and
suspended in PBS to a nal OD
625
of 1.0 (1 10
8
CFU/
ml). Three milliliters of cell suspension were mixed with
0.6 ml of n-hexadecane (Merk, Hohenbrunn, Germany),
vortexed vigorously for 2 min and incubated for 1 h at
37 8C. The lower aqueous phase was carefully transferred
to clean tubes and the absorbance measured as before. The
percent of hydrophobicity (% H) was obtained from the
following calculation:
%H
OD
before
OD
after
OD
before
100
2.7. Adhesion capacity of the lactococcal strains to bovine
mammary epithelial cells
Lactococcal strains were grown overnight at 30 8C in
M17 broth for 18 h. After incubation, bacterial cells were
collected by centrifugation, washed twice with PBS and
suspended in RPMI 1640 (Invitrogen Corporation, Carls-
bad, CA, USA) to an appropriate dilution (2 10
7
CFU/ml).
The bovine mammary epithelial cell line (BME-UV1)
described earlier (Van Oostveldt et al., 2002) was grown in
75 cm
3
cell culture bottles (Sarstedt, Inc., Newton, NC, USA)
using Hams F12 medium (40%) (Invitrogen) made up of
23% RPMI 1640 (Invitrogen), 20% NCTC 135 (Invitrogen),
10% fetal bovine serum (SigmaAldrich), 1% lactose
(SigmaAldrich), 1% lactoalbumin hydrolysate (Sigma
Aldrich), 2% antibiotic-antimycotic solution (Sigma
Aldrich), 2% hydrocortisone (SigmaAldrich), 0.5%
L-ascorbic acid (SigmaAldrich), 0.03% L-glutathione
(SigmaAldrich) and 0.01% insulin (SigmaAldrich). Cells
were incubated at 37 8C in a 5% CO
2
atmosphere. The
culture medium was replaced every 23 days.
BME-UV1 cells were seeded to 24-well culture plates at
a concentration of 2.5 10
5
cells per well and left to attach
for 24 h. One milliliter of the lactococcal strains
(1 10
7
CFU/ml) were added to each well and incubated
for 30 min. After incubation, the cells were washed three
times and lysed with 0.1% Triton X-100 (SigmaAldrich).
Cell lysates were serially diluted and plated in duplicate on
M17 agar plates. Plates were then incubated at 30 8C for 2
days and the bacteria counted. The adhesion capacity of
the strains is calculated as percentage of the bacteria
counted from the cell lysates divided by the total bacteria
added to the well.
2.8. Nucleic sequence accession numbers
The sequences of the 16S rRNA gene amplicons have
been deposited in the GenBank database under accession
numbers JQ953668JQ953697 and JQ965752JQ965756.
3. Results
3.1. Bacterial identication and phenotypic characterization
Twenty seven mastitis isolates and eight starter strains,
which all were Gram-positive, oxidase- and catalase-
negative cocci were identied by partial sequencing of the
16S rRNA gene. The identication at species level showed
99% similarity or higher with the corresponding species.
Most of the mastitis isolates (17) were L. lactis subsp. lactis;
ve isolates were L. lactis subsp. cremoris; one isolate was
identied as L. garvieae; one as Streptococcus parauberis;
and three as Aerococcus viridans. The seven bulk starters
were identied as L. lactis subsp. cremoris and the DVS
starter as L. lactis subsp. lactis. The list of the identied
strains and their accession numbers is shown in the
supplementary material 1.
All mastitis isolates, L. garvieae and the DVS starter
strain were able to grow at 37 8C, whereas the bulk starter
strains did not grow at higher temperatures than 32 8C
(Table 1). The bulk starter strains were only able to ferment
galactose, glucose, fructose, mannose, N-acetyl-D-glucosa-
mine and lactose. In contrast, all mastitis isolates and the
DVS starter were able to ferment a wide variety of
carbohydrates (Table 1).
The antimicrobial susceptibility testing of the isolates is
shown in Table 2. Only two mastitis isolates (one L. lactis
subsp. lactis and one L. lactis subsp. cremoris) were non-
wildtype with substantially higher MICs for tetracycline,
while ve mastitis isolates slightly exceeded the strepto-
mycin cut-off values. All the other isolates were wildtype
regarding the susceptibility to the antibiotics tested.
However, the breakpoints to gentamicin, kanamycin,
streptomycin, chloramphenicol and neomycin of all the
mastitis isolates and DVS starter strains were clearly
higher than those of the bulk starter strains (see the
complete antibiotic resistance proles in supplementary
material 2).
C. Plumed-Ferrer et al. / Veterinary Microbiology 167 (2013) 592599 594
3.2. Phylogenetic analysis
The phylogenetic tree generated from the 16S rRNA
gene sequence of the L. lactis strains/isolates included in
this study showed, as expected, that all L. lactis subsp. lactis
were clustered in one clade, whereas all L. lactis subsp.
cremoris were clustered in another clade (Fig. 1), regardless
of the origin (mastitis isolate or starter strain). Two L. lactis
subsp. lactis isolates, Mast40 and Mast35, clustered
separately from the rest of the lactis subcluster.
3.3. RAPD-PCR analysis
Three different primers were used to create nger-
prints from the total genome of all L. lactis strains/isolates
included in this work (Fig. 2). It was conrmed that the
mastitis isolates were different strains with a division
separating the strains of L. lactis subsp. lactis from all the
strains of L. lactis subsp. cremoris, without discriminating
between starters or mastitis isolates. At around 60%
similarity, the strains were separated into 5 clusters,
three of them containing the L. lactis subsp. lactis. One
of the L. lactis subsp. lactis subclusters consisted of
Mast40 and the DVS starter strain (a mastitis and a starter
strain respectively). One of the L. lactis subsp. cremoris
clusters likewise contained both mastitis and starter
strains.
3.4. Sensitivity to pH, lysozyme and bile, hydrophobicity
All the bulk starter strains were completely inhibited
(>95%) at 0.2 mg/ml or lower concentration of lysozyme,
whereas the mastitis isolates and the DVS strain tolerating
even the top concentrations (3 mg/ml). The results of
representative strains are shown in Table 3.
The survival of the bulk starter strains exposed to bile
was reduced, some strains being unable to survive after
24 h. However, the mastitis isolates and DVS starter strain
were able to survive for 24 h, some being able to even grow
(representative strains shown in Table 4).
Table 1
Phenotypic properties of bovine mastitis isolates compared to the starters strains.
Mastitis isolates Starter strains
L. lactis subsp. lactis (n = 22) L. lactis subsp. cremoris (n = 7) Bulk starters (n = 7) DVS starter (n = 1)
Growth at 37 8C
a
+ + +
Fermentation of carbohydrates
b,c
L-Arabinose /+ /+
Ribose + + +
D-Xylose + + +
Mannitol + + +
Amygdalin + + +
Arbutin + + +
Salicin + + +
Cellobiose + + +
Maltose + + +
Lactose + + + +
Saccharose + +
Trehalose + + +
Strach + + +
b-Gentiobiose + + +
D-Turanose /(+)
d

D-Lyxose /(+)
d

D-Tagatose /(+)
d

Gluconate /+ /+
a
All strains were also cocci, gram positive, and catalase and oxidase negative.
b
All strains fermented galactose, D-glucose, D-fructose, D-mannose and N-acetyl-D-glucoseamine.
c
None of the strains fermented glycerol, erythritol, D-arabinose, L-xylose, adonitol, b-methyl-xyloside, L-sorbose, rhamnose, dulcitol, inositol, sorbitol, a-
methyl-D-mannoside, a-methyl-D-glucoside, esculin, melibiose, inulin, melezitose, D-rafnose, glycogen, xylitol, D-fucose, L-fucose, D-arabitol, L-arabitol, 2-
keto-gluconate and 2-keto-gluconate.
d
/(+), most negative but occasional positive.
Table 2
The antimicrobial susceptibility testing of all L. lactis strains/isolates.
Cut-off values (mg/ml)
Antibiotic gen kan strep ery chl tet van amp neo pen
Cut-off values (mg/ml)
a
32 64 32 1 8 4 4 2 ng
b
ng
b
Mastitis isolates (n = 22) 48 864 1664/64 (n = 5)
c
<0.5 4 <0.5/>128 (n = 2)
d
<0.5 <0.5 864 <0.5
Bulk starters (n = 7) <0.5 0.5 0.52 <0.5 1 <0.5 <0.5 <0.5 <0.50.5 <0.5
DVS starter (n = 1) 2 8 8 <0.5 4 <0.5 <0.5 <0.5 8 <0.5
a
Cut-off values according to the committee on Antimicrobial Susceptibility testing (EUCAST).
b
ng, not given for L. lactis.
c
Mast6, Mast34, Mast36, Mast38 and Mast40.
d
Mast19, Mast26.
C. Plumed-Ferrer et al. / Veterinary Microbiology 167 (2013) 592599 595
None of the bulk starter strains were able to survive at
pH 2.5 after 1 h, whereas the mastitis and DVS starter
survived up to 3 h, with time-dependent decrease of viable
counts (representative strains shown in Table 5). The
differences in hydrophobicity between the strains were
only slight and not related to the origin or subspecies of the
isolates (data not shown).
3.5. Adhesion capacity of the lactococcal strains to bovine
mammary epithelial cells
The capacity of the mastitis isolates and the DVS starter
strain to adhere to a primary bovine mammary epithelial
cell line was substantially higher in comparison to bulk
starter strains (representative strains shown in Fig. 3).
4. Discussion
The phenotypic tests used to identify bovine mastitis
associated isolates have probably resulted in some under-
estimation of the frequency of lactococci in clinical cases
over the years. Devriese et al. (1999) found isolates of L.
lactis subsp. lactis incorrectly identied in subclinical
intramammary infections in dairy cows. A study per-
formed by Fortin et al. (2003) on a great number of
tentatively identied gram-positive, catalase-negative
bacteria from milk samples, showed that 11% of Enter-
ococcus ssp., 24% of S. uberis, 3% of S. bovis and 1% of S.
dysgalactiae were actually Lactococcus ssp. when identied
using the tests recommended by the National Mastitis
council (NMC, 1999). In this work, from the 27 bovine
Fig. 1. Phylogenetic tree constructed by the neighbor-joining method based on the partial nucleotide sequences of the 16S rRNA gene from the mastitis
isolated and the control strains. Bootstrap values were obtained from 1000 replicates.
C. Plumed-Ferrer et al. / Veterinary Microbiology 167 (2013) 592599 596
mastitis isolates preliminarily identied as L. lactis, 17
isolates were L. lactis subsp. lactis, ve L. lactis subsp.
cremoris, three A. viridans, one L. garvieae and one S.
parauberis. Although for practical purposes regarding the
treatment and prevention an accurate identication may
not be necessary, these results conrm the need to use
PCR-based methods in cases where an exact species
designation is required.
The numerous cases where L. lactis is the cause of
infection, in different animals as well as in humans indicate
a safety concern, particularly as strains isolated from
diseased prawns can transmit an infection to healthy
prawns in experimental conditions (Wang et al., 2008).
Therefore, it is crucial to elucidate the differences between
pathogenic and innocuous strains, and this work
represents an attempt to phenotypic and genotypic
comparison of strains isolated from infections and some
strains widely used as starters in fermented milk products.
No genotypic differences between the mastitis isolates
and the starter strains were identiable by the methods
applied in this study. Although RAPD-PCR is not a very
Fig. 2. RAPD-PCR dendrogram analysis with three different primers for genotyping lactococcal strains of different origin.
Table 3
Lysosyme sensitivity (% of inhibition) of lactococcal strains representative of bulk starter strains (ARH53, ARH74), DVS starter strain (R604E), and mastitis
strains (Mast34, Mast36).
Strains % of Inhibition (average SD)
0.05 0.1 0.2 1 3
(Lysozyme concentration in mg/ml)
ARH53 44.6 4.5 81.2 4.5 97.1 1.0 nd nd
ARH74 60.6 3.3 97.8 0.5 97.4 0.7 nd nd
DVS starter nd nd 67.5 2.5 72.3 0.6 81.9 1.1
Mast34 nd nd 14.4 1.0 58.8 2.3 73.3 0.6
Mast36 nd nd 13.8 2.9 74.6 3.7 77.8 2.3
nd, not determined.
Table 4
Bile sensitivity of lactococcal strains representative of bulk starter strains
(ARH53, ARH74), DVS starter strain (R604E), and mastitis strains (Mast34,
Mast36).
Strains Log cfu/ml (average SD)
0 (Time in hours) 4 (Time in hours) 24 (Time in hours)
ARH53 6.1 0.2 3.6 0.0 0
ARH74 6.5 0.1 4.5 0.3 1.0 0.0
DVS starter 6.8 0.0 6.7 0.0 7.2 0.0
Mast34 6.8 0.0 6.4 0.0 6.9 0.0
Mast36 7.9 0.0 7.4 0.0 6.8 0.1
C. Plumed-Ferrer et al. / Veterinary Microbiology 167 (2013) 592599 597
accurate taxonomic tool, our results are in accordance with
Passerini et al. (2010), which did not nd genotypic
difference between strains of L. lactis originated from
different ecotypes (dairy, plant, animal skin and sour-
dough).
Despite the similarity of the genotypic proles between
L. lactis strains of different origin, the phenotypic proper-
ties showed consistent differences (higher tolerance to
temperature, lysozyme and bile, wider carbohydrate
fermentation capacity, better adhesion to mammary
epithelial cells and higher MICs for certain antibiotics)
between strains isolated from bovine mastitis and the
starter strains (with the exception of the DVS starter strain
which shared several characteristics of the mastitis
isolates). It is worth noticing that all the phenotypic
properties that were characteristic to the mastitis isolates,
are considered desirable while screening for robust
starters that could be used as human or animal probiotics
(Nomura et al., 2006; Fernandez et al., 2011), should be
kept in mind while assessing the safety of such strains.
Moreover, the fact that some proposed virulence factors
(such as capsule polysaccharide on the cell surface) found
in L. garvieae, E. faecalis, S. pneumonia and S. mutans have
also been found in L. lactis strains (Dabour and LaPointe,
2005; Chapot-Chartier et al., 2010; Morita et al., 2011) may
be of certain concern.
Some previous studies on strains of L. lactis isolated
from different environments indicate that the ability to
ferment carbohydrates is not related to the genotype but
reects the adaptation of the strain to the specic
environment (Fernandez et al., 2011). Strains are usually
separated in two groups: domesticated and environ-
mental, the dairy starters representing the former and the
plant- or animal-derived strains (including raw milk) the
latter, respectively (Passerini et al., 2010). Generally, the
environmental strains showed greater ability to ferment
carbohydrates and tolerate various stresses than the
domesticated ones. The assumption is that domesti-
cated strains that have been for thousands of years
propagated by back-slopping, have either lost or down
regulated functions not necessary in dairy fermentations,
such as the ability to ferment many plant carbohydrates or
tolerate different stress factors (Nomura et al., 2006;
Passerini et al., 2010; Fernandez et al., 2011). This
hypothesis ts also to our results, since mastitis isolates
are typical environmental strains and the bulk starters
domesticated ones. As the history and origin of the DVS
starter is not known, it cannot be stated whether its
phenotypic similarity to the mastitis is accidental or
whether it reects a relatively recent domestication.
In conclusion, while the typical domesticated L. lactis
strains are safe and apparently share certain phenotypic
characteristics, the environmental strains represent a
large and heterogeneous group that includes pathogenic
and non-pathogenic strains with no reliable genotypic or
phenotypic characteristics to differentiate between them.
This invites further studies with a representative number
of human and veterinary clinical isolates to provide
reliable safety criteria for the selection of strains for either
starter or probiotic use.
Acknowledgements
We thank Valio Oy and The Finnish Food Safety
Authority (Evira) for kindly providing the experimental
strains.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.vetmic.2013.09.011.
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Table 5
pH sensitivity of lactococcal strains representative of bulk starter strains (ARH53, ARH74), DVS starter strain (R604E), and mastitis strains (Mast34, Mast36).
Strains Log cfu/ml (average SD)
0 (Time in hours) 1 (Time in hours) 2 (Time in hours) 3 (Time in hours) 4 (Time in hours)
ARH53 6.0 0.3 0 0 0 0
ARH74 5.4 0.2 0 0 0 0
DVS starter 9.2 0.0 4.4 0.0 3.2 0.0 1.0 0.0 0.3 0.0
Mast34 9.3 0.0 4.0 0.0 2.8 0.1 1.4 0.1 0
Mast36 8.5 0.0 1.4 0.1 2.2 0.0 0.3 0.0 0
Fig. 3. Adhesion percentage of strains representative of bulk starter
strains (ARH53, ARH74), DVS starter strain (R604E), and mastitis strains
(Mast_34, Mast_36).
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