Lactobacillus fermentum CECT 5716 Reduces Staphylococcus Load in the Breastmilk of Lactating Mothers Suffering Breast Pain: A Randomized Controlled Trial

Abstract

Introduction:
Recent results indicate that human mastitis and painful breastfeeding may be characterized by a mammary bacterial dysbiosis, a process in which the population of potential pathogens increases at the expense of the normal mammary microbiota. The objective of the present study is the evaluation of three different doses of Lactobacillus fermentum CECT5716 to reduce the load of Staphylococcus in the breastmilk of women suffering from painful breastfeeding.

Materials and methods:
A randomized double-blinded controlled study with four study groups was performed. Three groups received the probiotic strain for 3 weeks at doses of 3×10(9) colony-forming units (CFU)/day, 6×10(9) CFU/day, or 9×10(9) CFU/day. The fourth group received a placebo of maltodextrin. The main outcome of the study was Staphylococcus counts in breastmilk. The secondary outcomes were Streptococcus, Lactobacillus, and total bacteria counts in breastmilk, immunoglobulin A and interleukin 8 concentrations in breastmilk, and breast pain scores.

Results:
At the end of the study, a significant decrease in the Staphylococcus load was observed in the probiotic groups compared with the baseline loads (p=0.045), whereas the control group maintained similar levels over time. A significant difference in the pain score was observed among the groups receiving the three probiotic doses compared with the control group (p=0.035, p=0.000, and p=0.028, respectively). A dose-response effect could not be observed because the three doses tested induced similar effects, and no significant differences were detected.

Conclusions:
We conclude that L. fermentum CECT5716 is an efficient treatment for breast pain during lactation associated with a high level of Staphylococcus in breastmilk.

Full text

Lactobacillus fermentum CECT 5716
Reduces Staphylococcus Load in the Breastmilk
of Lactating Mothers Suffering Breast Pain:
A Randomized Controlled Trial
Jose A. Maldonado-Lobo´n,
1,2
Miguel A. Dı´az-Lo´pez,
3
Raffaele Carputo,
3
Pilar Duarte,
3
Maria Paz Dı´az-Ropero,
1
Antonio D. Valero,
1
Ana San˜udo,
1
Lluis Sempere,
1
Marı´a Dolores Ruiz-Lo´ pez,
4,5
O
´
scar Ban˜uelos,
1
Juristo Fonolla´,
1
and Mo´nica Olivares Martı´n
1
Abstract
Introduction: Recent results indicate that human mastitis and painful breastfeeding may be characterized by a
mammary bacterial dysbiosis, a process in which the population of potential pathogens increases at the expense
of the normal mammary microbiota. The objective of the present study is the evaluation of three different doses
of Lactobacillus fermentum CECT5716 to reduce the load of Staphylococcus in the breastmilk of women
suffering from painful breastfeeding.
Materials and Methods: A randomized double-blinded controlled study with four study groups was performed.
Three groups received the probiotic strain for 3 weeks at doses of 3 · 10
9
colony-forming units (CFU)/day,
6 · 10
9
CFU/day, or 9 · 10
9
CFU/day. The fourth group received a placebo of maltodextrin. The main outcome
of the study was Staphylococcus counts in breastmilk. The secondary outcomes were Streptococcus, Lacto-
bacillus, and total bacteria counts in breastmilk, immunoglobulin A and interleukin 8 concentrations in
breastmilk, and breast pain scores.
Results: At the end of the study, a significant decrease in the Staphylococcus load was observed in the probiotic
groups compared with the baseline loads (p = 0.045), whereas the control group maintained similar levels over time.
A significant difference in the pain score was observed among the groups receiving the three probiotic doses
compared with the control group (p = 0.035, p = 0.000, and p = 0.028, respectively). A dose–response effect could
not be observed because the three doses tested induced similar effects, and no significant differences were detected.
Conclusions: We conclude that L. fermentum CECT5716 is an efficient treatment for breast pain during
lactation associated with a high level of Staphylococcus in breastmilk.
Introduction
E
xclusive breastfeeding is recommended up to 6
months of age by the World Health Organization
1
be-
cause breastmilk provides all the nutrients and components
necessary for proper infant development and growth. Espe-
cially during the first weeks of breastfe eding, women can
experien ce a range o f b rea st fee di ng problems, such as b reas t
and nipple pain, nipple cracks, and mastitis. These problems
make lactation difficult and, in some cases, lead to the
cessation of breastfeeding. Indeed, mastitis is the primary
medical cause of early weaning.
2
Mastitis is an inflammatory condition of the breast that is
usually associated with lactation. The reported incidence
varies from 3% to 33% and may or may not be accompa-
nied by infection.
3
Staphylococcus has been considered the
most common etiological agent of mastitis.
2
Staphylococcus
aureus is related primarily to acute mastitis cases, which
1
Biosearch Life S.A., Granada, Spain.
2
PhD Program in Nutrition and Food Science, University of Granada, Granada, Spain.
3
Virgen de las Nieves University Hospital, Granada, Spain.
4
Nutrition and Bromatology Department, University of Granada, Granada, Spain.
5
Institute of Nutrition and Food Technology, University of Granada, Granada, Spain.
This study is registered at www.ClinicalTrials.gov with clinical trial registration number NCT02093338.
BREASTFEEDING MEDICINE
Volume 10, Number 9, 2015
ª Mary Ann Liebert, Inc.
DOI: 10.1089/bfm.2015.0070
425

usually include both local (breast redness, engorgement, and
pain) and systemic symptoms. However, breastfeeding wo-
men may also experience local symptoms, such as nipple/
breast pain and breast engorgement not associated with fever,
or other systemic symptoms. These sym ptoms occur in ap-
proximately 10% of all breastfeeding women an d are
termed subacute mastitis.
2,4
This type of mas titis is typi-
cally caused by coagulase-negative s taphylococci, mainly
Staphylococcus epidermidis , and other bacterial agents
such as Str eptococcus and is g enerally misdiagnosed a s
‘‘noninfectious’’ or ‘‘mammary candidiasis.’’
2
In healthy
conditions, breastmilk has been shown to contain physiolog-
ical microbiota
5–7
in which Lactobacillus, Streptococcus,
Staphylococcus, Enterococcus spp., Bifidobacterium,and
Enterococcus represent the predominant bacterial groups.
5,8,9
Recent results indicate that human mastitis may be charac-
terized by a mammary bacterial dysbiosis, a process in which
the population of mastitis agents increases at the expense of
the normal mammary microbiota.
2
Some of the lactic acid bacteria isolated from the breast-
milk of healthy women exhibit the capability to inhibit the
growth of a wide spectrum of pathogenic bacteria through the
production of antibacterial compounds, competition for adhe-
sion locations, and co-aggregation events, among others,
10,11
and also exhibited immunomodulatory properties.
12
A previous
study demonstrated that Lactobacillus fermentum CECT5716,
a probiotic strain previously isolated from breastmilk, can be
used as an effective treatment of mastitis by reducing pathogen
counts in breastmilk.
13
The objective of the present study is the evaluation of three
different doses of L. fermentum CECT5716 to reduce the
Staphylococcus load in the breastmilk of women suffering
breast pain not associated with acute mastitis.
Materials and Methods
Study design and protocol
A randomized double-blinded controlled study with four
study groups was performed. Women suffering breast pain
with lactation were recruited. Written informed consent was
obtained from the women. The inclusion criteria were as
follows: women with painful breastfeeding and milk bacterial
counts at least 3 log
10
colony-forming units (CFU)/mL. The
exclusion criteria included fever, antibiotic treatment, mam-
mary abscesses, Raynaud’s syndrome, or any other mammary
pathology. Exclusion criteria during the study were lack of
compliance with the study protocol, antibiotic treatment, ad-
verse effects, or voluntary decision by the women to cease
participation.
The sample size was estimated based on the effect on the
main outcome of the study, the bacterial counts in breastmilk.
Based on previous results,
13
the study was designed to exhibit
sufficient power (80%) to detect a reduction of 1 log in total
bacterial counts in breastmilk after treatment with a 0.05
significance level. The number of women necessary was 20
per group.
One hundred forty-eight women were selected and dis-
tributed into four study groups, according to a randomization
generated by a computer program (SIGESMU

; Ediciones
DiazdeSantos, Madrid, Spain). Three groups received the
probiotic strain for 3 weeks at doses of 3 · 10
9
CFU/day for
Experimental Group I (EGI), 6 · 10
9
CFU/day for Experi-
mental Group II (EGII), and 9 · 10
9
CFU/day for Experimental
Group III (EGIII). The fourth group, the control group (CG),
received a placebo of maltodextrin. Pills containing 3 · 10
9
CFU per pill of L. fermentum CECT5716 or maltodextrin were
prepared. EGI consumed one probiotic pill per day with food,
EGII consumed two probiotic pills (one pill every 12 hours
taken with food), EGIII consumed three probiotic pills (taken
with breakfast, lunch, and dinner), and CG consumed three
pills of placebo (breakfast, lunch, and dinner). The probiotic
was provided in identical pills labeled in plain white with
a code number that referred to the manufacturing batch.
Pills were kept at 4C throughout the study. The concentra-
tion of viable Lactobacillus in the probiotic pills was stable
throughout the study.
This study was performed in accordance with the De-
claration of Helsinki, and the protocol was approved by the
Regional Ethics Committee of the Sistema Andaluz de Salud,
based in Seville, Spain.
Study outcomes and data collection
The main outcome of the study was Staphylococcus counts
in breastmilk. Secondary outcomes were Streptococcus,
Lactobacillus, and total bacteria counts, immunoglobulin A
(IgA) and interleukin 8 (IL-8) concentrations in breastmilk,
and breast pain scores.
Breastmilk samples were collected after 0, 1, 2, and 3
weeks of treatment. For breastmilk sample collection, the
nipple and mammary areola were cleaned with soap and
water, and an antibacterial (chlorhexidine) solution was ap-
plied. Breastmilk samples were obtained by manual expres-
sion; after the first drops were discarded, milk samples were
collected in sterile tubes. Samples were preserved at -20C
and processed within 1 week.
A Food Frequency Questionnaire was completed by the
women at the beginning and at the end of the intervention.
Breastmilk bacteria quantification
To estimate the concentration of total bacteria in breast-
milk, appropriate dilutions of breastmilk in a peptone saline
solution were spread in quadruplicate onto plates of PC agar
(Oxoid, Basingstoke, United Kingdom). The cultures were in-
cubated in anaerobiosis at 37C for 48 hours. After the incu-
bation, the colonies grown on the culture medium were counted,
and the number of viable microorganism (in CFU) per milliliter
of milk was calculated.
Staphylococcus, Streptococcus, and Lactobacillus counts
were measured by quantitative polymerase chain reaction.
The E.Z.N.A.

Stool DNA kit (Omega Bio-Tek, Norcross,
GA) was used for bacterial DNA isolation. In brief, 0.5 mL of
0.5 M EDTA Tergitol (Dow Chemical Co., Midland, MI)
surfactant (0.1% [wt/vol]) was added to 1 mL of breastmilk,
and the solution was mixed and incubated for 10 minutes at
room temperature. After incubation, samples were centri-
fuged at 14,000 g for 10 minutes at room temperature, and the
supernatants and fat residues were completely removed.
14
Bacterial pellets were homogenized with 200 lL of sterile
water, and each sample was transferred to a tube containing
200 mg of glass beads for DNA extraction following the in-
structions of the E.Z.N.A. Stool DNA kit protocol.
DNA quantification was performed by quantitative poly-
merase chain reaction using SYBR

green as a fluorophore
426 MALDONADO-LOBO
´
N ET AL.

(PerfeCTa

SYBR Green SuperMix with ROX [Quanta
Biosciences, Gaithersburg, MD] for Streptococcus and Sta-
phylococcus and Brilliant III Ultra-Fast SYBR Green QPCR
Master Mix [Agilent Technologies, Santa Clara, CA] for
Lactobacillus) and specific primers for each group.
15–17
A common thermal profile was applied for all amplifications:
95C for 5 minutes followed by 40 cycles of 95C for 30
seconds, 55C for 30 seconds, and 72C for 35 seconds.
A final melting curve was performed from 55Cto95C. For
amplification reactions, 2 lL of DNA from every sample and
bacterial group was used as template in 20 lL of final volume.
C
t
values (threshold cycle) were interpolated in specific
calibration curves to calculate the bacterial concentrations (in
CFU/mL).
IgA and IL-8 quantification in breastmilk
IgA and IL-8 concentrations were measured in breastmilk
samples by enzyme-linked immunosorbent assay quantifi-
cation kits following the manufacturer’s instructions (Bethyl
Laboratories [Montgomery, TX] and Diaclone [Besanc¸on,
France], respectively).
Pain questionnaire
The evolution of the symptoms was evaluated at 0, 1, 2,
and 3 weeks of treatment. Women were asked to score their
breast pain from 1 (extremely painful) to 10 (no pain).
An adaptation of the McGill Pain Questionnaire
18
validated
in Spain was used.
19
The Questionnaire has four dimensions
or scales of measures of intensity: Sensory Intensity Value,
Affective Intensity Value, Current Intensity Value, and Eva-
luative Intensity Value. The scales of the Questionnaire were
calculated as the sum of their items. Those items were re-
corded as 1 if the participant described the pain or as 0 if the
participant did not report that type of pain. Thus, the sensory
intensity scale can range from 0 to 33, as the full scale is
formed by 33 items. The Affective scale varies from 0 to 19,
and the scales of Current Intensity and Evaluative Intensity
can range from 0 to 6 and 5, respectively.
Statistical analysis
Initially, statistical tests for differences in the effect o f the
treatment were performed using parametric (analysis of
variance test) and nonparametric (Kruskal–Wallis) test
statistics (depending on the assumptions that were met)
when data we re c onti n uous or using the c hi -s qua re d te st f or
binary or categorical responses. Finally, a more robust and
accurate analysis via statistical modeling was performed to
determine the effect of the treatment adjusted by the time
and the covariates stated in the previous paragraph. Fur-
thermore, the association of the biochemical parameters
with the pain scores was studied by including these pa-
rameters as covariates when modeling outcomes related to
pain. The models applied to the data were linear mixed
models
20
for continuous data when the residuals were nor-
mally distributed, Poisson mixed models when the data
were relat ed to t he nu mb er of ev en ts ob se rve d, a nd lo gist i c
mixed m od els when t he outcomes to be analyzed were bi-
nary responses.
21
The te st s were pe rf or med at the two-sid ed
5% significance level, and 95% confidence intervals were
obtained for the estimates. The statistical software used to
perform the analysis were SPSS version 19 (IBM, Armonk,
NY) and R version 3.0 ( http://www.R-project.org/).
FIG. 1. Flow chart of participants. EGI, EGII, and EGIII, the three experimental groups receiving 3 · 10
9
,6· 10
9
, and
9 · 10
9
colony-forming units (cfu)/day, respectively.
PROBIOTIC TREATMENT OF BREAST PAIN IN LACTATION 427

Results
Population
Two hundred women were informed about the study, but
only 148 women agreed to participate and were randomized
for inclusion in the study. Finally, 113 women (30 in CG, 26
in EGI, 27 in EGII, and 30 in EGIII) re ceived the t reatment.
Women with total bacteria counts in breastmilk of < 10
3
CFU/mL were excluded. Before completion of the 3-w eek
intervention, 17 women discontinued treatment due to the
following: antibiotic treatment for mastitis (n = 1inCG,2in
EGI, 2 in EGII, and 2 in EGIII), own perception of lack of
improvement (n = 2 in CG, 2 in EGI, and 1 in EGII), antibiotic
treatment for tonsillitis (n = 1 in EGII), or loss to follow-up
(n = 1 in CG, 1 in EGI, and 2 in EGIII). The data for these
women were included in the analysis.
Therefore, data from 98 women were finally included in
the analysis (27 in CG, 23 in EGI, 24 in EGII, and 24 in
EGIII). A flow chart of the participants in the study is pre-
sented in Figure 1. The baseline characteristics of women of
all groups were similar with the exception of the time of
lactation, which was significantly lower in EGI: it was ap-
proximately half of the mean days for EGII and EGIII and
with a difference of 40 days on average with respect to CG
(Table 1). Interaction between treatment and this covariate
was included in the modeling data analysis.
Participants were asked to fill in a questionnaire regard-
ing their diets during the study period. In general, no sig-
nificant differences were noted in the diets of women among
the study groups or between baseline and the end of the
intervention. Therefore the significant effects observed for
outcomes cannot be attributed to the participants’ different
dietary habits.
Bacterial counts
At the end of intervention, a significant decrease in the
bacterial load, estimated as 0.764, was observed in EGI
compared with CG and with respect the initial point of the
study (p = 0.011). This result contrasts with that observed in
CG, which exhibited a significant increase in the total bac-
terial load at 21 days of intervention with respect to the
starting point of the study (Table 2). It is interesting that we
noted an interaction between days of breastfeeding and
bacterial load; as breastfeeding duration increased, there was
a significant decrease in the bacterial load (p = 0.027). Ad-
ditionally, the levels of IL-8 also appear to be closely related
to the bacterial load, as the latter increased as the IL-8 level
significantly increased (p = 0.000).
A significant decrease in the Staphylococcus load was
observed at the end of the study compared with baseline loads
(p = 0.045) in the experimental groups, whereas CG main-
tained similar levels (Table 2). The estimated mean value of
reduction was -1.387 for EGI, -1.736 for EGII, and slightly
lower for EGIII (–0.589). Higher values of IL-8 were sig-
nificantly associated with higher Staphylococcus loads
(p = 0.029). An interaction with the presence of nipple cracks
was detected in EGI and EGII. In these groups, the reduction
in the load of Staphylococcus was greater among women
Table 1. Baseline Characteristics of the Subjects of the Study
Control group EGI EGII EGIII p value
Mother’s age (years) 33.4 – 4.5 33.3 – 5.2 34.3 – 4.3 36.0 – 2.8 0.102
a
Cesarean section (%) 32.0 25.0 12.5 20.8 0.443
b
Previous children (%) 41.7 57.1 37.5 25.0 0.231
b
Previous mastitis events (%) 38.5 21.7 8.3 16.7 0.063
b
Breastfeeding time (days) 93.1 (56.3 – 137.7) 54.0 (27.7 – 87.8) 109.2 (74.0 – 155.9) 106.2 (59.2 – 175.7) 0.007
c
Mixed breastfeeding (%) 23.1 21.7 25.0 25.0 1.000
b
Days with pain symptoms (days) 56.6 (23.8 – 99.4) 36.2 (9.4 – 75.7) 46.2 (23.1 – 71.9) 84.7 (30.8 – 158.7) 0.305
c
Previous antibiotic treatment (%) 4.2 10.5 4.5 4.8 0.806
b
Nipple cracks (%) 28.0 47.8 54.2 29.2 0.159
b
Data are mean – standard deviation values, percentages, or mean (confidence interval) as indicated.
a
Analysis of variance.
b
Chi-squared test.
c
Kruskal–Wallis test.
EGI, EGII, and EGIII, the three experimental groups receiving 3 · 10
9
,6· 10
9
, and 9 · 10
9
colony-forming units/day, respectively.
Table 2. Bacterial Counts
CG EGI EGI II EGI III
Total bacteria
Time 0 4.41 – 0.93 4.55 – 1.32 4.31 – 0.75 4.39 – 1.02
7 days 4.65 – 0.94 4.37 – 0.88 4.15 – 0.88 4.53 – 1.20
14 days 4.61 – 1.10 4.58 – 0.97 4.38 – 0.97 4.62 – 1.11
21 days 4.66 – 0.99 4.18 – 0.73
a,b
4.36 – 0.93 4.33 – 1.15
Staphylococcus
Time 0 3.61 – 1.06 3.85 – 1.63 3.70 – 0.89 3.61 – 1.07
7 days 3.71 – 1.44 3.23 – 1.33 3.17 – 1.32 3.66 – 1.17
14 days 3.83 – 1.40 3.58 – 1.20 3.21 – 1.11 3.41 – 1.40
21 days 3.60 – 1.14 3.33 – 1.18
a,b
2.93 – 1.31
a,b
3.15 – 0.54
a
Streptococcus
Time 0 3.12 – 0.99 3.38 – 0.93 2.99 – 0.69 3.08 – 0.96
7 days 2.91 – 0.86 3.34 – 0.92 3.39 – 0.76 3.20 – 0.78
14 days 3.31 – 0.86 3.51 – 1.09 3.17 – 0.89 3.03 – 0.82
21 days 3.29 – 0.76 3.33 – 0.55 3.04 – 0.93
b
3.15 – 0.74
Lactobacillus
Time 0 2.11 – 0.51 1.96 – 0.19 1.95 – 0.37 2.05 – 0.30
7 days 2.30 – 0.37 2.10 – 0.38 1.97 – 0.47 2.19 – 0.42
14 days 2.19 – 0.34 2.12 – 0.41 1.86 – 0.45 2.03 – 0.35
21 days 2.20 – 0.42 1.94 – 0.34 1.88 – 0.31 1.98 – 0.40
Data are mean – standard deviation values, in log
10
colony-
forming units/mL of milk).
a
p < 0.05 versus basal (time 0).
b
p < 0.05 versus control group (CG)
EGI, EGII, and EGIII, the three experimental groups receiving
3 · 10
9
,6· 10
9
, and 9 · 10
9
colony-forming units/day, respectively.
428 MALDONADO-LOBO
´
N ET AL.

without nipple cracks than among women with nipple cracks
(p = 0.017 for EGI and p = 0.039 for EGII). This interaction
was also observed in EGIII, although it did not reach statis-
tical significance (p = 0.191).
With respect to Streptococcus load in breastmilk, although
lower loads were observed on avera ge for women in EGI,
EGII, and EGIII versus CG, differences were statistically
significant only for EGII (p = 0.044) (Table 2). Higher levels
of IL-8 was significantly correlated with higher Strepto-
coccus loads (p = 0.007).
No significant changes were detected in the Lactobacillus
counts.
Immune parameters in breastmilk
IL-8 was measured in the breastmilk samples. In EGI, a
progressive significant decrease over time was observed with
respect to the IL-8 values, and the difference was significant
compared with CG (p = 0.003). In EGII, a decreasing trend
was observed at 21 days of treatment (p = 0.067) (Table 3). A
strong association was detected between bacterial load and
IL-8 levels. This relationship was observed for total bacteria,
Staphylococcus loads, and Streptococcus loads (p = 0.000,
p = 0.008, and p = 0.007, respectively); as bacterial load in-
creased, the level of IL-8 significantly increased between
0.125 and 0.082 units for each unit of bacterial count. Women
with nipple cracks exhibited higher values of IL-8 than those
without (p = 0.020), and this association was independent of
group.
IgA values were only recorded at the beginning of the
treatment and at the end of the intervention (Table 3). At the
beginning of the study, IgA levels in EGI were significantly
higher than in CG (p = 0.027), but at the end of the intervention,
a decrease in the values was detected in EGI (p = 0.036).
Evaluation of breast pain
A score of the discomfort on a scale from 1 to 10 was
obtained from participants at each visit, where 1 indicated
significant pain and 10 indicated no pain (Fig. 2). At baseline,
there is no difference in the pain scores for the four groups
(Fig. 2). Each individual group showed improvement in the
pain score over time, measured on Days 7, 14 and 21, com-
pared with baseline score (p = 0.000). The three experimental
groups, EGI, EGII, and EGIII, showed improvement in the
pain scores over time compared with CG (P = 0.035,
p = 0.000, and p = 0.028, respectively). The effect of probiotic
treatment was noticeable from the first week. An association
between bacterial load and pain score was detected, as an
increase in total bacterial loads led to a decrease in the score
pain (p = 0.029). This association was also detected for Sta-
phylococcus load, although the interaction did not reach
statistical significance (p = 0.057).
An adaptation of the McGill Pain Questionnaire was used
to evaluate the pain perception. This questionnaire evaluates
four dimensions of pain: Sensory Intensity Value, Affective
Intensity Value, Current Intensity Value, and Evaluative In-
tensity Value (Table 4).
For the Sensory Intensity scale, the estimated mean values
indicated significantly lower pain for the experimental groups
EGI, EGII, and EGIII than for the CG (p = 0.000, p = 0.001,
and p = 0.040, respectively). IL-8 was identified as a covari-
ate that influences the Sensory Intensity scale, as the load of
IL-8 increased 1 unit as the average value of the pain scale
increased by 0.63 (p = 0.037). At the beginning of the study,
women mainly described their pain on the punctate/incisive
Table 3. Immune Parameters in Breastmilk
CG EGI EGI II EGI III
IL-8 (log
10
pg/mL)
Time 0 2.53 – 0.49 2.72 – 0.56 2.52 – 0.43 2.38 – 0.62
7 days 2.61 – 0.48 2.44 – 0.61
a
2.45 – 0.42 2.40 – 0.56
14 days 2.58 – 0.56 2.43 – 0.63
a
2.39 – 0.36 2.40 – 0.50
21 days 2.75 – 0.61 2.31 – 0.57
a
2.32 – 0.36
b
2.41 – 0.52
IgA (log
10
ng/mL)
Time 0 5.4 – 0.6 5.9 – 0.2
a
5.4 – 0.5 5.6 – 0.5
21 days 5.4 – 0.6 5.6 – 0.5
c
5.4 – 1.31 5.5 – 0.5
Data are mean – standard deviation values.
a
p < 0.05 versus control group (CG).
b
p = 0.067 versus CG.
c
p < 0.05 versus basal (time 0).
EGI, EGII, and EGIII, the three experimental groups receiving
3 · 10
9
,6· 10
9
, and 9 · 10
9
colony-forming units/day, respectively;
IgA, immunoglobulin A; IL-8, interleukin 8.
FIG. 2. Evolution of pain over time from the
pain rating scale. Data are mean – standard er-
ror of the mean values. *Significant difference,
p < 0.05 versus basal (time 0).
#
Significant dif-
ference, p < 0.05 for each probiotic treatment
versus the control group (CG) at each time.
EGI, EGII, and EGIII, the three experimental
groups receiving 3 · 10
9
,6· 10
9
, and 9 · 10
9
colony-forming units/day, respectively.
PROBIOTIC TREATMENT OF BREAST PAIN IN LACTATION 429

pressure subscale; between 83% and 92% of women selected
at least one item in this subscale.
For the Affective Intensity scale, a significant effect of the
treatment was observed in EGI and EGII (p = 0.011 and
p = 0.010, respectively). The subscale of anger/disgust was
typically used to describe the feelings related to the pain;
between 78% and 100% of women in the study described this
feeling at the beginning of the study. At the end of the in-
tervention the percentage of women reporting this item in the
experimental groups was 42% lower than in CG. Items on the
subscale of fear (fearful, frightful, terrifying) were selected
by 57–85% of women at the beginning of the intervention. At
the end of the treatment the percentage of women reporting
this item in the experimental groups was 62.3% lower than in
the CG. Similar results were observed in the case of feelings
related to grief and anxiety.
The treatment exhibited a significant effect on the evalu-
ative intensity of pain (temporary, intermittent, increasing,
constant, persistent). In particular, EGI and EGII reported a
30% reduced incidence on this scale versus CG (p = 0.035).
No significant changes were detected on the Current In-
tensity scale.
Occurrence of gastrointestinal disorders
and pharmacological treatments
The occurrence of diarrhea or flatulence was recorded in
the questionnaire completed by women. No significant dif-
ferences were detected with respect to diarrhea (p = 0.718) or
flatulence (p = 0.905) incidence among the groups.
Analgesic consumption decreased progressively through-
out the study in all groups; however, women in EGIII con-
sumed significantly fewer analgesics than women in CG
(p = 0.046).
Discussion
Traditionally, S. aureus has been considered the most
common etiological agent of mastitis; however, recent studies
have suggested that other microorganisms, such as coagulase-
negative Staphylococcus, are related to mastitis and breast pain
during lactation.
4,22
Previous studies demonstrated the capa-
bility of certain Lactobacillus strains to reduce the bacterial
load in the breastmilk of women suffering mastitis.
13
One of
these strains is L. fermentum CECT5716, a strain isolated from
human milk. The present study evaluated the effect of three
different escalating doses of the L. fermentum CECT5716
strain on bacterial load in the breastmilk of women suffering
breast pain symptoms compatible with subacute mastitis. The
Lactobacillus consumption significantly reduced the Staphy-
lococcus load in breastmilk. The total bacteria and Staphylo-
coccus loads were significantly correlated with the perception
of breast pain of the women, demonstrating that alterations in
breastmilk microbiota are directly related to the symptoms of
pain in this disease. The bacterial load was also strongly cor-
related with the level of IL-8 in breastmilk. This cytokine
elicits the infiltration of immune cells to the site of infection,
and its concentration in human milk has been proposed as an
effective indicator of mastitis.
23
The presence of physiological
microbiota in human milk is a relatively recent discovery,
5,6
and the number of studies dealing with the microbiological
aspects of human mastitis is few. The present work demon-
strates the correlation between the bacterial load of certain
bacterial groups in breastmilk and the occurrence of breast
pain during lactation.
According to these associations between Staphylococcus
load and breast pain, the reduction in the bacterial load induced
by the administration of L. fermentum CECT5716 significantly
reduced breast pain in women. A strength of this study
Table 4. Results of the Spanish Adaptation of the McGill Pain Questionnaire
Time, scale CG EGI EGII EGIII
Kruskal–Walllis
p value
Time 0
Sensory Intensity Value 7.88 – 3.56 5.91 – 2.31 6.22 – 2.33 6.50 – 2.04 0.293
Affective Intensity Value 5.19 – 2.45 3.87 – 1.96 3.70 – 2.30 4.08 – 2.24 0.131
Current Intensity Value 1.08 – 0.27 0.91 – 0.29 1.04 – 0.21 0.92 – 0.28 0.067
Evaluative Intensity Value 1.12 – 0.43 0.91 – 0.29 0.96 – 0.37 0.92 – 0.50 0.254
7 days
Sensory Intensity Value 6.92 – 2.17 4.35 – 2.95 4.48 – 2.86 5.71 – 2.71 0.023
Affective Intensity Value 3.81 – 2.00 1.91 – 2.19 1.70 – 2.01 2.92 – 2.32 0.004
Current Intensity Value 1.00 – 0.40 0.87 – 0.34 0.96 – 0.21 1.00 – 0.29 0.465
Evaluative Intensity Value 1.12 – 0.43 0.74 – 0.45 0.78 – 0.42 0.92 – 0.41 0.016
14 days
Sensory Intensity Value 6.40 – 3.71 3.89 – 2.40 3.77 – 2.51 4.86 – 2.82 0.011
Affective Intensity Value 2.80 – 2.50 1.11 – 1.85 1.32 – 1.89 2.41 – 2.32 0.015
Current Intensity Value 0.96
– 0.35 0.84 – 0.37 0.91 – 0.29 0.86 – 0.35 0.679
Evaluative Intensity Value 1.00 – 0.41 0.68 – 0.48 0.73 – 0.46 0.91 – 0.43 0.069
21 days
Sensory Intensity Value 5.27 – 4.24 3.06 – 2.56 2.85 – 2.56 3.70 – 2.90 0.070
Affective Intensity Value 2.05 – 2.36 0.53 – 1.23 0.95 – 1.88 1.40 – 2.01 0.032
Current Intensity Value 1.05 – 0.38 0.71 – 0.47 0.85 – 0.37 0.80 – 0.52 0.104
Evaluative Intensity Value 0.95 – 0.58 0.58 – 0.53 0.51 – 0.45 0.51 – 0.75 0.025
Data are mean – standard deviation values for each scale at each time of the study and for each treatment.
CG, control group; EGI, EGII, and EGIII, the three experimental groups receiving 3 · 10
9
,6· 10
9
, and 9 · 10
9
colony-forming units/day,
respectively.
430 MALDONADO-LOBO
´
N ET AL.

compared with previous research is the inclusion in the pro-
tocol of intermediate measures of different parameters. This
design, besides the advantages of a model of repeated mea-
sures, has allowed us to demonstrate that the effects were
noticeable from the first week of treatment. Another strength
of the study is the inclusion of a validated pain questionnaire
that evaluated the sensorial aspects of pain as well as the af-
fective dimension of pain. In this sense, women in the study
described their pain before intervention with adjectives related
to anger, fear, and anxiety. The Lactobacillus treatment sig-
nificantly improved this assessment in the women. This is an
important aspect of the study because the emotional distress
causes by mastitis can lead to the cessation of breastfeeding.
Although a significant increase in Lactobacillus counts in
breastmilk could not be detected in women consuming the
Lactobacillus strain, the presence of the strain in breastmilk
after oral administration has been previously demonstrated.
13
The relatively low counts of Lactobacillus versus Staphylo-
coccus in the breastmilk samples suggest that the mechanism
for the decreased pain is unlikely to be simply a ‘‘competition
phenomenon.’’ Some authors have suggested that host innate
immune responses activated in the mammary gland are a
critical feature of mastitis disease and that mastitis could be
the result of an inflammatory response to an otherwise in-
nocuous bacterium.
24
In fact, although S. epidermidis was
common in the breastmilk of women suffering mastitis,
strains of this species can also be found in milk of healthy
women.
6,8,22
With respect to the role of the immune system in
mastitis, the effect of probiotic strains on this pathology has
been related to an effect on the immune response.
25,26
L. fermentum CECT5716 has been shown in previous studies
to possess immunomodulatory capabilities. For example, the
strain exhibited anti-inflammatory activity in animal models
of inflammation
27,28
and immuno-enhancing activity in an
intervention study in humans.
29
The immunomodulatory
properties of L. fermentum CECT5716 may contribute to the
effect of the strain on mastitis.
A dose–response effect could not be observed because the
three doses tested induced similar effects, and no statistically
significant differences were detected. The fact that three
doses tested range over the same magnitude order, 9 log,
could be a limitation of the study. We cannot exclude the
possibility that a significantly higher dose of Lactobacillus
could lead to a dose-related response. We conclude that the
current dosing of lactobacilli, at a level of 3 · 10
9
CFU/day is
effective and sufficient to produce a decrease in staphylo-
coccal load in breastmilk leading to improvement in pain
symptoms in women suffering from breast pain associated
with Staphylococcus in the breastmilk.
Acknowledgments
The authors wish to thank Matilde Palomares Cuadrados,
Maria Jose
´
Goma
´
riz, Rosa Hoyas Romero, Isaura Gonza
´
lez,
Maria Jesu
´
s Gil, and Carmela Santamarı
´
a Palacios, midwives
of Spanish Health Services and B reastf eedin g Support
Associations (Mamilactancia and Grupo Apoyo Multi-
lactancia), for their collaboration in the study and Llenalia
Garcı
´
a Ferna
´
ndez (SEPLIN, Soluciones Estadı
´
sticas) for
the statistical analysis of the data. J.A.M.-L. was the recipient
of a fellowship from the Fundacio
´
n Universidad-Empresa
(Universidad de Granada, Granada, Spain). This work is part
of his doctoral thesis entitled ‘‘Lactobacillus fermentum
CECT5716: An ally during lactation’’ (Programa de Doctor-
ado en Nutricio
´
n y Ciencias de los Alimentos [B14.56.1]).
Disclosure Statement
J.A.M.-L., M.P.D.-R., A.D.V., A.S., L.S., O.B., J.F., and
M.O.M. are employees of Biosearch, which is the owner of
the patent for L. fermentum CECT5716. M.A.D.-L., R.C.,
P.D., and M.D.R.-L. declare no competing financial interests
exist.
References
1. Kramer MS, Kamuka R. The Optimal Duration of Ex-
clusive Breastfeeding. A Systematic Review. World Health
Organization, Geneva, 2001.
2. Contreras GA, Rodrı
´
guez JM. Mastitis: Comparative eti-
ology and epidemiology. J Mammary Gland Biol Neoplasia
2011;16:339–356.
3. World Health Organization. Mastitis: Causes and Man-
agement. World Health Organization, Geneva, 2000.
4. Amir LH, Cullinane M, Garland SM, et al. The role of
micro-organisms (Staphylococcus aureus and Candida al-
bicans) in the pathogenesis of breast pain and infection in
lactating women: Study protocol. BMC Pregnancy Child-
birth 2011;11:54.
5. Martı
´
n R, Langa S, Reviriego C, et al. Human milk is a
source of lactic acid bacteria for the infant gut. J Pediatr
2003;143:754–758.
6. Heikkila
¨
MP, Saris PE. Inhibition of Staphylococcus aureus
by the commensal bacteria of human milk. J Appl Micro-
biol 2003;95:471–478.
7. Perez PF, Dore
´
J, Leclerc M, et al. Bacterial imprinting of
the neonatal immune system: Lessons from maternal cells?
Pediatrics 2007;119:e724–e732.
8. Martı
´
n R, Heilig HG, Zoetendal EG, et al. Cultivation-
independent assessment of the bacterial diversity of breast
milk among healthy women. Res Microbiol 2007;158:
31–37.
9. Collado MC, Delgado S, Maldonado A, et al. Assessment
of the bacterial diversity of breast milk of healthy women
by quantitative real-time PCR. Lett Appl Microbiol
2009;48:523–528.
10. Martı
´
n R, Olivares M, Marin ML, et al. Probiotic potential
of three lactobacilli strains isolated from human breast
milk. J Hum Lact 2005;21:8–17.
11. Olivares M, Dı
´
az-Ropero MP, Martin R, et al. Anti-
microbial potential of four Lactobacillus strains isolated
from breast milk. J Appl Microbiol 2006;101:72–79.
12. Dı
´
az-Ropero MP, Martin R, Sierra S, et al. Two Lacto-
bacillus strains, isolated from breast milk, differently
modulate the immune response. J Appl Microbiol 2007;
102:337–343.
13. Arroyo R, Martı
´
n V, Maldonado A, et al. Treatment of
infectious mastitis during lactation: Antibiotics versus oral
administration of Lactobacilli isolated from breast milk.
Clin Infect Dis 2010;50:1551–1558.
14. Grattepanche F, Lacroix C, Audet P, et al. Quantification
by real-time PCR of Lactococcus lactis subsp. cremoris in
milk fermented by a mixed culture. Appl Microbiol Bio-
technol
2005;66:414–421.
15. Heilig HG, Zoetendal EG, Vaughan EE, et al. Molecular
diversity of Lactobacillus spp. and other lactic acid bac-
teria in the human intestine as determined by specific
PROBIOTIC TREATMENT OF BREAST PAIN IN LACTATION 431

amplification of 16S ribosomal DNA. Appl Environ Mi-
crobiol 2002;68:114–123.
16. Dalwai F, Spratt DA, Pratten J. Use of quantitative PCR
and culture methods to characterize ecological flux in
bacterial biofilms. J Clin Microbiol 2007;45:3072–3076.
17. Martineau F, Picard, FJ, Ke D, et al. Development of a PCR
assay for identification of staphylococci at genus and spe-
cies levels. J Clin Microbiol 2001;39:2541–2547.
18. Melzack R. The McGill Pain Questionnaire: Major prop-
erties and scoring methods. Pain 1975;1:277–299.
19. La
´
zaro C, Caseras X, Whizar-Lugo VM, et al. Psycho-
metric properties of a Spanish version of the McGill Pain
Questionnaire in several Spanish-speaking countries. Clin J
Pain 2001;17:365–374.
20. West BT, Welch KB, Galecki AT. Linear Mixed Models. A
Practical Guide Using Statistical Software. Chapman &
Hall/CRC, Boca Raton, FL, 2007.
21. Gelman A, Hill J. Data Analysis Using Regression and
Multilevel/Hierarchical Models. Cambridge University
Press, Cambridge, United Kingdom, 2007.
22. Delgado S, Arroyo R, Jime
´
nez E, et al. Staphylococcus
epidermidis strains isolated from breast milk of women
suffering infectious mastitis: Potential virulence traits and
resistance to antibiotics. BMC Microbiol 2009;9:82.
23. Hunt KM, Williams JE, Shafii B, et al. Mastitis is associ-
ated with increased free fatty acids, somatic cell count, and
interleukin-8 concentrations in human milk. Breastfeed
Med 2013;8:105–110.
24. Glynn DJ, Hutchinson MR, Ingman WV. Toll-like receptor
4 regulates lipopolysaccharide-induced inflammation and
lactation insufficiency in a mouse model of mastitis. Biol
Reprod 2014;90:91.
25. Klostermann K, Crispie F, Flynn J, et al. Intramammary
infusion of a live culture of Lactococcus lactis for treatment
of bovine mastitis: Comparison with antibiotic treatment in
field trials. J Dairy Res 2008;75:365–373.
26. Crispie F, Alonso-Go
´
mez M, O’Loughlin C, et al. In-
tramammary infusion of a live culture for treatment of
bovine mastitis: Effect of live lactococci on the mammary
immune response. J Dairy Res 2008;75:374–384.
27. Peran L, Camuesco D, Comalada M, et al. Lactobacillus
fermentum, a probiotic capable to release glutathione, pre-
vents colonic inflammation in the TNBS model of rat co-
litis. Int J Colorectal Dis 2006;21:737–746.
28. Arribas B, Elena Rodrı
´
guez-Cabezas M, Comalada M,
et al. Evaluation of the preventative effects exerted by
Lactobacillus fermentum in an experimental model of
septic shock induced in mice. Br J Nutr 2008;29:1–8.
29. Olivares M, Diaz-Ropero MP, Sierra S, et al. Oral intake of
Lactobacillus fermentum CECT5716 enhances the effect of
influenza vaccination. Nutrition 2007;23:254–260.
Address correspondence to:
Mo
´
nica Olivares Martı
´
n, PhD
Biosearch Life S.A.
Camino de Purchil 66
18004, Granada, Spain
E-mail: molivares@biosearchlife.com
432 MALDONADO-LOBO
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