Clinical Infectious Diseases Advance Access published November 26, 2015 1
Prevention of infectious mastitis by oral administration of Lactobacillus
cr ipt
salivarius PS2 during late pregnancy
Leónides Fernández, Nivia Cárdenas, Rebeca Arroyo, Susana Manzano, Esther Jiménez, Virginia Martín, and Juan Miguel Rodríguez*
*
us
an
Complutense de Madrid, 28040 Madrid, Spain
Corresponding author and reprint requests: Dr. Juan Miguel Rodríguez, Departamento de
M
Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid,
pt ed
28040 Madrid, Spain. Phone: 34-91-3943837. Fax: 34-91-3943743. E-mail: [email protected]
Summary: Oral administration of Lactobacillus salivarius PS2, a strain isolated from breast milk
Ac
ce
of healthy women, during late pregnancy and early lactation can prevent infectious mastitis.
© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail [email protected].
Downloaded from http://cid.oxfordjournals.org/ at University of Sussex on November 30, 2015
Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad
2
ABSTRACT
cr ipt
Background. Previous studies have shown that oral administration of lactobacilli can be an efficient approach to treat lactational infectious mastitis. In this trial, we have evaluated the
potential of Lactobacillus salivarius PS2 to prevent this condition when orally administered
during late pregnancy to women that had suffered infectious mastitis after previous pregnancies.
us
probiotic group (n = 55) ingested daily 9 log10 colony-forming units of L. salivarius PS2 from
an
approximately week 30 of pregnancy until delivery while those in the placebo group (n = 53) received a placebo. The occurrence of mastitis was evaluated during the first 3 months after
M
delivery.
Results. Globally, 44 out of 108 women (59%) suffered mastitis; however, the percentage of women suffering mastitis in the probiotic group (25%, n = 14) was significantly lower than in the
pt ed
control group (57%, n = 30). When mastitis occurred, the milk bacterial counts in the probiotic group were significantly lower than those obtained in the placebo one. Conclusion. Oral administration of L. salivarius PS2 during late pregnancy appears to be an
ce
efficient method to prevent infectious mastitis in a susceptible population.
Ac
ClinicalTrials.gov identifier: NCT01505361.
Downloaded from http://cid.oxfordjournals.org/ at University of Sussex on November 30, 2015
Methods. In this study, 108 pregnant women were randomly divided in two groups. Those in the
3
INTRODUCTION The health effects of breastfeeding are well recognized and apply to mothers and infants in
cr ipt
developed and developing countries [1]; therefore, this infant feeding option has been
recommended by several prominent health organizations, including the World Health Organization, the American Academy of Pediatrics, the American College of Obstetricians and Gynecologists, or the American Public Health Association. However, numerous barriers to breastfeeding remain and,
us
generally used to define an infectious process of the mammary gland characterized by a variety of
an
local and, in acute cases, systemic symptoms [3].
The lactating mammary gland harbours many microorganisms in dynamic equilibrium, including potential mastitis-causing agents [4]. Upon disturbance of this balanced state, infection
M
can occur and recent studies suggest that mastitis is related to a mammary bacterial dysbiosis [5, 6]. Since resistance to antibiotics and formation of biofilms are common properties among
pt ed
mastitis-causing bacteria, many cases are refractory to antibiotic therapy. Therefore, alternative strategies are required to decrease mastitis incidence rates. In previous studies, we reported that oral administration of selected lactobacilli strains to lactating women was an effective alternative to antibiotics for treating infectious mastitis [7-9]. In
ce
this context, the objective of this work was to evaluate the efficacy of oral administration of one of these strains, Lactobacillus salivarius PS2, to prevent the development of lactational mastitis
Ac
in a population of pregnant women with a history of mastitis after previous pregnancies.
MATERIALS AND METHODS Design of the study and definition of mastitis. A total of 108 pregnant women, aged 24-35 and at 27-32 weeks of gestation, participated in this study. All met the following criteria: normal
Downloaded from http://cid.oxfordjournals.org/ at University of Sussex on November 30, 2015
among them, mastitis represents the first medical cause of undesired weaning [2]. Mastitis is
4
pregnancy, healthy status, and a history of lactational mastitis after, at least, a previous pregnancy. Women ingesting probiotic supplements or receiving antibiotic treatment in the
cr ipt
previous 30 days were excluded. All volunteers gave written informed consent to the protocol,
which had been approved by the Ethical Committee of Clinical Research of Hospital Clínico San Carlos Madrid (Spain). The study was registered in the ClinicalTrials.gov database
(NCT01505361). The study was designed as a double-blind randomized placebo-controlled trial.
us
equal size, using a computer-generated allocation sequence. Volunteers in the probiotic group (n
an
= 55) consumed a daily capsule with ~50 mg of freeze-dried probiotic (~9 log10 colony forming units [CFU] of L. salivarius PS2) from ~30 week of pregnancy until birth. The probiotic was
M
manufactured by Biopolis (Valencia, Spain), and kept at 4ºC throughout the study. The women in the placebo group (n = 55) only received the probiotic excipient (powdered milk). Two dropouts were registered in this group by factors that were related to the study. All volunteers were
pt ed
provided with diaries to record compliance with study product intake. Minimum compliance rate (% of the total treatment doses) was set at 86%.
Diagnosis of mastitis and breast pain score (BPS). Mastitis symptoms were assessed by
ce
midwifes during the first 3 months after birth. “Acute mastitis” was defined as an acute inflammation of the breast with local (ingurgitation, pain, redness, reduced milk secretion) and
Ac
systemic (flu-like) symptoms, while “subacute mastitis” was defined as a breast inflammation accompanied by other local symptoms (ingurgitation, needle-like and/or burning pain, reduced
milk secretion) without systemic symptoms [5]. Both definitions include cases associated with high milk bacterial and leukocyte counts (>3 log10 CFU/mL and >4 log10 cells/mL, respectively). Leukocyte quantification was performed by optical microscopy after May-Grünwald-Giemsa
Downloaded from http://cid.oxfordjournals.org/ at University of Sussex on November 30, 2015
Volunteers were randomly assigned, in a 1:1 ratio, into 2 groups (“probiotic” and “placebo”) of
5
differential staining. When mastitis occurred, volunteers were asked to score breast pain from 0
cr ipt
(extremely painful) to 10 (no pain), following a standard procedure [10].
Collection and microbiological analysis of the milk samples. Milk samples (10 mL) were
collected and stored as described previously [10]. Samples were obtained between 91 and 100
days after birth from women that did not suffer mastitis during the 3-month follow-up period. A
us
of such condition.
an
Samples were spread onto Baird Parker, Columbia Nadilixic Acid, Mac Conkey and Sabouraud Dextrose Chloramphenicol agar plates (BioMerieux, Marcy l'Etoile, France) and
M
incubated for 48 h at 37ºC in aerobic conditions for selective isolation and quantification of the main agents involved in infectious mastitis. After bacterial counting, representatives of each colony morphology type were identified by MALDI-TOF (VITEK MS, BioMerieux) in
pt ed
Probisearch (Tres Cantos, Spain).
Isolation and identification of L. salivarius PS2 in milk samples. Women were asked to provide a sample of milk at day 7 (± 2 days) after birth. These samples were inoculated onto
ce
MRS (Oxoid, Basingstoke, UK) plates supplemented with L-cysteine (0.5 g/L) (MRS-Cys). Plates were incubated as described previously [7, 8]. Isolates identified as L. salivarius by
Ac
MALDI-TOF were submitted to pulsed-field gel electrophoresis (PFGE) genotyping [8], and their profiles were compared with that of L. salivarius PS2.
Statistical analysis. Per protocol analyses were performed in the assessment of the effectiveness of the probiotic treatment. Microbiological data were recorded as CFU/mL, and transformed to
Downloaded from http://cid.oxfordjournals.org/ at University of Sussex on November 30, 2015
sample was collected from women developing mastitis as soon as possible (
Prevention of infectious mastitis by oral administration of Lactobacillus
cr ipt
salivarius PS2 during late pregnancy
Leónides Fernández, Nivia Cárdenas, Rebeca Arroyo, Susana Manzano, Esther Jiménez, Virginia Martín, and Juan Miguel Rodríguez*
*
us
an
Complutense de Madrid, 28040 Madrid, Spain
Corresponding author and reprint requests: Dr. Juan Miguel Rodríguez, Departamento de
M
Nutrición, Bromatología y Tecnología de los Alimentos, Universidad Complutense de Madrid,
pt ed
28040 Madrid, Spain. Phone: 34-91-3943837. Fax: 34-91-3943743. E-mail: [email protected]
Summary: Oral administration of Lactobacillus salivarius PS2, a strain isolated from breast milk
Ac
ce
of healthy women, during late pregnancy and early lactation can prevent infectious mastitis.
© The Author 2015. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail [email protected].
Downloaded from http://cid.oxfordjournals.org/ at University of Sussex on November 30, 2015
Departamento de Nutrición, Bromatología y Tecnología de los Alimentos, Universidad
2
ABSTRACT
cr ipt
Background. Previous studies have shown that oral administration of lactobacilli can be an efficient approach to treat lactational infectious mastitis. In this trial, we have evaluated the
potential of Lactobacillus salivarius PS2 to prevent this condition when orally administered
during late pregnancy to women that had suffered infectious mastitis after previous pregnancies.
us
probiotic group (n = 55) ingested daily 9 log10 colony-forming units of L. salivarius PS2 from
an
approximately week 30 of pregnancy until delivery while those in the placebo group (n = 53) received a placebo. The occurrence of mastitis was evaluated during the first 3 months after
M
delivery.
Results. Globally, 44 out of 108 women (59%) suffered mastitis; however, the percentage of women suffering mastitis in the probiotic group (25%, n = 14) was significantly lower than in the
pt ed
control group (57%, n = 30). When mastitis occurred, the milk bacterial counts in the probiotic group were significantly lower than those obtained in the placebo one. Conclusion. Oral administration of L. salivarius PS2 during late pregnancy appears to be an
ce
efficient method to prevent infectious mastitis in a susceptible population.
Ac
ClinicalTrials.gov identifier: NCT01505361.
Downloaded from http://cid.oxfordjournals.org/ at University of Sussex on November 30, 2015
Methods. In this study, 108 pregnant women were randomly divided in two groups. Those in the
3
INTRODUCTION The health effects of breastfeeding are well recognized and apply to mothers and infants in
cr ipt
developed and developing countries [1]; therefore, this infant feeding option has been
recommended by several prominent health organizations, including the World Health Organization, the American Academy of Pediatrics, the American College of Obstetricians and Gynecologists, or the American Public Health Association. However, numerous barriers to breastfeeding remain and,
us
generally used to define an infectious process of the mammary gland characterized by a variety of
an
local and, in acute cases, systemic symptoms [3].
The lactating mammary gland harbours many microorganisms in dynamic equilibrium, including potential mastitis-causing agents [4]. Upon disturbance of this balanced state, infection
M
can occur and recent studies suggest that mastitis is related to a mammary bacterial dysbiosis [5, 6]. Since resistance to antibiotics and formation of biofilms are common properties among
pt ed
mastitis-causing bacteria, many cases are refractory to antibiotic therapy. Therefore, alternative strategies are required to decrease mastitis incidence rates. In previous studies, we reported that oral administration of selected lactobacilli strains to lactating women was an effective alternative to antibiotics for treating infectious mastitis [7-9]. In
ce
this context, the objective of this work was to evaluate the efficacy of oral administration of one of these strains, Lactobacillus salivarius PS2, to prevent the development of lactational mastitis
Ac
in a population of pregnant women with a history of mastitis after previous pregnancies.
MATERIALS AND METHODS Design of the study and definition of mastitis. A total of 108 pregnant women, aged 24-35 and at 27-32 weeks of gestation, participated in this study. All met the following criteria: normal
Downloaded from http://cid.oxfordjournals.org/ at University of Sussex on November 30, 2015
among them, mastitis represents the first medical cause of undesired weaning [2]. Mastitis is
4
pregnancy, healthy status, and a history of lactational mastitis after, at least, a previous pregnancy. Women ingesting probiotic supplements or receiving antibiotic treatment in the
cr ipt
previous 30 days were excluded. All volunteers gave written informed consent to the protocol,
which had been approved by the Ethical Committee of Clinical Research of Hospital Clínico San Carlos Madrid (Spain). The study was registered in the ClinicalTrials.gov database
(NCT01505361). The study was designed as a double-blind randomized placebo-controlled trial.
us
equal size, using a computer-generated allocation sequence. Volunteers in the probiotic group (n
an
= 55) consumed a daily capsule with ~50 mg of freeze-dried probiotic (~9 log10 colony forming units [CFU] of L. salivarius PS2) from ~30 week of pregnancy until birth. The probiotic was
M
manufactured by Biopolis (Valencia, Spain), and kept at 4ºC throughout the study. The women in the placebo group (n = 55) only received the probiotic excipient (powdered milk). Two dropouts were registered in this group by factors that were related to the study. All volunteers were
pt ed
provided with diaries to record compliance with study product intake. Minimum compliance rate (% of the total treatment doses) was set at 86%.
Diagnosis of mastitis and breast pain score (BPS). Mastitis symptoms were assessed by
ce
midwifes during the first 3 months after birth. “Acute mastitis” was defined as an acute inflammation of the breast with local (ingurgitation, pain, redness, reduced milk secretion) and
Ac
systemic (flu-like) symptoms, while “subacute mastitis” was defined as a breast inflammation accompanied by other local symptoms (ingurgitation, needle-like and/or burning pain, reduced
milk secretion) without systemic symptoms [5]. Both definitions include cases associated with high milk bacterial and leukocyte counts (>3 log10 CFU/mL and >4 log10 cells/mL, respectively). Leukocyte quantification was performed by optical microscopy after May-Grünwald-Giemsa
Downloaded from http://cid.oxfordjournals.org/ at University of Sussex on November 30, 2015
Volunteers were randomly assigned, in a 1:1 ratio, into 2 groups (“probiotic” and “placebo”) of
5
differential staining. When mastitis occurred, volunteers were asked to score breast pain from 0
cr ipt
(extremely painful) to 10 (no pain), following a standard procedure [10].
Collection and microbiological analysis of the milk samples. Milk samples (10 mL) were
collected and stored as described previously [10]. Samples were obtained between 91 and 100
days after birth from women that did not suffer mastitis during the 3-month follow-up period. A
us
of such condition.
an
Samples were spread onto Baird Parker, Columbia Nadilixic Acid, Mac Conkey and Sabouraud Dextrose Chloramphenicol agar plates (BioMerieux, Marcy l'Etoile, France) and
M
incubated for 48 h at 37ºC in aerobic conditions for selective isolation and quantification of the main agents involved in infectious mastitis. After bacterial counting, representatives of each colony morphology type were identified by MALDI-TOF (VITEK MS, BioMerieux) in
pt ed
Probisearch (Tres Cantos, Spain).
Isolation and identification of L. salivarius PS2 in milk samples. Women were asked to provide a sample of milk at day 7 (± 2 days) after birth. These samples were inoculated onto
ce
MRS (Oxoid, Basingstoke, UK) plates supplemented with L-cysteine (0.5 g/L) (MRS-Cys). Plates were incubated as described previously [7, 8]. Isolates identified as L. salivarius by
Ac
MALDI-TOF were submitted to pulsed-field gel electrophoresis (PFGE) genotyping [8], and their profiles were compared with that of L. salivarius PS2.
Statistical analysis. Per protocol analyses were performed in the assessment of the effectiveness of the probiotic treatment. Microbiological data were recorded as CFU/mL, and transformed to
Downloaded from http://cid.oxfordjournals.org/ at University of Sussex on November 30, 2015
sample was collected from women developing mastitis as soon as possible (
