The breast microbiota: role in breastfeeding succe and women's health
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1 The breast microbiota: role in breastfeeding succe and women's health Leonides Fernández Álvarez Dpt. Nutrition, Food Science and Food Technology Complutense University of Madrid, Spain
2 El Cuerpo Humano y su Universo Bacteriano. Fuente: ScienceMagazine Is there a breast microbiota??
3 The Body s Ecosystem Research on the human microbiome is booming, and scientists have moved from simply taking stock of gut flora to understanding the influence of microbes throughout the body. By The Scientist Staff August 1, 2014
4 Human mammary gland
5 Human milk microbiota Bacterial diversity How do they reach the mammary gland? What are their biological functions? What happens in special situations?
6 Human milk microbiota: natural diversity Enterococcus 10 3 ufc/ml Streptococcus Roseburia Kocuria >50 genera >200 species Staphylococcus Lactobacillus Corynebacterium Shigella Lactococcus Klebsiella Bifidobacterium Escherichia Ruminococcus Propionibacterium Faecalibacterium Veillonella 12 predominant genera Detected in >90% of samples Heikkila and Saris, 2003 Martin et al., 2003, 2006, 2007a,b, 2009 Martin et al., 2010 Hunt et al, 2011 Jost et al, 2013
7 Human milk microbiota: it s dynamic! Hunt et al (2011) PLoS ONE 6:e21313 Cabrera-Rubio R et al. (2012). Am J Clin Nutr 96:
8 Human milk microbiota: origin
9 Human milk microbiota: biological function Infant gut colonization 800 ml/day bacteria/day
10 Benefits of breastfeeding obesity necrotizing enterocolitis acute otitis media Diabetes mellitus type II gastrointestinal infections infecciones respiratory infections BREAST CANCER OVARIAN
11 Lactation problems? Mastitis: la gran desconocida MASTITIS? 30-35%!!!
12 Lactation problems: mastitis Mastitis: por qué se desconoce? Routine breast milk analysis is considered Laboratory investigations unnecessary are NOT routinely NEEDED for MASTITIS Mastitis aguda Recolección muestras No standarized protocols Amir & ABM Protocol Commitee Interpretación resultados
13 Mastitis: an altered microbiota N = (100%) S. epidermidis S. aureus Staphylococcus spp. Rothia spp. Kocuria spp. 92% 30% 4% 29% 1% Staphylococcus et rel. 98 Streptococcus 70 Corynebacterium Enterococcus Enterobacteriaceae Candida spp. 2 4 <1 17 Grupo Mitis Grupo Salivarius Otros estreptococos 51% 32% 10% No identificados Muestras analizadas (%)
14 Mastitis: a bacterial dysbiosis Mastitis infecciosas: una disbiosis bacteriana Staphylococcus Streptococcus Corynebacterium Staphylococcus Streptococcus Lactobacillus Leuconostoc Bifidobacterium Corynebacterium Total CFU/mL 1 species/strain: Up Enterococcus to CFU/mL
15 Mastitis: a bacterial dysbiosis Infectious mastitis: a dysbiosis of the mammary microbiota Samples: 50 ml Acute mastitis (Staphylococcus aureus) No mastitis Subacute mastitis (dilution 0; sample 100 µl) (Staphylococcus epidermidis)
16 Healthy breast Staphyloccus epidermidis Lymphocyte Epithelial cell Subacute mastitis by S. epidermidis (misdiagnosed as mammary candidiasis after visual assessment!!!!! of the breast)
17 Relative abundance (%) Infectious mastitis: Effect on the microbiome Time (week) Mastitis Recovering Well Worsening Mastitis Somatic cell count (cells/μl) Dr. M. McGuire (unpublished results)
18 Factors that modulate the mammary microbiota Host Breastfeeding Microbial Medical
19 Infectious mastitis: different types El iceberg de las mastitis Sensación de hipogalactia Staphylococcus aureus Acute mastitis Subacute mastitis Staph. epidermidis Strep. mitis Strep. salivarius et rel. Granulomatous mastitis Corynebacterium spp.
20 Acute mastitis: Staphylococcus aureus Acute inflammation Colonization Infection Toxins Toxins Systemic symptoms (flu-like symptoms)
21 Subacute mastitis: Coagulase-negative staphylococci (S. epidermidis ) Streptococci (S. mitis, S. salivarius ) Subacute mastitis: Colonization Infection Biofilm formation Antibiotic resistance Recurrent/chronic infection Pain Engorgement NO Toxins No breast redness No systemic symptoms Widely underrated/underdiagnosed
22 Mastitis and biofilms There is a perception that single-celled organisms are asocial, but that is misguided. When bacteria are under stress which is the story of their lives they team up and form this collective called a biofilm. If you look at naturally occurring biofilms, they have very complicated architecture. They are like cities with channels for nutrients to go in and waste to go out Symptoms Infectious mastitis Inflammation Obstruction Antibiotic resistance Immune response Evasion Dissemination (progression of the infection) Andre Levchenko, Johns Hopkins University 3. Maturation 4. Detachment Physiology 1. Attached to the protein matrix of the epithelium 2. Cell-cell adhesion and proliferation Staphylococci /Streptococci Mammary epithelium
23 Mastitis and biofilms Mammary epithelium Mammary areola Bacteria in milk: 10 3 /ml Heterogeneous population Bacteria Normal milk flow Bacteria Predisposing factors Inflammation Mammary epithelium cracking Milky spots Bacteria in milk: >10 4 /ml Homogeneous population Bacteria Anomalous milk flow Bacteria Inflammation obstruction
24 Mastitis and biofilms: consequences Increased inflammation Increased feeding times Increased number of feedings More aggresive feeding ( baby fighnting against my breast ) Obstruction Ingurgitation Engorgement Retention Milk stasis Decreased milk secretion
25 Staphylococcus epidermidis is a commensal species!!! Staphylococcus epidermidis is one of the most important opportunistic pathogens Host/Ecology Strain (biofilm, antibiotic resistance) Concentration Lactating mammary gland is a very complex ductal system (a multi-catheter-like system ) produces a lactose/hmo-rich biological fluid S. epidermidis: main cause of catheter-associated infections Staphylococci: the most efficient galactose/lactose metabolism Highly efficient in using HMOs Hunt KM et al., Appl Environ Microbiol 78:
26 Mastitis: approaches to the treatment Antibiotics Probiotics High (and increasing) rates of antibiotic-resistant strains Antibiotic-resistance: STRAIN-specific property Two similar cases may have a rather different response to the same antibiotic Empirical treatment + culture Culture: species concentration antibiogram Alteration mammary immunity Infection Paracetamol Ibuprofen Inflammation = PAIN Management measures Very mild Mild Severe Very severe
27 Mastitis: approaches to the treatment MRSA-MRSE Biofilms Probiotics
28 Human milk: source of probiotics? PROBIOTICS Live microorganisms which when administered in adequate amounts confer a health benefit on the host Join FAO/WHO Expert Consultation on Evolution of Health and Nutritional Properties of Probiotics in Food (2001)
29 Probiotics for mastitis Identification (strain level) GRAS, QPS Safety PROBIOTICS Target: mastitis Efficacy in clinical trials Inhibition of Staphylococcus Streptococcus Target: mammary gland Viability and survival Good industrial traits Cost
30 Probiotics from human milk Breast milk lactobacilli to treat infectious lactational mastitis GROUP A Lb. gasseri CECT5714 Lb. salivarius CECT5713 ~ ufc GROUP B excipient 20 women Clinical signs of bilateral mastitis Staphylococci > 10 4 cfu ml -1 Previous non-efficient antibiotherapy Non other concomitant disease 4 weeks Milk samples (days 0, 7, 14, 28) Staphylococci and lactobacilli counts Medical examination (midwife/pediatrician/gynecologist)
31 Milk bacterial concentration (log 10 cfu/ml) Pain Lactobacilli in milk (% women) Milk bacterial concentration (log 10 cfu/ml) Pain Lactobacilli in milk (% women) L. fermentum CECT5716, group A (n=124) L. salivarius CECT5713, group B (n=127) Antibiotics, group C (n=101) 5 (No pain) 100 Before treatment (Very painful) 5 10 (No pain) 100 After treatment (Very painful) Arroyo R, Martín V, Maldonado A, Jiménez E, Fernández L, Rodríguez JM. Treatment of infectious mastitis during lactation: antibiotics versus oral administration of lactobacilli isolated from breast milk. Clin Infect Dis 2010, 50:
32 Effectiveness of probiotics: metabolomics Vázquez-Fresno et al. Urinary metabolomic fingerprinting after consumption of a probiotic strain in women with mastitis. Pharmacological Research, 87, 2014, L. salivarius PS2
33 Effectiveness of probiotics: transcriptomics Transcriptomics: Initial vs. Final (unpublished) L. salivarius PS2 Pathway s involved in cell death and inflamm ation INITIAL FINAL Pathways involved in normal cell processes
34 Mastitis: an unresolved issue Mastitis is common but poorly understood. Our study highlights how little is known about its epidemiology and pathogenesis
35 Conclusions Breast milk contains its own microbiota Lactational mastitis is a disbiosis Early diagnosis of mastitis must be improved Bacteria isolated from breast milk can be used as probiotics for mastitis
36 PROBILAC: Microbiota comensal en el periodo perinatal UCM-PROBISEARCH Juan Miguel Rodríguez Leonides Fernández María L. Marín Rebeca Arroyo Nivia Cárdenas Javier de Andrés Susana Delgado Diana Escuder Irene Espinosa Esther Jiménez Susana Langa Antonio Maldonado Irene Martín Rocío Martín Virginia Martín Marta Gómez Arantza Gómez Susana Manzano Laura Moles Pilar Mediano Carlota Reviriego Ana Soto M. Antonia Villar Irene Chico Elena Montoya Mireia Vallespinós Dpto. Nutrición, Bromatología y Tecnología de los Alimentos Universidad Complutense de Madrid
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