M. Carmen Collado

The human milk microbiome changes over lactation and is shaped by maternal weight and mode of delivery

BACKGROUND Breast milk is recognized as the most important postpartum element in metabolic and immunologic programming of health of neonates. The factors influencing the milk microbiome and the potential impact of microbes on infant health have not yet been uncovered. OBJECTIVE Our objective was to identify pre- and postnatal factors that can potentially influence the bacterial communities inhabiting human milk. DESIGN We characterized the milk microbial community at 3 different time points by pyrosequencing and quantitative polymerase chain reaction in mothers (n = 18) who varied in BMI, weight gain, and mode of delivery. RESULTS We found that the human milk microbiome changes over lactation. Weisella, Leuconostoc, Staphylococcus, Streptococcus, and Lactococcus were predominant in colostrum samples, whereas in 1- and 6-mo milk samples the typical inhabitants of the oral cavity (eg, Veillonella, Leptotrichia, and Prevotella) increased significantly. Milk from obese mothers tended to contain a different and less diverse bacterial community compared with milk from normal-weight mothers. Milk samples from elective but not from nonelective mothers who underwent cesarean delivery contained a different bacterial community than did milk samples from individuals giving birth by vaginal delivery, suggesting that it is not the operation per se but rather the absence of physiological stress or hormonal signals that could influence the microbial transmission process to milk. CONCLUSIONS Our results indicate that milk bacteria are not contaminants and suggest that the milk microbiome is influenced by several factors that significantly skew its composition. Because bacteria present in breast milk are among the very first microbes entering the human body, our data emphasize the necessity to understand the biological role that the milk microbiome could potentially play for human health.

The Mucin Degrader Akkermansia muciniphila Is an Abundant Resident of the Human Intestinal Tract

ABSTRACT A 16S rRNA-targeted probe, MUC-1437, was designed and validated in order to determine the presence and numbers of cells of Akkermansia muciniphila, a mucin degrader, in the human intestinal tract. As determined by fluorescent in situ hybridization, A. muciniphila accounted more than 1% of the total fecal cells and was shown to be a common bacterial component of the human intestinal tract.

Intestinal integrity and Akkermansia muciniphila, a mucin-degrading member of the intestinal microbiota present in infants, adults, and the elderly.

ABSTRACT Fluorescence in situ hybridization and real-time PCR analysis targeting the 16S rRNA gene of Akkermansia muciniphila were performed to determine its presence in the human intestinal tract. These techniques revealed that an A. muciniphila-like bacterium is a common member of the human intestinal tract and that its colonization starts in early life and develops within a year to a level close to that observed in adults (108 cells/g) but decreases (P < 0.05) in the elderly.

Adhesion of Selected Bifidobacterium Strains to Human Intestinal Mucus and the Role of Adhesion in Enteropathogen Exclusion

The ability of potential probiotic strains to adhere to the intestinal mucosa and exclude and displace pathogens is of utmost importance for therapeutic manipulation of the enteric microbiota. The ability of seven selected human bifidobacterial strains and five human enteropathogenic strains to adhere to human intestinal mucus was analyzed and compared with that of four strains isolated from chicken intestines. The adhesion of the bifidobacterial strains ranged from 3 to 16% depending on the strain. Bifidobacterium strains of animal origin adhered significantly better than did strains of human origin. Of the pathogenic bacteria, Escherichia coli NCTC 8603 had the highest adhesion value (20%), Salmonella Typhimurium ATCC 29631, Enterobacter sakazakii ATCC 29544, and Clostridium difficile ATCC 9689 had adhesion values ranging from 10 to 15%, and Listeria monocytogenes ATCC 15313 had the lowest adhesive value (3%). The ability of these bifidobacteria to inhibit pathogen adhesion and to displace pathogens previously adhering to mucus was also tested. The inhibition of pathogens adhesion by these bifidobacterial strains was variable and clearly strain dependent. In general, bifidobacterial strains of animal origin were better able to inhibit and displace pathogens than were human strains. Preliminary characterization of bacterial adhesion was accomplished using different pretreatments to explore adhesion mechanisms. The results indicate that different molecules are implicated in the adhesion of bifidobacteria to the human intestinal mucus, constituting a multifactorial process.

Production of Bacteriocin-Like Inhibitory Compounds by Human Fecal Bifidobacterium Strains

Acid- and bile-resistant Bifidobacterium strains were isolated from human feces and identified by genus-specific PCR and randomly amplified polymorphic DNA PCR. Twenty-four different strains were screened for possible production of proteinaceous antimicrobial compounds by assaying the inhibitory effects of their neutralized culture supernatants. Six Bifidobacterium strains (BIR-0304, BIR-0307, BIR-0312, BIR-0324, BIR-0326, and BIR-0349) were selected on the basis of their broad inhibitory spectra. These strains were active against gram-positive and gram-negative bacteria and yeasts relevant to food safety and human health. The antagonistic effects of the six selected Bifidobacterium strains were related to bacteriocin-like compounds, which were active at pH values between 3 and 10, stable at 100 degrees C for 10 min, resistant to alpha-amylase and lipase A, but sensitive to proteinases (trypsin, proteinase K, protease A, pepsin, and cathepsin B). The molecular masses of the antimicrobial compounds produced by Bifidobacterium BIR-0312 and BIR-0324 were in the range of 10 to 30 kDa, and those of the compounds produced by Bifidobacterium BIR-0304, BIR-0307, BIR-0326, and BIR-0349 were less than 10 kDa. All Bifidobacterium strains produced maximum antimicrobial activities in the late logarithmic phase of growth and in the presence of Tween 80. These results confirm that the synthesis of bacteriocin-like inhibitory compounds is a key factor in the in vitro inhibition of pathogen and spoilage bacteria by Bifidobacterium strains.

Adhesion properties and competitive pathogen exclusion ability of bifidobacteria with acquired acid resistance.

The adhesion properties of Bifidobacterium longum and Bifidobacterium catenulatum strains with an acquired resistance to acid and their ability to competitively exclude Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, Enterobacter sakazakii, and Clostridium difficile from adhering to human intestinal mucus were evaluated and compared with the results when the same experiments were run with the original acid-sensitive strains. In half of the four studied cases, the acid-resistant derivative showed a greater ability to adhere to human intestinal mucus than the original strain. The ability of bifidobacteria to inhibit pathogen adhesion to mucus was not generally improved by the acquisition of acid resistance. In contrast, three of the four acid-resistant strains showed a greater ability to displace preadhered pathogens than the original strains, especially preadhered Salmonella Typhimurium and C. difficile. Overall, the induction of acid resistance in bifidobacteria could be a strategy when selecting strains with enhanced stability and improved surface properties that favor their potential functionality as probiotics against specific pathogens.

Potential probiotic characteristics of Lactobacillus and enterococcus strains isolated from traditional dadih fermented milk against pathogen intestinal colonization

Traditional fermented buffalo milk in Indonesia (dadih) has been believed to have a beneficial impact on human health, which could be related to the properties of the lactic acid bacteria (LAB) involved in its fermentation process. In previous studies, it was discovered that strains of dadih lactic isolates possessed some beneficial properties in vitro. In the present study, the adhesion capacity of specific LAB isolates from dadih to intestinal mucus was analyzed. Further, the ability to inhibit model human pathogens and displace them from mucus was assessed. The adhesion of tested LAB strains was strain-dependent and varied from 1.4 to 9.8%. The most adhesive Lactobacillus plantarum strain was IS-10506, with 9.8% adhesion. The competition assay between dadih LAB isolates and pathogens showed that a 2-h preincubation with L. plantarum at 37 degrees C significantly reduced pathogen adhesion to mucus. All tested LAB strains displaced and inhibited pathogen adhesion, but the results were strain-specific and dependent on time and pathogen strains. In general, L. plantarum IS-10506 showed the best ability against pathogen adhesion.

Effect of glucose in removal of microcystin-LR by viable commercial probiotic strains and strains isolated from dadih fermented milk.

The removal of the cyanobacterial peptide toxin microcystin-LR at 4 and 37 degrees C by six commercial probiotic strains and Lactobacillus plantarum strains IS-10506 and IS-20506 isolated from dadih, Indonesian traditional fermented milk, was assessed in this study. The aim was to evaluate the main factors influencing the viability and metabolic activity of the probiotic strains, as well as their capacity to remove microcystin-LR. Both L. plantarum strains isolated from dadih, as well as Bifidobacterium lactis strains Bb12 and 420, were shown to be more resistant, and >85% remained viable in phosphate-buffered saline (PBS) solution for 48 h of incubation at either temperature, while the viability of the other four commercial bacteria decreased markedly over time. The effect of glucose on viability and removal of toxin was shown to be a strain-specific and strain-dependent property, but in general, the efficiency of microcystin-LR removal increased when glucose was added to the solution. A maximum removal of 95% was observed for L. plantarum strain IS-20506 (37 degrees C, 10 (11) colony-forming units mL(-1)) with 1-2% glucose supplementation and 75% in PBS alone.

Changes in Cecal Microbiota and Mucosal Gene Expression Revealed New Aspects of Epizootic Rabbit Enteropathy

Epizootic Rabbit Enteropathy (ERE) is a severe disease of unknown aetiology that mainly affects post-weaning animals. Its incidence can be prevented by antibiotic treatment suggesting that bacterial elements are crucial for the development of the disease. Microbial dynamics and host responses during the disease were studied. Cecal microbiota was characterized in three rabbit groups (ERE-affected, healthy and healthy pretreated with antibiotics), followed by transcriptional analysis of cytokines and mucins in the cecal mucosa and vermix by q-rtPCR. In healthy animals, cecal microbiota with or without antibiotic pretreatment was very similar and dominated by Alistipes and Ruminococcus. Proportions of both genera decreased in ERE rabbits whereas Bacteroides, Akkermansia and Rikenella increased, as well as Clostridium, γ-Proteobacteria and other opportunistic and pathogenic species. The ERE group displayed remarkable dysbiosis and reduced taxonomic diversity. Transcription rate of mucins and inflammatory cytokines was very high in ERE rabbits, except IL-2, and its analysis revealed the existence of two clearly different gene expression patterns corresponding to Inflammatory and (mucin) Secretory Profiles. Furthermore, these profiles were associated to different bacterial species, suggesting that they may correspond to different stages of the disease. Other data obtained in this work reinforced the notion that ERE morbidity and mortality is possibly caused by an overgrowth of different pathogens in the gut of animals whose immune defence mechanisms seem not to be adequately responding.

Breast Milk Polyamines and Microbiota Interactions: Impact of Mode of Delivery and Geographical Location

Background/Aims: The aim of the present study was to identify and quantify the polyamine levels in human milk obtained from different countries and through different modes of delivery, and to investigate their association with breast milk microbes. Methods: Mature breast milk samples were obtained from 78 healthy mothers after 1 month of lactation from 4 different geographical locations: Finland, Spain (Europe); South Africa (Africa); and China (Asia). Polyamines were determined using HPLC after dansyl derivatization and milk microbiota was obtained by 16S rRNA gene sequencing. Results: The mean values of polyamines in breast milk were 70.0, 424.2, and 610.0 nmol/dL for putrescine, spermidine and spermine, respectively, and 1,170.9 nmol/dL of total polyamines. The levels of putrescine were significantly higher in Spain (p < 0.05) and spermidine levels were significantly higher in Finland (p < 0.05) compared with other countries. Cesarean delivery had an impact on polyamine levels and it was related to an increase in the putrescine concentration being significant in Spanish samples (p < 0.01). Furthermore, putrescine levels were correlated positively with Gammaproteobacteria (r = 0.46, p < 0.001), especially with Pseudomonas fragi (r = 0.40, p < 0.001). Conclusions: The results demonstrate significant effect of geographical variations in human milk polyamine concentrations, being correlated with human milk microbiota composition. These differences may have an impact on infant development during lactation.