A proteomic approach towards understanding the cross talk between Bacteroides fragilis and Bifidobacterium longum in coculture.

A better understanding of the interactions among intestinal microbes is needed to decipher the complex cross talk that takes place within the human gut. Bacteroides and Bifidobacterium genera are among the most relevant intestinal bacteria, and it has been previously reported that coculturing of these 2 microorganisms affects their survival. Therefore, coculturing of Bifidobacterium longum NB667 and Bacteroides fragilis DSMZ2151 was performed with the aim of unravelling the mechanisms involved in their interaction. To this end, we applied proteomic (2D-DIGE) analyses, and by chromatographic techniques we quantified the bacterial metabolites produced during coincubation. Coculture stimulated the growth of B. longum, retarding that of B. fragilis, with concomitant changes in the production of some proteins and metabolites of both bacteria. The combined culture promoted upregulation of the bifidobacterial pyruvate kinase and downregulation of the Bacteroides phosphoenolpyruvate carboxykinase - 2 enzymes involved in the catabolism of carbohydrates. Moreover, B. fragilis FKBP-type peptidyl-prolyl cis-trans isomerase, a protein with chaperone-like activity, was found to be overproduced in coculture, suggesting the induction of a stress response in this microorganism. This study provides mechanistic data to deepen our understanding of the interaction between Bacteroides and Bifidobacterium intestinal populations.

Impact of Prematurity and Perinatal Antibiotics on the Developing Intestinal Microbiota: A Functional Inference Study.

BACKGROUND: The microbial colonization of the neonatal gut provides a critical stimulus for normal maturation and development. This process of early microbiota establishment, known to be affected by several factors, constitutes an important determinant for later health. METHODS: We studied the establishment of the microbiota in preterm and full-term infants and the impact of perinatal antibiotics upon this process in premature babies. To this end, 16S rRNA gene sequence-based microbiota assessment was performed at phylum level and functional inference analyses were conducted. Moreover, the levels of the main intestinal microbial metabolites, the short-chain fatty acids (SCFA) acetate, propionate and butyrate, were measured by Gas-Chromatography Flame ionization/Mass spectrometry detection. RESULTS: Prematurity affects microbiota composition at phylum level, leading to increases of Proteobacteria and reduction of other intestinal microorganisms. Perinatal antibiotic use further affected the microbiota of the preterm infant. These changes involved a concomitant alteration in the levels of intestinal SCFA. Moreover, functional inference analyses allowed for identifying metabolic pathways potentially affected by prematurity and perinatal antibiotics use. CONCLUSION: A deficiency or delay in the establishment of normal microbiota function seems to be present in preterm infants. Perinatal antibiotic use, such as intrapartum prophylaxis, affected the early life microbiota establishment in preterm newborns, which may have consequences for later health.

Interactions between Bifidobacterium and Bacteroides species in cofermentations are affected by carbon sources, including exopolysaccharides produced by bifidobacteria.

Cocultures of strains from two Bifidobacterium and two Bacteroides species were performed with exopolysaccharides (EPS) previously purified from bifidobacteria, with inulin, or with glucose as the carbon source. Bifidobacterium longum NB667 and Bifidobacterium breve IPLA20004 grew in glucose but showed poor or no growth in complex carbohydrates (inulin, EPS E44, and EPS R1), whereas Bacteroides grew well in the four carbon sources tested. In the presence of glucose, the growth of Bacteroides thetaiotaomicron DSM-2079 was inhibited by B. breve, whereas it remained unaffected in the presence of B. longum. Ba. fragilis DSM-2151 contributed to a greater survival of B. longum, promoting changes in the synthesis of short-chain fatty acids (SCFA) and organic acids in coculture with respect to monocultures. In complex carbohydrates, cocultures of bifidobacterium strains with Ba. thetaiotaomicron did not modify the behavior of Bacteroides nor improve the poor growth of bifidobacteria. The metabolic activity of Ba. fragilis in coculture with bifidobacteria was not affected by EPS, but greater survival of bifidobacteria at late stages of incubation occurred in cocultures than in monocultures, leading to a higher production of acetic acid than in monocultures. Therefore, cocultures of Bifidobacterium and Bacteroides can behave differently against fermentable carbohydrates as a function of the specific characteristics of the strains from each species. These results stress the importance of considering specific species and strain interactions and not simply higher taxonomic divisions in the relationship among intestinal microbial populations and their different responses to probiotics and prebiotics.

Assessment of intestinal microbiota modulation ability of Bifidobacterium strains in in vitro fecal batch cultures from preterm neonates.

Microbial colonization of the infant gut is essential for the development of the intestine and the immune system. The intestinal microbiota of full-term breast-fed infants is considered as the health standard for newborns. A culture medium containing formula milk was designed, which allowed a balanced growth of intestinal microorganisms and was used to perform fecal batch cultures from preterm babies. Sixteen Bifidobacterium strains and fructooligosaccharides (FOS) were tested for their ability to modulate in vitro the intestinal microbiota. The production of short chain fatty acids (SCFA) was measured by Gas Chromatography and the levels of some anaerobe (Bifidobacterium and Bacteroides groups) and facultative anaerobes (Enterobacteriaceae, Enterococcaceae, Weissella group, and Klebsiella pneumoniae) were determined by quantitative PCR. Results were referred to a fecal negative control culture without microorganisms or FOS added. Strains that in fecal cultures counteracted better the aberrancies previously found in feces of preterm babies, as compared with full-term breast-fed infants, were selected. The three Bifidobacterium bifidum strains tested in this work promoted the most suitable shifts in SCFA and in the ratio of variables facultative anaerobes to anaerobes. Two Bifidobacterium breve strains complied with the requirement for facultative anaerobes and anaerobes and one of them also promoted a suitable shift of SCFA. Bifidobacteria behaved similarly as FOS regarding the microbial profiles in fecal cultures but the production of lactic and acetic acid was much lower. B. breve and B. bifidum strains selected represent promising candidates for their assessment in more complex in vitro and in vivo models.

Impact of intrapartum antimicrobial prophylaxis upon the intestinal microbiota and the prevalence of antibiotic resistance genes in vaginally delivered full-term neonates.

BACKGROUND: Disturbances in the early establishment of the intestinal microbiota may produce important implications for the infant's health and for the risk of disease later on. Different perinatal conditions may be affecting the development of the gut microbiota. Some of them, such as delivery mode or feeding habits, have been extensively assessed whereas others remain to be studied, being critical to identify their impact on the microbiota and, if any, to minimize it. Antibiotics are among the drugs most frequently used in early life, the use of intrapartum antimicrobial prophylaxis (IAP), present in over 30% of deliveries, being the most frequent source of exposure. However, our knowledge on the effects of IAP on the microbiota establishment is still limited. The aim of the present work was to evaluate the impact of IAP investigating a cohort of 40 full-term vaginally delivered infants born after an uncomplicated pregnancy, 18 of which were born from mothers receiving IAP. RESULTS: Fecal samples were collected at 2, 10, 30, and 90 days of age. We analyzed the composition of the fecal microbiota during the first 3 months of life by 16S rRNA gene sequencing and quantified fecal short chain fatty acids by gas chromatography. The presence of genes for resistance to antibiotics was determined by PCR in the samples from 1-month-old infants. Our results showed an altered pattern of intestinal microbiota establishment in IAP infants during the first weeks of life, with lower relative proportions of Actinobacteria and Bacteroidetes and increased of Preoteobacteria and Firmicutes. A delay in the increase on the levels of acetate was observed in IAP infants. The analyses of specific antibiotic resistance genes showed a higher occurrence of some ß-lactamase coding genes in infants whose mothers received IAP. CONCLUSIONS: Our results indicate an effect of IAP on the establishing early microbiota during the first months of life, which represent a key moment for the development of the microbiota-induced host homeostasis. Understanding the impact of IAP in the gut microbiota development is essential for developing treatments to minimize it, favoring a proper gut microbiota development in IAP-exposed neonates.

Effect of Bifidobacterium upon Clostridium difficile Growth and Toxicity When Co-cultured in Different Prebiotic Substrates.

The intestinal overgrowth of Clostridium difficile, often after disturbance of the gut microbiota by antibiotic treatment, leads to C. difficile infection (CDI) which manifestation ranges from mild diarrhea to life-threatening conditions. The increasing CDI incidence, not only in compromised subjects but also in traditionally considered low-risk populations, together with the frequent relapses of the disease, has attracted the interest for prevention/therapeutic options. Among these, probiotics, prebiotics, or synbiotics constitute a promising approach. In this study we determined the potential of selected Bifidobacterium strains for the inhibition of C. difficile growth and toxicity in different carbon sources. We conducted co-cultures of the toxigenic strain C. difficile LMG21717 with four Bifidobacterium strains (Bifidobacterium longum IPLA20022, Bifidobacterium breve IPLA20006, Bifidobacterium bifidum IPLA20015, and Bifidobacterium animalis subsp. lactis Bb12) in the presence of various prebiotic substrates (Inulin, Synergy, and Actilight) or glucose, and compared the results with those obtained for the corresponding mono-cultures. C. difficile and bifidobacteria levels were quantified by qPCR; the pH and the production of short chain fatty acids was also determined. Moreover, supernatants of the cultures were collected to evaluate their toxicity using a recently developed model. Results showed that co-culture with B. longum IPLA20022 and B. breve IPLA20006 in the presence of short-chain fructooligosaccharides, but not of Inulin, as carbon source significantly reduced the growth of the pathogen. With the sole exception of B. animalis Bb12, whose growth was enhanced, the presence of C. difficile did not show major effects upon the growth of the bifidobacteria. In accordance with the growth data, B. longum and B. breve were the strains showing higher reduction in the toxicity of the co-culture supernatants.