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1.  Bifidobacteria strains isolated from stools of iron deficient infants can efficiently sequester iron 
BMC Microbiology  2015;15(1):3.
Bifidobacteria is one of the major gut commensal groups found in infants. Their colonization is commonly associated with beneficial effects to the host through mechanisms like niche occupation and nutrient competition against pathogenic bacteria. Iron is an essential element necessary for most microorganisms, including bifidobacteria and efficient competition for this micronutrient is linked to proliferation and persistence. For this research we hypothesized that bifidobacteria in the gut of iron deficient infants can efficiently sequester iron. The aim of the present study was to isolate bifidobacteria in fecal samples of iron deficient Kenyan infants and to characterize siderophore production and iron internalization capacity.
Fifty-six bifidobacterial strains were isolated by streaking twenty-eight stool samples from Kenyan infants, in enrichment media. To target strains with high iron sequestration mechanisms, a strong iron chelator 2,2-dipyridyl was supplemented to the agar media. Bifidobacterial isolates were first identified to species level by 16S rRNA sequencing, yielding B. bifidum (19 isolates), B. longum (15), B. breve (11), B. kashiwanohense (7), B. pseudolongum (3) and B. pseudocatenulatum (1). While most isolated bifidobacterial species are commonly encountered in the infantile gut, B. kashiwanohense was not frequently reported in infant feces. Thirty strains from culture collections and 56 isolates were characterized for their siderophore production, tested by the CAS assay. Siderophore activity ranged from 3 to 89% siderophore units, with 35 strains (41%) exhibiting high siderophore activity, and 31 (36%) and 20 (23%) showing intermediate or low activity. The amount of internalized iron of 60 bifidobacteria strains selected for their siderophore activity, was in a broad range from 8 to118 μM Fe. Four strains, B. pseudolongum PV8-2, B. kashiwanohense PV20-2, B. bifidum PV28-2a and B. longum PV5-1 isolated from infant stool samples were selected for both high siderophore activity and iron internalization.
A broad diversity of bifidobacteria were isolated in infant stools using iron limited conditions, with some strains exhibiting high iron sequestration properties. The ability of bifidobacteria to efficiently utilize iron sequestration mechanism such as siderophore production and iron internalization may confer an ecological advantage and be the basis for enhanced competition against enteropathogens.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-014-0334-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4320568  PMID: 25591860
Bifidobacteria; Iron; Siderophore; Nutrient competition; CAS assay
2.  Protective effect of probiotics on Salmonella infectivity assessed with combined in vitro gut fermentation-cellular models 
BMC Microbiology  2011;11:264.
Accurate assessment of probiotics with targeted anti-Salmonella activity requires suitable models accounting for both, microbe-microbe and host-microbe interactions in gut environments. Here we report the combination of two original in vitro intestinal models closely mimicking the complex in vivo conditions of the large intestine. Effluents from continuous in vitro three-stage fermentation colonic models of Salmonella Typhimurium infection inoculated with immobilized child microbiota and Salmonella were directly applied to confluent mucus-secreting HT29-MTX cell layers. The effects of Salmonella, addition of two bacteriocinogenic strains, Bifidobacterium thermophilum RBL67 (thermophilicin B67) and Escherichia coli L1000 (microcin B17), and inulin were tested on Salmonella growth and interactions with epithelial cell layers. Salmonella adhesion and invasion were investigated and epithelial integrity assessed by transepithelial electrical resistance (TER) measurements and confocal microscopy observation. Data from complex effluents were compared with pure Salmonella cultures.
Salmonella in effluents of all reactors of the colonic fermentation model stabilized at mean values of 5.3 ± 0.8 log10 cfu/ml effluent. Invasion of cell-associated Salmonella was up to 50-fold lower in complex reactor samples compared to pure Salmonella cultures. It further depended on environmental factors, with 0.2 ± 0.1% being measured with proximal, 0.6 ± 0.2% with transverse and 1.3 ± 0.7% with distal reactor effluents, accompanied by a similar high decrease of TER across cell monolayers (minus 45%) and disruption of tight junctions. Subsequent addition of E. coli L1000 stimulated Salmonella growth (6.4 ± 0.6 log10 cfu/ml effluent of all 3 reactors) and further decreased TER, but led to 10-fold decreased invasion efficiency when tested with distal reactor samples. In contrast, presence of B. thermophilum RBL67 revealed a protective effect on epithelial integrity compared to previous E. coli L1000 periods, as reflected by a significant mean increase of TER by 58% in all reactors. Inulin addition enhanced Salmonella growth and invasion when tested with distal and proximal reactor samples, respectively, but induced a limited decrease of TER (minus 18%) in all reactors.
Our results highlight the benefits of combining suitable cellular and colonic fermentation models to assess strain-specific first-level host protection properties of probiotics during Salmonella infection, providing an efficient system biology tool for preclinical development of new antimicrobials.
PMCID: PMC3295705  PMID: 22171685
3.  Population dynamics of two antilisterial cheese surface consortia revealed by temporal temperature gradient gel electrophoresis 
BMC Microbiology  2010;10:74.
Surface contamination of smear cheese by Listeria spp. is of major concern for the industry. Complex smear ecosystems have been shown to harbor antilisterial potential but the microorganisms and mechanisms involved in the inhibition mostly remain unclear, and are likely related to complex interactions than to production of single antimicrobial compounds. Bacterial biodiversity and population dynamics of complex smear ecosystems exhibiting antilisterial properties in situ were investigated by Temporal temperature gradient gel electrophoresis (TTGE), a culture independent technique, for two microbial consortia isolated from commercial Raclette type cheeses inoculated with defined commercial ripening cultures (F) or produced with an old-young smearing process (M).
TTGE revealed nine bacterial species common to both F and M consortia, but consortium F exhibited a higher diversity than consortium M, with thirteen and ten species, respectively. Population dynamics were studied after application of the consortia on fresh-produced Raclette cheeses. TTGE analyses revealed a similar sequential development of the nine species common to both consortia. Beside common cheese surface bacteria (Staphylococcus equorum, Corynebacterium spp., Brevibacterium linens, Microbacterium gubbeenense, Agrococcus casei), the two consortia contained marine lactic acid bacteria (Alkalibacterium kapii, Marinilactibacillus psychrotolerans) that developed early in ripening (day 14 to 20), shortly after the growth of staphylococci (day 7). A decrease of Listeria counts was observed on cheese surface inoculated at day 7 with 0.1-1 × 102 CFU cm-2, when cheeses were smeared with consortium F or M. Listeria counts went below the detection limit of the method between day 14 and 28 and no subsequent regrowth was detected over 60 to 80 ripening days. In contrast, Listeria grew to high counts (105 CFU cm-2) on cheeses smeared with a defined surface culture.
This work reports the first population dynamics study of complex smear ecosystems exhibiting in situ antilisterial activity. TTGE revealed the presence of marine lactic acid bacteria that are likely related to the strong Listeria inhibition, as their early development in the smear occurred simultaneously with a decrease in Listeria cell count.
PMCID: PMC2907837  PMID: 20222967
4.  PCR and real-time PCR primers developed for detection and identification of Bifidobacterium thermophilum in faeces 
BMC Microbiology  2008;8:179.
Culture-independent methods based on the 16S ribosomal RNA molecule are nowadays widely used for assessment of the composition of the intestinal microbiota, in relation to host health or probiotic efficacy. Because Bifidobacterium thermophilum was only recently isolated from human faeces until now, no specific real-time PCR (qPCR) assay has been developed for detection of this species as component of the bifidobacterial community of the human intestinal flora.
Design of specific primers and probe was achieved based on comparison of 108 published bifidobacterial 16S rDNA sequences with the recently published sequence of the human faecal isolate B. thermophilum RBL67. Specificity of the primer was tested in silico by similarity search against the sequence database and confirmed experimentally by PCR amplification on 17 Bifidobacterium strains, representing 12 different species, and two Lactobacillus strains. The qPCR assay developed was linear for B. thermophilum RBL67 DNA quantities ranging from 0.02 ng/μl to 200 ng/μl and showed a detection limit of 105 cells per gram faeces. The application of this new qPCR assay allowed to detect the presence of B. thermophilum in one sample from a 6-month old breast-fed baby among 17 human faecal samples tested. Additionally, the specific qPCR primers in combination with selective plating experiments led to the isolation of F9K9, a faecal isolate from a 4-month old breast-fed baby. The 16S rDNA sequence of this isolate is 99.93% similar to that of B. thermophilum RBL67 and confirmed the applicability of the new qPCR assay in faecal samples.
A new B. thermophilum-specific qPCR assay was developed based on species-specific target nucleotides in the 16S rDNA. It can be used to further characterize the composition of the bifidobacterial community in the human gastrointestinal tract. Until recently, B. thermophilum was considered as a species of animal origin, but here we confirm with the application of this new PCR assay the presence of B. thermophilum strains in the human gut.
PMCID: PMC2588598  PMID: 18847469
5.  Inhibitory activity spectrum of reuterin produced by Lactobacillus reuteri against intestinal bacteria 
BMC Microbiology  2007;7:101.
Reuterin produced from glycerol by Lactobacillus reuteri, a normal inhabitant of the human intestine, is a broad-spectrum antimicrobial agent. It has been postulated that reuterin could play a role in the probiotic effects of Lb. reuteri. Reuterin is active toward enteropathogens, yeasts, fungi, protozoa and viruses, but its effect on commensal intestinal bacteria is unknown. Moreover reuterin's mode of action has not yet been elucidated. Glutathione, a powerful antioxidant, which also plays a key role in detoxifying reactive aldehydes, protects certain bacteria from oxidative stress, and could also be implicated in resistance to reuterin.
The aim of this work was to test the activity of reuterin against a representative panel of intestinal bacteria and to study a possible correlation between intracellular low molecular weight thiols (LMW-SH) such as glutathione, hydrogen peroxide and/or reuterin sensitivity. Reuterin was produced by Lb. reuteri SD2112 in pure glycerol solution, purified and used to test the minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC). Hydrogen peroxide sensitivity and intracellular LMW-SH concentration were also analysed.
Our data showed that most tested intestinal bacteria showed MIC below that for a sensitive indicator Escherichia coli (7.5–15 mM). Lactobacilli and Clostridium clostridioforme were more resistant with MIC ranging from 15 to 50 mM. No correlation between bacterial intracellular concentrations of LMW-SH, including glutathione, and reuterin or hydrogen peroxide sensitivities were found.
Our data showed that intestinal bacteria were very sensitive to reuterin and that their intracellular concentration of LMW-SH was not directly linked to their capacity to resist reuterin or hydrogen peroxide. This suggests that detoxification by LMW-SH such as glutathione is not a general mechanism and that other mechanisms are probably involved in bacterial tolerance to reuterin and hydrogene peroxide.
PMCID: PMC2222629  PMID: 17997816
6.  Classification of a moderately oxygen-tolerant isolate from baby faeces as Bifidobacterium thermophilum 
BMC Microbiology  2007;7:79.
Bifidobacteria are found at varying prevalence in human microbiota and seem to play an important role in the human gastrointestinal tract (GIT). Bifidobacteria are highly adapted to the human GIT which is reflected in the genome sequence of a Bifidobacterim longum isolate. The competitiveness against other bacteria is not fully understood yet but may be related to the production of antimicrobial compounds such as bacteriocins. In a previous study, 34 Bifidobacterium isolates have been isolated from baby faeces among which six showed proteinaceous antilisterial activity against Listeria monocytogenes. In this study, one of these isolates, RBL67, was further identified and characterized.
Bifidobacterium isolate RBL67 was classified and characterized using a polyphasic approach. RBL67 was classified as Bifidobacterium thermophilum based on phenotypic and DNA-DNA hybridization characteristics, although 16S rDNA analyses and partial groEL sequences showed higher homology with B. thermacidophilum subsp. porcinum and B. thermacidophilum subsp. thermacidophilum, respectively. RBL67 was moderately oxygen-tolerant and was able to grow at pH 4 and at a temperature of 47°C.
In order to assign RBL67 to a species, a polyphasic approach was used. This resulted in the classification of RBL67 as a Bifidobacterium thermophilum strain. To our knowledge, this is the first report about B. thermophilum isolated from baby faeces since the B. thermophilum strains were related to ruminants and swine faeces before. B. thermophilum was previously only isolated from animal sources and was therefore suggested to be used as differential species between animal and human contamination. Our findings may disapprove this suggestion and further studies are now conducted to determine whether B. thermophilum is distributed broader in human faeces. Furthermore, the postulated differentiation between human and animal strains by growth above 45°C is no longer valid since B. thermophilum is able to grow at 47°C. In our study, 16S rDNA and partial groEL sequence analysis were not able to clearly assign RBL67 to a species and were contradictory. Our study suggests that partial groEL sequences may not be reliable as a single tool for species differentiation.
PMCID: PMC2045100  PMID: 17711586

Results 1-6 (6)