In vitro effects of phosphatidylcholine and transgalactooligosaccharides on the production of 1,2-sn-diacylglycerol by Bifidobacterium longum biovar infantis

Aims: To investigate the production of the tumour promoter 1,2-sn-diacylglycerol (DAG) by a human gut isolate of Bifidobacterium longum biovar infantis. Methods and Results: Bifidobacterium longum biovar infantis was grown in vitro using anaerobic static batch cultures in the presence of phosphatidylcholine (PC) and trans-galactooligosaccharides (TOS). Production of DAG was found to be dependent upon the presence of PC, while TOS had a reducing effect. Considerable differences in morphology, growth and metabolic end products from the micro-organism were observed under the different culture conditions. Conclusions: Our results have provided evidence that B. longum biovar infantis can produce DAG in vitro and that a prebiotic exerted a reducing effect upon this production. Significance and Impact of the Study: The results presented in this study demonstrate an ability of ostensibly beneficial member of the colonic environment to produce unwanted compounds under certain conditions. Therefore, it may be important that a combination of substrates and other factors are assessed when studying the behaviour of any bacterial group or species, especially when designing the dietary interventions.

The use of gaseous ozone and gas packaging to control populations of Salmonella infantis and Pseudomonas aeruginosa on the skin of chicken portions

Chilled breasts of chicken were inoculated with Salmonella infantis or Pseudomonas aeruginosa and then given one of the following treatments: (i) exposure to gaseous ozone (>2000 ppm for up to 30 min); (ii) storage under 70% CO2:30% N-2; and (iii) exposure to gaseous ozone (>2000 ppm for 15 min) followed by storage under 70% CO2:30% N-2; all storage at 7degreesC. Gaseous ozone reduced the counts of samnonellae by 97(Y,, and pseudomonads by 95%, but indigenous coliforms were unaffected. Under the modified atmosphere, the cell count of S. infantis was reduced by 72% following initial exposure and then stabilised, coliforms grew, but Ps. aeruginosa behaved like S. infantis-initial reduction (58%) followed by stability. Exposure to gaseous ozone followed by gas packaging allowed survival of S. infantis, Ps. aeruginosa and coliforms over 9 days at 7degreesC, but there was no evidence of any sensory deterioration. It is proposed that the latter treatment could, in a modified form perhaps, be used to reduce the contamination of chicken carcasses with salmonellae and improve their shelf-life. (C) 2004 Elsevier Ltd. All rights reserved.

Phenotype MicroArray (TM) in the metabolic characterisation of Salmonella serotypes Agona, Enteritidis, Give, Hvittingfoss, Infantis, Newport and Typhimurium

The Phenotype MicroArray (TM) (PM) technology was used to study the metabolic characteristics of 29 Salmonella strains belonging to seven serotypes of S. enterica spp. enterica. Strains of serotypes Typhimurium (six strains among definite phage types DTs 1, 40 and 104) and Agona (two strains) were tested for 949 substrates, Enteritidis (six strains of phage type PT1), Give, Hvittingfoss, Infantis and Newport strains (two of each) were tested for 190 substrates and seven other Agona strains for 95 substrates. The strains represented 18 genotypes in pulsed-field gel electrophoresis (PFGE). Among 949 substrates, 18 were identified that could be used to differentiate between the strains of those seven serotypes or within a single serotype. Unique metabolic differences between the Finnish endemic Typhimurium DT1 and Agona strains were detected, for example, in the metabolism of d-tagatose, d-galactonic acid gamma-lactone and l-proline as a carbon source. Thus, the PM technique is a useful tool for identifying potential differential markers on a metabolic basis that could be used for epidemiological surveillance.

Emulsifying, rheological and physicochemical properties of exopolysaccharide produced by Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205

The rheological, emulsification and certain physicochemical properties of purified exopolysaccharides (EPS) of Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205 were studied and compared with those of guar gum and xanthan gum. The two strains were grown in skim milk supplemented with 1.5% (w/v) casein hydrolysate at 37 ◦C for 24 h; they both produced heteropolysaccharides with different molecular mass and composition. The carbohydrate content of both polymers was more than 92% and no protein was detected. The EPS of B. longum subsp. infantis CCUG 52486 showed highly branched entangled porous structure under scanning electron microscopy. Higher intrinsic viscosity was observed for the EPS of B. longum subsp. infantis CCUG 52486 compared to the EPS of B. infantis NCIMB 702205 and guar gum. Both polymers showed pseudoplastic non-Newtonian fluid behaviour in an aqueous solution. The EPS of B. infantis NCIMB 702205 and B. longum subsp. infantis CCUG 52486 produced more stable emulsions with orange oil, sunflower seed oil, coconut oil and xylene compared to guar and xanthan gum. The EPS of B. longum subsp. infantis CCUG 52486 is the most promising one for applications in the food industry, as it had higher intrinsic viscosity, higher apparent viscosity in aqueous solution, porous dense entangled structure and good emulsification activity.

Evidence of immunomodulatory effects of a novel probiotic, Bifidobacterium longum bv. infantis CCUG 52486

Bifidobacterium longum bv. infantis CCUG 52486 was originally isolated from healthy elderly subjects and demonstrated to have particular ecological fitness and anti-pathogenic effects. Bifidobacteria are commonly associated with immunomodulatory properties, especially in older people, but this strain has not been investigated for effects on immune function. This study aimed to explore the immunomodulatory effects of this novel probiotic, compared with three commercial strains, B. longum SP 07/3, L. rhamnosus GG (L.GG) and L. casei Shirota (LcS). Human peripheral blood mononuclear cells (PBMCs) were isolated from fasting blood of young or older volunteers and exposed to probiotic strains or Con A. NK activity and activation, and cytokine release were enhanced by all probiotics with strain-specificities. The effect of B. infantis on NK activity was influenced by ageing. Except for L.GG, probiotics increased IFN-γ production to a much greater degree in young subjects, and increased IL-6 production to a much greater degree in older subjects. Based on IL-10/IL-12 ratios, B. infantis resulted in the most anti-inflammatory profile of all of the probiotics. These results suggest that B. infantis CCUG 52486 has strong immunomodulatory potential compared with well-known commercial strains, and that the immune response to probiotics may be influenced by ageing.

Microbial species involved in production of 1,2-sn-diacylglycerol and effects of phosphatidylcholine on human fecal microbiota

1,2-sn-Diacylglycerols (DAGs) are activators of protein kinase C (PKQ, which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and Escherichia coli multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of Bifidobacterium spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly Clostridium bifermentans (8 of 12), followed by Escherichia coli (2 of 12). Interestingly, one DAG-producing strain was Bifidobacterium infantis, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.

Polydextrose, lactitol, and fructo-oligosaccharide fermentation by colonic bacteria in a three-stage continuous culture system

In vitro fermentations were carried out by using a model of the human colon to simulate microbial activities of lower gut bacteria. Bacterial populations (and their metabolic products) were evaluated under the effects of various fermentable substrates. Carbohydrates tested were polydextrose, lactitol, and fructo-oligosaccharide (FOS). Bacterial groups of interest were evaluated by fluorescence in situ hybridization as well as by species-specific PCR to determine bifidobacterial species and percent-G+C profiling of the bacterial communities present. Short-chain fatty acids (SCFA) produced during the fermentations were also evaluated. Polydextrose had a stimulatory effect upon colonic bifidobacteria at concentrations of 1 and 2% (using a single and pooled human fecal inoculum, respectively). The bifidogenic effect was sustained throughout all three vessels of the in vitro system (P = 0.01 seen in vessel 3), as corroborated by the bacterial community profile revealed by %G+C analysis. This substrate supported a wide variety of bifidobacteria and was the only substrate where Bifidobacterium infantis was detected. The fermentation of lactitol had a deleterious effect on both bifidobacterial and bacteroides populations (P = 0.01) and decreased total cell numbers. SCFA production was stimulated, however, particularly butyrate (beneficial for host colonocytes). FOS also had a stimulatory effect upon bifidobacterial and lactobacilli populations that used a single inoculum (P = 0.01 for all vessels) as well as a bifidogenic effect in vessels 2 and 3 (P = 0.01) when a pooled inoculum was used. A decrease in bifidobacteria throughout the model was reflected in the percent-G+C profiles.

Molecular identification and anti-pathogenic activities of putative probiotic bacteria isolated from faeces of healthy elderly individuals

One hundred and nine lactic acid bacterial strains (56 bifidobacteria-like and 53 lactobacilli-like) were isolated from faecal samples donated by healthy elderly individuals (>65 years old). Isolates were identified to species level by phenotypic analysis (by API) and by 16S rDNA sequencing. Eleven species of Lactobacillus and six species of Bifidobacterium were identified. The most frequently isolated lactobacillus was L. fermentum and the most frequently isolated bifidobacterium was closely related to B. infantis by 16S rDNA sequence alignment. The isolates were characterized for their antimicrobial activity against Clostridium difficile, enteropathogenic Escherichia coli (EPEC), verocytotoxigenic E. coli (VTEC) and Campylobacter jejuni. The lactobacilli displayed variations in their antimicrobial activity with few strains showing inhibitory activity against all pathogens. The bifidobacteria displayed higher levels of inhibitory activity against C. jejuni and Cl. difficile than against the E. coli strains. Keywords: Lactobacillus, Bifidobacterium, elderly, gastrointestinal microbiota, inhibition, Clostridium difficile, enteropathogenic Escherichia coli (EPEC), verocytotoxigenic E. coli (VTEC), Campylobacter jejuni.

Molecular cloning and comparative analysis of four beta-galactosidase genes from Bifidobacterium bifidum NCIMB41171

Bifidobacterium bifidum NCIMB41171 carries four genes encoding different beta-galactosidases. One of them, named bbgIII, consisted of an open reading frame of 1,935 amino acid (a.a.) residues encoding a protein with a multidomain structure, commonly identified on cell wall bound enzymes, having a signal peptide, a membrane anchor, FIVAR domains, immunoglobulin Ig-like and discoidin-like domains. The other three genes, termed bbgI, bbgII and bbgIV, encoded proteins of 1,291, 689 and 1,052 a.a. residues, respectively, which were most probably intracellularly located. Two cases of protein evolution between strains of the same species were identified when the a.a. sequences of the BbgI and BbgIII were compared with homologous proteins from B. bifidum DSM20215. The homologous proteins were found to be differentiated at the C-terminal a.a. part either due to a single nucleotide insertion or to a whole DNA sequence insertion, respectively. The bbgIV gene was located in a gene organisation surrounded by divergently transcribed genes putatively for sugar transport (galactoside-symporter) and gene regulation (LacI-transcriptional regulator), a structure that was found to be highly conserved in B. longum, B. adolescentis and B. infantis, suggesting optimal organisation shared amongst those species.

Examination of faecal Bifidobacterium populations in breast- and formula-fed infants during the first 18 months of life.

Bifidobacteria in the infant faecal microbiota have been the focus of much interest, especially during the exclusive milk-feeding period and in relation to the fortification of infant formulae to better mimic breast milk. However, longitudinal studies examining the diversity and dynamics of the Bifidobacterium population of infants are lacking, particularly in relation to the effects of weaning. Using a polyphasic strategy, the Bifidobacterium populations of breast- and formula-fed infants were examined during the first 18 months of life. Bifidobacterium-specific denaturing gradient gel electrophoresis demonstrated that breast-fed infants harboured greater diversity than formula-fed infants and the diversity of the infants' Bifidobacterium populations increased with weaning. Twenty-seven distinctive banding profiles were observed from ∼1100 infant isolates using ribosomal intergenic spacer analysis, 14 biotypes of which were confirmed to be members of the genus Bifidobacterium. Two profiles (H, Bifidobacterium longum subsp. infantis; and I, Bifidobacterium bifidum) were common culturable biotypes, seen in 9/10 infants, while profile E (Bifidobacterium breve) was common among breast-fed infants. Overall, inter- and intra-individual differences were observed in the Bifidobacterium populations of infants between 1 and 18 months of age, although weaning was associated with increased diversity of the infant Bifidobacterium populations. Breast-fed infants generally harboured a more complex Bifidobacterium microbiota than formula-fed infants.

Screening human intestinal Bifidobacterium strains for growth, acidification, EPS production and viscosity potential in low-fat milk

Bifidobacterium strains of human origin were screened for their ability to grow in milk and produce exopolysaccharides (EPS). Bifidobacterium strains were grown in low-fat UHT milk and were evaluated for their growth, acidification properties, EPS production and ability to increase the viscosity of fermented milk. The strains that grew well in milk were strains of Bifidobacterium breve and Bifidobacterium longum and B. longum subsp. longum. Among the 22 strains, EPS was produced by Bifidobacterium bifidum ALM 35, B. breve NCIMB 8807 (UCC 2003), B. longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205 at concentrations ranging from 25 to 140 . The molecular mass and the composition varied considerably, depending on the strain. Analysis of the correlation between the apparent viscosity of the fermented milk and pH indicated that the EPS produced during the acidification of milk possibly contributed to the viscosity of the milk products.

Effect of dairy-based protein sources and temperature on growth, acidification and exopolysaccharide production of Bifidobacterium strains in skim milk

The aim of the present study was to find out the best growing conditions for exopolysaccharide (EPS) producing bifidobacteria, which improve their functionality in yoghurt-like products. Two Bifidobacterium strains were used in this study, Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205. In the first part of the study the effect of casein hydrolysate, lactalbumin hydrolysate, whey protein concentrate and whey protein isolate, added at 1.5% w/v in skim milk, was evaluated in terms of cell growth and EPS production; skim milk supplemented with yeast extract served as the control. Among the various nitrogen sources, casein hydrolysate (CH) showed the highest cell growth and EPS production for both strains after 18 h incubation and therefore it was selected for subsequent work. Based on fermentation experiments using different levels of CH (from 0.5 to 2.5% w/v) it was deduced that 1.5% (w/v) CH resulted in the highest EPS production, yielding 102 and 285 mg L− 1 for B. infantis NCIMB 702205 and B. longum subsp. infantis CCUG 52486, respectively. The influence of temperature on growth and EPS production of both strains was further evaluated at 25, 30, 37 and 42 °C for up to 48 h in milk supplemented with 1.5% (w/v) CH. The temperature had a significant effect on growth, acidification and EPS production. The maximum growth and EPS production were recorded at 37 °C for both strains, whereas no EPS production was observed at 25 °C. Lower EPS production for both strains were observed at 42 °C, which is the common temperature used in yoghurt manufacturing compared to that at 37 °C. The results showed that the culture conditions have a clear effect on the growth, acidification and EPS production, and more specifically, that skim milk supplemented with 1.5% (w/v) CH could be used as a substrate for the growth of EPS-producing bifidobacteria, at 37 °C for 24 h, resulting in the production of a low fat yoghurt-like product with improved functionality.

Virulotyping and antimicrobial resistance typing of salmonella enterica serovars relevant to human health in Europe

The combination of virulence gene and antimicrobial resistance gene typing using DNA arrays is a recently developed genomics-based approach to bacterial molecular epidemiology. We have now applied this technology to 523 Salmonella enterica subsp. enterica strains collected from various host sources and public health and veterinary institutes across nine European countries. The strain set included the five predominant Salmonella serovars isolated in Europe (Enteritidis, Typhimurium, Infantis, Virchow, and Hadar). Initially, these strains were screened for 10 potential virulence factors (avrA, ssaQ, mgtC, siiD, sopB, gipA, sodC1, sopE1, spvC, and bcfC) by polymerase chain reaction. The results indicated that only 14 profiles comprising these genes (virulotypes) were observed throughout Europe. Moreover, most of these virulotypes were restricted to only one (n = 9) or two (n = 4) serovars. The data also indicated that the virulotype did not vary significantly with host source or geographical location. Subsequently, a representative subset of 77 strains was investigated using a microarray designed to detect 102 virulence and 49 resistance determinants. The results confirmed and extended the previous observations using the virulo-polymerase chain reaction screen. Strains belonging to the same serovar grouped together, indicating that the broader virulence-associated gene complement corresponded with the serovar. There were, however, some differences in the virulence gene profiles between strains belonging to an individual serovar. This variation occurred primarily within those virulence genes that were prophage encoded, in fimbrial clusters or in the virulence plasmid. It seems likely that such changes enable Salmonella to adapt to different environmental conditions, which might be reflected in serovar-specific ecology. In this strain subset a number of resistance genes were detected and were serovar restricted to a varying degree. Once again the profiles of those genes encoding resistance were similar or the same for each serovar in all hosts and countries investigated.

Microbiological, chemical and rheological properties of low fat set yoghurt produced with exopolysaccharide (EPS) producing Bifidobacterium strains.

In this work, the microbiological and physicochemical differences of three types of low fat set yoghurts were studied, as well as the changes taking place during storage at 4 °C for 28 days. The first yoghurt was produced with yoghurt starters and exopolysaccharide (EPS) producing Bifidobacterium longum subsp. infantis CCUG 52486 (CCUGY), the second with yoghurt starters and Bifidobacterium infantis NCIMB 702205 (NCIMBY) and the third with just yoghurt starters (control yoghurt). No significant differences were observed in terms of cell concentrations; for all three yoghurts, similar final cell concentrations were obtained for the yoghurt starter cultures (~7.5 log cfu g−1) and the Bifidobacterium strains (~7.8 log cfu g−1). Both Bifidobacterium survived well during storage, as in both cases the cell viability decreased by less than 0.5 log cfu g−1after 28 days of storage. A decrease in pH followed by an increase in lactic acid was observed during storage for all three yoghurts, which was mostly attributed to the activity of the yoghurt starter cultures. The two yoghurts with the EPS producing Bifidobacterium strains exhibited lower syneresis than the control yoghurt. The lowest was shown by CCUGY, which also exhibited the highest storage modulus and firmness, and a well defined porous web-like structure in cryo-SEM. Examination of the micro-structure of the yoghurts using cryo-scanning electron microscopy (cryo-SEM) indicated that the above observations were due to the interaction between the EPS and the milk proteins. Overall, the results indicated that the EPS producing Bifidobacterium longum subsp. infantis CCUG 52486 is the most promising strain, and can be used with yoghurt starter cultures to manufacture low fat set yoghurt with probiotic activities and at the same time enhanced physicochemical and rheological properties.

Probiotic modulation of dendritic cell function is influenced by ageing

Dendritic cells (DCs) are critical for the generation of T-cell responses. DC function may be modulated by probiotics, which confer health benefits in immunocompromised individuals, such as the elderly. This study investigated the effects of four probiotics, Bifidobacterium longum bv. infantis CCUG 52486, B. longum SP 07/3, L. rhamnosus GG (L.GG) and L. casei Shirota (LcS) on DC function in an allogeneic mixed leucocyte reaction (MLR) model, using DCs and T-cells from young and older donors in different combinations. All four probiotics enhanced expression of CD40, CD80 and CCR7 on both young and older DCs, but enhanced cytokine production (TGF-β, TNF-α) by old DCs only. LcS induced IL-12 and IFNγ production by DC to a greater degree than other strains, while Bifidobacterium longum bv. infantis CCUG 52486 favoured IL-10 production. Stimulation of young T cells in an allogeneic MLR with DC was enhanced by probiotic pretreatment of old DCs, which demonstrated greater activation (CD25) than untreated controls. However, pretreatment of young or old DCs with LPS or probiotics failed to enhance the proliferation of T-cells derived from older donors. In conclusion, this study demonstrates that ageing increases the responsiveness of DCs to probiotics, but this is not sufficient to overcome the impact of immunosenescence in the MLR.