Bacteriocin production by Bifidobacterium spp. A review

Bacteriocins are ribosomally-synthesized antibacterial peptides. These compounds are produced by a broad variety of different bacteria belonging mainly to the genus Bifidobacterium, to which health promoting properties have frequently been attributed. However, despite the fact that the identification of Bifidobacterium-associated bacteriocins was first reported in 1980 and that they exhibit antimicrobial activity against pathogenic microorganisms such as Listeria monocytogenes, Clostridium perfringens, and Escherichia coli, relatively little information is still available about the antimicrobial compounds produced by strains of this genus. More detailed understanding of the action mechanisms of these antimicrobials could allow us to determine the extent to which their production contributes to the probiotic properties of specific bifidobacteria strains and, potentially, be of crucial significance for ultimate preservation of functional foods or pharmaceutical applications. Here we review what is already known about their structure, classification, mode of action, functionality, immunity, production and purification.     

Application of molecular methods to classification and identification of bacteria of the genus <Emphasis Type="Italic">Bifidobacterium</Emphasis>

The molecular methods currently used in the classification and identification of bifidobacteria are reviewed. The sequencing of the 16S rRNA gene and some other genes considered to be phylogenetic markers is a universal and effective approach for taxonomic characterization of members of the genus Bifidobacterium and to reliable identification of new isolates. Various techniques of obtaining DNA fingerprints (PFGE, RAPD, rep-PCR) are widely used for solving particular problems in identifying bifidobacteria. Bacteria of the genus Bifidobacterium are important organisms in biotechnology and medicine. The research in the field of molecular systematics of bifidobacteria provides a basis not only for the solution of taxonomic problems, but also for monitoring of individual species in the environment and for more detailed study of the genetics and ecology of this group of microorganisms.     

Enumeration of Bifidobacteria in Gastrointestinal Samples from Piglets

The population of Bifidobacterium spp. in fecal samples from suckling piglets was investigated, and Beerens, raffinose-bifidobacterium (RB), and modified Wilkins-Chalgren (MW) agar media were evaluated with regard to the enumeration of bifidobacteria in porcine intestinal samples. The results demonstrated that the population of bifidobacteria in the feces of suckling piglets is numerically low, and a phylogenetic analysis of the 16S rRNA gene from bifidobacterial isolates suggested that a possibly new Bifidobacterium species was isolated. Beerens, RB, and MW agar media were not selective for bifidobacteria in the fecal samples. The highest recovery and diversity of bifidobacteria were obtained for MW agar. Nonbifidobacterial isolates from the three agar media were identified and may contribute to the future formulation of improved selective media for the enumeration of bifidobacteria.     

Characterization of Bifidobacterium spp. strains for the treatment of enteric disorders in newborns

Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastrointestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to nontumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify three Bifidobacterium breve strains and a Bifidobacterium longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics. A validation clinical trial involving the selected strains is being planned.     

Investigation of the Evolutionary Development of the Genus Bifidobacterium by Comparative Genomics

The Bifidobacterium genus currently encompasses 48 recognized taxa, which have been isolated from different ecosystems. However, the current phylogeny of bifidobacteria is hampered by the relative paucity of genotypic data. Here, we reassessed the taxonomy of this bacterial genus using genome-based approaches, which demonstrated that the previous taxonomic view of bifidobacteria contained several inconsistencies. In particular, high levels of genetic relatedness were shown to exist between particular Bifidobacterium taxa which would not justify their status as separate species. The results presented are here based on average nucleotide identity analysis involving the genome sequences for each type strain of the 48 bifidobacterial taxa, as well as phylogenetic comparative analysis of the predicted core genome of the Bifidobacterium genus. The results of this study demonstrate that the availability of complete genome sequences allows the reconstruction of a more robust bifidobacterial phylogeny than that obtained from a single gene-based sequence comparison, thus discouraging the assignment of a new or separate bifidobacterial taxon without such a genome-based validation.     

In Silico Assigned Resistance Genes Confer Bifidobacterium with Partial Resistance to Aminoglycosides but Not to Β-Lactams

Bifidobacteria have received significant attention due to their contribution to human gut health and the use of specific strains as probiotics. It is thus not surprising that there has also been significant interest with respect to their antibiotic resistance profile. Numerous culture-based studies have demonstrated that bifidobacteria are resistant to the majority of aminoglycosides, but are sensitive to β-lactams. However, limited research exists with respect to the genetic basis for the resistance of bifidobacteria to aminoglycosides. Here we performed an in-depth in silico analysis of putative Bifidobacterium-encoded aminoglycoside resistance proteins and β-lactamases and assess the contribution of these proteins to antibiotic resistance. The in silico-based screen detected putative aminoglycoside and β-lactam resistance proteins across the Bifidobacterium genus. Laboratory-based investigations of a number of representative bifidobacteria strains confirmed that despite containing putative β-lactamases, these strains were sensitive to β-lactams. In contrast, all strains were resistant to the aminoglycosides tested. To assess the contribution of genes encoding putative aminoglycoside resistance proteins in Bifidobacterium sp. two genes, namely Bbr_0651 and Bbr_1586, were targeted for insertional inactivation in B. breve UCC2003. As compared to the wild-type, the UCC2003 insertion mutant strains exhibited decreased resistance to gentamycin, kanamycin and streptomycin. This study highlights the associated risks of relying on the in silico assignment of gene function. Although several putative β-lactam resistance proteins are located in bifidobacteria, their presence does not coincide with resistance to these antibiotics. In contrast however, this approach has resulted in the identification of two loci that contribute to the aminoglycoside resistance of B. breve UCC2003 and, potentially, many other bifidobacteria.     

Development of lyophilization procedure ensuring survival of bifidobacteria and preservation of their probiotic potential upon long-term storage

Survival of bifidobacteria and preservation of their morphological characteristics after 12-month storage of lyophilized cells was studied for the strains of Bifidobacterium bifidum and B. animalis isolated and maintained in the microbial collection of the Department of Microbiology, Moscow State University. A combined approach to pre-lyophilization treatment of microorganisms and subsequent storage was developed in order to improve cell survival. Compared to the standard cryoprotector concentrations, sucrose and glucose (5% and higher) in skim milk, as well as freezing at?70°C with subsequent storage at the same temperature resulted in improved survival of bifidobacteria. Under such conditions, the number of viable cells (CFU) after 12 months of storage was two to three orders of magnitude higher than in the case of the standard lyophilization procedure. Investigation of dynamics of resistance of reactivated clones to such gastrointestinal stress factors as gastric juice and bile acids revealed preservation of these properties at all storage modes. However, since the number of surviving cells decreased during storage according to the standard procedure, the number of stress-affected cells was correspondingly lower. Reactivated cultures exhibited high resistance to oxygen, with survival decreasing to 35% of the initial level.     

High Abundance of Escherichia During the Establishment of Fecal Microbiota in Brazilian Children

The sequence of bacterial events that occurs during the colonization of the gastrointestinal tract may affect the future health of the host. A clear understanding of the colonization process of the human neonatal gut in developing countries is lacking because the few available studies were mostly performed using culture techniques. Using molecular approaches, this study analyzed the fecal microbiota of children of low socioeconomic status in São Paulo, Brazil, during their first year of life. We collected fecal samples of healthy children at 3, 6, and 12 months of life. Total DNA was extracted directly from feces, and the bacteria-specific primers 27F-1492R were used to construct 16S rRNA libraries. Clones were randomly selected and partially sequenced. The main phylogenetic groups identified at 3 months were Streptococcus, unidentified bacteria, and Escherichia. At 6 months, Escherichia remained predominant, while the unidentified bacterial population increased significantly. At 12 months, a more complex composition of fecal microbiota was observed, represented by unidentified bacteria and microorganisms found at low rates at earlier ages. The genus Escherichia remained the most abundant microorganism (34 % relative abundance and 75 % prevalence). Principal component analysis (PCA) revealed changes in the composition of the microbiota at 6 months and an increase of diversity at 12 months of life. Bifidobacterium was identified by quantitative PCR (qPCR) and showed a high incidence in the microbiota at 3 months. The present results corroborate the global observation of inter-individual variability with an early establishment of microbial complexity at the end of the first year of life and highlight the presence of the Escherichia as abundant in microbiota composition of this group of children.     

Specific Identification and Targeted Characterization of Bifidobacterium lactis from Different Environmental Isolates by a Combined Multiplex-PCR Approach

The species Bifidobacterium lactis, with its main representative strain Bb12 (DSM 10140), is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29 Bifidobacterium and 9 Lactobacillus species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus Bifidobacterium and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of B. lactis were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.     

Mining the Microbiota of the Neonatal Gastrointestinal Tract for Conjugated Linoleic Acid-Producing Bifidobacteria

This study was designed to isolate different strains of the genus Bifidobacterium from the fecal material of neonates and to assess their ability to produce the cis-9, trans-11 conjugated linoleic acid (CLA) isomer from free linoleic acid. Fecal material was collected from 24 neonates aged between 3 days and 2 months in a neonatal unit (Erinville Hospital, Cork, Ireland). A total of 46 isolates from six neonates were confirmed to be Bifidobacterium species based on a combination of the fructose-6-phosphate phosphoketolase assay, RAPD [random(ly) amplified polymorphic DNA] PCR, pulsed-field gel electrophoresis (PFGE), and partial 16S ribosomal DNA sequencing. Interestingly, only 1 of the 11 neonates that had received antibiotic treatment produced bifidobacteria. PFGE after genomic digestion with the restriction enzyme XbaI demonstrated that the bifidobacteria population displayed considerable genomic diversity among the neonates, with each containing between one and five dominant strains, whereas 11 different macro restriction patterns were obtained. In only one case did a single strain appear in two neonates. All genetically distinct strains were then screened for CLA production after 72 h of incubation with 0.5 mg of free linoleic acid ml⁻¹ by using gas-liquid chromatography. The most efficient producers belonged to the species Bifidobacterium breve, of which two different strains converted 29 and 27% of the free linoleic acid to the cis-9, trans-11 isomer per microgram of dry cells, respectively. In addition, a strain of Bifidobacterium bifidum showed a conversion rate of 18%/μg dry cells. The ability of some Bifidobacterium strains to produce CLA could be another human health-promoting property linked to members of the genus, given that this metabolite has demonstrated anticarcinogenic activity in vitro and in vivo.     

Quantitative PCR with 16S rRNA-Gene-Targeted Species-Specific Primers for Analysis of Human Intestinal Bifidobacteria

A highly sensitive quantitative PCR detection method has been developed and applied to the distribution analysis of human intestinal bifidobacteria by combining real-time PCR with Bifidobacterium genus- and species-specific primers. Real-time PCR detection of serially diluted DNA extracted from cultured bifidobacteria was linear for cell counts ranging from 10⁶ to 10 cells per PCR assay. It was also found that the method was applicable to the detection of Bifidobacterium in feces when it was present at concentrations of >10⁶ cells per g of feces. Concerning the distribution of Bifidobacterium species in intestinal flora, the Bifidobacterium adolescentis group, the Bifidobacterium catenulatum group, and Bifidobacterium longum were found to be the three predominant species by examination of DNA extracted from the feces of 46 healthy adults. We also examined changes in the population and composition of Bifidobacterium species in human intestinal flora of six healthy adults over an 8-month period. The results showed that the composition of bifidobacterial flora was basically stable throughout the test period.     

Comparative Genomic Analysis of 45 Type Strains of the Genus Bifidobacterium: A Snapshot of Its Genetic Diversity and Evolution

Bifidobacteria are well known for their human health-promoting effects and are therefore widely applied in the food industry. Members of the Bifidobacterium genus were first identified from the human gastrointestinal tract and were then found to be widely distributed across various ecological niches. Although the genetic diversity of Bifidobacterium has been determined based on several marker genes or a few genomes, the global diversity and evolution scenario for the entire genus remain unresolved. The present study comparatively analyzed the genomes of 45 type strains. We built a robust genealogy for Bifidobacterium based on 402 core genes and defined its root according to the phylogeny of the tree of bacteria. Our results support that all human isolates are of younger lineages, and although species isolated from bees dominate the more ancient lineages, the bee was not necessarily the original host for bifidobacteria. Moreover, the species isolated from different hosts are enriched with specific gene sets, suggesting host-specific adaptation. Notably, bee-specific genes are strongly associated with respiratory metabolism and are potential in helping those bacteria adapt to the oxygen-rich gut environment in bees. This study provides a snapshot of the genetic diversity and evolution of Bifidobacterium, paving the way for future studies on the taxonomy and functional genomics of the genus.     

Fermentation of Fructooligosaccharides by Lactic Acid Bacteria and Bifidobacteria

Lactic acid bacteria and bifidobacteria were screened of their ability to ferment fructooligosaccharides (FOS) on MRS agar. Of 28 strains of lactic acid bacteria and bifidobacteria examined, 12 of 16 Lactobacillus strains and 7 of 8 Bifidobacterium strains fermented FOS. Only strains that gave a positive reaction by the agar method reached high cell densities in broth containing FOS.     

Characterization of Bifidobacterium Strains Using Box Primers

Twenty-five Bifidobacterium strains isolated from infants' faeces were identified by Rep-PCR. Using BOX-PCR, characteristic bands of Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adolescentis were found in 40 strains of bidfidobacteria. These bands were not found in lactobacilli. By computerized numerical analysis strains were grouped in two major clusters. Strains of B. bifidum fell into a well-differentiated cluster that joined the cluster of the remaining species at 0.771 of similarity. The predominant species among the isolated strains were Bifidobacterium bifidum, Bifidobacterium longum andBifidobacterium breve . In another set of experiments, DNA was extracted from bacteria harvested from fermented milks to which different concentrations of bifidobacteria had been added. In all cases characteristic bands in the agarose gel belonging to lactobacilli and streptococci were detected. Bifidobacterium was detected only when 10⁸CFU/ml were added to the fermented milks. On the basis of our results, we propose this methodology as another tool in the polyphasic taxonomy.     

Hormone-Dependent Bacterial Growth,Persistence and Biofilm Formation – A Pilot Study Investigating Human Follicular Fluid Collected during IVF Cycles

Human follicular fluid, considered sterile, is aspirated as part of an in vitro fertilization (IVF) cycle. However, it is easily contaminated by the trans-vaginal collection route and little information exists in its potential to support the growth of microorganisms. The objectives of this study were to determine whether human follicular fluid can support bacterial growth over time, whether the steroid hormones estradiol and progesterone (present at high levels within follicular fluid) contribute to the in vitro growth of bacterial species, and whether species isolated from follicular fluid form biofilms. We found that bacteria in follicular fluid could persist for at least 28 weeks in vitro and that the steroid hormones stimulated the growth of some bacterial species, specifically Lactobacillus spp., Bifidobacterium spp. Streptococcus spp. and E. coli. Several species, Lactobacillus spp., Propionibacterium spp., and Streptococcus spp., formed biofilms when incubated in native follicular fluids in vitro (18/24, 75%). We conclude that bacteria aspirated along with follicular fluid during IVF cycles demonstrate a persistent pattern of growth. This discovery is important since it can offer a new avenue for investigation in infertile couples.     

Bifidobacteria strains isolated from stools of iron deficient infants can efficiently sequester iron

Background

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.

Results

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.

Conclusions

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.     

In Vitro Antibacterial Mechanism of Action of Crude Garlic (Allium sativum) Clove Extract on Selected Probiotic Bifidobacterium Species as Revealed by SEM,TEM, and SDS-PAGE Analysis

There has been much research on the effects of garlic (Allium sativum) on numerous pathogens, but very few, if any, studies on its effect on beneficial, probiotic bifidobacteria. We have recently shown that garlic exhibits antibacterial activity against bifidobacteria. The mechanism by which garlic kills bifidobacteria is yet to be elucidated. This study sought to determine the mechanism of action of garlic clove extract on selected Bifidobacterium species using scanning and transmission electron microscopy and SDS-PAGE analysis. SEM micrographs revealed unusual morphological changes such as cell elongation, cocci-shaped cells with cross-walls, and distorted cells with bulbous ends. With TEM, observed changes included among others, condensation of cytoplasmic material, disintegration of membranes, and loss of structural integrity. SDS-PAGE analysis did not reveal any differences in whole-cell protein profiles of untreated and garlic clove extract-treated cells. The current study is the first to reveal the mechanism of action of garlic clove extract on probiotic Bifidobacterium species. The results indicate that garlic affects these beneficial bacteria in a manner similar to that exhibited in pathogens. These results therefore further highlight that caution should be taken especially when using raw garlic and probiotic bifidobacteria simultaneously as viability of these bacteria could be reduced by allicin released upon crushing of garlic cloves, thereby limiting the health benefits that the consumer anticipate to gain from probiotics.     

Influence of intrapartum antibiotic prophylaxis against group B Streptococcus on the early newborn gut composition and evaluation of the anti-Streptococcus activity of Bifidobacterium strains

Several factors are known to influence the early colonization of the gut in newborns. Among them, the use of antibiotics on the mother during labor, referred to as intrapartum antibiotic prophylaxis (IAP), has scarcely been investigated, although this practice is routinely used in group B Streptococcus (GBS)-positive women. This work is therefore aimed at verifying whether IAP can influence the main microbial groups of the newborn gut microbiota at an early stage of microbial establishment. Fifty-two newborns were recruited: 26 born by mothers negative to GBS (control group) and 26 by mothers positive to GBS and subjected to IAP with ampicillin (IAP group). Selected microbial groups (Lactobacillus spp., Bidobacterium spp., Bacteroides fragilis, Clostridium difficile, and Escherichia coli) were quantified with real-time PCR on DNA extracted from newborn feces. Further analysis was performed within the Bidobacterium genus by using DGGE after amplification with genus-specific primers. Results obtained showed a significant decrease of the bifidobacteria counts after antibiotic treatment of the mother. Bifidobacteria were found to be affected by IAP not only quantitatively but also qualitatively. In fact, IAP determined a decrement in the frequency of Bidobacterium breve, Bidobacterium bifidum, and Bidobacterium dentium with respect to the control group. Moreover, this study has preliminarily evaluated that some bifidobacterial strains, previously selected for use in infants, have antibacterial properties against GBS and are therefore potential candidates for being applied as probiotics for the prevention of GBS infections.     

Gnotobiotic Mouse Immune Response Induced by Bifidobacterium sp. Strains Isolated from Infants

Bifidobacterium, which is a dominant genus in infants’ fecal flora and can be used as a probiotic, has shown beneficial effects in various pathologies, including allergic diseases, but its role in immunity has so far been little known. Numerous studies have shown the crucial role of the initial intestinal colonization in the development of the intestinal immune system, and bifidobacteria could play a major role in this process. For a better understanding of the effect of Bifidobacterium on the immune system, we aimed at determining the impact of Bifidobacterium on the T-helper 1 (T_H1)/T_H2 balance by using gnotobiotic mice. Germfree mice were inoculated with Bifidobacterium longum NCC2705, whose genome is sequenced, and with nine Bifidobacterium strains isolated from infants’ fecal flora. Five days after inoculation, mice were killed. Transforming growth factor β1 (TGF-β1), interleukin-4 (IL-4), IL-10, and gamma interferon (IFN-γ) gene expressions in the ileum and IFN-γ, tumor necrosis factor alpha (TNF-α), IL-10, IL-4, and IL-5 secretions by splenocytes cultivated for 48 h with concanavalin A were quantified. Two Bifidobacterium species had no effect (B. adolescentis) or little effect (B. breve) on the immune system. Bifidobacterium bifidum, Bifidobacterium dentium, and one B. longum strain induced T_H1 and T_H2 cytokines at the systemic and intestinal levels. One B. longum strain induced a T_H2 orientation with high levels of IL-4 and IL-10, both secreted by splenocytes, and of TGF-β gene expression in the ileum. The other two strains induced T_H1 orientations with high levels of IFN-γ and TNF-α splenocyte secretions. Bifidobacterium's capacity to stimulate immunity is species specific, but its influence on the orientation of the immune system is strain specific.