A multifaceted investigation of lactococcal strain diversity in undefined mesophilic starter cultures.

The dairy fermentation industry relies on the activity of lactic acid bacteria in robust starter cultures to accomplish milk acidification. Maintenance of the composition of these starter cultures, whether defined or undefined, is essential to ensure consistent and high-quality fermentation end products. To date, limited information exists regarding the microbial composition of undefined starter culture systems. Here, we describe a culture-based analysis combined with a metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, we describe a qPCR-based genotype detection assay, which is capable of discerning nine distinct lactococcal genotypes to characterize these undefined starter cultures, and which can be applied to monitor compositional changes in an undefined starter culture during a fermentation. IMPORTANCE: This study reports on the development of a combined culture-based analysis and metagenomics approach to evaluate the composition of two undefined mesophilic starter cultures. In addition, a novel qPCR-based genotype detection assay, capable of discerning nine distinct lactococcal genotypes (based on lactococcal cell wall polysaccharide biosynthesis gene clusters), was used to monitor compositional changes in an undefined starter culture following phage attack. These analytical approaches facilitate a multifaceted assessment of starter culture compositional stability during milk fermentation, which has become an important QC aspect due to the increasing demand for consistent and high-quality dairy products.

Galacto-oligosaccharides as infant prebiotics: production, application, bioactive activities and future perspectives.

Galacto-oligosaccharides (GOS) are non-digestible oligosaccharides characterized by a mix of structures that vary in their degree of polymerization (DP) and glycosidic linkage between the galactose moieties or between galactose and glucose. They have enjoyed extensive scientific scrutiny, and their health-promoting effects are supported by a large number of scientific and clinical studies. A variety of GOS-associated health-promoting effects have been reported, such as growth promotion of beneficial bacteria, in particular bifidobacteria and lactobacilli, inhibition of pathogen adhesion and improvement of gut barrier function. GOS have attracted significant interest from food industries for their versatility as a bioactive ingredient and in particular as a functional component of infant formulations. These oligosaccharides are produced in a kinetically-controlled reaction involving lactose transgalactosylation, being catalyzed by particular ß-galactosidases of bacterial or fungal origin. Despite the well-established technology applied for GOS production, this process may still meet with technological challenges when employed at an industrial scale. The current review will cover relevant scientific literature on the beneficial physiological properties of GOS as a prebiotic for the infant gut microbiota, details of GOS structures, the associated reaction mechanism of ß-galactosidase, and its (large-scale) production.

A cell wall-associated polysaccharide is required for bacteriophage adsorption to the Streptococcus thermophilus cell surface.

Streptococcus thermophilus strain ST64987 was exposed to a member of a recently discovered group of S. thermophilus phages (the 987 phage group), generating phage-insensitive mutants, which were then characterized phenotypically and genomically. Decreased phage adsorption was observed in selected bacteriophage-insensitive mutants, and was partnered with a sedimenting phenotype and increased cell chain length or aggregation. Whole genome sequencing of several bacteriophage-insensitive mutants identified mutations located in a gene cluster presumed to be responsible for cell wall polysaccharide production in this strain. Analysis of cell surface-associated glycans by methylation and NMR spectroscopy revealed a complex branched rhamno-polysaccharide in both ST64987 and phage-insensitive mutant BIM3. In addition, a second cell wall-associated polysaccharide of ST64987, composed of hexasaccharide branched repeating units containing galactose and glucose, was absent in the cell wall of mutant BIM3. Genetic complementation of three phage-resistant mutants was shown to restore the carbohydrate and phage resistance profiles of the wild-type strain, establishing the role of this gene cluster in cell wall polysaccharide production and phage adsorption and, thus, infection.

Comparative genome and methylome analysis reveals restriction/modification system diversity in the gut commensal Bifidobacterium breve.

Bifidobacterium breve represents one of the most abundant bifidobacterial species in the gastro-intestinal tract of breast-fed infants, where their presence is believed to exert beneficial effects. In the present study whole genome sequencing, employing the PacBio Single Molecule, Real-Time (SMRT) sequencing platform, combined with comparative genome analysis allowed the most extensive genetic investigation of this taxon. Our findings demonstrate that genes encoding Restriction/Modification (R/M) systems constitute a substantial part of the B. breve variable gene content (or variome). Using the methylome data generated by SMRT sequencing, combined with targeted Illumina bisulfite sequencing (BS-seq) and comparative genome analysis, we were able to detect methylation recognition motifs and assign these to identified B. breve R/M systems, where in several cases such assignments were confirmed by restriction analysis. Furthermore, we show that R/M systems typically impose a very significant barrier to genetic accessibility of B. breve strains, and that cloning of a methyltransferase-encoding gene may overcome such a barrier, thus allowing future functional investigations of members of this species.

Gene-trait matching across the Bifidobacterium longum pan-genome reveals considerable diversity in carbohydrate catabolism among human infant strains.

BACKGROUND: Bifidobacterium longum is a common member of the human gut microbiota and is frequently present at high numbers in the gut microbiota of humans throughout life, thus indicative of a close symbiotic host-microbe relationship. Different mechanisms may be responsible for the high competitiveness of this taxon in its human host to allow stable establishment in the complex and dynamic intestinal microbiota environment. The objective of this study was to assess the genetic and metabolic diversity in a set of 20 B. longum strains, most of which had previously been isolated from infants, by performing whole genome sequencing and comparative analysis, and to analyse their carbohydrate utilization abilities using a gene-trait matching approach. RESULTS: We analysed their pan-genome and their phylogenetic relatedness. All strains clustered in the B. longum ssp. longum phylogenetic subgroup, except for one individual strain which was found to cluster in the B. longum ssp. suis phylogenetic group. The examined strains exhibit genomic diversity, while they also varied in their sugar utilization profiles. This allowed us to perform a gene-trait matching exercise enabling the identification of five gene clusters involved in the utilization of xylo-oligosaccharides, arabinan, arabinoxylan, galactan and fucosyllactose, the latter of which is an abundant human milk oligosaccharide (HMO). CONCLUSIONS: The results showed high diversity in terms of genes and predicted glycosyl-hydrolases, as well as the ability to metabolize a large range of sugars. Moreover, we corroborate the capability of B. longum ssp. longum to metabolise HMOs. Ultimately, their intraspecific genomic diversity and the ability to consume a wide assortment of carbohydrates, ranging from plant-derived carbohydrates to HMOs, may provide an explanation for the competitive advantage and persistence of B. longum in the human gut microbiome.

Staying alive: growth and survival of Bifidobacterium animalis subsp. animalis under in vitro and in vivo conditions.

Members of the Bifidobacterium genus are widely used as probiotics in fermented milk products. Bifidobacterium animalis subsp. animalis CNCM I-4602 grows and survives poorly in reconstituted skimmed milk (RSM). Availing of genome and transcriptome information, this poor growth and survival phenotype in milk was substantially improved by the addition of certain compounds, such as yeast extract, uric acid, glutathione, cysteine, ferrous sulfate, and a combination of magnesium sulfate and manganese sulfate. Carbohydrate utilization of CNCM I-4602 was also investigated, allowing the identification of several carbohydrate utilization gene clusters, and highlighting this strain's inability to utilize lactose, unlike the type strain of this subspecies, B. animalis subsp. animalis ATCC25527 and the B. animalis subsp. lactis subspecies. In addition, the ability of B. animalis subsp. animalis CNCM I-4602 to colonize a murine model was investigated, which showed that this strain persists in the murine gut for a period of at least 4 weeks. Associated in vivo transcriptome analysis revealed that, among other genes, a gene cluster encoding a predicted type IVb tight adherence (Tad) pilus was upregulated, indicating that this extracellular structure plays a role in the colonization/adaptation of the murine gastrointestinal tract by this strain.

Omics of bifidobacteria: research and insights into their health-promoting activities.

Members of the genus Bifidobacterium include gut commensals that are particularly abundant among the microbial communities residing in the gut of healthy breast-fed infants, where their presence has been linked to many beneficial host effects. Next-generation DNA sequencing and comparative and functional genome methodologies have been shown to be particularly useful in exploring the diversity of this genus. These combined approaches have allowed the identification of genetic features related to bifidobacterial establishment in the gut, involving host-microbe as well as microbe-microbe interactions. Among these, proteinaceous structures, which protrude from the bacterial surface, i.e. pili or fimbriae, and exopolysaccharidic cell surface layers or capsules represent crucial features that assist in their colonization and persistence in the gut. As bifidobacteria are colonizers of the large intestine, they have to be able to cope with various sources of osmotic, oxidative, bile and acid stress during their transit across the gastric barrier and the small intestine. Bifidobacterial genomes thus encode various survival mechanisms, such as molecular chaperones and efflux pumps, to overcome such challenges. Bifidobacteria represent part of an anaerobic gut community, and feed on nondigestible carbohydrates through a specialized fermentative metabolic pathway, which in turn produces growth substrates for other members of the gut community. Conversely, bifidobacteria may also be dependent on other (bifido)bacteria to access host- and diet-derived glycans, and these complex co-operative interactions, based on resource sharing and cross-feeding strategies, represent powerful driving forces that shape gut microbiota composition.

Comparative and functional genomics of the Lactococcus lactis taxon; insights into evolution and niche adaptation.

BACKGROUND: Lactococcus lactis is among the most widely studied lactic acid bacterial species due to its long history of safe use and economic importance to the dairy industry, where it is exploited as a starter culture in cheese production. RESULTS: In the current study, we report on the complete sequencing of 16 L. lactis subsp. lactis and L. lactis subsp. cremoris genomes. The chromosomal features of these 16 L. lactis strains in conjunction with 14 completely sequenced, publicly available lactococcal chromosomes were assessed with particular emphasis on discerning the L. lactis subspecies division, evolution and niche adaptation. The deduced pan-genome of L. lactis was found to be closed, indicating that the representative data sets employed for this analysis are sufficient to fully describe the genetic diversity of the taxon. CONCLUSIONS: Niche adaptation appears to play a significant role in governing the genetic content of each L. lactis subspecies, while (differential) genome decay and redundancy in the dairy niche is also highlighted.

Global transcriptional landscape and promoter mapping of the gut commensal Bifidobacterium breve UCC2003.

BACKGROUND: Bifidobacterium breve represents a common member of the infant gut microbiota and its presence in the gut has been associated with host well being. For this reason it is relevant to investigate and understand the molecular mechanisms underlying the establishment, persistence and activities of this gut commensal in the host environment. RESULTS: The assessment of vegetative promoters in the bifidobacterial prototype Bifidobacterium breve UCC2003 was performed employing a combination of RNA tiling array analysis and cDNA sequencing. Canonical -10 (TATAAT) and -35 (TTGACA) sequences were identified upstream of transcribed genes or operons, where deviations from this consensus correspond to transcription level variations. A Random Forest analysis assigned the -10 region of B. breve promoters as the element most impacting on the level of transcription, followed by the spacer length and the 5'-UTR length of transcripts. Furthermore, our transcriptome study also identified rho-independent termination as the most common and effective termination signal of highly and moderately transcribed operons in B. breve. CONCLUSION: The present study allowed us to identify genes and operons that are actively transcribed in this organism during logarithmic growth, and link promoter elements with levels of transcription of essential genes in this organism. As homologs of many of our identified genes are present across the whole genus Bifidobacterium, our dataset constitutes a transcriptomic reference to be used for future investigations of gene expression in members of this genus.

Discovery of a conjugative megaplasmid in Bifidobacterium breve.

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains.

Comparative genomics of the Bifidobacterium breve taxon.

BACKGROUND: Bifidobacteria are commonly found as part of the microbiota of the gastrointestinal tract (GIT) of a broad range of hosts, where their presence is positively correlated with the host's health status. In this study, we assessed the genomes of thirteen representatives of Bifidobacterium breve, which is not only a frequently encountered component of the (adult and infant) human gut microbiota, but can also be isolated from human milk and vagina. RESULTS: In silico analysis of genome sequences from thirteen B. breve strains isolated from different environments (infant and adult faeces, human milk, human vagina) shows that the genetic variability of this species principally consists of hypothetical genes and mobile elements, but, interestingly, also genes correlated with the adaptation to host environment and gut colonization. These latter genes specify the biosynthetic machinery for sortase-dependent pili and exopolysaccharide production, as well as genes that provide protection against invasion of foreign DNA (i.e. CRISPR loci and restriction/modification systems), and genes that encode enzymes responsible for carbohydrate fermentation. Gene-trait matching analysis showed clear correlations between known metabolic capabilities and characterized genes, and it also allowed the identification of a gene cluster involved in the utilization of the alcohol-sugar sorbitol. CONCLUSIONS: Genome analysis of thirteen representatives of the B. breve species revealed that the deduced pan-genome exhibits an essentially close trend. For this reason our analyses suggest that this number of B. breve representatives is sufficient to fully describe the pan-genome of this species. Comparative genomics also facilitated the genetic explanation for differential carbon source utilization phenotypes previously observed in different strains of B. breve.

Genomic encyclopedia of type strains of the genus Bifidobacterium.

Bifidobacteria represent one of the dominant microbial groups that are present in the gut of various animals, being particularly prevalent during the suckling stage of life of humans and other mammals. However, the overall genome structure of this group of microorganisms remains largely unexplored. Here, we sequenced the genomes of 42 representative (sub)species across the Bifidobacterium genus and used this information to explore the overall genetic picture of this bacterial group. Furthermore, the genomic data described here were used to reconstruct the evolutionary development of the Bifidobacterium genus. This reconstruction suggests that its evolution was substantially influenced by genetic adaptations to obtain access to glycans, thereby representing a common and potent evolutionary force in shaping bifidobacterial genomes.

Molecular characterization of three Lactobacillus delbrueckii subsp. bulgaricus phages.

In this study, three phages infecting Lactobacillus delbrueckii subsp. bulgaricus, named Ld3, Ld17, and Ld25A, were isolated from whey samples obtained from various industrial fermentations. These phages were further characterized in a multifaceted approach: (i) biological and physical characterization through host range analysis and electron microscopy; (ii) genetic assessment through genome analysis; (iii) mass spectrometry analysis of the structural components of the phages; and (iv), for one phage, transcriptional analysis by Northern hybridization, reverse transcription-PCR, and primer extension. The three obtained phage genomes display high levels of sequence identity to each other and to genomes of the so-called group b L. delbrueckii phages c5, LL-Ku, and phiLdb, where some of the observed differences are believed to be responsible for host range variations.

Progress in lactic acid bacterial phage research.

Research on lactic acid bacteria (LAB) has advanced significantly over the past number of decades and these developments have been driven by the parallel advances in technologies such as genomics, bioinformatics, protein expression systems and structural biology, combined with the ever increasing commercial relevance of this group of microorganisms. Some of the more significant and impressive outputs have been in the domain of bacteriophage-host interactions which provides a prime example of the cutting-edge model systems represented by LAB research. Here, we present a retrospective overview of the key advances in LAB phage research including phage-host interactions and co-evolution. We describe how in many instances this knowledge can be pivotal in creating real improvements in the application of LAB cultures in commercial practice.

Diversity, ecology and intestinal function of bifidobacteria.

The human gastrointestinal tract represents an environment which is a densely populated home for a microbiota that has evolved to positively contribute to host health. At birth the essentially sterile gastrointestinal tract (GIT) is rapidly colonized by microorganisms that originate from the mother and the surrounding environment. Within a short timeframe a microbiota establishes within the (breastfed) infant's GIT where bifidobacteria are among the dominant members, although their numerical dominance disappears following weaning. The numerous health benefits associated with bifidobacteria, and the consequent commercial relevance resulting from their incorporation into functional foods, has led to intensified research aimed at the molecular understanding of claimed probiotic attributes of this genus. In this review we provide the current status on the diversity and ecology of bifidobacteria. In addition, we will discuss the molecular mechanisms that allow this intriguing group of bacteria to colonize and persist in the GIT, so as to facilitate interaction with its host.

Functional genome analysis of Bifidobacterium breve UCC2003 reveals type IVb tight adherence (Tad) pili as an essential and conserved host-colonization factor.

Development of the human gut microbiota commences at birth, with bifidobacteria being among the first colonizers of the sterile newborn gastrointestinal tract. To date, the genetic basis of Bifidobacterium colonization and persistence remains poorly understood. Transcriptome analysis of the Bifidobacterium breve UCC2003 2.42-Mb genome in a murine colonization model revealed differential expression of a type IVb tight adherence (Tad) pilus-encoding gene cluster designated "tad(2003)." Mutational analysis demonstrated that the tad(2003) gene cluster is essential for efficient in vivo murine gut colonization, and immunogold transmission electron microscopy confirmed the presence of Tad pili at the poles of B. breve UCC2003 cells. Conservation of the Tad pilus-encoding locus among other B. breve strains and among sequenced Bifidobacterium genomes supports the notion of a ubiquitous pili-mediated host colonization and persistence mechanism for bifidobacteria.

Transcriptional landscape of the pMP7017 megaplasmid and its impact on the Bifidobacterium breve UCC2003 transcriptome.

The 190 kb megaplasmid pMP7017 of Bifidobacterium breve JCM7017 represents the first conjugative and largest plasmid characterised within this genus to date. In the current study, we adopted an integrated approach combining transcriptomics, whole genome comparative analysis and metagenomic data mining to understand the biology of pMP7017 and related megaplasmids, and to assess the impact of plasmid-carriage on the host strain. The data generated revealed variations within basic features of promoter elements which correlate with a high level of transcription on the plasmid and highlight the transcriptional activity of genes encoding both offensive and defensive adaptations, including a Type IIL restriction-modification system, an anti-restriction system and four Type II toxin-antitoxin systems. Furthermore, a highly transcribed tmRNA, which likely provides translational support to the host strain, was identified, making pMP7017 the first plasmid of the Bifidobacterium genus and the smallest plasmid known to express a tmRNA. Analyses of synteny and variability among pMP7017 and related plasmids indicate substantial diversity in gene organisation and accessory gene cargo highlighting diverse (co-)evolution and potential host-specific rearrangements and adaptations. Systematic analysis of the codon usage profile of transcriptionally active pMP7017-encoded genes suggests that pMP7017 originated from (sub)species of Bifidobacterium longum. Furthermore, mining of metagenomic data suggests the presence of pMP7017-homologues in ~10% of microbiome samples, mostly infants and/or mothers from various geographical locations. Comparative transcriptome analysis of the B. breve UCC2003 chromosome in the presence or absence of pMP7017 revealed differential expression of genes representing 8% of the total gene pool. Genes involved in genetic information processing were exclusively upregulated, while altered expression of genes involved in biofilm production and polysaccharide biosynthesis was also observed.

Whole genome sequencing of uropathogenic E. coli from Ireland reveals diverse resistance mechanisms and strong correlation with phenotypic (EUCAST) susceptibility testing.

Urinary tract infections (UTI) caused by uropathogenic Escherichia coli (UPEC) pose a global health concern. Resistance mechanisms, including genetic mutations in antimicrobial target genes, efflux pumps, and drug deactivating enzymes, hinder clinical treatment. These resistance factors often spread through mobile genetic elements. Molecular techniques like whole genome sequencing (WGS), multilocus sequence typing (MLST), and phylotyping help decode bacterial genomes and categorise resistance genes. In this study, we analysed 57 UPEC isolates from different UTI patients following EUCAST guidelines. A selection of 17 representative strains underwent WGS, phylotyping, MLST, and comparative analysis to connect laboratory susceptibility data with predictive genomics based on key resistance genes and chromosomal mutations in antimicrobial targets. Trimethoprim resistance consistently correlated with dfr genes, with six different alleles detected among the isolates. These dfr genes often coexisted with class 1 integrons, with the most common gene cassette combining dfr and aadA. Furthermore, 52.9% of isolates harboured the blaTem-1 gene, rendering resistance to ampicillin and amoxicillin. Ciprofloxacin-resistant strains exhibited mutations in GyrA, GyrB and ParC, plasmid-mediated quinolone resistance genes (qnrb10), and aac(6')-Ib-cr5. Nitrofurantoin resistance in one isolate stemmed from a four amino acid deletion in NfsB. These findings illustrate the varied strategies employed by UPEC to resist antibiotics and the correlation between clinical susceptibility testing and molecular determinants. As molecular testing gains prominence in clinical applications, understanding key resistance determinants becomes crucial for accurate susceptibility testing and guiding effective antimicrobial therapy.

Two extracellular α-arabinofuranosidases are required for cereal-derived arabinoxylan metabolism by Bifidobacterium longum subsp. longum.

Members of the genus Bifidobacterium are commonly found in the human gut and are known to utilize complex carbohydrates that are indigestible by the human host. Members of the Bifidobacterium longum subsp. longum taxon can metabolize various plant-derived carbohydrates common to the human diet. To metabolize such polysaccharides, which include arabinoxylan, bifidobacteria need to encode appropriate carbohydrate-active enzymes in their genome. In the current study, we describe two GH43 family enzymes, denoted here as AxuA and AxuB, which are encoded by B. longum subsp. longum NCIMB 8809 and are shown to be required for cereal-derived arabinoxylan metabolism by this strain. Based on the observed hydrolytic activity of AxuA and AxuB, assessed by employing various synthetic and natural substrates, and based on in silico analyses, it is proposed that both AxuA and AxuB represent extracellular α-L-arabinofuranosidases with distinct substrate preferences. The variable presence of the axuA and axuB genes and other genes previously described to be involved in the metabolism of arabinose-containing glycans can in the majority cases explain the (in)ability of individual B. longum subsp. longum strains to grow on cereal-derived arabinoxylans and arabinan.

Exploration of the genomic diversity and core genome of the Bifidobacterium adolescentis phylogenetic group by means of a polyphasic approach.

In the current work, we describe genome diversity and core genome sequences among representatives of three bifidobacterial species, i.e., Bifidobacterium adolescentis, Bifidobacterium catenulatum, and Bifidobacterium pseudocatenulatum, by employing a polyphasic approach involving analysis of 16S rRNA gene and 16S-23S internal transcribed spacer (ITS) sequences, pulsed-field gel electrophoresis (PFGE), and comparative genomic hybridization (CGH) assays.