Reduced synthesis of phospho-polysaccharide in Lactococcus as a strategy to evade phage infection.

Lactococcus spp. are applied routinely in dairy fermentations and their consistent growth and associated acidification activity is critical to ensure the quality and safety of fermented dairy foods. Bacteriophages pose a significant threat to such fermentations and thus it is imperative to study how these bacteria may evade their viral predators in the relevant confined settings. Many lactococcal phages are known to specifically recognise and bind to cell wall polysaccharides (CWPSs) and particularly the phospho-polysaccharide (PSP) side chain component that is exposed on the host cell surface. In the present study, we generated derivatives of a lactococcal strain with reduced phage sensitivity to establish the mode of phage evasion. The resulting mutants were characterized using a combination of comparative genome analysis, microbiological and chemical analyses. Using these approaches, it was established that the phage-resistant derivatives incorporated mutations in genes within the cluster associated with CWPS biosynthesis resulting in growth and morphological defects that could revert when the selective pressure of phages was removed. Furthermore, the cell wall extracts of selected mutants revealed that the phage-resistant strains produced intact PSP but in significantly reduced amounts. The reduced availability of the PSP and the ability of lactococcal strains to revert rapidly to wild type growth and activity in the absence of phage pressure provides Lactococcus with the means to survive and evade phage attack.

Bipartite rgp Locus Diversity in Streptococcus thermophilus Corresponds to Backbone and Side Chain Differences of Its Rhamnose-Containing Cell Wall Polysaccharide.

The rhamnose-glucose polysaccharide (Rgp) of Streptococcus thermophilus represents a major cell wall component, and the gene cluster responsible for its biosynthesis (termed rgp) has recently been identified. Significant genetic diversity among these loci has previously been reported, with five distinct rgp genotypes identified (designated rgp1 through -5). In the present study, two additional genotypes were identified (designated rgp6 and rgp7) through comparative analysis of the rgp loci of 78 Streptococcus thermophilus genomes. The rgp locus of a given S. thermophilus strain encoded the biosynthetic machinery for a rhamnan-rich backbone and a variable side chain component, the latter being associated with the highly specific interactions with many bacteriophages that infect this species. The chemical structure of the Rgp from three S. thermophilus strains, representing the rgp2, -3, and -4 genotypes, was elucidated, and based on bioinformatic and biochemical analyses we propose a model for Rgp biosynthesis in dairy streptococci. Furthermore, we exploited the genetic diversity within the S. thermophilus bipartite rgp locus to develop a two-step multiplex PCR system to classify strains based on gene content associated with the biosynthesis of the variable side chain structure as well as the rhamnan backbone. IMPORTANCE Streptococcus thermophilus is present and applied in industrial and artisanal dairy fermentations for the production of various cheeses and yogurt. During these fermentations, S. thermophilus is vulnerable to phage predation, and recent studies have identified the rhamnose-glucose polymer (Rgp) as the definitive receptor for at least one problematic phage species. Detailed analysis of S. thermophilus rgp loci has revealed an unprecedented level of genetic diversity, particularly within the glycosyltransferase-encoding gene content of a given locus. Our study shows that this genetic diversity reflects the biochemical structure(s) of S. thermophilus Rgp. As such, we harnessed the genetic diversity of S. thermophilus rgp loci to develop a two-step multiplex PCR method for the classification of strain collections and, ultimately, the formation of phage-robust rational starter sets.

Needle in a Whey-Stack: PhRACS as a Discovery Tool for Unknown Phage-Host Combinations.

The field of metagenomics has rapidly expanded to become the go-to method for complex microbial community analyses. However, there is currently no straightforward route from metagenomics to traditional culture-based methods of strain isolation, particularly in (bacterio)phage biology, leading to an investigative bottleneck. Here, we describe a method that exploits specific phage receptor binding protein (RBP)-host cell surface receptor interaction enabling isolation of phage-host combinations from an environmental sample. The method was successfully applied to two complex sample types-a dairy-derived whey sample and an infant fecal sample, enabling retrieval of specific and culturable phage hosts. IMPORTANCE PhRACS aims to bridge the current divide between in silico genetic analyses (i.e., phageomic studies) and traditional culture-based methodology. Through the labeling of specific bacterial hosts with fluorescently tagged recombinant phage receptor binding proteins and the isolation of tagged cells using flow cytometry, PhRACS allows the full potential of phageomic data to be realized in the wet laboratory.

Brussowvirus SW13 Requires a Cell Surface-Associated Polysaccharide To Recognize Its Streptococcus thermophilus Host.

Four bacteriophage-insensitive mutants (BIMs) of the dairy starter bacterium Streptococcus thermophilus UCCSt50 were isolated following challenge with Brussowvirus SW13. The BIMs displayed an altered sedimentation phenotype. Whole-genome sequencing and comparative genomic analysis of the BIMs uncovered mutations within a family 2 glycosyltransferase-encoding gene (orf06955UCCSt50) located within the variable region of the cell wall-associated rhamnose-glucose polymer (Rgp) biosynthesis locus (designated the rgp gene cluster here). Complementation of a representative BIM, S. thermophilus B1, with native orf06955UCCSt50 restored phage sensitivity comparable to that of the parent strain. Detailed bioinformatic analysis of the gene product of orf06955UCCSt50 identified it as a functional homolog of the Lactococcus lactis polysaccharide pellicle (PSP) initiator WpsA. Biochemical analysis of cell wall fractions of strains UCCSt50 and B1 determined that mutations within orf06955UCCSt50 result in the loss of the side chain decoration from the Rgp backbone structure. Furthermore, it was demonstrated that the intact Rgp structure incorporating the side chain structure is essential for phage binding through fluorescence labeling studies. Overall, this study confirms that the rgp gene cluster of S. thermophilus encodes the biosynthetic machinery for a cell surface-associated polysaccharide that is essential for binding and subsequent infection by Brussowviruses, thus enhancing our understanding of S. thermophilus phage-host dynamics. IMPORTANCE Streptococcus thermophilus is an important starter culture bacterium in global dairy fermentation processes, where it is used for the production of various cheeses and yogurt. Bacteriophage predation of the species can result in substandard product quality and, in rare cases, complete fermentation collapse. To mitigate these risks, it is necessary to understand the phage-host interaction process, which commences with the recognition of, and adsorption to, specific host-encoded cell surface receptors by bacteriophage(s). As new groups of S. thermophilus phages are being discovered, the importance of underpinning the genomic elements that specify the surface receptor(s) is apparent. Our research identifies a single gene that is critical for the biosynthesis of a saccharidic moiety required for phage adsorption to its S. thermophilus host. The acquired knowledge provides novel insights into phage-host interactions for this economically important starter species.

In Vitro and In Vivo Assessment of the Potential of Escherichia coli Phages to Treat Infections and Survive Gastric Conditions.

Enterotoxigenic Escherichia coli (ETEC) and Shigella ssp. infections are associated with high rates of mortality, especially in infants in developing countries. Due to increasing levels of global antibiotic resistance exhibited by many pathogenic organisms, alternative strategies to combat such infections are urgently required. In this study, we evaluated the stability of five coliphages (four Myoviridae and one Siphoviridae phage) over a range of pH conditions and in simulated gastric conditions. The Myoviridae phages were stable across the range of pH 2 to 7, while the Siphoviridae phage, JK16, exhibited higher sensitivity to low pH. A composite mixture of these five phages was tested in vivo in a Galleria mellonella model. The obtained data clearly shows potential in treating E. coli infections prophylactically.

Lactic Acid Bacteria Diversity and Characterization of Probiotic Candidates in Fermented Meats.

Probiotics are defined as live microorganisms which confer health benefits to the host when administered in adequate amounts. Many lactic acid bacteria (LAB) strains have been classified as probiotics and fermented foods are an excellent source of such LAB. In this study, novel probiotic candidates from two fermented meats (pancetta and prosciutto) were isolated and characterized. LAB populations present in pancetta and prosciutto were evaluated and Lactiplantibacillus plantarum was found to be the dominant species. The antagonistic ability of selected isolates against LAB and non-LAB strains was investigated, in particular, the ability to produce anti-microbial compounds including organic acids and bacteriocins. Probiotic characteristics including antibiotic susceptibility, hydrophobicity and autoaggregation capacity; and ability to withstand simulated gastric juice, bile salt, phenol and NaCl were assessed. Among the characterized strains, L. plantarum 41G isolated from prosciutto was identified as the most robust probiotic candidate compared. Results from this study demonstrate that artisanal fermented meat is a rich source of novel strains with probiotic potential.

Lysogenization of a Lactococcal Host with Three Distinct Temperate Phages Provides Homologous and Heterologous Phage Resistance.

Lactococcus lactis is the most widely exploited microorganism in global dairy fermentations. Lactococcal strains are described as typically harboring a number of prophages in their chromosomes. The presence of such prophages may provide both advantages and disadvantages to the carrying host. Here, we describe the deliberate generation of three distinct lysogens of the model lactococcal strain 3107 and the impact of additional prophage carriage on phage-resistance and anti-microbial susceptibility. Lysogen-specific responses were observed, highlighting the unique relationship and impact of each lysogenic phage on its host. Both homologous and heterologous phage-resistance profiles were observed, highlighting the presence of possible prophage-encoded phage-resistance factors. Superinfection exclusion was among the most notable causes of heterologous phage-resistance profiles with resistance observed against members of the Skunavirus, P335, P087, and 949 lactococcal phage groups. Through these analyses, it is now possible to identify phages that may pursue similar DNA injection pathways. The generated lysogenic strains exhibited increased sensitivity to the antimicrobial compounds, nisin and lysozyme, relative to the parent strain, although it is noteworthy that the degree of sensitivity was specific to the individual (pro)phages. Overall, the findings highlight the unique impact of each prophage on a given strain and the requirement for strain-level analysis when considering the implications of lysogeny.

A Quest of Great Importance-Developing a Broad Spectrum Escherichiacoli Phage Collection.

Shigella ssp. and enterotoxigenic Escherichiacoli are the most common etiological agents of diarrheal diseases in malnourished children under five years of age in developing countries. The ever-growing issue of antibiotic resistance and the potential negative impact of antibiotic use on infant commensal microbiota are significant challenges to current therapeutic approaches. Bacteriophages (or phages) represent an alternative treatment that can be used to treat specific bacterial infections. In the present study, we screened water samples from both environmental and industrial sources for phages capable of infecting E. coli laboratory strains within our collection. Nineteen phages were isolatedand tested for their ability to infect strains within the ECOR collection and E. coli O157:H7 Δstx. Furthermore, since coliphages have been reported to cross-infect certain Shigella spp., we also evaluated the ability of the nineteen phages to infect a representative Shigella sonnei strain from our collection. Based on having distinct (although overlapping in some cases) host ranges, ten phage isolates were selected for genome sequence and morphological characterization. Together, these ten selected phages were shown to infect most of the ECOR library, with 61 of the 72 strains infected by at least one phage from our collection. Genome analysis of the ten phages allowed classification into five previously described genetic subgroups plus one previously underrepresented subgroup.

Isolation and Characterization of Lactobacillus brevis Phages.

Lactobacillus brevis has been widely used in industry for fermentation purposes. However, it is also associated with the spoilage of foods and beverages, in particular, beer. There is an increasing demand for natural food preservation methods, and in this context, bacteriophages possess the potential to control such spoilage bacteria. Just a few studies on phages infecting Lactobacillus brevis have been performed to date and in the present study, we report the isolation and characterization of five virulent phages capable of infecting Lb. brevis strains. The analysis reveals a high diversity among the isolates, with members belonging to both, the Myoviridae and Siphoviridae families. One isolate, designated phage 3-521, possesses a genome of 140.8 kb, thus representing the largest Lb. brevis phage genome sequenced to date. While the isolated phages do not propagate on Lb. brevis beer-spoiling strains, phages showed activity against these strains, impairing the growth of some Lb. brevis strains. The results highlight the potential of bacteriophage-based treatments as an effective approach to prevent bacterial spoilage of beer.

The Lactococcus lactis Pan-Plasmidome.

Plasmids are autonomous, self-replicating, extrachromosomal genetic elements that are typically not essential for growth of their host. They may encode metabolic capabilities, which promote the maintenance of these genetic elements, and may allow adaption to specific ecological niches and consequently enhance survival. Genome sequencing of 16 Lactococcus lactis strains revealed the presence of 83 plasmids, including two megaplasmids. The limitations of Pacific Biosciences SMRT sequencing in detecting the total plasmid complement of lactococcal strains is examined, while a combined Illumina/SMRT sequencing approach is proposed to combat these issues. Comparative genome analysis of these plasmid sequences combined with other publicly available plasmid sequence data allowed the definition of the lactococcal plasmidome, and facilitated an investigation into (bio) technologically important plasmid-encoded traits such as conjugation, bacteriocin production, exopolysaccharide (EPS) production, and (bacterio) phage resistance.

Metagenomic dissection of the canine gut microbiota: insights into taxonomic, metabolic and nutritional features.

Domestication of dogs from wolves is the oldest known example of ongoing animal selection, responsible for generating more than 300 dog breeds worldwide. In order to investigate the taxonomic and functional evolution of the canine gut microbiota, a multi-omics approach was applied to six wild wolves and 169 dog faecal samples, the latter encompassing 51 breeds, which fully covers currently known canine genetic biodiversity. Specifically, 16S rRNA gene and bifidobacterial Internally Transcribed Spacer (ITS) profiling were employed to reconstruct and then compare the canine core gut microbiota to those of wolves and humans, revealing that artificial selection and subsequent cohabitation of dogs with their owners influenced the microbial population of canine gut through loss and acquisition of specific bacterial taxa. Moreover, comparative analysis of the intestinal bacterial population of dogs fed on Bones and Raw Food (BARF) or commercial food (CF) diet, coupled with shotgun metagenomics, highlighted that both bacterial composition and metabolic repertoire of the canine gut microbiota have evolved to adapt to high-protein or high-carbohydrates intake. Altogether, these data indicate that artificial selection and domestication not only affected the canine genome, but also shaped extensively the bacterial population harboured by the canine gut.

Health benefits conferred by the human gut microbiota during infancy.

Development of the human gut throughout the entire life.

Biodiversity of Streptococcus thermophilus Phages in Global Dairy Fermentations.

Streptococcus thermophilus strains are among the most widely employed starter cultures in dairy fermentations, second only to those of Lactococcus lactis. The extensive application of this species provides considerable opportunity for the proliferation of its infecting (bacterio)phages. Until recently, dairy streptococcal phages were classified into two groups (cos and pac groups), while more recently, two additional groups have been identified (5093 and 987 groups). This highlights the requirement for consistent monitoring of phage populations in the industry. Here, we report a survey of 35 samples of whey derived from 27 dairy fermentation facilities in ten countries against a panel of S. thermophilus strains. This culminated in the identification of 172 plaque isolates, which were characterized by multiplex PCR, restriction fragment length polymorphism analysis, and host range profiling. Based on this characterisation, 39 distinct isolates representing all four phage groups were selected for genome sequencing. Genetic diversity was observed among the cos isolates and correlations between receptor binding protein phylogeny and host range were also clear within this phage group. The 987 phages isolated within this study shared high levels of sequence similarity, yet displayed reduced levels of similarity to those identified in previous studies, indicating that they are subject to ongoing genetic diversification.

Functional carbohydrate binding modules identified in evolved dits from siphophages infecting various Gram-positive bacteria.

With increasing numbers of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and associated functionality of phage adhesion devices. Recently, decorations have been reported in the tail and neck passage structures of members of the so-called 936 group of lactococcal siphophages. In the current report, using bioinformatic analysis we identified a conserved carbohydrate binding module (CBM) among many of the virion baseplate Dit components, in addition to the CBM present in the 'classical' receptor binding proteins (RBPs). We observed that, within these so-called 'evolved' Dit proteins, the identified CBMs have structurally conserved folds, yet can be grouped into four distinct classes. We expressed such modules in fusion with GFP, and demonstrated their binding capability to their specific host using fluorescent binding assays with confocal microscopy. We detected evolved Dits in several phages infecting various Gram-positive bacterial species, including mycobacteria. The omnipresence of CBM domains in siphophages indicates their auxiliary role in infection, as they can assist in the specific recognition of and attachment to their host, thus ensuring a highly efficient and specific phage-host adhesion process as a prelude to DNA injection.

A Decade of Streptococcus thermophilus Phage Evolution in an Irish Dairy Plant.

Phages of Streptococcus thermophilus present a major threat to the production of many fermented dairy products. To date, only a few studies have assessed the biodiversity of S. thermophilus phages in dairy fermentations. In order to develop strategies to limit phage predation in this important industrial environment, it is imperative that such studies are undertaken and that phage-host interactions of this species are better defined. The present study investigated the biodiversity and evolution of phages within an Irish dairy fermentation facility over an 11-year period. This resulted in the isolation of 17 genetically distinct phages, all of which belong to the so-called cos group. The evolution of phages within the factory appears to be influenced by phages from other dairy plants introduced into the factory for whey protein powder production. Modular exchange, primarily within the regions encoding lysogeny and replication functions, was the major observation among the phages isolated between 2006 and 2016. Furthermore, the genotype of the first isolate in 2006 was observed continuously across the following decade, highlighting the ability of these phages to prevail in the factory setting for extended periods of time. The proteins responsible for host recognition were analyzed, and carbohydrate-binding domains (CBDs) were identified in the distal tail (Dit), the baseplate proteins, and the Tail-associated lysin (Tal) variable regions (VR1 and VR2) of many isolates. This supports the notion that S. thermophilus phages recognize a carbohydrate receptor on the cell surface of their host.IMPORTANCE Dairy fermentations are consistently threatened by the presence of bacterial viruses (bacteriophages or phages), which may lead to a reduction in acidification rates or even complete loss of the fermentate. These phages may persist in factories for long periods of time. The objective of the current study was to monitor the progression of phages infecting the dairy bacterium Streptococcus thermophilus over a period of 11 years in an Irish dairy plant so as to understand how these phages evolve. A focused analysis of the genomic region that encodes host recognition functions highlighted that the associated proteins harbor a variety of carbohydrate-binding domains, which corroborates the notion that phages of S. thermophilus recognize carbohydrate receptors at the initial stages of the phage cycle.

Impact of gut-associated bifidobacteria and their phages on health: two sides of the same coin?

Bifidobacteria are among the first microbial colonisers of the human infant gut post-partum. Their early appearance and dominance in the human infant gut and the reported health-promoting or probiotic status of several bifidobacterial strains has culminated in intensive research efforts that focus on their activities as part of the gut microbiota and the concomitant implications for human health. In this mini-review, we evaluate current knowledge on the genomics of this diverse bacterial genus, and on the genetic and functional adaptations that have underpinned the success of bifidobacteria in colonising the infant gut. The growing interest in functional genomics of bifidobacteria has also created interest in the interactions of bifidobacteria and their (bacterio)phages. While virulent phages of bifidobacteria have yet to be isolated, the incidence of integrated (pro)phages in bifidobacterial genomes are widely reported and this mini-review considers the role of these so-called bifidoprophages in modulating bifidobacterial populations in the human gastrointestinal tract and the implications for existing and future development of probiotic therapies.

How to Feed the Mammalian Gut Microbiota: Bacterial and Metabolic Modulation by Dietary Fibers.

The composition of the gut microbiota of mammals is greatly influenced by diet. Therefore, evaluation of different food ingredients that may promote changes in the gut microbiota composition is an attractive approach to treat microbiota disturbances. In this study, three dietary fibers, such as inulin (I, 10%), resistant starch (RS, 10%), and citrus pectin (3%), were employed as supplements to normal chow diet of adult male rats for 2 weeks. Fecal microbiota composition and corresponding metabolite profiles were assessed before and after prebiotics supplementation. A general increase in the Bacteroidetes phylum was detected with a concurrent reduction in Firmicutes, in particular for I and RS experiments, while additional changes in the microbiota composition were evident at lower taxonomic levels for all the three substrates. Such modifications in the microbiota composition were correlated with changes in metabolic profiles of animals, in particular changes in acetate and succinate levels. This study represents a first attempt to modulate selectively the abundance and/or metabolic activity of various members of the gut microbiota by means of dietary fiber.

Global Survey and Genome Exploration of Bacteriophages Infecting the Lactic Acid Bacterium Streptococcus thermophilus.

Despite the persistent and costly problem caused by (bacterio)phage predation of Streptococcus thermophilus in dairy plants, DNA sequence information relating to these phages remains limited. Genome sequencing is necessary to better understand the diversity and proliferative strategies of virulent phages. In this report, whole genome sequences of 40 distinct bacteriophages infecting S. thermophilus were analyzed for general characteristics, genomic structure and novel features. The bacteriophage genomes display a high degree of conservation within defined groupings, particularly across the structural modules. Supporting this observation, four novel members of a recently discovered third group of S. thermophilus phages (termed the 5093 group) were found to be conserved relative to both phage 5093 and to each other. Replication modules of S. thermophilus phages generally fall within two main groups, while such phage genomes typically encode one putative transcriptional regulator. Such features are indicative of widespread functional synteny across genetically distinct phage groups. Phage genomes also display nucleotide divergence between groups, and between individual phages of the same group (within replication modules and at the 3' end of the lysis module)-through various insertions and/or deletions. A previously described multiplex PCR phage detection system was updated to reflect current knowledge on S. thermophilus phages. Furthermore, the structural protein complement as well as the antireceptor (responsible for the initial attachment of the phage to the host cell) of a representative of the 5093 group was defined. Our data more than triples the currently available genomic information on S. thermophilus phages, being of significant value to the dairy industry, where genetic knowledge of lytic phages is crucial for phage detection and monitoring purposes. In particular, the updated PCR detection methodology for S. thermophilus phages is highly useful in monitoring particular phage group(s) present in a given whey sample. Studies of this nature therefore not only provide information on the prevalence and associated threat of known S. thermophilus phages, but may also uncover newly emerging and genomically distinct phages infecting this dairy starter bacterium.

Unveiling bifidobacterial biogeography across the mammalian branch of the tree of life.

Internally transcribed spacer (ITS) rRNA profiling is a novel tool for detailed analysis of microbial populations at low taxonomic ranks. Here we exploited this approach to explore species-level biogeography of the Bifidobacterium genus across 291 adult mammals. These include humans and 13 other primates, domesticated animals, such as dogs, cats, cows, sheep, goats, horses and pigs, and 46 additional species. The collected profiles revealed the presence of 89 putative novel bifidobacterial taxa in addition to 45 previously described species. Remarkably, in contrast to what is currently known for many gut commensals, we did not observe host-specialization among bifidobacterial species but rather their widespread distribution across mammals. Moreover, ITS rRNA profiling of wild relatives of domesticated dogs, rabbits and pigs clearly indicates that domestication and close contact with humans have impacted on the composition of the fecal bifidobacterial population. These data were complemented by analysis of bifidobacterial communities in milk of eight mammalian families, showing that bifidobacteria represent prototypical early gut microbiota members which are inherited by newborns from their lactating mother. Thus this study highlights the role of bifidobacteria as pioneering gut colonizers of a wide range of mammals.