Microbiome networking analysis has emerged as a powerful tool for studying the complex interactions among microorganisms in various ecological niches, including the human body and several environments. This analysis has been used extensively in both human and environmental studies, revealing key taxa and functional units peculiar to the ecosystem considered. In particular, it has been mainly used to investigate the effects of environmental stressors, such as pollution, climate change or therapies, on host-associated microbial communities and ecosystem function. In this review, we discuss the latest advances in microbiome networking analysis, including methods for constructing and analyzing microbiome networks, and provide a case study on how to use these tools. These analyses typically involve constructing a network that represents interactions among microbial taxa or functional units, such as genes or metabolic pathways. Such networks can be based on a variety of data sources, including 16S rRNA sequencing, metagenomic sequencing, and metabolomics data. Once constructed, these networks can be analyzed to identify key nodes or modules important for the stability and function of the microbiome. By providing insights into essential ecological features of microbial communities, microbiome networking analysis has the potential to transform our understanding of the microbial world and its impact on human health and the environment.
Aim: Lactococcal skunaviruses are diverse and problematic in the industrial dairy environment. Host recognition involves the specific interaction of phage-encoded proteins with saccharidic host cell surface structures. Lactococcal plasmid pEPS6073 encodes genes required for the biosynthesis of a cell surface-associated exopolysaccharide (EPS), designated 6073-like. Here, the impact of this EPS on Skunavirus sensitivity was assessed.
Methods: Conjugal transfer of pEPS6073 into two model strains followed by phage plaque assays and adsorption assays were performed to assess its effect on phage sensitivity. Phage distal tail proteins were analyzed bioinformatically using HHpred and modeling with AlphaFold. Construction of recombinant phages carrying evolved Dits was performed by supplying a plasmid-encoded template for homologous recombination.
Results: pEPS6073 confers resistance against a subset of skunaviruses via adsorption inhibition. IFF collection skunaviruses that infect strains encoding the 6073-like eps gene cluster carry insertions in their distal tail protein-encoding (dit) genes that result in longer Dit proteins (so-called evolved Dits), which encode carbohydrate-binding domains. Three skunaviruses with classical Dits (no insertion) were unable to fully infect their hosts following the conjugal introduction of pEPS6073, showing reductions in both adsorption and efficiency of plaquing. Cloning the evolved Dit into these phages enabled full infectivity on their host strains, both wild type and transconjugant carrying pEPS6073, with recombinant phages adsorbing slightly better to the EPS+ host than wild type.
Conclusion: The 6073-like EPS potentially occludes the phage receptor for skunaviruses that encode a classical Dit protein. Skunaviruses that infect strains encoding the 6073-like EPS harbor evolved Dits, which likely help promote phage adsorption rather than just allow the phage to circumvent the putative EPS barrier. This work furthers our knowledge of phage-host interactions in Lactococcus and proposes a role for insertions in the Dit proteins of a subset of skunaviruses.
Aim: Comparative metagenomic analysis requires measuring a pairwise similarity between metagenomes in the dataset. Reference-based methods that compute a beta-diversity distance between two metagenomes are highly dependent on the quality and completeness of the reference database, and their application on less studied microbiota can be challenging. On the other hand, de-novo comparative metagenomic methods only rely on the sequence composition of metagenomes to compare datasets. While each one of these approaches has its strengths and limitations, their comparison is currently limited.
Methods: We developed sets of simulated short-reads metagenomes to (1) compare k-mer-based and taxonomy-based distances and evaluate the impact of technical and biological variables on these metrics and (2) evaluate the effect of k-mer sketching and filtering. We used a real-world metagenomic dataset to provide an overview of the currently available tools for de novo metagenomic comparative analysis.
Results: Using simulated metagenomes of known composition and controlled error rate, we showed that k-mer-based distance metrics were well correlated to the taxonomic distance metric for quantitative Beta-diversity metrics, but the correlation was low for presence/absence distances. The community complexity in terms of taxa richness and the sequencing depth significantly affected the quality of the k-mer-based distances, while the impact of low amounts of sequence contamination and sequencing error was limited. Finally, we benchmarked currently available de-novo comparative metagenomic tools and compared their output on two datasets of fecal metagenomes and showed that most k-mer-based tools were able to recapitulate the data structure observed using taxonomic approaches.
Conclusion: This study expands our understanding of the strength and limitations of k-mer-based de novo comparative metagenomic approaches and aims to provide concrete guidelines for researchers interested in applying these approaches to their metagenomic datasets.
Background: The peptide MS2-L represents toxins of the ssRNA Leviviridae phage family and consists of a predicted N-terminal soluble domain followed by a transmembrane domain. MS2-L mediates bacterial cell lysis through the formation of large lesions in the cell envelope, but further details of this mechanism as a prerequisite for applied bioengineering studies are lacking. The chaperone DnaJ is proposed to modulate MS2-L activity, whereas other cellular targets of MS2-L are unknown.
Methods: Here, we provide a combined in vitro and in vivo overexpression approach to reveal molecular insights into MS2-L action and its interaction with DnaJ. Full-length MS2-L and truncated derivatives were synthesized cell-free and co-translationally inserted into nanodiscs or solubilized in detergent micelles. By native liquid bead ion desorption mass spectrometry, we demonstrate that MS2-L assembles into high oligomeric states after membrane insertion.
Results: Oligomerization is directed by the transmembrane domain and is impaired in detergent environments. Studies with truncated MS2-L derivatives provide evidence that the soluble domain acts as a modulator of oligomer formation. DnaJ strongly interacts with MS2-L in membranes as well as in detergent environments. However, this interaction affects neither the MS2-L membrane insertion efficiency nor its oligomerization in nanodisc membranes. In accordance with the in vitro data, the assembly of MS2-L derivatives into large membrane located clusters was monitored by overexpression of corresponding fusions with fluorescent monitors in E. coli cells. Analysis by cryo-electron microscopy indicates that lesion formation is initiated in the outer membrane, followed by disruption of the peptidoglycan layer and disintegration of the inner membrane.
Conclusion: MS2-L forms oligomeric complexes similar to the related phage toxin ΦX174-E. The oligomeric interface of both peptides is located within their transmembrane domains. We propose a potential function of the higher-order assembly of small phage toxins in membrane disintegration and cell lysis.
Background: The microbiota acquired at birth is known to play an intimate role in later life health and disease and has been shown to be affected by the mode of birth. There has been recent interest in microbiota correction by maternal vaginal seeding in Cesarean section-born infants; however, the safety of this practice has been debated. The aim of this study was to assess how other factors, such as timing of sampling, maternal obesity, vaginal Group B Streptococcus colonization (GBS), and antibiotic exposure, affect the maternal and infant microbiota.
Methods: Maternal vaginal and saliva samples were collected at three time periods: 35-37 weeks gestation (prenatal), within 24-36 hours after birth (birth), and at ~6 weeks postpartum. Infant saliva and stool samples were collected at ~6 weeks postpartum. 16S rRNA amplicon sequencing was utilized to assess the taxonomic and inferred functional compositions of the bacterial communities from both mothers and infants.
Results: Samples from 36 mothers and 32 infants were obtained. Gestational age, breastfeeding, mode of birth, and gravidity were associated with taxonomic alterations in the infant samples, while obesity, antibiotic use, and GBS status were not. Maternal samples were predominantly affected by time, whereby significant alterations including increased microbial diversity were seen at birth and persisted to 6 weeks postpartum.
Conclusion: This study provides information on the relationship between health and delivery factors and changes in vaginal and infant microbiota. These results may better direct clinicians and mothers in optimizing the infant microbiota towards health during infancy and later life.
Background: American foulbrood (AFB) is a devastating disease of the European honey bee (Apis mellifera) and is found throughout the world. AFB is caused by the bacterium Paenibacillus larvae (P. larvae). Treatment with antibiotics is strictly forbidden in many regions, including New Zealand. Safe and natural prophylactic solutions to protect honey bees from AFB are needed. Bacteriophages are a well-studied alternative to antibiotics and have been shown to be effective against P. larvae in other countries.
Methods: We employed a community science approach to obtaining samples from around New Zealand to discover novel bacteriophages. Standard isolation approaches were employed for both bacteria and bacteriophages. Host range testing was performed by agar overlay spot tests, and cocktail formulation and in vitro testing were performed in 96-well plate assays, followed by sub-sampling and CFU visualization on agar plates.
Results: Herein, we describe the discovery and isolation of eight P. larvae bacterial isolates and 26 P. larvae bacteriophages that are novel and native to New Zealand. The phage genomes were sequenced and annotated, and their genomes were compared to extant sequenced P. larvae phage genomes. We test the host ranges of the bacteriophages and formulate cocktails to undertake in vitro testing on a set of representative bacterial strains. These results form the basis of a promising solution for protecting honey bees in New Zealand from AFB.
Aim: Bifidobacteria benefit host health and homeostasis by breaking down diet- and host-derived carbohydrates to produce organic acids in the intestine. However, the sugar utilization preference of bifidobacterial species is poorly understood. Thus, this study aimed to investigate the sugar utilization preference (i.e., glucose or lactose) of various bifidobacterial species.
Methods: Strains belonging to 40 bifidobacterial species/subspecies were cultured on a modified MRS medium supplemented with glucose and/or lactose, and their preferential sugar utilization was assessed using high-performance thin-layer chromatography. Comparative genomic analysis was conducted with a focus on genes involved in lactose and glucose uptake and genes encoding for carbohydrate-active enzymes.
Results: Strains that preferentially utilized glucose or lactose were identified. Almost all the lactose-preferring strains harbored the lactose symporter lacS gene. However, the comparative genomic analysis could not explain all their differences in sugar utilization preference. Analysis based on isolate source revealed that all 10 strains isolated from humans preferentially utilized lactose, whereas all four strains isolated from insects preferentially utilized glucose. In addition, bifidobacterial species isolated from hosts whose milk contained higher lactose amounts preferentially utilized lactose. Lactose was also detected in the feces of human infants, suggesting that lactose serves as a carbon source not only for infants but also for gut microbes in vivo.
Conclusion: The different sugar preference phenotypes of Bifidobacterium species may be ascribed to the residential environment affected by the dietary habits of their host. This study is the first to systematically evaluate the sugar uptake preference of various bifidobacterial species.
The microbiota-gut-brain axis refers to the intricate bidirectional communication between commensal microorganisms residing in the digestive tract and the central nervous system, along neuroendocrine, metabolic, immune, and inflammatory pathways. This axis has been suggested to play a role in several neurological disorders, such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, and epilepsy, paving the way for microbiome-based intervention strategies for the mitigation and treatment of symptoms. Epilepsy is a multifaceted neurological condition affecting more than 50 million individuals worldwide, 30% of whom do not respond to conventional pharmacological therapies. Among the first-hand microbiota modulation strategies, nutritional interventions represent an easily applicable option in both clinical and home settings. In this narrative review, we summarize the mechanisms underlying the microbiota-gut-brain axis involvement in epilepsy, discuss the impact of antiepileptic drugs on the gut microbiome, and then the impact of a particular dietary pattern, the ketogenic diet, on the microbiota-gut-brain axis in epileptic patients. The investigation of the microbiota response to non-pharmacological therapies is an ever-expanding field with the potential to allow the design of increasingly accessible and successful intervention strategies.
Background: Lytic bacteriophages infect and lyse bacteria and, as a by-product, may affect diversity in microbial communities through selective predation on abundant bacterial strains. We used a complex dairy starter named Ur to investigate population dynamics of Lactococcus lactis, Lactococcus cremoris and Leuconostoc mesenteroides strains in terms of constant-diversity and periodic selection models.
Methods: To mimic the starter Ur, we designed blends of 24 strains representing all eight previously identified genetic lineages in the starter culture. The blends were propagated by daily transfers in milk for over 500 generations in the presence or absence of a cocktail of lytic bacteriophages. The relative abundance of genetic lineages of L. lactis, L. cremoris and Lc. mesenteroides strains present in the complex blend, as well as phage presence, were monitored.
Results: Control blends without phage predation showed decreased strain diversity, leading to a stable state due to the domination of the fittest strain(s) of a particular lineage according to periodic selection dynamics. However, in phage-challenged blends, predation caused a large shift in the microbial composition by killing the fittest and sensitive strains.
Conclusion: It was demonstrated that phage-challenged blends maintained their diversity at the level of genetic lineages, thus providing experimental support for the constant-diversity dynamics model in a complex microbial community.
Aim: Temperate phages are known to heavily impact the growth of their host, be it in a positive way, e.g., when beneficial genes are provided by the phage, or negatively when lysis occurs after prophage induction. This study provides an in-depth look into the distribution and variety of prophages in Latilactobacillus curvatus (L. curvatus). This species is found in a wide variety of ecological niches and is routinely used as a meat starter culture.
Methods: Fourty five L. curvatus genomes were screened for prophages. The intact predicted prophages and their chromosomal integration loci were described. Six L. curvatus lysogens were analysed for phage-mediated lysis post induction via UV light and/or mitomycin C. Their lysates were analysed for phage particles via viral DNA sequencing and transmission electron microscopy.
Results: Two hundred and six prophage sequences of any completeness were detected within L. curvatus genomes. The 50 as intact predicted prophages show high levels of genetic diversity on an intraspecies level with conserved regions mostly in the replication and head/tail gene clusters. Twelve chromosomal loci, mostly tRNA genes, were identified, where intact L. curvatus phages were integrated. The six analysed L. curvatus lysogens showed strain-dependent lysis in various degrees after induction, yet only four of their lysates appeared to contain fully assembled virions with the siphovirus morphotype.
Conclusion: Our data demonstrate that L. curvatus is a (pro)phage-susceptible species, harbouring multiple intact prophages and remnant sequences thereof. This knowledge provides a basis to study phage-host interaction influencing microbial communities in food fermentations.
Inflammatory bowel disease (IBD) is a complex heterogeneous disorder defined by recurring chronic inflammation of the gastrointestinal tract, attributed to a combination of factors including genetic susceptibility, altered immune response, a shift in microbial composition/microbial insults (infection/exposure), and environmental influences. Therapeutics generally used to treat IBD mainly focus on the immune response and include non-specific
Bifidobacterium species are integral members of the human gut microbiota and these microbes have significant interactions with the intestinal mucus layer. This review delves into Bifidobacterium-mucus dynamics, shedding light on the multifaceted nature of this relationship. We cover conserved features of Bifidobacterium-mucus interactions, such as mucus adhesion and positive regulation of goblet cell and mucus production, as well as species and strain-specific attributes of mucus degradation. For each interface, we explore the molecular mechanisms underlying these interactions and their potential implications for human health. Notably, we emphasize the ability of Bifidobacterium species to positively influence the mucus layer, shedding light on its potential as a mucin-builder and a therapeutic agent for diseases associated with disrupted mucus barriers. By elucidating the complex interplay between Bifidobacterium and intestinal mucus, we aim to contribute to a deeper understanding of the gut microbiota-host interface and pave the way for novel therapeutic strategies.