Molecular and evolutionary basis of O-antigenic polysaccharide driven phage sensitivity in environmental pseudomonads

Pseudomonas protegens CHA0, a bacterial strain able to suppress plant pathogens as well as efficiently kill lepidopteran pest insects, has been studied as biocontrol agent to prevent ensuing agricultural damage. However, the success of this method is dependent on the efficient plant colonization by the bacterial inoculant while it faces competition from the resident microbiota as well as predators such as bacteriophages. One of these naturally occurring phages, ΦGP100, was found to drastically reduce the abundance of CHA0 once inoculated into plant microcosms, resulting in the loss of plant protection against a phytopathogen. Here, we investigated the molecular determinants implicated in the interaction between CHA0 and the phage ΦGP100 using a high-density transposon-sequencing approach. We show that lipopolysaccharide cell surface decorations, specifically the longer OBC3-type O-antigenic polysaccharide (O-PS, O-antigen) of the two dominant O-PS of CHA0 is essential for the attachment and infection of ΦGP100. Moreover, when exploring the distribution of the OBC3 cluster in bacterial genomes, we identified several parts of this gene cluster that are conserved in phylogenetically distant bacteria. Through heterologous complementation, we integrated an OBC3-type gene copy from a phylogenetically distant bacterium and were able to restore the phage sensitivity of a CHA0 mutant which lacked the ability to form long O-PS. Finally, we evidence that the OBC3 gene cluster of CHA0 displays a high genomic plasticity and likely underwent several horizontal acquisitions and genomic rearrangements. Collectively, this study underlines the complexity of phage-bacteria interaction and the multifunctional aspect of bacterial cell surface decorations

Enrichment, Sequencing, and Identification of DNA Bacteriophages from Fecal Samples

Research on individual viruses and phages, as well as viral populations (viromes), is greatly expanding. Phages and viromes are increasingly suspected to have numerous impacts on the ecosystem in which they reside by interacting directly or indirectly with the other organisms present in their environment. In particular, phage communities of the gut microbiota have been associated with a wide range of diseases. However, properly investigating intestinal viromes is still very challenging, both experimentally and analytically. This chapter proposes a simple and reproducible protocol to separate and enrich DNA phage particles from fecal samples, to sequence them, and finally obtain a basic but robust bioinformatic characterization and classification of the global bacteriophage community

High Prevalence and Genetic Diversity of Large phiCD211 (phiCDIF1296T)-Like Prophages in Clostridioides difficile.

We thank Christiane Bouchier and Laurence Ma from the Genomics Platform and the Center of Informatics for Biology (Institut Pasteur).International audienceClostridioides difficile (formerly Clostridium difficile) is a pathogenic bacterium displaying great genetic diversity. A significant proportion of this diversity is due to the presence of integrated prophages. Here, we provide an in-depth analysis of phiCD211, also known as phiCDIF1296T, the largest phage identified in C. difficile so far, with a genome of 131 kbp. It shares morphological and genomic similarity with other large siphophages, like phage 949, infecting Lactococcus lactis, and phage c-st, infecting Clostridium botulinum A PhageTerm analysis indicated the presence of 378-bp direct terminal repeats at the phiCD211 genome termini. Among striking features of phiCD211, the presence of several transposase and integrase genes suggests past recombination events with other mobile genetic elements. Several gene products potentially influence the bacterial lifestyle and fitness, including a putative AcrB/AcrD/AcrF multidrug resistance protein, an EzrA septation ring formation regulator, and a spore protease. We also identified a CRISPR locus and a cas3 gene. We screened 2,584 C. difficile genomes available and detected 149 prophages sharing ≥80% nucleotide identity with phiCD211 (5% prevalence). Overall, phiCD211-like phages were detected in C. difficile strains corresponding to 21 different multilocus sequence type groups, showing their high prevalence. Comparative genomic analyses revealed the existence of several clusters of highly similar phiCD211-like phages. Of note, large chromosome inversions were observed in some members, as well as multiple gene insertions and module exchanges. This highlights the great plasticity and gene coding potential of the phiCD211/phiCDIF1296T genome. Our analyses also suggest active evolution involving recombination with other mobile genetic elements.IMPORTANCEClostridioides difficile is a clinically important pathogen representing a serious threat to human health. Our hypothesis is that genetic differences between strains caused by the presence of integrated prophages could explain the apparent differences observed in the virulence of different C. difficile strains. In this study, we provide a full characterization of phiCD211, also known as phiCDIF1296T, the largest phage known to infect C. difficile so far. Screening 2,584 C. difficile genomes revealed the presence of highly similar phiCD211-like phages in 5% of the strains analyzed, showing their high prevalence. Multiple-genome comparisons suggest that evolution of the phiCD211-like phage community is dynamic, and some members have acquired genes that could influence bacterial biology and fitness. Our study further supports the relevance of studying phages in C. difficile to better understand the epidemiology of this clinically important human pathogen

Gut microbiomes of agropastoral children from the Adadle region of Ethiopia reflect their unique dietary habits

The composition and function of the intestinal microbiota are major determinants of human health and are strongly influenced by diet, antibiotic treatment, lifestyle and geography. Nevertheless, we currently have only little data on microbiomes of non-westernized communities. We assess the stool microbiota composition in 59 children aged 2–5 years from the Adadle district of Ethiopia, Somali Regional State. Here, milk and starch-rich food are predominant components of the local diet, where the inhabitants live a remote, traditional agropastoral lifestyle. Microbiota composition, function and the resistome were characterized by both 16S rRNA gene amplicon and shotgun metagenomic sequencing and compared to 1471 publicly available datasets from children living in traditional, transitional, and industrial communities with different subsistence strategies. Samples from the Adadle district are low in Bacteroidaceae, and Prevotellaceae, the main bacterial representatives in the feces of children living in industrialized and non-industrialized communities, respectively. In contrast, they had a higher relative abundance in Streptococcaceae, Bifidobacteriaceae and Erysipelatoclostridiaceae. Further, genes involved in degradation pathways of lactose, d-galactose and simple carbohydrates were enriched. Overall, our study revealed a unique composition of the fecal microbiota of these agropastoral children, highlighting the need to further characterize the fecal bacterial composition of human populations living different lifestyles.ISSN:2045-232

Chromosome folding and prophage activation reveal specific genomic architecture for intestinal bacteria

International audienceBackground Bacteria and their viruses, bacteriophages, are the most abundant entities of the gut microbiota, a complex community of microorganisms associated with human health and disease. In this ecosystem, the interactions between these two key components are still largely unknown. In particular, the impact of the gut environment on bacteria and their associated prophages is yet to be deciphered. Results To gain insight into the activity of lysogenic bacteriophages within the context of their host genomes, we performed proximity ligation-based sequencing (Hi-C) in both in vitro and in vivo conditions on the 12 bacterial strains of the OMM 12 synthetic bacterial community stably associated within mice gut (gnotobiotic mouse line OMM 12 ). High-resolution contact maps of the chromosome 3D organization of the bacterial genomes revealed a wide diversity of architectures, differences between environments, and an overall stability over time in the gut of mice. The DNA contacts pointed at 3D signatures of prophages leading to 16 of them being predicted as functional. We also identified circularization signals and observed different 3D patterns between in vitro and in vivo conditions. Concurrent virome analysis showed that 11 of these prophages produced viral particles and that OMM 12 mice do not carry other intestinal viruses. Conclusions The precise identification by Hi-C of functional and active prophages within bacterial communities will unlock the study of interactions between bacteriophages and bacteria across conditions (healthy vs disease)

Virulence and genomic diversity among clinical isolates of ST1 (BI/NAP1/027) Clostridioides difficile

Summary: Clostridioides difficile produces toxins that damage the colonic epithelium, causing colitis. Variation in disease severity is poorly understood and has been attributed to host factors and virulence differences between C. difficile strains. We test 23 epidemic ST1 C. difficile clinical isolates for their virulence in mice. All isolates encode a complete Tcd pathogenicity locus and achieve similar colonization densities. However, disease severity varies from lethal to avirulent infections. Genomic analysis of avirulent isolates reveals a 69-bp deletion in the cdtR gene, which encodes a response regulator for binary toxin expression. Deleting the 69-bp sequence in virulent R20291 strain renders it avirulent in mice with reduced toxin gene transcription. Our study demonstrates that a natural deletion within cdtR attenuates virulence in the epidemic ST1 C. difficile isolates without reducing colonization and persistence. Distinguishing strains on the basis of cdtR may enhance the specificity of diagnostic tests for C. difficile colitis