Marion Dalmasso

Exploiting gut bacteriophages for human health

The human gut contains approximately 10(15) bacteriophages (the phageome), probably the richest concentration of biological entities on earth. Mining and exploiting these potential agents of change is an attractive prospect. For many years, phages have been used to treat bacterial infections in humans and more recently have been approved to reduce pathogens in the food chain. Phages have also been studied as drug or vaccine delivery vectors to help treat and prevent diseases such as cancer and chronic neurodegenerative conditions. Individual phageomes vary depending on age and health, thus providing a useful biomarker of human health as well as suggesting potential interventions targeted at the gut microbiota.

Three New Escherichia coli Phages from the Human Gut Show Promising Potential for Phage Therapy

With the emergence of multi-drug resistant bacteria the use of bacteriophages (phages) is gaining renewed interest as promising anti-microbial agents. The aim of this study was to isolate and characterize phages from human fecal samples. Three new coliphages, ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03, were isolated. Their phenotypic and genomic characteristics, and lytic activity against biofilm, and in combination with ciprofloxacin, were investigated. All three phages reduced the growth of E. coli strain DPC6051 at multiplicity of infection (MOI) between 10−3 and 105. A cocktail of all three phages completely inhibited the growth of E. coli. The phage cocktail also reduced biofilm formation and prevented the emergence of phage-resistant mutants which occurred with single phage. When combined with ciprofloxacin, phage alone or in cocktail inhibited the growth of E. coli and prevented the emergence of resistant mutants. These three new phages are promising biocontrol agents for E. coli infections.

Isolation of a Novel Phage with Activity against Streptococcus mutans Biofilms.

Streptococcus mutans is one of the principal agents of caries formation mainly, because of its ability to form biofilms at the tooth surface. Bacteriophages (phages) are promising antimicrobial agents that could be used to prevent or treat caries formation by S. mutans. The aim of this study was to isolate new S. mutans phages and to characterize their antimicrobial properties. A new phage, ɸAPCM01, was isolated from a human saliva sample. Its genome was closely related to the only two other available S. mutans phage genomes, M102 and M102AD. ɸAPCM01 inhibited the growth of S. mutans strain DPC6143 within hours in broth and in artificial saliva at multiplicity of infections as low as 2.5x10-5. In the presence of phage ɸAPCM01 the metabolic activity of a S. mutans biofilm was reduced after 24 h of contact and did not increased again after 48 h, and the live cells in the biofilm decreased by at least 5 log cfu/ml. Despite its narrow host range, this newly isolated S. mutans phage exhibits promising antimicrobial properties.

Microorganisms in Fermented Apple Beverages: Current Knowledge and Future Directions

Production of fermented apple beverages is spread all around the world with specificities in each country. ‘French ciders’ refer to fermented apple juice mainly produced in the northwest of France and often associated with short periods of consumption. Research articles on this kind of product are scarce compared to wine, especially on phenomena associated with microbial activities. The wine fermentation microbiome and its dynamics, organoleptic improvement for healthy and pleasant products and development of starters are now widely studied. Even if both beverages seem close in terms of microbiome and process (with both alcoholic and malolactic fermentations), the inherent properties of the raw materials and different production and environmental parameters make research on the specificities of apple fermentation beverages worthwhile. This review summarizes current knowledge on the cider microbial ecosystem, associated activities and the influence of process parameters. In addition, available data on cider quality and safety is reviewed. Finally, we focus on the future role of lactic acid bacteria and yeasts in the development of even better or new beverages made from apples.

Viromes of one year old infants reveal the impact of birth mode on microbiome diversity

Establishing a diverse gut microbiota after birth is being increasingly recognised as important for preventing illnesses later in life. It is well established that bacterial diversity rapidly increases post-partum; however, few studies have examined the infant gut virome/phageome during this developmental period. We performed a metagenomic analysis of 20 infant faecal viromes at one year of age to determine whether spontaneous vaginal delivery (SVD) or caesarean section (CS) influenced viral composition. We find that birth mode results in distinctly different viral communities, with SVD infants having greater viral and bacteriophage diversity. We demonstrate that CrAssphage is acquired early in life, both in this cohort and two others, although no difference in birth mode is detected. A previous study has shown that bacterial OTU’s (operational taxonomic units) identified in the same infants could not discriminate between birth mode at 12 months of age. Therefore, our results indicate that vertical transmission of viral communities from mother to child may play a role in shaping the early life microbiome, and that birth mode should be considered when studying the early life gut virome.

Reproducible protocols for metagenomic analysis of human faecal phageomes

BackgroundRecent studies have demonstrated that the human gut is populated by complex, highly individual and stable communities of viruses, the majority of which are bacteriophages. While disease-specific alterations in the gut phageome have been observed in IBD, AIDS and acute malnutrition, the human gut phageome remains poorly characterised. One important obstacle in metagenomic studies of the human gut phageome is a high level of discrepancy between results obtained by different research groups. This is often due to the use of different protocols for enriching virus-like particles, nucleic acid purification and sequencing.The goal of the present study is to develop a relatively simple, reproducible and cost-efficient protocol for the extraction of viral nucleic acids from human faecal samples, suitable for high-throughput studies. We also analyse the effect of certain potential confounding factors, such as storage conditions, repeated freeze-thaw cycles, and operator bias on the resultant phageome profile. Additionally, spiking of faecal samples with an exogenous phage standard was employed to quantitatively analyse phageomes following metagenomic sequencing. Comparative analysis of phageome profiles to bacteriome profiles was also performed following 16S rRNA amplicon sequencing.ResultsFaecal phageome profiles exhibit an overall greater individual specificity when compared to bacteriome profiles. The phageome and bacteriome both exhibited moderate change when stored at +u20094xa0°C or room temperature. Phageome profiles were less impacted by multiple freeze-thaw cycles than bacteriome profiles, but there was a greater chance for operator effect in phageome processing. The successful spiking of faecal samples with exogenous bacteriophage demonstrated large variations in the total viral load between individual samples.ConclusionsThe faecal phageome sequencing protocol developed in this study provides a valuable additional view of the human gut microbiota that is complementary to 16S amplicon sequencing and/or metagenomic sequencing of total faecal DNA. The protocol was optimised for several confounding factors that are encountered while processing faecal samples, to reduce discrepancies observed within and between research groups studying the human gut phageome. Rapid storage, limited freeze-thaw cycling and spiking of faecal samples with an exogenous phage standard are recommended for optimum results.

PFGE as a tool to track Listeria monocytogenes in food processing facilities: case studies.

The use of PFGE to track Listeria monocytogenes strains from the food processing environment to the food is explained in this chapter through two case studies. This illustrates the usefulness of this method to identify putative routes of contamination and persistent strains and to help in the implementation of corrective actions in food processing facilities to control the occurrence of this pathogenic bacterium.

A long and abundant non-coding RNA in Lactobacillus salivarius

Lactobacillus salivarius, found in the intestinal microbiota of humans and animals, is studied as an example of the sub-dominant intestinal commensals that may impart benefits upon their host. Strains typically harbour at least one megaplasmid that encodes functions contributing to contingency metabolism and environmental adaptation. RNA sequencing (RNA-seq)transcriptomic analysis of L. salivarius strain UCC118 identified the presence of a novel unusually abundant long non-coding RNA (lncRNA) encoded by the megaplasmid, and which represented more than 75u200a% of the total RNA-seq reads after depletion of rRNA species. The expression level of this 520 nt lncRNA in L. salivarius UCC118 exceeded that of the 16S rRNA, it accumulated during growth, was very stable over time and was also expressed during intestinal transit in a mouse. This lncRNA sequence is specific to the L. salivarius species; however, among 45 L . salivarius genomes analysed, not all (only 34) harboured the sequence for the lncRNA. This lncRNA was produced in 27 tested L. salivarius strains, but at strain-specific expression levels. High-level lncRNA expression correlated with high megaplasmid copy number. Transcriptome analysis of a deletion mutant lacking this lncRNA identified altered expression levels of genes in a number of pathways, but a definitive function of this new lncRNA was not identified. This lncRNA presents distinctive and unique properties, and suggests potential basic and applied scientific developments of this phenomenon.

Pulsed-Field Gel Electrophoresis for Leuconostoc mesenteroides and L. pseudomesenteroides

Pulsed-field gel electrophoresis (PFGE) is a technique using alternating electric fields to help the separation of high molecular weight DNA fragments with a high resolution. This method consists of the digestion of bacterial chromosomal DNA with rare-cutting restriction enzymes and of applying an alternating electrical current between spatially distinct pairs of electrodes. DNA molecules migrate at different speeds according to the size of the fragment. This method is considered as the gold standard for genotyping, genetic fingerprinting, epidemiological studies, genome size estimation, and studying radiation-induced DNA damage and repair.

Pulsed field gel electrophoresis for dairy propionibacteria.

Pulsed field gel electrophoresis (PFGE) is a technique using alternating electric fields to migrate high molecular weight DNA fragments with a high resolution. This method consists of the digestion of bacterial chromosomal DNA with rare-cutting restriction enzymes and in applying an alternating electrical current between spatially distinct pairs of electrodes. DNA molecules migrate at different speeds according to the size of the fragments. Among other things, this technique is considered as the gold standard for genotyping, genetic fingerprinting, epidemiological studies, genome size estimation, and studying radiation-induced DNA damage and repair. This chapter describes a PFGE method that can be used to differentiate dairy propionibacteria.