In Silico Characterisation of Putative Prophages in Lactobacillaceae Used in Probiotics for Vaginal Health.

While live biotherapeutics offer a promising approach to optimizing vaginal microbiota, the presence of functional prophages within introduced Lactobacillaceae strains could impact their safety and efficacy. We evaluated the presence of prophages in 895 publicly available Lactobacillaceae genomes using Phaster, Phigaro, Phispy, Prophet and Virsorter. Prophages were identified according to stringent (detected by ≥4 methods) or lenient criteria (detected by ≥2 methods), both with >80% reciprocal sequence overlap. The stringent approach identified 448 prophages within 359 genomes, with 40.1% genomes harbouring at least one prophage, while the lenient approach identified 1671 prophages within 83.7% of the genomes. To confirm our in silico estimates in vitro, we tested for inducible prophages in 57 vaginally-derived and commercial Lactobacillaceae isolates and found inducible prophages in 61.4% of the isolates. We characterised the in silico predicted prophages based on weighted gene repertoire relatedness and found that most belonged to the Siphoviridae or Myoviridae families. ResFam and eggNOG identified four potential antimicrobial resistance genes within the predicted prophages. Our results suggest that while Lactobacillaceae prophages seldomly carry clinically concerning genes and thus unlikely a pose a direct risk to human vaginal microbiomes, their high prevalence warrants the characterisation of Lactobacillaceae prophages in live biotherapeutics.

Presence and Persistence of Putative Lytic and Temperate Bacteriophages in Vaginal Metagenomes from South African Adolescents.

The interaction between gut bacterial and viral microbiota is thought to be important in human health. While fluctuations in female genital tract (FGT) bacterial microbiota similarly determine sexual health, little is known about the presence, persistence, and function of vaginal bacteriophages. We conducted shotgun metagenome sequencing of cervicovaginal samples from South African adolescents collected longitudinally, who received no antibiotics. We annotated viral reads and circular bacteriophages, identified CRISPR loci and putative prophages, and assessed their diversity, persistence, and associations with bacterial microbiota composition. Siphoviridae was the most prevalent bacteriophage family, followed by Myoviridae, Podoviridae, Herelleviridae, and Inoviridae. Full-length siphoviruses targeting bacterial vaginosis (BV)-associated bacteria were identified, suggesting their presence in vivo. CRISPR loci and prophage-like elements were common, and genomic analysis suggested higher diversity among Gardnerella than Lactobacillus prophages. We found that some prophages were highly persistent within participants, and identical prophages were present in cervicovaginal secretions of multiple participants, suggesting that prophages, and thus bacterial strains, are shared between adolescents. The number of CRISPR loci and prophages were associated with vaginal microbiota stability and absence of BV. Our analysis suggests that (pro)phages are common in the FGT and vaginal bacteria and (pro)phages may interact.

Evolutionary biology and development model of medicines: A necessary 'pas de deux' for future successful bacteriophage therapy.

The increase in frequency of multidrug-resistant bacteria worldwide is largely the result of the massive use of antibiotics in the second half of the 20th century. These relatively recent changes in human societies revealed the great evolutionary capacities of bacteria towards drug resistance. In this article, we hypothesize that the success of future antibacterial strategies lies in taking into account both these evolutionary processes and the way human activities influence them. Faced with the increasing prevalence of multidrug-resistant bacteria and the scarcity of new antibacterial chemical molecules, the use of bacteriophages is considered as a complementary and/or alternative therapy. After presenting the evolutionary capacities of bacteriophages and bacteria, we show how the development model currently envisaged (based on the classification of bacteriophages as medicinal products similar to antibacterial chemical molecules) ignores the evolutionary processes inherent in bacteriophage therapy. This categorization imposes to bacteriophage therapy a specific conception of what a treatment and a therapeutic scheme should be as well as its mode of production and prescription. We argue that a new development model is needed that would allow the use of therapeutic bacteriophages fully adapted (after in vitro 'bacteriophage training') to the aetiologic bacteria and/or aimed at rendering bacteria either avirulent or antibiotic-susceptible ('bacteriophage steering'). To not repeat the mistakes made with antibiotics, we must now think about and learn from the ways in which the materialities of microbes (e.g. evolutionary capacities of both bacteriophages and bacteria) are intertwined with those of societies.

Characterisation of the Viral Community Associated with the Alfalfa Weevil (Hypera postica) and Its Host Plant, Alfalfa (Medicago sativa).

Advances in viral metagenomics have paved the way of virus discovery by making the exploration of viruses in any ecosystem possible. Applied to agroecosystems, such an approach opens new possibilities to explore how viruses circulate between insects and plants, which may help to optimise their management. It could also lead to identifying novel entomopathogenic viral resources potentially suitable for biocontrol strategies. We sampled the larvae of a natural population of alfalfa weevils (Hypera postica), a major herbivorous pest feeding on legumes, and its host plant alfalfa (Medicago sativa). Insect and plant samples were collected from a crop field and an adjacent meadow. We characterised the diversity and abundance of viruses associated with weevils and alfalfa, and described nine putative new virus species, including four associated with alfalfa and five with weevils. In addition, we found that trophic accumulation may result in a higher diversity of plant viruses in phytophagous pests compared to host plants.

Exploring potential of vaginal Lactobacillus isolates from South African women for enhancing treatment for bacterial vaginosis.

Antibiotics continue to be the standard-of-care for bacterial vaginosis (BV), although recurrence rates are high. Vaginal probiotics may improve durability of BV treatment, although few probiotics for vaginal health contain Lactobacillus spp. that commonly colonize the lower female genital tract. Characteristics of vaginal Lactobacillus strains from South African women were evaluated for their probiotic potential in vitro compared to strains from commercial vaginal products, including growth at varying pHs, ability to lower pH, produce D-/L-lactate and H2O2, influence growth of BV-associated Gardnerella vaginalis and Prevotella bivia, adherence to cervical cells and susceptibility to antibiotics. Fifty-seven Lactobacillus strains were purified from cervico-vaginal fluid, including L. crispatus, L. jensenii, L. gasseri, L. mucosae, and L. vaginalis. L crispatus strains grew better at pHs below 4.5 and lowered pH more effectively than other strains. Production of D-/L-lactate and H2O2 varied between Lactobacillus species and strains. Lactobacillus strains generally inhibited P. bivia more uniformly than G. vaginalis isolates. All vaginal Lactobacillus isolates were resistant to metronidazole while susceptibility to clindamycin varied. Furthermore, vaginal Lactobacillus strains tended to be broadly susceptible to penicillin, amoxicillin, rifampicin and rifabutin. Whole-genome-sequencing of five of the best-performing vaginal Lactobacillus strains confirmed their likely safety, due to antimicrobial resistance elements being largely absent, while putative intact prophages were present in the genomes of two of the five strains. Overall, vaginal Lactobacillus strains largely performed better in these in vitro assays than probiotic strains currently used in probiotics for vaginal health. Including the best-performing vaginal Lactobacillus isolates in a region-specific probiotic for vaginal health may result in improved BV treatment options.

Inflammatory and antimicrobial properties differ between vaginal Lactobacillus isolates from South African women with non-optimal versus optimal microbiota.

Female genital tract (FGT) inflammation increases HIV infection susceptibility. Non-optimal cervicovaginal microbiota, characterized by depletion of Lactobacillus species and increased bacterial diversity, is associated with increased FGT cytokine production. Lactobacillus species may protect against HIV partly by reducing FGT inflammation. We isolated 80 lactobacilli from South African women with non-optimal (Nugent 4-10; n = 18) and optimal microbiota (Nugent 0-3; n = 14). Cytokine production by vaginal epithelial cells in response to lactobacilli in the presence and absence of Gardnerella vaginalis was measured using Luminex. Adhesion to vaginal epithelial cells, pH, D/L-lactate production and lactate dehydrogenase relative abundance were assessed. Lactobacilli from women with non-optimal produced less lactic acid and induced greater inflammatory cytokine production than those from women with optimal microbiota, with IL-6, IL-8, IL-1α, IL-1ß and MIP-1α/ß production significantly elevated. Overall, lactobacilli suppressed IL-6 (adjusted p < 0.001) and IL-8 (adjusted p = 0.0170) responses to G. vaginalis. Cytokine responses to the lactobacilli were inversely associated with lactobacilli adhesion to epithelial cells and D-lactate dehydrogenase relative abundance. Thus, while cervicovaginal lactobacilli reduced the production of the majority of inflammatory cytokines in response to G. vaginalis, isolates from women with non-optimal microbiota were more inflammatory and produced less lactic acid than isolates from women with optimal microbiota.

[Antibacterial applications of bacteriophages]. / Les applications antibactériennes des bactériophages.

In the 1917 article in which Félix d'Hérelle describes his first observations and proposes the name of bacteriophage, he also reports the first use of these viruses to treat bacterial infections, thus giving birth to phage therapy. Soon after antibiotics supplanted bacteriophages. Today, bacteria resistant to multiple antibiotics become a growing public health issue worldwide. This situation has revived research aiming at developing the antibacterial activity of bacteriophages to treat patients as well as diseases in animals and plants. In fact, the areas of applications of bacteriophages as antibacterial are widening as current solutions of chemical nature are questioned. This review summarizes the basic principles of therapeutic applications of bacteriophages and presents recent data in areas where commercial exploitation is occurring or about to emerge.

[A century of research on bacteriophages]. / Un siècle de recherche sur les bactériophages.

Bacteriophages have a prominent place in the living world. They participate to our understanding of the living world through three main aspects : (i) the dissection of the most intimist aspects of viral infection molecular mechanisms (molecular biology), (ii) the description and functioning mechanisms of ecosystems (ecology), and (iii) the adaptive dynamics of integrated viral and host-cell populations (evolution). This review looks back at the genesis of these fundamental findings and draws a picture of the most active fields of current research.

Symptom evolution following the emergence of maize streak virus.

For pathogens infecting single host species evolutionary trade-offs have previously been demonstrated between pathogen-induced mortality rates and transmission rates. It remains unclear, however, how such trade-offs impact sub-lethal pathogen-inflicted damage, and whether these trade-offs even occur in broad host-range pathogens. Here, we examine changes over the past 110 years in symptoms induced in maize by the broad host-range pathogen, maize streak virus (MSV). Specifically, we use the quantified symptom intensities of cloned MSV isolates in differentially resistant maize genotypes to phylogenetically infer ancestral symptom intensities and check for phylogenetic signal associated with these symptom intensities. We show that whereas symptoms reflecting harm to the host have remained constant or decreased, there has been an increase in how extensively MSV colonizes the cells upon which transmission vectors feed. This demonstrates an evolutionary trade-off between amounts of pathogen-inflicted harm and how effectively viruses position themselves within plants to enable onward transmission.

Natural history, dynamics, and ecology of human papillomaviruses in genital infections of young women: protocol of the PAPCLEAR cohort study.

INTRODUCTION: Human papillomaviruses (HPVs) are responsible for one-third of all cancers caused by infections. Most HPV studies focus on chronic infections and cancers, and we know little about the early stages of the infection. Our main objective is to better understand the course and natural history of cervical HPV infections in healthy, unvaccinated and vaccinated, young women, by characterising the dynamics of various infection-related populations (virus, epithelial cells, vaginal microbiota and immune effectors). Another objective is to analyse HPV diversity within hosts, and in the study population, in relation to co-factors (lifestyle characteristics, vaccination status, vaginal microbiota, human genetics). METHODS AND ANALYSIS: The PAPCLEAR study is a single center longitudinal study following 150 women, aged 18-25 years, for up to 2 years. Visits occur every 2 or 4 months (depending on HPV status) during which several variables are measured, such as behaviours (via questionnaires), vaginal pH, HPV presence and viral load (via qPCR), local concentrations of cytokines (via MesoScale Discovery technology) and immune cells (via flow cytometry). Additional analyses are outsourced, such as titration of circulating anti-HPV antibodies, vaginal microbiota sequencing (16S and ITS1 loci) and human genotyping. To increase the statistical power of the epidemiological arm of the study, an additional 150 women are screened cross-sectionally. Finally, to maximise the resolution of the time series, participants are asked to perform weekly self-samples at home. Statistical analyses will involve classical tools in epidemiology, genomics and virus kinetics, and will be performed or coordinated by the Centre National de la Recherche Scientifique (CNRS) in Montpellier. ETHICS AND DISSEMINATION: This study has been approved by the Comité de Protection des Personnes Sud Méditerranée I (reference number 2016-A00712-49); by the Comité Consultatif sur le Traitement de l'Information en matière de Recherche dans le domaine de la Santé (reference number 16.504); by the Commission Nationale Informatique et Libertés (reference number MMS/ABD/AR1612278, decision number DR-2016-488) and by the Agence Nationale de Sécurité du Médicament et des Produits de Santé (reference 20160072000007). Results will be published in preprint servers, peer-reviewed journals and disseminated through conferences. TRIAL REGISTRATION NUMBER: NCT02946346; Pre-results.

"French Phage Network" Annual Conference 2018-Fourth Meeting Report.

The present meeting report aims to cover the scientific activities of the 4th French Bacteriophage Network (Phages.fr) symposium which took place during 24th-25th September 2018, at the Agora du Haut-Carré in Talence (France). The hosting institute was University Bordeaux and 72 participants attended the meeting from both public and private sectors, coming from France, Belgium, Ireland, Germany, Portugal and Canada. The scientific program was structured in three themed oral sessions entitled "ecology and evolution", "bacteriophage-host molecular interaction", and "therapy and biotechnology applications" consisting of 21 oral presentations, including three keynote lectures, and a presentation of the activities of the Spanish bacteriophage network. A poster session included 22 presentations.

A New Prevalent Densovirus Discovered in Acari. Insight from Metagenomics in Viral Communities Associated with Two-Spotted Mite (Tetranychus urticae) Populations.

Viral metagenomics and high throughput sequence mining have revealed unexpected diversity, and the potential presence, of parvoviruses in animals from all phyla. Among arthropods, this diversity highlights the poor knowledge that we have regarding the evolutionary history of densoviruses. The aim of this study was to explore densovirus diversity in a small arthropod pest belonging to Acari, the two-spotted spider mite Tetranychus urticae, while using viral metagenomics based on virus-enrichment. Here, we present the viromes obtained from T. urticae laboratory populations made of contigs that are attributed to nine new potential viral species, including the complete sequence of a novel densovirus. The genome of this densovirus has an ambisens genomic organization and an unusually compact size with particularly small non-structural proteins and a predicted major capsid protein that lacks the typical PLA2 motif that is common to all ambidensoviruses described so far. In addition, we showed that this new densovirus had a wide prevalence across populations of mite species tested and a genomic diversity that likely correlates with the host phylogeny. In particular, we observed a low densovirus genomic diversity between the laboratory and natural populations, which suggests that virus within-species evolution is probably slower than initially thought. Lastly, we showed that this novel densovirus can be inoculated to the host plant following feeding by infected mites, and circulate through the plant vascular system. These findings offer new insights into densovirus prevalence, evolution, and ecology.

Antimicrobial and inflammatory properties of South African clinical Lactobacillus isolates and vaginal probiotics.

Bacterial vaginosis (BV) causes genital inflammation and increased HIV acquisition risk. The standard-of-care for BV, antibiotic therapy, is associated with high recurrence rates. Probiotics may improve treatment outcomes, although substantial heterogeneity in efficacy has been observed during clinical trials. To evaluate the potential to improve existing probiotics, we compared the inflammatory and antimicrobial (adhesion, H2O2, D-lactate and L-lactate production) characteristics of 23 vaginal Lactobacillus isolates from South African women, commercial vaginal probiotics (L. casei rhamnosus, L. acidophilus) and 4 reference strains. All lactobacilli induced inflammatory cytokine production by genital epithelial cells and produced D-lactate. Of six isolates assessed, five suppressed inflammatory responses to Gardnerella vaginalis. Although the L. acidophilus probiotic was the most adherent, many clinical isolates produced greater amounts of H2O2, D-lactate and L-lactate than the probiotics. The most L-lactate and H2O2 were produced by L. jensenii (adjusted p = 0.0091) and L. mucosae (adjusted p = 0.0308) species, respectively. According to the characteristics evaluated, the top 10 isolates included 4 L. jensenii, 2 L. crispatus, 1 L. mucosae, 1 L. vaginalis and the L. acidophilus probiotic. There is potential to develop an improved vaginal probiotic using clinical Lactobacillus isolates. Inflammatory profiles are critical to evaluate as some isolates induced substantial cytokine production.

Experimental demonstration of the impact of hard and soft selection regimes on polymorphism maintenance in spatially heterogeneous environments.

Predicting and managing contemporary adaption requires a proper understanding of the determinants of genetic variation. Spatial heterogeneity of the environment may stably maintain polymorphism when habitat contribution to the next generation can be considered independent of the degree of adaptation of local populations within habitats (i.e., under soft selection). In contrast, when habitats contribute proportionally to the mean fitness of the populations they host (hard selection), polymorphism is not expected to be maintained by selection. Although mathematically established decades ago, this prediction had never been demonstrated experimentally. Here, we provide an experimental test in which polymorphic populations of Escherichia coli growing in heterogeneous habitats were exposed to hard and soft selection regimes. As predicted by theory, polymorphism was preserved longer under soft selection. Complementary tests established that soft selection slowed fixation processes and could even protect polymorphism in the long term by providing a systematic advantage to rare genotypes.

Determinants of host species range in plant viruses.

Prediction of pathogen emergence is an important field of research, both in human health and in agronomy. Most studies of pathogen emergence have focused on the ecological or anthropic factors involved rather than on the role of intrinsic pathogen properties. The capacity of pathogens to infect a large set of host species, i.e. to possess a large host range breadth (HRB), is tightly linked to their emergence propensity. Using an extensive plant virus database, we found that four traits related to virus genome or transmission properties were strongly and robustly linked to virus HRB. Broader host ranges were observed for viruses with single-stranded genomes, those with three genome segments and nematode-transmitted viruses. Also, two contrasted groups of seed-transmitted viruses were evidenced. Those with a single-stranded genome had larger HRB than non-seed-transmitted viruses, whereas those with a double-stranded genome (almost exclusively RNA) had an extremely small HRB. From the plant side, the family taxonomic rank appeared as a critical threshold for virus host range, with a highly significant increase in barriers to infection between plant families. Accordingly, the plant-virus infectivity matrix shows a dual structure pattern: a modular pattern mainly due to viruses specialized to infect plants of a given family and a nested pattern due to generalist viruses. These results contribute to a better prediction of virus host jumps and emergence risks.

"French Phage Network"-Second Meeting Report.

The study of bacteriophages (viruses of bacteria) includes a variety of approaches, such as structural biology, genetics, ecology, and evolution, with increasingly important implications for therapeutic and industrial uses. Researchers working with phages in France have recently established a network to facilitate the exchange on complementary approaches, but also to engage new collaborations. Here, we provide a summary of the topics presented during the second meeting of the French Phage Network that took place in Marseille in November 2016.

Probiotics for vaginal health in South Africa: what is on retailers' shelves?

BACKGROUND: Probiotics are widely used to improve gastrointestinal (GI) health, but they may also be useful to prevent or treat gynaecological disorders, including bacterial vaginosis (BV) and candidiasis. BV prevalence is high in South Africa and is associated with increased HIV risk and pregnancy complications. We aimed to assess the availability of probiotics for vaginal health in retail stores (pharmacies, supermarkets and health stores) in two major cities in South Africa. METHODS: A two-stage cluster sampling strategy was used in the Durban and Cape Town metropoles. Instructions for use, microbial composition, dose, storage and manufacturers' details were recorded. RESULTS: A total of 104 unique probiotics were identified in Cape Town and Durban (66.4% manufactured locally). Cape Town had more products than Durban (94 versus 59 probiotics), although 47% were common between cities (49/104). Only four products were explicitly for vaginal health. The remainder were for GI health in adults (51.0%) or infants (17.3%). The predominant species seen overall included Lactobacillus acidophilus (53.5%), L. rhamnosus (37.6%), Bifidobacterium longum ssp. longum (35.6%) and B. animalis ssp. lactis (33.7%). Products for vaginal health contained only common GI probiotic species, with a combination of L. acidophilus/B. longum ssp. longum/B. bifidum, L. rhamnosus/L. reuteri or L. rhamnosus alone, despite L. crispatus, L. gasseri, and L. jensenii being the most common commensals found in the lower female reproductive tract. CONCLUSION: This survey highlights the paucity of vaginal probiotics available in South Africa, where vaginal dysbiosis is common. Most vaginal products contained organisms other than female genital tract commensals.

Reduction of leaf area and symptom severity as proxies of disease-induced plant mortality: the example of the Cauliflower mosaic virus infecting two Brassicaceae hosts.

Disease induced effects on host survival are important to understand the evolution of parasitic virulence and host resistance/tolerance. Unfortunately, experiments evaluating such effects are in most cases logistically demanding justifying the measurement of survival proxies. For plant hosts commonly used proxies are leaf area and the nature and severity of visual qualitative disease symptoms. In this study we tested whether these traits are indeed correlated to the host mortality rate induced by viral infection. We infected Brassica rapa and Arabidopsis thaliana plants with different natural isolates of Cauliflower mosaic virus (CaMV) and estimated over time the development of symptoms and the relative reduction of leaf area compared to healthy plants and followed plant mortality. We observed that the mortality of infected plants was correlated with the relative reduction of leaf area of both B. rapa and A. thaliana. Measures of mortality were also correlated with the severity of visual qualitative symptoms but the magnitude of the correlations and the time frame at which they were significant depended on the host plant: stronger and earlier correlations were observed on A. thaliana.

Evolution of virulence in emerging epidemics.

Theory predicts that selection for pathogen virulence and horizontal transmission is highest at the onset of an epidemic but decreases thereafter, as the epidemic depletes the pool of susceptible hosts. We tested this prediction by tracking the competition between the latent bacteriophage λ and its virulent mutant λcI857 throughout experimental epidemics taking place in continuous cultures of Escherichia coli. As expected, the virulent λcI857 is strongly favored in the early stage of the epidemic, but loses competition with the latent virus as prevalence increases. We show that the observed transient selection for virulence and horizontal transmission can be fully explained within the framework of evolutionary epidemiology theory. This experimental validation of our predictions is a key step towards a predictive theory for the evolution of virulence in emerging infectious diseases.