K1 capsule-dependent phage-driven evolution in Escherichia coli leading to phage resistance and biofilm production.

AIMS: Understanding bacterial phage resistance mechanisms has implications for developing phage-based therapies. This study aimed to explore the development of phage resistance in Escherichia coli K1 isolates' to K1-ULINTec4, a K1-dependent bacteriophage. METHODS AND RESULTS: Resistant colonies were isolated from two different strains (APEC 45 and C5), both previously exposed to K1-ULINTec4. Genome analysis and several parameters were assessed, including growth capacity, phage adsorption, phenotypic impact at capsular level, biofilm production, and virulence in the in vivo Galleria mellonella larvae model. One out of the six resistant isolates exhibited a significantly slower growth rate, suggesting the presence of a resistance mechanism altering its fitness. Comparative genomic analysis revealed insertion sequences in the region 2 of the kps gene cluster involved in the capsule biosynthesis. In addition, an immunoassay targeting the K1 capsule showed a very low positive reaction compared to the control. Nevertheless, microscopic images of resistant strains revealed the presence of capsules with a clustered organization of bacterial cells and biofilm assessment showed an increased biofilm production compared to the sensitive strains. In the G. mellonella model, larvae infected with phage-resistant isolates showed better survival rates than larvae infected with phage-sensitive strains. CONCLUSIONS: A phage resistance mechanism was identified at the genomic level and had a negative impact on the K1 capsule production. The resistant isolates showed an increased biofilm production and a decreased virulence in vivo.

Phage Targeting Neonatal Meningitis E. coli K1 In Vitro in the Intestinal Microbiota of Pregnant Donors and Impact on Bacterial Populations.

Escherichia coli K1 is a leading cause of neonatal meningitis. The asymptomatic carriage of these strains in the maternal intestinal microbiota constitutes a risk of vertical transmission to the infant at birth. The aim of this work was to evaluate the efficacy of phage therapy against E. coli K1 in an intestinal environment and its impact on the intestinal microbiota. For this purpose, three independent experiments were conducted on the SHIME® system, the first one with only the phage vB_EcoP_K1_ULINTec4, the second experiment with only E. coli K1 and the last experiment with both E. coli K1 and the phage. Microbiota monitoring was performed using metagenetics, qPCR, SCFA analysis and the induction of AhR. The results showed that phage vB_EcoP_K1_ULINTec4, inoculated alone, was progressively cleared by the system and replicates in the presence of its host. E. coli K1 persisted in the microbiota but decreased in the presence of the phage. The impact on the microbiota was revealed to be donor dependent, and the bacterial populations were not dramatically affected by vB_K1_ULINTec4, either alone or with its host. In conclusion, these experiments showed that the phage was able to infect the E. coli K1 in the system but did not completely eliminate the bacterial load.

Shear strength of wet granular materials: Macroscopic cohesion and effective stress : Discrete numerical simulations, confronted to experimental measurements.

Rheometric measurements on assemblies of wet polystyrene beads, in steady uniform quasistatic shear flow, for varying liquid content within the small saturation (pendular) range of isolated liquid bridges, are supplemented with a systematic study by discrete numerical simulations. The numerical results agree quantitatively with the experimental ones provided that the intergranular friction coefficient is set to the value [Formula: see text], identified from the behaviour of the dry material. Shear resistance and solid fraction [Formula: see text] are recorded as functions of the reduced pressure [Formula: see text], which, defined as [Formula: see text], compares stress [Formula: see text], applied in the velocity gradient direction, to the tensile strength [Formula: see text] of the capillary bridges between grains of diameter a, and characterizes cohesion effects. The simplest Mohr-Coulomb relation with [Formula: see text]-independent cohesion c applies as a good approximation for large enough [Formula: see text] (typically [Formula: see text]. Numerical simulations extend to different values of µ and, compared to experiments, to a wider range of [Formula: see text]. The assumption that capillary stresses act similarly to externally applied ones onto the dry granular contact network (effective stresses) leads to very good (although not exact) predictions of the shear strength, throughout the numerically investigated range [Formula: see text] and [Formula: see text]. Thus, the internal friction coefficient [Formula: see text] of the dry material still relates the contact force contribution to stresses, [Formula: see text], while the capillary force contribution to stresses, [Formula: see text], defines a generalized Mohr-Coulomb cohesion c, depending on [Formula: see text] in general. c relates to [Formula: see text] , coordination numbers and capillary force network anisotropy. c increases with liquid content through the pendular regime interval, to a larger extent, the smaller the friction coefficient. The simple approximation ignoring capillary shear stress [Formula: see text] (referred to as the Rumpf formula) leads to correct approximations for the larger saturation range within the pendular regime, but fails to capture the decrease of cohesion for smaller liquid contents.

Contribution of arbuscular mycorrhizal fungi to the bioavailability of micronutrients (iron and zinc) in millet accessions.

Introduction: Micronutrient deficiencies, particularly iron (Fe) and zinc (Zn) deficiencies, are prevalent public health issues in developing countries, with children under 5 years old and breastfeeding women being the most affected in Senegal. Agronomic approaches, including soil fertilization and microbial biotechnology, are used to alleviate these deficiencies, yet challenges persist due to poor nutrient availability in staple food crops like pearl millet (Pennisetum glaucum L.). Methods: This study aimed to assess the contribution of one arbuscular mycorrhizal fungal (AMF) strain, Glomus mosseae, to the bioavailability of micronutrients (zinc and iron) in pearl millet biomass. Four pearl millet accessions from the National Laboratory for Research on Plant Production (LNRPV) collection were inoculated with G. mosseae obtained from the Common Microbiology Laboratory (LCM), with four replications. Gaussian regression tests were employed to analyze the data and determine correlations between AMF concentration and micronutrient bioavailability. Results: The results indicate that the combination of Glomus mosseae inoculation and organic residual products improved growth parameters and micronutrient absorption in pearl millet accessions. Analysis revealed significantly greater iron, zinc, phosphorus, and potassium contents in the foliar biomass of mycorrhizal pearl millet plants compared to non-mycorrhizal plants (control). Inoculation with AMF facilitated micronutrient absorption, particularly iron and zinc, not only in roots but also in aerial parts of the plants. Discussion: These findings suggest that incorporating AMF and organic residual products in millet cultivation could be a viable strategy for enhancing plant development and increasing iron and zinc contents in varieties. Further research is needed to elucidate the mechanisms underlying AMF-mediated nutrient uptake and optimize their use in agricultural practices.

Impact Assessment of vB_KpnP_K1-ULIP33 Bacteriophage on the Human Gut Microbiota Using a Dynamic In Vitro Model.

New control methods are needed to counter antimicrobial resistances and the use of bacteriophages as an alternative treatment seems promising. To that end, the effect of the phage vB_KpnP_K1-ULIP33, whose host is the hypervirulent Klebsiella pneumoniae SA12 (ST23 and capsular type K1), was assessed on intestinal microbiota, using an in vitro model: the SHIME® system (Simulator of the Human Intestinal Microbial Ecosystem). After stabilization of the system, the phage was inoculated for 7 days and its persistence in the different colons was studied until its disappearance from the system. The concentration of short chain fatty acids in the colons showed good colonization of the bioreactors by the microbiota and no significant effect related to the phage treatment. Diversity (α and ß), the relative abundance of bacteria, and qPCR analysis targeting different genera of interest showed no significant variation following phage administration. Even if further in vitro studies are needed to assess the efficacy of this phage against its bacterial host within the human intestinal ecosystem, the phage ULIP33 exerted no significant change on the global colonic microbiota.

Unexpected Slow Kinetics of Poly(Methacrylic Acid) Phase Separation in the Semi-Dilute Regime.

Poly (methacrylic acid) (PMAA) solutions are known to exhibit a lower critical solution temperature (LCST). A temperature-composition phase diagram of PMAA has been constructed by standard cloud point determination through transmittance measurements, and also by studying the steady states reached under phase separation. This allows us to reconstruct the binodal curve describing the phase behavior of PMAA for both low and high concentration regimes, and to determine accurately the LCST temperature. In a second step, the structures formed following a temperature jump above the cloud point and their evolution in time have been investigated at the nanoscale using small angle neutron scattering (SANS). This approach shows that the formation of phase-separated nanostructures is a slow process, requiring more than 12 h. The formed structures are then shown to depend on the amplitude of the temperature jump above the cloud point. An original mechanism of phase separation is identified in the semi-dilute regime. The growth of micrometric-size droplets with an inner structure displaying the rheological properties of a gel leads to the formation of a percolating network which hinders the influence of gravity. Such a result can explain the slow kinetics of the PMAA LCST transition.

In Vitro and In Vivo Assessments of Two Newly Isolated Bacteriophages against an ST13 Urinary Tract Infection Klebsiella pneumoniae.

Antibiotic resistance represents a major public health concern requiring new alternatives including phage therapy. Klebsiella pneumoniae belongs to the ESKAPE bacteria and can cause urinary tract infections (UTIs). The aims of this study were to isolate and characterize new bacteriophages against a K. pneumoniae strain isolated from UTIs and to assess their efficacy in vitro and in vivo in a Galleria (G.) mellonella larvae model. For this purpose, two bacteriophages were newly isolated against an ST13 K. pneumoniae strain isolated from a UTI and identified as K3 capsular types by wzi gene PCR. Genomic analysis showed that these bacteriophages, named vB_KpnP_K3-ULINTkp1 and vB_KpnP_K3-ULINTkp2, belong to the Drulisvirus genus. Bacteriophage vB_KpnP_K3-ULINTkp1 had the narrowest host spectrum (targeting only K3), while vB_KpnP_K3-ULINTkp2 also infected other Klebsiella types. Short adsorption times and latent periods were observed for both bacteriophages. In vivo experiments showed their ability to replicate in G. mellonella larvae and to decrease host bacterial titers. Moreover, both bacteriophages improved the survival of the infected larvae. In conclusion, these two bacteriophages had different in vitro properties and showed in vivo efficacy in a G. mellonella model with a better efficiency for vB_KpnP_K3-ULINTkp2.

Roles of Arbuscular Mycorrhizal Fungi on Soil Fertility: Contribution in the Improvement of Physical, Chemical, and Biological Properties of the Soil.

Many of the world's soils are experiencing degradation at an alarming rate. Climate change and some agricultural management practices, such as tillage and excessive use of chemicals, have all contributed to the degradation of soil fertility. Arbuscular Mycorrhizal Fungi (AMFs) contribute to the improvement of soil fertility. Here, a short review focusing on the role of AMF in improving soil fertility is presented. The aim of this review was to explore the role of AMF in improving the chemical, physical, and biological properties of the soil. We highlight some beneficial effects of AMF on soil carbon sequestration, nutrient contents, microbial activities, and soil structure. AMF has a positive impact on the soil by producing organic acids and glomalin, which protect from soil erosion, chelate heavy metals, improve carbon sequestration, and stabilize soil macro-aggregation. AMF also recruits bacteria that produce alkaline phosphatase, a mineralization soil enzyme associated with organic phosphorus availability. Moreover, AMFs influence the composition, diversity, and activity of microbial communities in the soil through mechanisms of antagonism or cooperation. All of these AMF activities contribute to improve soil fertility. Knowledge gaps are identified and discussed in the context of future research in this review. This will help us better understand AMF, stimulate further research, and help in sustaining the soil fertility.

Supranational Assessment of the Quality of Probiotics: Collaborative Initiative between Independent Accredited Testing Laboratories.

Recent acquisitions about the role of the microbiota in the functioning of the human body make it possible to envisage an increasing use of beneficial microbes, and more particularly of probiotics as well as their metabolites, as nutritional supplements. National and EU authorities are engaged in assuring the safety and quality of food supplements and in defining rules to assess and communicate their efficacy on human health. The quality of probiotics, intended as strains' identification, viability, and stability over time, is a crucial factor of credibility with consumers and health professionals. Analytical technologies for the quality control of probiotics must also be adapted to new preparations, such as those including new multistrains complex combinations. Accredited laboratories face this relevant challenge on a daily basis. Through its close collaboration with the laboratory commissioned to produce the specifications for its ESLP quality label (identification and quantitative analyses) together with its scientific committee, the ESLP has been focusing on this issue for 10 years. Recently, as part of the internationalization of the ESLP quality label, a new and unique initiative in Europe for the evaluation of the quality of probiotic preparations has been carried out. The collaboration between two accredited laboratories in Belgium and in Italy represented a concrete example of supranational collaboration in the assessment of the quality of probiotic preparations. Results show that both laboratories are in line as expected in terms of performance. Common approaches to the qualitative assessment of probiotic preparations, especially for complex and composite recipes, in terms of number of strains and included substances, should be encouraged and promoted all over the EU.

In Vitro Characterization and In Vivo Efficacy Assessment in Galleria mellonella Larvae of Newly Isolated Bacteriophages against Escherichia coli K1.

Extra-intestinal Escherichia coli express several virulence factors that increase their ability to colonize and survive in different localizations. The K1 capsular type is involved in several infections, including meningitis, urinary tract, and bloodstream infections. The aims of this work were to isolate, characterize, and assess the in vivo efficacy of phages targeting avian pathogenic E. coli (APEC) O18:K1, which shares many similarities with the human strains responsible for neonatal meningitis. Eleven phages were isolated against APEC O18:K1, and four of them presenting a narrow spectrum targeting E. coli K1 strains were further studied. The newly isolated phages vB_EcoS_K1-ULINTec2 were similar to the Siphoviridae family, and vB_EcoP_K1-ULINTec4, vB_EcoP_K1-ULINTec6, and vB_EcoP_K1-ULINTec7 to the Autographiviridae family. They are capsular type (K1) dependent and present several advantages characteristic of lytic phages, such as a short adsorption time and latent period. vB_EcoP_K1-ULINTec7 is able to target both K1 and K5 strains. This study shows that these phages replicate efficiently, both in vitro and in vivo in the Galleria mellonella model. Phage treatment increases the larvae survival rates, even though none of the phages were able to eliminate the bacterial load.

Yielding and shear banding in soft glassy materials.

Yield stress fluids have proven difficult to characterize, and a reproducible determination of the yield stress is difficult. We study two types of yield stress fluids (YSF) in a single system: simple and thixotropic ones. This allows us to show that simple YSF are simply a special case of thixotropic ones, and to pinpoint the difference between static and dynamic yield stresses, one of the major problems in the field. The thixotropic systems show a strong time dependence of the viscosity due to the existence of an internal percolated structure that confers the yield stress to the material. Using loaded emulsions to control the thixotropy, we show that the transition to flow at the yield stress is discontinuous for thixotropic materials, and continuous for ideal ones. The discontinuity leads to a critical shear rate below which no steady flows can be observed, accounting for the ubiquitous shear banding observed in these materials.

Shear thickening and migration in granular suspensions.

We study the emergence of shear thickening in dense suspensions of non-Brownian particles. We combine local velocity and concentration measurements using magnetic resonance imaging with macroscopic rheometry experiments. In steady state, we observe that the material is heterogeneous, and we find that the local rheology presents a continuous transition at low shear rate from a viscous to a shear thickening, Bagnoldian, behavior with shear stresses proportional to the shear rate squared, as predicted by a scaling analysis. We show that the heterogeneity results from an unexpectedly fast migration of grains, which we attribute to the emergence of the Bagnoldian rheology. The migration process is observed to be accompanied by macroscopic transient discontinuous shear thickening, which is consequently not an intrinsic property of granular suspensions.

Evaluation of Enzymatic Cleaning on Food Processing Installations and Food Products Bacterial Microflora.

Biofilms are a permanent source of contamination in food industries and could harbor various types of microorganisms, such as spoiling bacteria. New strategies, such as enzymatic cleaning, have been proposed to eradicate them. The purpose of this study was to evaluate the impact of enzymatic cleaning on the microbial flora of installations in a processing food industry and of the final food product throughout its shelf life. A total of 189 samples were analyzed by classical microbiology and 16S rDNA metagenetics, including surface samples, cleaning-in-place (CIP) systems, and food products (at D0, Dend of the shelf life, and Dend of the shelf life +7 days). Some surfaces were highly contaminated with spoiling bacteria during conventional cleaning while the concentration of the total flora decreased during enzymatic cleaning. Although the closed circuits were cleaned with conventional cleaning before enzymatic cleaning, there was a significant release of microorganisms from some parts of the installations during enzymatic treatment. A significant difference in the total flora in the food products at the beginning of the shelf life was observed during enzymatic cleaning compared to the conventional cleaning, with a reduction of up to 2 log CFU/g. Metagenetic analysis of the food samples at the end of their shelf life showed significant differences in bacterial flora between conventional and enzymatic cleaning, with a decrease of spoiling bacteria (Leuconostoc sp.). Enzymatic cleaning has improved the hygiene of the food processing instillations and the microbial quality of the food throughout the shelf life. Although enzymatic cleaning is not yet commonly used in the food industry, it should be considered in combination with conventional sanitizing methods to improve plant hygiene.

Density waves in shear-thickening suspensions.

Shear thickening corresponds to an increase of the viscosity as a function of the shear rate. It is observed in many concentrated suspensions in nature and industry: water or oil saturated sediments, crystal-bearing magma, fresh concrete, silica suspensions, and cornstarch mixtures. Here, we reveal how shear-thickening suspensions flow, shedding light onto as yet non-understood complex dynamics reported in the literature. When shear thickening is important, we show the existence of density fluctuations that appear as periodic waves moving in the direction of flow and breaking azimuthal symmetry. They come with strong normal stress fluctuations of the same periodicity. The flow includes small areas of normal stresses of the order of tens of kilopascals and areas of normal stresses of the order of hundreds of pascals. These stress inhomogeneities could play an important role in the damage caused by thickening fluids in the industry.

Characterization of microcrystalline cellulose spheres and prediction of hopper flow based on a µ(I)-rheology model.

The objective of this study was to characterize the rheology of a pharmaceutical material in the context of the µ(I)-rheology model and to use this model to predict powder flow in a manufacturing operation that is relevant to pharmaceutical manufacturing. The rheology of microcrystalline cellulose spheres was therefore characterized in terms of the µ(I)-rheology model using a modified Malvern Kinexus rheometer. As an example of an important problem in pharmaceutical manufacturing, the flow of these particles from a hopper was studied experimentally and numerically using a continuum Navier-Stokes solver based on the Volume-Of-Fluid (VOF) interface-capturing numerical method. The work shows that the rheology of this typical pharmaceutical material can be measured using a modified annular shear rheometer and that the results can be interpreted in terms of the µ(I)-rheology model. It is demonstrated that both the simulation results and the experimental data show a constant hopper discharge rate. It is noted that the model can suffer from ill-posedness and it is shown how an increasingly fine grid resolution can result in predictions that are not entirely physically realistic. This shortcoming of the numerical framework implies that caution is required when making a one-to-one comparison with experimental data.

Yield stress and shear banding in granular suspensions.

We study the emergence of a yield stress in dense suspensions of non-Brownian particles by combining local velocity and concentration measurements using magnetic resonance imaging with macroscopic rheometric experiments. We show that the competition between gravity and viscous stresses is at the origin of the development of a yield stress in these systems at relatively low volume fractions. Moreover, it is accompanied by a shear-banding phenomenon that is the signature of this competition. However, if the system is carefully density matched, no yield stress is encountered until a volume fraction of 62.7 +/- 0.3%.

Quick clay and landslides of clayey soils.

We study the rheology of quick clay, an unstable soil responsible for many landslides. We show that above a critical stress the material starts flowing abruptly with a very large viscosity decrease caused by the flow. This leads to avalanche behavior that accounts for the instability of quick clay soils. Reproducing landslides on a small scale in the laboratory shows that an additional factor that determines the violence of the slides is the inhomogeneity of the flow. We propose a simple yield stress model capable of reproducing the laboratory landslide data, allowing us to relate landslides to the measured rheology.

Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods.

Kombucha, historically an Asian tea-based fermented drink, has recently become trendy in Western countries. Producers claim it bears health-enhancing properties that may come from the tea or metabolites produced by its microbiome. Despite its long history of production, microbial richness and dynamics have not been fully unraveled, especially at an industrial scale. Moreover, the impact of tea type (green or black) on microbial ecology was not studied. Here, we compared microbial communities from industrial-scale black and green tea fermentations, still traditionally carried out by a microbial biofilm, using culture-dependent and metabarcoding approaches. Dominant bacterial species belonged to Acetobacteraceae and to a lesser extent Lactobacteriaceae, while the main identified yeasts corresponded to Dekkera, Hanseniaspora and Zygosaccharomyces during all fermentations. Species richness decreased over the 8-day fermentation. Among acetic acid bacteria, Gluconacetobacter europaeus, Gluconobacter oxydans, G. saccharivorans and Acetobacter peroxydans emerged as dominant species. The main lactic acid bacteria, Oenococcus oeni, was strongly associated with green tea fermentations. Tea type did not influence yeast community, with Dekkera bruxellensis, D. anomala, Zygosaccharomyces bailii and Hanseniaspora valbyensis as most dominant. This study unraveled a distinctive core microbial community which is essential for fermentation control and could lead to Kombucha quality standardization.

An attempt to categorize yield stress fluid behaviour.

We propose a new view on yield stress materials. Dense suspensions and many other materials have a yield stress-they flow only if a large enough shear stress is exerted on them. There has been an ongoing debate in the literature on whether true yield stress fluids exist, and even whether the concept is useful. This is mainly due to the experimental difficulties in determining the yield stress. We show that most if not all of these difficulties disappear when a clear distinction is made between two types of yield stress fluids: thixotropic and simple ones. For the former, adequate experimental protocols need to be employed that take into account the time evolution of these materials: ageing and shear rejuvenation. This solves the problem of experimental determination of the yield stress. Also, we show that true yield stress materials indeed exist, and in addition, we account for shear banding that is generically observed in yield stress fluids.

Shear thickening of cornstarch suspensions as a reentrant jamming transition.

We study the rheology of cornstarch suspensions, a non-Brownian particle system that exhibits shear thickening. From magnetic resonance imaging velocimetry and classical rheology it follows that as a function of the applied stress the suspension is first solid (yield stress), then liquid, and then solid again when it shear thickens. For the onset of thickening we find that the smaller the gap of the shear cell, the lower the shear rate at which thickening occurs. Shear thickening can then be interpreted as the consequence of dilatancy: the system under flow wants to dilate but instead undergoes a jamming transition because it is confined, as confirmed by measurement of the dilation of the suspension as a function of the shear rate.