Investigating the Metabolism of Estrogens in Ligilactobacillus salivarius Strains Isolated from Human Milk and Vaginal Microbiota.

The interplay between enterohepatic circulation and the gut microbiota is the main driver determining systemic levels of estrogens and their metabolites. Nevertheless, the role of potentially probiotic microorganisms in estrogen metabolism has not been investigated so far. In this work, we have explored the ability of six Ligilactobacillus salivarius strains isolated from human milk and vaginal samples to degrade and/or conjugate parental estrogens in vitro and under aerobic conditions. The quantification of estrogens and their derivatives was carried out in cell-free supernatants by LC-QQQ-MS. All the tested L. salivarius strains achieved an average degradation rate of estrone and estriol of 98% and 55%, respectively, whereas 17ß-estradiol was preferentially conjugated (up to 40%). The presence of seven out of ten genes encoding enzymes relevant for estrogen metabolism was further confirmed by PCR, highlighting their genetic potential for degrading, conjugating and/or deconjugating estrogens. The tested L. salivarius strains may be considered potential probiotics affecting the fate of endogenous estrogens. Clinical trials targeting populations with estrogen-dependent conditions will be required to elucidate the true potential of these strains for the restoration and maintenance of a healthy host estrobolome.

Expansion microscopy applied to mono- and dual-species biofilms.

Expansion microscopy (ExM) is a new super-resolution technique based on embedding the biological sample within a hydrogel and its physical expansion after swelling. This allows increasing its size by several times while preserving its structural details. Applied to prokaryotic cells, ExM requires digestion steps for efficient expansion as bacteria are surrounded by a rigid cell wall. Furthermore, bacteria can live in social groups forming biofilms, where cells are protected from environmental stresses by a self-produced matrix. The extracellular matrix represents an additional impenetrable barrier for ExM. Here we optimize the current protocols of ExM and apply them to mono- and dual-species biofilms formed by clinical isolates of Limosilactobacillus reuteri, Enterococcus faecalis, Serratia marcescens and Staphylococcus aureus. Using scanning electron microscopy for comparison, our results demonstrate that embedded bacteria expanded 3-fold. Moreover, ExM allowed visualizing the three-dimensional architecture of the biofilm and identifying the distribution of different microbial species and their interactions. We also detected the presence of the extracellular matrix after expansion with a specific stain of the polysaccharide component. The potential applications of ExM in biofilms will improve our understanding of these complex communities and have far-reaching implications for industrial and clinical research.

A randomized pilot trial assessing the reduction of gout episodes in hyperuricemic patients by oral administration of Ligilactobacillus salivarius CECT 30632, a strain with the ability to degrade purines.

Introduction: Hyperuricemia and gout are receiving an increasing scientific and medical attention because of their relatively high prevalence and their association with relevant co-morbidities. Recently, it has been suggested that gout patients have an altered gut microbiota. The first objective of this study was to investigate the potential of some Ligilactobacillus salivarius strains to metabolize purine-related metabolites. The second objective was to evaluate the effect of administering a selected potential probiotic strain in individuals with a history of hyperuricemia. Methods: Inosine, guanosine, hypoxanthine, guanine, xanthine, and uric acid were identified and quantified by high-performance liquid chromatography analysis. The uptake and biotransformation of these compounds by a selection of L. salivarius strains were assessed using bacterial whole cells and cell-free extracts, respectively. The efficacy of L. salivarius CECT 30632 to prevent gout was assessed in a pilot randomized controlled clinical trial involving 30 patients with hyperuricemia and a history of recurrent gout episodes. Half of the patients consumed L. salivarius CECT 30632 (9 log10 CFU/day; probiotic group; n = 15) for 6 months while the remaining patients consumed allopurinol (100-300 mg/daily; control group; n = 15) for the same period. The clinical evolution and medical treatment received by the participants were followed, as well as the changes in several blood biochemical parameters. Results: L. salivarius CECT 30632 was the most efficient strain for inosine (100%), guanosine (100%) and uric acid (50%) conversion and, therefore, it was selected for the pilot clinical trial. In comparison with the control group, administration of L. salivarius CECT 30632 resulted in a significant reduction in the number of gout episodes and in the use of gout-related drugs as well as an improvement in some blood parameters related to oxidative stress, liver damage or metabolic syndrome. Conclusion: Regular administration of L. salivarius CECT 30632 reduced serum urate levels, the number of gout episodes and the pharmacological therapy required to control both hyperuricemia and gout episodes in individuals with a history of hyperuricemia and suffering from repeated episodes of gout.

Rheology of Pseudomonas fluorescens biofilms: From experiments to predictive DPD mesoscopic modeling.

Bacterial biofilms mechanically behave as viscoelastic media consisting of micron-sized bacteria cross-linked to a self-produced network of extracellular polymeric substances (EPSs) embedded in water. Structural principles for numerical modeling aim at describing mesoscopic viscoelasticity without losing details on the underlying interactions existing in wide regimes of deformation under hydrodynamic stress. Here, we approach the computational challenge to model bacterial biofilms for predictive mechanics in silico under variable stress conditions. Up-to-date models are not entirely satisfactory due to the plethora of parameters required to make them functioning under the effects of stress. As guided by the structural depiction gained in a previous work with Pseudomonas fluorescens [Jara et al., Front. Microbiol. 11, 588884 (2021)], we propose a mechanical modeling by means of Dissipative Particle Dynamics (DPD), which captures the essentials of topological and compositional interactions between bacterial particles and cross-linked EPS-embedding under imposed shear. The P. fluorescens biofilms have been modeled under mechanical stress mimicking shear stresses as undergone in vitro. The predictive capacity for mechanical features in DPD-simulated biofilms has been investigated by varying the externally imposed field of shear strain at variable amplitude and frequency. The parametric map of essential biofilm ingredients has been explored by making the rheological responses to emerge among conservative mesoscopic interactions and frictional dissipation in the underlying microscale. The proposed coarse grained DPD simulation qualitatively catches the rheology of the P. fluorescens biofilm over several decades of dynamic scaling.

Interspecies relationships between nosocomial pathogens associated to preterm infants and lactic acid bacteria in dual-species biofilms.

The nasogastric enteral feeding tubes (NEFTs) used to feed preterm infants are commonly colonized by bacteria with the ability to form complex biofilms in their inner surfaces. Among them, staphylococci (mainly Staphylococcus epidermidis and Staphylococcus aureus) and some species belonging to the Family Enterobacteriaceae are of special concern since they can cause nosocomial infections in this population. NETF-associated biofilms can also include lactic acid bacteria (LAB), with the ability to compete with pathogenic species for nutrients and space. Ecological interactions among the main colonizers of these devices have not been explored yet; however, such approach could guide future strategies involving the pre-coating of the inner surfaces of NEFTs with well adapted LAB strains in order to reduce the rates of nosocomial infections in neonatal intensive care units (NICUs). In this context, this work implied the formation of dual-species biofilms involving one LAB strain (either Ligilactobacillus salivarius 20SNG2 or Limosilactobacillus reuteri 7SNG3) and one nosocomial strain (either Klebsiella pneumoniae 9SNG3, Serratia marcescens 10SNG3, Staphylococcus aureus 45SNG3 or Staphylococcus epidermidis 46SNG3). The six strains used in this study had been isolated from the inner surface of NEFTs. Changes in adhesion ability of the pathogens were characterized using a culturomic approach. Species interactions and structural changes of the resulting biofilms were analyzed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). No aggregation was observed in dual-species biofilms between any of the two LAB strains and either K. pneumoniae 9SNG3 or S. marcescens 10SNG3. In addition, biofilm thickness and volume were reduced, suggesting that both LAB strains can control the capacity to form biofilms of these enterobacteria. In contrast, a positive ecological relationship was observed in the combination L. reuteri 7SNG3-S. aureus 45SNG3. This relationship was accompanied by a stimulation of S. aureus matrix production when compared with its respective monospecies biofilm. The knowledge provided by this study may guide the selection of potentially probiotic strains that share the same niche with nosocomial pathogens, enabling the establishment of a healthier microbial community inside NEFTs.

Role of Lactobacillus biofilms in Listeria monocytogenes adhesion to glass surfaces.

Listeria monocytogenes can form long-lasting biofilms on food-contact surfaces. Lactic acid bacteria (LAB) have shown promise in antagonizing this microorganism in liquid media. However, the ecological relationships differ when cells are forming biofilms. In this work, we propose the use of Lactobacillus biofilms as surface "conditioners" to modulate the adhesion of L. monocytogenes. For this, the biofilm formation ability of Lactobacillus fermentum MP26 and Lactobacillus salivarius MP14 (human milk origin), fluorescently labeled by transfer of the mCherry-encoding pRCR12 plasmid, was first evaluated. Then, mature biofilms of these strains transformed with pRCR12 for expressing the fluorescent protein mCherry were used as adhesion substrate for GFP-tagged L. monocytogenes Scott A. The resulting biofilms were studied in terms of cellular population and attached biomass (cells plus matrix). Species distribution inside the biofilm structure was revealed by confocal laser scanning microscopy (CLSM). Although none of the Lactobacillus spp. strains reduced the adhesion of L. monocytogenes Scott A, species interactions seem to interfere with the synthesis of extracellular polymeric substances and species distribution inside the biofilms. In dual-species biofilms, CLSM images revealed that Lactobacillus cells were trapping those of L. monocytogenes Scott A. When surfaces were conditioned with Lactobacillus biofilms, the spatial distribution of L. monocytogenes Scott A cells was species-specific, suggesting these interactions are governing the ultimate biofilm structure. The results here obtained open new possibilities for controlling L. monocytogenes dispersal using these Lactobacillus spp. biofilms as a "natural" immobilization way. Whether species interactions could modify the virulence of L. monocytogenes still remains unclear.

Self-Adaptation of Pseudomonas fluorescens Biofilms to Hydrodynamic Stress.

In some conditions, bacteria self-organize into biofilms, supracellular structures made of a self-produced embedding matrix, mainly composed of polysaccharides, DNA, proteins, and lipids. It is known that bacteria change their colony/matrix ratio in the presence of external stimuli such as hydrodynamic stress. However, little is still known about the molecular mechanisms driving this self-adaptation. In this work, we monitor structural features of Pseudomonas fluorescens biofilms grown with and without hydrodynamic stress. Our measurements show that the hydrodynamic stress concomitantly increases the cell density population and the matrix production. At short growth timescales, the matrix mediates a weak cell-cell attractive interaction due to the depletion forces originated by the polymer constituents. Using a population dynamics model, we conclude that hydrodynamic stress causes a faster diffusion of nutrients and a higher incorporation of planktonic bacteria to the already formed microcolonies. This results in the formation of more mechanically stable biofilms due to an increase of the number of crosslinks, as shown by computer simulations. The mechanical stability also relies on a change in the chemical compositions of the matrix, which becomes enriched in carbohydrates, known to display adhering properties. Overall, we demonstrate that bacteria are capable of self-adapting to hostile hydrodynamic stress by tailoring the biofilm chemical composition, thus affecting both the mesoscale structure of the matrix and its viscoelastic properties that ultimately regulate the bacteria-polymer interactions.

Strategies for the Preservation, Restoration and Modulation of the Human Milk Microbiota. Implications for Human Milk Banks and Neonatal Intensive Care Units.

Studies carried in the last years have revealed that human milk contains a site-specific microbiota and constitutes a source of potentially beneficial bacteria to the infant gut. Once in the infant gut, these bacteria contribute to the assembly of a physiological gut microbiota and may play several functions, contributing to infant metabolism, protection against infections, immunomodulation or neuromodulation. Many preterm neonates are fed with pasteurized donor's human milk (DHM) or formula and, therefore, are devoid of contact with human milk microbes. As a consequence, new strategies are required to allow the exposition of a higher number of preterm infants to the human milk microbiota early in life. The first strategy would be to promote and to increase the use of own mother's milk (OMM) in Neonatal Intensive Care Units (NICUs). Even small quantities of OMM can be very valuable since they would be added to DHM in order to microbiologically "customize" it. When OMM is not available, a better screening of donor women, including routine cytomegalovirus (CMV) screening of milk, may help to avoid the pasteurization of the milk provided by, at least, a relevant proportion of donors. Finally, when pasteurized DHM or formula are the only feeding option, their supplementation with probiotic bacteria isolated from human milk, such as lactic acid bacteria or bifidobacteria, may be an alternative to try to restore a human milk-like microbiota before feeding the babies. In the future, the design of human milk bacterial consortia (minimal human milk microbiotas), including well characterized strains representative of a healthy human milk microbiota, may be an attractive strategy to provide a complex mix of strains specifically tailored to this target population.

Listeria monocytogenes Colonizes Pseudomonas fluorescens Biofilms and Induces Matrix Over-Production.

In food facilities, biofilms or their debris might act as helpers for attracting free floating microorganisms. In this sense, Pseudomonas fluorescens, a dense biofilm producer frequently isolated from food contact surfaces, could be a good candidate for sheltering other microorganisms, such as Listeria monocytogenes. The main objective of this work was to evaluate the ability of L. monocytogenes to colonize pre-established Pseudomonas biofilms. For this, the movement throughout mature Pseudomonas biofilms of a green fluorescent protein (GFP) - tagged strain of L. monocytogenes was tracked for 24 h by confocal laser scanning microscopy (CLSM). Moreover, in order to check the effect of the incorporation of Listeria on the overall matrix production, attached populations of both microorganisms and total biomass (cells + matrix) of the resulting biofilms were measured over time. Planktonic cells of L. monocytogenes efficiently migrated to preformed P. fluorescens biofilms. Moreover, they moved preferentially toward the bottom layers of these structures, suggesting some kind of tropism. When preformed P. fluorescens biofilms were conditioning the surfaces, the L. monocytogenes attached population was on average, 1-2 Log higher than when this organism grew on bare coupons. Furthermore, the arrival of L. monocytogenes to the already established P. fluorescens biofilms led to a matrix over-production. Indeed, biomass values [optical density (OD595nm)] of the resulting biofilms were double those of the ordinary L. monocytogenes-P. fluorescens mixed biofilms (1.40 vs. 0.6). The fact that L. monocytogenes cells accumulate in the bottom layers of preformed biofilms provides this microorganism an extra protection toward physical-chemical damages. This might partly explain why this microorganism can persist in food industry environments.

Quantifying the combined effects of pronase and benzalkonium chloride in removing late-stage Listeria monocytogenes-Escherichia coli dual-species biofilms.

This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium chloride (BAC) sequential treatment in removing Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel (SS) using fluorescence microscopy and plate count assays. The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-species samples were determined using a first-order factorial design. Empirical equations significantly (r2 = 0.927) described a negative individual effect of BAC and a negative interactive effect of PRN-BAC achieving OA reductions up to 46%. After treatment, high numbers of remaining attached and released viable and cultivable E. coli cells were detected in PRN-BAC combinations when low BAC concentrations were used. Therefore, at appropriate BAC doses, in addition to biofilm removal, sequential application of PRN and BAC represents an appealing strategy for pathogen control on SS surfaces while hindering the dispersion of live cells into the environment.

Listeria monocytogenes Impact on Mature or Old Pseudomonas fluorescens Biofilms During Growth at 4 and 20°C.

Changes in spatial organization, as observed by confocal laser scanning microscopy (CLSM), viable cell content, biovolume, and substratum surface coverage of the biofilms formed on glass by Pseudomonas fluorescens resulting from co-culture with Listeria monocytogenes, were examined. Two strains of L. monocytogenes, two culture temperatures and two biofilm developmental stages were investigated. Both L. monocytogenes strains, a persistently sampled isolate (collected repeatedly along 3 years from a meat factory) and Scott A, induced shrinkage in matrix volume, both at 20°C and 4°C, in mature or old biofilms, without loss of P. fluorescens cell count per surface unit. The nearly homogeneous pattern of surface coverage shown by mono-species P. fluorescens biofilms, turned into more irregular layouts in co-culture with L. monocytogenes. The upper layer of both mono and dual-species biofilms turned to predominantly consist of matrix, with plenty of viable cells underneath, in old biofilms cultured at 20°C, but not in those grown at 4°C. Between 15 and 56% of the substratum area was covered by biofilm, the extent depending on temperature, time and L. monocytogenes strain. Real biofilms in food-related surfaces may thus be very heterogeneous regarding their superficial components, i.e., those more accessible to disinfectants. It is therefore a hygienic challenge to choose an adequate agent to disrupt them.

Pathogens protection against the action of disinfectants in multispecies biofilms.

Biofilms constitute the prevalent way of life for microorganisms in both natural and man-made environments. Biofilm-dwelling cells display greater tolerance to antimicrobial agents than those that are free-living, and the mechanisms by which this occurs have been investigated extensively using single-strain axenic models. However, there is growing evidence that interspecies interactions may profoundly alter the response of the community to such toxic exposure. In this paper, we propose an overview of the studies dealing with multispecies biofilms resistance to biocides, with particular reference to the protection of pathogenic species by resident surface flora when subjected to disinfectants treatments. The mechanisms involved in such protection include interspecies signaling, interference between biocides molecules and public goods in the matrix, or the physiology and genetic plasticity associated with a structural spatial arrangement. After describing these different mechanisms, we will discuss the experimental methods available for their analysis in the context of complex multispecies biofilms.

Effectiveness of chitosan against mature biofilms formed by food related bacteria.

Chitosan has proven antimicrobial properties against planktonic cell growth. Little is known, however, about its effects on already established biofilms. Oriented for application in food industry disinfection, the effectiveness of both medium molecular weight (MMW) chitosan and its enzymatically hydrolyzed product was tested against mature biofilms of four pathogenic strains, Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus and Salmonella enterica, and a food spoilage species, Pseudomonas fluorescens. Unexpectedly, log reductions were in some cases higher for biofilm than for planktonic cells. One hour exposure to MMW chitosan (1% w/v) caused a 6 log viable cell reduction on L. monocytogenes monospecies mature biofilms and reduced significantly (3-5 log reductions) the attached population of the other organisms tested, except S. aureus. Pronase-treated chitosan was more effective than MMW chitosan on all tested microorganisms, also with the exception of S. aureus, offering best results (8 log units) against the attached cells of B. cereus. These treatments open a new possibility to fight against mature biofilms in the food industry.

Polysaccharide differences between planktonic and biofilm-associated EPS from Pseudomonas fluorescens B52.

The polysaccharides associated with free (planktonic) and surface-attached (biofilm) cells from cultures of Pseudomonas fluorescens strain B52 were compared. Variations in the attached matrix due to surface material (glass or stainless steel) were also analyzed. Two digestion methods were used to optimize the recoveries of sugars, uronic acids and acidic substituents. The yield of analyzable material after digestion reached 90% for the material associated to the biofilms, though only 20-30% for that bound to planktonic cells. The polysaccharide(s) in the biofilm had glucuronic and guluronic acids as main components, besides rhamnose, glucose and glucosamine. The proportion of glucuronic to guluronic acid was higher in the polysaccharide(s) found in biofilms formed on stainless steel than in those on glass.