A new killing assay with the Caenorhabditis elegans PX627 mutant to assess the virulence of uropathogenic Escherichia coli strains.

The nematode Caenorhabditis elegans (C. elegans) is a prime invertebrate host model for studying uropathogenic Escherichia coli (UPEC) pathogenesis. The aim of this work was to develop a new C. elegans killing assay based on feeding bacteria by the nematode throughout its life from the egg. With this model, the lifespan of C. elegans rrf-3, temperature-sterile, mutant, and PX627, auxin-inducible infertile, mutant fed UPEC strains, was compared. The behavior of three clinical UPEC strains and the non-pathogenic Escherichia coli OP50 strain was analyzed. Survival curves were generated by the Kaplan-Meier method and compared by the log-rank test over 10 days of follow-up. There was no significant difference between the survival curves obtained with each of the two C. elegans mutants (PX627 and rrf-3) fed with each of the strains of E. coli (OP50, G1722, G1473 or ER41). The UPEC strains were classified according to their virulence in vivo in the C. elegans PX627 mutant. The most virulent strain was ER41 which harbored the virulence genes fimA, papC and hlyA, expressed hemolysis in vitro and showed no antibiotic resistance. The least virulent strain was G1722 which only harbored the two adhesion factor genes, was not hemolytic and was resistant to multiple antibiotics. The C. elegans PX627 mutant fed with UPEC bacteria from the egg stage is a simple and inexpensive invertebrate animal model for assessing the in vivo virulence of different strains. The early exposure of C. elegans to pathogenic bacteria at the egg stage, without the need to change the incubation temperature, is an advantage over previously described C. elegans killing assays.

Fungal Biodegradation of Polyurethanes.

Polyurethanes (PURs) are versatile polymers used in a wide variety of fields, such as the medical, automotive, textile, thermal insulation, and coating industries as well as many everyday objects. Many PURs have applications that require a long service life, sometimes with exposure to aggressive conditions. They can undergo different types of physicochemical and biological degradation, but they are not compostable, and many of them constitute persistent waste in the environment. Although both bacteria and fungi can be involved in the degradation of PURs, fungi are often the main biodegradation agents. The chemical structure of PURs determines their degree of biodegradation. Fungal biodegradation of PURs is linked to the production of enzymes, mainly esterases and proteases, alongside laccases, peroxidases, and tyrosinases, which can modify the structure of polyurethane compounds by forming carbonyl groups. The experimental analysis of the biodegradation of PUR can be carried out by bringing the polymer into contact with a mold in pure culture or with a microbial consortium. Then, global measurements can be taken, such as weight loss, tensile tests, or the ability of microorganisms to grow in the presence of PUR as the sole carbon source. The analysis of the chemical structure of the polymer and its degradation products after fungal growth can confirm biodegradation and specify the mechanism. The main avenues of future research are directed towards the development of fully biodegradable PURs and, on the contrary, towards the development of PURs that are more resistant to degradation phenomena, in particular biodegradation, for applications where the material is in contact with living organisms.

Adherence of Uropathogenic Escherichia Coli in Dog Urine After Consumption of Food Supplemented with Cranberry (Vaccinium Macrocarpon).

Introduction: Escherichia coli is the most common pathogen isolated from the urine of dogs with urinary tract infections (UTIs). While there are many studies in humans investigating the potential for the prevention of UTIs by dietary consumption of cranberry, few analogous studies have been carried out in dogs. Material and Methods: Eight dogs, four male and four female, were successively fed two diets, first a control without cranberry, and then the second diet containing cranberry extracts. Naturally excreted urine was collected on the tenth day after the start of each diet for 24 h and used for bacterial growth. Madin-Darby canine kidney cell adherence by the uropathogenic E. coli G1473 strain expressing type 1 pili and positive for P pili and haemolysin gene markers was quantified after growth in urine samples. Results: Significant reductions in bacterial adherence to MDCK cells (from -16.5 to -73.4%, P < 0.05) were observed in the four females but not in the males after consumption of the cranberry extracts compared to the same animals consuming the control diet. Conclusion: Dietary supplementation with cranberry may provide some degree of protection to female dogs against adhesion of uropathogenic E. coli to urinary epithelial cells.

Molecular Docking and In-Silico Analysis of Natural Biomolecules against Dengue, Ebola, Zika, SARS-CoV-2 Variants of Concern and Monkeypox Virus.

The emergence and rapid evolution of human pathogenic viruses, combined with the difficulties in developing effective vaccines, underline the need to develop innovative broad-spectrum antiviral therapeutic agents. The present study aims to determine the in silico antiviral potential of six bacterial antimicrobial peptides (AMPs), two phytochemicals (silvestrol, andrographolide), and two bacterial secondary metabolites (lyngbyabellin A, hapalindole H) against dengue virus, Zika virus, Ebola virus, the major variants of SARS-CoV-2 and monkeypox virus. The comparison of docking scores obtained with natural biomolecules was performed with specific neutralizing antibodies (positive controls for ClusPro) and antiviral drugs (negative controls for Autodock Vina). Glycocin F was the only natural biomolecule tested to show high binding energies to all viral surface proteins and the corresponding viral cell receptors. Lactococcin G and plantaricin ASM1 also achieved high docking scores with all viral surface proteins and most corresponding cell surface receptors. Silvestrol, andrographolide, hapalindole H, and lyngbyabellin A showed variable docking scores depending on the viral surface proteins and cell receptors tested. Three glycocin F mutants with amino acid modifications showed an increase in their docking energy to the spike proteins of SARS-CoV-2 B.1.617.2 Indian variant, and of the SARS-CoV-2 P.1 Japan/Brazil variant, and the dengue DENV envelope protein. All mutant AMPs indicated a frequent occurrence of valine and proline amino acid rotamers. AMPs and glycocin F in particular are the most promising biomolecules for the development of broad-spectrum antiviral treatments targeting the attachment and entry of viruses into their target cell.

Antimicrobial agents and microbial ecology.

Antimicrobials are therapeutic substances used to prevent or treat infections. Disinfectants are antimicrobial agents applied to non-living surfaces. Every year, several thousand tonnes of antimicrobials and their by-products are released into the environment and in particular into the aquatic environment. This type of xenobiotic has ecological consequences in the natural environment but also in technological environments such as wastewater treatment plants and methane fermentation sewage sludge treatment plants. The constant exposure of microbial communities not only to high concentrations but also to sub-inhibitory concentrations of antibiotics is a key element in the development of antibiotic resistance in aquatic environments and in soils. The future of antimicrobials lies in the development of biosourced or bioinspired molecules. The observation and deciphering of interactions between living organisms is the key to this development.

Current and future chemical treatments to fight biodeterioration of outdoor building materials and associated biofilms: Moving away from ecotoxic and towards efficient, sustainable solutions.

All types of building materials are rapidly colonized by microorganisms, initially through an invisible and then later a visible biofilm that leads to their biodeterioration. Over centuries, this natural phenomenon has been managed using mechanical procedures, oils, or even wax. In modern history, many treatments such as high-pressure cleaners, biocides (mainly isothiazolinones and quaternary ammonium compounds) are commercially available, as well as preventive ones, such as the use of water-repellent coatings in the fabrication process. While all these cleaning techniques offer excellent cost-benefit ratios, their limitations are numerous. Indeed, building materials are often quickly recolonized after application, and microorganisms are increasingly reported as resistant to chemical treatments. Furthermore, many antifouling compounds are ecotoxic, harmful to human health and the environment, and new regulations tend to limit their use and constrain their commercialization. The current state-of-the-art highlights an urgent need to develop innovative antifouling strategies and the widespread use of safe and eco-friendly solutions to biodeterioration. Interestingly, innovative approaches and compounds have recently been identified, including the use of photocatalysts or natural compounds such as essential oils or quorum sensing inhibitors. Most of these solutions developed in laboratory settings appear very promising, although their efficiency and ecotoxicological features remain to be further tested before being widely marketed. This review highlights the complexity of choosing the adequate antifouling compounds when fighting biodeterioration and proposes developing case-to-case innovative strategies to raise this challenge, relying on integrative and multidisciplinary approaches.

Cranberry (Vaccinium macrocarpon) dietary supplementation and fecal microbiota of Wistar rats.

Cranberry (Vaccinium macrocarpon) dietary supplementation can help prevention of urinary tract infections through the supply of proanthocyanidin-type polyphenols (PAC). The main uropathogenic bacteria are members of the intestinal microbiota. A randomized cross-over experiment was done to investigate whether cranberry dietary supplementation affects concentrations of thermotolerant coliforms, Enterococcus spp. and Lactobacillus spp. in rat faeces. Thirteen rats, housed in individual cages, received successively two diets as pellets during 7 days each: a standard diet without polyphenols and the standard diet supplemented with cranberry powder containing 10.9 mg/100 g of PAC. There was a 7 days wash-out period in between with standard diet without polyphenols. Body weight and feed intake were recorded. Faeces were collected on the last day of treatment, and crushed to count the different bacterial populations using the most probable number method. Thermotolerant coliforms were grown in BGBLB tubes and on MacConkey agar. Enterococcus spp. were grown in Rothe and Litsky broths and on KF Streptococcus agar. Lactobacillus spp. were grown in Man Rogosa Sharpe broth. Body mass gains were not affected by cranberry supplementation. This is consistent with equal food intake, cranberry powder not providing significant energy supplement. Cranberry dietary supplementation was associated with changes in fecal concentrations of thermotolerant coliforms, and Enterococcus spp. in some rats, but did not induce significant changes in bacterial fecal concentrations in a global population of 13 rats. In conclusion, we did not observe any significant effect of dietary cranberry supplementation on the fecal microbiota of Wistars rats for a 7-day diet.

Ways to improve biocides for metalworking fluid.

Metalworking fluids (MWF) are mainly emulsions of oil in water containing additives such as corrosion inhibitors, emulsifiers, defoamers, and biocides. Microbial contamination of MWF is almost systematic, and some of their constituents serve as nutrients for contaminating microorganisms. Biocides for MWF are protection products used to counter microbial contaminations and growth. Ideally, a biocide for MWF should have the following non-exhaustive criteria: have a broad-spectrum activity, be usable at low concentrations, be compatible with the formulation and the physical-chemical properties of MWF, be stable over time, retain its effectiveness in the presence of soiling, have no corrosive action on metals, present no danger to humans and the environment, be inexpensive. The future lies in the development of new molecules with biocidal activity corresponding to these ideal specifications, but in the meantime, it is possible to improve the performance of existing molecules currently on the market. Different strategies for potentiation of the activity of existing biocides are possible. The compatibility of the potentiation strategies with their use in metal working fluids is discussed.

Rheological Behaviour of Cementitious Materials Incorporating Solid-Solid Phase Change Materials.

Nowadays, thermal regulation of the indoor environment is mandatory to reduce greenhouse gas emissions. The incorporation of Phase Change Materials (PCMs) and especially solid-solid PCMs (s/s PCMs) into building materials can be a major step forward in reducing energy consumption. Such materials are used for their high latent heat to save and release heat during phase change. To integrate these products in the fabrication of cementitious materials, it is essential to predict their influence on the rheological behaviour of construction materials. In this work, rheological measurements were carried out on composite suspensions made of cement or mortar plus s/s PCMs. Results showed that the fitting of the Herschel-Bulkley model with a constant value of flow exponent was reliable. The s/s PCMs influenced the consistency and the yield strength values, with the yield strength value being only slightly affected. The adaptation of an existing viscosity model is proposed to predict the consistency value of suspensions. Finally, an innovative approach to predict the flow behaviour is proposed and we highlight the research needs to mainstream the use of s/s PCMs in construction materials.

Polysaccharide-hydrolysing enzymes enhance the in vitro cleaning efficiency of Nanofiltration membranes.

The development of biofilm on the surface of filtration membranes is the main fouling component of water filtration systems. Chemical cleaning is only partially effective in removing biofilm components from the membrane surface. In order to identify opportunities to improve the efficiency of commercial cleaning solutions used in nanofiltration, we compared the in vitro efficacy of different commercial treatments, with or without the addition of polysaccharidases, to clean fouled membrane samples. The treatments were tested at two stages of biofilm development corresponding to 80 (D80) and 475 (D475) days of filtration in an industrial plant. The cleaning efficiency was evaluated by comparing the ATR-FTIR spectra before and after cleaning. At D80 and D475, all cleaning solutions led to a reduction of infrared signals from the biofilm. At D80, enzymatic alkaline detergent (AEDT) treatment was significantly more effective than alkaline detergent (ADT) treatment in removing proteins, but no significant difference in efficacy between the two treatments was observed for polysaccharides. The addition of polysaccharidases to AEDT did not bring any significant efficiency gain. At D475, ADT and AEDT treatments had the same efficacy, but the addition of polysaccharidases to the AEDT treatment significantly increased the removal of polysaccharides and proteins from the membrane surface. In conclusion, polysaccharidases can increase the in vitro efficacy of a commercially available alkaline enzymatic detergent cleaning solution against sufficiently developed biofilms. These results pave the way for the development of new cleaning solutions containing polysaccharide degrading enzymes for the cleaning of membranes used in the production of drinking water. Further experiments are needed to characterize the mechanism of this polysaccharidase effect and to confirm this increase in cleaning efficiency in an industrial context.

Anti-persister activity of squalamine against Acinetobacter baumannii.

Squalamine is a natural polycationic aminosterol extracted from the shark Squalus acanthias. Squalamine displays remarkable efficacy against antimicrobial-resistant Gram-negative and Gram-positive bacteria. Its membranolytic activity and low cytotoxicity make squalamine one of the most promising agents to fight nosocomial pathogens such as Acinetobacter baumannii. In the context of chronic diseases and therapeutic failures associated with this pathogen, the presence of dormant cells, i.e. persisters and viable but non-culturable cells (VBNCs), highly tolerant to antimicrobial compounds is problematic. The aim of this study was to investigate the antibacterial activity of squalamine against this bacterial population of A. baumannii. Bacterial dormancy was induced by cold shock and nutrient starvation in the presence of high doses of either colistin, ciprofloxacin or squalamine. Persisters and VBNCs induced by these treatments were then challenged with 100 mg/L squalamine. The efficacy of each treatment was determined by evaluating culturability on agar medium, membrane integrity (LIVE/DEAD®BacLightTM staining) and respiratory activity (BacLightTM RedoxSensorTM CTC staining) of bacteria. A. baumannii ATCC 17978 generated persisters as well as VBNCs in the presence of high doses of ciprofloxacin but not colistin or squalamine. Squalamine at 100 mg/L (below its haemolytic concentration) was able to kill dormant cells. Squalamine did not induce persister cell or VBNC formation in A. baumannii ATCC 17978. Interestingly, squalamine was significantly active against this type of dormant population generated by ciprofloxacin, making it a very promising anti-persister agent.

Assessment of flow cytometry for microbial water quality monitoring in cooling tower water and oxidizing biocide treatment efficiency.

The control of Legionella proliferation in cooling tower water circuits requires regular monitoring of water contamination and effective disinfection procedures. In this study, flow cytometry was assessed to monitor water contamination and disinfection treatment efficiency on bacterial cells regarding nucleic acid injury (SYBR® Green II), cell integrity (SYBR® Green II and propidium iodide) and metabolism activity (ChemChrome V6). A total of 27 cooling tower water samples were analyzed in order to assess water contamination levels regarding viable populations: standard culture, ATP measurement and flow cytometry methods were compared. Flow cytometry and plate counts methods showed a significant correlation for changes in concentrations despite a 1 to 2-log difference regarding absolute quantification. Concerning intracellular activity, the use of two different flow cytometers (FACSCanto™ II and Accuri™ C6) showed no statistical difference while a difference was observed between flow cytometry and usual methods (culture and ATP measurement). The standard culture and flow cytometry methods were also compared for in vitro bacteria inactivation measurements in the presence of 3 different types of oxidizing biocides commonly used for cooling tower disinfection. Reductions observed ranged between 1 and 2 log depending on (1) the detection method, (2) the bacterial population origin and/or (3) the active biocide molecule used. In conclusion, flow cytometry represents an efficient, accurate and fast approach to monitor water contamination and biocide treatment efficiency in cooling towers.

Extracellular polymeric substances, a key element in understanding biofilm phenotype.

One of the key elements in the establishment and maintenance of the biofilm structure and properties is the extracellular matrix. The extracellular matrix is composed of water and extracellular polymeric substances (EPS): primarily polysaccharides, proteins and DNA. Characterization of the matrix requires component identification, as well as determination of the relative concentration of EPS constituents, including their physicochemical properties and descriptions of their interactions. Several types of experimental approaches with varying degrees of destructiveness can be utilized for this characterization. The analysis of biofilm by infrared spectroscopy gives information about the chemical content of the matrix and the proportions of different EPS. The sensitivity of a biofilm to hydrolytic enzymes targeting different EPS gives insight into the composition of the matrix and the involvement of matrix components in the integrity of the structure. Using both chemical and physical treatments, extraction and purification of EPS from the biofilm also provides a means of determining matrix composition. Purified and/or artificial EPS can be used to obtain artificial matrices and to study their properties. Using examples from the literature, this review will illustrate selected technologies useful in the study of EPS that provide a better understanding of the structure-function relationships in extracellular matrix, and thus the structure-function relationships of the biofilm phenotype.

Effects of Fibronectin Coating on Bacterial and Osteoblast Progenitor Cells Adherence in a Co-culture Assay.

Bacterial adherence to the surface of implants functionalized with cell-adhesive biomolecules is a critical first step of infection development. This study was designed to determine how the immobilization of human plasmatic fibronectin (pFN) could impact bacterial and osteoblast cells interaction with the surface during concomitant exposition to the two cell-types. Calibrated suspensions of P. aeruginosa PAOI or S. aureus CIP4.83 bacteria and STRO-1+A osteoblast progenitor cells were mixed, co-seeded on glass coverslips coated or not with pFN and incubated at 37 °C. After 3 h of co-culture, the presence of bacteria did not modify the STRO-1+A cells adherence to glass. pFN coating significantly enhanced STRO-1+A cells, CIP4.83 and PAOI adherence to glass and bacterial interaction with STRO-1+A cells. Confocal laser scanning microscopy observations revealed that cells on the pFN-coated substrate exhibited a greater spreading, better organized network of cytoskeletal filaments, and an increased cellular FN expression than cells on the uncoated substrate. The use of fluorescently labeled pFN showed that adherent STRO-1+A cells were able to remodel and to concentrate coated pFN at the cells surface. Thus, the use of FN coating could increase the risk of bacterial adherence to the material surface, acting either directly onto the coating layer or indirectly on adherent osteoblastic cells. This may increase the infection risk in the presence of bacterial contamination.

Synergistic antibiofilm efficacy of various commercial antiseptics, enzymes and EDTA: a study of Pseudomonas aeruginosa and Staphylococcus aureus biofilms.

A multistep strategy was used to generate a combined antibiofilm treatment that could efficiently decrease the biomass of dense biofilms (≥6 × 10(7) CFU/cm(2)). Several compounds that exhibited activity against various targets were tested individually and in combination to search for possible synergistic effects. First, the antibiofilm activity of various commercially available antiseptics was tested on Pseudomonas aeruginosa and Staphylococcus aureus. Second, antiseptics were mixed with ethylene diamine tetra-acetic acid (EDTA), which is an ion chelator that can disturb biofilm organisation, and additive effects on biofilm biomass degradation were found for both strains. Then, enzymes with the ability to destabilise the biofilm matrix by hydrolysing either its proteins or its polysaccharides were used; as expected, they did not decrease bacterial viability but were revealed as efficient biomass reducers. The combination of antiseptics, EDTA and proteases, all at low concentrations, revealed a synergistic effect leading to total eradication of dense biofilms both of P. aeruginosa and S. aureus.

Bap: A New Type of Functional Amyloid.

Bacteria can build a biofilm matrix scaffold from exopolysaccharides or proteins, and DNA. In a recent report, Taglialegna and colleagues show that pathogenic Staphylococcus aureus produces a protein scaffold based on amyloid assembly of fragments from the biofilm-associated protein. Amyloidogenesis occurs in response to environmental signals.

The Hexapeptide Repeated Segment LIAGY is a Hot Spot of Aggregation of the Pseudomonas syringae Ice Nucleation Protein.

Ice nucleation proteins (INPs) form oligomeric structures by self-assembly and aggregation. We looked for the presence of potential aggregating sequences inside the INP from Pseudomonas syringae by a computational approach with the AGGRESCAN, FOMDAMYLOID and TANGO softwares. A total of 38 hot spots of aggregation were predicted in the INP sequence: 7 localized in the Nterminal domain, 2 in the C-terminal region, 28 in the highly repetitive central (HRC) region and 1 shared between the HRC and the Carboxyl-terminus regions of the protein. All the hot spots of aggregation identified in the HRC domain overlapped a 8-residue low fidelity repeat including a LIAGYrelated sequence. We confirmed the predictions by an experimental approach using synthetic peptides corresponding to different parts of the INP central sequence, absorbance spectroscopy and fluorescence spectroscopy in the presence of Congo red (CR) or Thioflavin T (ThT), respectively. Peptide 620-SFIIAGYG-627 predicted to aggregate by the three softwares induced an increase in fluorescence of ThT. Peptide 729-GFKSILTAGY-738 predicted to aggregate by AGGRESCAN and FOLDAMYLOID induced a shift in the maximum of absorbance of CR. Peptide 1124-SVLTAGA-1130 predicted to aggregate only by TANGO did not interfere with CR absorbance or ThT fluorescence. In conclusion, the use of three aggregation prediction algorithms and two biochemical assays showed that the hexapeptide repeated segment LIAGY, previously shown to form a hairpin loop may be involved in the aggregation of the P. syringae INP.