Bacterial Wall Research Articles

The Impact of an Extended Bleed-to-evisceration Interval on the Microbiological Quality of On-farm Slaughtered Cattle Carcasses

The uncertainties surrounding the microbiological risks of an extended exsanguination-to-evisceration interval have limited the implementation of on-farm slaughter in Europe. On-farm slaughter is increasingly advocated by farmers, consumers, and policymakers as a humane alternative to traditional slaughterhouse operations. However, concerns about hygiene and food safety, particularly bacterial contamination, have led to stringent time limits imposed by Member States on the interval between bleeding and evisceration. Microbiological standards for bovine carcasses in the European Union are governed by Commission Regulation (EC) No 2073/2005, which sets process hygiene criteria for aerobic colony count and Enterobacteriaceae. To investigate whether extending the bleed-to-evisceration interval compromises meat safety, five Holstein dairy cattle were slaughtered on-farm, with samples collected from the internal paralumbar area in contact with the intestines for up to four hours postmortem. The samples were analyzed for Enterobacteriaceae, aerobic colony count, and Escherichia coli. None of the samples exceeded the established thresholds of 1.5 and 3.5 log CFU/cm2 for aerobic colony count and Enterobacteriaceae, respectively, as defined by Belgian health authorities for the nondestructive sampling method. These preliminary findings suggest that on-farm slaughter with evisceration occurring up to four hours postmortem does not pose increased microbiological risks to human health. However, further research is necessary, particularly under warmer environmental conditions and with a larger sample size, to confirm these results and to explore additional factors that may influence bacterial translocation and digestive tract wall integrity.

Preparation, in vitro and in vivo assessment of novel carvacrol@pro-phytomicelles for the treatment of Salmonella enteritidis infection in mice.

In livestock and poultry farming, the use of antibiotics has been abused, which seriously endangers human health. Thus, antibiotic alternatives are urgently needed. The phytochemical carvacrol (CAR) has attracted attention as an antibiotic alternative due to its excellent antibacterial activity and anti-inflammatory activity. However, CAR has high volatility and low water solubility, which seriously affect its antibacterial activity. In this study, two plant-derived small-molecule phytochemicals-glycyrrhizin and rebaudioside A-were selected as nanocarriers for the preparation of a novel solid pro-phytomicelle formulation named as CAR@PP. Using a simple fabrication method, the encapsulation efficiency of CAR reached 98.74 ± 1.14 %. CAR@PP was found to rapidly dissolve in water, resulting in a transparent solution (named as CAR@M) and a 59-fold increase in solubility compared to CAR. CAR@M contained uniform nanoparticles with a particle size, polydispersity index, and zeta potential of 3.52 ± 0.93 nm, 0.17 ± 0.01, and -10.63 ± 0.45 mV, respectively. The in vitro antibacterial activity of CAR@M was evaluated, and the minimum inhibitory concentration for the tested strains was 125-250 μg/ml. The antibacterial mechanisms were found that CAR@M disrupted the bacterial wall and biomembranes and efficiently inhibited bacterial biofilm growth. To the in vivo activity evaluation, treatment with 50 mg/kg CAR@M could effectively improve bacterial liver abscesses, decrease the inflammatory cytokine levels in the liver and cecum, and reduce the bacterial load in the liver and feces in Salmonella enteritidis-infected mice. In conclusion, CAR@PP is a promising alternative to antibiotics in livestock and poultry farming warranting further research.

Golden Guardians: Advanced pH Sensors with Built‐in Antimicrobial Defense

AbstractGold nanoclusters (Au NCs) capped with a small biomolecule, namely (+)‐norephedrine, (Au@Nore) display exceptional performance as fluorescent pH sensors. They display high emission quantum yield (ΦPL of ≈33% in strongly basic media); absence of nanocluster (NC) aggregation and/or induced rigidification of the organic shell; a linear dependency of their emission on the pH in the 8.2–9.5 range; and reversible pH sensing. In addition, excitation at 310 nm, where both the NC and the ligand absorb, enables ratiometric sensing. Studies on the fungus Candida albicans and bacterium Bacillus subtilis demonstrate that while Au@Nore do not cause any impact on the growth of the fungus, it inhibited the growth of the Bacillus, showing the lowest cell viability of 18% at the highest pH values via a mechanism of action involving the disruption of the bacterial wall.

The Effect of Lipopolysaccharide Challenge in RAW 264.7 Cells on Nitric Oxide Release and Cell Viability

Lipopolysaccharide (LPS) is a major component of a gram-negative bacterial wall that is widely used and well-established to induce inflammation in vitro. In addition, the in vitro model using RAW 264.7 cells is the most commonly applied in screening the anti-inflammatory and elucidating the pathophysiology of inflammation-based disease, as well. However, there is still limited data on the efficacy of different doses of LPS in inducing inflammation in RAW 264.7 cells. This study aimed to evaluate the effect and safety of LPS at various doses in RAW 264.7 cells. RAW 264.7 cells were exposed to LPS at different dose ranges (10ng/mL-10µg/mL) for 24 hours. The nitric oxide (NO) release as inflammatory responses and viability test were evaluated using Griess assay and CCK-8 assays, respectively. The result showed that NO production was increased at different doses of LPS compared to the control although not significant. Whereas, All LPS-treated RAW 264.7 cells tended to increase but not significantly compared to the control groups. This study showed that the LPS treatment effectively induced inflammation in RAW 264.7 cells as shown by NO production and was considerably safe as the viability was comparable between LPS and control group for RAW cells 264.7 at least up to 10µg/mL for 24 hours.

Synergism of colistin and globular endolysins against multidrug-resistant gram-negative bacteria

Endolysins (lysins), a novel class of antibacterial agents derived from bacteriophages, efficiently lyse bacteria by degrading the peptidoglycan layer within the bacterial wall. Colistin, a classic peptide antibiotic with the ability to permeabilize the outer membrane, has recently shown great promise in synergizing with lysins against gram-negative bacteria. However, the exact mechanisms responsible for their synergy remain unclear. Here, we first demonstrated the synergistic bacterial killing of various lysin and colistin combinations. With a model lysin, LysAB2, we then confirmed that there is a threshold concentration of colistin causing sufficient permeabilization of the outer membrane for lysin to access the peptidoglycan layer and subsequently exert its lytic ability. The threshold colistin concentrations were found to range 0.2−0.8 μM for the tested bacteria, with the exact value largely depending on the density of lipopolysaccharides on the outer membrane. Beyond the threshold colistin level, LysAB2 could synergize with colistin at a concentration as low as 0.31 μM. Next, we proved for the first time that lysin-induced degradation of the peptidoglycan layer facilitated the disruption of cytoplasmic membrane by colistin, elevated the level of reactive oxygen species in bacterial cells, and boosted the killing effect of colistin. Additionally, the colistin-lysin combination could effectively eliminate established biofilms due to the biofilm dispersal ability of lysin. The in-vivo efficacy was preliminary confirmed in a Galleria mellonella infection model for combination with colistin doses (≥ 1.8 μg/larvae), which could reach beyond the threshold concentration, and a fixed LysAB2 dose (10 μg/larvae). In summary, our study provided the first experimental evidence unravelling the mechanisms behind the synergy of colistin and lysins. All these findings provided important insights in guiding the dosing strategy for applying this combination in future development.

Abstract Tu039: Intestinal atrophy contributes to low-grade inflammation, cardiac dysfunction and metabolic syndrome-like phenotype in a model of heart failure with preserved ejection fraction.

Introduction: Heart failure with preserved ejection fraction (HFpEF) comprises half of all HF cases and lacks effective treatment. HFpEF is now seen as a systemic syndrome often linked to comorbidities like hypertension, obesity, and diabetes. These conditions are commonly associated with unhealthy diets rich in fats but lacking in fiber. We explored the effects of a 3-week exposure to excess mineralocorticoids, salt, and high fat diet (HFD) deficient in fiber. We hypothesize that these challenges lead to intestinal atrophy, contributing to low-grade inflammation that promotes the progression of heart failure and metabolic disease. Methods: We used a mix of 1% saline in drinking water and subcutaneous implantation of controlled-release DOCA pellets (0.83 mg/day) alongside a high-fat diet (60% fat) in 8-week-old female and male C57BL6/J mice. Control mice received a standard chow diet and regular water. Results: The challenge resulted in significant reductions in colon mass, colon length, crypt length, and mucus layer thickness compared to the control group. Additionally, elevated levels of C-reactive protein and IL-6 were detected in the plasma. These changes were accompanied by alterations in diastolic function, including decreased peak myocardial relaxation velocity (e'), elevated E/e' ratio, increased E/A ratio, and prolonged isovolumetric relaxation time (IVRT), while the ejection fraction (EF) remained unchanged. Contractility parameters, such as peak myocardial contraction velocity (s'), declined in challenged mice, along with diminished stroke volume (SV). We also found increased organ weight in the liver, spleen, and lungs indicative of organ congestion. Furthermore, signs of metabolic syndrome were identified, including increased body weight, increased adiposity characterized by gonadal and subcutaneous fat accumulation, and dysglycemia (see Data Table). Conclusions: Our data suggests that in this model, heart failure triggers intestinal hypoperfusion, resulting in intestinal atrophy and compromising the integrity of the intestinal lining. Additionally, a diet rich in fat and lacking in fiber alters the gut environment by diminishing mucus production and promoting a closer interaction between the microbiome and host cells. This compromised mucus layer and intestinal barrier facilitates bacterial wall products to move into the circulation, resulting in a pro-inflammatory state that subsequently affects both cardiac function and metabolism.

Click chemistry-based fluorescence polarization sensor for sensitive detection of ampicillin

Ampicillin (AMP) is a β-lactam antibiotic that can inhibit bacterial wall synthesis. The overuse and misuse of AMP makes it micropollutant that commonly found in food and various environmental media. In this work, a fluorescence polarization sensor was designed to sensitive detection of trace ampicillin based on click chemistry, using graphene oxide (GO) as a fluorescence polarization (FP) signal enhancement element. First, when ampicillin binds to its aptamer (apt), the adjacent alkyne and azide groups are separated, hindering the click-linking reaction. When Carboxyfluorescein (FAM) fluorophore-labeled probe (C-FAM) is added, its protruding 3-terminal FAM is recognized and cleaved by exonuclease I (EXO I), releasing fluorophores free that could not be adsorbed on GO, resulting in a lo0wer polarization signal. If there is no AMP in the system, aptamer probe is connected to its complementary chain ends by a click reaction. After C-FAM hybridizes with apt, the apt/P duplex is opened and the prominent single-stranded ends adsorb on the GO, leading a significantly enhanced FP signal. According to the relationship between the difference in FP values and the concentrations of AMP, the limit of detection of proposed method is as low as 80 pg/mL. This assay has a wide linear range plus excellent selectivity, and has been applied to detect AMP in milk and river water samples with satisfactory results, which demonstrates that the FP sensor has great potential for practical applications in food safety and environmental protection fields.

Ultrasmall Ag6Cu2 clusters for accelerating wound healing through the activation of antibacterial activity and endogenous anti-inflammatory property

Significal challenges exist to bacterial infection wound healing. The bacterial infection and inflammation generated by oxidative stress inevitably hinder the process of wound healing. Ag6Cu2 nanoclusters in this study were successfully prepared and displayed excellent physiological stability. Noticeably, Ag6Cu2 nanoclusters exhibited efficient therapeutic potentials for accelerating the process of wound healing, which had good biosafety under the appropriate concentration. The treatment of Ag6Cu2 was able to suppress the bacterial proliferation by destructing the bacterial, resulting in the secondary release of bacterial contents and to exert anti-inflammatory properties via scavenging the overproduction of reactive oxygen species and upregulating the expression of Nrf2 as well as its downstream genes including HO-1 and NQO1. In-vivo studies further validated the efficient therapeutic effects of Ag6Cu2 nanoclusters by inhibiting the activation of the cascade of inflammatory factors and the proliferation of bacteria as a novel agent in a nano scale for accelerating the process of wound healing.

In Silico Design and In Vitro Assessment of Bicyclic Trifluoromethylated Pyrroles as New Antibacterial and Antifungal Agents.

QSAR studies on the number of compounds tested as S. aureus inhibitors were performed using an interactive Online Chemical Database and Modeling Environment (OCHEM) web platform. The predictive ability of the developed consensus QSAR model was q2=0.79±0.02. The consensus prediction for the external evaluation set afforded high predictive power (q2=0.82±0.03). The models were applied to screen a virtual chemical library with anti-S. aureus activity. Six promising new bicyclic trifluoromethylated pyrroles were identified, synthesized and evaluated in vitro against S. aureus, E. coli, and A. baumannii for their antibacterial activity and against C. albicans, C. krusei and C. glabrata for their antifungal activity. The synthesized compounds were characterized by 1H, 19F, and 13C NMR and elemental analysis. The antimicrobial activity assessment indicated that trifluoromethylated pyrroles 9 and 11 demonstrated the greatest antibacterial and antifungal effects against all the tested pathogens, especially against multidrug-resistant strains. The acute toxicity of the compounds to Daphnia magna ranged from 1.21 to 33.39 mg/L (moderately and slightly toxic). Based on the docking results, it can be suggested that the antibacterial and antifungal effects of the compounds can be explained by the inhibition of bacterial wall component synthesis.

White light-driven enhanced cerium-doped carbon dots activity to combat multidrug-resistant bacterial infection

Infections caused by multidrug-resistant (MDR) bacteria are increasing and becoming an urgent global health crisis. The discovery and development of novel antibacterial agents to combat MDR are highly desirable. Here, we report the fabrication of cerium-doped carbon dots (CeCDs) with a simple hydrothermal method, which exhibit intrinsic broad efficacy against MDR bacteria including clinical isolates while maintaining low cytotoxicity and hemolytic effects. Importantly, the antibacterial activity of CeCDs is dramatically improved owing to the generation of reactive oxygen species (ROS) upon white light irradiation. Comprehensive analyses revealed that the CeCDs can penetrate the bacterial wall, disrupt the cell membrane, and prevent the biofilm formation, possibly hindering the bacterial resistance development. And the interaction of CeCDs with lipopolysaccharide (LPS) may contribute to the higher activity against Gram-negative bacteria strains. The treatment of CeCDs in a murine skin infection model can significantly reduce the number of bacteria on infected sites and accelerate wound healing by irradiation with light. Overall, CeCDs show great promise as low-cost and efficient antibacterial agents for chronic wounds and may be served as a powerful weapon to fight against the growing threat of MDR bacterial infection.

Expression and basic biochemical characteristics of recombinant surfactant protein D of bottlenose dolphin (Tursiopstruncatus)

We previously identified surfactant protein D (SP-D) in the bottlenose dolphin Tursiops truncatus as a unique evolutionary factor of the cetacean pulmonary immune system. In this short report, recombinant SP-D of bottlenose dolphin (dSP-D) was synthesized in mammalian cells, and its properties were analyzed in vitro. The recombinant proteins were purified using Ni-carrier or Co-carrier. Sodium dodecyl sulfate poly-acrylamide gel electrophoresis and western blotting revealed a 50 kDa major band with minor secondary bands. Enzyme-linked immunosorbent assay-like methods revealed that recombinant dSP-D bonded to gram-positive and gram-negative bacterial walls. Our findings suggest the clinical usefulness of dSP-D for cetacean pneumonia.

Concanavalin A Delivers a Photoactive Protein to the Bacterial Wall.

Modular supramolecular complexes, where different proteins are assembled to gather targeting capability and photofunctional properties within the same structures, are of special interest for bacterial photodynamic inactivation, given their inherent biocompatibility and flexibility. We have recently proposed one such structure, exploiting the tetrameric bacterial protein streptavidin as the main building block, to target S. aureus protein A. To expand the palette of targets, we have linked biotinylated Concanavalin A, a sugar-binding protein, to a methylene blue-labelled streptavidin. By applying a combination of spectroscopy and microscopy, we demonstrate the binding of Concanavalin A to the walls of Gram-positive S. aureus and Gram-negative E. coli. Photoinactivation is observed for both bacterial strains in the low micromolar range, although the moderate affinity for the molecular targets and the low singlet oxygen yields limit the overall efficiency. Finally, we apply a maximum entropy method to the analysis of autocorrelation traces, which proves particularly useful when interpreting signals measured for diffusing systems heterogeneous in size, such as fluorescent species bound to bacteria.

Visible-Light-Responsive Novel Ru(II)-Metallo-Antibiotics with Potential Antibiofilm and Antibacterial Activity.

Growing challenges with antibiotic resistance pose immense challenges in combating microbial infections and biofilm prevention on medical devices. Lately, antibacterial photodynamic therapy (aPDT) is now emerging as an alternative therapy to overcome this problem. Herein, we synthesized and characterized four Ru(II)-complexes, viz., [Ru(ph-tpy)(bpy)Cl]PF6 (Ru1), [Ru(ph-tpy)(dpq)Cl]PF6 (Ru2), [Ru(ph-tpy)(dppz)Cl]PF6 (Ru3), and [Ru(ph-tpy)(dppn)Cl]PF6 (Ru4) (where 4'-phenyl-2,2':6',2″-terpyridine = ph-tpy; 2,2'-bipyridine = bpy; dipyrido[3,2-f:2',3'-h]quinoxaline = dpq; dipyrido[3,2-a:2',3'-c]phenazine = dppz; and Benzo[I]dipyrido[3,2-a:2',3'-c]phenazine = dppn), among which Ru2-Ru4 are novel. Octahedral geometry of the complexes with a RuN5Cl core was evident from the crystal structure of Ru2. Ru1-Ru4 showed an MLCT absorption band in the 450-600 nm region, useful for aPDT performances. Further, optimum triplet excited state energy and excellent photostability of Ru1-Ru4 made them good photosensitizers for aPDT. Ru1-Ru4 demonstrated enhanced antimicrobial activity on visible-light exposure (400-700 nm, 10 J cm-2), confirmed using different antibacterial assays. Mechanistic studies revealed that inhibition of bacterial growth was due to the generation of oxidative stress (via NADH oxidation and ROS generation) upon treatment with Ru2-Ru4, resulting in destruction of the bacterial wall. Ru2 performed best killing performance against both Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtilis) bacteria when exposed to light. Ru2-Ru4, when coated on a polydimethylsiloxane (PDMS) disk, showed long-term reusability and durable antibiofilm properties. Molecular docking confirmed the efficient interaction of Ru2-Ru4 with FabH (regulates fatty acid biosynthesis of E. coli) and PgaB (gives structural stability and helps biofilm formation of E. coli), resulting in probable downregulation. In vivo studies with healthy Wistar rats confirmed the biocompatibility of Ru2. This study shows that these lead complexes (Ru2-Ru4) can be used as potent alternative antimicrobial agents in low concentrations toward bacterial eradication with photodynamic therapy (PDT).

Label-free aptasensor targeting Staphylococcus aureus surface Protein A

The rapid detection of Staphylococcus aureus (S. aureus) is of major importance for the biomedical world in the context where the antimicrobial resistance (AMR), the “silent pandemic”, is considered a global public health threat to humanity. There are various approaches to detect S. aureus either as a whole bacterium or via different biomarkers that indicate the presence of this microorganism. Herein, a label-free aptasensor was designed for the detection of a cellular bacterial wall component, protein A (PrA), as a biomarker for the bacteria presence. A rapid procedure, under 5 min, that involves the deposition of the aptamer at the surface of a gold-based screen-printed electrode was applied followed by a blocking step to prevent non-specific adsorption of the target. After the incubation with PrA, the electrochemical detection was achieved, the aptasensor exhibiting good sensitivity across a wide linear range from 25 nM to 1000 nM, with a limit of detection (LOD) of 8.33 nM in standard solutions of the target. S. aureus was detected in commercial human serum and culture samples displaying a good correlation of the signal and the number of bacteria colonies, in the latter case.

Ambient long-term exposure to organophosphorus pesticides and the human gut microbiome: an observational study

BackgroundOrganophosphorus pesticides (OP) have been associated with various human health conditions. Animal experiments and in-vitro models suggested that OP may also affect the gut microbiota. We examined associations between ambient chronic exposure to OP and gut microbial changes in humans.MethodsWe recruited 190 participants from a community-based epidemiologic study of Parkinson’s disease living in a region known for heavy agricultural pesticide use in California. Of these, 61% of participants had Parkinson’s disease and their mean age was 72 years. Microbiome and predicted metagenome data were generated by 16S rRNA gene sequencing of fecal samples. Ambient long-term OP exposures were assessed using pesticide application records combined with residential addresses in a geographic information system. We examined gut microbiome differences due to OP exposures, specifically differences in microbial diversity based on the Shannon index and Bray–Curtis dissimilarities, and differential taxa abundance and predicted Metacyc pathway expression relying on regression models and adjusting for potential confounders.ResultsOP exposure was not associated with alpha or beta diversity of the gut microbiome. However, the predicted metagenome was sparser and less evenly expressed among those highly exposed to OP (p = 0.04). Additionally, we found that the abundance of two bacterial families, 22 genera, and the predicted expression of 34 Metacyc pathways were associated with long-term OP exposure. These pathways included perturbed processes related to cellular respiration, increased biosynthesis and degradation of compounds related to bacterial wall structure, increased biosynthesis of RNA/DNA precursors, and decreased synthesis of Vitamin B1 and B6.ConclusionIn support of previous animal studies and in-vitro findings, our results suggest that ambient chronic OP pesticide exposure alters gut microbiome composition and its predicted metabolism in humans.

Gut microbiota – a new link in the pathogenesis of osteoarthritis (literature review)

The intestinal microbiota (IMB) can indirectly affect the course of ostearthritis (OA) at the systemic level by stimulating a chronic nonspecific inflammatory reaction in the synovial membrane and subchondral bone, the cause of which is an increase in the amount of circulating lipopolysaccharides (LPS) of the bacterial wall, as well as provoke the development of metabolic syndrome, which links the two necessary components of the pathogenesis of OA. The result of direct exposure is the formation of leaky gut syndrome with the activation of LPS of the bacterial wall of mild inflammation, provoking the production of proinflammatory cytokines, the effect of which on synoviocytes and chondrocytes leads to their activation with subsequent production of IL-6 and IL-8, which contributes to the persistence of inflammation. By correlation analysis, the relationship of three taxa with OA joint damage was proved, namely, the order Desulfovibrionales and the genus Ruminiclostridium 5 – with knee joint OA, Methanobacteriaceae – with knee joint OA, and OA of any localization, and the appearance and degree of contamination with the genus Streptococcus correlated with the severity of pain syndrome. The metabolic syndrome itself can provoke the development of dysbiosis, so it can also be its consequence. A change in the composition of the microbiota in the form of the predominance of the genus Clostridium and the species Staphylococcus aureus with a decrease in the diversity of microorganisms is associated with an increase in the amount of adipose tissue in the body, dyslipidemia, insulin resistance with impaired carbohydrate metabolism. Low levels of LPS in the blood are found in obese patients even in the absence of an obvious focus of infection due to violations in the CMB, they signal TLR-4, triggering systemic inflammation. Studies have shown a positive effect of prescribing proand prebiotics on the course of OA, which makes IMB a promising target for the treatment and prevention of OA.

Convolutional Neural Network-Driven Impedance Flow Cytometry for Accurate Bacterial Differentiation.

Impedance flow cytometry (IFC) has been demonstrated to be an efficient tool for label-free bacterial investigation to obtain the electrical properties in real time. However, the accurate differentiation of different species of bacteria by IFC technology remains a challenge owing to the insignificant differences in data. Here, we developed a convolutional neural networks (ConvNet) deep learning approach to enhance the accuracy and efficiency of the IFC toward distinguishing various species of bacteria. First, more than 1 million sets of impedance data (comprising 42 characteristic features for each set) of various groups of bacteria were trained by the ConvNet model. To improve the efficiency for data analysis, the Spearman correlation coefficient and the mean decrease accuracy of the random forest algorithm were introduced to eliminate feature interaction and extract the opacity of impedance related to the bacterial wall and membrane structure as the predominant features in bacterial differentiation. Moreover, the 25 optimized features were selected with differentiation accuracies of >96% for three groups of bacteria (bacilli, cocci, and vibrio) and >95% for two species of bacilli (Escherichia coli and Salmonella enteritidis), compared to machine learning algorithms (complex tree, linear discriminant, and K-nearest neighbor algorithms) with a maximum accuracy of 76.4%. Furthermore, bacterial differentiation was achieved on spiked samples of different species with different mixing ratios. The proposed ConvNet deep learning-assisted data analysis method of IFC exhibits advantages in analyzing a huge number of data sets with capacity for extracting predominant features within multicomponent information and will bring about progress and advances in the fields of both biosensing and data analysis.

The interplay between bacterial biofilms, encrustation, and wall shear stress in ureteral stents: a review across scales

Ureteral stents are hollow tubes that are inserted into the ureter to maintain the flow of urine from the kidney to the bladder. However, the use of these indwelling stents is associated with potential complications. Biofilm, an organized consortium of bacterial species embedded within a self-producing extracellular matrix, can attach to the outer and inner surfaces of ureteral stents. Furthermore, encrustation - defined as the buildup of mineral deposits on the stent surface - can occur independently or in parallel with biofilm formation. Both phenomena can cause stent obstruction, which can lead to obstructive pyelonephritis and make stent removal difficult. Understanding the influence of flow on the development of biofilm and encrustation and the impact of small mechanical environmental changes (e.g., wall shear stress distribution) is key to improve the long-term performance of stents. Identifying the optimal stent properties to prevent early bacterial attachment and/or crystal deposition and their growth, would represent a breakthrough in reducing biofilm-/encrustation-associated complications. This review identifies the most prevalent bacterial strains and crystal types associated with ureteral stents, and the process of their association with the stent surface, which often depends on patient comorbidities, stent material, and indwelling time. Furthermore, we focus on the often-overlooked role of fluid dynamics on biofilm and encrustation development in ureteral stents, across a range of physical scales (i.e., from micro- to macro-scale) with the aim of providing a knowledge base to inform the development of safer and more effective ureteral stents.

Chemical inhibition of cell surface modification sensitizes bacteria to phage infection.

Many bacteriophages that infect Gram-positive bacteria rely on the bacterial cell surface polymer wall teichoic acid (WTA) as a receptor. However, some bacteria modulate their cell wall with d-alanine residues, which can disrupt phage adsorption. The prevalence and significance of WTA alanylation as an anti-phage defense is unknown. A chemical inhibitor of WTA d-alanylation could be employed to efficiently screen phage-host combinations for those that exhibit alanylation-dependent infections. Since the incorporation of d-alanine residues into the cell wall requires the activity of d-alanine:alanyl carrier protein ligase (DltA), a DltA inhibitor was employed as this tool. Herein, we found that a chemical probe inhibiting DltA activity impeded bacterial cell wall alanylation and enhanced infectivity of many phages against Bacillus subtilis, including phages Phi29, SPP1, SPO1, SP50, and Goe2. This finding reveals the breadth of immunity conferred by WTA alanylation in B. subtilis, which was previously known to impact only phages Phi29 and SPP1, but not SPO1, SP50, or Goe2. DltA inhibition selectively promoted infection by several phages that bind WTA, having no impact on the flagellotropic phage PBS1. Unexpectedly, DltA inhibition also had no effect on phage SP10, which binds to WTA. This selective chemical tool has the potential to unravel bacteriophage interactions with bacteria, leading to improved phage therapies in the future.

A unique combination of natural fatty acids from Hermetia illucens fly larvae fat effectively combats virulence factors and biofilms of MDR hypervirulent mucoviscus Klebsiella pneumoniae strains by increasing Lewis acid-base/van der Waals interactions in bacterial wall membranes.

Hypervirulent Klebsiella pneumoniae (hvKp) and carbapenem-resistant K. pneumoniae (CR-Kp) are rapidly emerging as opportunistic pathogens that have a global impact leading to a significant increase in mortality rates among clinical patients. Anti-virulence strategies that target bacterial behavior, such as adhesion and biofilm formation, have been proposed as alternatives to biocidal antibiotic treatments to reduce the rapid emergence of bacterial resistance. The main objective of this study was to examine the efficacy of fatty acid-enriched extract (AWME3) derived from the fat of Black Soldier Fly larvae (Hermetia illucens) in fighting against biofilms of multi-drug resistant (MDR) and highly virulent Klebsiella pneumoniae (hvKp) pathogens. Additionally, the study also aimed to investigate the potential mechanisms underlying this effect. Crystal violet (CV) and ethidium bromide (EtBr) assays show how AWME3 affects the formation of mixed and mature biofilms by the KP ATCC BAA-2473, KPi1627, and KPM9 strains. AWME3 has shown exceptional efficacy in combating the hypermucoviscosity (HMV) virulent factors of KPi1627 and KPM9 strains when tested using the string assay. The rudimentary motility of MDR KPM9 and KP ATCC BAA-2473 strains was detected through swimming, swarming, and twitching assays. The cell wall membrane disturbances induced by AWME3 were detected by light and scanning electron microscopy and further validated by an increase in the bacterial cell wall permeability and Lewis acid-base/van der Waals characteristics of K. pneumoniae strains tested by MATS (microbial adhesion to solvents) method. After being exposed to 0.5 MIC (0.125 mg/ml) of AWME3, a significant reduction in the rudimentary motility of MDR KPM9 and KP ATCC BAA-2473 strains, whereas the treated bacterial strains exhibited motility between 4.23 ± 0.25 and 4.47 ± 0.25 mm, while the non-treated control groups showed significantly higher motility ranging from 8.5 ± 0.5 to 10.5 ± 0.5 mm. In conclusion, this study demonstrates the exceptional capability of the natural AWME3 extract enriched with a unique combination of fatty acids to effectively eliminate the biofilms formed by the highly drug-resistant and highly virulent K. pneumoniae (hvKp) pathogens. Our results highlight the opportunity to control and minimize the rapid emergence of bacterial resistance through the treatment using AWME3 of biofilm-associated infections caused by hvKp and CRKp pathogens.