Preformed Biofilms Research Articles

The Synergistic and Chimeric Mechanism of Bacteriophage Endolysins: Opportunities for Application in Biotherapeutics, Food, and Health Sectors.

A major growing concern in the human and animal health sector is the emergence of antibiotic-resistant pathogenic bacteria due to the indiscriminate use of antibiotics. The exogenous application of bacteriophage endolysins causes abrupt lysis of the bacterial cell wall, which computes them as alternatives to antibiotics. Although naturally occurring endolysins may display limitations in solubility, lytic activity, and narrow lytic spectrum, novel strategies like developing chimeric endolysins and using endolysins in synergism with other antimicrobial agents are required to improve the lytic activity of natural endolysins. The modular structure of endolysins led to the development of novel chimeric endolysins via shuffling enzymatic and cell wall binding domains of different endolysins, using endolysins in a synergistic approach, and their applications in various in vitro and in vivo experiments and different applicable areas. This article aims to review the role of chimeric endolysins and their use in synergistic mode with other biofilm-reducing agents to control biofilm formation and deteriorating pre-formed biofilms in food, dairy, and medical industries. Promoting further development of phage technology and innovation in antibiotic therapy can achieve long-term sustainable development and economic returns.

Inhibitory Effect of DNase–Chitosan–Nisin Nanoparticles on Cell Viability, Motility, and Spatial Structures of Listeria monocytogenes Biofilms

Listeria monocytogenes biofilm contamination on food contact surfaces is a major concern for the food industry. Nanoparticle encapsulation appears as a novel strategy for food surface disinfection to prevent biofilm formation. Chitosan nanoparticles loaded with nisin and DNase I (DNase-CS-N) have been constructed to exhibit antimicrobial activity against L. monocytogenes. This study aimed to investigate their ability to inhibit L. monocytogenes biofilm formation and eliminate preformed biofilms on food contact surfaces (polystyrene, polyurethane, and stainless steel). DNase-CS-N could decrease 99% and 99.5% biofilm cell numbers at 1/2 MIC and MIC, respectively. At sub-MICs, DNase-CS-N could reduce cell motility (swimming and swarming) and slime production of L. monocytogenes. In terms of effect on biofilm elimination, DNase-CS-N at the concentration of 4 MIC led to 3–4 log reduction in biofilm cells in preformed biofilms, performing higher efficiency compared with other treatments (CSNPs, CS-N). Furthermore, the three-dimensional structure of L. monocytogenes biofilms was severely disrupted after DNase-CS-N treatment, with bacterial cells scattered on the surface. The morphology of biofilm cells was also greatly damaged with wrinkled surfaces, disrupted cell membranes, and leakage of intracellular nucleic acids and proteins. These results indicate the potential applicability of DNase-CS-N for inhibiting and eliminating L. monocytogenes biofilms on food contact surfaces.

Enhanced Antibacterial, Anti-Inflammatory, and Antibiofilm Activities of Tryptophan-Substituted Peptides Derived from Cecropin A-Melittin Hybrid Peptide BP100

The emergence of multidrug-resistant pathogens necessitates the development of novel antimicrobial agents. BP100, a short α-helical antimicrobial peptide (AMP) derived from cecropin A and melittin, has shown promise as a potential therapeutic. To enhance its efficacy, we designed and synthesized 16 tryptophan-substituted BP100 analogs based on helical wheel projections. Among these, BP5, BP6, BP8, BP11, and BP13 exhibited 1.5- to 5.5-fold higher antibacterial activity and improved cell selectivity compared to BP100. These analogs demonstrated superior efficacy in suppressing pro-inflammatory cytokine release in LPS-stimulated RAW 264.7 cells and eradicating preformed biofilms of multidrug-resistant Pseudomonas aeruginosa (MDRPA). Additionally, these analogs showed greater resistance to physiological salts and serum compared to BP100. Mechanistic studies revealed that BP100 and its analogs exert their antibacterial effects through membrane disruption, depolarization, and permeabilization. Notably, these analogs showed synergistic antimicrobial activity with ciprofloxacin against MDRPA. Our findings suggest that these tryptophan-substituted BP100 analogs represent promising candidates for combating multidrug-resistant bacterial infections, offering a multifaceted approach through their antibacterial, anti-inflammatory, and antibiofilm activities.

Batzelladine D, a Marine Natural Product, Reverses the Fluconazole Resistance Phenotype Mediated by Transmembrane Transporters in Candida albicans and Interferes with Its Biofilm: An In Vitro and In Silico Study

Numerous Candida species are responsible for fungal infections; however, Candida albicans stands out among the others. Treatment with fluconazole is often ineffective due to the resistance phenotype mediated by transmembrane transporters and/or biofilm formation, mechanisms of resistance commonly found in C. albicans strains. A previous study by our group demonstrated that batzelladine D can inhibit the Pdr5p transporter in Saccharomyces cerevisiae. In the present study, our aim was to investigate the efficacy of batzelladine D in inhibiting the main efflux pumps of Candida albicans, CaCdr1p and CaCdr2p, as well as to evaluate the effect of the compound on C. albicans biofilm. Assays were conducted using a clinical isolate of Candida albicans expressing both transporters. Additionally, to allow the study of each transporter, S. cerevisiae mutant strains overexpressing CaCdr1p or CaCdr2p were used. Batzelladine D was able to reverse the fluconazole resistance phenotype by acting on both transporters. The compound synergistically improved the effect of fluconazole against the clinical isolate when tested in the Caenorhabditis elegans animal model. Moreover, the compound disrupted the preformed biofilm. Based on the obtained data, the continuation of batzelladine D studies as a potential new antifungal agent and/or chemosensitizer in Candida albicans infections can be suggested.

Trans, Trans-Farnesol Enhances the Anti-Bacterial and Anti-Biofilm Effect of Arachidonic Acid on the Cariogenic Bacteria Streptococcus mutans and Streptococcus sobrinus.

Streptococcus mutans and Streptococcus sobrinus are Gram-positive bacteria involved in the development of dental caries, as they are able to form biofilms on tooth enamel, ferment sugars into acids, and survive under acidic conditions. This ultimately leads to a local lowering of the pH value on the tooth surface, which causes enamel cavities. One measure to reduce caries is to limit the growth of cariogenic bacteria by using two anti-bacterial agents with different mechanisms of action. The hypothesis of this study was that the anti-bacterial activity of ω-6 polyunsaturated arachidonic acid (AA) against S. mutans and S. sobrinus can be enhanced by the sesquiterpene alcohol trans, trans-farnesol (t,t-farnesol). The anti-bacterial activity of single and combined treatment was determined by the checkerboard assay. Bacterial viability was assessed by live/dead SYTO 9/propidium iodide (PI) staining on flow cytometry. Anti-biofilm activity was determined by MTT metabolic assay, crystal violet staining of biofilm biomass, SYTO 9/PI staining by spinning disk confocal microscopy (SDCM) and high-resolution scanning electron microscopy (HR-SEM). t,t-Farnesol lowered the minimum inhibitory concentration (MIC) and the minimum biofilm inhibitory concentration (MBIC) of AA at sub-MICs. AA reduced the metabolic activity of preformed mature biofilms, while t,t-farnesol had no significant effect. The enhanced anti-bacterial effect of the combined t,t-farnesol/AA treatment was further evidenced by increased PI uptake, indicating membrane perforation. The enhanced anti-biofilm effect was further verified by SDCM and HR-SEM. Gene expression studies showed reduced expression of some biofilm-related genes. Altogether, our study suggests a potential use of the two naturally occurring compounds arachidonic acid and t,t-farnesol for preventing biofilm formation by the cariogenic bacteria S. mutans and S. sobrinus. These findings have implications for caries prevention.

Anti-biofilm potential of red pitahaya betacyanins against Streptococcus mutans, Actinomyces viscosus and Aggregatibacter actinomycetemcomitans

This study investigated the antibiofilm effects of betacyanin fraction (BF) from red pitahaya against Streptococcus mutans, Actinomyces viscosus, and Aggregatibacter actinomycetemcomitans, key pathogens in oral biofilm formation. Betacyanin purification employed column chromatography, and characterization was performed using liquid chromatography-mass spectrometry. Crystal violet assay and scanning electron microscopy demonstrated significant inhibition of early biofilm formation and disruption of preformed biofilms using BF (0.94–15 mg/mL). Anti-adhesion assays on artificial teeth indicated potent betacyanin-mediated inhibition of both sucrose-dependent and -independent bacterial attachment. Notably, growth curve and pH drop assays revealed that BF hindered pH reduction for all tested bacteria without suppressing their growth. Tetrazolium-based cytotoxicity assays showed minimal toxicity towards normal human gingival fibroblasts (HGF-1) at 0.78–12.5 mg/mL. These findings demonstrate the potential of red pitahaya betacyanins as promising antibiofilm agents for oral health, targeting both initial and mature stages of biofilm development.

An Eco-friendly β-cyclodextrin/Stilbene-Integrated supramolecular material Realizes the effective treatment of bacterial diseases via enhancing the biofilm eradication and Agrochemical bioavailability

Bacterial biofilm-associated diseases have become the leading challenge for global food security. The inherently dense biofilm matrix provides bacteria with violent resistance to the host immune response and chemical attack. Current antimicrobials with weak biofilm-targeting options and inadequate foliar adhesion severely degrade the disruptor-biofilm interactions. To tackle these issues, the host–guest supramolecular technique was employed to optimize a bactericidal stilbene-decorated ingredient (StA8) by β-cyclodextrin (β-CD). Thus, a potent supramolecular bactericidal material (StA8@β-CD), owning both enhancive biofilm eradication and excellent foliar deposition properties, was successfully fabricated. This inclusion effectively disrupts pre-formed biofilms of Xanthomonas oryzae pv. oryzae (Xoo) and definitely kills planktonic and biofilm-enclosed bacteria. After aged Xoo cells for 48 h, subsequent StA8@β-CD treatment provides excellent biofilm eradication rate of 82.8 % at 15.68 μg mL−1. Mechanism researches disclose that StA8@β-CD inhibits exopolysaccharides (EPS) production, the transcription of swimming-associated genes, bacterial quorum sensing, and cellulase/amylase activity, all pivotal for biofilm formation and bacterial virulence. Also, this self-assembled material dramatically relieves droplet splash and rebound on hydrophobic rice leaves, thereby improving efficient utilization of agrochemicals. Combining these advantages, in vivo anti-Xoo experiments reveal that StA8@β-CD has excellent control efficiency of 50.8 % at 200 μg mL−1, quite superior to the thiodiazole-copper-20 %SC formulation (34.5 %). Besides, StA8@β-CD exhibits broad-spectrum bioactivities against citrus and kiwifruit cankers with outstanding control efficacies of 73.3 % and 73.7 %, respectively. This attempt provides a new direction for the exploitation of multipurpose supramolecular bactericidal materials.

Antimicrobial Activity of Corynebacterium amycolatum ICIS 53 and Corynebacterium amycolatum ICIS 82 Against Urogenital Isolates of Multidrug-Resistant Staphylococcus aureus.

Intermicrobial interactions play a key role in the regulation of microbial populations and the colonization of various ecological niches. In the present study, we assessed the effect of cell-free supernatants (CFSs) from the vaginal isolates Corynebacterium amycolatum ICIS 53 and Corynebacterium amycolatum ICIS 82 on urogenital test strain biofilm formation of Staphylococcus aureus. Our studies showed that the CFSs of both C. amycolatum strains significantly reduced biofilm formation and disrupted preformed S. aureus biofilms. Pretreatment with C. amycolatum ICIS 53 or C. amycolatum ICIS 82 CFSs decreased the cell surface hydrophobicity and exopolysaccharide production of all the test S. aureus isolates. The scanning electron microscopy (SEM) results showed that the CFSs of corynebacteria caused the S. aureus biofilms to be small clusters scattered across the surface, there were no fibres or adhesions between cells, and the cell membrane was not damaged. Treatment of preformed biofilms with CFSs from both C. amycolatum strains resulted in a flat, scattered, and unstructured architecture. The S. aureus cell membrane was damaged. GC‒MS analysis of the CFS of C. amycolatum ICIS 53 revealed the presence of 22 chemical compounds, including long-chain fatty alcohols, esters, fatty acids and heterocyclic pyrrolizines and pyrazoles, that, according to the literature, exhibit a wide range of biological activities. The results of the present work provide insight for the study of Corynebacterium microorganisms as a source of multifunctional bioactive compounds, which may find promising applications in the medical, biotechnological and pharmaceutical industries.

Inhibitory effect of Bacillus velezensis 1273 strain cell-free supernatant against developing and preformed biofilms of Staphylococcus aureus and MRSA

Microbial biofilms constitute a significant virulence factor and a substantial challenge in clinical environments due to their role in promoting antimicrobial resistance and their resilience to eradication efforts. Methicillin-resistant Staphylococcus aureus (MRSA) infections substantially increase healthcare costs, extend hospitalizations, and elevate morbidity and mortality rates. Therefore, developing innovative strategies to target and eliminate these bacteria and their biofilms effectively is imperative for robust epidemiological control. In this study, we evaluated the antibacterial and antibiofilm activities of cell-free supernatant (CFS) obtained from the Bacillus velezensis 1273 strain culture. Our data showed that CFS inhibited the growth of S. aureus ATCC 29213 and MRSA (clinical strain), with greater efficacy observed against S. aureus (1:16 dilution). Furthermore, CFS showed substantial potential in reducing biofilm formation in both strains (∼30 %) at subinhibitory concentrations. Additionally, the antibacterial activity against biofilm-formed cells showed that pure CFS treatment decreased the viability of S. aureus (60 %) and MRSA (45 %) sessile cells. We further demonstrated that CFS treatment induces the production of reactive oxygen species (ROS) and damages the membranes and cell walls of the pathogen cells. Genome analysis revealed the presence of genes encoding bacteriocins and secondary metabolites with antibacterial activity in the B. velezensis 1273 genome. These findings highlight the potential of probiotic bacterial metabolites as antibiofilm and anti-multidrug-resistant pathogens.

Activity of polymyxin B combined with cefepime-avibactam against the biofilms of polymyxin B-resistant Pseudomonas aeruginosa and Klebsiella pneumoniae in in vitro and in vivo models

Bacterial biofilms, often forming on medical devices, can lead to treatment failure due to their increased antimicrobial resistance. Cefepime-avibactam (CFP-AVI) exhibits potent activities against Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) when used with polymyxin B (PMB). However, its efficacy in biofilm-related infections is unknown. The present study aimed to evaluate the activity of PMB combined with CFP-AVI against the biofilms of PMB-resistant Gram-negative bacteria. Five K. pneumoniae strains and three P. aeruginosa strains known to be PMB-resistant and prone to biofilm formation were selected and evaluated. Antimicrobial susceptibility assays demonstrated that the minimal biofilm inhibitory and eradication concentrations of PMB and CFP-AVI for biofilms formed by the eight strains were significantly higher than the minimal inhibitory concentrations of the antibiotics for planktonic cells. The biofilm formation inhibition and eradication assays showed that PMB combined with CFP-AVI cannot only suppress the formation of biofilm but also effectively eradicate the preformed mature biofilms. In a modified in vitro pharmacokinetic/pharmacodynamic biofilm model, CFP-AVI monotherapy exhibited a bacteriostatic or effective activity against the biofilms of seven strains, whereas PMB monotherapy did not have any activity at 72 h. However, PMB combined with CFP-AVI demonstrated bactericidal activity against the biofilms of all strains at 72 h. In an in vivo Galleria mellonella infection model, the 7-day survival rates of larvae infected with biofilm implants of K. pneumoniae or P. aeruginosa were 0-6.7%, 40.0-63.3%, and 46.7–90.0%, respectively, for PMB alone, CFP-AVI alone, and PMB combined with CFP-AVI; the combination therapy increased the rate by 6.7–33.3% (P < 0.05, n = 6), compared to CFP-AVI monotherapy. It is concluded that PMB combined with CFP-AVI exhibits effective anti-biofilm activities against PMB-resistant K. pneumoniae and P. aeruginosa both in vitro and in vivo, and thus may be a promising therapeutic strategy to treat biofilm-related infections.

Disrupting Mycobacterium smegmatis biofilm using enzyme-immobilized rifampicin loaded silk fibroin nanoparticles for dual anti-bacterial and anti-biofilm action

Biofilm formation is a major challenge in the treatment of tuberculosis, leading to poor treatment outcomes and latent infections. The complex and dense extracellular polymeric substances (EPS) of the biofilm provides safe harbour for bacterium enabling persistence against anti-TB antibiotics. In this study, we demonstrated that rifampicin-encapsulated silk fibroin nanoparticles immobilized with antibiofilm enzymes can disrupt the Mycobacterium smegmatis biofilm and facilitate the anti-bacterial action of Rifampicin (RIF). The EPS of M.smegmatis biofilm predominantly comprised of lipids (48.8 ± 1.32 %) and carbohydrates (34.8 ± 4.70 %), similar to tuberculosis biofilms. Pre-formed biofilm eradication screening revealed that hydrolytic enzymes such as β-Glucosidase, Glucose oxidase, ɑ-Amylase, Acylase, and Phytase can exhibit biofilm eradication of M.smegmatis biofilms. The enzyme-mediated biofilm disruption was associated with a decrease in hydrophobicity of biofilm surfaces. Treatment with β-glucosidase and Phytase demonstrated a putative biofilm eradication by reducing the total carbohydrates and lipid composition without causing any significant bactericidal activity. Further, Phytase (250 μg/ml) and β-Glucosidase (112.5 ± 17.6 μg/ml) conjugated rifampicin-loaded silk fibroin nanoparticles (R-SFNs) exhibited an enhanced anti-bacterial activity against pre-formed M.smegmatis biofilms, compared to free rifampicin (32.5±7 μg/ml). Notably, treatment with β-glucosidase, Phytase and ɑ-amylase immobilized SFNs decreased the biofilm thickness by ∼98.84 % at 6h, compared to control. Thus, the study highlights that coupling anti-mycobacterial drugs with biofilm-eradicating enzymes such as amylase, phytase or β-glucosidase can be a potential strategy to improve the TB therapeutic outcomes.

Inhibition of Streptococcus pyogenes biofilm by Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus.

The human pathobiont Streptococcus pyogenes forms biofilms and causes infections, such as pharyngotonsillitis and necrotizing fasciitis. Bacterial biofilms are more resilient to antibiotic treatment, and new therapeutic strategies are needed to control biofilm-associated infections, such as recurrent pharyngotonsillitis. Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus are two bacterial commensals used for their probiotic properties. This study aimed to elucidate the anti-biofilm properties of L. plantarum and L. rhamnosus cell-free supernatants (LPSN and LRSN, respectively) on S. pyogenes biofilms grown in vitro in supplemented minimal medium. When planktonic or biofilm S. pyogenes were exposed to LPSN or LRSN, S. pyogenes survival was reduced significantly in a concentration-dependent manner, and the effect was more pronounced on preformed biofilms. Enzymatic digestion of LPSN and LRSN suggested that glycolipid compounds might cause the antimicrobial effect. In conclusion, this study indicates that L. plantarum and L. rhamnosus produce glycolipid bioactive compounds that reduce the viability of S. pyogenes in planktonic and biofilm cultures.IMPORTANCEStreptococcus pyogenes infections are a significant concern for populations at risk, such as children and the elderly, as non-invasive conditions such as impetigo and strep throat can lead to severe invasive diseases such as necrotizing fasciitis. Despite its susceptibility to current antibiotics, the formation of biofilm by this pathogen decreases the efficacy of antibiotic treatment alone. The ability of commensal lactobacillus to kill S. pyogenes has been documented by previous studies using in vitro settings. The relevance of our study is in using a physiological setup and a more detailed understanding of the nature of the lactobacillus molecule affecting the viability of S. pyogenes. This additional knowledge will help for a better comprehension of the molecules' characteristics and kinetics, which in turn will facilitate new avenues of research for its translation to new therapies.

Exploration of Antibiofilm Activities of Pediococci Strains Against Diarrheal Multi-Drug Resistant E.coli Strains

ABSTRACT The present study reports the potential of cell-free supernatant (CFS) from different probiotics for its antimicrobial and antibiofilm activity against Multi-Drug Resistant Diarrheal E. coli (MDR-DEC) strains. The CFS from Pediococcus acidilactici 100 exhibited maximum antimicrobial activity with Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of 23.33 mg/mL and 33.33 mg/mL, respectively. The CFS was able to inhibit the process of biofilm formation as well as eradication of preformed biofilms by 72–73%. A complete disintegration of aggregation of cells was observed in treated biofilms through advanced microscopic techniques. Atomic force microscopy (AFM) revealed a decrease in roughness (30%) and height of treated biofilms (2–17%). Organic acids, fatty acids, phenolic compounds and other derivatives were determined in CFS fractions using Liquid Chromatography Mass Spectrometry. The findings demonstrated the potential of CFS components from Pediococci strains against biofilms of MDR-DEC strains.

Thermosensitive multivesicular liposomal hydrogel: a potential platform for loco-regional drug delivery in the treatment of osteomyelitis caused by antibiotic-resistant biofilm-forming bacteria.

Biofilm-mediated osteomyelitis presents significant therapeutic challenges. Given the limitations of existing osteomyelitis treatment approaches, there is a distinct need to develop a localized drug delivery system that is biocompatible, biodegradable, and capable of controlled antibiotic release. Multivesicular liposomes (MVLs), characterized by their non-concentric vesicular structure, distinct composition, and enhanced stability, serve as the system for a robust sustained-release drug delivery platform. In this study, various hydrogel formulations composed of poloxamer 407 and other hydrogels, incorporating vancomycin hydrochloride (VAN HL)-loaded MVLs (VAN HL-MVLs), were prepared and evaluated. The optimized VAN HL-MVL sol-gel system, consisting of poloxamer 407 and hyaluronic acid, successfully maintained drug release for up to 3 weeks and exhibited shear-thinning behavior at 37°C. While complete drug release from MVLs alone took place in 312 h, the hydrogel formulation extended this release to 504 h. The released drug effectively inhibited the Staphylococcus aureus biofilms growth within 24 h and methicillin-resistant S. aureus biofilms within 72 h. It also eradicated preformed biofilms of S. aureus and methicillin-resistant S. aureus in 96 and 120 h, respectively. This injectable in situ gel system incorporating VAN HL-MVLs holds potential as an alternative to undergoing multiple surgeries for osteomyelitis treatment and warrants further studies.

Efficacy of orange terpene against Escherichia coli biofilm on beef and food contact surfaces

Foodborne pathogen Escherichia coli frequently causes foodborne infections. In our study, we investigated the antibiofilm activity of orange terpene (OT) against E. coli biofilms on a food surface (beef) and different surfaces that come into touch with food, including stainless steel (SS), polyethylene terephthalate (PET), low-density polyethylene (LDPE), and rubber (SR). The study findings revealed that OT significantly (P < 0.05) eliminated 48-h-old biofilms from all food contact surfaces (SS: 2.09 log CFU/cm2, PET: 1.95 log CFU/cm2, LDPE: 1.94 log CFU/cm2, and SR: 1.4 log CFU/cm2). Additionally, on beef surfaces, OT at a minimum inhibitory concentration (MIC) of 0.13 % demonstrated the ability to inhibit biofilm development by approximately 1.5 log CFU/cm2 and reduced pre-formed biofilms by 2.02 log CFU/cm2. Our sensory evaluations showed that it had no adverse impacts on beef color and texture, although it slightly altered the natural odor of beef. Quantitative and qualitative assessments showed that OT has strong bactericidal actions on biofilm populations. It significantly altered cell surface hydrophobicity, reduced cellular ATP levels, and inhibited cell auto-aggregation in planktonic cells (P < 0.05). As a result, our findings emphasize the antibacterial potentiality of OT in reducing the biofilm of E. coli in the food sector.

Combinatorial Effects of Ursodeoxycholic Acid and Antibiotic in Combating Staphylococcus aureus Biofilm: The Roles of ROS and Virulence Factors.

Staphylococcus aureus is a biofilm-forming bacterium responsible for various human infections, one particularly challenging to treat due to its antibiotic resistance. Biofilms can form on both soft tissues and medical devices, leading to persistent and hard-to-treat infections. Combining multiple antimicrobials is a potential approach to overcoming this resistance. This study explored the effects of ursodeoxycholic acid (UDCA) combined with the antibiotic ciprofloxacin against S. aureus biofilms, aiming to evaluate any synergistic effects. Results showed that UDCA and ciprofloxacin co-treatment significantly reduced biofilm formation and disrupted pre-formed biofilms more effectively than either agent alone (p < 0.01). The combination also displayed a slight synergistic effect, with a fractional inhibitory concentration of 0.65. Additionally, the treatment reduced the production of extracellular polymeric substances, increased reactive oxygen species production, decreased metabolic activity, altered cell membrane permeability, and lowered cell surface hydrophobicity in S. aureus. Furthermore, it diminished biofilm-associated pathogenic factors, including proteolytic activity and staphyloxanthin production. Overall, the UDCA-ciprofloxacin combination shows considerable promise as a strategy to combat infections related to staphylococcal biofilms, offering a potential solution to the healthcare challenges posed by antibiotic-resistant S. aureus.

Elucidating the molecular properties and anti-mycobacterial activity of cysteine peptidase domain of D29 mycobacteriophage endolysin.

Emergence of antibiotic resistance in pathogenic Mycobacterium tuberculosis (Mtb) has elevated tuberculosis to a serious global threat, necessitating alternate solutions for its eradication. D29 mycobacteriophage can infect and kill several mycobacterial species including Mtb. It encodes an endolysin LysA to hydrolyze host bacteria peptidoglycan for progeny release. We previously showed that out of the two catalytically active domains of LysA [N-terminal domain (NTD) and lysozyme-like domain], NTD, when ectopically expressed in Mycobacterium smegmatis (Msm), is able to kill the bacterium nearly as efficiently as full-length LysA. Here, we dissected the functioning of NTD to develop it as a phage-derived small molecule anti-mycobacterial therapeutic. We performed a large-scale site-directed mutagenesis of the conserved residues in NTD and examined its structure, stability, and function using molecular dynamic simulations coupled with biophysical and biochemical experiments. Our data show that NTD functions as a putative cysteine peptidase with a catalytic triad composed of Cys41, His112, and Glu137, acting as nucleophile, base, and acid, respectively, and showing characteristics similar to the NlpC/P60 family of cysteine peptidases. Additionally, our peptidoglycan hydrolysis assays suggested that NTD hydrolyzes only mycobacterial peptidoglycan and does not act on Gram-positive and Gram-negative bacterial peptidoglycans. More importantly, the combined activity of exogenously added NTD and sub-lethal doses of anti-mycobacterial drugs kills Msm in vitro and exhibits disruption of pre-formed mycobacterial biofilm. We additionally show that NTD treatment increases the permeability of antibiotics in Msm, which reduces the minimum inhibitory concentration of the antibiotics. Collectively, we present NTD as a promising phage-derived therapeutic against mycobacteria.IMPORTANCEMycobacteriophages are the viruses that use mycobacteria as host for their progeny production and, in the process, kill them. Mycobacteriophages are, therefore, considered as promising alternatives to antibiotics for killing pathogenic Mycobacterium tuberculosis. The endolysin LysA produced by mycobacteriophage D29 plays an important role in host cell lysis and virion release. Our work presented here highlights the functioning of LysA's N-terminal catalytic domain (NTD) in order to develop it as phage-derived small molecule therapeutics. We show that combined treatment of exogenously added NTD and sub-lethal doses of anti-mycobacterial drugs kills M. smegmatis, shows synergism by reducing the minimum inhibitory concentration of these antibiotics, and exhibits disruption of pre-formed mature biofilm. These outcomes and our detailed biochemical and biophysical dissection of the protein further pave the way toward engineering and development of NTD as a promising therapeutic against mycobacterial infections such as tuberculosis.

Assessment of the potential susceptibility of planktonic cells and bacterial biofilms by diosgenin

Bacterial biofilms are formed by a complex community of microorganisms, held together by a matrix that provides structural support and stability to microbial communities, making infections associated with biofilms more difficult to treat. In the face of growing research in the health area in search of antibiotics with action against biofilms, natural products present an effective alternative strategy. In this context, the aim of this study was to evaluate bacterial biofilm formation, assess the susceptibility of planktonic cells to diosgenin, and examine its efficacy on inhibiting and treating biofilms. The broth microdilution method was used to determine the Minimum Inhibitory Concentration (MIC). Six bacterial strains were tested: Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 9027, Pseudomonas aeruginosa 24, Enterococcus faecalis INCQS 0017, Enterococcus faecalis INCQS 00018, and Enterococcus faecalis INCQS 654. The crystal violet method was employed to evaluate the potential of diosgenin in biofilm inhibition and eradication. Chlorhexidine gluconate served as a reference standard for the antibiofilm assays, along with the antibiotics norfloxacin, ampicillin, and gentamicin. MIC determination revealed that diosgenin exhibited intrinsic antimicrobial activity, with an MIC of 406 μg/mL against S. aureus ATCC 25923. The antibiofilm activity showed significant inhibition for E. faecalis 17 and moderate inhibition against E. faecalis 654 and S. aureus 25923. Furthermore, diosgenin effectively eradicated preformed biofilms of S. aureus 25923 and P. aeruginosa 9027, indicating its ability to interfere with biofilm biomass. These results suggest that diosgenin not only inhibits bacterial biofilm formation but also eradicates established biofilms, highlighting it as a promising candidate for the development of products aimed at combating bacterial biofilms.

Screening different solid supports for Pseudomonas aeruginosa biofilm formation and determining its efficiency for decolorization and degradation of congo red.

A global water crisis is emerging due to increasing levels of contaminated water and decreasing clean water supply on Earth. This study aims to address the removal of azo dye from wastewater to enable its reuse. Recently, utilizing microorganisms has been proven to be a practical choice for the remediation of azo dyes in wastewater. Hence, in this study, we employed a preformed biofilm of Pseudomonas aeruginosa on a solid support (called substrate) to degrade azo dyes. This process offers several advantages, such as stability, substrate portability, more biofilm production in less time, and efficient utilization of enzymes for remediation. From 50 ppm of initial Congo Red concentration, 75.74% decolorization was achieved within ten h using a preformed biofilm on a coverslip. A maximum of 52.27% decolorization was achieved using biofilm during its formation after 72h of incubation. The Fourier-transform infrared (FTIR) spectroscopic analysis of Congo Red dye before and after remediation revealed a significant change in peak intensity, indicating dye degradation. Phytotoxicity studies performed by seed germination with Vigna radiata revealed that, after 5-7 days, almost 40% more seeds with longer root and shoot lengths were germinated in the presence of treated dye compared to the untreated one. This data indicated that the harmful Congo Red was successfully degraded to a non-toxic product by Pseudomonas aeruginosa biofilm grown on a glass substrate.

Novel Teixobactin Analogues Show Promising In Vitro Activity on Biofilm Formation by Staphylococcus aureus and Enterococcus faecalis

The treatment of infections caused by biofilm-forming organisms is challenging. The newly discovered antibiotic teixobactin shows activity against a wide range of biofilm-forming bacteria. However, the laborious and low-yield chemical synthesis of teixobactin complicates its further development for clinical application. The use of more easily synthesized teixobactin analogues may offer promise in this regard. In this article, three newly developed analogues were tested for efficacy against Staphylococcus aureus and Enterococcus faecalis. Minimum inhibitory and -bactericidal concentrations were investigated. MIC values for S. aureus and E. faecalis ranged from 0.5–2 and 2–4 μg/mL, respectively. Moreover, the ability of the analogues to prevent biofilm formation and to inactivate bacterial cells in already established S. aureus biofilm on medical grade materials (PVC and PTFE) used in the production of infusion tubing and catheters were also tested. The analogues showed an ability to prevent biofilm formation and inactivate bacterial cells in established biofilms at concentrations as low as 1–2 μg/mL. Confocal laser scanning microscopy showed that the most promising analogue (TB3) inactivated S. aureus cells in a preformed biofilm and gave a reduction in biovolume. The relative ease of synthesis of the analogues and their in vitro efficacy, makes them promising candidates for pharmaceutical development.Graphical