Biofilm Cell Viability Research Articles

Effects of a Mouthrinse Containing Silver Nanoparticles on Polymicrobial Oral Biofilms.

To investigate the antimicrobial effects of a mouthrinse containing silver nanoparticles (AgNP) on polymicrobial biofilms in vitro. Polymicrobial biofilms were grown on glass cover slips following the method of Exterkate. Saliva collected from a healthy human was added to McBain medium (including 0.2% sucrose) to achieve a 50-fold dilution. Glass coverslips were attached to the lid of a 24-well culture plate and suspended in the medium of each well. After 24 h of cultivating, coverslips with biofilms were immersed in each of four treatment solutions or sterile deionized water for 5 min. The control and four treatment groups were as follows: 1) control: sterile deionized water; 2) nanosilver (NS): mouthrinse containing AgNP; 3) 0.05C: 0.05% chlorhexidine gluconate; 4) 0.2C: 0.2% chlorhexidine gluconate; 5) Xyl: 25% xylitol. The biofilms were further regrown for 48 h. After removing the biofilms ultrasonically, they were cultured on blood agar, viable cells were counted, and the amount of lactic acid in the biofilms was analysed using a colorimetric assay. Mouthrinse containing AgNP suppressed viable cells in the biofilm to the same degree or more than with chlorhexidine gluconate. Amounts of lactic acid after 72 h cultivation of biofilms treated with 0.2C and NS showed consistently low values. The mouthrinse containing AgNP suppressed viable cells in polymicrobial biofilms to the same level as 0.2% chlorhexidine or higher.

Harnessing anti-infective efficacy of Cinnamomum verum in synergy with β-lactam and fluoroquinolones drugs to combat virulence and biofilms of Pseudomonas aeruginosa PAO1

Multidrug resistance (MDR) Gram-negative bacteria are increasingly resistant to multiple antibiotics, posing a serious challenge to infection control and treatment. Combining plant-derived bioactives with antibiotics offers a promising approach to overcome the challenges posed by MDR pathogens like Pseudomonas aeruginosa. This study investigated the synergistic effects of Cinnamomum verum with beta-lactam and fluoroquinolones against P. aeruginosa PAO1. The ethyl acetate fraction of C. verum (CVEF) was obtained through fractionation in organic solvents with progressively higher polarity. The interaction of CVEF with selected antibiotics was assessed by checkerboard synergy assay. The effects of synergistic combinations on pyocyanin, pyoverdine, protease, EPS production, and biofilm development were measured using spectroscopic assays. CVEF combined with cefepime, ceftazidime, and levofloxacin significantly enhanced antibacterial efficacy with FICIs between 0.156 to 0.5. The most active combinations i.e., CVEF-cefepime and CVEF-ceftazidime inhibited viable cell count of growth by 3.6 and 4.2 log10 CFU/ml respectively. The combination also inhibited virulence factors (>75%) and biofilms (>80%) at lower 1/2×FICs. The viable count of biofilm cells was also reduced from 6.4 to 3.3 and 3.6 log10 CFU/ml. Membrane permeability was decreased by 60.34% and biofilm cell viability by 22.53-38.44%. Key phytochemicals analyzed by GC/MS and LC/MS/MS, include cinnamaldehyde, trans-chlorogenic acid, quercetin, and quercetin 3'-O-glucuronide. In molecular docking investigations, quercetin 3'-O-glucuronide had the highest binding affinity with quorum sensing (QS) and biofilm-associated protein. The findings suggest CVEF, in combination with antibiotics, effectively targets resistance phenotypes of P. aeruginosa, impairing growth, virulence, and biofilms. This supports further research into natural compounds alongside antibiotics to treat drug-resistant infections.

Effect of amikacin-humic acid combination on Acinetobacter baumannii biofilm: an in vitro and in silico study.

Aim: Acinetobacter baumannii (AB) is a clinically important bacterial pathogen responsible for nosocomial infections. The biofilm-forming capability of these pathogens reduces the antibiotic penetration and its efficacy, thereby complicating the treatment. The current work aims to isolate the most potent biofilm-forming Acinetobacter species from clinical isolates of the patient samples and to evaluate the efficacy of the amikacin-humic acid combination against it.Methods: The combination effect of Amikacin-Humic (AMK-HUM) acid against the highest biofilm-producing A. baumannii SLMK001 was studied via in-vitro (microscopic analysis) and in-silico (Network Pharmacology) analysis.Results: The amikacin-humic acid combination significantly inhibited both the biofilm formation and cell viability of A. baumannii SLMK001. The images observed via Scanning Electron Microscope (SEM) showed a significant decrease in the biofilm matrix. Confocal Laser Scanning Microscope (CLSM) confirmed a reduction of the Z value of its three-dimensional structure. Further, the Network Pharmacology approach supported these experimental findings by identifying the key targets of the amikacin-humic acid combination against the biofilm pathways of A. baumannii.Conclusion: The in-vitro results aligned with the in-silico findings, indicating that the AMK-HUM combination is a promising treatment that significantly activates the key proteins against A. baumannii biofilm formation and pathogenesis.

Efficacy of silver diamine fluoride (SDF) in arresting dentin caries against inter-kingdom biofilms of Streptococcus mutans and Candida albicans.

To compare, in vitro, the efficacy of three proprietary silver diamine fluoride (SDF) products in mitigating progression of dentinal caries induced by an inter-kingdom, dual-species, bacterial-yeast biofilm. Human dentin blocks were demineralized to create artificial caries lesions and randomized into three SDF test groups: Saforide, Topamine, T-SDF, and an aqueous control (n = 26 per group). After application of foregoing SDF variants, the blocks were incubated with Streptococcus mutans and Candida albicans for 24 h for biofilm development, and subsequently subjected to a microbe-induced, pH-cycling process for 7 days, to mimic the oral eco-system. The biofilm cell viability and surface topography were assessed by colony-forming units (CFUs) and scanning electron microscopy respectively. The lesion depth and mineral density were evaluated by micro-computer tomography. SDF precipitate and matrix-to-mineral ratio were evaluated by X-ray diffraction and Fourier transform infrared spectroscopy, respectively. Standard, accepted methodology was used for all these evaluations and procedures. After pH cycling, the SDF groups demonstrated comparable inhibition of the biofilm relative to the control. the log CFU of S. mutans for Saforide, Topamine, T-SDF, and control were 6.69±0.73, 6.48±0.56, 6.63±0.66, and 8.01±0.45, respectively. For C. albicans, the log CFU were 4.86±0.44, 4.72±0.53, 4.92±0.29, and 5.60±0.27, respectively. The log CFU of S. mutans and C. albicans in the SDF groups were significantly lower than the control group (p<0.001). Further, the lesion depth decreased by approximately 14.79±7.00% in the SDF groups, while it increased by 11.07±8.61% in the control (p<0.001), and the mineral density increased by 16.36±4.58% in the SDF group, as opposed to a 5.59±2.64% reduction in the control (p<0.001) implying their caries mitigating effect. These findings were corroborated by SEM images of the lesions. SDF significantly mitigated dentin caries due to an assault by a polymicrobial plaque biofilm whilst arresting mineral loss and lesion growth. There was no difference in the caries-arresting efficacy of the compared SDF variants.

Beyond Harmful: Exploring Biofilm Formation by Enterococci Isolated from Portuguese Traditional Cheeses.

This study investigated the biofilm-forming capabilities of Enterococcus isolates from Portuguese traditional cheeses with protected designation of origin (PDO) status, specifically Azeitão and Nisa. Given the absence of added starter cultures in the cheesemaking process, the characteristics of these cheeses are intrinsically linked to the autochthonous microbiota present in the raw materials and the production environment. Our findings demonstrate that all isolates possess biofilm production abilities, which are crucial for their colonization and persistence within cheese factories, thereby maintaining factory-specific microbial heritage. Through an integrated analysis utilizing principal component analysis (PCA), a direct correlation between biofilm formation and cell viability was established. Notably, these results underscore the adaptive capacity of enterococci to survive environmental fluctuations and their role in the unique characteristics of Portuguese traditional cheeses. Overall, this research enhances our understanding of the microbial dynamics in cheese production and highlights the importance of enterococci in preserving cheese quality and heritage.

Characterization of Two Novel Endolysins from Bacteriophage PEF1 and Evaluation of Their Combined Effects on the Control of Enterococcus faecalis Planktonic and Biofilm Cells.

Endolysin, a bacteriophage-derived lytic enzyme, has emerged as a promising alternative antimicrobial agent against rising multidrug-resistant bacterial infections. Two novel endolysins LysPEF1-1 and LysPEF1-2 derived from Enterococcus phage PEF1 were cloned and overexpressed in Escherichia coli to test their antimicrobial efficacy against multidrug-resistant E. faecalis strains and their biofilms. LysPEF1-1 comprises an enzymatically active domain and a cell-wall-binding domain originating from the NLPC-P60 and SH3 superfamilies, while LysPEF1-2 contains a putative peptidoglycan recognition domain that belongs to the PGRP superfamily. LysPEF1-1 was active against 89.86% (62/69) of Enterococcus spp. tested, displaying a wider antibacterial spectrum than phage PEF1. Moreover, two endolysins demonstrated lytic activity against additional gram-positive and gram-negative species pretreated with chloroform. LysPEF1-1 showed higher activity against multidrug-resistant E. faecalis strain E5 than LysPEF1-2. The combination of two endolysins effectively reduced planktonic cells of E5 in broth and was more efficient at inhibiting biofilm formation and removing biofilm cells of E. faecalis JCM 7783T than used individually. Especially at 4 °C, they reduced viable biofilm cells by 4.5 log after 2 h of treatment on glass slide surfaces. The results suggest that two novel endolysins could be alternative antimicrobial agents for controlling E. faecalis infections.

Inhibitory Effects of Lactobionic Acid on Biofilm Formation and Virulence of Staphylococcus aureus.

Staphylococcus aureus biofilm is a common bio-contaminant source that leads to food cross-contamination and foodborne disease outbreaks. Hence, there is a need for searching novel antibiofilm agents with potential anti-virulence properties to control S. aureus contamination and infections in food systems. In this study, the antibiofilm effects of lactobionic acid (LBA) against S. aureus and its influence on virulence were explored. The minimum inhibition concentration of LBA on S. aureus was 8 mg/mL. Viable count and crystal violet assays revealed that LBA inhibited and inactivated S. aureus biofilms. Microscopic observations further confirmed the antibiofilm activity of LBA on S. aureus that disrupted the biofilm architecture and inactivated the viable cells in biofilms. Moreover, LBA decreased the release of extracellular DNA (eDNA) and extracellular polysaccharide (EPS) in S. aureus biofilms. LBA suppressed biofilm formation by intervening metabolic activity and reduced virulence secretion by repressing the hemolytic activity of S. aureus. Furthermore, LBA altered the expressions of biofilm- and virulence-related genes in S. aureus, further confirming that LBA suppressed biofilm formation and reduced the virulence secretion of S. aureus. The results suggest that LBA might be useful in preventing and controlling biofilm formation and the virulence of S. aureus to ensure food safety.

Dual-function antimicrobial-antibiofilm peptide hybrid to tackle biofilm-forming Staphylococcus epidermidis

BackgroundDue to their resistance and difficulty in treatment, biofilm-associated infections are problematic among hospitalized patients globally and account for 60% of all bacterial infections in humans. Antibiofilm peptides have recently emerged as an alternative treatment since they can be effectively designed and exert a different mode of biofilm inhibition and eradication.MethodsA novel antibiofilm peptide, BiF, was designed from the conserved sequence of 18 α-helical antibiofilm peptides by template-assisted technique and its activity was improved by hybridization with a lipid binding motif (KILRR). Novel antibiofilm peptide derivatives were modified by substituting hydrophobic amino acids at positions 5 or 7, and both, with positively charged lysines (L5K, L7K). These peptide derivatives were tested for antibiofilm and antimicrobial activities against biofilm-forming Staphylococcus epidermidis and multiple other microbes using crystal violet and broth microdilution assays, respectively. To assess their impact on mammalian cells, the toxicity of peptides was determined through hemolysis and cytotoxicity assays. The stability of candidate peptide, BiF2_5K7K, was assessed in human serum and its secondary structure in bacterial membrane-like environments was analyzed using circular dichroism. The action of BiF2_5K7K on planktonic S. epidermidis and its effect on biofilm cell viability were assessed via viable counting assays. Its biofilm inhibition mechanism was investigated through confocal laser scanning microscopy and transcription analysis. Additionally, its ability to eradicate mature biofilms was examined using colony counting. Finally, a preliminary evaluation involved coating a catheter with BiF2_5K7K to assess its preventive efficacy against S. epidermidis biofilm formation on the catheter and its surrounding area.ResultsBiF2_5K7K, the modified antibiofilm peptide, exhibited dose-dependent antibiofilm activity against S. epidermidis. It inhibited biofilm formation at subinhibitory concentrations by altering S. epidermidis extracellular polysaccharide production and quorum-sensing gene expression. Additionally, it exhibited broad-spectrum antimicrobial activity and no significant hemolysis or toxicity against mammalian cell lines was observed. Its activity is retained when exposed to human serum. In bacterial membrane-like environments, this peptide formed an α-helix amphipathic structure. Within 4 h, a reduction in the number of S. epidermidis colonies was observed, demonstrating the fast action of this peptide. As a preliminary test, a BiF2_5K7K-coated catheter was able to prevent the development of S. epidermidis biofilm both on the catheter surface and in its surrounding area.ConclusionsDue to the safety and effectiveness of BiF2_5K7K, we suggest that this peptide be further developed to combat biofilm infections, particularly those of biofilm-forming S. epidermidis.

Cold Atmospheric Helium Plasma in the Post-COVID-19 Era: A Promising Tool for the Disinfection of Silicone Endotracheal Prostheses.

Despite the excellent properties of silicone endotracheal prostheses, their main limitation is the formation of a polymicrobial biofilm on their surfaces. It can cause local inflammation, interfering with the local healing process and leading to further complications in the clinical scenario. The present study evaluated the inhibitory effect of cold atmospheric plasma (CAP) on multispecies biofilms grown on the silicone protheses' surfaces. In addition to silicone characterization before and after CAP exposure, CAP cytotoxicity on immortalized human bronchial epithelium cell line (BEAS-2B) was evaluated. The aging time test reported that CAP could temporarily change the silicone surface wetting characteristics from hydrophilic (80.5°) to highly hydrophilic (<5°). ATR-FTIR showed no significant alterations in the silicone surficial chemical composition after CAP exposure for 5 min. A significant log reduction in viable cells in monospecies biofilms (log CFU/mL) of C. albicans, S. aureus, and P. aeruginosa (0.636, 0.738, and 1.445, respectively) was detected after CAP exposure. Multispecies biofilms exposed to CAP showed significant viability reduction for C. albicans and S. aureus (1.385 and 0.831, respectively). The protocol was not cytotoxic to BEAS-2B. CAP can be a simple and effective method to delay multispecies biofilm formation inside the endotracheal prosthesis.

Comparative evaluation of disinfectant efficacy against biofilm-residing microorganisms

Introduction. Bacteria in biofilms (BFs) have increased resistance to antibacterial agents, including disinfectants; however, the efficacy level varies depending on the chosen treatment. Therefore, evaluation of efficacy of main disinfectants against BF-residing microorganisms is of scientific and practical interest.&#x0D; The purpose of the study was to explore the effect of disinfectants from various chemical groups on gram-positive and gram-negative bacteria residing in BFs.&#x0D; Materials and methods. The effect of the following disinfectants has been evaluated: alkyldimethylbenzylammonium chloride (ADBAC), tertiary amine (TA), polyhexamethylene guanidine chloride (PHMG), hydrogen peroxide (HP), chloramine (CA), dichloroisocyanuric acid sodium salt (Na DCC), sodium hypochlorite (HC), ethyl alcohol (EA), glutaraldehyde (GA)) against Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus ATCC 6538-P BFs. BFs were grown in 96-well plates at 37ºC for 24 hours and then exposed to biocide solutions. The efficacy of disinfectants was evaluated by the number of remaining viable cells and BF relative density.&#x0D; Results. The analyzed bacterial strains formed moderate BFs; the average number of viable cells in BFs was 6.51 ± 0.19 lg. The viable bacterial cell counts in BFs reduced by more than 4 lg when exposed to HP solutions at a concentration of 6%, Na DCC solution — 0.1% (by active chlorine), HC — 1% (by active chlorine), CA – 1% (by product), PHMG — 0.05%, TA — 1.0 %. The BF density decreased by more than 70%. ADBAC solutions at concentrations of 0.1–1.0%, TA — 0.05%, HP — 3%, Na DCC solution — 0.05% (by active chlorine) caused a 2-lg reduction in viable cell counts in BFs. The efficacy of chlorine-active compounds and HP increased when 0.5% sulfonol was added. GA (0.25–1.00%) and EA (40–70%) solutions were ineffective against BF microorganisms.&#x0D; Conclusion. A promising potential in combating microbial biofilms is demonstrated by disinfectants from the group of oxidizing agents (chlorine-active and oxygen-containing), TA and PHMG; using ADBAC as an individual compound is ineffective; aldehydes and alcohols are unable to destroy BFs and eliminate microorganisms in them.

Identification of VP0143 gene which modulates biofilm formation of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a Gram-negative halophilic foodborne pathogen associated with seafood consumption and can cause acute inflammatory gastroenteritis. The biofilm formed by V. parahaemolyticus facilitates its colonization and persistence in the food production chain, posing a continuous threat to food safety. In this study, through transposon mutants screening, we identified a novel gene, VP0143, which positively regulated biofilm formation of V. parahaemolyticus. The VP0143 deletion mutant (ΔVP0143) exhibited less biofilm formation as determined by crystal violet staining assay and showed decreased viability of biofilm cells by XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) assay compared to the wild type strain. The ΔVP0143 strain also showed weakened flagella-mediated motility and surface hydrophobicity. Moreover, VP0143 deletion significantly affected the production of autoinducer-2 and exopolysaccharides in biofilm, and reduced Raman peak intensity of biofilm components. Microscopic analysis confirmed that biofilm formed by ΔVP0143 strain showed loosened structure. In addition, ΔVP0143 strain exhibited compromised ability to form biofilm on silicon wafers, glass and stainless-steel plates, as well as on shrimp and crab surfaces. All these defects were reversed in the complementation strain ΔVP0143C. RT-qPCR further indicated that expression of genes associated with biofilm formation was decreased after VP0143 deletion. Therefore, our data suggest that VP0143 is involved in regulating biofilm formation of V. parahaemolyticus and extend our understanding of genetic determinants of V. parahaemolyticus biofilm formation.

Anti-Biofilm Activity of Laurel Essential Oil against Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a primary seafood-associated pathogen that could cause gastroenteritis. It can attach to various surfaces and form a biofilm, which poses serious threats to food safety. Hence, an effective strategy is urgently needed to control the biofilm formation of V. parahaemolyticus. Laurel essential oil (LEO) is used in food, pharmaceutical and other industries, and is commonly used as a flavoring agent and valuable spice in food industries. The potential antibiofilm effects of LEO against V. parahaemolyticus were examined in this study. LEO obviously reduced biofilm biomass at subinhibitory concentrations (SICs). It decreased the metabolic activity and viability of biofilm cells. Microscopic images and Raman spectrum indicted that LEO interfered with the structure and biochemical compositions of biofilms. Moreover, it also impaired swimming motility, decreased hydrophobicity, inhibited auto-aggregation and reduced attachment to different food-contact surfaces. RT-qPCR revealed that LEO significantly downregulated transcription levels of biofilm-associated genes of V. parahaemolyticus. These findings demonstrate that LEO could be potentially developed as an antibiofilm strategy to control V. parahaemolyticus biofilms in food industries.

Efficacy of Plasma-Treated Water against Salmonella Typhimurium: Antibacterial Activity, Inhibition of Invasion, and Biofilm Disruption.

Plasma-treated water (PTW) has emerged as a potential sanitizing agent. This study evaluated antibacterial activity, inhibition of invasion, and biofilm disruption effects of PTW against Salmonella Typhimurium. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for different PTW types. Time-kill assays were conducted to assess bactericidal effects, while polarized Caco-2 cells were used to evaluate invasion inhibition. Biofilm formation and cell viability were examined following PTW treatment using Salmonella Typhimurium isolates, while biofilm disruption and regrowth prevention were investigated using the Bioflux system. PTW exhibited antibacterial activity against all Salmonella Typhimurium isolates, with MICs of 25% for PTW1 and PTW2, and 50% for PTW3, PTW4, and PTW5. MBCs of 50% in media were observed for all PTW types. Undiluted PTW1 and PTW2 showed the highest bactericidal capacity, significantly reduced Salmonella viability, and completely inhibited bacterial invasion, while PTW3 and PTW5 also showed significant invasion reduction. Bioflux experiments confirmed the eradication of biofilms by PTW1 and PTW2, with no regrowth observed 72 h after PTW was removed. PTW demonstrated significant antibacterial activity, inhibition of invasion, biofilm disruption, and reduction of bacterial viability against Salmonella Typhimurium. This highlights PTW's potential as an effective sanitizer for reducing Salmonella contaminations.

Effects of synthetic peptide RP557 and its origin, LL-37, on carbapenem-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a common bacterium in nosocomial infection. The biofilm-forming ability and antimicrobial resistance make P. aeruginosa biofilm infection refractory to patients requiring hospitalization, especially patients in the intensive care unit. Therefore, many alternative compounds have been developed. A newly synthesized peptide, RP557, derived from human cathelicidin LL-37, was evaluated for its antimicrobial and antibiofilm effect toward carbapenem-resistant P. aeruginosa (CRPA). The results showed that regardless of the resistance to carbapenems, the minimal inhibition concentrations of RP557 and LL-37 against P. aeruginosa were 32 µg/mL and 256 µg/mL, respectively. Both RP557 and LL-37 significantly reduced the P. aeruginosa biofilm mass at subMICs, while subMICs of carbapenems induced biofilm formation. RP557 could also remove approximately 50% of the mature biofilm at a concentration of 64 µg/mL, while 256 µg/mL LL-37 was needed to remove it. A quarter MIC of RP557 and LL-37 was used together with carbapenems (ertapenem, imipenem, and meropenem). The results show that both RP-557 and LL-37 might increase the susceptibility to CRPA by 4-16 times. Significant gene expression level changes were observed in RP557- or LL-37-treated CRPA. Confocal images showed that biofilm structures and biofilm cell viability were significantly reduced in the LL-37- or RP557-treated groups. Therefore, RP557 and its structural origin, LL-37, could be potential treatments for carbapenem-resistant P. aeruginosa infection, especially for chronic biofilm infection. IMPORTANCE Pseudomonas aeruginosa is one of the major pathogens of nosocomial infection. Combined its biofilm-forming ability with carbapenem-resistance, it is hard to handle P. aeruginosa infection, especially for patients requiring hospitalization. Antimicrobial peptide is a type of potential compound for bacterial infection treatment. Among these, RP557 was found effective in inhibiting biofilm previously. By assessing its effect on both carbapenem-resistant P. aeruginosa planktonic cells and biofilm, our results offered a potential treatment for carbapenem-resistant P. aeruginosa infection. It could be helpful to treat severe nosocomial infection related to carbapenem-resistant bacteria and increase the patients' survival rate.

Cladosporium sphaerospermum extract inhibits quorum sensing associated virulence factors of Serratia marcescens

Serratia marcescens is now becoming a propensity for its highly antimicrobial-resistant clinical infections. Currently, it provides a novel strategy to prevent and control microbial infection by regulating S. marcescens quorum sensing (QS). Deep-sea-derived fungi are rich in QS bioactive constituents. In this work, the extracts from Cladosporium sphaerospermum SCSGAF0054 showed potent QS-related virulence factors and biofilm-inhibiting activities against S. marcescens NJ01. The swimming motility and multiple virulence factors such as prodigiosin, exopolysaccharide (EPS), lipase, protease and hemolysin were moderately inhibited by the extracts at varied concentrations. The confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) images revealed that C. sphaerospermum extracts moderately arrested biofilm formation and cell viability. Further, real-time quantitative PCR (RT-qPCR) analysis revealed that expressions of genes associated with virulence factors, flhD, fimA, fimC, bsmA, bsmB, pigA, pigC, and shlA, were significantly down-regulated compared with control. In addition, the extracts combined with imipenem inhibited the QS system of S. marcescens NJ01, disrupted its preformed biofilm, released the intra-biofilm bacteria and killed the bacteria gradually. Therefore, the extracts combined with imipenem can partially restore bacterial drug sensitivity. These results suggest that the extracts from SCSGAF0054 effectively interfere with the QS system to treat S. marcescens infection alone or combining with classical antimicrobial drugs.

In Vitro Antifungal Activity of Three Synthetic Peptides against Candida auris and Other Candida Species of Medical Importance.

Candidiasis is an opportunistic infection affecting immunosuppressed and hospitalized patients, with mortality rates approaching 40% in Colombia. The growing pharmacological resistance of Candida species and the emergence of multidrug-resistant Candida auris are major public health problems. Therefore, different antimicrobial peptides (AMPs) are being investigated as therapeutic alternatives to control candidiasis effectively and safely. This work aimed to evaluate the in vitro antifungal activity of three synthetic AMPs, PNR20, PNR20-1, and 35409, against ATCC reference strains of Candida albicans, Candida glabrata, Candida parapsilosis, Candida krusei, and Candida tropicalis, and clinical isolates of C. auris. Antifungal susceptibility testing, determined by broth microdilution, showed that the AMPs have antifungal activity against planktonic cells of all Candida species evaluated. In C. auris and C. albicans, the peptides had an effect on biofilm formation and cell viability, as determined by the XTT assay and flow cytometry, respectively. Also, morphological alterations in the membrane and at the intracellular level of these species were induced by the peptides, as observed by transmission electron microscopy. In vitro, the AMPs had no cytotoxicity against L929 murine fibroblasts. Our results showed that the evaluated AMPs are potential therapeutic alternatives against the most important Candida species in Colombia and the world.

Assessment of the growth inhibition and anti-biofilm activity of aptamer (PmA2G02) against Proteus mirabilis 1429T

Proteus mirabilis is known to cause Catheter-associated urinary tract infections (CAUTIs), which exhibit virulence factors linked to forming biofilms. Aptamers have recently been explored as potential anti-biofilm agents. This study demonstrates the anti-biofilm activity of aptamer (PmA2G02) targeting P. mirabilis 1429T, a pathogenic bacteria known to cause Catheter-associated urinary tract infections (CAUTIs). The studied aptamer inhibited biofilm formation, swarming motility, and cell viability at a concentration of 3 μM. The study also showed that the PmA2G02 had a binding affinity towards fimbrial outer membrane usher protein (PMI1466), flagellin protein (PMI1619), and regulator of swarming behavior (rsbA), which are responsible for adhesion, motility, and quorum sensing, respectively. Crystal violet assay, SEM, and confocal imaging confirmed the effectiveness of the PmA2G02 as an anti-biofilm agent. Moreover, as verified by qPCR, the expression levels of fimD, fliC2, and rsbA were significantly reduced compared to the untreated group. This study suggests that aptamer may be a potential alternative to traditional antibiotics for the treatment of CAUTIs caused by P. mirabilis. These findings shed light on the mechanisms by which the aptamer inhibits biofilm formation.

Design, Synthesis and Evaluation of a Series of Zinc(II) Complexes of Anthracene-Affixed Multifunctional Organic Assembly as Potential Antibacterial and Antibiofilm Agents against Methicillin-Resistant Staphylococcus aureus

A novel class of zinc(II)-based metal complexes, i.e., [Zn2(acdp)(μ-Cl)]·2H2O (1), [Zn2(acdp)(μ-NO3)]·2H2O (2), and [Zn2(acdp)(μ-O2CCF3)]·2H2O (3) (Cl- = chloride; NO3- = nitrate; CF3CO2- = trifluoroacetate) of anthracene-affixed multifunctional organic assembly, H3acdp (H3acdp = N,N'-bis[anthracene-2-ylmethyl]-N,N'-bis[carboxymethyl]-1,3-diaminopropan-2-ol), have emerged as promising antibacterial and antibiofilm agents in the domain of medicinal chemistry. Accordingly, complexes 1-3 were synthesized by utilizing H3acdp in combination with ZnCl2, Zn(NO3)2·6H2O, and Zn(CF3CO2)2·H2O respectively, in the presence of NaOH at ambient temperature. The complexation between H3acdp and Zn2+ was delineated by a combined approach of spectrophotometric and spectrofluorometric titration studies. The stoichiometry of acdp3-/Zn2+ in all three complexes is observed to be 1:2, as confirmed by spectrophotometric/spectrofluorometric titration data. Elemental analysis (C, H, N, Zn), molar conductance, FTIR, UV-vis, and thermoanalytical (TGA/DTA) data were effectively used to characterize these complexes. Besides, the structures of 1-3 were established by density functional theory (DFT) calculation using B3LYP/6-311G, specifying a self-assembled compact geometry with average Zn···Zn separation of 3.4629 Å. All three zinc complexes exhibited significantly high antibacterial and antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA BAA1717). However, complex 1 showed a more recognizable activity than 2 and 3, with minimum inhibitory concentration (MIC) values of 200, 350, and 450 μg/mL, respectively. The antimicrobial activity was tested by employing the minimum inhibitory concentration (MIC) and time-kill assay. The crystal violet (CV) assay and microscopic study were performed to examine the antibiofilm activity. As observed, complexes 1-3 had an effect on the production of extracellular polymeric substance (EPS), biofilm cell-viability, and other virulence factors such as staphyloxanthin and hemolysin production, autoaggregation ability, and microbial cell-surface hydrophobicity. Reactive oxygen species (ROS) generated due to inhibition of staphyloxanthin production in response to 1-3 were also analyzed. Moreover, complexes 1-3 showed an ability to damage the bacterial cell membrane due to accumulation of ROS resulting in DNA leakage. In addition, complexes 1-3 displayed a synergistic/additive activity with a commercially available antibiotic drug, vancomycin, with enhanced antibacterial activity. On the whole, our investigation disclosed that complex 1 could be a promising drug lead and attract much attention to medicinal chemists compared to 2 and 3 from therapeutic aspects.

Lytic Bacteriophage Is a Promising Adjunct to Common Antibiotics across Cystic Fibrosis Clinical Strains and Culture Models of Pseudomonas aeruginosa Infection

Bacteriophages (phages) are antimicrobials with resurgent interest that are being investigated for the treatment of antibiotic refractory infection, including for Pseudomonas aeruginosa (Pa) lung infection in cystic fibrosis (CF). In vitro work supports the use of this therapy in planktonic and biofilm culture models; however, consistent data are lacking for efficacy across different clinical Pa strains, culture models, and in combination with antibiotics in clinical use. We first examined the efficacy of a 4-phage cocktail as an adjunct to our CF centre's first-line systemic combination antibiotic therapy (ceftazidime + tobramycin) for 16 different clinical Pa strains and then determined subinhibitory interactions for a subset of these strains with each antibiotic in planktonic and biofilm culture. When a 4-phage cocktail (4 × 108 PFU/mL) was added to a ceftazidime-tobramycin combination (ceftazidime 16 mg/mL + tobramycin 8 mg/mL), we observed a 1.7-fold and 1.3-fold reduction in biofilm biomass and cell viability, respectively. The four most antibiotic resistant strains in biofilm were very susceptible to phage treatment. When subinhibitory concentrations of antibiotics and phages were investigated, we observed additivity/synergy as well as antagonism/inhibition of effect that varied across the clinical strains and culture model. In general, more additivity was seen with the phage-ceftazidime combination than with phage-tobramycin, particularly in biofilm culture, where no instances of additivity were seen when phages were combined with tobramycin. The fact that different bacterial strains were susceptible to phage treatment when compared to standard antibiotics is promising and these results may be relevant to ongoing clinical trials exploring the use of phages, in particular in the selection of subjects for clinical trials.

The alginate polymer OligoG alters susceptibility of biofilm-embedded non-typeable Haemophilus influenzae to ampicillin and ciprofloxacin.

Treatment of respiratory infections with non-typeable Haemophilus influenzae (NTHi) in COPD patients is complicated by biofilm formation, protecting the bacteria against the hosts' immune response and antibiotics. We investigated the antibiofilm and antibacterial effects of the alginate polymer OligoG, alone or combined with ampicillin or ciprofloxacin, on mature NTHi biofilms. Two unrelated COPD strains with PBP3-mediated β-lactam resistance, with additional TEM-1 β-lactamase (Hi-022) or quinolone resistance due to altered GyrA and ParC (Hi-072) were used. Antibiofilm and antibacterial effects were assessed macroscopically, by measurement of biofilm biomass (OD), and by viable cell counts, with determination of minimum biofilm inhibitory concentration (MBIC) and the novel parameter 'minimum concentration for 2 log10 drop in viable cells in biofilm' (MB2LDC). Drug interactions between OligoG and antibiotics were assessed by comparing expected and observed inhibitory effects (percent inhibition of no-treatment control) of combined treatment. OligoG had dose-dependent biofilm disruptive abilities and a weak inhibitory effect on viable cells. Combination with OligoG (64 g/L) significantly lowered MBIC for ampicillin (both strains) and MB2LDC for ciprofloxacin (Hi-022). For Hi-022, there was significant synergism between OligoG and both antibiotics. For Hi-072, interactions were subtle, but a tendency in direction of antagonism was significant at two concentrations of ciprofloxacin. OligoG shows promise as a potential adjuvant to antibiotics in NTHi infections, but strain-specific factors appear to affect drug interactions and may lead to antagonism. More research is needed to clarify the mechanisms of action of OligoG and interactions with antibiotics.