Reduced Biofilm Biomass Research Articles

Investigating Sortase A inhibitory potential of herbal compounds using integrated computational and biochemical approaches

Multi-drug resistance in bacteria is emerging as a major global health challenge, causing substantial harm in terms of mortality, morbidity, and financial strain on healthcare systems. These bacteria are constantly acquiring new virulence factors and drug-resistance mechanisms, which highlights the critical need for innovative antimicrobial medicines and identification of new therapeutic targets, such as Sortase A (EfSrtAΔN59). EfSrtAΔN59, a transpeptidase significant for the adhesion and virulence of Enterococcus faecalis (E. faecalis), presents an attractive target for disrupting biofilm formation—a key factor in persistent infections. This study investigates the inhibitory effects of two natural flavonoids- Rutin Trihydrate and Quercetin, on EfSrtAΔN59 and biofilm formation in E. faecalis. With in vitro enzymatic assays and biofilm quantification techniques, we demonstrate that both compounds significantly attenuate EfSrtAΔN59 activity, thereby hindering bacterial biofilm formation. Rutin Trihydrate and Quercetin exhibited strong binding affinities to the EfSrtAΔN59 enzyme, as confirmed by molecular docking and MD simulation studies. This was further substantiated by a notable reduction in biofilm biomass in bacterial cultures treated with these compounds. These findings highlight the potential of Rutin Trihydrate and Quercetin as promising candidates for the development of novel anti-virulence therapies aimed at mitigating E. faecalis infections, thereby offering a compelling alternative to traditional antibiotics.

Patterning Bacterial Cells on Quasi‐Liquid Surfaces for Biofilm Morphological Control

AbstractBacterial cells within biofilms exhibit resistance to antibiotics, presenting persistent health risks. Current approaches to inhibit biofilm formation have limitations due to their poor biofilm morphological control. For instance, bactericidal surfaces suffer from the accumulation of dead cells that compromise their antibacterial efficacy, and existing antifouling surfaces fail to inhibit biofilm formation. In this work, exceptional biofilm suppression is achieved on quasi‐liquid surfaces (QLS) with patterned surface chemistry where live bacterial cells are guided from slippery to sticky patterned destinations. These surfaces consist of 50 µm slippery and 10 µm sticky stripes. Live bacterial cells are directed to congregate on the sticky patterns, resulting in reduced biofilm biomass and surface coverage compared to uniform slippery surfaces. The patterned biofilm produces sparser extracellular matrix, thus reducing the barrier for antibiotic penetration and treatment. The innovative approach to direct cell migration on patterned QLS represents a promising strategy for inhibiting biofilm formation and combating biofilm‐associated infections.

A Strain of Streptococcus mitis Inhibits Biofilm Formation of Caries Pathogens via Abundant Hydrogen Peroxide Production.

Commensal oral streptococci that colonize supragingival biofilms deploy mechanisms to combat competitors within their niche. Here, we determined that Streptococcus mitis more effectively inhibited biofilm formation of Streptococcus mutans within a seven species panel. This phenotype was common amongst all assayed isolates of S. mutans, but was specific to a single strain of S. mitis, ATCC 49456. The growth inhibitory factor was not effectively carried in spent supernatants of S. mitis. However, we documented ATCC 49456 to accumulate 4-5 times more hydrogen peroxide (H2O2) than other species tested, and 5-18 times more than other S. mitis strains assayed. The S. mutans biofilm formation inhibitory phenotype was reduced when grown in media containing catalase or with a S. mitis mutant of pyruvate oxidase (spxB; pox), confirming that SpxB-dependent H2O2 production was the main antagonistic factor. Addition of S. mitis within hours after S. mutans inoculation was effective at reducing biofilm biomass, but not for 24 h pre-formed biofilms. Transcriptome analysis revealed responses for both S. mitis and S. mutans, with several S. mutans differentially expressed genes following a gene expression pattern previously described, while others being unique to the interaction with S. mitis. Finally, we show that S. mitis also affected coculture biofilm formation of several other commensal streptococci. Our study shows that strains with abundant H2O2 production are effective at inhibiting initial growth of caries pathogens like S. mutans, but are less effective at disrupting pre-formed biofilms and have the potential to influence the stability of other oral commensal strains.

Bacteriophages as potential antibiotic potentiators in cystic fibrosis: A new model to study the combination of antibiotics with a bacteriophage cocktail targeting dual species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa

Staphylococcus aureus and Pseudomonas aeruginosa co-infections in patients with cystic fibrosis (CF) are associated with disease severity. Their treatment is complicated by biofilm formation in the sticky mucus obstructing the airways. We investigated the activity of phages-antibiotics combinations using a dual species biofilm (P. aeruginosa/S. aureus) formed in artificial sputum medium. Biofilmswere incubated with broad-spectrum antibiotics (meropenem, ceftazidime, ciprofloxacin, tobramycin) combined with a cocktail of two (bacterio)phages (PSP3 and ISP) proven active via spot tests and double agar on P. aeruginosa PAO1 and S. aureus ATCC 25923. At the highest tested concentrations (100 x MIC), antibiotics alone caused a 20-50% reduction in biomass and reduced S. aureus and P. aeruginosa CFU of 2.3 to 2.8 and 2.1 to 3.6 log10, respectively. Phages alone reduced biofilm biomass by 23% and reduced P. aeruginosa CFU of 2.1 log10, but did not affect S. aureus viability. Phages enhanced antibiotic effects on biomass and exhibited additive effects with antibiotics against P. aeruginosa, but not against S. aureus. Following inhibition of bacterial respiration by phages in planktonic cultures rationalised these observations by demonstrating that PSP3 was effective at multiplicities of infection (MOI) as low as 10-4 plaque forming units (PFU)/CFU on P. aeruginosa, but ISP, at higher MOI (> 0.1) against S. aureus. Pre-screening inhibition of bacterial respiration by phages may assist in selecting those showing activity at sufficiently low titers to showcase anti-biofilm activity in this complex but clinically-relevant in vitro model of biofilm.

A New Casjensviridae Bacteriophage Isolated from Hospital Sewage for Inactivation of Biofilms of Carbapenem Resistant Klebsiella pneumoniae Clinical Isolates.

Klebsiella pneumoniae, a member of the ESKAPE pathogen group, is a prominent cause of hospital-acquired infections. The WHO has recognized carbapenem-resistant K. pneumoniae as a critical-one priority pathogen. These resilient superbugs have the ability to form biofilms and present a significant global threat. In the present study, we isolated and characterized a bacteriophage SAKp02, from hospital sewage, infectious to carbapenem-resistant K. pneumoniae patient isolates. SAKp02 could infect 43 of 72 clinical isolates, indicating a broad host spectrum. Whole genome analysis classified SAKp02 within the family Casjensviridae, with a 59,343 bp genome encoding 82 ORFs. Comparative genomic analysis revealed significant differences between SAKp02 and its closest viruses, indicating a distinct genetic makeup positioning it as a novel phage strain within the lineage. The SAKp02 genome comprises bacteriolytic enzymes, including holin, endolysin, and phage depolymerase, crucial for bacterial lysis and biofilm disruption. It reduced biofilm biomass by over threefold compared to the control and eradicated 99% of viable cells within a 4 h treatment period. Scanning electron microscopy corroborated the ability of the phage to dismantle biofilm matrices and lyse bacterial cells. Safe and effective treatments are warranted, and hence, the fully characterized lytic phages with therapeutic potential against drug-resistant clinical isolates of bacteria are needed. Our study is the first to report the antibacterial and antibiofilm activity of Casjensviridae phages, and our discovery of a novel K. pneumoniae phage broadens the arsenal against the bacteria.

Light-activated conjugated polymer nanoparticles to defeat pathogens associated with bovine mastitis

Bovine mastitis (BM) represents a significant challenge in the dairy industry. Limitations of conventional treatments have prompted the exploration of alternative approaches, such as photodynamic inactivation (PDI). In this study, we developed a PDI protocol to eliminate BM-associated pathogens using porphyrin-doped conjugated polymer nanoparticles (CPN). The PDI-CPN protocol was evaluated in four mastitis isolates of Staphylococcus and in a hyper-biofilm-forming reference strain. The results in planktonic cultures demonstrated that PDI-CPN exhibited a bactericidal profile upon relatively low light doses (∼9.6 J/cm2). Furthermore, following a seven-hour incubation period, no evidence of cellular reactivation was observed, indicating a highly efficient post-photodynamic inactivation effect. The successful elimination of bacterial suspensions encouraged us to test the PDI-CPN protocol on mature biofilms. Treatment using moderate light dose (∼64.8 J/cm2) reduced biofilm biomass and metabolic activity by up to 74% and 88%, respectively. The impact of PDI-CPN therapy on biofilms was investigated using scanning electron microscopy (SEM), which revealed nearly complete removal of the extracellular matrix and cocci. Moreover, ex vivo studies conducted on bovine udder skin demonstrated the efficacy of the therapy in eliminating bacteria from these scaffolds and its potential as a prophylactic method. Notably, the histological analysis of skin revealed no signs of cellular degeneration, suggesting that the protocol is safe and effective for BM treatment. Overall, this study demonstrates the potential of PDI-CPN in treating and preventing BM pathogens. It also provides insights into the effects of PDI-CPN on bacterial growth, metabolism, and survival over extended periods, aiding the development of effective control strategies and the optimization of future treatments.

Analysis of a new phage, KZag1, infecting biofilm of Klebsiella pneumoniae: genome sequence and characterization

BackgroundThis study investigates the effectiveness of the bacteriophage KZag1 against drug-resistant Klebsiella pneumoniae, aiming to assess its potential as a therapeutic agent. The novelty lies in the characterization of KZag1, a Myovirus with specific efficacy against multidrug-resistant K. pneumoniae strains. This highlights the significance of exploring alternative strategies, particularly phage therapy, in addressing biofilm-associated infections.MethodsKZag1, characterized by a typical Myovirus structure with a 75 ± 5 nm diameter icosahedral head and a 15 ± 5 nm short tail, was evaluated in experimental trials against 15 strains of K. pneumoniae. The infection cycle duration was determined to be 50 min, resulting in an estimated burst size of approximately 83 plaque-forming units per colony-forming unit (PFU/CFU). Stability assessments were conducted within a pH range of 4 to 12 and temperatures ranging from 45°C to 60°C. Biofilm biomass reduction was observed, particularly at a multiplicity of infection (MOI) of 10.ResultsKZag1 demonstrated infection efficacy against 12 out of 15 tested K. pneumoniae strains. The phage exhibited stability across a broad pH range and at elevated temperatures. Notably, treatment with KZag1 significantly reduced K. pneumoniae biofilm biomass, emphasizing its potential in combating biofilm formation. Genomic analysis revealed a complete genome of 157,089 base pairs with a GC content of 46.38%, encompassing 203 open reading frames (ORFs) and a cysteine-specific tRNA sequence. Comparison with phage GP4 highlighted similarities, with KZag1 having a longer genome by approximately 4829 base pairs and a higher GC content by approximately 0.93%. Phylogenetic analysis classified KZag1 within the Myoviridae family.ConclusionThe efficacy of KZag1 against K. pneumoniae biofilm suggests its potential as a therapeutic candidate, especially for drug-resistant infections. Further clinical research is warranted to explore its synergy with other treatments, elucidate genomic traits, compare with Myoviridae phages, and understand its host interactions. These findings underscore the promising role of KZag1 in addressing drug-resistant bacterial infections.

Responses of colonization and development of periphytic biofilms to three typical tire wear particles with or without incubation-aging in migrating aqueous phases

Understanding the behavior of tire wear particles (TWPs) and their impact on aquatic environments after aging is essential. This study explored the characteristics of TWPs generated using different methods (rolling friction, sliding friction, and cryogenic milling) and their transformation after exposure to environmental conditions mimicking runoff and sewage, focusing on their effects on river water and periphytic biofilms. Laboratory experiments indicate that at low exposure levels (0.1 mg/L), TWPs promoted biofilm growth, likely due to zinc release acting as a nutrient and the aggregation of particles serving as biofilm scaffolds. However, at higher concentrations (100 mg/L), TWPs inhibited biofilm development. This inhibition is linked to toxic byproducts like N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine-quinone and environmentally persistent free radicals, which reduce biofilm biomass, alter algal diversity, and decrease the production of essential biofilm components such as proteins and polysaccharides, consistent with the inhibitory behavior of TWPs on bis-(3′-5′)-cyclic diguanosine monophosphate and quorum sensing signals, including acyl-homoserine lactone and autoinducer-2. Aging processes, particularly after simulated sewage treatment, further affect ecological impacts of TWPs, reducing the benefits observed at low concentrations and intensifying the negative effects at high concentrations. Contribution of here lies in systematically revealing the impact of TWPs on the development of aquatic biofilms, emphasizing the logical relationship between their aging characteristics, environmental behavior, and ecological risks. It assesses not only the release effects of typical additives and conventional size effects but also highlights the emerging photochemical toxicity (persistent free radicals), thus providing valuable insights into the aquatic ecological risk assessment of TWPs.

Anti-Biofilm and Anti-Quorum-Sensing Activity of Inula Extracts: A Strategy for Modulating Chromobacterium violaceum Virulence Factors.

The formation of microbial biofilm is a self-organizing process among bacterial cells, regulated by quorum-sensing (QS) mechanisms, contributing to development of infections. These processes, either separately or in combination, significantly contribute to bacterial resistance to antibiotics and disinfectants. A novel approach to addressing the challenge of treating infections due to antibacterial resistance involves the use of plant metabolites. In recent years, there has been increasing recognition of different phytochemicals as potential modulators. In our study, we evaluated the synergistic effect of chloroform and methanol extracts from Inula species against key virulence factors, including biofilm formation, violacein production, and swarming motility. Each of the 11 examined plant extracts demonstrated the ability to reduce biofilms and pigment synthesis in C. violaceum. Two of the extracts from I. britannica exhibited significant anti-biofilm and anti-quorum-sensing effects with over 80% inhibition. Their inhibitory effect on violacein synthesis indicates their potential as anti-QS agents, likely attributed to their high concentration of terpenoids (triterpenoids, sesquiterpene lactones, and diterpenoids). Scanning electron microscopy revealed a notable reduction in biofilm biomass, along with changes in biofilm architecture and cell morphology. Additionally, fluorescence microscopy revealed the presence of metabolically inactive cells, indicating the potent activity of the extracts during treatment. These new findings underscore the effectiveness of the plant extracts from the genus Inula as potential anti-virulent agents against C. violaceum. They also propose a promising strategy for preventing or treating its biofilm formation.

Insights into betulinic acid as a promising molecule to fight the interkingdom biofilm Staphylococcus aureus–Candida albicans

The demand for antibiofilm molecules has increased over several years due to their potential to fight biofilm-associated infections, such as those including the interkingdom Staphylococcus aureus-Candida albicans occurring in clinical settings worldwide. Recently, we identified a pentacyclic triterpenoid compound, betulinic acid, from invasive macrophytes, with interesting antibiofilm properties. The aim of the present study was to provide insights into the mechanism of action of betulinic acid against the clinically relevant bi-species S. aureus-C. albicans biofilms. Microscopy examinations, flow cytometry and crystal violet assays confirmed that betulinic acid was effective at damaging mature S. aureus-C. albicans biofilms or inhibiting their formation, reducing biofilm biomass by 70% on average and without microbicidal activity. The results suggested an action of betulinic acid on cell membranes, inducing changes in properties such as composition, hydrophobicity and fluidity as observed in C. albicans, which may hinder the early adhesion step, biofilm growth and the physical interactions of both microbial species. Further results of real-time polymerase chain reaction argued in favour of a reduction in S. aureus-C. albicans physical interaction due to betulinic acid by the modulation of biofilm-related gene expression, as observed in early stages of biofilm formation. This study revealed the potential of betulinic acid as a candidate agent for the prevention and treatment of S. aureus-C. albicans biofilm-related infections.

Common food preservatives impose distinct selective pressures on Salmonella Typhimurium planktonic and biofilm populations

Food preservatives are crucial in controlling microbial growth in processed foods to maintain food safety. Bacterial biofilms pose a threat in the food chain by facilitating persistence on a range of surfaces and food products. Cells in a biofilm are often highly tolerant of antimicrobials and can evolve in response to antimicrobial exposure. Little is known about the efficacy of preservatives against biofilms and their potential impact on the evolution of antimicrobial resistance. In this study we investigated how Salmonella enterica serovar Typhimurium responded to subinhibitory concentrations of four food preservatives (sodium chloride, potassium chloride, sodium nitrite or sodium lactate) when grown planktonically and in biofilms. We found that each preservative exerted a unique selective pressure on S. Typhimurium populations. There was a trade-off between biofilm formation and growth in the presence of three of the four preservatives, where prolonged preservative exposure resulted in reduced biofilm biomass and matrix production over time. All three preservatives selected for mutations in global stress response regulators rpoS and crp. There was no evidence for any selection of cross-resistance to antibiotics after preservative exposure. In conclusion, we showed that preservatives affect biofilm formation and bacterial growth in a compound specific manner. We showed trade-offs between biofilm formation and preservative tolerance, but no antibiotic cross-tolerance. This indicates that bacterial adaptation to continuous preservative exposure, is unlikely to affect food safety or contribute to antibiotic resistance.

Combination antimicrobial therapy: in vitro synergistic effect of anti-staphylococcal drug oxacillin with antimicrobial peptide nisin against Staphylococcus epidermidis clinical isolates and Staphylococcus aureus biofilms

The ability of Staphylococcus epidermidis and S. aureus to form strong biofilm on plastic devices makes them the major pathogens associated with device-related infections (DRIs). Biofilm-embedded bacteria are more resistant to antibiotics, making biofilm infections very difficult to effectively treat. Here, we evaluate the in vitro activities of anti-staphylococcal drug oxacillin and antimicrobial peptide nisin, alone and in combination, against methicillin-resistant S. epidermidis (MRSE) clinical isolates and the methicillin-resistant S. aureus ATCC 43,300. The minimum inhibitory concentrations (MIC) and minimum biofilm eradication concentrations (MBEC) of oxacillin and nisin were determined using the microbroth dilution method. The anti-biofilm activities of oxacillin and nisin, alone or in combination, were evaluated. In addition, the effects of antimicrobial agents on the expression of icaA gene were examined by quantitative real-time PCR. MIC values for oxacillin and nisin ranged 4–8 µg/mL and 64–128 µg/mL, respectively. Oxacillin and nisin reduced biofilm biomass in all bacteria in a dose-dependent manner and this inhibitory effect was enhanced with combinatorial treatment. MBEC ranges for oxacillin and nisin were 2048–8192 µg/mL and 2048–4096 µg/mL, respectively. The addition of nisin significantly decreased the oxacillin MBECs from 8- to 32-fold in all bacteria. At the 1× MIC and 1/2× MIC, both oxacillin and nisin decreased significantly the expression of icaA gene in comparison with untreated control. When two antimicrobial agents were combined at 1/2× MIC concentration, the expression of icaA were significantly lower than when were used alone. Nisin/conventional oxacillin combination showed considerable anti-biofilm effects, including inhibition of biofilm formation, eradication of mature biofilm, and down-regulation of biofilm-related genes, proposing its applications for treating or preventing staphylococcal biofilm-associated infections, including device-related infections.

In vitro effects of selective serotonin reuptake inhibitors on Cryptococcus gattii capsule and biofilm.

Infections caused by Cryptococcus gattii mainly affect immunocompetent individuals and the treatment presents important limitations. This study aimed to validate the efficacy of selective serotonin reuptake inhibitors (SSRI), fluoxetine hydrochloride (FLH), and paroxetine hydrochloride (PAH) in vitro against C. gattii. The antifungal activity of SSRI using the microdilution method revealed a minimal inhibitory concentration (MIC) of 31.25µg/ml. The combination of FLH or PAH with amphotericin B (AmB) was analyzed using the checkerboard assay and the synergistic effect of SSRI in combination with AmB was able to reduce the SSRI or AmB MIC values 4-8-fold. When examining the effect of SSRI on the induced capsules, we observed that FLH and PAH significantly decreased the size of C. gattii capsules. In addition, the effects of FLH and PAH were evaluated in biofilm biomass and viability. The SSRI were able to reduce biofilm biomass and biofilm viability. In conclusion, our results indicate the use of FLH and PAH exhibited in vitro anticryptococcal activity, representing a possible future alternative for the cryptococcosis treatment.

Investigation of the Effect of Farnesol on Biofilm Formation by Candida albicans and Candida parapsilosis Complex Isolates

The incidence of infections caused by Candida species has significantly increased over the past three decades. Candida albicans is commonly recognized as the primary causative agent in cases of candidiasis; however, non-albicans Candida species, including Candida parapsilosis, are also frequently defined as pathogens. Treatment-resistant infections arise as a result of biofilm formation, which is one of the effective mechanisms in the pathogenesis of Candida infections. However, the mechanisms of action of farnesol, a quorum sensing (QS) system molecule, on biofilm formation by Candida species remain unclear. This study aimed to demonstrate the changes in the biofilm biomass of C.albicans and C.parapsilosis complex isolates in the presence of farnesol and reveal the expression of the EFG1 and BCR1 genes, which are believed to play a role in the production of QS molecules, using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis. C.albicans (n= 91) and C.parapsilosis complex (n= 29) isolates obtained from different clinical samples were included in the study. The minimum inhibitory concentration (MIC) values of farnesol were determined using the broth microdilution method according to the M27-A3 protocol of the Clinical and Laboratory Standards Institute (CLSI). The biofilm biomass of the isolates was examined without farnesol and at the MIC-0 and MIC-2 concentrations of farnesol. Changes in the expression of the biofilm-associated EFG1 and BCR1 genes were investigated using qRT-PCR. According to the results of the study, the MIC values of farnesol were detected in the range of 1-2 mM in 82.4% (n= 75) of the C.albicans isolates and in the range of 0.5-1 mM in 72.4% (n= 21) of the C.parapsilosis complex isolates. Of the C.albicans isolates, 27 (29.7%) exhibited a strong biofilm formation and 58 (63.7%) demonstrated a weaker biofilm formation, while these rates were 34.4% (n= 10) and 62.1% (n= 18), respectively, for the C.parapsilosis complex isolates. At the MIC-0 and MIC-2 concentrations, farnesol was observed to reduce biofilm biomass among C.albicans (n= 24, 88.9%) and C.parapsilosis complex (n= 8, 80.0%) isolates that formed strong biofilms and observed to increase biofilm biomass among those that formed weak biofilms [60.3% (n= 35) and 55.6% (n= 10), respectively]. On completion of the qRT-PCR analysis supporting the results of the biofilm experiment, it was determined that the expressions of the EFG1 and BCR1 genes decreased at the MIC-0 and MIC-2 concentrations of farnesol among the strong biofilm-forming C.albicans and C.parapsilosis complex isolates, but there was an increase in gene expressions among the weak biofilm-forming isolates. In addition to the antifungal effect of farnesol on Candida species, this study provided data on the efficacy of the MIC-0 and MIC-2 concentrations of farnesol against Candida biofilm biomass. Although our results suggest that farnesol can be used as an alternative agent to reduce biofilm formation in Candida infections, they need to be supported by further studies. Moreover, this research has significance as it represents the first study to determine the EFG1 and BCR1 gene expressions among C.parapsilosis complex isolates in the presence of farnesol.

Cold atmospheric plasma for surface disinfection: a promising weapon against deleterious meticillin-resistant Staphylococcus aureus biofilms

Bacteria are becoming increasingly resistant to classical antimicrobial agents, so new approaches need to be explored. To assess the potential of cold atmospheric plasma for the management of meticillin-resistant Staphylococcus aureus (MRSA). The 24, 48, and 72h resistant and susceptible S.aureus biofilms were exposed to 60, 120, and 180s treatment with plasma. Increasing the treatment time results in higher cell reduction for both susceptible and resistant strains of S.aureus (P < 0.05). Up to log10 reduction factor of 5.24cfu/cm2 can be achieved in 180s of plasma treatment. Furthermore, plasma can substantially alter the cell's metabolisms and impact cell membrane integrity. However, it has not been shown that plasma can reduce biofilm biomass in the case of 24h and 48h biofilms, although the 72h biofilm was more susceptible, and its biomass was decreased (P < 0.05). The accumulation of intrabacterial reactive oxygen species was also observed, which confirms the plasma's induction of oxidative stress. Finally, it was shown that continuous plasma exposure of bacterial cells does not cause resistance to plasma, nor is resistance developed to cefoxitin. Cold atmospheric plasma is a good candidate for S.aureus and MRSA biofilm treatment and may therefore be of value in the bacterial resistance crisis.

Enhancing the Functional Properties of Tea Tree Oil: In Vitro Antimicrobial Activity and Microencapsulation Strategy.

In the context of addressing antimicrobial drug resistance in periocular infections, Tea Tree Oil (TTO) has emerged as a promising therapeutic option. This study aimed to assess the efficacy of TTO against bacterial strains isolated from ocular infections, with a particular focus on its ability to inhibit biofilm formation. Additionally, we designed and analyzed microcapsules containing TTO to overcome certain unfavorable physicochemical properties and enhance its inherent biological attributes. The quality of TTO was confirmed through rigorous analysis using GC-MS and UV-Vis techniques. Our agar diffusion assay demonstrated the effectiveness of Tea Tree Oil (TTO) against ocular bacterial strains, including Corynebacterium spp., coagulase-negative Staphylococcus spp., and Staphylococcus aureus, as well as a reference strain of Staphylococcus aureus (ATCC 25923). Notably, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for all tested microorganisms were found to be 0.2% and 0.4%, respectively, with the exception of Corynebacterium spp., which exhibited resistance to TTO. Furthermore, TTO exhibited a substantial reduction in biofilm biomass, ranging from 30% to 70%, as determined by the MTT method. Through the spray-drying technique, we successfully prepared two TTO-containing formulations with high encapsulation yields (80-85%), microencapsulation efficiency (90-95%), and embedding rates (approximately 40%). These formulations yielded microcapsules with diameters of 6-12 μm, as determined by laser scattering particle size distribution analysis, and exhibited regular, spherical morphologies under scanning electron microscopy. Importantly, UV-Vis analysis post-encapsulation confirmed the presence of TTO within the capsules, with preserved antioxidant and antimicrobial activities. In summary, our findings underscore the substantial therapeutic potential of TTO and its microcapsules for treating ocular infections.

Inhibition of Acinetobacter baumannii Biofilm Formation by Terpenes from Oregano (Lippia graveolens) Essential Oil.

Acinetobacter baumannii is a nosocomial pathogen known for its ability to form biofilms, leading to persistent infections and antibiotic resistance. The limited effective antibiotics have encouraged the development of innovative strategies such as using essential oils and their constituents. This study evaluated the efficacy of oregano (Lippia graveolens) essential oil (OEO) and its terpene compounds, carvacrol and thymol, in inhibiting A. baumannii biofilms. These treatments showed a minimum inhibitory concentration of 0.6, 0.3, and 2.5 mg/mL and a minimum bactericidal concentration of 1.2, 0.6, and 5 mg/mL, respectively. Sub-inhibitory doses of each treatment and the OEO significantly reduced biofilm biomass and the covered area of A. baumannii biofilms as measured by fluorescence microscopy. Carvacrol at 0.15 mg/mL exhibited the most potent efficacy, achieving a remarkable 95% reduction. Sub-inhibitory concentrations of carvacrol significantly reduced the biofilm formation of A. baumannii in stainless steel surfaces by up to 1.15 log CFU/cm2 compared to untreated bacteria. The OEO and thymol exhibited reductions of 0.6 log CFU/cm2 and 0.4 log CFU/cm2, respectively, without affecting cell viability. Moreover, the terpenes inhibited twitching motility, a crucial step in biofilm establishment, with carvacrol exhibiting the highest inhibition, followed by OEO and thymol. The study provides valuable insights into the potential of terpenes as effective agents against A. baumannii biofilms, offering promising avenues for developing novel strategies to prevent persistent infections and overcome antibiotic resistance.

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.

Response Surface Methodology Application for Bacteriophage-Antibiotic Antibiofilm Activity Optimization.

Phage-antibiotic combination-based protocols are presently under heightened investigation. This paradigm extends to engagements with bacterial biofilms, necessitating novel computational approaches to comprehensively characterize and optimize the outcomes achievable via these combinations. This study aimed to explore the Response Surface Methodology (RSM) in optimizing the antibiofilm activity of bacteriophage-antibiotic combinations. We employ a combination of antibiotics (gentamicin, meropenem, amikacin, ceftazidime, fosfomycin, imipenem, and colistin) alongside the bacteriophage vB_AbaP_AGC01 to combat Acinetobacter baumannii biofilm. Based on the conducted biofilm challenge assays analyzed using the RSM, the optimal points of antibiofilm activity efficacy were effectively selected by applying this methodology, enabling the quantifiable mathematical representations. Subsequent optimization showed the synergistic potential of the anti-biofilm that arises when antibiotics are judiciously combined with the AGC01 bacteriophage, reducing biofilm biomass by up to 80% depending on the antibiotic used. The data suggest that the phage-imipenem combination demonstrates the highest efficacy, with an 88.74% reduction. Notably, the lower concentrations characterized by a high maximum reduction in biofilm biomass were observed in the phage-amikacin combination at cA = 0.00195 and cP = 0.38 as the option that required minimum resources. It is worth noting that only gentamicin antagonism between the phage and the antibiotic was detected.

Mutation of putative glycosyl transferases PslC and PslI confers susceptibility to antibiotics and leads to drastic reduction in biofilm formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic, multidrug-resistant pathogen capable of adapting to numerous environmental conditions and causing fatal infections in immunocompromised patients. The predominant lifestyle of P. aeruginosa is in the form of biofilms, which are structured communities of bacteria encapsulated in a matrix containing exopolysaccharides, extracellular DNA (eDNA) and proteins. The matrix is impervious to antibiotics, rendering the bacteria tolerant to antimicrobials. P. aeruginosa also produces a plethora of virulence factors such as pyocyanin, rhamnolipids and lipopolysaccharides among others. In this study we present the molecular characterization of pslC and pslI genes, of the exopolysaccharide operon, that code for putative glycosyltransferases. PslC is a 303 amino acid containing putative GT2 glycosyltrasferase, whereas PslI is a 367 aa long protein, possibly functioning as a GT4 glycosyltransferase. Mutation in either of these two genes results in a significant reduction in biofilm biomass with concomitant decline in c-di-GMP levels in the bacterial cells. Moreover, mutation in pslC and pslI dramatically increased susceptibility of P. aeruginosa to tobramycin, colistin and ciprofloxacin. Additionally, these mutations also resulted in an increase in rhamnolipids and pyocyanin formation. We demonstrate that elevated rhamnolipids promote a swarming phenotype in the mutant strains. Together these results highlight the importance of PslC and PslI in the biogenesis of biofilms and their potential as targets for increased antibiotic susceptibility and biofilm inhibition.