Minimum Inhibitory Concentration Assay Research Articles

Stability of the antimicrobial activity of selected essential oils and nature identical compounds and their interaction with Tween 20 against reference bacterial strains of zootechnical interest

Essential oils (EOs) and nature identical compounds (NICs) express different antimicrobial activity (AAc) which can be affected by variability in composition, stability over time, and the carrier employed for their inclusion in feedstuff and products for animal treatments. The aim of the present study was to evaluate the stability over time of the AAc of 8 EOs and 5 NICs, alone and in combination with Tween 20, on four major bacterial livestock pathogens (E. coli, S. Typhimurium, S. aureus and MRSA). For this purpose, minimal inhibitory concentration (MIC) and checkerboard assay (CkA) tests were performed on EOs and NICs at batch first use (T0 – only MIC), and after 12 (T1) and 24 (T2) months. Several EOs showed initial MICs lower than 2% (v/v) and, except for clove bud and cinnamon EOs, reduced their efficacy over time. The best NICs’ AAc was showed at T1 by cinnamic aldehyde (against MRSA), carvacrol (against E. coli), and thymol (except against MRSA), while at T2 the MIC values decreased for most of the NICs. The CkA between EOs and Tween 20 showed different interactions (Fractional Inhibitory Concentration -FIC- index from 0.06 to 32.04) based on bacteria and time. No interactions were found between NICs and Tween 20 (FIC index from 1.0 to 3.0). The AAc of EOs and NICs changes over time. Therefore, the preservation interval of these additives is critical for the AAc of feedstuff and products intended for livestock animals. Moreover, the NICs seem more suitable for the association with Tween 20.

The antimicrobial and antibiofilm potential of the Piper nigrum L. essential oil: in vitro, in situ, and in silico study

The modern era has raised awareness of healthier lifestyles and food safety, creating new goals for scientists and industry. Here, pathogen microorganisms represent one of the difficulties in ensuring food quality. Microorganism’s resistance to broad-spectrum antibiotics has increased, especially with the planktonic cells' ability to switch into sessile mode and form biofilms. In the endeavor for efficient natural food preservatives, essential oils represent one of the possible solutions for controlling food spoilage, due to their recognized antimicrobial properties. In this study, the assessment of the chemical profile of Piper nigrum L. essential oil (PNEO) was performed. The GC-MS analysis identified (E)-caryophyllene, limonene, and sabinene as prevalent constituents of PNEO, which prompted further antimicrobial and antibiofilm investigations. The minimal inhibitory concentration (MIC) assays revealed that PNEO generally expressed better antimicrobial properties in comparison to the common antibiotics. Moreover, the in situ antimicrobial analysis uncovered the ability of the PNEO’s vapor phase to inhibit the growth of L. monocytogenes, S. aureus, and H. influenzae on selected food models, exposing PNEO as a promising preservation agent. Antibiofilm effects of PNEO were evaluated against biofilm-forming P. fluorescens and S. maltophilia employing MIC assays, crystal violet assays, MALDI-TOF MS analysis, and molecular docking. The obtained results in MIC and crystal violet assays indicated significant antibiofilm activity of PNEO. The MALDI-TOF MS analysis revealed that PNEO altered the protein profiles of the investigated bacteria on stainless steel and glass surfaces, especially in the case of S. maltophilia. In silico antibiofilm investigations indicated a potential involvement of the PNEO components in the interference of quorum-sensing and drug-resistance mechanisms. These findings underline the antimicrobial and antibiofilm virtues of PNEO, indicating its promising usage in food preservation.

Macrofungal Mediated Biosynthesis of Silver Nanoparticles and Evaluation of Its Antibacterial and Wound-Healing Efficacy.

Recently, the utilization of biological agents in the green synthesis of nanoparticles has been given interest. In this study, silver nanoparticles were synthesized from an aqueous extract of macrofungus (mushroom), namely Phellinus adamantinus, in a dark room using 20 µL of silver nitrate. Biosynthesized silver nanoparticles were confirmed by analyzing them using a UV-Vis (ultraviolet-visible) spectrophotometer. The synthesized silver nanoparticles were optimized at different pH and temperatures with various dosages of AgNO3 (silver nitrate) and fungal extracts. The synthesized AgNPs (silver nanoparticles) were characterized using TEM (transmission electron microscopy) and EDX (energy-dispersive X-ray) analyses, which confirmed the presence of silver nanoparticles. The size of the nanosilver particles was found to be 50 nm with higher stability. The mycosynthesized AgNPs showed effective antibacterial activity against strains of Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (E. coli and Pseudomonas aeruginosa) bacteria. The minimum inhibitory concentration (MIC) was found to be 3.125 μg/mL by MIC assay. The MTT assay (3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl-2H-tetrazolium bromide) was performed to study cytotoxicity, and reduced cell viability was recorded at 100 μg/mL. Silver-Polygalacturonic acid-Polyvinyl alcohol ((Ag-PGA)-PVA) nanofiber was prepared using the electrospinning method. The in vitro wound scratch assay was demonstrated to study the wound-healing efficacy of the prepared nanofiber. The wound-healing efficacy of the AgNP-incorporated nanofiber was found to be 20% after 24 h. This study will lay a platform to establish a unique route to the development of a novel nanobiomaterial and its application in antibacterial and wound-healing therapy.

Toxicological Studies and Antibacterial Efficacy of Sorghum bicolor Fermented Liquor Extracts of Bambusa tuldoides cv. ventricosa Leaf against Salmonella typhi

Abstract Background: Bamboo leaf extracted with steep liquor from fermented cereal grains has been used as an ethnomedicine for the treatment of severe typhoid fever in Africa without the proper documentation of its efficacy and side effects. Aim: This study embarks on the evaluation of the toxicity and pharmacological safety profile of fermented Sorghum bicolor liquor extract of Bambusa tuldoides cv. ventricosa leaf as an ethnomedicine against Salmonella typhi infection. Settings and Design: Reconstituted concentrates of various fermented extracts were used for the minimum inhibitory concentration (MIC) assay. Acute toxicity assay comprised two phases. The bioassay constitutes a total of 25 albino mice randomly distributed into 5 test groups. Materials and Methods: Steep liquor from fermented S. bicolor seeds served as the extracting solvent for the leaf of B. tuldoides using Soxhlet apparatus. The determination of both the sensitivity test and MIC assay used different levels. Extracts were orally administered for acute toxicity and dosage determination studies. Extracts were administered at concentrations 25, 50, and 100 mg/mL per day for the bioassays. Application of one-way analysis of variance was used for the determination of the statistical significance of the variations at 0.05 and 0.01 levels. Results and Conclusions: The average zones of inhibition were between 15.00 ± 2.34 and 20.00 ± 4.64 mm, and MIC was 12.5 mg/mL for the growth of S. typhi. The extract administered was found to be safe up to 5000 mg/kg body weight dosage. The agglutination result showed a concentration-dependent effectiveness of fermented liquor extract against S. typhi. Therefore, the bamboo leaf extract is efficient for the treatment of S. typhi infection.

Non-traditional small companion mammals in Spain as reservoirs of antimicrobial-resistant Staphylococci.

The increasing prevalence of antimicrobial resistance (AMR) and multidrug resistance (MDR) in microorganisms poses a significant concern in both human and veterinary medicine. Non-traditional companion animals (NTCAs), particularly popular amongst households with children, play a crucial role in AMR epidemiology due to their rising population. Indeed, it is known that some of these animals may act as reservoirs of zoonotic pathogens and thus be able to spread and transmit them to family members, along with their AMR, through their shared environment. It is therefore imperative to address this concern with the involvement of human, animal and environmental health professionals. This pilot study aimed to assess the prevalence and AMR patterns of Staphylococcus spp. strains obtained from commensal mucosal and skin infection samples in NTC small mammals, with a focus on strains like methicillin-resistant Staphylococcus spp. (MRS) that are critical in public health. For this purpose, 81 animals of different small mammal species were sampled, assessing antimicrobial susceptibility to 27 relevant antimicrobial agents (AMAs) in human health using minimum inhibitory concentration assays, and interpreting them according to EUCAST and CLSI guidelines. The isolated Staphylococci strains were identified by MALDI-TOF, with the predominant species being Mammalicoccus sciuri and Staphylococcus aureus. Including all strains isolated, AMR was observed against all 27 AMAs, including six last-resort AMAs in human medicine. Additionally, over 85% of the strains exhibited MDR. These findings underscore the need to monitor AMR and MDR trends in companion animals and emphasise the potential role of NTCAs in spreading resistance to humans, other animals, and their shared environment, calling for a comprehensive "One Health" approach.

Novel anionic surfactant-modified chlorhexidine and its potent antimicrobial properties.

Chlorhexidine dodecyl sulfate (CHX-DS) was synthesized and characterized via single-crystal X-ray diffraction (SC-XRD), 1H nuclear magnetic resonance (NMR) spectroscopy, 1H nuclear Overhauser effect spectroscopy (NOESY), and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR). The solid-state structure, comprising a 1 : 2 stoichiometric ratio of chlorhexidine cations [C22H30Cl2N10]2+ to dodecyl sulfate anions [C12H25SO4]-, is the first report of chlorhexidine isolated with a surfactant. CHX-DS exhibits broad-spectrum antibacterial activity and demonstrates superior efficacy for reducing bacteria-generated volatile sulfur compounds (VSCs) as compared to chlorhexidine gluconate (CHG). The minimum inhibitory concentrations (MICs) of CHX-DS were 7.5, 2.5, 2.5, and 10 μM for S. enterica, E. coli, S. aureus, and S. mutans, respectively. Furthermore, MIC assays for E. coli and S. mutans demonstrate that CHX-DS and CHX exhibit a statistically significant efficacy enhancement in 2.5 μM treatment as compared to CHG. CHX-DS was incorporated into SBA-15, a mesoporous silica nanoparticle (MSN) framework, and its release was qualitatively measured via UV-vis in aqueous media, which suggests its potential as an advanced functional material for drug delivery applications.

Co-culture of benzalkonium chloride promotes the biofilm formation and decreases the antibiotic susceptibility of a Pseudomonas aeruginosa strain.

Benzalkonium chloride (BAC) is a disinfectant with broad-spectrum antibacterial properties, yet despite its widespread use and detection in the environment, the effects of BAC exposure on microorganisms remain poorly documented. Herein, the impacts of BAC on a Pseudomonas aeruginosa strain Jade-X were systematically investigated. The results demonstrated that the minimum inhibitory concentration (MIC) of BAC against strain Jade-X was 64 mg L-1. Exposure to BAC concentrations of 8, 16, 32, and 64 mg L-1 significantly augmented biofilm formation by 2.03-, 2.43-, 2.96-, and 2.56-fold respectively. The swimming and twitching abilities, along with the virulence factor production, were inhibited. Consistently, quantitative reverse transcription PCR assays revealed significant downregulation of genes related to flagellate- and pili-mediated motilities (flgD, flgE, pilB, pilQ, and motB), as well as phzA and phzB genes involved in pyocyanin production. The results of disk diffusion and MIC assays demonstrated that BAC decreased the antibiotic susceptibility of ciprofloxacin, levofloxacin, norfloxacin, and tetracycline. Conversely, an opposite trend was observed for polymyxin B and ceftriaxone. Genomic analysis revealed that strain Jade-X harbored eleven resistance-nodulation-cell division efflux pumps, with mexCD-oprJ exhibiting significant upregulation while mexEF-oprN and mexGHI-opmD were downregulated. In addition, the quorum sensing-related regulators LasR and RhlR were also suppressed, implying that BAC might modulate the physiological and biochemical behaviors of strain Jade-X by attenuating the quorum sensing system. This study enhances our understanding of interactions between BAC and P. aeruginosa, providing valuable insights to guide the regulation and rational use of BAC.

Graphene oxide-modified polythiophene nanohybrids: antibacterial properties and sonophotocatalytic degradation of dyes under visible-light irradiation

The present work highlights the synthesis of graphene oxide (GO) modified polythiophene (PTh) for its application as an antibacterial agent as well as a visible light active photocatalyst. PTh was synthesized via the interfacial polymerization method using FeCl3 as initiator and then formulated as nanohybrids by mixing it in different weight ratios with graphene oxide (GO) i.e., 1 wt.%,3 wt.% and 5 wt. %. PTh and GO/PTh nanohybrids were characterized using FTIR, UV-visible, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) techniques. The polymerization of PTh as well as modification of PTh with GO was confirmed by IR studies, while the shifting of electronic transitions of PTH upon modification with GO were confirmed by the UV-visible studies. The degradation of dyes, Alizarin Yellow (AY), Basic Blue (BB) and Congo Red (CR) was carried out via sonophotocatalysis under visible light using PTh and GO/PTh as catalysts for a period of 60 min. The GO/PTh revealed higher degradation efficiency as compared to PTh. To evaluate its effectiveness as an antibacterial agent against Bacillus subtilis, disk diffusion inhibitory assay was carried out under dark and light conditions. The results showed that PTh and GO/PTh nanohybrids had bactericidal properties, regardless of light exposure. The results of the minimal inhibitory concentration (MIC) assay indicated the nanohybrid of 3-GO/PTh had the lowest MIC, followed by 1-GO/PTh and 5-GO/PTh.

Antibacterial and antifungal activity of curcumin and methylene blue associated with laser on bacterial and fungal strains

Objective: To analyze the effect of methylene blue and 10% curcumin in fungi and bacteria through an in vitro study using photodynamic therapy (PDT). Methods: Curcumin and methylene blue were photosensitized by a Photon Lase III laser applied for 90 s in a dark environment within a laminar flow chamber. Enterococcus faecalis and Candida albicans strains were cultured and standardized. Then, a minimum inhibitory concentration (MIC) assay was conducted for these photosensitizers, with concentration variations and incubation to evaluate their antimicrobial activity. Results: With PDT, Curcumin had significant antibacterial activity against E. faecalis (MIC = 250 µg/mL). In contrast, methylene blue had antibacterial activity against E. faecalis (MIC < 12.5 µg/mL with PDT) and antifungal activity against C. albicans (MIC <12.5 µg/mL with or without PDT). Both agents showed greater efficacy in the presence of the laser. The results suggest that curcumin and methylene blue associated with laser may effectively treat microbial infections. Conclusion: Further research is needed to evaluate the efficacy and safety of using these agents in animal and human models and their effectiveness against different bacterial and fungal strains.

Synthesis and Antibacterial Activity of Mono- and Bi-Cationic Pyridinium 1,2,4-Oxadiazoles and Triazoles.

One of the main causes of mortality in humans continues to be infectious diseases. Scientists are searching for new alternatives due to the fast increase in resistance of some harmful bacteria to the frontline antibiotics. To effectively treat pathogenic infections, it is crucial to design antibiotics that can prevent the development of pathogenic resistance. For this purpose, a set of 39 quaternary pyridinium and bis-pyridinium salts with different lengths of side alkyl or fluorinated chains, heterocyclic spacers, and counter ions were tested on diverse reference bacterial ATCC (American Type Culture Collection) strains, such as S. aureus and E. coli. Subsequently, 6 out of the 39 pyridinium salts showing relevant MIC (Minimum Inhibitory Concentration) values were tested on clinically isolated, resistant strains of S. aureus, S. epidermids, S. haemolyticus, K. pneumoniae, A. baumannii, and P. aeruginosa. Additional tests have been performed to assess if the minimum concentration detected through MIC assay may limit the growth of biofilms.

Antibiotic Resistant Escherichia coli Isolated from Faecal Samples of Cow at Livestock Farm in Kedah, Malaysia

Introduction: Escherichia coli is a typical microflora found in the intestines of livestock, but regular exposure to antibiotics puts them under selection pressure to acquire antibiotic resistance. This study aimed to identify and characterise the antibiotic resistance profile of E. coli isolated in cow faeces collected from Tobiar Modern farm located in Kedah, Malaysia. Materials and methods: The antibiotic susceptibility test (AST) was conducted to assess the sensitivity of E. coli isolates to tetracycline (30 µg/mL), erythromycin (15 µg/mL) and ampicillin (10 µg/mL) using disk diffusion method followed by Minimum Inhibitory Concentration (MIC) assay. For molecular identification of selected resistant isolates, 16S rDNA gene sequencing was carried out. Results: Six (Isolates A1, P1, P2, P3, P4, and P5) out of 30 isolates were identified as E. coli based on their colonial morphological characteristics. The tests for catalase, indole, MR, TSI, and lactose fermentation all yielded positive results for the isolates, whereas the tests for oxidase, citrate, and VP yielded negative results. All six isolates were found to be erythromycin resistant. The Isolate P4 was observed as a multidrug resistant (MDR) bacterial strain since it exhibited resistance to all tested antibiotics. The MDR Isolate P4 is identified as E. coli strain LWY24 using molecular identification with a 99.7% identity rate. Conclusion: This study offers important preliminary information on the incidence of antibiotic-resistant bacteria (ARB) on this particular local livestock farm. This data is useful for developing plans to reduce the prevalence of ARB in livestock.

UPLC–QTOF-MS/MS analysis of saponin-enriched fractions from Calliandra umbellifera Benth and evaluation of antibacterial activity against multidrug-resistant bacteria

The genus Calliandra, rich in diterpenes, flavonoids, tannins and saponins, has several species with medicinal use. The species Calliandra umbellifera has abundant saponins in its composition, it is a glycoside that acts in defense against insects and pathogens. Due to these properties, saponins have been described to have antibacterial, antifungal, antiparasitic, anti-inflammatory and antiviral activity. Thus, the present work aimed to isolate the saponin-rich fraction of the Calliandra umbellifera species (FRSCu) and to evaluate the antibacterial activity against standard and multidrug-resistant bacterial species. The fraction was obtained from the preparation of the ethanolic extract of the species, being characterized by chromatography, using the method ultra-efficient liquid chromatography coupled to quadrupole/time of flight system (UPLC–QTOF-MS/MS). Minimum inhibitory concentration (MIC) assays were performed to verify the inhibition of bacterial growth for standard and multiresistant strains of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, as well as the evaluation of the activity when combined with the antibiotics: gentamicin, norfloxacin and ampicillin. The chemical analysis of FRSCu allowed the identification of the presence of phenolic compounds, flavonoids, condensed tannins and saponins. The intrinsic antibacterial activity of FRSCu did not show relevant results against the bacteria used. However, it showed promising results when combined with antibiotics, enhancing the effect of gentamicin against two gram-negative strains E. coli and P. aeruginosa and also when combined with norfloxacin against P. aeruginosa. Thus, this work presents an innovative and biotechnological potential that can contribute to future pharmaceutical formulations and knowledge about the C. umbellifera species.

An In-Depth Study on the Chemical Composition and Biological Effects of Pelargonium graveolens Essential Oil.

The essential oil of Pelargonium graveolens (PGEO) is identified in the literature as a rich source of bioactive compounds with a high level of biological activity. This study aimed to examine the chemical profile of PGEO as well as its antioxidant, antibacterial, antibiofilm, and insecticidal properties. Its chemical composition was analyzed using gas chromatography-mass spectrometry (GC-MS), achieving comprehensive identification of 99.2% of volatile compounds. The predominant identified compounds were β-citronellol (29.7%) and geraniol (14.6%). PGEO's antioxidant potential was determined by means of DPPH radical and ABTS radical cation neutralization. The results indicate a higher capacity of PGEO to neutralize the ABTS radical cation, with an IC50 value of 0.26 ± 0.02 mg/mL. Two techniques were used to assess antimicrobial activity: minimum inhibitory concentration (MIC) and disk diffusion. Antimicrobial evaluation using the disk diffusion method revealed that Salmonella enterica (14.33 ± 0.58 mm), which forms biofilms, and Priestia megaterium (14.67 ± 0.58 mm) were most susceptible to exposure to PGEO. The MIC assay demonstrated the highest performance of this EO against biofilm-forming S. enterica (MIC 50 0.57 ± 0.006; MIC 90 0.169 ± 0.08 mg/mL). In contrast to contact application, the assessment of the in situ vapor phase antibacterial activity of PGEO revealed significantly more potent effects. An analysis of antibiofilm activity using MALDI-TOF MS demonstrated PGEO's capacity to disrupt the biofilm homeostasis of S. enterica growing on plastic and stainless steel. Additionally, insecticidal evaluations indicated that treatment with PGEO at doses of 100% and 50% resulted in the complete mortality of all Harmonia axyridis individuals.

Bioprospecting of secondary metabolites produced by endophytic actinomycete isolated from Aloe vera

The aim of this work to evaluate the antibacterial action of secondary metabolites produced by endophytic actinomycetes, against isolates of medical interest. For such, the bacteria were isolated from the botanical sample and classical morphological identification was performed. The induction of metabolites production was carried out using the Submerged Fermentation technique, followed by purification of the extract. The evaluation of antibacterial activity was accomplished by using agar diffusion and Minimum Inhibitory Concentration (MIC) assays. Macro and microscopic analysis of the grown microorganism allowed it to be identified as belonging to the genus Streptomyces. Test results demonstrated that the extract was able to inhibit the growth of C. propinquum 431024, with an inhibition zone of 27±0 mm and of C. propinquum 36063, with an inhibition zone of 24±0 mm and MIC of 32.1 µg/mL for boths. The evaluation of biofilm formation resulted in: C. propinquun 421024 as a weak producer and C. propinquun 36063 as a moderate producer. The biofilm interference test showed that compared to the control there was a reduction in biofilm formation of 22% for C. propinquun 421024, as well as a reduction in pre-formed biofilm of 27%. There was a 48% reduction in the formation and 42% in the pre-formed biofilm of C. propinquun 36063. Lastly, it was concluded that the secondary metabolites produced by the endophytic actinomycetes demonstrated an antibiotic potential against clinical strains (nasopharynx) of Corynebacterium propinquum, a rare pathogen described for causing infections in patients with pulmonary involvement, pleural effusion, bacteremia and endocarditis and which presents an emerging antibiotic resistance.

Whole-transcriptome analysis reveals mechanisms underlying antibacterial activity and biofilm inhibition by a malic acid combination (MAC) in Pseudomonas aeruginosa

Background Pseudomonas aeruginosa is a highly prevalent bacterial species known for its ability to cause various infections and its remarkable adaptability and biofilm-forming capabilities. In earlier work, we conducted research involving the screening of 33 metabolites obtained from a commercial source against two prevalent bacterial strains, Escherichia coli and Staphylococcus aureus. Through screening assays, we discovered a novel malic acid combination (MAC) consisting of malic acid, citric acid, glycine, and hippuric acid, which displayed significant inhibitory effects. However, the precise underlying mechanism and the potential impact of the MAC on bacterial biofilm formation remain unknown and warrant further investigation. Methods To determine the antibacterial effectiveness of the MAC against Pseudomonas aeruginosa, we conducted minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) techniques were employed to observe bacterial morphology and biofilm formation. We further performed a biofilm inhibition assay to assess the effect of the MAC on biofilm formation. Whole-transcriptome sequencing and bioinformatics analysis were employed to elucidate the antibacterial mechanism of the MAC. Additionally, the expression levels of differentially expressed genes were validated using the real-time PCR approach. Results Our findings demonstrated the antibacterial activity of the MAC against P. aeruginosa. SEM analysis revealed that the MAC can induce morphological changes in bacterial cells. The biofilm assay showed that the MAC could reduce biofilm formation. Whole-transcriptome analysis revealed 1093 differentially expressed genes consisting of 659 upregulated genes and 434 downregulated genes, in response to the MAC treatment. Mechanistically, the MAC inhibited P. aeruginosa growth by targeting metabolic processes, secretion system, signal transduction, and cell membrane functions, thereby potentially compromising the survival of this human pathogen. This study provides valuable insights into the antibacterial and antibiofilm activities of the MAC, a synergistic and cost-effective malic acid combination, which holds promise as a potential therapeutic drug cocktail for treating human infectious diseases in the future.

Sensitivity of 240 Listeria monocytogenes isolates to common industrial biocides is more dependent on the presence of residual organic matter or biofilm than on genetic determinants

Listeria monocytogenes continues to be a problem in ready-to-eat foods. Tolerance and adaptation to biocides have been proposed to contribute to the persistence of L. monocytogenes in the food processing industry. This study aimed to determine if the sensitivity to common biocides varies among 240 L. monocytogenes isolates. The impact of residual organic matter (soils), biofilm, and ability to adapt to biocides was also evaluated. Food and food processing related isolates of L. monocytogenes (n = 240) were whole genome sequenced and assigned to sequence types (n = 53) and clonal complexes (n = 32). Isolates were screened for genetic elements associated with biocide tolerance and tested for their minimum inhibitory concentration (MIC) to benzalkonium chloride (BC), peracetic acid (PAA), sodium hypochlorite (SH) and ethanol (ET). Biocide tolerance under soiled conditions, in biofilm and in a broth suspension test (BST) was further tested for 19 representative isolates. Adaptation to sub-MICs of BC, PAA or SH (18 days, 15 °C) was tested for 15 isolates. MIC for PAA (62 mg/L) showed no variation, while for SH (47–94 mg/L) and ET (4.7–9.4% v/v) values differed 2 × among isolates. For BC, an 8 × difference (0.3–2.5 mg/L) was observed. Eighty-seven of 88 BC tolerant isolates (MIC of 2.5 mg/L) harbored known BC tolerance genes. Soiled conditions increased MIC-values 8–33 × for BC and 8 × for SH but not for PAA and ET. Biofilm increased minimum bactericidal concentrations 4–8 × for BC but not for PAA and SH. Survival of isolates with BC tolerance genes was not significantly (p > 0.05) improved in BSTs with PAA, SH or BC. Lineage I isolates (n = 7 of 19) showed significantly (p < 0.05) better survival than lineage II isolates (n = 12) when exposed to PAA (100 mg/L) in BSTs. Adaptation to BC (8 × MIC increase) occurred exclusively for originally sensitive isolates, while no adaptation to SH and PAA were observed. In conclusion, L. monocytogenes isolates showed limited variation in sensitivity to biocides in MIC assays. However, lineage I isolates were less sensitive to PAA than lineage II isolates in BSTs. The efficacy of SH and BC were affected by residual organic matter and/or biofilm, demonstrating the need for proper cleaning prior to biocide use with PAA being less perturbed.

Synthesis, spectroscopic and structural characterizations and in vitro antimycobacterial activity of silver(I) complexes with sulfapyridine (SPY) and sulfamerazine (SM)

Sulfonamides (sulfas) are a specific class of organic compounds that possesses antibacterial, anti-inflammatory, antiviral and antitumor activities. This class of molecules is also used in the synthesis of silver(I) complexes, which usually present significant biological activity as antimicrobial agents. In this context, the present work describes the synthesis, structural characterization and antibacterial assays of two silver(I) complexes with the sulfa drugs sulfapyridine (SPY) and sulfamerazine (SM). Chemical analyses and high-resolution mass spectrometric studies led to 1:1 metal/ligand compositions, suggesting the minimal formulas C11H10N3O2SAg·2H2O for AgSPY and C11H11N4O2SAg·2H2O for AgSM. Spectroscopic techniques such as 1H, 13C and {1H–15N} nuclear magnetic resonance (NMR), and also infrared measurements (IR) suggest the coordination of the ligands to silver(I) ions by the nitrogen atom of the SO2N moiety for both complexes. Crystal structures of AgSPY and AgSM were solved and refined by X-ray powder diffraction studies, where dimeric structures with Ag(I) ions bridging between two ligand molecules by the usual eight membered ring [AgNCN]2 and strong argentophilic interactions were observed. Minimum Inhibitory Concentration (MIC90) assays showed the promising activities of the free sulfonamides and the respective complexes against Mycobacterium tuberculosis H37Rv. MIC90 values, in µmol L–1, were 2.47, 3.60, 1.33 and 1.61 for SPY, AgSPY, SM and AgSM, respectively. The obtained MIC90 values were lower than those found for silver sulfadiazine and silver nitrate in the same experimental conditions.

Tetraclinis articulata (Vahl) Mast. essential oil as a promising source of bioactive compounds with antimicrobial, antioxidant, anti-inflammatory and dermatoprotective properties: In vitro and in silico evidence

Tetraclinis articulata is a known traditional medicinal plant used to manage various ailments, such as diabetes, rheumatism and infectious diseases. This study aims to determine the chemical constituents of T. articulata essential oil (EO) and to evaluate its in vitro antibacterial, anti-candidal, antioxidant, anti-inflammatory and dermatoprotective properties. In addition, a computational docking approach was used to predict the potential antioxidant, antibacterial, antifungal, anti-inflammatory, and cytotoxic properties of the identified compounds. The volatile oil obtained by hydrodistillation was characterized using gas chromatography-mass spectrometry (GC-MS). The antioxidant activity of T. articulata EO was investigated using three complementary assays: DPPH, ABTS and FRAP. Lipoxygenase (5-LOX) and tyrosinase enzymes were used to assess the anti-inflammatory and dermatoprotective effects of this oil. Moreover, disc-diffusion technique, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were employed for the antimicrobial screening. The GC-MS analysis revealed that bornyl acetate (41.80 %), α-pinene (17.97 %) and camphor (15.97 %) are the major components of the studied EO. Moreover, T. articulata EO has exhibited promising antioxidant effect on FRAP, DPPH, and ABTS experiments. It also significantly inhibited 5-LOX (IC50 = 67.82 ± 0.03 μg/mL) and tyrosinase (IC50 = 211.93 ± 0.02 μg/mL). The results of MIC and MBC assays indicated that T. articulata EO is able to inhibit the growth of all tested bacteria (Gram + and Gram -) and Candida species. The ratio of tolerance level indicated that the tested oil was bactericidal against the Gram + bacteria and Candida species, whereas it has a bacteriostatic behavior against the Gram− bacteria. In light of these findings, T. articulata EO may be suggested as a potential pharmaceutical agent to prevent inflammation and skin problems and may serve as a natural antimicrobial and antioxidant alternative for sustainable application in food products.

Electrospun PVA Nanofibers Loaded with Antimicrobial Herbal Extracts for Healing the Infectious Wound

Background: Low-cost, soluble polyvinyl alcohol (PVA) polymers enhance the efficacy of herbal formulations with established antimicrobial properties. Objectives: This study investigates the healing process of Staphylococcus aureus (S. aureus)-infected wounds using PVA loaded with herbal extracts, including Arnebia euchroma, Allium sativum, and Echinacea purpurea. Methods: minimal bactericidal concentration (MBC) and minimal inhibitory concentration (MIC) assays, Disk Diffusion Method (DDM) tests, and Gas Chromatography-Mass Spectrometry (GC/MS) analyses were conducted on herbal extract samples. Twelve male Wistar rats were divided into G1: Negative control group (healthy mice), G2: Wound model + S. aureus (Positive control), G3: wound model + S. aureus + Povidone-iodine, and G4: Wound model + S. aureus + PVA/formulation. Hematoxylin-eosin and immunofluorescent staining were employed to assess wound healing. Results: The ethanolic herbal extract exhibited potent antibacterial activity against S. aureus, with MIC and MBC values in the 1.87 mg/mL range. The PVA/formulation displayed a zone of inhibition with a diameter of 7 mm against S. aureus. Histopathological investigations indicated that the infected wound treated with Nanofibers extract experienced a significant reduction in inflammation between days 7 and 14. Notably, the increased CD3 expression at this site was remarkable for the healing process. Conclusions: Consequently, this experimental study suggests combining PVA and herbal extracts enhances antibacterial properties and promotes CD3 expression and re-epithelialization effects.

2065. Efficacy of D-peptide-conjugated Antisense Compounds Against Pseudomonas aeruginosa

Abstract Background Pseudomonas aeruginosa (PA) causes significant morbidity and mortality in immunocompromised hosts and those with cystic fibrosis. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are antisense compounds designed to target specific genes and prevent translation. PPMOs with L-isomer peptides (L-PPMOs) were previously shown to have in vitro and in vivo activity. However, L-PPMOs are vulnerable to degradation by host proteases. We hypothesize that PPMOs with a D-isomer peptide (D-PPMOs) may be a more stable and potent alternative. We aimed to characterize the activity of L- and D-isomers in PA strains using planktonic and biofilm assays as well as an infected mouse model. Methods All L- and D-PPMOs were synthesized by Sarepta Therapeutics, Inc. We tested L- and D-isomers of the (RXR)4XB (X for 6-aminohexanoic acid and B for β-Ala) and R6G peptide [(Arg)6-Gly]. PMOs for rpsJ (ribosomal protein) and acpP (fatty acid synthesis protein) were conjugated to the 3’ end of each peptide. Minimum inhibitory concentration (MIC) assays were performed in unique lab and clinical (including MDR) isolates using MOPS medium. Strain PAO1 biofilms were grown in MBEC plates for 24 hours, with PPMOs then dosed every 8 hours for 24 hours, followed by colony enumeration. For in vivo studies, mice were infected with strain PA103 intratracheally and given intranasal PPMO at 6 hours post-infection. Lung burden was determined at 24 hours post-infection. Results MIC values of D-R6G-RpsJ and D-R6G-AcpP were comparable to their L-isomers, but D-RXR-RpsJ showed an improved MIC90 of 0.5 μM compared to the L-RXR-RpsJ MIC90 of 16 μM (Table 1). Both isomers demonstrated reduction of 48-hour biofilm to various degrees. D-PPMO biofilm reduction ranged from 1.5 logs to &amp;gt; 3 logs (D-R6G-AcpP and D-RXR-RpsJ, respectively). L-PPMO biofilm reduction ranged from 2 logs to &amp;gt; 4 logs. D-PPMOs were superior to their L-PPMO counterparts in our delayed in vivo acute pneumonia model, with most D-PPMOs resulting in an additional 50% decrease in lung burden. Conclusion D-PPMOs show similar or improved activity in vitro and in vivo when compared to L-PPMOs at the same dosage. Future studies will investigate the pharmacology of D-PPMOs, as they may represent a more biologically robust alternative to L-PPMOs for PA infections. Disclosures David E. Greenberg, MD, University of Texas Southwestern Medical Center: Dr. Greenberg has numerous patents on PPMOs