Minimum Inhibitory Concentration Research Articles

Valorisation of Refosco grape pomace: exploring its antioxidant, antimicrobial, and antiadhesive properties

Abstract Agricultural by-products, including winery waste, represent both an environmental challenge and a valuable source of bioactive compounds. This study investigated the antioxidative, antimicrobial, and antiadhesive properties of grape seed and peel extracts derived from the red wine variety Refosco. Water and methanol extracts were prepared from dried, ground seeds and peels, both before and after supercritical extraction (SFE). The total antioxidant capacity (TAC) of water- and lipid-soluble compounds was measured. Minimal inhibitory concentrations (MICs) and antiadhesion effects of water extracts were then determined for Campylobacter jejuni, Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The antimicrobial effects were further evaluated in pasta dough as a model food matrix. Our results showed that some methanol extracts exhibited significantly higher TAC values compared to water extracts. However, the TAC of water-soluble compounds was significantly higher than that of lipid-soluble compounds, regardless of the solvent used. Although the TAC of dried seed and peel samples (SDRY; PDRY) was higher than that of SFE residues, the difference was not statistically significant. The most potent antimicrobial effect was observed for SDRY extracts, with MICs as low as 0.625 mg/mL. Additionally, both SDRY and PDRY water extracts, at 1/8 of the MIC, exhibited antiadhesion activity. In conclusion, red grape pomace from the Refosco variety proves to be a cost-effective source of valuable water-soluble compounds with antioxidative, antimicrobial, and antiadhesive properties. Importantly, results showed that the SFE residue retained some bioactive properties, further highlighting the potential of this by-product.

Chemical characterization of Eucalyptus (Eucalyptus globulus) leaf essential oil and evaluation of its antifungal, antibacterial and antioxidant activities.

This study investigates the chemical composition of the essential oil (EO) extracted by hydrodistillation from dry Eucalyptus leaves (Eucalyptus globulus) and its antifungal, antibacterial and antioxidant potential. The Eucalyptus leaves were harvested in the commune of Seraïdi (north-eastern Algeria). Chemical analysis carried out by chromatography coupled with mass spectrophotometry (GC-SM) revealed the presence of 20 molecules representing approximately 100% of the overall component, with a yield of 1.58%. This oil is composed mainly of linalool (30.09%), followed by b-Linalyl oxide (13.93%), Camphor (12.09%), 1,8-Cineole=eucalyptol (10.95%) and Bergamol (10.03%). Other constituents were identified at relatively medium (Epoxylinalol - 8.82%, Borneol - 5.71%) and low (alpha-Terpinol - 1.11) levels. This result shows that this EO differs from those usually extracted from eucalyptus leaves because it is of linalool chemotype and not eucalyptol. The determination of the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) was carried out to evaluate the antifungal activity of Eucalyptus EO on the growth of Fusarium roseum mycelium. The values recorded are 2500µg/ml for the MIC and 4000µg/ml for the MFC. The results obtained revealed an antifungal activity of this oil for practically all doses applied against Fusarium mycelial proliferation despite the low reported levels of 1,8-cineole compared to the other components. The antibacterial activity against the Pseudomonas savastanoi strain was also examined which revealed effectiveness of this oil. In parallel, the DPPH test revealed a moderate antioxidant activity of the studied EO compared to Vit C with an IC50 17mg/ml probably due to its components' antagonistic or synergistic effect.

Cinnamic Acid Compounds (p-Coumaric, Ferulic, and p-Methoxycinnamic Acid) as Effective Antibacterial Agents Against Colistin-Resistant Acinetobacter baumannii

Colistin-resistant Acinetobacter baumannii (COLR-Ab) is an opportunistic pathogen commonly associated with nosocomial infections, and it is difficult to treat with current antibiotics. Therefore, new antimicrobial agents need to be developed for treatment. Based on this information, we investigated the antimicrobial, antibiofilm, and combination activities of p-coumaric acid (p-CA), ferulic acid (FA), and p-methoxycinnamic acid (p-MCA) against five COLR-Ab isolates. p-CA, FA, and p-MCA exhibited antimicrobial activity against COLR-Ab isolates, with minimum inhibitory concentration (MIC) values in the range of 256–128 µg/mL, 1024–512 µg/mL, and 512–128 µg/mL, respectively. The combination effects of the compounds with colistin (COL) were evaluated using a checkerboard synergy test. The combinations exhibited a synergistic effect and caused a 128- to 16-fold decrease in COL MIC values. In addition, the biofilm production capacities of the COLR-Ab isolates and the antibiofilm activities of the compounds were determined using the microtitre plate-based crystal violet (CV) technique. The compounds showed effective antibiofilm activity against strong and moderate biofilm-producing isolates, inhibiting biofilm formation by 77.5% and 19.7%. Spectrometric measurements were used to examine the effect of compounds on membrane permeability; 1.9-, 1.66-, and 1.34-fold increases in absorbance values were observed at MIC concentrations of p-CA, FA, and p-MCA, respectively. Furthermore, morphological changes caused by the compounds in the isolate were observed using scanning electron microscopy (SEM) micrographs. According to the WST assay, the compounds did not show any statistically significant cytotoxic effect on the cells (p > 0.05). These findings indicate that p-CA, FA, and p-MCA may be potential new alternative candidates against resistant A. baumannii.

Seasonal Variations in Chemical Composition and Antibacterial and Antioxidant Activities of Rosmarinus officinalis L. Essential Oil from Southwestern Romania

Our study reports for the first time, over a 12-month period, the seasonal variations in chemical composition and antibacterial and antioxidant activity of Rosmarinus officinalis L. essential oil (RoEO) from Southwestern Romania (Oltenia region). To analyze the constituents of RoEO, a comprehensive gas chromatography/mass spectrometry (GC/MS) method was employed. The analysis aimed to identify and quantify the various components by comparing their mass spectra with reference spectra from the National Institute of Standards and Technology (NIST) Library 2020. The minimum inhibitory concentration (MIC) values of Staphylococcus aureus minimum were determined using the microdilution method (96-well plates). The antioxidant activity was analyzed using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and hydrogen peroxide (H2O2) radical scavenging assays. This analysis provided a detailed profile of RoEO’s constituents, revealing significant monthly variations. Key compounds, such as camphor, eucalyptol, α-pinene, camphene, and α-myrcene, were quantified, alongside lesser-studied constituents like β-pinene, α-terpinene, linalool, terpinolene, and carvacrol. Comparisons were made with a reference sample from Tunisia. Oxygenated monoterpenes reach the highest concentration (56.82–66.94%), followed by monoterpene hydrocarbons (30.06–40.28%), sesquiterpene hydrocarbons (0.90–2.44%), and oxygenated sesquiterpenes (0.02–0.23%). Camphor was found in high concentrations ranging from 29.41% to 40.03%. 1,8-Cineole was another dominant compound, ranging from 13.07% to 16.16%, significantly lower compared to the Tunisian reference (52.77%). α-Pinene ranged from 11.36% to 19.33%, while α-myrcene ranged from 1.65% to 3.08%. Correlations between specific compounds and their bioactivity were explored to understand their contributions to the overall efficacy of RoEO. This comprehensive analysis provides valuable insights into the potential applications and seasonal variability of RoEO from Romania.

Synthesis and structural insights of bis(2-methoxy-6-{[(2-methylpropyl)imino]methyl}phenolato) nickel (II) complex through DFT and docking investigations

Nickel complexes are a potential candidate for antibacterial and antifungal activity. A new Ni (II) complex, bis(2-methoxy-6-{[(2-methylpropyl)imino]methyl}phenolato)nickel (II) (2), was synthesised by reacting, bis(3-methoxy-salicylaldehyde)nickel (II) (1) with isobutylamine. It was characterised by single crystal X-ray diffraction (ScXRD), UV-Vis, NMR, IR, mass spectrometry, and thermogravimetry (TG) to study its structure and physico-chemical properties. The ScXRD showed a square planar geometry, and monoclinic crystal system with a space group P21/n. The TG analysis revealed its thermal durability pre and post-melting up to 225 oC with a weight loss of only 2%. The optimized molecular structure, energy gap between HOMO and LUMO, and intermolecular interactions were studied by computational methods. The microbial activity evaluation showed significant anti-bacterial activity against E. coli and S. aureus when the concentration exceeded 40 µg/mL, and a prominent anti-fungal activity over C. albicans and C. tropicalis above 30 µg/mL. The values of minimum inhibitory concentration (MIC) for bacteria (MIB) and fungi (MIF) implied its potential to inhibit the growth of microbes. Docking studies revealed that the molecule binds well with proteins such as PDB: 2W9H for Dihydrofolate Reductase of S.aureus as shown by its binding energy of -8.62 kcal.mol− 1.

Antioxidant, Antimicrobial, Anticancer, and Molecular Docking Insights into Pancratium maritimum Seeds and Flowers: A Phytochemical Approach.

This study investigates the antioxidant, antimicrobial, and anticancer properties of Pancratium maritimum L. in Sp. Pl.: 291 (1753) seeds and flowers. Antioxidant activity was assessed using DPPH free radical scavenging and iron chelation assays. Antimicrobial evaluations assessed the efficacy of the extracts against diverse microorganisms. Cell viability assays were conducted on the dukes c colon cancer (SW480), while gas chromatography-mass spectrometry (GC-MS) analysis facilitated the identification of bioactive compounds. The ethanol extract of P. maritimum seeds exhibited a total phenolic content of 296.89±14.53 mg GAE/g extract DW and a total flavonoid content of 361.03±20.18 mg QE/g extract DW. Conversely, the flower extract showed a total phenolic content of 95.03±7.22 mg GAE/g extract DW and a total flavonoid content of 272.12±16.42 mg QE/g extract DW. As a result, the ethanol extract of P. maritimum seeds contains higher phenolic and flavonoid contents than the flower extract. Antimicrobial evaluations demonstrated significant inhibitory effects of both seed and flower extracts, with minimum inhibitory concentration (MIC) values ranging from 25 to >50 mg/mL. Notably, the seed extract showed greater activity against E. coli and C. krusei. GC-MS analysis identified 18 bioactive compounds in the seed extract and 16 in the flower extract, with crucial components including ethyl oleate and 5-hydroxymethylfurfural. Additionally, cell viability assays revealed that ethanol extracts from seeds and flowers significantly reduced SW480 cell viability, particularly at doses of 750 μg/mL and 250 μg/mL, respectively. These findings underscore the therapeutic potential of P. maritimum in terms of its antioxidant, antimicrobial, and anticancer properties, highlighting its value as a natural source of antioxidants and antimicrobial agents. Furthermore, the molecular docking study emphasises strong binding interactions of key compounds, particularly ethyl oleate and hexadecanoic acid ethyl ester, with the human STARD10 protein. The biological interactions and health implications of P. maritimum provide a significant foundation for future research in drug development and therapeutic applications.

Effect of Chirality and Amphiphilicity on the Antimicrobial Activity of Tripodal Lysine-Based Peptides.

A series of tripodal (three-arm) lysine-based peptides were designed and synthesized and their self-assembly properties in aqueous solution and antimicrobial activity were investigated. We compare the behaviors of homochiral tripodal peptides (KKY)3K and a homologue containing the bulky aromatic fluorenylmethoxycarbonyl (Fmoc) group Fmoc-(KKY)3K, and heterochiral analogues containing k (d-Lys), (kkY)3K and Fmoc-(kkY)3K. The molecular conformation and self-assembly in aqueous solutions were probed using various spectroscopic techniques, along with small-angle X-ray scattering (SAXS) and cryogenic-transmission electron microscopy (cryo-TEM). In cell viability assays using fibroblast cell lines, the tripodal peptides without Fmoc were observed to be noncytotoxic over the concentration range studied, and the Fmoc functionalized tripodal peptides were only cytotoxic at the highest concentrations (above the critical aggregation concentration of the lipopeptides). The molecules also show good hemocompatibility at sufficiently low concentration, and antimicrobial activity was assessed via MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) assays. These revealed that the Fmoc-functionalized tripodal peptides had significant activity against both Gram-negative and Gram-positive bacteria, and in the case of Gram-positive Staphylococcus aureus, the antimicrobial activity for Fmoc-(kkY)3K was improved compared to polymyxin B. The mechanism of the antimicrobial assay was found to involve rupture of the bacterial membrane as evident from fluorescence microscopy live/dead cell assays, and scanning electron microscopy images.

In-cell NMR reveals metabolic adaptations in central carbon pathways driving antibiotic tolerance in Salmonella Typhimurium.

Antibiotic tolerance presents a significant challenge in eradicating bacterial infections, as tolerant strains can survive antibiotic treatment, contributing to the recurrence of infections and the development of resistance. However, unlike antibiotic resistance, tolerance is not detectable by standard susceptibility assays such as minimal inhibitory concentration (MIC) tests. Consequently, antibiotic tolerance often goes unnoticed in clinical settings. Bacterial metabolism is closely linked to antibiotic efficacy, and thus presents as a potential target for novel diagnostic methods. Recent advancements in nuclear magnetic resonance (NMR) spectroscopy, including dynamic nuclear polarization (DNP-NMR), enable a non-invasive real-time approach to analyzing bacterial metabolism. In this study, we applied both 1H and in-cell 13C NMR spectroscopy to investigate metabolic adaptations in a tolerance-evolved Salmonella Typhimurium strain, C10, developed through ten cycles of ampicillin treatment. Our results demonstrated that despite similar MICs and growth rates, the C10 strain exhibited a 25-fold increase in tolerance compared to the wild-type, while exhibiting lower metabolic activity. Under ampicillin stress, however, the C10 strain maintained higher metabolic activity and demonstrated greater resilience in glucose consumption and metabolite production relative to the wild-type. Using DNP-NMR, rapid metabolic shifts in the C10 strain were identified within 10 minutes of exposure to high concentrations of ampicillin, characterized by accumulation of key metabolites such as pyruvate and acetate. Overall, our findings underscore the potential of real-time NMR-based analyses to provide deeper insights into antibiotic tolerance and distinguish between susceptible and tolerant bacterial strains.

Anti-Mycobacterial Activity of Bacterial Topoisomerase Inhibitors with Dioxygenated Linkers.

Developing new classes of drugs that are active against infections caused by Mycobacterium tuberculosis is a priority for treating and managing this deadly disease. Here, we describe screening a small library of 20 DNA gyrase inhibitors and identifying new lead compounds. Three structurally diverse analogues were identified with minimal inhibitory concentrations of 0.125 μg/mL against both drug-susceptible and drug-resistant strains of M. tuberculosis. These lead compounds also demonstrated antitubercular activity in ex vivo studies using infected THP-1 macrophages with minimal cytotoxicity in THP-1, HeLa, and HepG2 cells (IC50 ≥ 128 μg/mL). The molecular target of the lead compounds was validated through biochemical studies of select analogues with purified M. tuberculosis gyrase and the generation of resistant mutants. The lead compounds were assessed in combination with bedaquiline and pretomanid to determine the clinical potential, and the select lead (158) demonstrated in vivo efficacy in an acute model of TB infection in mice, reducing the lung bacterial burden by approximately 3 log10 versus untreated control mice. The advancement of DNA gyrase inhibitors expands the field of innovative therapies for tuberculosis and may offer an alternative to fluoroquinolones in future therapeutic regimens.

Fabrication and characterization of chitosan-based bionanocomposite coating reinforced with TiO2 nanoparticles and carbon quantum dots for enhanced antimicrobial efficacy.

Polymer-based nanocomposite coatings that are enhanced with nanoparticles have gained recognition as effective materials for antibacterial purposes, providing improved durability and biocidal effectiveness. This research introduces an innovative chitosan-based polymer nanocomposite, enhanced with titanium oxide nanopowders and carbon quantum dots. The material was synthesized via the sol-gel process and applied to 316L stainless steel through dip-coating. Structural and morphological properties, including crystal structure, microstructure, elemental dispersion, particle size distribution, chemical composition, and surface morphology, were thoroughly characterized. The results demonstrated that carbon quantum dots and titanium oxide nanopowders were uniformly dispersed within the chitosan matrix, forming a homogeneous, non-agglomerated coating. The antibacterial efficacy of the synthesized samples against 7 different Gram-positive and Gram-negative bacteria was assessed through disk diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) tests. The results confirmed the antibacterial activity of synthesized samples against most of the bacterial pathogens tested but exhibited stronger antibacterial effects on Gram-negative bacteria compared to Gram-positive bacteria. The largest inhibition zones, measuring 21 mm and 16 mm, were observed for Pseudomonas and E. coli for titanium dioxide nanoparticles and the final nanocomposite, respectively. Additionally, the MIC and MBC values for all 7 bacteria were determined.

Investigating the inhibitory effect of nettle (Urtica dioica L.) essential oil and Pickering nanoemulsion on some pathogenic bacteria inoculated into pizza cheese.

The aim of the present research was to evaluate the effect of Urtica dioica L. (nettle) essential oil (in the forms of Pickering nanoemulsion (NEO) and free (EO)) on microbial, chemical and sensory changes of pizza cheese stored at 4°C for 12days. For this purpose, Escherichia coli and Listeria monocytogenes were inoculated into pizza cheese. In all tests, the control group had the lowest score after 12days of storage. In the antimicrobial assay test in different treatments, NEO4% treatment decreased the growth of E.coli from 4 (0th day) to 3.3 log CFU/g (12th day) and the growth of L. monocytogenes from 3.8 (0th day) to 3.1 log CFU/g (12th day). The minimum inhibitory concentration (MIC) of NEO and EO for E.coli and L. monocytogenes was 0.62±0.01mg/mL. Additionally, the minimum bactericidal concentration (MBC) of EO and NEO for E. coli was 25±0.1mg/mL, and for L. monocytogenes was 1.25±0.1mg/mL. At day 12, almost all treatments (free form and nano) had relatively similar pH. In our study, the minimum and maximum value of DPPH was detected in the treatment of NEO1% (31.25±1.50%) and BHT200 (96.40±2.5%), respectively. Also, on the 12th day of the test, the NEO treatment obtained the highest score in all sensory tests (appearance & color, body & texture, odor and overall acceptability). According to the findings of the present study, Pickering emulsion form of nettle EO increases the storage period of pizza cheese.

Comparative antibacterial activity of clove extract against Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen belonging to the γ-proteobacteria family, known to cause pneumonia linked with ventilator use and nosocomial infections. With the increasing prevalence of antibiotic-resistant bacteria, there is a pressing need to identify alternatives to conventional antibiotics. Plant-derived substances (PDSs) offer potential not only as antibacterial agents but also as modulators of antibiotic resistance. In this study, diffusion assay using disc agar, and the minimum inhibitory concentration (MIC) assay of each imipenem, amoxicillin-clavulanic acid, and clove extract was determined. The clove extract was analyzed via the UHPLC/MS, and the checkerboard assay was employed to evaluate the potential synergistic effects of combining clove extract with both antibiotics. The combination of clove extract to each antibiotic led to a significant reduction in their respective MICs. Moreover, each antibiotic exhibited synergistic effects on the fractional inhibitory concentration value (FIC) of clove extract. The analysis identified seventeen components in the clove extract, predominantly flavonoids and phenolic compounds. The antibacterial efficacy of the Syzygium aromaticum Myrtaceae extract against P. aeruginosa indicated its potential as a promising antibacterial agent capable of enhancing the effectiveness of existing medications.

Antibacterial activity of Nigella sativa against multi-drug resistant bacteria

Background: Multidrug Resistant (MDR) bacteria are considered as the one of the most dangerous pathogens causing a high mortality rate globally. Medicines from the natural source might be the best therapeutic option in current emerging trends of resistance. Methods: We determined the antibacterial activity of Nigella sativa seeds extracts against the clinical isolates of MDR Staphylococcus aureus and Escherichia coli. The collection of different isolates was done from the different hospitals’ settings in Faisalabad. Identifications of these isolates were done phenotypically and through the conventional PCR via genus and species-specific primers. The isolates were then tested for antimicrobial susceptibility using agar well diffusion technique. The activity of N. sativa seed oil was also evaluated in DMSO at concentrations (25%, 50% and 75%). Results: Against S. aureus, 50% oil concentration in DMSO exhibited highest inhibitory zone of 15 mm followed by 90% ethanol extract showed clear zone of 13 mm and 12 mm zone of inhibition showed by the methanol-based extract. Against E. coli 50% oil concentration in Dimethyl sulfoxide (DMSO) and 90% ethanol extract both showed clear zone of 12 mm and 11 mm zone of inhibition showed by the methanol-based extract. However, Minimum inhibitory concentration (MIC) of 50% oil concentration is 0.4 μl followed by 90% ethanol extract showed 0.4 mg and 0.8 mg showed by the methanol-based extract 90% on S. aureus. The oil concentration MIC value of 50% is 0.4 μl then followed by the ethanol extract at 90% and revealed 0.4mg and methanol was followed by 90% extract showed 0.4 mg to the E. coli. Conclusion: We concluded that N. sativa can be used as a pharmaceutical and new therapy for action against MDR bacteria.

Isolation of New Strains of Lactic Acid Bacteria from the Vaginal Microbiome of Postmenopausal Women and their Probiotic Characteristics

Lactic acid bacteria (LAB), traditionally consumed as fermented foods, are now being applied to the medical field beyond health-functional food as probiotics. Therefore, it is necessary to continuously discover and evaluate new strains with suitable probiotic characteristics, mainly focusing on safety. In this study, we isolated eight new strains from postmenopausal vaginal fluid using culturomics approaches, an emerging area of interest. Data showed that most strains possessed significant cell surface hydrophobicity (≥ 76%), auto-aggregation capacity (17 to 61%), strong adhesion activity (8 to 34%), and excellent resistance to gastric acid, bile salt, and digestive enzyme, enhancing their survival in the gastrointestinal tract. Moreover, the strains exhibited functional characteristics, including substantial antibacterial activity with a minimal inhibitory concentration (MIC) ranging from 12.5 to 50%. They also harbored bacteriocins genes, produced short-chain fatty acids (acetate and propionate), exhibited significant phagocytic activity, possessed high antioxidative properties, rapidly depleted sodium nitrite, and exhibited proteolysis and β-glucosidase activity. In addition, heat-killed LAB strains significantly reduced the gene expressions of proinflammatory cytokines such as IL-β, IL-6, and iNOS in macrophages. Safety assessment revealed no cytotoxicity in macrophage cell lines. All strains tested negative for biogenic amine or H2O2 production, displayed no gelatinase or hemolytic activity, lacked virulence genes or detrimental enzymes, and displayed antibiotic susceptibility. In summary, these newly isolated strains demonstrate excellent probiotic functionality with a strong focus on safety, making them promising candidates for future drug development in the relevant fields.

Whole Genome Sequence Analysis of Terbinafine Resistant and Susceptible Trichophyton Isolates from Human and Animal Origin.

Trichophyton indotineae, first identified in India, has increasingly been reported in Asia, the Middle East, Europe, and recently in the USA. The global spread of terbinafine-resistant T. indotineae underscores the urgency of the issue. With its ability for human-to-human transmission, it can be considered anthropophilic. However, its highly virulent nature suggests a possible link to zoophilic species, raising the potential for disease transmission from animals. In this study, we have performed whole genome sequencing (WGS) of terbinafine susceptible and resistant Trichophyton species from animal and human origin to understand transmission dynamics of this species. Thirteen isolates of Trichophyton spp. from human (n = 9) and canine (n = 4) origin, respectively from Chandigarh and Bareilly, India, were included in this study. Isolate identification based on ITS extracted from WGS data identified six T. indotineae (ITS genotype VIII) and seven T. interdigitale (ITS genotype II) isolates. WGS single nucleotide polymorhpism (SNP) analysis separated the isolates into two distinct groups, T. indotineae and T. interdigitale and showed the clonal nature of both species. For both species, low SNP differences between isolates from humans and dogs were observed as well as low differences between isolates from Chandigarh and Bareilly, cities >350km apart from each other. These findings suggest zoonotic transmission, next to fast spread across large distances. The T. indotineae terbinafine-resistant strains exhibitedthe SQLEF397L substitution while susceptible strains had the SQLES395P substitution or demonstrated a wild-type (WT) SQLE sequence. However, all T. interdigitale strains displayed a WT SQLE sequence despite terbinafine minimum inhibitory concentrations (MICs) ranging between 0.031 to64µg/mL.

In vitro evolution of ciprofloxacin resistance in Neisseria commensals and derived mutation population dynamics in natural Neisseria populations.

Commensal Neisseria are members of a healthy human oropharyngeal microbiome; however, they also serve as a reservoir of antimicrobial resistance for their pathogenic relatives. Despite their known importance as sources of novel genetic variation for pathogens, we still do not understand the full suite of resistance mutations commensal species can harbor. Here, we use in vitro selection to assess the mutations that emerge in response to ciprofloxacin selection in commensal Neisseria by passaging 4 replicates of 4 different species in the presence of a selective antibiotic gradient for 20 days; then categorized derived mutations with whole genome sequencing. 10/16 selected cells lines across the 4 species evolved ciprofloxacin resistance (≥ 1 ug/ml); with resistance-contributing mutations primarily emerging in DNA gyrase subunit A and B (gyrA and gyrB), topoisomerase IV subunits C and E (parC and parE), and the multiple transferable efflux pump repressor (mtrR). Of note, these derived mutations appeared in the same loci responsible for ciprofloxacin reduced susceptibility in the pathogenic Neisseria, suggesting conserved mechanisms of resistance across the genus. Additionally, we tested for zoliflodacin cross-resistance in evolved strain lines and found 6 lineages with elevated zoliflodacin minimum inhibitory concentrations. Finally, to interrogate the likelihood of experimentally derived mutations emerging and contributing to resistance in natural Neisseria, we used a population-based approach and identified GyrA 91I as a substitution circulating within commensal Neisseria populations and ParC 85C in a single gonococcal isolate. A small cluster of gonococcal isolates shared commensal alleles at parE, suggesting recent cross-species recombination events.

A Conjugated Oligomer with Drug Efflux Pump Inhibition and Photodynamic Therapy for Synergistically Combating Resistant Bacteria.

High expression of drug efflux pump makes antibiotics ineffective against bacteria, leading to drug-resistant strains and even the emergence of "superbugs". Herein, we design and synthesize a dual functional o-nitrobenzene (NB)-modified conjugated oligo-polyfluorene vinylene (OPFV) photosensitizer, OPFV-NB, which can depress efflux pump activity and also possesses photodynamic therapy (PDT) for synergistically overcoming drug-resistant bacteria. Upon light irradiation, the OPFV-NB can produce aldehyde active groups to covalently bind outer membrane proteins, such as tolerant colicin (TolC), blocking drug efflux of bacteria. The minimum inhibitory concentration of antibiotic model chloramphenicol (CHL) is reduced about 64 times, significantly resensitizing drug-resistant bacteria to antibiotics. Also, the probe can produce highly efficient reactive oxygen species (ROS) under light irradiation. Consequently, the unimolecular OPFV-NB-based system demonstrates insusceptibility to antibiotic resistance while maintaining significant antimicrobial effects (100%) against drug-resistant bacteria. More importantly, in vivo assays corroborate that the combined system greatly accelerates wound healing by eradicating the bacterial population, dampening inflammation, and promoting angiogenesis. Overall, the OPFV-NB not only counteracts antibiotic resistance but also holds tremendous PDT efficiency, which provides a promising therapeutic strategy for combating drug-resistant bacteria and treating bacteria-infected wounds.

Novel antimicrobial defensin peptides from different coleopteran insects (Coleoptera: Insecta): identification, characterisation and antimicrobial properties

Antimicrobial peptides are crucial components of the immune systems of both vertebrates and invertebrates. Here, defensins, the most studied class of antimicrobial molecules in arthropods were investigated in four coleopteran insect species: Harpalus rufipes (DeGeer, 1774), Mylabris quadripunctata (Linnaeus, 1767), Sphaeridium marginatum (Linnaeus, 1758), and Ocypus mus (Brullé, 1832). The peptides synthesized with over 95% purity and their antimicrobial activities were evaluated by MIC test method. As a result, it was determined that Mylabris quadripunctata defensin (MqDef) showed high antimicrobial activity against Staphylococcus aureus and MRSA, whereas Sphaeridium marginatum (SmDef) and Harpalus rufipes (HrDef) defensins against Candida tropicalis.

Dissemination of arr-2 and arr-3 is associated with class 1 integrons in Klebsiella pneumoniae clinical isolates from Portugal.

\nKlebsiella pneumoniae is a common pathogen of healthcare-associated infections expressing a plethora of antimicrobial resistance loci, including ADP-ribosyltransferase coding genes (arr), able to mediate rifampicin resistance. The latter has activity against a broad range of microorganisms by inhibiting DNA-dependent RNA polymerases. This study aims to characterise the arr distribution and genetic context in 138 clinical isolates of K. pneumoniae and correlate these with rifampicin resistance. All isolates were subjected to whole-genome sequencing for species identification, typing and AMR genes identification, along with the determination of the minimum inhibitory concentration (MIC) of rifampicin. Molecular detection of arr genes and class 1 integrons was performed for rifampicin-resistant isolates. Efflux activity was investigated as a possible determinant of rifampicin resistance in isolates devoid of known genetic determinants. Twelve isolates exhibited high rifampicin MICs (≥ 64mg/L), 124 showed intermediate MICs (16-32mg/L) and two displayed low (8mg/L) MICs. Two arr allelic variants, arr-2 and arr-3, were found across one and nine K. pneumoniae isolates, respectively, all within class 1 integrons, including a newly described integron, and all associated with high rifampicin MICs (≥ 64mg/L). Elevated resistance levels were additionally linked to increased arr-2/3 expression and closer proximity to the promoter. No arr gene or rpoB mutations were found across the remaining two isolates and no correlation between efflux activity and high-level rifampicin resistance was found for both isolates. In conclusion, this study demonstrates that arr genes confer high levels of rifampicin resistance in K. pneumoniae highlighting its widespread dissemination within class 1 integrons.

Exploring caffeine as a disruptor of membrane integrity and genomic stability in Staphylococcus aureus: functional and in silico analysis.

To explore the mechanistic underpinnings of caffeine as a potent antibacterial against Staphylococcus aureus ATCC 25923 via in vitro functional assays, whole-genome sequencing, and in silico docking studies. In vitro studies established that caffeine's minimum inhibitory concentration (MIC) against S. aureus ATCC 25923 is 0.01544mmol/mL. Functional assays along with Scanning Electron Microscopy confirmed that caffeine at 0.030089mmol/mL (2MIC) released nucleotide constituents (nucleotide leakage assay) and effluxed potassium ions (potassium efflux assay) thereby, further validating caffeine's role as a membrane-active antimicrobial agent. Whole genome sequencing of control versus caffeine treated samples revealed a significant drop in read mapping percentage from 99.96 to 23.68% and GC content from 30.69 to 6.93%. This massive reduction in the treated sample was a consequence of single nucleotide polymorphisms (SNPs, 50,303), along with insertions and deletions (InDels, 62). Several of these caffeine-induced mutations were found to be harbouring the coding regions of genes involved in processes such as cell membrane organization, bacterial virulence, and DNA repair processes. Thus, implying a caffeine-mediated genomic rearrangement and instability. In silico docking studies revealed a strong binding affinity of caffeine to key cell wall proteins ltaA (-6.9kcal/mol) and ltaS (-6.5kcal/mol) respectively. The dynamic simulation studies revealed caffeine's interaction with receptor ltaS remained stable, with low deviations and minimal fluctuations. Although caffeine has been widely investigated for its antibacterial properties, its specific mechanisms of action, notably its effects on the cell membrane and genomic integrity in S. aureus ATCC 25923, are little understood. This study thus offers a comprehensive functional genomic analysis of caffeine as an antibacterial against S. aureus.