Reduction In Minimum Inhibitory Concentration Research Articles

Determination of antimicrobial and modulatory activity of bacterial resistance by nettle (Cnidoscolus urens) extracts in multiresistant bacteria isolated from bovine mastitis.

The present study aimed to evaluate the antimicrobial and modulating activity of the ethanol extract obtained from the leaves, stems, and roots of Cnidoscolus urens in multiresistant bacteria. The Minimum Inhibitory Concentration (MIC) values obtained for the extracts of leaves, stems, and roots were greater than 1024µg/mL for all isolates. In the antimicrobial resistance modulation test, the extract of the leaves of C. urens showed a better modulating effect than that of the stems and roots for gentamicin, highlighting the reduction of MIC for Escherichia coli, Lactococcus garvieae and Staphylococcus sciuri. For erythromycin, a reduction of MIC was observed in L. garvieae, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus agalactiae. The extract from the leaves of C. urens has an important modulating effect on resistance in multiresistant bacteria, especially with gentamicin and erythromycin.

Potentiation of the Antimicrobial Effect of Oxytetracycline Combined with Cinnamon, Clove, Oregano, and Red Thyme Essential Oils against MDR Salmonella enterica Strains.

Tetracyclines have a high resistance percentage in Salmonella spp. of both human and animal origin. Essential oils, such as cinnamon (Cinnamomum zeylanicum), clove (Eugenia caryophyllata), oregano (Origanum vulgare), and red thyme (Thymus zygis), have shown bactericidal activity against this bacterium. However, in many cases, the minimum inhibitory concentration (MIC) exceeds the cytotoxicity limits. The objective of this study was to assess the in vitro efficacy of combining oxytetracycline with essential these oils against field multidrug-resistant (MDR) Salmonella enterica strains. The MIC of each product was determined using the broth microdilution method. The interaction was evaluated using the checkerboard method, by means of the fractional inhibitory concentration index (FICindex) determination. The results showed a positive interaction (synergy and additivity) between oxytetracycline and the four oils tested, resulting in a reduction in both products' MICs by 2 to 4 times their initial value, in the case of oils, and by 2 to 1024 times in the case of the antibiotic. The combination of oxytetracycline and cinnamon achieved the best results (FICindex 0.5), with a decrease in the antibiotic effective concentration to below the sensitivity threshold (MIC of the combined oxytetracycline 0.5 µg/mL). There was no antagonistic effect in any case, although differences in response were observed depending on the bacterial strain. The results of this study suggest that combining oxytetracycline with cinnamon oil could be an effective alternative for controlling tetracycline-resistant strains of Salmonella. However, its individual use should be further evaluated through in vitro susceptibility tests.

Antibiotic potentiating effect of Bauhinia purpurea L. against multidrug resistant Staphylococcus aureus.

Bauhinia purpurea L. is a medium-sized tree from the family Fabaceae. The plant is traditionally used as medicine by different tribes in Sikkim. The present study aimed to evaluate the modulation in minimum inhibitory concentration (MIC) of the bark methanol extract of Bauhinia purpurea L. against the clinical isolates of multidrug resistant Staphylococcus aureus. The synergistic activity of the test plant extract with different classes of antibiotics was also evaluated. The methanol extract of Bauhinia purpurea exhibited modulation by a 16-fold reduction in the MIC of clindamycin against both resistant and susceptible isolates, followed by penicillin and gentamicin, whereas a maximum of only a 4-fold MIC reduction was observed with ciprofloxacin. The lowest minimum inhibitory concentration and minimum bactericidal concentration showed by the plant extract was 0.48 and 0.97 mg/mL, respectively. The methanol extract of Bauhinia purpurea exhibited synergistic activity with penicillin, gentamicin, ciprofloxacin, and clindamycin against most of the tested isolates of multidrug-resistant Staphylococcus aureus (MDR-SA). Gas chromatography-mass spectrometry analysis of Bauhinia purpurea L. bark methanol extract revealed 16 phytocompounds. The results provide an insight into the potential antibacterial property of the plant extract in terms of its antibiotic MIC modulation and synergistic properties with the selected antibiotics. This is the first report of the antibiotic potentiation property of Bauhinia purpurea L., collected from Sikkim, India.

Synergistic Potential Sof Cinnamon Extracts, Antibiotics, and Alum on Selected Clinical Microbes

Antibiotics are natural or synthetic substances used to inhibit or kill susceptible microorganisms. Due to the increase rate of bacterial resistance to antibiotics, there is the need to venture in other methods involving the coalescence effect of antibiotics and other substances such as cinnamon and alum to effectively eradicate these pathogenic organisms using less concentrated conventional antibiotics. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the selected antimicrobial agents were determined using the broth dilution method, following standards and procedures set by the national council of clinical laboratory standards (NCCLS). The synergistic potentials of all the antimicrobics used were analyzed by the checkerboard assay. The MIC values of 25, 0.25, 0.75,25mg/ml were obtained for cinnamon extracts, gentamicin, penicillin G, and aq. Alum respectively against S. aureus. For E. coli O157H:H7, the MIC values of cinnamon extracts, gentamicin, penicillin G, and aq. Alum were 12.5, 0.25, 0.75 and 25mg/ml respectively,s while the MIC values of cinnamon extracts alum and fluconazole for C. albicans were 12.5, 25 and 0.05mg/ml respectively. There is a reduction in the MIC values for the various combinations against the same clinical microbes. All the antimicrobics used in this study inhibited the growth of the clinical microbes. All the microbes used in this study inhibited the tested microbes in dose dependent manner. The combinations of these agents resulted in reduction in MIC and MBC values, with an additive, indifference, and antagonistic effects using checkerboard assay which resulted to a fractional inhibitory concentration index which ranged from 0.56 to 7.9. Therefore, application of cinnamon, alum, and their combinations with conventional antibiotics can be used as treatment measure against infections caused by S. aureus, E. coli O157H:H7, and C. albicans.

Efficacy of a Novel Antibiotic Drug Combination Toward Multidrug-Resistant Ocular Pathogens.

Antimicrobial resistance is a global health threat, compounded by the reduction in the discovery of new antibiotics. A repurposed drugs-based approach could provide a viable alternative for the treatment of multidrug-resistant (MDR) bacterial infections. In this study, we sought to evaluate the in vitro efficacy of a novel drug combination, polymyxin B/trimethoprim (PT) + rifampin on MDR isolates from patients with bacterial keratitis in India. Forty-three isolates, which included 20 Staphylococcus aureus, 19 Pseudomonas aeruginosa, 3 Pseudomonas stutzeri, and 1 Acinetobacter baumannii, were evaluated for their antibiotic resistance by minimum inhibitory concentration (MIC). Fractional Inhibitory Concentration Index (FICI) testing was performed to measure the antimicrobial impact of PT + rifampin in combination. Among S. aureus isolates, 100% were resistant to at least 1 antibiotic class, 12 (60%) were MDR, and 14 (70%) were classified as methicillin-resistant. Among the gram-negative isolates, >90% were classified as MDR. Fractional Inhibitory Concentration (FIC) testing revealed that PT + rifampin was effective in completely inhibiting growth of all isolates while also displaying additive or synergistic activity in approximately 70% of the strains. Mean FICI values were 0.753 ± 0.311 and 0.791 ± 0.369 for S. aureus and gram-negative isolates, respectively, and a >2-fold reduction in MIC was measured for both PT and rifampin when tested in combination versus alone. Our data demonstrate the ability of PT + rifampin to eliminate all isolates tested, even those conferring MDR, highlighting the promise of this drug combination for the treatment of bacterial keratitis.

Enhancing Antibiotic Efficacy: Exploring Synergistic Interactions between Plant Extracts and Conventional Antibiotics

Medicinal herbs including Senna alata, Ricinus communis, and Lannea barteri have been utilized for centuries to cure a variety of illnesses caused by microbial infections. This study looked at the synergistic effects of these drugs with traditional antibiotics on clinical isolates of Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The test bacteria were chosen based on their minimal potential for monotherapy and susceptibility to at least one antibiotic with a known genetic basis. The interactions of plant extracts with antibiotics against the chosen pathogenic microorganisms were investigated using the agar well diffusion, broth microdilution, and checkerboard methods. Calculated fractional inhibitory concentration index (FICI) values were used to describe how the extracts and antibiotics interacted. All the extracts from the three plants combined with fluconazole exhibited a synergistic interaction against C. albicans (FICI < 0.5). The minimum inhibitory concentration (MIC) of ampicillin against E. coli was demonstrated to be reduced by the combination of the ethanol extract of S. alata with ampicillin, with a FICI value of 0.4 indicating a synergistic effect. With a synergistic action (FICI ˂ 0.5) against P. aeruginosa, the ethanol extract of S. alata and amoxicillin were successful in reducing the MIC of amoxicillin from 0.32 to 0.17 mg/mL. Aqueous L. barteri extract combined with amoxicillin exhibited synergism (FICI < 0.2) against S. aureus with a reduction of MIC from 0.20 to 0.03 mg/mL. The current study is the first to investigate the aforementioned plants in combination with conventional antibiotics for their antimicrobial activities. The findings of this study could be used to create a useful, applicable, feasible, and alternative source of novel antimicrobial agents.

Synergistic Activity of 3-Hydrazinoquinoxaline-2-Thiol in Combination with Penicillin Against MRSA.

The growing resistance seen in various antibiotics, including those considered as last-resort options, underscores the pressing need for novel approaches and new substances to address MRSA infections. Combining antibiotics as a treatment approach can enhance effectiveness, expand the range of targeted bacteria, and minimize the likelihood of resistance emergence. This approach holds promise in addressing the escalating issue of antibiotic resistance. This study seeks to investigate the potential synergy between 3-hydrazinoquinoxaline-2-thiol and penicillin against a diverse array of MRSA isolates, thereby providing insights into their combined antimicrobial action. Twenty-two clinical MRSA isolates subjected to broth microdilution to determine the Minimum Inhibitory Concentrations (MICs) of 3-hydrazinoquinoxaline-2-thiol and penicillin. Subsequently, a checkerboard assay was employed to evaluate the interaction between 3-hydrazinoquinoxaline-2-thiol and penicillin, focusing on the Fractional Inhibitory Concentration Index (FICI). The MICs of penicillin and 3-hydrazinoquinoxaline-2-thiol were determined for 22 clinical MRSA strains. Penicillin exhibited MICs within a range of 1024 to 128 µg/mL, while 3-hydrazinoquinoxaline-2-thiol displayed MICs varying from 64 to 8 µg/mL. Remarkably, the combination of 3-hydrazinoquinoxaline-2-thiol and penicillin yielded a synergistic effect, resulting in a significant reduction of MICs by up to 64-fold. The potential of 3-hydrazinoquinoxaline-2-thiol in combination with penicillin as a viable solution against MRSA appears promising. However, to establish its practical utility, further extensive testing and experiments are essential.

Unveiling Synergistic Potency: Exploring Butyrolactone I to Enhance Gentamicin Efficacy against Methicillin-Resistant Staphylococcus aureus (MRSA) Strain USA300.

Staphylococcus aureus, including MRSA strains, poses significant health risks, imposing a significant disease burden and mortality. We investigate butyrolactone I (BL-1), a marine-derived metabolite from Aspergillus terreus, enhancing aminoglycoside efficacy against MRSA. A promising synergy is observed with BL-1 and various aminoglycosides, marked by low fractional inhibitory concentration indexes (FICIs < 0.5). Comprehensive studies utilizing USA300 MRSA and gentamicin reveal a remarkable one-fourth reduction in minimum inhibitory concentration (MIC) with 20 μg/mL BL-1. A relative abundance assay indicates that BL-1 enhances gentamicin uptake while restraining extracellular presence, involving intricate transmembrane signaling and molecular interactions. RNA-Seq analysis yielded an unexpected revelation, unveiling a distinctive gene expression profile and distinguishing it from other treatment approaches. Furthermore, meticulous analyses validated the extensive perturbations induced by BL-1 exposure, affecting diverse biological functions, encompassing glycolysis, amino acid metabolisms, substance transmembrane transport, and virulence generation. These valuable insights inspired further confirmation of bacterial virulence and the modulation of membrane permeability resulting from BL-1 treatment. Phenotypic validations corroborated our observations, revealing reduced membrane permeability and hemolytic toxicity, albeit demanding a deeper comprehension of the intricate interplay underlying these actions. Our study contributes crucial mechanistic insights to the development of therapeutic strategies against this notorious pathogen and the judicious employment of aminoglycosides. Additionally, it elucidates marine-derived metabolites' ecological and functional roles, exemplified by fungal quorum sensing signals. These compounds could give producers a competitive edge, inhibiting microorganism proliferation and suggesting novel approaches for combating resistant pathogens.

Antibacterial and Antibiofilm Activity of Endophytic Alternaria sp. Isolated from Eremophila longifolia.

The threat to public health resulting from the emergence of antimicrobial resistance (AMR) is ever rising. One of the major bacterial pathogens at the forefront of this problem is methicillin-resistant Staphylococcus aureus, or MRSA, for which there is a great need to find alternative treatments. One of the most promising alternatives is endophytic fungi, which were shown to produce a vast array of bioactive compounds, including many novel antibacterial compounds. In this study, two endophytic Alternaria sp., EL 24 and EL 35, were identified from the leaves of Eremophila longifolia. Ethyl acetate (EtOAc) extracts of their culture filtrates were found to inhibit both methicillin-sensitive S. aureus ATCC 25923 and MRSA strains M173525 and M180920. The activity of each extract was shown to be greatly affected by the growth medium, with considerable reductions in minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) observed when tested in tryptic soy broth with glucose (TSBG) compared with Mueller-Hinton broth (MHB). Both extracts displayed significant (p ≤ 0.05) antibiofilm activity against all three S. aureus strains, the greatest of which was that of EL 35, which reduced biofilm formation by M180920 by 72%, while that of EL 24 resulted in a 57% reduction against ATCC 25923. Both extracts also disrupted established biofilms, of which the most effective was EL 35, which reduced the M180920 biofilm by 64%, while EL 24 also performed best against M180920, reducing biofilm by 54%. Gas chromatography-mass spectrometry (GC-MS) analysis of the EL 24 EtOAc extract revealed five known compounds. This study highlights the promise of endophytic fungi from Australian plants as a potential source of substances effective against important bacterial pathogens. Further understanding of the responsible compounds and their mechanisms could lead to the development of treatments effective against MRSA, as well as novel biofilm-resistant biomedical materials, contributing towards reducing the burden of AMR.

Mycobacterium tuberculosis Gene Expression Associated With Fluoroquinolone Resistance and Efflux Pump Inhibition.

We evaluated the relationship between response to efflux pump inhibition in fluoroquinolone-resistant Mycobacterium tuberculosis (Mtb) isolates and differences in gene expression and expression quantitative trait loci (eQTL). We determined ofloxacin minimum inhibitory concentration (MIC) for ofloxacin-resistant and -susceptible Mtb isolates without and with the efflux pump inhibitor verapamil. We performed RNA sequencing (RNA-seq), whole genome sequencing (WGS), and eQTL analysis, focusing on efflux pump, transport, and secretion-associated genes. Of 42 ofloxacin-resistant Mtb isolates, 27 had adequate WGS coverage and acceptable RNA-seq quality. Of these 27, 7 had >2-fold reduction in ofloxacin MIC with verapamil; 6 had 2-fold reduction, and 14 had <2-fold reduction. Five genes (including Rv0191) had significantly increased expression in the MIC fold change >2 compared to <2 groups. Among regulated genes, 31 eQTLs (without ofloxacin) and 35 eQTLs (with ofloxacin) had significant allele frequency differences between MIC fold change >2 and <2 groups. Of these, Rv1410c, Rv2459, and Rv3756c (without ofloxacin) and Rv0191 and Rv3756c (with ofloxacin) have previously been associated with antituberculosis drug resistance. In this first reported eQTL analysis in Mtb, Rv0191 had increased gene expression and significance in eQTL analysis, making it a candidate for functional evaluation of efflux-mediated fluoroquinolone resistance in Mtb.

Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis.

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as "resistant" to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.

Synergy Potential of Ursolic Acid-Based Hybrid Molecules

Background: Ursolic acid (UA, 3β-hydroxy-urs-12-en-28-oic acid), a pentacyclic triterpenoid from various medicinal plants, has been blessed with proven biological effects, including anti-inflammatory, anticancer, antidiabetic, antioxidant and antibacterial, but its bioavailability and solubility limit its clinical application. Objective: Synthesis of UA-based hybrid molecules to explore their antibacterial and synergy potential in combination with azithromycin (AZT) for the treatment of multidrug-resistant (MDR) bacterial infections. Methods: Hybrid molecules of UA with menthol, eugenol, and nalidixic acid (NAL) along with some other ester derivatives were synthesized, and evaluated for their antibacterial and synergy potential in combination with AZT against the clinical isolate of Escherichia coli in terms of their minimum inhibitory concentration (MIC), fold reduction in MIC, fractional inhibitory concentration index (FICI) and type of interaction. In silico screening of pharmacokinetic parameters, docking affinity against efflux pump proteins AcrA, AcrB, and TolC was performed on the most potent derivative 7 (3-O-nalidixoyl UA). Results: Derivative 7 showed MIC of 62.5 µg/mL and a strong synergistic effect with AZT reducing the MIC of AZT from 100 to 0.19 µg/mL (512-fold reduction) against E. coli at a concentration of 12.5 µg/mL. Other derivatives neither showed antibacterial activity of their own (MIC &gt; 1000 µg/mL) nor any significant synergistic interaction in combination with AZT. The in silico studies on 7 revealed improved druggability parameters over the parent UA and NAL. Conclusion: The findings highlight derivative 7 as strong synergistic agent in combination with AZT which may be further investigated to render its efficient use for the treatment of MDR bacterial infections.

The In vitro activity of carbapenems alone and in combination with β-lactamase inhibitors against difficult-to-treat mycobacteria; Mycobacterium tuberculosis, Mycobacterium abscessus, and Mycobacterium avium complex: A systematic review.

Difficult-to-treat mycobacterial infections are increasing globally. There is an urgent need of new treatment alternatives for multidrug-resistant Mycobacterium tuberculosis (MTB), as well as nontuberculous mycobacteria such as the Mycobacterium abscessus complex (MABC) and Mycobacterium avium complex (MAC). Recently, new carbapenems and combinations of carbapenems with β-lactamase inhibitors have become available, but activity data in vitro against mycobacteria are so far scarce. Therefore, we performed a systematic review collating the minimum inhibitory concentrations (MICs) of carbapenems, with or without a β-lactamase inhibitors for MTB, MABC, and MAC. The databases PubMed and Web of Science were searched for the relevant articles in English up until September 21, 2022. Screening of studies was performed by two independent reviewers. MIC data by recommended methods with at least five individual MICs were included. Data were reported as MIC range, MIC50, modal MIC, and/or histograms when individual MICs were available. The study protocol was registered at PROSPERO (CRD42021258537). After screening, a total of 75 studies with MIC data for carbapenems with or without β-lactamase inhibitors were included in the review. For MTB, the oral carbapenem tebipenem combined with the β-lactamase inhibitor clavulanic acid resulted in the most significant reduction of MICs. For MABC, the addition of avibactam to tebipenem resulted in a 64-fold reduction of modal MIC. Data were insufficient for the analysis of MAC. Carbapenems, and in particular the novel oral compound tebipenem, in combination with clavulanic acid for MTB and avibactam for MABC may be an untapped potential for difficult-to-treat mycobacterial infections.

Alginate oligosaccharides enhance the antifungal activity of nystatin against candidal biofilms.

The increasing prevalence of invasive fungal infections in immuno-compromised patients is a considerable cause of morbidity and mortality. With the rapid emergence of antifungal resistance and an inadequate pipeline of new therapies, novel treatment strategies are now urgently required. The antifungal activity of the alginate oligosaccharide OligoG in conjunction with nystatin was tested against a range of Candida spp. (C. albicans, C. glabrata, C. parapsilosis, C. auris, C. tropicalis and C. dubliniensis), in both planktonic and biofilm assays, to determine its potential clinical utility to enhance the treatment of candidal infections. The effect of OligoG (0-6%) ± nystatin on Candida spp. was examined in minimum inhibitory concentration (MIC) and growth curve assays. Antifungal effects of OligoG and nystatin treatment on biofilm formation and disruption were characterized using confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and ATP cellular viability assays. Effects on the cell membrane were determined using permeability assays and transmission electron microscopy (TEM). MIC and growth curve assays demonstrated the synergistic effects of OligoG (0-6%) with nystatin, resulting in an up to 32-fold reduction in MIC, and a significant reduction in the growth of C. parapsilosis and C. auris (minimum significant difference = 0.2 and 0.12 respectively). CLSM and SEM imaging demonstrated that the combination treatment of OligoG (4%) with nystatin (1 µg/ml) resulted in significant inhibition of candidal biofilm formation on glass and clinical grade silicone surfaces (p < 0.001), with increased cell death (p < 0.0001). The ATP biofilm disruption assay demonstrated a significant reduction in cell viability with OligoG (4%) alone and the combined OligoG/nystatin (MIC value) treatment (p < 0.04) for all Candida strains tested. TEM studies revealed the combined OligoG/nystatin treatment induced structural reorganization of the Candida cell membrane, with increased permeability when compared to the untreated control (p < 0.001). Antimicrobial synergy between OligoG and nystatin against Candida spp. highlights the potential utility of this combination therapy in the prevention and topical treatment of candidal biofilm infections, to overcome the inherent tolerance of biofilm structures to antifungal agents.

Physicochemical mechanisms of bacterial response in the photodynamic potentiation of antibiotic effects

Antibiotic failures in treatments of bacterial infections from resistant strains have been a global health concern, mainly due to the proportions they can reach in the coming years. Making microorganisms susceptible to existing antibiotics is an alternative to solve this problem. This study applies a physicochemical method to the standard treatment for modulating the synergistic response towards circumventing the mechanisms of bacterial resistance. Photodynamic inactivation protocols (curcumina 10 µM, 10 J/cm2) and their cellular behavior in the presence of amoxicillin, erythromycin, and gentamicin antibiotics were analyzed from the dynamics of bacterial interaction of a molecule that produces only toxic effects after the absorption of a specific wavelength of light. In addition to bacterial viability, the interaction of curcumin, antibiotics and bacteria were imaged and chemically analyzed using confocal fluorescence microscopy and Fourier-transform infrared spectroscopy (FTIR). The interaction between therapies depended on the sequential order of application, metabolic activity, and binding of bacterial cell surface biomolecules. The results demonstrated a potentiating effect of the antibiotic with up to to 32-fold reduction in minimum inhibitory concentrations and mean reductions of 7 log CFU/ml by physicochemical action at bacterial level after the photodynamic treatment. The changes observed as a result of bacteria-antibiotic interactions, such as membrane permeabilization and increase in susceptibility, may be a possibility for solving the problem of microbial multidrug resistance.

Enhancement of Antimycobacterial Activity of Rifampicin Using Mannose-Anchored Lipid Nanoparticles against Intramacrophage Mycobacteria.

Tuberculosis treatment requires a multidrug combination for the long-term, associated with adverse effects which lead to nonpatient compliance and the emergence of drug-resistant strains. Thus, mannose-anchored rifampicin-loaded solid lipid nanoparticles (M-RIF-SLNs) were developed to enhance the effect of rifampicin by selectively delivering to the macrophage, which led to the high intracellular killing of mycobacteria. The synthesized M-RIF-SLNs show a particle size of ∼100 nm and a drug loading of ∼8%. Cytotoxicity assay confirms that M-RIF-SLNs are not toxic up to 16 μg/mL (equivalent to incorporated rifampicin in SLN) toward THP-1-differentiated macrophages. An antimicrobial assay exhibits a reduction of minimum inhibitory concentration by 4-fold and 8-fold against wild-type and laboratory drug-resistant strains of M. smegmatis, respectively, compared to free rifampicin. Furthermore, mannose-functionalized SLNs loaded with coumarin-6 exhibit a higher macrophage uptake than that of unfunctionalized SLNs. Finally, higher intramacrophage clearance of M. tuberculosis H37Ra was observed with M-RIF-SLNs compared to RIF-SLNs and free rifampicin. Hence, the overall results support that the developed M-RIF-SLNs can be a promising approach for improving the antibacterial activity of rifampicin against intracellular mycobacteria residing in the alveolar macrophages.

Potentiation of Antibiotic Action and Efflux Pump Inhibitory Effect on Staphylococcus aureus Strains by Solasodine.

A worrisome fact is the increase in microbial resistance, which has as its main cause the indiscriminate use of antibiotics. Scientific studies have investigated bioactive compounds such as steroidal sapogenins, in the perspective of new beneficial alternatives for the control of bacterial resistance. Therefore, the objective of this work was to verify the antibacterial activity as well as the modifying action of antibiotics associated with solasodine and its ability to inhibit the efflux pump mechanism in strains of Staphylococcus aureus. Tests were performed to verify the minimum inhibitory concentration (MIC). In addition, the action-modifying potential of antibiotics and the inhibitory capacity of the efflux pump NorA and MepA through synergistic effects on the antibiotic and ethidium bromide were evaluated. Solasodine showed significant results for the standard bacteria with an MIC of 512 μg/mL, and when associated with the antibiotics gentamicin and nofloxacin for the multidrug-resistant bacteria S. aureus 10, Escherichia coli 06, and Pseudomonas aeruginosa 24, it showed a 50% reduction in MIC. The association of solasodine with the antibiotic ciprofloxacin against S. aureus K2068 (MepA) showed synergism, with a reduction in the MIC of the antibiotic from 64 μg/mL to 40 μg/mL, and also a reduction in the MIC when the antibiotic was used in conjunction with the efflux pump inhibitors. Solasodine may be acting on the mechanism of action of the antibiotic, as it has shown a potentiating effect when associated with antibiotics, inducing a reduction in the MIC against Gram-positive and Gram-negative bacteria. Therefore, this study demonstrated significant results for the potentiating action of solasodine when associated with antibiotics of clinical importance.

P059 The overexpression of efflux pump gene cdr1B resulting in voriconazole- and isavuconazole- resistance in Aspergillus fumigatus recovered from a patient with chronic pulmonary aspergillosis in China

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesTriazole resistance in the pathogenic Aspergillus fumigatus has been increasing worldwide, posing a growing therapeutic challenge. To date, triazole resistance in clinical isolates of A. fumigatus causing pulmonary aspergillosis has been mainly attributed to the mutations in the cyp51A gene or its promoter, followed by mutations in cyp51B and hmg1 gene encoding 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. From chronic pulmonary aspergillosis (CPA) patient, we isolated a strain of A. fumigatus (BMU10672) with resistance to voriconazole (VRC) and isavuconazole (ISZ), which was caused by overexpression of efflux pump Cdr1B.MethodsAntifungal susceptibility testing of the isolate of A. fumigatus BMU10672 was performed using the broth microdilution method (CLSI M38-A3), E-test and disk diffusion method. The promoter region and open reading frame of the cyp51A, cyp51B, and hmg1 gene were amplified and sequenced. Then, the expression levels of cyp51A, cyp51B, and efflux pump gene cdr1B with or without being exposed to VRC or ISZ were quantified using real-time PCR, compared with triazole-susceptible A. fumigatus Af293. And the function of efflux pump Cdr1B was tested by efflux pump substrate (Nile red) accumulation assay and efflux pump inhibitor (FK520) assay.ResultsThe minimum inhibitory concentration (MIC) of itraconazole (ITC), VRC, posaconazole (POS), ISZ and amphotericin B (AMB), and the minimal effective concentration (MEC) of caspofungin (CAS) against A. fumigatus BMU10672 was 1 μg/ml, 2 μg/mL, 0.5 μg/mL, 2 μg/mL, 1 μg/mL and 0.125 μg/mL, respectively. The results of E-test and disk diffusion assay were consistent with those of the broth microdilution method (Figs. 1a and b). Together, these results indicate that A. fumigatus BMU10672 is resistant to VRC and ISZ, while being susceptible to ITC, POS, AMB, and CAS. Sequencing of the cyp51A, cyp51B and hmg1 gene of A. fumigatus BMU10672 were all intact. The basal and VRC- or ISZ- induced expression levels of efflux pumps gene cdr1B in A. fumigatus BMU10672 were all higher (> 4-fold) than those in triazole-susceptible A. fumigatus Af293. However, no differences in basal and VRC- or ISZ- induced expression levels of cyp51A gene and cyp51B gene were observed between A. fumigatus BMU10672 and Af293. The efflux pump substrate Nile red accumulation assay showed the A. fumigatus BMU10672 accumulated less Nile red than Af293, confirming that Cdr1B was active at exporting Nile red, while efflux pumps inhibitor FK520 can increase the accumulation of the Nile red in A. fumigatus BMU10672 (Fig. 1c). Inhibition of efflux pumps activity by inhibitor FK520 resulted in a MIC reduction of 4-fold in VRC and ISZ MICs, and 2-fold in ITC and POS, against A. fumigatus BMU10672 (Figs. 1a and b).ConclusionOverexpression of efflux pumps gene cdr1B resulting in VRC- and ISZ- resistance in the clinical isolate of A. fumigatus BMU10672.

Evaluation of Sodium Alginate Stabilized Nanoparticles and Antibiotics against Drug Resistant Escherichia coli Isolated from Gut of Houbara Bustard Bird.

Alternative approaches and/or modified approaches to tackle resistance in gut microbes are need of the hour. The current study was planned to find the resistance modulation and toxicity potential of sodium alginate stabilized MgO nanoparticles and antibiotics against Escherichia coli (E. coli) isolated from the gut of Houbara bustard bird (n = 105 fecal samples). The preparations consisted of gel stabilized ampicillin (G+A), gel stabilized MgO and ampicillin (G+M+A), gel stabilized MgO and cefoxitin (G+M+C), gel stabilized tylosin (G+T), gel stabilized MgO and tylosin (G+M+T), and gel stabilized MgO (M+G). The fecal samples showed 53% (56/105) prevalence of E. coli which was found to be significantly (p < 0.05) associated with most of the assumed factors and resistant to multiple drugs. G+M+T showed the lowest (4.883 ± 0.00μg/mL) minimum inhibitory concentration (MIC) followed G+M+C, G+M+A, G+A, M+G, and G+T. Significant reduction (p < 0.05) in MIC with respect to incubation interval found at the 16th hr for G+M+A, G+A, and G+M+C that further remained nonsignificant (p > 0.05) onwards until the 24th hr of incubation. In the case of G+T and M+G, significant reduction in MIC was found at the 20th hr and 24th hr of incubation. Ecotoxicology and histopathology trials on snails showed mild changes in MICs of the preparations. The study thus concluded increasing drug resistance in E. coli of houbara bird while sodium alginate stabilized MgO nanoparticles and antibiotics were effective alternative antibacterial composites with mild toxicity.

Determination of efflux activity in Enterococci by Hoechst accumulation assay and the role of zinc oxide nanoparticles in inhibition of this activity

BackgroundContribution of efflux pumps in development of antimicrobial resistance has been largely addressed in Gram negative and to a much lesser extent in Gram positive bacteria. Measuring accumulation of Hoechst (H) dye is known as a safe and rapid method for monitoring efflux activity in bacteria. Antimicrobial effects of metal nanoparticles have been attributed in part to inhibition of efflux pumps. This study aimed to first determine efflux activity in enterococci by Hoechst accumulation assay, and to second characterize the role of zinc oxide nanoparticles (ZnONPs) in inhibition of these pumps.ResultsIncreased accumulation of Hoechst dye showed more potential of ZnONPs in efflux inhibition compared with CCCP. H33258 represented more suitability for accumulation studies in enterococci. Two to six-fold reduction in minimum inhibitory concentration (MIC) values of antimicrobial agents in the presence of ZnONPs was observed.ConclusionsEfflux activity in enterococcal strains can be measured by H33258 accumulation assay. Application of ZnONPs as an efflux inhibitor, may rejuvenate the use of conventional antimicrobial agents against these bacteria.