Broth Microdilution Assay Research Articles

ZnO-Rutin nanostructure as a potent antibiofilm agent against Pseudomonas aeruginosa

Pseudomonas aeruginosa is a common human pathogen that is resistant to multiple antibiotics due to its ability to form biofilms. Developing novel nanoformulations capable of inhibiting and removing biofilms offers a promising solution for controlling biofilm-related infections. In this study, we investigated the anti-biofilm activity of rutin-conjugated ZnO nanoparticles (ZnO-Rutin NPs) in pathogenic strains of P. aeruginosa. The synthesized ZnO-Rutin NPs had amorphous shapes with sizes ranging from 14 to 100 nm. The broth microdilution assay revealed that ZnO-Rutin NPs, with an MIC value of 2 mg/mL, exhibit greater antimicrobial activity than ZnO NPs and rutin alone. Based on crystal violet staining, the biofilm inhibition rate by ½ MIC of the conjugated nanoparticles was recorded at above 90%. The significant reduction in exopolysaccharide (62.75–66.37%) and alginate (38.3–57.61%) levels, as well as the formation of thin biofilms in the ZnO-Rutin NP-treated group, confirmed the anti-biofilm potential of these nanoparticles. Additionally, a significant decrease in the metabolic activity and viable cells of mature biofilms was observed after exposure to the conjugated nanoparticles. Furthermore, ZnO-Rutin NPs considerably attenuated the expression of the Las-Rhl quorum-sensing transcriptional regulator genes (lasR and rhlR) in P. aeruginosa by 0.39–0.40 and 0.25–0.42 folds, respectively. This work demonstrated that ZnO-Rutin NPs are remarkably capable of inhibiting the initial stage of biofilm formation and eradicating mature biofilms, suggesting they could be a useful agent for treating P. aeruginosa biofilm-related infections.

FL058, a novel β-lactamase inhibitor, increases the anti-Mycobacterium abscessus activity of imipenem.

β-lactams are crucial for anti-Mycobacterium abscessus complex (MABC) therapy. Treating infections is challenging since MABC produces a class A β-lactamase (BlaMab) , which is capable of hydrolyzing β-lactams thus causing drug resistance. Diazabicyclooctane (DBO) β-lactamase inhibitors (BLIs) can inhibit BlaMab. FL058 is a novel DBO BLI; the anti-MABC activity of FL058 combined with β-lactams remains unknown. The activities of ten β-lactams (imipenem, meropenem, faropenem, tebipenem, cefoxitin, cefepime, ceftazidime, cefdinir, cefuroxime, and amoxicillin) combined with three DBO BLIs (FL058, avibactam, and relebactam) toward two MABC reference strains were determined by broth microdilution assay. The anti-MABC activities of imipenem combined with three BLIs against 193 clinical isolates were also evaluated. The activity of imipenem combined with FL058 was also tested against intracellular MABC residing in macrophages and in a mouse model. Finally, the BlaMab mutations in clinical isolates were analyzed using sequence alignment to determine whether BlaMab mutations are associated with DBO BLIs sensitivity. FL058, avibactam and relebactam significantly increased the anti-MABC activity of β-lactams, especially imipenem, against reference strains and clinical isolates. The anti-MABC activity of imipenem combined with FL058 was superior to its activity when combined with either avibactam or relebactam. The combination of imipenem and FL058 significantly reduced the numbers of intracellular organisms in cultured macrophages, and of viable bacteria in the lungs of MABC-infected mice. Rough morphotypes tended to be more resistant than smooth morphotype. A BlaMab T141A mutation may reduce the susceptibility of MABC to imipenem-BLIs. The elevated anti-MABC activity exhibited by imipenem combined with FL058 suggests a potential new approach to treating MABC infections.

Performance of a broth microdilution assay for routine minimum inhibitory concentration determination of 14 anti-tuberculous drugs against the Mycobacterium tuberculosis complex based on the EUCAST reference protocol.

This comparative study aimed at qualifying a broth microdilution (BMD) assay for phenotypic drug susceptibility testing (pDST) of Mycobacterium tuberculosis complex (MTBC) strains for implementation in a routine DST workflow. The assay was developed based on the EUCAST (European Committee on Antimicrobial Susceptibility Testing) reference protocol for determination of the minimum inhibitory concentration (MIC) of 14 anti-tuberculous drugs (isoniazid [INH], rifampicin [RIF], ethambutol [EMB], amikacin [AMI], moxifloxacin [MFX], levofloxacin [LFX], bedaquiline [BDQ], clofazimine [CFZ], delamanid [DLM], pretomanid [PA], para-aminosalicylic acid [PAS], linezolid [LZD], ethionamide [ETH], and cycloserine [CS]). Forty MTBC strains with various drug resistance profiles were tested to determine the agreement between MIC results and genotypic drug susceptibility testing (gDST) results derived from whole-genome sequencing (WGS). The agreement between the BMD and gDST results was solid for the majority of the drugs (average agreement 98%, range 90%-100%), including key drugs such as INH, RIF, MFX, LFX, BDQ, DLM, and PA. Ten discrepancies were identified (corresponding to 1.8% of the total number of MIC determinations) and most (8/10) were characterized by MICs equal or close to the potential critical concentration (pCC) applied in the BMD assay. Importantly, the assay can be adjusted to new drug recommendations and concentrations, tailored to local needs. We conclude that the BMD assay provides reliable results, and its implementation in our MTBC routine workflow will produce valuable data that improve our understanding and management of MTBC drug resistance.

Heteroleptic Coumarin-Based Silver(I) Complexes: Possible New Antimicrobial Agents.

Heteroleptic coumarin-based silver(I) complexes with improved solubility profiles were synthesised using either triphenylphosphine or an N-heterocyclic carbene as adduct ligands, and were fully characterised using IR and NMR spectroscopy, elemental analysis, and, where possible, X-ray crystallography. The triphenylphosphine adducts formed well-resolved structures, where the oxyacetate ligands asymmetrically chelated the silver(I) ion in a bidentate chelating mode, and the silver(I) ion was also bound to two triphenylphosphine ligands. The solubility profile and photostability of the adducts were considerably improved compared to those of previously isolated simple coumarin silver(I) complexes. Analysis of the coumarin N-heterocyclic carbene(NHC) silver(I) adduct indicated that it likely formed as a complex aggregate species with an overall stoichiometry of 1:1:1 coumarin:Ag(I):NHC. The Kirby Bauer assay and broth microdilution assays were used to assess the silver(I) complexes' and adducts' antimicrobial activity against pathogenic strains of Pseudomonas aeruginosa, Escherichia coli, and MRSA. Interestingly, the formation of more soluble complexes did not increase the activity of the silver(I) complexes and, in effect, made them less effective antimicrobial agents, particularly against Escherichia coli and Pseudomonas aeruginosa, although they retained their activity against MRSA.

Revisiting mutational resistance to ampicillin and cefotaxime in Haemophilus influenzae

BackgroundHaemophilus influenzae is an opportunistic bacterial pathogen that can cause severe respiratory tract and invasive infections. The emergence of β-lactamase-negative ampicillin-resistant (BLNAR) strains and unclear correlations between genotypic (i.e., gBLNAR) and phenotypic resistance are challenging empirical treatments and patient management. Thus, we sought to revisit molecular resistance mechanisms and to identify new resistance determinants of H. influenzae.MethodsWe performed a systematic meta-analysis of H. influenzae isolates (n = 291) to quantify the association of phenotypic ampicillin and cefotaxime resistance with previously defined resistance groups, i.e., specific substitution patterns of the penicillin binding protein PBP3, encoded by ftsI. Using phylogenomics and a genome-wide association study (GWAS), we investigated evolutionary trajectories and novel resistance determinants in a public global cohort (n = 555) and a new clinical cohort from three European centers (n = 298), respectively.ResultsOur meta-analysis confirmed that PBP3 group II- and group III-related isolates were significantly associated with phenotypic resistance to ampicillin (p < 0.001), while only group III-related isolates were associated with resistance to cefotaxime (p = 0.02). The vast majority of H. influenzae isolates not classified into a PBP3 resistance group were ampicillin and cefotaxime susceptible. However, particularly group II isolates had low specificities (< 16%) to rule in ampicillin resistance due to clinical breakpoints classifying many of them as phenotypically susceptible. We found indications for positive selection of multiple PBP3 substitutions, which evolved independently and often step-wise in different phylogenetic clades. Beyond ftsI, other possible candidate genes (e.g., oppA, ridA, and ompP2) were moderately associated with ampicillin resistance in the GWAS. The PBP3 substitutions M377I, A502V, N526K, V547I, and N569S were most strongly related to ampicillin resistance and occurred in combination in the most prevalent resistant haplotype H1 in our clinical cohort.ConclusionsGradient agar diffusion strips and broth microdilution assays do not consistently classify isolates from PBP3 groups as phenotypically resistant. Consequently, when the minimum inhibitory concentration is close to the clinical breakpoints, and genotypic data is available, PBP3 resistance groups should be prioritized over susceptible phenotypic results for ampicillin. The implications on treatment outcome and bacterial fitness of other extended PBP3 substitution patterns and novel candidate genes need to be determined.

In-vitro Interactions between Fluconazole and Diphenyl Diselenide against Various Candida Species

Background: In the immunocompromised population, Candida species are the most aetiologic agents causing severe nosocomial fungal infections. Candida species, irrespective of being commensals in the human microbiome, are the fourth most prevalent source of potentially fatal yeast infections. Monotherapy is frequently employed to treat invasive fungal infections, but sometimes, patients do not favor the monotherapy treatment regime. It may be because of the reduced susceptibility of the pathogen toward traditional antimycotic drugs. Antimycotic drug combination therapy could be a better choice in such specific circumstances. In our study, we evaluated the interactions of fluconazole with diphenyl diselenide. Methods: The antimycotic susceptibilities of Candida species for fluconazole and diphenyl diselenide were determined by broth microdilution assay, and the in-vitro interactions of fluconazole with diphenyl diselenide were studied by using disc diffusion assay and chequerboard assay. The nature of the interactions was assessed by calculating the fractional inhibitory concentration index (FICI). The interactions were also analyzed by the response surface approach. Results: The minimum inhibitory concentrations (MICs) for fluconazole and diphenyl diselenide as determined by the broth microdilution assay against Candida species were 4 μg/ml-512 μg/ml and 1 μg/ml-32 μg/ml, respectively. The FICI values varied from 0.375 to 2. Conclusion: Our finding demonstrated that there is no antagonism interaction between fluconazole and diphenyl diselenide in Candida species. Thus, this innovative combination should be explored in the future.

Evaluation of Antibacterial Activity and Synergistic Interaction between Punica granatum peels and Synthetic Antibiotics on Multidrug-Resistant Bacteria

IntroductionAntibiotic resistance threatens global health, necessitating the development of new antimicrobial medications. Novel drugs can reverse drug resistance and revitalize the antibiotic arsenal. Therefore, this study aimed to evaluate the synergistic antibacterial effect of Punica granatum (P. granatum) extract and antibiotics against multidrug-resistant bacteria (MDR). MethodsPreliminary susceptibility profiling of the antibiotics, the antibacterial activity of the extract, and synergistic interactions between the extract and the three antibiotics were conducted using well diffusion and micro-broth dilution assays with resazurin dye. The inhibition zones (IZs), minimum inhibitory concentrations (MICs), and Fractional Inhibition Concentration Index (FICI) were determined. The data were analyzed using statistical software program (SPSS 26). ResultsThe bacterial isolates exhibited resistance to all antibiotics, except E. coli and P. aeruginosa were sensitive to Ciprofloxacin. P. granatum extract demonstrated an inhibitory effect on all tested bacteria. The FICI values for MDR strains ranged from (0.12 to 0.53), and the best synergistic effect was observed when Augmentin and Ciprofloxacin were combined against these strains. The FICI indices for the combination of P. granatum and Azithromycin ranged from (0.12 to 0.53). ConclusionThis study identified P. granatum extract as a potential adjunctive therapy to antibiotics for the treatment of multidrug-resistant bacteria.

Green coffee infusion: An endodontic elixir

Context: Irrigation is a critical component in accessing the labyrinth of the pulp space. Although traditional irrigants such as sodium hypochlorite (NaOCl) are widely employed, they exhibit several limitations, including cytotoxicity and complete microbial eradication. In contrast, phytochemicals derived from plants, including coffee, present promising alternatives due to their established antimicrobial properties. Aims: The aim of this study was to evaluate the efficacy of intracanal irrigants derived from locally cultivated Coffea arabica and Coffea canephora (Robusta) varieties, both recognized for their antimicrobial attributes. Subjects and Methods: A total of 70 extracted human single-rooted teeth were prepared. Coffee extracts were prepared through a decoction method, and the minimum inhibitory concentrations (MICs) of these extracts were determined using microbroth dilution assays. Microbial growth was quantitatively assessed at predetermined intervals. Results: Green Robusta extracts exhibited antimicrobial effectiveness at lower concentrations of 25% and 12.5%. All coffee extract groups demonstrated substantial reductions in microbial counts compared to the negative control, with Robusta extracts proving particularly efficacious. Notably, the Robusta green 12.5% extract exhibited sustained antimicrobial efficacy, suggesting its potential as an alternative to NaOCl. Conclusions: The findings of this study suggest a novel approach to improving endodontic therapy outcomes through the utilization of coffee extracts, potentially enhancing dental health.

Synthesis and Anti-bacterial Activity of New Substituted 2-trifluoromethyl-4-quinolinylhydrazone Analogs against Mycobacterium tuberculosis Strains.

Tuberculosis (TB) is a serious disease that still affects humanity, despite being old, caused by the bacterium Mycobacterium tuberculosis (Mtb). The emergence of drug-resistant strains has alarmed governments and international organizations, such as the World Health Organization (WHO). The need for research on new drugs that are effective in a shorter treatment time and active against resistant strains still persists. The objective of this study is to synthesize and evaluate forty-four substituted 2-trifluoromethyl-4-quinolinylhydrazone analogs, as probable inhibitors of Mycobacterium tuberculosis growth. The anti-mycobacterial activities of all tested compounds against Mycobacterium tuberculosis strains, as well as the cytotoxicity test, were evaluated using the in vitro microplate procedure with broth microdilution assay. Thirteen compounds exhibited some activity against sensitive strain ATCC 27294, six of which were the most active: 4a, 4c, 6a, 6b, 6c, and 6g; with MIC around 7 - 8 μM, close to that presented by ethambutol (15.9 μM), a drug used in the treatment of tuberculosis. These same compounds also were active against a resistant strain of Mtb (T113), with MIC around 7 - 8 μM. Three of these compounds 4a, 6a, and 6c were not cytotoxic against Vero cells at concentrations near the MIC. This study indicates the importance of the hydrazone function to obtain promising anti-TB compounds and open new perspectives for drug development.

The in vitro Activity of Omadacycline Alone and in Combination Against Carbapenem-Resistant Klebsiella pneumoniae.

This study aimed to evaluate the in vitro activity of omadacycline (OMC) and OMC-based combination therapy against carbapenem-resistant Klebsiella pneumoniae (CRKP). The broth microdilution assay assessed the in vitro susceptibility of CRKP to OMC. The checkerboard assay was performed to evaluate the activity of OMC combined with polymyxin B (PB), amikacin (AN), or meropenem (MEM) against KPC-producing (class A) CRKP strains, and OMC combined with PB, aztreonam (ATM), MEM, or AN against class B and class A plus class B CRKP strains. Synergistic effects of OMC and PB were further evaluated by time-kill assays in the KPC-producing CRKP strains. Broth microdilution assays revealed a notable variation in susceptibility between KPC-producing and class B CRKP strains, with MIC50/90 of 32/32 mg/L and 0.5/8 mg/L, respectively. Although KPC-producing CRKP strains were resistant to OMC, a synergistic effect was observed in 37.5% of KPC-producing CRKP strains when OMC was combined with PB. In the nine KPC-producing CRKP strains, time-kill assays found that cell densities of six strains (66.7%) decreased by 3.61 ± 0.23 log10 CFU/mL compared to the initial inoculum after 2hours of PB exposure. The cell densities further decreased by an average of 2.38 ± 0.23 log10 CFU/mL when the six strains were exposed to OMC plus PB, confirming their potent synergism. OMC monotherapy is ineffective against KPC-producing CRKP strains, but OMC plus PB has a potent synergistic effect on them, suggesting that OMC plus PB is the preferred combination therapy against KPC-producing CRKP in vitro.

In vitro antibacterial and antibiofilm effects of mupirocin spray against Staphylococcus pseudintermedius.

Mupirocin is an effective antibiotic for infectious skin diseases. However, mupirocin is formulated as an ointment and is difficult to apply in canine systemic pyoderma. Therefore, many clinicians reformulate mupirocin off-label ointment into a spray. This study aimed to evaluate the antibacterial and antibiofilm effects of different concentrations of mupirocin spray (2%, 1%, and 0.5%) on Staphylococcus pseudintermedius over 21 days. Mupirocin spray was prepared by mixing mupirocin ointment and distilled water. The antibacterial effects were evaluated by measuring the optical density using broth microdilution assay and by live/dead staining. The antibiofilm activity of mupirocin spray was measured using a crystal violet staining method. All concentrations of mupirocin spray inhibited the growth of S. pseudintermedius. Mupirocin spray also inhibited biofilm formation of each isolate, although the degree of inhibition was influenced by the mupirocin concentration. The antibacterial and antibiofilm effects of mupirocin spray were maintained for 21 days. The 2% and 1% mupirocin sprays exhibited significantly better antibacterial and antibiofilm efficacy than the 0.5% mupirocin spray. Thus, 1-2% mupirocin spray may be effective for clinical use. Mupirocin spray is convenient and effective for the treatment of canine systemic pyoderma caused by S. pseudintermedius infection.

Novel Benzothiazole-Benzonitrile-Based Fluorescent Chromophore: Synthesis, DFT, Antimicrobial Properties.

The synthetic strategy for the benzothiazole-benzonitrile chromophore 6 involved three sequences of reactions. It was initiated by condensation of 2-amino-4-nitrothiophenol (2) with 4-(piperidin-1-yl)benzaldehyde (1) to produce the corresponding 5-nitrobenzothiazole compound 3 followed by reduction of compound 3 into the 5-aminobenzothiazole compound 4 and then ended by condensation of 5-aminobenzothiazole compound 4 with 4-formylbenzonitrile (5). The chromophore's absorption and emission spectra have been measured in EtOH and presented good Stokes' shift ( = 8363 cm-1). The DFT configuration of the frontier molecular orbits in gas and solvated ground state (So) were compared with the solvated excited state (S1). The synthesized chromophore 6 was assessed for its antimicrobic properties against a group of bacterial and fungal strains. The minimum inhibitory concentration (MIC) values were determined using standard broth microdilution assay. Notably, chromophore 6 exhibited remarkable MIC values against Staphylococcus aureus and Escherichia coli (< 48 and < 118 μg/mL). Regarding the antibacterial effectiveness against both S. aureus and E. coli, molecular docking was performed to simulate their binding interactions with two protein structures, PDB:2eg7 and PDB:3u2k. The SwissADME study has been applied to explore the pharmacokinetic and pharmacodynamic characteristics of chromophore 6.

Purified α-Amylase from Bacillus cereus exhibits antibiofilm and antiquorum sensing activities against uropathogenic Escherichia coli, Downregulating fimH, and papC virulence genes: implications for urinary tract infections

Background and aimPathogenic Escherichia coli is a known harmful microorganism that takes advantage of favorable conditions to cause various infections in healthcare settings, such as bloodstream infections related to catheters, as well as infections in the urinary and respiratory tracts. E. coli utilizes biofilm development as a means to enhance its virulence and pathogenicity. This work aims to investigate the antibiofilm activity of α-amylase enzyme against uropathogenic E. coli (UPEC) and its effect on biofilm-regulatory genes.MethodologyIn this study, we evaluated the antibiofilm activity of α-amylase enzyme by spectrophotometric microtiter plate analysis and confocal laser scanning microscopy. Also, the antibacterial activity of the test enzyme was evaluated by measuring the MIC and MBC levels against UPEC. The quorum-quenching activity of α-amylase enzyme was assessed using a qRT-PCR to evaluate the impact on biofilm-regulatory genes.ResultsBased on our results, purified α-amylase showed MIC and MBC levels ranged between 128 and 512 µg /ml against UPEC isolates using broth microdilution assay. Crystal violet assay revealed MBIC of 128 µg/ml and MBEC of 256 µg/ml for the purified α-amylase. When the biofilm was analyzed by confocal laser scanning microscope, our results showed inhibition of biofilm thickness (56%) and live/dead cell percentages (43/55%). Furthermore, analysis of the effect on the expression of biofilm-encoding genes showed downregulation of both fimH and papC genes by 57 and 25%, respectively, upon treatment of UPEC with ½ of the MIC (64 µg/ml).ConclusionsThe results demonstrate that our purified α-amylase from B. cereus exhibits promising antibiofilm activities against UPEC at both phenotypic as well as genotypic levels. These findings suggest that this enzyme may serve as a natural effective tool for removing bacterial biofilms, potentially offering new therapeutic avenues for treating infections caused by UPEC.

Antibacterial potency of mid-polar extracts obtained from Malaysian plant Parkia speciosa against human pathogenic bacteria

Background and objectivesPlants contain a wide variety of bioactive compounds, which have attracted the interest of researchers in finding novel sources of natural medicine. In the following paper, we aim to evaluate the antibacterial potential of extract fractions associated with Parkia speciosa pods and beans against human pathogenic bacteria. MethodsAntimicrobial activity was determined with disc diffusion and broth microdilution assays against eight skin colonising microorganisms including Staphylococcus aureus, Staphylococcus epidermidis, Salmonella enterica, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia followed by further fractionation of the pods ethyl acetate fraction by column chromatography along with preparative thin-layer chromatography. Quantification of bacterial death mechanism was elucidated by the measurement of hole size in cell wall that has been induced by extract constituents via field-emission scanning electron microscopy (FESEM). ResultsFour fractions showed significant antimicrobial activity against the six microorganisms tested (p < 0.01), with inhibition zones ranging from 35.67 to 17.00 mm, and minimum inhibitory concentration ranging from 6.25 to 50.00 mg/ml in which the pods ethyl acetate fraction was the most effective. The methanol fraction isolated from the pods ethyl acetate fraction was much more effective with a four-fold increase from 6.25 to 1.25 mg/ml against S. epidermidis. The disintegration of S. aureus was due to chronic cell wall alterations with pore creation, invaginations and morphological disorganisation. Autolysis in bacterial cells via the expression of peptidoglycan-disrupting lysozyme or bacterial murein hydrolase was postulated. A significantly large pore with a mean diameter of 293.7 nm was detected in the cell wall of S. aureus. ConclusionP. speciosa fraction could be a potential novel source for the development of a natural antibacterial agent.

Volatiles extracted from Melaleuca Rugulosa (Link) Craven leaves: comparative profiling, bioactivity screening, and metabolomic analysis

BackgroundMelaleuca species (family Myrtaceae) are characterized by their wide-ranging applications as antimicrobials and in skin-related conditions. Herein, we estimated the volatile profile and biological significance of M. rugulosa (Link) leaves for the first time supported by a dereplication protocol.Materials and methodsVolatile components were extracted using hydrodistillation (HD), supercritical fluid (SF), and headspace (HS) techniques and identified using GC/MS. The variations among the three extracts were assessed using principal component analysis and orthogonal partial least square discriminant analysis (OPLS-DA). The extracted volatiles were tested for radical scavenging activity, anti-aging, and anti-hyperpigmentation potential. Finally, disc diffusion and broth microdilution assays were implemented to explore the antibacterial capacity against Streptococcus pyogenes, Staphylococcus aureus, Clostridium perfringens, and Pseudomonas aeruginosa.ResultsThe yield of the SF technique (0.8%) was three times higher than HD. GC/MS analysis revealed that the oxygenated compounds are the most proponents in the three extracts being 95.93% (HD), 80.94% (HS), and 48.4% (SF). Moreover, eucalyptol (1,8-cineol) represents the major component in the HD-EO (89.60%) and HS (73.13%) volatiles, while dl-α-tocopherol (16.27%) and α-terpineol (11.89%) represent the highest percentage in SF extract. Regarding the bioactivity profile, the HD-EO and SF-extract showed antioxidant potential in terms of oxygen radical absorbance capacity, and β- carotene assays, while exerting weak activity towards DPPH. In addition, they displayed potent anti-elastase and moderate anti-collagenase activities. The HD-EO exhibited potent anti-tyrosinase activity, while the SF extract showed a moderate level compared to tested controls. OPLS-DA and dereplication studies predicted that the selective antibacterial activity of HD-EO to S. aureus was related to eucalyptol, while SF extract to C. perfringens was related to α-tocopherol.ConclusionsM. rugulosa leaves are considered a vital source of bioactive volatile components that are promoted for controlling skin aging and infection. However, further safety and clinical studies are recommended.

Evaluating the Antifungal Potential of Cinnamaldehyde: A Study of its Efficacy against Candida Species

Candida species exist as commensals in nature, colonizing the mucous membranes, gastrointestinal tract, vagina as well as the skin and usually cause infections in immunocompromised patients. C. albicans are known to be the most prevalent Candida species associated with infections, while there has been a significant surge in the incidence of Non-Candida albicans Candida species (NCAC) recently. The recent occurrences of the antifungal resistance in Candida, especially in NCAC species are quite alarming which raises the need for a safe and efficient alternative antimycotic drug. This study analyses the efficacy of cinnamaldehyde against Candida species, which is known to cause the majority of the fungal infections in humans. Cinnamaldehyde is a natural antimicrobial compound derived from cinnamon and has demonstrated significant antimycotic properties. Antifungal susceptibility profiles of cinnamaldehyde against Candida species were studied by disc diffusion as well as by broth microdilution assays. The mean diameter of the inhibition zone (IZ) formed by direct contact and disc volatilization assays were 61.26 mM and 65.20 mM, respectively. Both the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) of cinnamaldehyde ranged from 16-256 mg/L with mean MIC of 60.61 mg/L and a mean MFC of 81.94 mg/L. Co-incubation of Candida cells with cinnamaldehyde resulted in the loss of viable cells within 4 hours of incubation. Cinnamaldehyde was found to exhibit both fungistatic and fungicidal properties, making it a potent natural alternative for conventional antifungal agents.

Odontites linkii subsp. cyprius Ethanolic Extract Indicated In Vitro Anti-Acanthamoeba Effect.

This study aims to investigate three endemic ethanolic leaf extracts from Cyprus for anti-Acanthamoeba activities: Odontites linkii subsp. cyprius (Boiss.) Bolliger, Ptilostemon chamaepeuce subsp. cyprius (Greuter) Chrtek & B. Slavík, and Quercus alnifolia Poech. Screening for radical scavenging activity, total phenolic content (TPC), and total flavonoid content (TFC) were performed by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABST) methods, Folin-Ciocalteu method, and aluminum chloride method, respectively. An antibacterial-susceptibility test (AST) was performed using a broth microdilution assay to estimate the minimum inhibitory concentration (MIC) using iodonitrotetrazolium chloride (INT). Trypan blue (0.5%) was used to assess in vitro anti-Acanthamoeba cell viability of the ethanolic leaf extracts after 24-, 48-, and 72-h exposure-screening of ethanolic leaf extracts with liquid chromatography-mass spectrometry (LC-MS) for known compounds with biological activity. The ethanolic leaf extract of Odontites linkii subsp. cyprius demonstrated the highest anti-Acanthamoeba activity, with an inhibitory concentration (IC50) of 7.02 mg/mL after 72 h. This extract also showed an in vitro minimum inhibitory concentration (MIC) of 0.625 mg/mL against Enterococcus faecalis, a common nosocomial pathogen. The LC-MS analysis revealed the presence of bioactive iridoid compounds in O. linkii subsp. cyprius, further highlighting its potential as a source for new drug compounds. The ethanolic extract of O. linkii subsp. cyprius demonstrated a dose-dependent and time-dependent anti-Acanthamoeba effect in vitro. This study is the first to report the presence of iridoid compounds and anti-Acanthamoeba activities in the ethanolic extract of O. linkii subsp. cyprius. These promising findings highlight the potential of plant extracts, particularly O. linkii subsp. cyprius, as a source for new drug compounds for Acanthamoeba infections.

The mammalian Ire1 inhibitor, 4µ8C, exhibits broad anti-Aspergillus activity in vitro and in a treatment model of fungal keratitis.

The fungal unfolded protein response consists of a two-component relay in which the ER-bound sensor, IreA, splices and activates the mRNA of the transcription factor, HacA. Previously, we demonstrated that hacA is essential for Aspergillus fumigatus virulence in a murine model of fungal keratitis (FK), suggesting the pathway could serve as a therapeutic target. Here we investigate the antifungal properties of known inhibitors of the mammalian Ire1 protein both in vitro and in a treatment model of FK. The antifungal activity of Ire1 inhibitors was tested against conidia of several A. fumigatus isolates by a broth microdilution assay and against fungal biofilm by XTT reduction. The influence of 4μ8C on hacA mRNA splicing in A. fumigatus was assessed through gel electrophoresis and qRT-PCR of UPR regulatory genes. The toxicity and antifungal profile of 4μ8C in the cornea was assessed by applying drops to uninfected or A. fumigatus-infected corneas 3 times daily starting 4 hours post-inoculation. Corneas were evaluated daily through slit-lamp imaging and optical coherence tomography, or at endpoint through histology or fungal burden quantification via colony forming units. Among six Ire1 inhibitors screened, the endonuclease inhibitor 4μ8C displayed the strongest antifungal profile with an apparent fungicidal action. The compound both blocked conidial germination and hyphal metabolism of A. fumigatus Af293 in the same concentration range that blocked hacA splicing and UPR gene induction (60-120 µM). Topical treatment of sham-inoculated corneas with 0.5 and 2.5 mM 4μ8C did not impact corneal clarity, but did transiently inhibit epithelialization of corneal ulcers. Relative to vehicle-treated Af293-infected corneas, treatment with 0.5 and 2.5 mM drug resulted in a 50% and >90% reduction in fungal load, respectively, the latter of which corresponded to an absence of clinical signs of infection or corneal pathology. The in vitro data suggest that 4μ8C displays antifungal activity against A. fumigatus through the specific inhibition of IreA. Topical application of the compound to the murine cornea can furthermore block the establishment of infection, suggesting this class of drugs can be developed as novel antifungals that improve visual outcomes in FK patients.

Bioactive Properties of Campomanesia lineatifolia: Correlation Between Anti-Helicobacter pylori Activity, Antioxidant Potential and Chemical Composition.

Helicobacter pylori is found in the stomach of patients with chronic gastritis and peptic ulcers, infecting approximately half of the world's population. Current treatment for H. pylori infection involves a multi-drug therapeutic regime with various adverse effects, which leads to treatment abandonment and contributes to the emergence of resistant strains of H. pylori. Previously, we demonstrated that the essential oil of Campomanesia lineatifolia leaves exhibited an anti-H. pylori activity. In this study, we aimed to evaluate the phenolic content of the phenolic-rich ethanol extract (PEE) from C. lineatifolia and its anti-H. pylori and antioxidant properties. Additionally, the anti-H. pylori activity was assessed in polar and non-polar fractions from PEE, isolated myricitrin (MYR) and a mixture of myricitrin and quercitrin (MYR/QUER) from polar fractions, and aqueous extract (tea) to correlate the responsible fractions or compounds with the observed activity. Broth microdilution assays were performed to assess the anti-H. pylori activity using type cultures (ATCC 49503, NCTC 11638, both clarithromycin-sensitive) and clinical isolate strains (SSR359, clarithromycin-sensitive, and SSR366, clarithromycin-resistant). The antioxidant activity was evaluated using the DPPH assay. The total tannin and flavonoid contents were determined using the hide-powder method, the Folin-Ciocalteu reagent, and the aluminium chloride colourimetric assay, respectively. The tea (MIC 1:100), PEE, polar and non-polar fractions, MYR, and MYR/QUER inhibited the growth of H. pylori strains tested (MIC values ranging from 0.49 to 250 μg/mL). The antioxidant assays revealed that PEE exhibited a higher antioxidant activity (EC50 = 18.47 μg/mL), which correlated to the high phenolic content (tannin and flavonoid, 22.31 and 0.15% w/w, respectively). These findings support the traditional use of C. lineatifolia as a multitarget medicinal plant for treating gastric ulcers and reinforce the potential use of the species as a coadjuvant in therapeutic regimes involving patients with resistant H. pylori infection.

Repurposing the non-steroidal anti-inflammatory drug diflunisal as an adjunct therapy with amphotericin B against mucoralean fungi.

Introduction. Mucormycosis is an aggressive, angioinvasive infection associated with high morbidity and mortality. The disease remains difficult to treat, with limited available antifungal drugs. Consequently, there is an urgent need to develop alternate therapeutics against mucormycosis. In an earlier study, we demonstrated that the non-steroidal anti-inflammatory drug diflunisal impacted the actin cytoskeleton and quorum sensing and inhibited the formation of filopodia-/cytoneme-like extensions in Rhizopus arrhizus.Hypothesis. The non-steroidal anti-inflammatory drug diflunisal could exhibit potential antifungal activity.Aim. This study aimed to investigate the plausible antifungal activity of diflunisal against a range of medically important Mucorales and its combination effect with antifungal drugs.Methodology. The antifungal activity of diflunisal against Rhizopus arrhizus, Lichtheimia corymbifera, Rhizomucor pusillus, Cunninghamella bertholletiae, Mucor indicus, Mucor irregularis and Apophysomyces elegans was evaluated by broth microdilution assay. Allied salicylates were also screened. A combination assay with amphotericin B deoxycholate and posaconazole was performed by fractional inhibitory concentration test.Results. Exposure to diflunisal inhibited Rhizopus arrhizus spore germination in a dose-dependent manner. MICs of diflunisal against different Mucorales ranged from 64 to 2048 µg ml-1. Remarkably low levels of diflunisal (0.03-2 µg ml-1), depending on the strain/species tested, improved the antifungal activity of amphotericin B against mucoralean fungi by twofold (ΣFIC ≈ 0.5-0.508; P<0.01). Field-emission scanning electron micrographs further confirmed these observations. MICs of posaconazole were unchanged by this compound.Conclusion. Considering that amphotericin B remains the first-line drug against mucormycosis and exhibits dose-dependent side effects in clinical practice, especially nephrotoxicity, the observed additive interaction at remarkably low, clinically achievable levels of diflunisal demonstrates its potential utility as an adjunct therapy against mucoralean fungi.