Checkerboard Microdilution Method Research Articles

Ascorbic Acid Enhances the Inhibitory Effect of Theasaponins against Candida albicans

Candida albicans (C. albicans) is a main cause of hospital-acquired fungal infections. Combination therapy is promising as a novel anti-C. albicans strategy because of its better efficacy. Theasaponins are pentacyclic triterpenes in the Camellia genus with multiple biological activities. Our previous studies prove that theasaponins display inhibitory activity against C. albicans. Ascorbic acid (VC) is a vitamin found in many plants that shows potential in combination therapy. However, whether VC enhances the activity of theasaponins remains unclear. In this study, the checkerboard micro-dilution method was used to assess the effect of VC (0–80 mmol/L) on the anti-C. albicans effect of theasaponins (0–1000 μg/mL). Then, the effects of theasaponins (31.25 μg/mL), VC (80 mmol/L), and theasaponins (31.25 μg/mL) + VC (80 mmol/L) on C. albicans planktonic cells and different stages of biofilm formation were assessed. Transcriptomic analysis was conducted to investigate the molecular mechanisms. According to the results, VC enhanced the anti-planktonic and anti-biofilm effect of theasaponins against C. albicans. The minimum inhibitory concentration of theasaponins was significantly decreased and the fungicidal efficiency was increased with the addition of VC. VC remarkably aggravated the suppression of theasaponins with regard to various virulence factors of C. albicans, including adhesion, early biofilm formation, mature biofilm, cell surface hydrophobicity, and phospholipase activity. Compared with the theasaponins or VC groups, the level of intracellular reactive oxygen species was higher, while the levels of mitochondrial membrane potential and adenosine triphosphate were lower in the combination group, suggesting more severe oxidative stress, mitochondrial injury, and energy deficiency. Transcriptomic analysis revealed that the combination predominantly suppressed the pathways of glycolysis, glycerophospholipid metabolism, glutathione metabolism, and cysteine and methionine metabolism. This implied that energy deficiency and redox imbalance were associated with the anti-C. albicans activity of the combination. These results prove that VC enhances the inhibitory effect of theasaponins against C. albicans and that the combination has the potential to be used as a topical antifungal therapy or disinfectant.

Fluopsin C Promotes Biofilm Removal of XDR Acinetobacter baumannii and Presents an Additive Effect with Polymyxin B on Planktonic Cells.

Acinetobacter baumannii emerged as one of the most important pathogens for the development of new antimicrobials due to the worldwide detection of isolates resistant to all commercial antibiotics, especially in nosocomial infections. Biofilm formation enhances A. baumannii survival by impairing antimicrobial action, being an important target for new antimicrobials. Fluopsin C (FlpC) is an organocupric secondary metabolite with broad-spectrum antimicrobial activity. This study aimed to evaluate the antibiofilm activity of FlpC in established biofilms of extensively drug-resistant A. baumannii (XDRAb) and the effects of its combination with polymyxin B (PolB) on planktonic cells. XDRAb susceptibility profiles were determined by Vitek 2 Compact, disk diffusion, and broth microdilution. FlpC and PolB interaction was assessed using the microdilution checkerboard method and time-kill kinetics. Biofilms of XDRAb characterization and removal by FlpC exposure were assessed by biomass staining with crystal violet. Confocal Laser Scanning Microscopy was used to determine the temporal removal of the biofilms using DAPI, and cell viability using live/dead staining. The minimum inhibitory concentration (MIC) of FlpC on XDRAb was 3.5 µg mL-1. Combining FlpC + PolB culminated in an additive effect, increasing bacterial susceptibility to both antibiotics. FlpC-treated 24 h biofilms reached a major biomass removal of 92.40 ± 3.38% (isolate 230) using 7.0 µg mL-1 FlpC. Biomass removal occurred significantly over time through the dispersion of the extracellular matrix and decreasing cell number and viability. This is the first report of FlpC's activity on XDRAb and the compound showed a promissory response on planktonic and sessile cells, making it a candidate for the development of a new antimicrobial product.

Antimicrobial and synergistic effects of lemongrass and geranium essential oils against Streptococcus mutans, Staphylococcus aureus, and Candida spp.

The oral cavity harbors more than 700 species of bacteria, which play crucial roles in the development of various oral diseases including caries, endodontic infection, periodontal infection, and diverse oral diseases. To investigate the antimicrobial action of Cymbopogon Schoenanthus and Pelargonium graveolens essential oils against Streptococcus mutans, Staphylococcus aureus, Candida albicans, Ca. dubliniensis, and Ca. krusei. Minimum microbicidal concentration was determined following Clinical and Laboratory Standards Institute documents. The synergistic antimicrobial activity was evaluated using the Broth microdilution checkerboard method, and the antibiofilm activity was evaluated with the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay. Data were analyzed by one-way analysis of variance followed by the Tukey post-hoc test (P ≤ 0.05). C. schoenanthus and P. graveolens essential oils were as effective as 0.12% chlorhexidine against S. mutans and St. aureus monotypic biofilms after 24 h. After 24 h P. graveolens essential oil at 0.25% was more effective than the nystatin group, and C. schoenanthus essential oil at 0.25% was as effective as the nystatin group. C. schoenanthus and P. graveolens essential oils are effective against S. mutans, St. aureus, Ca. albicans, Ca. dubliniensis, and Ca. krusei at different concentrations after 5 min and 24 h.

Exploring the Antibiofilm Effect of Sertraline in Synergy with Cinnamomum verum Essential Oil to Counteract Candida Species.

The emergence and spread of drug-resistant pathogens, resulting in antimicrobial resistance, continue to compromise our capability to handle commonly occurring infectious diseases. The rapid global spread of multi-drug-resistant pathogens, particularly systemic fungal infections, presents a significant concern, as existing antimicrobial drugs are becoming ineffective against them. In recent decades, there has been a notable increase in systemic fungal infections, primarily caused by Candida species, which are progressively developing resistance to azoles. Moreover, Candida species biofilms are among the most common in clinical settings. In particular, they adhere to biomedical devices, growing as a resilient biofilm capable of withstanding extraordinarily high antifungal concentrations. In recent years, many research programs have concentrated on the development of novel compounds with possible antimicrobial effects to address this issue, and new sources, such as plant-derived antimicrobial compounds, have been thoroughly investigated. Essential oils (EOs), among their numerous pharmacological properties, exhibit antifungal, antibacterial, and antiviral activities and have been examined at a global scale as the possible origin of novel antimicrobial compounds. A recent work carried out by our research group concerned the synergistic antibacterial activities of commercially available and chemically characterized Cinnamomum verum L. essential oil (C. verum EO) in association with sertraline, a selective serotonin reuptake inhibitor whose repositioning as a non-antibiotic drug has been explored over the years with encouraging results. The aim of this work was to explore the synergistic effects of C. verum EO with sertraline on both planktonic and sessile Candida species cells. Susceptibility testing and testing of the synergism of sertraline and C. verum EO against planktonic and sessile cells were performed using a broth microdilution assay and checkerboard methods. A synergistic effect was evident in both the planktonic cells and mature biofilms, with significant reductions in fungal viability. Indeed, the fractional inhibitory concentration index (FICI) was lower than 0.5 for all the associations, thus indicating significant synergism of the associations with the Candida strains examined. Moreover, the concentrations of sertraline able to inhibit Candida spp. strain growth and biofilm formation significantly decreased when it was used in combination with C. verum EO for all the strains considered, with a reduction percentage in the amount of each associated component ranging from 87.5% to 97%.

Antimicrobial Effect of Lippia citriodora Extract in Combination with Gallic Acid or Octyl Gallate on Bacteria from Meat.

Chicken meat and its derivatives are easily alterable. They are a nutritionally healthy food, and their consumption has seen a remarkable increase worldwide in recent years. At the same time, consumer demand for the use of natural products to control microbial growth is increasing. In this context, the antimicrobial capacity of a commercial extract of the lemon verbena (Lippia citriodora) plant, (LCE) was tested in binary combination with gallic acid or octyl gallate against two strains of lactic acid bacteria (LAB) of meat origin: Carnobacterium divergens ATCC 35677 and Leuconostoc carnosum ATCC 49367. First, the antimicrobial potential was evaluated by the checkerboard microdilution method at the optimal growth temperature of each and at 4 °C, pH 5.7 and 6.7, in culture medium. Octyl gallate was the most effective antimicrobial against the two bacteria under all study conditions. At 4 °C, the combination of LCE with octyl gallate had a similar antimicrobial effect on the two LAB, being bactericidal at pH 6.7. In chicken breast, this effective combination was tested in normal or modified atmosphere and refrigerated (4-8 °C) for 9 days. LCE + OG in modified atmosphere reduced the different microbial groups studied, including the lactic acid bacteria as the main microorganisms responsible for the spoilage of fresh meat. Further research could pave the way for the development of novel strategies contributing to the technological stability, security, and functional properties of chicken meat.

Study on synergistic effects of curcumin and bixin against foodborne pathogens.

Various studies have shown that natural colorants, in addition to their coloring attributes, have valuable biological effects such as antioxidant, anti-inflammation, and anticarcinogenic properties. Moreover, their use as a food colorant can restrict the potential disadvantages of synthetic additives and turn foods into functional products. In this study, invitro antimicrobial activities of two natural colorants of bixin and curcumin against some important foodborne pathogens: Staphylococcus aureus (S. aureus), Listeria innocua (L. innocua), and Escherichia coli (E. coli) were investigated by disk diffusion method. Minimum inhibitory concentration and minimum bactericidal concentration values were determined by agar dilution and broth microdilution methods. The synergistic activity of the colorants against selected microorganisms was assayed by the checkerboard microdilution method. The results showed that the inhibitory effects of bixin against S. aureus were more pronounced than E. coli and L. innocua. The lowest concentration of curcumin (0.6 mg/mL) in the disk diffusion method was not inhibited by any tested bacteria. However, it was effective at the higher concentrations against three microorganisms, but its diameter of inhibition zones was lower than gentamicin in all concentrations. Synergetic effects were observed by curcumin and bixin combination against S. aureus (FICI ≤ 0.5), but they act as an antagonist against E. coli and L. innocua. The results of the synergy test were confirmed by the isobologram curves.

The Therapeutic Role and Mechanism of Glabridin Under Aspergillus fumigatus Infection.

Purpose: To characterize the efficiency of glabridin alone and in combination with clinical antifungals in Aspergillus fumigatus keratitis. Methods: The broth microdilution method was performed to investigate whether glabridin exerted an antifungal role on planktonic cells and immature and mature biofilm. Antifungal mechanism was evaluated by Sorbitol and Ergosterol Assays. The synergistic effect of glabridin and antifungals was assessed through the checkerboard microdilution method and time-killing test. Regarding anti-inflammatory role, inflammatory substances induced by A. fumigatus were assessed by real-time quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Drug toxicity was assessed by Draize test in vivo. Macrophage phenotypes were examined by flow cytometry. Results: Regarding antifungal activity, glabridin destroyed fungal cell wall and membrane on planktonic cells and suppressed immature and mature biofilm formation. After combining with natamycin or amphotericin B, glabridin possessed a potent synergistic effect against A. fumigatus. Regarding anti-inflammatory aspects, Dectin-1, toll‑like receptor (TLR)-2 and TLR-4 expression of human corneal epithelial cells were significantly elevated after A. fumigatus challenge and reduced by glabridin. The elevated expression of interleukin-1β and tumor necrosis factor-alpha induced by A. fumigatus or corresponding agonists were reversed by glabridin, equivalent to the effect of corresponding inhibitors. Glabridin could also contribute to anti-inflammation by downregulating inflammatory mediator expression to suppress macrophage infiltration. Conclusions: Glabridin contributed to fungal clearance by destroying fungal cell wall and membrane, and disrupting biofilm. Combining glabridin with clinical antifungals was superior in reducing A. fumigatus growth. Glabridin exerted an anti-inflammatory effect by downregulating proinflammatory substance expression and inhibiting macrophage infiltration, which provide a potential agent and treatment strategies for fungal keratitis.

Pythium insidiosum: In vitro oomicidal evaluation of telithromycin and interactions with azithromycin and amorolfine hydrochloride

This study evaluated the repositioning of the ketolide antibacterial telithromycin (TLT) against the oomycete Pythium insidiosum and verified the combination of TLT and the antimicrobials azithromycin (AZM) and amorolfine hydrochloride (AMR), which have known anti-P. insidiosum activity. Susceptibility tests of P. insidiosum isolates (n = 20) against the drugs were carried out according to CLSI protocol M38-A2, and their combinations were evaluated using the checkerboard microdilution method. The minimum inhibitory concentrations were 0.5–4 µg/mL for TLT, 2–32 µg/mL for AZM, and 16–64 µg/mL for AMR. For the TLT+AZM combination, 52.75 % of interactions were indifferent, 43.44 % were antagonistic, and 9.70 % were synergistic. As for interactions of the TLT+AMR combination, 60.43 % were indifferent, 39.12 % were antagonistic, and 10.44 % synergistic interactions. This study is the first to evaluate the repositioning of the antibacterial TLT against mammalian pathogenic oomycetes, and our results show that its isolated action is superior to its combinations with either AZM or AMR. Therefore, we recommend including TLT in future research to evaluate therapeutic approaches in different clinical forms of human and animal pythiosis.

Exploring Medicinal Plants for Antimicrobial Activity and Synergistic Effects With Doxycycline Against Bacterial Species.

Medicinal plants are rich sources of bioactive compounds with diverse pharmacological properties, including antimicrobial activities. This study aimed to assess the antibiofilm potential of methanol and ethanol extracts from nine selected medicinal plants, as well as their synergistic effects with doxycycline against Bacillus strains. Standard procedures were employed to determine the phytochemical composition, total phenolic, and flavonoid contents of the extracts. The antibacterial activity was evaluated using the broth microdilution method, while biofilm formation was assessed via the microtiter plate method. Antibiofilm activity was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium-bromide (MTT) assay. Combination studies were conducted using the checkerboard microdilution method. All extracts contained phenols, flavonoids, steroids, triterpenes, and anthraquinones. The methanolic extract of Psidium guajava exhibited the highest total phenolic and flavonoid contents (90.48 ± 0.55 mg GAE/g), while the ethanolic extract of Olax subscorpioidea showed the highest flavonoid content (6.48 ± 0.33 mg QE/g). Ethanol extracts of Eucalyptus globulus and Psidium guajava and methanolic extract of Syzygium jambos demonstrated significant antibacterial activity against Bacillus anthracis 34F2 Sterne strains, with a MIC value of 64 μg/mL. Biofilm formation in Bacillus strains was notably enhanced in the presence of glucose. The methanolic extract of O. subscorpioides exhibited the highest biofilm imbibition (85%), while Picralima nitida methanolic extract showed the most effective biofilm eradication (79%). The combination of Solanum torvum ethanol extract with doxycycline displayed synergistic effects against biofilm formation inhibition and eradication in all tested Bacillus strains. Taken together, Solanum torvum ethanol extract shows promise for developing new combination antibacterial therapies.

Effects of chitooligosaccharide on the in vitro antibacterial activity against avian Escherichia coli and the pharmacokinetics of florfenicol in healthy chickens

This study investigates the combined effects of chitooligosaccharide (COS) and florfenicol (FLO) on the inhibition of Escherichia coli in vitro, as well as the pharmacokinetic interactions between these compounds in healthy chickens. The minimum inhibitory concentration (MIC) of COS and FLO alone and the fractional inhibitory concentration index (FICI) after combined treatment were determined using the broth microdilution method and checkerboard method, respectively. Additionally, we evaluated the pharmacokinetic interactions between the 2 types of COS and FLO in healthy chickens. Thirty chickens were randomly divided into 3 groups: Florfenicol group (30 mg/kg), COS J85 group (COS J85 20 mg/kg + florfenicol 30 mg/kg), COS H85 group (COS H85 20 mg/kg + florfenicol 30 mg/kg). Either FLO or COS was orally administered by gavage. The concentrations of FLO in chicken plasma were measured at different time points after the drug withdrawal using high-performance liquid chromatography (HPLC), and pharmacokinetic parameters were calculated by a compartmental method. The results showed that COS J85 and COS H85, when combined with FLO, had FICI values of 0.1875 to 0.75 and 0.3125 to 1, respectively, indicating good synergistic or additive effects against Escherichia coli. The pharmacokinetics of FLO alone and in combination with COS followed a 1-compartment model with first-order absorption and elimination. Furthermore, the pharmacokinetic analysis revealed that the elimination half-life (t1/2ke) of florfenicol was significantly increased in the COS H85 group compared to oral administration of florfenicol alone (P < 0.05). Other pharmacokinetic parameters did not show significant changes (P > 0.05), except between the 2 combined treatment groups, where statistical differences were observed for various parameters, excluding the area under the concentration-time curve from the time of dosing to infinity (AUC) and peak concentration (Cmax).

Antifungal Biofilm Inhibitory Effects of Combinations of Diclofenac and Essential Oils.

Systemic fungal infections have risen in recent decades and most of them are caused by Candida species, which are becoming increasingly resistant to conventional antifungal drugs. Biofilm production has been considered the most common growth form of Candida cells and is associated with a high level of antifungal resistance. At present, international research reports on the antifungal activity of non-traditional antimicrobial drugs and their potential use against life-threatening resistant fungal infections. Indeed, drug repurposing has led to the consideration of well-known compounds as a last-line therapy. The goal of this work is to evaluate the potential synergistic antifungal biofilm activity of new combinations between diclofenac sodium salt (DSS), a widely used non-steroidal anti-inflammatory drug (NSAID), with the essential oils (EOs) of Mentha piperita, Pelargonium graveolens, and Melaleuca alternifolia, whose antifungal activity has been well documented over the years. The in vitro antifungal activity of DSS and EOs was determined on different Candida strains. Susceptibility testing and the synergism of DSS and EOs versus biofilm cells was performed by using the broth microdilution assay and checkerboard methods. Minimum inhibitory concentrations (sMIC50) of DSS alone ranged from 1.25 to 2.05 mg/mL for all the strains considered. These values significantly decreased when the drug was used in combination with the EOs. The fractional inhibitory concentration index (FICI) was lower than 0.5 for almost all the associations, thus indicating a significant synergism, particularly for the DSS-Pelargonium graveolens combination towards the Candida strains examined. These preliminary results show that the combination of the EOs with DSS improves the antifungal activity on all the tested Candida strains, significantly lowering the concentrations of the components used and thus allowing any toxic effects to be overcome.

Rapamycin and caspofungin show synergistic antifungal effects in caspofungin-susceptible and caspofungin-resistant Candida strains in vitro.

Caspofungin is an echinocandin antifungal agent that inhibits synthesis of glucan required for the fungal cell wall. Resistance is mediated by mutation of Fks1 glucan synthase, among which S645P is the most common resistance-associated polymorphism. Rapamycin is a macrolide that inhibits the mechanistic target of rapamycin (mTOR) protein kinase activity. This study investigated the interaction between rapamycin and caspofungin in inhibiting the growth of WT Candida albicans and Fks1 S645P mutant clinical isolate, and WT Candida lusitaniae and genetically engineered isogenic strain with Fks1 S645P mutation at equivalent position. Interactions between caspofungin and rapamycin were evaluated using the microdilution chequerboard method in liquid medium. The results were analysed using the Loewe additivity model (FIC index, FICI) and the Bliss independence model (response surface, RS, analysis). Synergy between rapamycin and caspofungin was shown for C. albicans and C. lusitaniae strains by RS analysis of the chequerboard tests. Synergy was observed in strains susceptible and resistant to caspofungin. Weak subinhibitory concentrations of rapamycin were sufficient to restore caspofungin susceptibility. We report here, for the first time, synergy between caspofungin and rapamycin in Candida species. Synergy was shown for strains susceptible and resistant to caspofungin. This study highlights the possible implication of the TOR pathway in sensing antifungal-mediated cell wall stress and in modulating the cellular response to echinocandins in Candida yeasts.

Molecular Epidemiology of Carbapenem Resistant Klebsiella Pneumoniae in Northern China: Clinical Characteristics, Antimicrobial Resistance, Virulence and Geographic Distribution.

In this article, we studied in detail 74 Carbapenem Resistant Klebsiella pneumoniae (CRKP) in Shanxi to provide essential insight into development of effective strategies for control of CRKP. From 2018 to 2021, we collected 74 clinical CRKP from 11 hospitals in Shanxi Province. Clinical data were obtained from medical records, and all isolates were subjected to antimicrobial susceptibility testing, multi locus sequence typing, capsular serotypes, resistant gene profiles and virulence gene profiles. The synergistic activity was performed by microdilution checkerboard method. Our study found differences in the clinical characteristics of CRKP between regions in Shanxi. Sequence type (ST) 11 was the dominant ST in Shanxi; however, the ST types in Shanxi had become more diverse over time and the proportion of STs showed a more balanced distribution with a significant decrease in ST11. NDM was the most common carbapenemase in Shanxi. In addition, the STs, carbapenemases, serotypes and virulence gene distribution varied by region in Shanxi. Moreover, tigecycline in combination with carbapenems and aztreonam had an excellent synergistic effect on CRKP in vitro. The results of this study provide essential insight into development of effective strategies for control of CRKP in Shanxi.

Photodynamic Activity of Chlorophyllin and Polyethylenimine on Pseudomonas aeruginosa Planktonic, Biofilm and Persister Cells.

Antimicrobial photodynamic inactivation is considered a promising antimicrobial approach that may not develop resistance in the near future. Here, we investigate the influence of the photosensitizer chlorophyllin (CHL) and the cationic permeabilizer polyethylenimine (PEI), exposed to a red light-emitting diode, on the human pathogen Pseudomonas aeruginosa free-living planktonic cells, the sessile biofilm and persister cells. The broth microdilution checkerboard method was used to test antimicrobial susceptibility. As a substrate for biofilms, the Calgary biofilm device was used, and the quantification of the biofilm biomass was carried out using a crystal violet assay. Serine hydroxamate was used for the induction of persisters. Our findings reveal that PEI ameliorates the antimicrobial activity of CHL against P. aeruginosa planktonic and biofilm states, and the concentration required to eradicate the bacteria in the biofilm is more than fourfold that is required to eradicate planktonic cells. Interestingly, the persister cells are more susceptible to CHL/PEI (31.25/100 µg mL-1) than the growing cells by 1.7 ± 0.12 and 0.4 ± 0.1 log10 reduction, respectively, after 15 min of illumination. These data demonstrate that CHL excited with red light together with PEI is promising for the eradication of P. aeruginosa, and the susceptibility of P. aeruginosa to CHL/PEI is influenced by the concentrations and the exposure time.

Synergistic activity of the combination of falcarindiol and itraconazole in vitro against dermatophytes.

Previous studies have shown that natural polyacetylene alcohols, such as falcarindiol (FADOH), have good antifungal effects on plant fungi. While its effect on fungi that infect humans remains to be explored. In our study, checkerboard microdilution, drop-plate assay, and time-growth method were employed to analyze the interactions between FADOH and itraconazole (ITC) in vitro against dermatophytes, including 12 Trichophyton rubrum (T. rubrum), 12 Trichophyton mentagrophytes (T. mentagrophytes), and 6 Microsporum canis (M. canis). The results showed that the combination of FADOH and ITC exhibited synergistic and additive activity against 86.7% of all tested dermatophytes. FADOH had an excellent synergistic effect on ITC against T. rubrum and T. mentagrophytes; the synergistic rates were 66.7% and 58.3%, respectively. On the contrary, FADOH combined with ITC showed poor synergistic inhibitory activity (16.7%) against M. canis. Moreover, the additive rates of these two drugs against T. rubrum, T. mentagrophytes, and M. canis were 25%, 41.7%, and 33.3%, respectively. No antagonistic interactions were observed. The drop-plate assay and time-growth curves confirmed that the combination of FADOH and ITC had a potent synergistic antifungal effect. The in vitro synergistic effect of FADOH and ITC against dermatophytes is reported here for the first time. Our findings suggest the potential use of FADOH as an effective antifungal drug in the combined therapy of dermatophytoses caused especially by T. rubrum and T. mentagrophytes.

Lower Concentrations of Amphotericin B Combined with Ent-Hardwickiic Acid Are Effective against Candida Strains

Life-threatening Candida infections have increased with the COVID-19 pandemic, and the already limited arsenal of antifungal drugs has become even more restricted due to its side effects associated with complications after SARS-CoV-2 infection. Drug combination strategies have the potential to reduce the risk of side effects without loss of therapeutic efficacy. The aim of this study was to evaluate the combination of ent-hardwickiic acid with low concentrations of amphotericin B against Candida strains. The minimum inhibitory concentration (MIC) values were determined for amphotericin B and ent-hardwickiic acid as isolated compounds and for 77 combinations of amphotericin B and ent-hardwickiic acid concentrations that were assessed by using the checkerboard microdilution method. Time-kill assays were performed in order to assess the fungistatic or fungicidal nature of the different combinations. The strategy of combining both compounds markedly reduced the MIC values from 16 µg/mL to 1 µg/mL of amphotericin B and from 12.5 µg/mL to 6.25 µg/mL of ent-hardwickiic acid, from isolated to combined, against C. albicans resistant to azoles. The combination of 1 µg/mL of amphotericin B with 6.25 µg/mL of ent-hardwickiic acid killed all the cells of the same strain within four hours of incubation.

665. Modified Superposition in vitro Susceptibility of Aztreonam and Ceftazidime/Avibactam for an NDM-5 Metallo-β-Lactamase-Producing Klebsiella pneumoniae Isolate

Abstract Background We describe a novel in vitro susceptibility testing approach of aztreonam and ceftazidime/avibactam using a superposition method paired with confirmatory testing and molecular sequencing of a New Delhi metallo-β-lactamase (NDM)-5-producing Klebsiella pneumoniae. Methods A modified strip superposition method with Epsilometer test (Etest) strips and Kirby Bauer disks was utilized to test the in vitro activity of aztreonam and ceftazidime/avibactam against the NDM-producing K. pneumoniae. The isolate was also sent for whole genome sequencing and tested for the in vitro activity of aztreonam and ceftazidime in combination with avibactam using a broth microdilution checkerboard assay. Aztreonam and ceftazidime/avibactam in vitro testing with modified superposition method Results All zones of inhibition from the Etest superposition method were measured to be 22 mm in diameter (Clinical and Laboratory Standards Institute [CLSI] zone of inhibition for aztreonam susceptibility is ≥ 21 mm for Enterobacterales). The results of whole genome sequencing showed the K. pneumoniae isolate carried a metallo-β-lactamase (blaNDM-5), extended-spectrum β-lactamase (blaCTX-M-15), and other β-lactamases (blaSHV-11, blaTEM-1). Through the broth microdilution checkerboard method, the isolate demonstrated inhibition by aztreonam/avibactam (fixed concentration of 4mg/L) at a minimum inhibitory concentration (MIC) of 0.25 mg/L but was resistant to ceftazidime/avibactam (MIC &amp;gt; 16 mg/L) and aztreonam alone (MIC &amp;gt; 16 mg/L) according to CLSI breakpoints. Conclusion The in vitro activity of aztreonam and ceftazidime/avibactam against an NDM-5-producing K. pneumoniae was demonstrated with a modified superposition method. This may serve as a simplistic and readily available technique for rapidly determining antimicrobial activity in these difficult to treat pathogens. Disclosures Justin Schmetterer, PharmD, PhC, BCIDP, AbbVie Speaker Bureau: Honoraria Yao Lin, MS, Abbvie Inc: employee|Abbvie Inc: Stocks/Bonds.

SINERGISTIC COMBINATION OF ALOE VERA AND KERSEN LEAF EXTRACTS FOR INHIBITION OF THE GROWTH OF Staphylococcus aureus 25923

Aloe vera leaf (Aloe vera L.) and cherry leaf (Muntingia calabura L.) contain bioactive compounds that serve as the potential antibacterial agent. The combination treatment of multiple extracts can produce a stronger effect than a single compound. This study aims to determine the synergistic combination of aloe vera leaf extract (EDLB) and cherry leaf extract (EDK) as an antibacterial agent against Staphylococcus aureus ATCC 25923. The extract was obtained by maceration using 96% ethanol. Chemical content in the extract was analyzed by tube test and thin layer chromatography (TLC). Antibacterial activity against S. aureus ATCC 25923 was measured by the checkerboard microdilution method on a 96 well microplate. The effect of the combination of extracts on antibacterial activity was based on the fractional inhibitory concentration index (FICI) value. EDLB and EDK contain flavonoids, tannins, alkaloids, and saponins. The second combination showed a synergistic effect with a FICI value of 0.5 in inhibiting the growth of S. aureus with minimal inhibitory levels in a mixture of 0.625 mg/mL EDLB and 2.5 mg/mL EDK.

In Vitro Combination of Terbinafine with Ketoconazole Against Aspergillus Species with Terbinafine High MIC Values Isolated From Otomycosis.

Otomycosis is a common mycotic infection of the external auditory canal, and Aspergillus species are one of the most frequent causative agents worldwide. The limited antifungal arsenal, the high toxicity and side effects of antifungal agents, and the growing resistance to the currently available antifungals underscore the need for new therapeutic strategies. The present study aimed to evaluate the combined in vitro efficacy of terbinafine and ketoconazole against Aspergillus species with terbinafine high MIC values isolated from patients with otomycosis.84 Aspergillus species with high MIC values to terbinafine (≥ 4µg/ml), consisting of A. flavus, A. tubingensis, A. niger, and A. terreus, were included in this study. The checkerboard microdilution method evaluated the in vitro interactions using the CLSI reference technique. Synergistic effects were observed for 66.67% (56/84) of all isolates (FICI ranging from 0.19 to 0.5). However, the interactions of terbinafine and ketoconazole exhibited indifference in 33.33% (28/84) of the isolates, and no antagonism was observed for any combination. The interaction of terbinafine and ketoconazole showed synergistic activity against Aspergillus species with high MIC values, suggesting that this is an alternative and promising approach for treating otomycosis.

Synergistic Effect of Clinically Available Beta-Lactamase Inhibitors Combined with Cefiderocol against Carbapenemase-Producing Gram-Negative Organisms.

The role of β-lactamases in reduced susceptibility or resistance to cefiderocol has been supported by recent reports. The purpose of this study was to investigate the in vitro impact of clinically available β-lactamase inhibitors on cefiderocol activity against characterized carbapenemase-producing Gram-negative isolates. A collection of 39 well-characterized Gram-negative isolates obtained from various clinical sources and countries were included. Cefiderocol antimicrobial susceptibility was evaluated via reference broth microdilution. The chequerboard microdilution method and time-kill assays were used to determine the synergy of tazobactam, avibactam, vaborbactam and relebactam in combination with cefiderocol. MICs of cefiderocol presented a 4- to 256-fold reduction against Klebsiella pneumoniae carbapenemase (KPC)-producing Gram-negative isolates (predominantly K. pneumoniae) when avibactam, vaborbactam and relebactam were combined individually. Notably, the KPC-inhibitors led to a 4- to 32-fold reduction in cefiderocol MICs in the four cefiderocol-resistant KPC-producing K. pneumoniae isolates, showing restoration of cefiderocol susceptibility (MIC ≤ 2 mg/L) in ten out of twelve cases. Tazobactam led to a 4- to 64-fold decrease in cefiderocol MICs only in K. pneumoniae strains harbouring blaKPC-41, blaKPC-31, blaKPC-53 and blaKPC-66. The synergistic effect of all serine-β-lactamase inhibitors on cefiderocol activity was also shown in OXA-48-like-producing Enterobacterales strains. Conversely, a combination of β-lactamases inhibitors with cefiderocol was not synergistic with all OXA-23-like-producing strains and most metallo-β-lactamases producers. In conclusion, the addition of clinically available serine β-lactamase inhibitors to cefiderocol might represent an important development in the formulation to increase its spectrum and therapeutic efficacy, and to limit in vivo resistance emergence.