Checkerboard Microdilution Assay Research Articles

Research Note: Synergistic effect of isopropoxy benzene guanidine and colistin against mcr-1-positive Escherichia coli in vitro and in duck intestine infection models

Colistin (CST) is considered as "agent of last resort" against gram-negative bacteria as feed additive. Its clinical effectiveness has reduced since the emergence of mcr-1 gene in ducks. Isopropoxy benzene guanidine (IBG), a new guanidine derivative, showed positive effects on improving animal weights and alleviating intestinal pathogens, therefore, the objective of this study was to evaluate the effect of this compound supplement with CST in ducks and explore the possibilities in feed additive. A total of fifteen duck-origin Escherichia coli carrying the mcr-1 gene were included in this study. A checkerboard microdilution assay was used to evaluate the in vitro antibacterial activity of IBG combined with CST against mcr-1-positive E. coli. A 3-by-2 time-kill array of IBG (16, 32, and 64 μg/mL) and CST (1/2 MIC and 1/4 MIC) over 24 hours was utilized to characterize the activity of the agents alone and in combination against E. coli strain 1 in vitro. The intestinal colonization model was used to evaluate the in vivo effect of IBG combined with CST. These results indicated that the combination of IBG plus CST showed a synergistic effect against all clinical isolates (FICI < 0.5). The bacterial burden was reduced by more than 2 log10 CFU/mL when E. coli strain 1 was tested with 1/2 MIC CST plus 64 μg/mL IBG for 24 h. Further experiments in vivo demonstrated that the CST combined with IBG was able to increase duck weights, reduced intestinal pathogenic E. coli and showed a synergistic antibacterial effect. Combination of CST (4 mg/kg b.w.) plus IBG (32 or 64 mg/kg b.w.) achieved 1.84 to 3.29 log10 CFU/g killing after 7 d of therapy, which was significantly different from that in the challenge control group (p<0.05). In summary, our study demonstrated the potential use of IBG as feed additive for veterinary purposes in ducks and provided new insights into overcoming resistance in the future.

Dissolving microneedle to improve transdermal delivery of terbinafine for treatment of onychomycosis

The topical formulation of antifungal agents has demonstrated prominent success in treating fungal infection. However, traditional topical cream or solution limited ability to penetrate the nail plate and target the infected area contributes to the inefficacy of antifungals in the treatment of onychomycosis. In this work, the problem of topical administration reaching only the skin's surface was addressed by dissolving microneedles (MN) to pierce the skin and deliver drugs directly to the site of infection. Terbinafine (TH) was encapsulated into polydopamine (PDA) nanoparticles (TH@PDA) to increase the antifungal efficacy of TH. The results of checkerboard microdilution assays showed that the antifungal efficacy of TH@PDA was increased up to 2–8 times in comparison to free TH. In animal experiment, TH@PDA was incorporated into MN to target the infected site and increase the therapeutic effect. Our results demonstrated TH@PDA MN significantly decreased the fungal burden in the nails and relieved inflammation at the site of infection when compared to TH MN, TH cream, and oral TH. These results suggested that the TH@PDA MN exhibited enhanced antifungal effects and can serve as a potentially promising approach for onychomycosis treatment.

The combination of allicin with domiphen is effective against microbial biofilm formation.

Microorganisms in biofilms are particularly difficult to control because of their increased survival and antibiotic resistance. Allicin and domiphen were employed to inhibit the microbial growth and biofilm formation of Staphylococcus aureus, Escherichia coli, and Candida albicans strains. Broth microdilution method and checkerboard assay were conducted to determine the efficacy of allicin combined with domiphen against S. aureus, E. coli, and C. albicans. Microbial biofilm formation was measured using the crystal violet staining method and fluorescence microscopy. And the total viable count of the biofilm cells on material surface after the treatment with antimicrobial reagents was calculated with the plate count technique. The two drugs showed synergistic effects against the pathogens with a fractional bactericidal concentration of less than 0.38. The combination of 64 μg/mL allicin with 1 μg/mL domiphen dispersed over 50% of the biofilm mass of S. aureus, E. coli, and C. albicans. In addition, the drug combination reduced the total viable counts of E. coli and C. albicans biofilm cells on stainless steel and polyethylene surfaces by more than 102 CFU/mL. The combination of allicin and domiphen is an effective strategy for efficiently decreasing biofilms formation on various industrial materials surfaces.

KOJİK ASİT İLE LAVANDULA ANGUSTIFOLIA L. UÇUCU YAĞININ ANTİMİKROBİYAL KOMBİNASYONU

Objective: Lavandula angustifolia L. (Lavender) is one of the most plants essential oils used in the cosmetic, food and biological activities. Kojic acid has been used in cosmetics for its whitening effect and pharmaceutical activity. The aim of this study to determine the composition of the Pharmacopoeia quality L. angustifolia essential oil (EO). Moreover, antimicrobial activities against skin pathogens and synergistic antibacterial activity were also examined of EO and kojic acid. Material and Method: In this work, chemical composition of the EO was defined. Linalyl acetate (43.3%) and linalool (38.6%) were determined as the major components by GC-MS and GC-FID, simultaneously. The antimicrobial activity was evaluated against Candida albicans, C. glabrata, Staphylococcus aureus and Salmonella typhimurium. Result and Discussion: EO and kojic acid showed weak antimicrobial effects. MIC values were determined as the EO 10 mg/ml and kojic acid 1.25 mg/ml against S. typhimurium. To assess the synergistic activity was evaluated by the checkerboard microdilution assay, EO was combined with kojic acid against S. typhimurium. Among the tested skin pathogen microorganisms, S. typhimurium was more sensitive to kojic acid. Therefore, synergic activity was investigated against S. typhimurium and found indifferent effect.

Synergistic potential of teriflunomide with fluconazole against resistant Candida albicans in vitro and in vivo.

Candida albicans is the primary cause of systemic candidiasis, which is involved in high morbidity and mortality. Drug resistance exacerbates these problems. In addition, there are limited antifungal drugs available. In order to solve these problems, combination therapy has aroused great interest. Teriflunomide is an immunosuppressant. In the present work, we aimed to identify whether teriflunomide can reverse the resistance of Candida albicans in the presence of sub-inhibitory concentrations of fluconazole in vitro and in vivo. Seven Candida albicans isolates were used in this study. Susceptibility of Candida albicans in vitro to the drugs was determined using a checkerboard microdilution assay in accordance with the recommendations of the Clinical and Laboratory Standards Institute. The effects of drugs on biofilm biomass of Candida albicans were determined by crystal violet staining. The development ability of Candida albicans hyphae was performed using a modified broth microdilution method. Galleria mellonella was used for testing the in vivo efficacy of the combination therapies. We found that the combination of teriflunomide (64 µg/mL) and fluconazole (0.5-1 µg/mL) has a significant synergistic effect in all resistant Candida albicans isolates (n=4). Also, this drug combination could inhibit the immature biofilm biomass and hyphae formation of resistant Candida albicans. Galleria mellonella was used for testing the in vivo efficacy of this combination therapies. As for the Galleria mellonella larvae infected by resistant Candida albicans, teriflunomide (1.6 µg/larvae) combined with fluconazole (1.6 µg/larvae) significantly increased their survival rates, and reduced the fungal burden, as well as damage of tissue in comparison to that in the control group or drug monotherapy group. These results expand our knowledge about the antifungal potential of teriflunomide as an adjuvant of existing antifungal drugs, and also open new perspectives in the treatment of resistant Candida albicans based on repurposing clinically available nonantifungal drugs.

Synergistic effect of eravacycline combined with fluconazole against resistant Candida albicans in vitro and in vivo

ABSTRACT Background The limited availability of antifungal drugs for candidiasis and the persistent problem of drug resistance, necessitates the urgent development of new antifungal drugs and alternative treatment options. Research design and methods This study examined the synergistic antifungal activity of the combination of eravacycline (ERV) and fluconazole (FLC) both in vitro by microdilution checkerboard assay and in vivo by Galleria mellonella model. The underlying synergistic mechanisms of this drug combination was investigated using RNA-sequencing and qPCR. Results ERV (2 μg/mL) + FLC (0.25–0.5 μg/mL) had strong synergistic antifungal activity against resistant Candida albicans (C. albicans) in vitro, as evidenced by a fractional inhibitory concentration index of 0.0044–0.0088. In vivo experiments in Galleria mellonella larvae infected with resistant C. albicans revealed that ERV (2 μg/larva) + FLC (1 μg/larva) improved survival rates and reduced fungal burden. The results of RNA-sequencing and qPCR showed that the mechanism of synergistic inhibition on resistant C. albicans was related to the inhibition of DNA replication and cell meiosis. Conclusions These results indicate that the combination of ERV and FLC effectively inhibits resistant C. albicans both in vitro and in vivo and lay the foundation for a potential novel treatment option for candidiasis.

Pseudolaric Acid A: A Promising Antifungal Agent Against Prevalent Non-albicans Candida Species.

The non-albicans Candida (NAC) species have recently gained great importance worldwide due to the increasing proportion in candida causing bloodstream infections. This investigation aimed to explore the efficacy of Pseudolaric acid A (PAA, a diterpenoid derived from Pseudolarix kaempferi) and its synergistic effect with fluconazole (FLC) against NAC species, including C. tropicalis, C. parapsilosis complex, and C. glabrata. The microdilution checkerboard assay and time-killing curves were performed to detect the antifungal efficiency. To examine the integrity of cell walls and membranes, calcofluor white stain and propidium iodide stain were used. The changes of intracellular ultrastructure in Candida cells after treatment were observed using transmission electron microscopy. Changes in cell viability with the autophagy inhibitor 3-MA were assessed by the XTT method. It was revealed that PAA alone is effective on C. tropicalis, C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis (MIC 8-128 µg/mL). Strong synergism against FLC-resistant C. tropicalis was observed (FICI 0.07-0.281), when PAA and FLC were combined. PAA had dose-dependently detrimental effects on C. tropicalis cell membranes. Moreover, increased vacuoles and autophagosome formation were found in C. tropicalis exposed to PAA. And the inhibitory effect of PAA against C. tropicalis can be relieved by autophagy inhibitor 3-MA in a certain concentration range. Ultrastructural alterations of C. tropicalis were more pronounced under the combination of PAA and FLC, including separation of the cell membrane from the cell wall, increased number of vacuoles, and degradation of organelles. These observations indicated that PAA and its combination with FLC could be a promising therapeutic candidate for treating infections caused by NAC species.

Synergistic effects of Thymus vulgaris essential oil in combination with antifungal agents and inhibition of virulence factors of Candida albicans

BackgroundCombination antifungal therapy has become a prominent approach in medical practice as it takes advantage of synergistic interactions by interfering with multiple targets of the pathogen, broadening the spectrum of activity, reducing the development of resistance, and improving treatment outcomes. Therefore, combining conventional antifungal drugs with natural products can increase antifungal activity, reduce side effects, and optimize therapeutic effects. PurposeThe present study aimed to evaluate the antifungal activity of thyme essential oil (ThyEO) in combination with amphotericin B, caspofungin, fluconazole, itraconazole, and posaconazole (PSZ), as well as its inhibitory effect on Candida albicans virulence factors. Materials and MethodsThe microbroth dilution assay was employed to determine the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC). Interactions were assessed using the microdilution checkerboard assay. Ergosterol and methylene blue assays were used to detect effects on fungal membrane, while the sorbitol assay was used to detect effects on fungal cell wall. Inhibition of yeast virulence factors (adherence to epithelial cells, germ tube and pseudomycelium formation, secretion of hydrolytic enzymes, and biofilm formation) was assessed with previously reported methods. ResultsAlmost all combinations showed additivism against C. albicans, except ThyEO/PSZ (31.25/0.0039 µg/ml), which showed partial synergism. Furthermore, all mixtures were fungicidal against C. albicans strains. ThyEO/PSZ, its components alone, and thymol were shown to disrupt the fungal cytoplasmic membrane, increasing its permeability. ThyEO/PSZ, at sub-inhibitory concentrations, significantly decreased the ability of C. albicans to adhere to buccal epithelial cells. ThyEO/PSZ, ThyEO, and PSZ were able to reduce the pseudomycelium production of C. albicans while thymol completely inhibited its formation. ThyEO/PSZ, each combination component on its own, and thymol inhibited biofilm formation and preformed biofilm of C. albicans. Notably, ThyEO/PSZ showed synergistic and fungicidal activity against a resistant strain of C. albicans, reducing the PSZ dose by 4-fold. ConclusionThese findings make ThyEO and ThyEO/PSZ mixture valuable candidates for the development of alternative antifungals with a lower incidence of adverse effects.

Synergistic activity of Coptis Chinensis and clotrimazole against Candida Albicans

IntroductionCandida albicans is an important and widespread opportunistic pathogen. In recent years, studies have found that traditional Chinese medicine (TCM) can inhibit the growth of various types of fungi without inducing drug resistance. This study aims to investigate the anti-C. albicans abilities of four types of TCM extracts, namely Oroxylum indicum, Coptis chinensis, Verbena officinalis and Ramulus cinnamomi. Materials and methodsOxford cup assays were employed to determine the sensitivity of C. albicans to the TCM extracts and establish the initial effective concentrations for further experiments. Broth microdilution methods and checkerboard microdilution assays were used to determine the C. albicans inhibition activities of the individual TCM extract and their combinations with clotrimazole and eugenol. ResultsAmong the four TCM extracts, C. chinensis had the highest potency against C. albicans, indicated by the minimum inhibitory concentration (MIC50) and minimum fungicidal concentration (MFC) values of 30 and 60 mg/mL, respectively. The C. chinensis extract also potentiated the anti-C. albicans activity of clotrimazole, indicated by a fractional Inhibitory concentration index (FICI) of 0.25. Both O. indicum and V. officinalis showed additive effects on C. chinensis in the TCM combinations. The combination of clotrimazole (a Western medicine compound) and eugenol (a TCM ingredient) had a weak additive effect with a FICI value of 1.0. ConclusionThis study supports further investigations of C. chinensis for the development of adjuvant therapeutic agents to current anti-fungal treatment against C. albicans infection.

Small Molecule IITR00693 (2-Aminoperimidine) Synergizes Polymyxin B Activity against Staphylococcus aureus and Pseudomonas aeruginosa.

The rise of antibiotic resistance among skin-infecting pathogens poses an urgent threat to public health and has fueled the search for new therapies. Enhancing the potency of currently used antibiotics is an alternative for the treatment of infections caused by drug-resistant pathogens. In this study, we aimed to identify a small molecule that can potentiate currently used antibiotics. IITR00693 (2-aminoperimidine), a novel antibacterial small molecule, potentiates the antibacterial activity of polymyxin B against Staphylococcus aureus and Pseudomonas aeruginosa. Herein, we investigated in detail the mode of action of this interaction and the molecule's capability to combat soft-tissue infections caused by S. aureus and P. aeruginosa. A microdilution checkerboard assay was performed to determine the synergistic interaction between polymyxin B and IITR00693 in clinical isolates of S. aureus and P. aeruginosa. Time-kill kinetics, post-antibiotic effect, and resistance generation studies were performed to assess the pharmacodynamics of the combination. Assays based on different fluorescent probes were performed to decipher the mechanism of action of this combination. The in vivo efficacy of the IITR00693-polymyxin B combination was determined in a murine acute wound infection model. IITR00693 exhibited broad-spectrum antibacterial activity. IITR00693 potentiated polymyxin B and colistin against polymyxin-resistant S. aureus. IITR00693 prevented the generation of resistant mutants against multiple antibiotics. The IITR00693-polymyxin B combination decreased the S. aureus count by >3log10 CFU in a murine acute wound infection model. IITR00693 is a potential and promising candidate for the treatment of soft-tissue infections along with polymyxins.

Siderophore Production and its Role as Therapeutic Agent

Siderophores are iron chelators, which are produced by bacteria under iron-deficient conditions required for their growth. Therefore, siderophores can be used as a carrier to direct drugs into the bacteria and kill them. The present study was designed to screen siderophore production using different bacteria using an iron-deficient medium and its synergistic capability to kill drug-resistant bacteria. Siderophore under iron-deprived condition was evaluated by chrome azurol S (CAS) assay. Whereas, broth micro-dilution method and checkerboard assay were used to determine the antimicrobial properties of selected drugs or epigallocatechin gallate (EGCG) individually or in combination with synthetic siderophore. Results demonstrated that the entire tested microorganisms produced siderophore under the iron-deprived condition as evidenced by orange halo zones in CAS agar plates. Gram-negative bacteria produced more siderophores as reflected by orange color with bacterial zone inhibition of 17-22mm as compared to Gram-positive bacteria (13-15mm). As compared to antibiotics and EGCG, acetohydroxamic acid (aHa; synthetic siderophore) showed no antibacterial properties (1500 - 6500 µg/ml). The synergism of aHa with tetracycline, ceftriaxone, and EGCG (FIC index &lt;0.5) against S. typhi, methicillin-resistant and sensitive Staphylococcus aureus, and E. coli were evident. In conclusion, siderophore may be considered a potential candidate to design different combination therapy against emerging antimicrobial-resistant pathogens.

421. In vitro and in vivo synergistic antimicrobial activities of the combinations of meropenem, colistin, tigecycline, and ceftolozane/tazobactam against carbapenem-resistant Klebsiella pneumoniae

Abstract Background The aim of this study was to evaluate the in vitro and in vivo activities of various antimicrobial combinations against carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods The in vitro activity of six two-drug combinations against CRKP isolates collected from the rectal swab samples of patients with intestinal CRKP colonization was evaluated using the checkerboard method and time-kill assay to identify potential synergistic and bactericidal two-drug combinations against CRKP isolates. The in vivo efficacy of the combinations of colistin, meropenem and ceftolozane/tazobactam (C/T) as therapeutic options, was assessed in a mouse model of intraperitoneal CRKP sepsis. Antimicrobial susceptibility and genotypes of carbapenem-resistance were also analyzed. Results A total of 5 clinical isolates of CRKP were collected from nonduplicate patients with CRKP infection. Of 5 CRKP isolates, 4 carried the blaKPC-2-type carbapenem-hydrolyzing enzymes except 1 blaNDM-1-type. Antimicrobial susceptibility rates were tigecycline 80%, colistin 40%, and C/T 0%, respectively. The checkerboard assay showed that in vitro synergistic activity against CRKP isolates was 90% for the meropenem-tigecycline combinations and 20% for colistin-C/T combinations. None of the various combinations of the six antibiotics showed a synergistic effect in the time-kill assay. However, the meropenem-colistin (100%) and colistin-C/T (100%) combinations showed bactericidal effects in the time-kill assay (using an antibiotic concentration of 1× MIC) (Table). The survival rates of the mice was 80% (4/5) at 48 h in the treated group using low dose combinations of colistin (10 mg/kg) and C/T (25 mg/kg), unlike both antibiotics, which were 60% in a monotherapy with the same dose (Figure). Comparison of checkerboard assay and time-kill assay Comparison of checkerboard assay and time-kill assay associated with the bactericidal activity of two-drug combinations (1 × MIC) against each carbapenem-resistant Klebsiella pneumoniae isolate In vivo survival tests for antimicrobial combinations Comparison of the survival rates of the mice in the treated group using antimicrobial combinations Conclusion In conclusion, the present in vivo study demonstrated that the low-dose combination of colistin and C/T may be a promising alternative to nephrotoxic high-dose colistin alone for treating CRKP infections. However, it was difficult to predict this possibility in in vitro tests, and even there is great discordance of antimicrobial synergistic activities between the checkerboard microdilution and time-kill assays. Disclosures All Authors: No reported disclosures.

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.

Statins and oral biofilm: Simvastatin as a promising drug to control periodontal dysbiosis.

This study evaluated antimicrobial activity of atorvastatin, pravastatin, rosuvastatin, and simvastatin against oral bacteria, and the interaction of simvastatin with standard antimicrobials (amoxicillin and metronidazole). Minimal inhibitory concentration assays were performed with Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, Actinomyces odontolyticus, Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, Streptococcus sanguinis, and Streptococcus gordonii; checkerboard microdilution assays between simvastatin and standard antimicrobials; monospecies and multispecies biofilms. Simvastatin showed the best antimicrobial activity against most species (MIC range from 3.12 to 25 μg/ml), highlighting the sensitivity of P.gingivalis. In the checkerboard assay, synergistic interaction was found between simvastatin and amoxicillin against S.oralis and S.sanguinis. P.gingivalis biofilm was inhibited by simvastatin at 10 and 50× Minimal inhibitory concentration, with similar effects to metronidazole. For multispecies biofilm, SMV reduced the biofilm metabolic activity (79%) and total counts (87%), comparable to amoxicillin. Simvastatin also reduced bacterial counts of Veilonnella parvula, P.gingivalis, Streptococcus mutans, Actinomyces naeslundii, P.intermedia, and Capnocytophaga ochracea in the multispecies biofilm. Simvastatin showed antimicrobial and antibiofilm activity against oral bacteria and may contribute to the control of dysbiosis, and may be considered in clinical studies as an adjuvant in the treatment of periodontitis.

The Anti-Multidrug-Resistant Acinetobacter baumannii Study on 1,3-diamino-7H-pyrrolo[3,2-f]quinazoline Compounds

As a major public health problem, the prevalence of Acinetobacter baumannii (A. baumannii) infections in hospitals due to the pathogen's multiple-antibiotic resistance has attracted extensive attention. We previously reported a series of 1,3-diamino-7H-pyrrolo[3,2-f]quinazoline (PQZ) compounds, which were designed by targeting Escherichia coli dihydrofolate reductase (ecDHFR), and exhibited potent antibacterial activities. In the current study, based on our molecular-modeling study, it was proposed that PQZ compounds may function as potent A. baumannii DHFR (abDHFR)-inhibitors as well, which inspired us to consider their anti-A. baumannii abilities. We further found that three PQZ compounds, OYYF-171, -172, and -175, showed significant antibacterial activities against A. baumannii, including multidrug-resistant (MDR) strains, which are significantly stronger than the typical DHFR-inhibitor, trimethoprim (TMP), and superior to, or comparable to, the other tested antibacterial agents belonging to β-lactam, aminoglycoside, and quinolone. The significant synergistic effect between the representative compound OYYF-171 and the dihydropteroate synthase (DHPS)-inhibitor sulfamethoxazole (SMZ) was observed in both the microdilution-checkerboard assay and time-killing assay, which indicated that using SMZ in combination with PQZ compounds could help to reduce the required dosage and forestall resistance. Our study shows that PQZ is a promising scaffold for the further development of folate-metabolism inhibitors against MDR A. baumannii.

In vitro and in vivo activity of meropenem+avibactam against MBL-producing carbapenem-resistant Klebsiella pneumoniae

Background Antibiotic resistance has become a public health problem to be solved worldwide and metallo-β-lactamase (MBL)-producing bacteria make this problem even more challenging. Methods : The interactions of meropenem (MEM) in combination with avibactam (AVI) in growth inhibition on MBL-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) strains were tested. In vitro interactions of MEM+AVI were tested using the microdilution chequerboard assay and time-kill curves. In vivo interactions of MEM+AVI were tested using the Galleria mellonella model. Results All strains were multi-drug resistant strains and six of them were proved to produce MBLs. We show that the combination of MEM+AVI generates profound synergistic effects on growth inhibition of all strains, which was better than that of MEM+vaborbactam or imipenem+relebactam. Time-kill curves further confirmed the potent synergistic antibacterial effects of MEM+AVI against MBL-producing CRKP strains. Galleria mellonella studies were consistent with in vitro analysis. Combining MEM with AVI improved survival rates and mean survival days were obviously prolonged compared to the drug alone and the untreated controls. Conclusions To our knowledge, this study is the first report of MEM+AVI collaborating against MBL-producing CRKP strains. Our findings showed that the combination of MEM+AVI has the potential for antibiotic drug development to combat MBL-producing pathogens.

Interaction of Amiodarone with Azoles Against Aspergillus Planktonic Cells and Biofilms in vitro.

Aspergillus spp. is the most common clinical pathogen of invasive fungal infection with high mortality. Existing treatments for Aspergillus spp. infection are still inefficient and accompanied by drug resistance, so it is still urgent to find new treatment approaches. The antiarrhythmic drug amiodarone (AMD) has demonstrated antifungal activity against a range of fungi. This study evaluated the efficacy of AMD in combination with triazoles for Aspergillus spp. infection. We tested the combined effect of AMD and three triazole drugs, namely, itraconazole (ITR), voriconazole (VRC), and posaconazole (POS), on the planktonic cells and biofilms of 20 strains of Aspergillus spp. via a checkerboard microdilution assay derived from 96-well plate-based method. Our results reveal that the combination of AMD with ITR or POS against Aspergillus biofilms has synergistic fungicidal effects. By contrast, the combination of AMD with VRC exhibits no antagonistic and synergistic effects. In this way, the use of AMD in combination with ITR or POS could be an effective adjunctive treatment for Aspergillus spp. infection.

Effect of Myrtenol and Its Synergistic Interactions with Antimicrobial Drugs in the Inhibition of Single and Mixed Biofilms of Candida auris and Klebsiella pneumoniae.

The increased incidence of mixed infections requires that the scientific community develop novel antimicrobial molecules. Essential oils and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities and are attracting more and more attention. Therefore, the aim of this study was to evaluate myrtenol (MYR), one of the constituents commonly found in some essential oils, for its potential to inhibit biofilms alone and in combination with antimicrobial drugs against Candida auris/Klebsiella pneumoniae single and mixed biofilms. The antimicrobial activity of MYR was evaluated by determining bactericidal/fungicidal concentrations (MIC), and biofilm formation at sub-MICs was analyzed in a 96-well microtiter plate by crystal violet, XTT reduction assay, and CFU counts. The synergistic interaction between MYR and antimicrobial drugs was evaluated by the checkerboard method. The study found that MYR exhibited antimicrobial activity at high concentrations while showing efficient antibiofilm activity against single and dual biofilms. To understand the underlying mechanism by which MYR promotes single/mixed-species biofilm inhibition, we observed a significant downregulation in the expression of mrkA, FKS1, ERG11, and ALS5 genes, which are associated with bacterial motility, adhesion, and biofilm formation as well as increased ROS production, which can play an important role in the inhibition of biofilm formation. In addition, the checkerboard microdilution assay showed that MYR was strongly synergistic with both caspofungin (CAS) and meropenem (MEM) in inhibiting the growth of Candida auris/Klebsiella pneumoniae-mixed biofilms. Furthermore, the tested concentrations showed an absence of toxicity for both mammalian cells in the in vitro and in vivo Galleria mellonella models. Thus, MYR could be considered as a potential agent for the management of polymicrobial biofilms.

Evaluation of the synergistic antifungal activity of micafungin and voriconazole plus sertraline against Candida auris.

Candida auris is an emerging global public health threat. It is an opportunistic yeast that usually affects critically ill patients in healthcare settings and is characterized by reduced susceptibility to multiple antifungal classes. Combination therapy with antifungals and repurposed drugs is a feasible alternative to overcome this problem. The aim of this study was to examine the in vitro interactions and potential synergy of micafungin (MFG) and voriconazole (VRC) plus the antidepressant sertraline (SRT) against clinical isolates of C. auris. Conventional antifungal testing was first performed with the three drugs according to the CLSI methodology. Drug interactions were determined by the checkerboard microdilution assay using the fractional inhibitory concentration (FIC) index. Synergistic interactions were noted with the combination of MFG and SRT plus VRC with FIC values of 0.37 to 0.49 for some strains. Indifferent interactions were observed when MFG was combined with SRT with just one exception (FIC 0.53). No antagonism was observed for any combination. The combination of VRC with MCF or SRT may be relevant for treating C. auris infections.

Promising Antimicrobial Activity and Synergy of Bacteriocins Against Mycobacterium tuberculosis.

In this study, we assessed the potential of bacteriocins and their in vitro synergistic effects in combination with anti-tuberculosis drugs against Mycobacterium tuberculosis. We evaluated the in vitro activity of chemically synthesized bacteriocins in combination with rifampicin (RIF), ofloxacin, and moxifloxacin against the reference M. tuberculosis H37Rv and a clinical-resistant strain. We first screened the bacteriocin PARAGEN collection and found active bacteriocins. We then determined their minimal inhibitory concentration (MIC), minimal bactericidal concentration, and their fractional inhibitory index by the checkerboard microdilution assay. Remarkably, we identified four bacteriocins with interesting antimycobacterial activity alone and in combinations with RIF, ofloxacin, and moxifloxacin, with significant reduction of the MIC that showed impressive synergistic effects against the susceptible and resistant clinical strains. In conclusion, our preliminary results show promising bacteriocins candidate used in a synergistic combination with anti-tuberculosis drugs and emphasize the need for combined therapy as a new strategy to enhance the activity of existing drugs, which may confer very promising therapeutic benefits against M. tuberculosis.