Checkerboard Microdilution Method Research Articles

Triple combination of meropenem, colistin and tigecycline was bactericidal in a dynamic model despite mere additive interactions in chequerboard assays against carbapenemase-producing Klebsiella pneumoniae isolates.

Combination schemes are commonly used for the treatment of infections due to carbapenemase-producing Klebsiella pneumoniae (CP-Kp). We therefore investigated the in vitro effectiveness of double and triple combinations of meropenem, colistin and tigecycline against CP-Kp isolates with different resistance mechanisms in a static broth microdilution model and a pharmacokinetic-pharmacodynamic model. One WT isolate and seven CP-Kp isolates with different carbapenem resistance mechanisms and increasing MICs of meropenem (4-512 mg/L), colistin (0.5-32 mg/L) and tigecycline (0.25-4 mg/L) were tested with a 3D chequerboard microdilution method. Combinations were then assessed in an in vitro pharmacokinetic-pharmacodynamic model simulating 50 and 100 mg of tigecycline q12h as a 1 h infusion, 4.5 million units of colistin q12h as a 1 h infusion and 1 g of meropenem q8h as 1 and 0.5 h infusions for 2 days. In the chequerboard assay, interactions within the triple combination were mainly additive with a median (range) fractional inhibitory index of 0.66 (0.22-1.26). In the dynamic model, meropenem alone was bactericidal against isolates with MICs up to 4 mg/L, whereas bactericidal activity was found with the double combination meropenem + colistin and the triple combination meropenem + colistin + tigecycline against CP-Kp isolates with meropenem MICs of 16 and 256 mg/L, respectively. A high dose (100 mg) of tigecycline and a prolonged infusion (1 h) of meropenem increased the efficacy of the triple combination. Despite the merely additive interactions in the chequerboard assay, the triple combination of meropenem, tigecycline and colistin was bactericidal in the dynamic model against highly resistant CP-Kp isolates. This effect was more pronounced if prolonged infusion of meropenem and high tigecycline dosing were used.

In vitro assessment of antifungal, antibacterial, and antiprotozoal drugs against clinical isolates of Conidiobolus lamprauges.

We have determined the in vitro activity of antifungal, antibacterial, and antiprotozoal drugs alone and in combination against seven Conidiobolus lamprauges clinical isolates. The assays were based on the M38-A2 protocol and the checkerboard microdilution method. The lowest inhibitory concentrations were observed for amphotericin B, miconazole (MCZ), terbinafine, and miltefosine (MTF) (MIC range 0.25-1; 2-8; 0.25-2; 2-16μg/ml, respectively). The main synergism observed was through the combination of azithromycin (AZI)+MTF and dapsone (DAP)+MTF (100%), AZI+DAP (85.7%), AZI+MCZ (57.1%) as well as MCZ plus CTX and DAP (42.9%). The in vitro activities suggest that the combination of MTF and AZI or DAP are promising candidate therapies for conidiobolomycosis.

Sulbactam enhances the in vitro activity of sitafloxacin against extensively-drug resistant Acinetobacter baumannii.

The present study aimed to determine the in vitro activities of sulbactam and sitafloxacin against extensively-drug resistant Acinetobacter baumannii (XDR-A. baumannii). A total of 50 strains of XDR-A. baumannii were isolated from clinical specimens. Broth microdilution assay was applied to determine the minimum inhibitory concentration (MIC) for sulbactam and sitafloxacin. Microdilution checkerboard method was used to determine the in vitro activity of this antimicrobial combination. Accordingly, the fractional inhibitory concentration (FIC) and FIC index (FICI) were calculated. Time-kill study was also carried out for four strains with different susceptibilities to determine the bactericidal activities of individual or combined use of sitafloxacin and sulbactam. Isolates with MICs of sitafloxacin ≤2 mg/l were considered to be susceptible to sitafloxacin. The susceptibility rate for sitafloxacin was 92% originally. When combined with sulbactam, this rate increased to 96%. Microdilution checkerboard results indicated that, when tested in combination, sulbactam/sitafloxacin exhibited marked synergistic and partial synergistic effects on 16 and 50% of the 50 strains, respectively. Time-kill assay suggested that sulbactam enhanced the bactericidal activity of sitafloxacin and the combination induced a synergistic effect. For strains that were not susceptible to sitafloxacin, the bactericidal activities of the combination of sitafloxacin and sulbactam at a sub-MIC concentration were impaired. However, this impairment could be overcome with the increase of the concentration to 1X MIC. The present study demonstrated that sulbactam enhanced the in vitro antimicrobial activity of sitafloxacin against XDR-A. baumannii.

Elucidation of the synergistic action of Mentha Piperita essential oil with common antimicrobials.

Mentha piperita L. essential oil (EO) is employed for external use as antipruritic, astringent, rubefacient and antiseptic. Several studies demonstrated its significant antiviral, antifungal and antibacterial properties. The aim of this work is the study of the synergistic effects of M. piperita EO with antibacterials and antifungals that are widely available and currently prescribed in therapies against infections. The observed strong synergy may constitute a potential new approach to counter the increasing phenomenon of multidrug resistant bacteria and fungi. In vitro efficacy of the association M. piperita EO/drugs was evaluated against a large panel of Gram-positive and Gram-negative bacteria and yeast strains. The antimicrobial effects were studied by checkerboard microdilution method. The synergistic effect of M. piperita EO with gentamicin resulted in a strong growth inhibition for all the bacterial species under study. The synergistic effect observed for M. piperita EO and antifungals was less pronounced.

In vitro combination of antifungal agents against Malassezia pachydermatis.

The yeast Malassezia pachydermatis is a common commensal and occasional opportunistic pathogen of theskin microbiota of animals and humans. In this study, the susceptibility of M. pachydermatis isolates to fluconazole (FLC), itraconazole (ITZ), ketoconazole (KTZ), clotrimazole (CLZ), and miconazole (MCZ) alone and in combination with terbinafine (TRB), nystatin (NYS), and caspofungin (CSP) was evaluated in vitro based on the M27-A3 technique and the checkerboard microdilution method using Sabouraud dextrose broth with 1% tween 80 (SDB). Based on the mean FICI values, the main synergies observed were combinations of ITZ+CSP and CLZ+CSP (55.17%). The most significant combinations deserve in vivo evaluations because might provide effective alternative treatments against M. pachydermatis due to their synergistic interactions.

Multi-azole-resistant strain of Malassezia pachydermatis isolated from a canine Malassezia dermatitis

In the case presented here, we describe the isolation of an azole-resistant strain of M. pachydermatis from a canine Malassezia dermatitis. The isolate (NUBS18001) from this case exhibited a minimum inhibitory concentration (MIC) of 320μg/ml to itraconazole (ITZ) by broth microdilution (BM) assay, >32μg/ml to ITZ by E-test, and >32μg/ml to KTZ by E-test. Synergistic effects between FK506 and ITZ in the azole-resistant strain was evaluated using the microdilution checker-board method. The ITZ-resistant strain exhibited MICs of 320μg/mL of ITZ alone and 5μg/ml of FK506 alone; the addition of FK506 attenuated the ITZ MIC to 2.5μg/ml, yielding an ITZ FICI value of 0.507. This result suggested that the combination of ITZ and FK506 exerted an additive effect against the ITZ-resistant strain. To understand the other mechanism inferred to be present in our multi-azole-resistant strain, we sequenced the ERG11 gene from this isolate, and detected missense mutations (A412G and C905T) in the sequence of the ERG11 open reading frame (ORF). To the best of our knowledge, this work is the first report that a multi-azole-resistant M. pacydermatis strain contains mutations in ERG11.

Potential effect of 2-isopropyl-5-methylphenol (thymol) alone and in combination with fluconazole against clinical isolates of Candida albicans, C. glabrata and C. krusei

Limitations of antifungals used in the treatment of candidiasis, as the development of resistant strains, are known by the scientific community. In this context, the aim of this study was to investigate the activity of 2-isopropyl-5-methylphenol (thymol) in combination with fluconazole (FLZ) against clinical Candida strains. The antifungal activity of thymol along with FLZ was evaluated by the Clinical Laboratory Standards Institute (CLSI) M27-A2 broth microdilution method. In addition, synergism was observed for clinical strains of Candida spp. with combination of thymol-FLZ evaluated by the chequerboard microdilution method. The mean of minimum inhibitory concentration (MIC) values of thymol and FLZ were 49.37 and 0.475μg/ml for C. albicans, 51.25 and 18.80μg/ml for C. glabrata and 70 and 179.20μg/ml for C. krusei strains, respectively. Thymol in combination with FLZ exhibited the synergistic effects against all species of Candida tested. FICI values for thymol plus FLZ ranged from 0.366 to 0.607 for C. albicans strains, 0.367 to 0.482 for C. glabrata strains, and 0.375 to 0.563 for C. krusei strains. No antagonistic activity was seen in the strains tested. Thymol was found to have a fungicidal effect on Candida species and a synergistic effect when combined with FLZ.

Bioactive compounds from the African medicinal plant Cleistochlamys kirkii as resistance modifiers in bacteria.

Cleistochlamys kirkii (Benth) Oliv. (Annonaceae) is a medicinal plant traditionally used in Mozambique to treat infectious diseases. The aim of this study was to find resistance modifiers in C.kirkii for Gram-positive and Gram-negative model bacterial strains. One of the most important resistance mechanisms in bacteria is the efflux pump-related multidrug resistance. Therefore, polycarpol (1), three C-benzylated flavanones (2-4), and acetylmelodorinol (5) were evaluated for their multidrug resistance-reverting activity on methicillin-susceptible and methicillin-resistant Staphylococcus aureus and Escherichia coli AG100 and AG100 A strains overexpressing and lacking the AcrAB-TolC efflux pump system. The combined effects of antibiotics and compounds (2 and 4) were also assessed by using the checkerboard microdilution method in both S.aureus strains. The relative gene expression of the efflux pump genes was determined by real-time reverse transcriptase quantitative polymerase chain reaction. The inhibition of quorum sensing was also investigated. The combined effect of the antibiotics and compound 2 or 4 on the methicillin-sensitive S.aureus resulted in synergism. The most active compounds 2 and 4 increased the expression of the efflux pump genes. These results suggested that C.kirkii constituents could be effective adjuvants in the antibiotic treatment of infections.

Antibacterial Effect of Red Betel (Piper crocatum Ruiz and Pav.) Extract in Combination with Vancomycin against Staphylococcus aureus

Objectives: The current study was performed to show the antibacterial effect of red betel ( Piper crocatum Ruiz and Pav.) in combination with vancomycin against Staphylococcus aureus. The reduction of S. aureus susceptibility to vancomycin was reported in 1997. The combination of isolated antibacterial compounds from plant with antibacterial drug were expected to increase antibacterial activity resulted in the reduction of resistance. Methods: The antibacterial effect of red betel extract and vancomycin separately and the combination of both materials were evaluated based on the zone of inhibition diameter using agar diffusion method. The interaction effect of extract and antibiotic combination was performed using microdilution checkerboard method. Results: There was an antibacterial effect of red betel extract started at the concentration of 150 mg/ml. The combination of red betel extract with vancomycin showed significantly greater inhibition growth of S. aureus compared to those of red betel or vancomycin alone. The addition of red betel extract to vancomycin reduced Minimum Inhibitory Concentration (MIC) vancomycin to be a-4 fold reduction against S. aureus . Moreover, the Fractional Inhibitory Concentration Index (FICI) of red betel extract and vancomycin combination was 0.3125. Conclusion: There was a synergistic effect of the red betel extract in combination with vancomycin against S. aureus . It is the potential result for the future research on the infectious treatment of S. aureus resistant to vancomycin. Key words: Piper crocatum , Vancomycin, Synergistic antibacterial, Staphylococcus aureus .

Combination of Epicatechin 3-Gallate from Euphorbia hirta and Cefepime Promotes Potential Synergistic Eradication Action against Resistant Clinical Isolate of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is naturally resistant to many classes of antipseudomonal antibiotics due to the species ability to easily acquire resistance. Plant-based antibacterial agent in combination with the existing antibiotic proposes an alternative treatment regimen for the eradication of resistant bacterial infections. The antibacterial effects of the isolated epicatechin 3-gallate compound from Euphorbia hirta in combination with cefepime were investigated in vitro against resistant P. aeruginosa. The fractional inhibitory concentration index of the combination was determined using checkerboard broth microdilution method. Epicatechin 3-gallate combined with cefepime had produced synergistic effect against P. aeruginosa (with average FIC index of 0.24). The MIC of epicatechin 3-gallate was effectively reduced to MIC/4, MIC/8, MIC/16, and MIC/32 in the presence of cefepime. Time-kill study of epicatechin 3-gallate combined with cefepime exhibited remarkable bactericidal activity where the eradication of P. aeruginosa occurred within 4 h of treatment. Scanning electron micrographs revealed apparent cell membrane damage and leakage of cytoplasmic contents from P. aeruginosa cells which eventually led to the cell lysis after the combination treatment of epicatechin 3-gallate and cefepime. The potential of epicatechin 3-gallate to act synergistically with cefepime against clinically resistant P. aeruginosa strain possibly will maximize the successful outcomes when choosing empirical antibiotic treatment in hospitals or health care institutions.

In Vitro Combination of Isavuconazole with Echinocandins against Azole-Susceptible and -Resistant Aspergillus spp.

In vitro combinations of isavuconazole with echinocandins were evaluated against 30 Aspergillus strains with a two-dimensional checkerboard microdilution method and an agar-based diffusion method. With the checkerboard method, the three combinations showed indifferent interactions for all strains. With the agar-based method, indifferent interactions were found for all strains for isavuconazole-micafungin and isavuconazole-anidulafungin. For the isavuconazole-caspofungin combination, indifference was found in 24/30 strains, synergism in 4/30 strains, and antagonism in 2/30 strains.

Combination Susceptibility Testing of Common Antimicrobials in Vitro and the Effects of Sub-MIC of Antimicrobials on Staphylococcus aureus Biofilm Formation.

The current study was conducted to evaluate the antibacterial combination efficacies, and whether the sub-inhibitory concentrations (sub-MIC) of antibiotics can influent on the biofilm formation of S. aureus. The minimum inhibitory concentration (MIC) of common antibacterial drugs was determined in vitro against clinical isolates of Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Pasteurella multocida (P. multocida) alone and in combination with each other by using the broth microdilution method and the checkerboard micro-dilution method analyzed with the fractional inhibitory concentration index (FICI), respectively. Regarding these results, antibacterial drug combinations were categorized as synergistic, interacting, antagonistic and indifferent, and most of the results were consistent with the previous reports. Additionally, the effects of sub-MIC of seven antimicrobials (kanamycin, acetylisovaleryltylosin tartrate, enrofloxacin, lincomycin, colistin sulfate, berberine, and clarithromycin) on S. aureus biofilm formation were determined via crystal violet staining, scanning electron microscopy (SEM) and real-time PCR. Our results demonstrate that all antibiotics, except acetylisovaleryltylosin tartrate, effectively reduced the S. aureus biofilm formation. In addition, real-time reverse transcriptase PCR was used to analyze the relative expression levels of S. aureus biofilm-related genes such as sarA, fnbA, rbf, lrgA, cidA, and eno after the treatment at sub-MIC with all of the six antimicrobials. All antibiotics significantly inhibited the expression of these biofilm-related genes except for acetylisovaleryltylosin tartrate, which efficiently up-regulated these transcripts. These results provide the theoretical parameters for the selection of effective antimicrobial combinations in clinical therapy and demonstrate how to correctly use antibiotics at sub-MIC as preventive drugs.

Anti-Candida Activity of Biosurfactant Produced by Rhodotorula paludigena

Background: Biosurfactants are natural extracellular products that are produced by several microorganisms. These compounds are highly developed during recent years for industrial, medical, and environmental applications. Although, the majority of microbial biosurfactants were originated from bacterial species, some studies have shown that Rhodotorula muciliginosa and Candida rugosa are fungal sources for biosurfactants production. The aim of the present study was to evaluate anti- Candida activity of biosurfactant produced by R. paludigena. In addition, the combination of biosurfactant and several antifungal drugs was also investigated against Candida isolates. Materials and Methods: In the present study, R. paludigena was subjected for the production of biosurfactant and yielded product was confirmed using several specific tests. Biosurfactant was produced using Sabouraud dextrose broth at 29°C for 5-7 days. Cell-free supernatants were collected and extracted using chloroform-methanol solvent. Checkerboard microdilution method was used to assess antifungal susceptibility and combination interaction. Results: Out of 80 suspected isolates to Rhodotorula, only one strain (R. paludigena) has been found to be predominant for biosurfactant production. Our results show that produced biosurfactant has anti- Candida activity at the range of 1-8 mg/mL. Furthermore, it showed a synergistic effect with antifungal drugs caspofungin. Conclusion: It is concluded that R. paludigena has great potential for biosurfactant production with valuable antifungal effect against C. albicans. On the other hand, the synergistic effect was found when a combination of biosurfactant and caspofungin were applied against C. albicans. Keywords: Antifungal drugs, biosurfactant, Candida albicans, combination therapy, synergistic effect.

Synergy of a combination of nisin and citric acid against Staphylococcus aureus and Listeria monocytogenes

ABSTRACTFood-borne diseases caused by pathogens, such as Staphylococcus aureus and Listeria monocytogenes, have long attracted attention globally from researchers, food industries, and food safety authorities. Nisin (NS) is the only bacteriocin used worldwide as a generally recognised as safe (GRAS) food preservative, while citric acid (CA) has an unrestricted use in foods since it has GRAS status. In this study, synergistic interactions of NS combined with CA against S. aureus and L. monocytogenes were studied by the chequerboard microdilution method, with fractional inhibitory concentration index values ranging from 0.25 to 0.375 and 0.19 to 0.375, respectively. The positive interactions were verified by time-kill studies in pasteurised milk and disk diffusion assays. The mechanism of the synergistic antibacterial of NS and CA is proposed following SEM analysis and the determination of release of cell constituents. These results suggest that the cell walls and membrane are the probable main targets of this antimicrobial combination. These findings indicated that the combination of NS and CA not only could be used as a new promising naturally sourced food preservative, but may also reduce the problem of bacterial resistance.

Gepotidacin (GSK2140944) In Vitro Activity against Gram-Positive and Gram-Negative Bacteria.

Gepotidacin is a first-in-class, novel triazaacenaphthylene antibiotic that inhibits bacterial DNA replication and has in vitro activity against susceptible and drug-resistant pathogens. Reference in vitro methods were used to investigate the MICs and minimum bactericidal concentrations (MBCs) of gepotidacin and comparator agents for Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli Gepotidacin in vitro activity was also evaluated by using time-kill kinetics and broth microdilution checkerboard methods for synergy testing and for postantibiotic and subinhibitory effects. The MIC90 of gepotidacin for 50 S. aureus (including methicillin-resistant S. aureus [MRSA]) and 50 S. pneumoniae (including penicillin-nonsusceptible) isolates was 0.5 μg/ml, and for E. coli (n = 25 isolates), it was 4 μg/ml. Gepotidacin was bactericidal against S. aureus, S. pneumoniae, and E. coli, with MBC/MIC ratios of ≤4 against 98, 98, and 88% of the isolates tested, respectively. Time-kill curves indicated that the bactericidal activity of gepotidacin was observed at 4× or 10× MIC at 24 h for all of the isolates. S. aureus regrowth was observed in the presence of gepotidacin, and the resulting gepotidacin MICs were 2- to 128-fold higher than the baseline gepotidacin MICs. Checkerboard analysis of gepotidacin combined with other antimicrobials demonstrated no occurrences of antagonism with agents from multiple antimicrobial classes. The most common interaction when testing gepotidacin was indifference (fractional inhibitory concentration index of >0.5 to ≤4; 82.7% for Gram-positive isolates and 82.6% for Gram-negative isolates). The postantibiotic effect (PAE) of gepotidacin was short when it was tested against S. aureus (≤0.6 h against MRSA and MSSA), and the PAE-sub-MIC effect (SME) was extended (>8 h; three isolates at 0.5× MIC). The PAE of levofloxacin was modest (0.0 to 2.4 h), and the PAE-SME observed varied from 1.2 to >9 h at 0.5× MIC. These in vitro data indicate that gepotidacin is a bactericidal agent that exhibits a modest PAE and an extended PAE-SME against Gram-positive and -negative bacteria and merits further study for potential use in treating infections caused by these pathogens.

Synergistic antimicrobial effects of nisin and p-Anisaldehyde on Staphylococcus aureus in pasteurized milk

Staphylococcus aureus (S. aureus) infections resistant to a variety of drugs have been increasingly documented in recent years. Drug resistance has appeared largely due to extensive use of antibiotics. Nisin has been widely used in food industry to prevent bacterial growth. However, nisin resistance has been found in several foodborne pathogenic bacteria. p-Anisaldehyde (AS), an extract from Pimpinella anisum seeds, is a very common digestive herb of north India. In this study, synergistic interactions of nisin combined with AS against S. aureus were observed. The synergy was confirmed by checkerboard microdilution method, with the fractional inhibitory concentration index values ranging from 0.25 to 0.375. The positive interactions were verified by the challenge tests in pasteurized milk and agar diffusion assays. In addition, when treatment with nisin or AS alone or in combination, an obvious loss of 260 nm absorbing materials was observed. And flow cytometry assay showed that nisin or AS alone or in combination caused bacterial membrane permeabilization. The LIVE/DEAD BacLight experiment, scanning electron microscope and transmission electron microscopy observation further confirmed that the compounds could kill bacteria via disrupting membranes. All results above verified that the combination of nisin and AS which showed antibacterial activity by a membrane damage mechanism could not only reduce drug resistance but also be used as a new promising naturally sourced food preservative.

In vitro antifungal activity of tacrolimus alone or in combination with itraconazole or terbinafine against Exophiala dermatitidis.

Objective To evaluate in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against Exophiala dermatitidis (E. dermatitidis) . Methods The minimum inhibitory concentrations (MICs) of itraconazole and terbinafine against 12 strains of E. dermatitidis were determined using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution susceptibility method (M38-A2 Document) . A broth microdilution checkerboard method was used to evaluate the in vitro antifungal activity of tacrolimus combined with itraconazole or terbinafine against E. dermatitidis. Results The MIC ranges of terbinafine and itraconazole against E. dermatitidis were 0.060-0.125 mg/L and 0.5-1 mg/L, respectively. The combination of tacrolimus with terbinafine showed synergistic inhibitory effects against 5 strains of E. dermatitidis, while the combination of tacrolimus with itraconazole revealed synergistic effects against 10 strains of E. dermatitidis. No antagonism was observed in either of the two combinations. Conclusion In vitro combination of tacrolimus with itraconazole or terbinafine can enhance the antifungal activity of itraconazole or terbinafine against E. dermatitidis. Key words: Itraconazole; Drug synergism; In vitro; Exophiala dermatitidis; Tacrolimus; Terbinafine

In vitro inhibitory effects of farnesol and interactions between farnesol and antifungals against biofilms of Candida albicans resistant strains

Antifungal resistance is a serious problem in clinical infections. Farnesol, which is a potential antifungal agent against biofilms formed by Candida albicans resistant strains (a fluconazole-resistant isolate derived from SC5314 and two clinical Candida resistant isolates), was investigated in this study. The inhibitory effects of farnesol on biofilms were examined by XTT assay. The morphological changes and biofilm thicknesses were analyzed by scanning electron microscopy and confocal laser scanning microscopy, respectively. Additionally, the checkerboard microdilution method was used to investigate the interactions between farnesol and antifungals (fluconazole, amphotericin B, caspofungin, itraconazole, terbinafine and 5-flurocytosine) against biofilms. The results showed decreased SMICs of farnesol and thinner biofilms in the farnesol-treated groups, indicating that farnesol inhibited the development of biofilms formed by the resistant strain. Furthermore, there were synergistic effects between farnesol and fluconazole/5-flurocytosine, while there were antagonistic effects between farnesol and terbinafine/itraconazole, respectively, on the biofilms formed by the resistant strains.

Synergistic action of amphotericin B and rhamnolipid in combination on Candida parapsilosis and Trichosporon cutaneum

Emerging resistance of microbial cells towards antibiotics or disinfectants leads to an increase of nosocomial diseases, often biofilm related, and gives rise to the need for research for new antimicrobial substances. These studies are often focused on substances that are able to support the efficacy of currently used antimicrobials, reduce their required dosage and reduce the probability of resistance development. This work brings new insights into the in vitro interaction of rhamnolipid biosurfactant and amphotericin B against Candida sp. or Trichosporon sp.. The minimum inhibitory concentrations and fractional inhibitory concentration indexes for both planktonic and biofilm cells were determined by the chequerboard microdilution method. Combination effect of rhamnolipid and amphotericin B was observed for both yeasts. Synergy, defined as a fractional inhibitory concentration (FICi) index of 0.50, was observed in both planktonic and biofilm cells of Trichosporon cutaneum. For C. parapsilosis, synergistic effect (FICi 0.5) was observed for planktonic cells, and additive effect (FICi 0.8) for biofilm cells. The influence of concentrations established as FICi on biofilm formation and susceptibility was studied by light microscopy. The highest inhibition (90% colonized area reduction) of initial adhesion of C. parapsilosis was observed when biofilm FIC was applied.

In Vitro Activity of Berberine Alone and in Combination with Antifungal Drugs Against Planktonic Forms and Biofilms of Trichosporon Asahii.

Trichosporon asahii (T. asahii) is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients, with high mortality rates up to 80% despite treated with antifungal drugs. The biofilms-forming ability of T. asahii on indwelling medical devices may account for the resistance to antifungal drugs. Berberine (BBR) has been demonstrated to have antifungal activity and synergistic effects in combination with antifungal drugs against pathogenic fungi. In the present study, the in vitro activities of BBR alone or combined with fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), caspofungin (CAS) and amphotericin B (AMB) against planktonic forms and biofilms of 21 clinical T. asahii isolates were evaluated using checkerboard microdilution method and XTT reduction assay, respectively. The fractional inhibitory concentration index (FICI) was used to interpret drug interactions. BBR alone did not exhibit significant antifungal activities against both T. asahii planktonic cells (MICs, 32→128μg/ml) and T. asahii biofilms (SMICs, >128μg/ml). However, BBR exhibited synergistic effects against T. asahii planktonic cells in combination with AMB, FLC and CAS (FICI≤0.5) and exhibited synergistic effects against T. asahii biofilms in combination with AMB and CAS (FICI≤0.5). BBR/ITC and BBR/VRC combinations yielded mainly indifferent interactions against T. asahii planktonic cells. BBR/FLC, BBR/ITC and BBR/VRC combinations also yielded indifferent interactions against T. asahii biofilms. Our study highlights the therapeutic potential of BBR to be used as an antifungal synergist in combination with antifungal drugs against T. asahii infections, especially BBR/AMB combination. Further in vivo studies are needed to validate our findings.