Flask and reactor scale production of plant growth regulators by Inonotus hispidus: optimization, immobilization and kinetic parameters.

The aims of the presented study are to compare submerged, static, and solid-state fermentation in the production of gibberellic acid (GA3), indole acetic acid (IAA), and abscisic acid (ABA) by Inonotus hispidus, to optimize with a statistical approach, and to determine the kinetic parameters under flask and reactor conditions. The maximum concentrations of GA3, (2478.85 ± 68.53 mg/L), ABA, (273.26 ± 6.17 mg/L) and IAA (30.67 ± 0.19 mg/L) were obtained in submerged conditions. After optimization, these values reached 2998.85 ± 28.85, 339.47 ± 5.50, and 34.56 ± 0.25 mg/L, respectively. Immobilization of fungal cells on synthetic fiber, polyurethane foam, and alginate beads resulted in an increase in plant growth regulators (PGR) production by 5.53%- 5.79% under optimized conditions. At the reactor scale, a significant increase was observed for GA3 concentration, 5441.54 mg/L, which was 2.14 and 1.45 times higher than non-optimized and optimized conditions in the flask scale, respectively. The maximum values for ABA and IAA were 390.39 and 44.79 mg/L, respectively. Although the specific growth rate (µ) decreases relatively from non-optimized flask conditions to optimized reactor conditions, it was observed that the PGR amounts produced per liter medium (rp) and per gram biomass (Qp) increased significantly. This is the first report on the synthesis of PGR by Inonotus hispidus which could be crucial for sustainable agriculture.

In vitro effect of farnesol on planktonic cells and dual biofilm formed by Candida albicans and Escherichia coli.

Many biofilm studies have focused on axial biofilms, however biofilms in nature and in vivo environment are multi-species. Farnesol is a sesquiterpene alcohol found in many essential oils. This study investigated the in vitro effects of farnesol on planktonic cells and biofilms of Candida albicans and Escherichia coli. The ultrastructural morphology of farnesol treated cells was evaluated by TEM. According to the XTT results, farnesol caused a significant decrease in metabolic activity and scanning electron microscope images confirmed a reduction in the preformed biofilm as a result of farnesol treatment for single species C. albicans and E. coli biofilms. Although farnesol has less effect on dual species biofilm compared to the single species biofilms, its effect on the dual biofilm was found to be stronger than amphotericin B or ampicillin. Further studies are needed to clarify the role of farnesol on fungal-bacterial biofilms.

The investigation of in vitro effects of farnesol at different cancer cell lines.

Farnesol (trans, trans-3,7,11-trimethyl-2,6,10-dodecatriene-1-ol) is an essential oil component that can be found in a variety of plants. In this study, in vitro effects of farnesol on human lung cancer A549 cell line, colon adenocarcinoma (Caco-2) cell line and healthy human lung epithelial BEAS-2B cell lines, WST-1 cytotoxicity test, dual staining of cell survival (DAPI-PI) analysis, micronucleus test, and transmission electron microscopy (TEM). Farnesol acted in a concentration-dependent manner at the dose ranges studied for cancer cell lines, and while at certain doses it reduced proliferation, interestingly at higher concentrations it induced growth more than the control. In the healthy BEAS-2B cell line, it was tested over a wide range of doses and at all studied concentrations, it did not suppress cellular growth, but rather increased. This seems promising in that farnesol harms cancer cell lines but does not cause significant damage to healthy cells. Obtained TEM data after treatment with farnesol at IC50 dose showed both autophagic and apoptotic findings in cancer cell lines compared to control, and normal findings exhibited in BEAS-2B cell line, cell survival, and micronucleus analyzes showed the presence of apoptotic findings and chromosomal damage as a result of farnesol application in cancer cell lines. RESEARCH HIGHLIGHTS: Farnesol has dose-dependent effects on human lung cancer and colon adenocarcinoma cell lines, with no significant damaging effects on healthy human lung epithelial cell lines. TEM, cell survival, and micronucleus findings support the findings of autophagic, apoptotic, and chromosomal damage on cancer cell lines.

Antifungal and antibiofilm efficacy of cinnamaldehyde-loaded poly(DL-lactide-co-glycolide) (PLGA) nanoparticles against Candida albicans.

Biofilm-associated Candida infections threaten public health and show high mortality. The drugs used in treatment are very limited due to reasons such as toxicity, low efficacy, and drug resistance, and new alternatives are needed. The use of natural products of plant origin in the biofilm management draws attention. CA (cinnamaldehyde, cinnamic aldehyde, or 3-phenyl-2-propenal) is an essential oil component that can also inhibit mold growth and mycotoxin production. However, there are some limitations in its use due to its poor solubility and volatility in water. Recently, the combination of natural components and nanoparticle-based drug delivery systems shows positive results. In this study, the effects of PLGA (poly(DL-lactide-co-glycolide)) nanoparticles arrested with CA (CA-PLGA NPs) on C. albicans planktonic and biofilm forms (prebiofilm and postbiofilm) were investigated. According to the results, the amount of active ingredient loaded in CA-PLGA NPs is much lower than the free CA and a strong antifungal effect was obtained even at this rate. Also, the postbiofilm application is more effective than prebiofilm application. PLGA NPs can also be a useful carrier for other essential oils, and their potential in various antifungal, antibiofilm, and biomedical applications should be investigated.

Research Into the Effect of Proton Pump Inhibitors on Lungs and Leukocytes.

BACKGROUND: Proton pump inhibitors (PPI) are the most commonly used medication in the world. They are prescribed as an effective treatment choice for gastrointestinal system diseases linked to hyperacidity, especially. Additionally, non-indication and unnecessary use are very common. Many publications in recent times have reported significant side effects. However, there are insufficient studies about the mechanism for these side effects. METHODS: Twenty-four Wistar albino rats were used in this study. Rats were divided into 3 groups of control, group-administered H2 receptor blockers and a group-administered PPI. Medications were administered for 30 days intraperitoneal. After 30 days, rats were euthanized and lung tissue was obtained. Lung was stained for immunohistochemical catalase, superoxide dismutase, Glutathione peroxidase, myeloperoxidase, and toluidine blue and investigated with a light microscope. Transmission electron microscopy (TEM) was used to investigate lung tissues and neutrophil leukocytes. Additionally, lung tissue had biochemical hydrogen peroxide (H2O2) levels researched. RESULTS: H2O2 amounts, produced by lysosomes with important duties for neutrophil functions in lung tissues, were found to be statistically significantly reduced in the group-administered PPI. Results from investigations of specimens obtained with immunohistochemical staining observed increases in antioxidant amounts in the PPI group. Investigation with TEM identified more inflammation findings in the lung tissue from the group-administered PPI compared to the control group and the group-administered H2 receptors. CONCLUSION: In conclusion, we identified long-term PPI use disrupts neutrophil leukocyte functions in the lung. All clinicians should be much more careful about PPI use.

An Enriched Environment Leads to Increased Synaptic Plasticity-Associated miRNA Levels after Experimental Subarachnoid Hemorrhage.

BACKGROUND AND PURPOSE: In subarachnoid hemorrhage (SAH), impairments in motor and cognitive functions may occur and continue in later periods. MicroRNAs (miRNAs) are small non-coding RNAs that can directly or indirectly affect synaptic reconstruction. mir-132, mir-134, and mir-138 are the leading miRNAs that can be effective on some neurological functions through its effects on synaptic plasticity in the relevant brain areas. In our study, it was aimed to determine the levels of miRNAs in the hippocampus and frontal lobe of rats exposed to different environmental conditions after the experimental SAH. METHODS: SAH was created using the cisterna magna double blood-injection method. Brain tissues were collected at different times after the last blood injection. Rats were grouped according to the different environmental conditions in which they were kept. Expression levels of miRNAs were performed by qPCR and ultrastructural changes in samples were determined by transmission electron microscopy (TEM). RESULTS: After SAH, miR-132, miR-134, and miR-138 expressions in the frontal lobes of rats increased in impoverished environment on the 7th day and in the enriched environment on the 14th day. It was observed that the myelin and microtubule structures in the axons that were disrupted after SAH were more organized and stable in the enriched environment. CONCLUSIONS: After SAH, different environmental conditions may affect the miRNA levels associated with synaptic plasticity and microtubule organization in the frontal lobe, and this might have some effects especially on cognitive and motor functions related to this brain area.

Synthesis of intracellular and extracellular gold nanoparticles with a green machine and its antifungal activity.

Green synthesis method is being increasingly used in the development of safe, stable, and eco-friendly nanostructures with biological resources. In this study, extracellular and intracellular synthesis of gold nanoparticles (AuNPs) was carried out using green algae Chlorella sorokiniana Shihira & R.W. Fresh algae were isolated and identified from Musaözü Pond located in the province of Eskisehir and then extraction process were performed. Optimization studies were studied using pH value, metal salt concentration, and time parameters for extracellular synthesis and using only time parameter for intrasellular synthesis. Since more controlled and optimum conditions can be achieved in the production of AuNPs by extracellular synthesis, these nanoparticles (NPs) were used for characterization and antifungal activity studies. Optical, physical, and chemical properties of synthesized NPs were characterized by UV visible spectrophotometer (UV-Vis), dynamic light scattering (DLS), Zetasizer, X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscope (FE-SEM), inductively coupled plasma mass spectrometer (ICP-MS) and transmission electron microscope (TEM) analysis. The optimum conditions for AuNPs synthesis were determined as 1 mM for HauCl4 concentration, 6 for pH value, and 60th min for time. AuNPs obtained from extracellular synthesis from C. sorokiniana extract are 5-15 nm in size and spherical shape. TEM images of extracellular synthesis show noticeable cell wall and membrane damages, cytoplasma dissolutions, and irregularities. AuNPs obtained by intracellular synthesis are in 20-40 nm size and localized in the cell wall and cytoplasm. These NPs exhibited significant antifungal activity against C. tropicalis, C. glabrata, and C. albicans isolates. AuNPs obtained by algae-mediated green synthesis have a significant potential for medical and industrial use, and this eco-friendly synthesis method can be easily scaled for future studies.

Effects of calcineurin inhibitors, cyclosporine A and tacrolimus (FK506), on the activity of antifungal drugs against Candida spp.

Introduction. The simultaneous use of antifungals with immunosuppressive agents has become a necessity for patients taking immunosuppressive therapy. However, antifungal drugs are problematic because of their limited target.Hypothesis. Scientists have been searching for new antifungals and some compounds with at least additive effects on antifungals. Calcineurin inhibitors used as immunosuppressive agents also attract attention due to their antifungal property.Aim. To evaluate the activity of two calcineurin inhibitors alone and in combination with amphotericin B (AMB), caspofungin (CAS), itraconazole (ITR), voriconazole (VOR) and fluconazole (FLU).Methodology. MICs of AMB, CAS, ITR, VOR, FLU and cyclosporine A (CsA) and tacrolimus (TAC) as calcineurin inhibitors were evaluated by the broth microdilution method against Candida albicans (n=13), C. krusei (n=7) and C. glabrata (n=10). Checkerboard and time-kill methods were performed to investigate the activity of combining calcineurin inhibitors with antifungal drugs.Results. The lowest MIC values were detected with VOR for all Candida isolates tested. Although we did not detect any inhibition for CsA or TAC alone at concentrations tested in this study, the combinations of CAS with CsA showed the highest synergistic activity (36.7%) by the checkerboard method, and CAS with CsA and ITR with TAC combinations exhibited apparent synergistic interaction by the time-kill method. However, the combinations of both CsA and TAC with AMB resulted in antagonistic interactions, especially against C. krusei isolate in time-kill testing.Conclusion. Synergistic interactions in the combinations of TAC or CsA with antifungal drugs, except for AMB, in many concentrations was found to be promising in terms of the treatment of patients with fungal infections.

In vitro antibiofilm efficacy of farnesol against Candida species.

Candida species are opportunistic fungi that can cause mucosal or invasive infections. Especially in biofilm-related infections, resistance is very high to anifungals; therefore more effective treatment strategies are needed. Farnesol(3,7,11-trimethyl-2,6,10-dodecatriene-1-ol) is the quorum sensing (QS) signal molecule and can interact with Candida species both as a QS molecule and as an exogenous agent. The aim of this study was to investigate the effects of farnesol on both the planktonic and biofilm forms of Candida species by colorimetric, microbiological, and electron microscopic methods. Obtained results demonstrated the inhibitory effect of farnesol on the planktonic and biofilm forms of Candida. Farnesol showed a biofilm-enhancing effect at lower concentrations. TEM findings showed the membrane and wall damage, vacuolization, or granulation in cells. SEM images confirmed biofilm reduction in pre-/post-biofilm applications as a result of farnesol treatment. In conclusion, farnesol can be used as an alternative agent to reduce the Candida biofilms, with future studies.

Bone Marrow Mesenchymal Stem Cell Transplantation Enhances Nerve Regeneration in a Rat Model of Hindlimb Replantation.

BACKGROUND: Successful limb replantation must be based not only on the viability of the amputated part but also on satisfactory long-term functional recovery. Once the vascular, skeletal, and soft-tissue problems have been taken care of, nerve recovery becomes the ultimate limiting factor. Unfortunately, nerve regeneration after limb replantation is impaired by several consequences. The authors tested the hypothesis that bone marrow mesenchymal stem cells could improve nerve regeneration outcomes in an experimental model of limb replantation. METHODS: Twenty rats underwent replantation after total hindlimb amputation. Animals were subdivided into two groups: a replanted but nontreated control group and a replanted and bone marrow mesenchymal stem cell-transplanted group. Three months after surgery, nerve regeneration was assessed using functional, electrophysiologic, histomorphologic, and immunohistochemical analyses. RESULTS: Bone marrow mesenchymal stem cell-treated animals showed significantly better sciatic functional index levels and higher compound muscle action potential amplitudes in comparison with the controls. Histomorphometric analysis revealed that the number of regenerating axons was approximately two-fold greater in the treated nerves. In addition, the mean g-ratio of these axons was within the optimal range. Immunohistochemical assessment revealed that expression of S-100 and myelin basic protein in the treated nerves was significantly higher than in controls. Correspondingly, the expression levels of anti-protein gene product 9.5 and vesicular acetylcholine transporter in motor endplates were also significantly higher. Finally, muscles in the bone marrow mesenchymal stem cell-transplanted group showed significantly larger average fiber areas. CONCLUSION: The authors' findings demonstrate that it is possible to improve the degree of nerve regeneration after limb replantation by bone marrow mesenchymal stem cell transplantation.

[Drug carrier nanosystems in malignant pleural mesothelioma]. / Malign plevral mezotelyomada ilaç tasiyici nanosistemler.

Malignant pleural mesothelioma (MPM), the incidence increased with each passing day, is an important lethal disease due to the limited survive with available treatment methods and with the lack of a standard treatment. Response and survive rates of cytotoxic agents which is used in MPM treatment are not good enough. Therefore, treatment studies of MPM seem to quite important and urgent. In cancer therapy, convensional chemotherapeutic agent applications, due to the lack of selectivity, lead to systemic toxicity. Besides the limited solubility of the agent used, the distribution between the cells is weak. It is very difficult to the pass through cellular barriers, particularly, drug resistance may develop to the treatment. All of these reasons lead to failure in the treatment process. Because of the fact that cytotoxic drugs either kill the rapidly growing and dividing cells or make them disfunctional by showing toxic effect on them, to avoid the side effects and to make an inherent effect for cytotoxic drug of active ingredient given for treatment on tumor, different studies have been under investigation. At the present time, nanocarriers as one of these solutions seem to have an important place. Nanocarriers are promising for the development of therapeutic effectiveness and safety. It seems that use of the nanocarrier in the treatment of mesothelioma has a potential, as effective alternative a method, with improve of the drug efficacy and reduce of toxicity in normal tissues.

Effects of ischemic preconditioning protocols on skeletal muscle ischemia-reperfusion injury.

BACKGROUND: Ischemic preconditioning (IPC) is described as brief ischemia-reperfusion (I/R) cycles to induce tolerance to subsequent in response to longer I/R insults. Various IPC protocols can be performed in four combinations as follows: at early or late phases and on local or distant organs. Although many experimental studies have been performed on IPC, no consensus has been established on which IPC protocol is most effective. The aims of the present study were as follows: (1) to compare the variables of preconditioning in different combinations (in early versus late phases; local versus remote organ implementations) and (2) to determine the most therapeutic IPC protocol(s). MATERIALS AND METHODS: A subtotal hind limb amputation model with clamping an intact femoral pedicle was used for I/R injury. IPC was induced using hind limb tourniquet with 3 × 10 min I/R cycles before longer I/R insult. Forty-nine rats were divided into seven groups (n = 7), sham, IsO (ischemia only), I/R, early ischemic preconditioning (e-IPC), late ischemic preconditioning (l-IPC), early remote ischemic preconditioning (e-RIPC), and l-RIPC (late-remote) groups, respectively. In the sham group, pedicle occlusion was not performed. Six hours ischemia was challenged in the IsO group. Three hours ischemia followed by 3 h reperfusion was performed in the I/R group. The e-IPC group was immediately preconditioned, whereas the l-IPC group was preconditioned 24 h before I/R injury on the same hind limb. In the e-RIPC and l-RIPC groups, the same protocols were performed on the contralateral hind limb. At the end of the experiments, skeletal muscle tissue samples were obtained for biochemical analysis (Malondialdehyde [MDA], catalase, myeloperoxidase [MPO], and nitric oxide end products [NOx]), light microscopy, and caspase-3 immunohistochemistry for determination of apoptosis. RESULTS: Tissue biochemical markers were improved in nearly all the IPC groups compared with IsO and I/R groups (P < 0.05). Similarly, the histologic damage scores were decreased in all the IPC groups (P < 0.05). The lowest damage score was in the e-RIPC group followed by the l-RIPC, e-IPC, and l-IPC groups, respectively. The apoptosis scores were significantly high in the I/R group compared with the e-RIPC and l-RIPC groups (P < 0.05). Although apoptosis scores of the e-IPC and l-IPC groups were lower than the I/R group, this finding was not statistically significant (P > 0.05). CONCLUSIONS: All IPC protocols were effective in reducing I/R injury. Among these protocols, e-RIPC achieved most protection.

Analysis of acute impact of oleoresin capsicum on rat nasal mucosa using scanning electron microscopy.

Analysis of acute cellular changes seen in nasal mucosa of Wistar-Albino rats exposed to different doses of oleoresin capsicum for various time periods by means of scanning electron microscopy. Thirty-five Wistar-Albino rats were divided into five groups of seven rats each. 6-gram oleoresin capsicum per second was sprayed into cages of the groups except group 1. Spray times and duration of exposure to pepper gasses were different for each group. Thirty minutes after the exposure, the animals were killed and specimens from their nasal mucosas were harvested and examined under scanning electron microscope. Mucosal damage was scored from 0-4 points. Mean values of nasal mucosa damage scores of the groups were calculated and compared statistically. Average damage scores of the groups exposed to identical doses of oleoresin capsicum for various exposure times were compared and a statistically significant difference was seen between Groups 2 and 3 (p < 0.05), however the difference between Groups 4 and 5 was insignificant (p > 0.05). Average damage scores of the groups exposed to various doses for identical exposure times were compared, and statistically significant differences were observed between Groups 2 and 4 and also Groups 3 and 5 (p < 0.05). Outcomes of our study have demonstrated that pepper gas exerts destructive changes on rat nasal mucosa. The extent of these destructive changes increases with the prolonged exposure to higher doses. Besides, exposure time also stands out as an influential factor on the extent of the destructive changes.

Effects of surfactant on biofilm formation on silicone nasal splints.

Biofilms are sessile communities of bacteria embedded in self-produced extracellular polysaccharide matrix and are considered to be responsible for bacterial infections in humans. Topical surfactant use on silicone nasal splints may have a preventive effect on biofilm formation. The objective of this study is to investigate the effect of surfactant-containing nasal solutions on biofilm formation over the surface of silicone nasal splints. Forty patients were randomized after septoplasty to receive surfactant-containing saline solution (group 1) or saline without surfactant (group 2). At the postoperative 48th, 72th and 96th hours, pieces of splint samples were taken and prepared for scanning electron microscopic evaluation. Biofilm formation was observed in 3, 6 and 14 of 20 samples in group 1 (surfactant used) and 3, 14 and 20 of 20 samples in group 2 (control) at 48th, 72th and 96th hours, respectively. Biofilm formation incidences of groups at 48th hour were similar (p > 0.05), whereas it was significantly lower at group 1 regarding 72th and 96th hours (p < 0.05). Surfactant-containing nasal solutions have an inhibitory effect on biofilm formation over the surface of silicone nasal splints especially after 48 h. Surfactant-containing nasal solutions may have an important role in nasal septal dressing in the future.

Microbial biofilm formation on silicone nasal splints: optimal time for splint removal.

OBJECTIVES: Biofilms are sessile communities associated with persistent infections and are resistant to conventional therapeutic strategies. They survive on the surface of various inorganic medical devices and cause serious medical problems. METHODS: We recruited 25 patients who underwent nasal surgery between January and May 2013. All patients received silicone splints at the conclusion of the procedure. Pieces of the splints were collected 48, 72 and 96 h post-surgery and prepared for scanning electron microscopy evaluation to assess biofilm formation. RESULTS: Biofilm was observed in 3, 14 and 25 of the 25 samples at 48, 72 and 96 h, respectively. The differences in the proportions of the samples with biofilm formation at each time point (48, 72, and 96 h) were statistically significant. CONCLUSION: Our data demonstrated that biofilm formation on silicone splints increases significantly after 48 h following placement. Although packing may reduce complications, surgeons must consider the potential hazards of packing materials, such as biofilm formation at 48 h post-surgery.

Influence of surface properties of Merocel® (polyvinyl acetal) and silicone nasal splints on biofilm formation.

The objective of the study was to investigate biofilm formation on Merocel(®) and silicone nasal splint after nasal septal surgery. 50 patients who were scheduled to undergo nasal septal surgery were included in this study. The patients were randomized into receiving an insert of Merocel(®) or silicone splint after septoplasty. In group 1 (8 females, 17 males) and group 2 (10 females, 15 males), Merocel (®) packs or silicone splints were inserted into nasal cavities at the end of the procedures, respectively. All packs were removed 48 h after insertion, and samples were taken from the packs under sterilized conditions. Scanning electron microscopy was performed to observe biofilm formation on the surfaces of Merocel(®) and silicone splints. Biofilm formation was observed in 25 (100%) and 3 (12%) of the Merocel(®) and silicone splint samples, respectively. Our study revealed that biofilm formation on Merocel(®) packs is significantly higher than silicone splints, mainly due to the different texture and surface properties of these materials. Considering the hazardous effects of biofilm formation on humans, our observations in this study may guide surgeons to choose the most appropriate packing material after nasal septal surgery.

Investigation of the presence of biofilms in chronic suppurative otitis media, nonsuppurative otitis media, and chronic otitis media with cholesteatoma by scanning electron microscopy.

OBJECTIVE: Biofilms have been shown to play a major role in the pathogenesis of otolaryngologic infections. However, very limited studies have been undertaken to demonstrate the presence of biofilms in tissues from patients with chronic otitis media (COM) with or without cholesteatoma. Our objective is to study the presence of biofilms in humans with chronic suppurative and nonsuppurative otitis media and cholesteatoma. Study Design. In all, 102 tissue specimens (middle ear, mastoid tissue, and ossicle samples) were collected during surgery from 34 patients. METHODS: The samples were processed for the investigation of biofilms by scanning electron microscopy (SEM). RESULTS: Our research supports the hypothesis in which biofilms are involved in chronic suppurative otitis media, cholesteatoma, and, to a lesser degree, chronic nonsuppurative otitis media. There were higher rates in hypertrophic and granulated tissue samples than in normal mucosa. In addition, the presence of biofilms was significantly higher in the middle ear mucosa compared with the mastoid and ossicle samples. CONCLUSION: In the clinic, the careful use of topical or systemic antimicrobials is essential, and, during surgery, hypertrophic tissue must be carefully removed from normal tissue.

Postantifungal effect of the combination of caspofungin with voriconazole and amphotericin B against clinical Candida krusei isolates.

We evaluated the postantifungal effects (PAFEs) of caspofungin (CAS), voriconazole (VOR), amphotericin B (AmB), and the combinations of CAS + VOR and CAS + AmB against 30 clinical Candida krusei isolates at 0.25, 1 and 4 times the MIC of each individually and in the indicated combinations. Antifungals were removed after 1 hour and colony counts were performed at 0, 2, 6, 24, and 48 h. VOR did not display any measurable PAFE regardless of antifungal concentrations, while AmB and CAS exhibited dose-dependent PAFE. The most effective agent producing a prolonged PAFE in this study was CAS. Although the combination of CAS with VOR generated longer PAFEs at 0.25 and 1 times their respective MICs in comparison with CAS alone, this combination was indifferent rather than synergistic. However, the combination of CAS with AmB at 4 times their MICs exhibited the best performance, reducing the colony counts during the 48 h after removal of drugs and resulted in synergic interaction in respect to 20 (67%) isolates. Consequently, CAS has a prolonged PAFE in vitro against C. krusei isolates, and the combination of AmB + CAS may increase significantly the efficacy of CAS. Our data may be useful in optimizing dosing regimens for these agents and their combinations, although further studies are needed to explore the clinical usefulness of our results.

Broth microdilution and time-kill testing of Caspofungin, voriconazole, amphotericin B and their combinations against clinical isolates of Candida krusei.

Treatment of invasive Candida krusei infections can be difficult due to its intrinsic fluconazole resistance and its reduced susceptibility to amphotericin B and flucytosine. Caspofungin (CAS) acts on a different cellular target, and its combination with voriconazole (VOR) or amphotericin B (AmB) appears promising. We evaluated the activity of CAS, VOR and AmB alone and in combination at 1/4, 1, 4xMIC concentrations by time-kill method against 30 C. krusei isolates. All isolates were susceptible to CAS and VOR; AmB MICs were 2 µg/ml for 50% of isolates by broth microdilution. CAS showed a fast killing activity at all concentrations; it was fungistatic at 1/4xMICs and fungicidal at 1-4xMICs in general. VOR displayed a concentration-independent fungistatic activity against all isolates. AmB exhibited a concentration-dependent activity; it was fungistatic at 1/4-1xMIC and fungicidal at 4xMIC. The most common interaction was indifference for both combinations. Frequency of synergic interaction for the VOR + CAS combination was 66.7% at 1/4xMIC after 48 h. The best results for CAS + AmB combination were obtained at 4xMIC in the first 4-8 h; synergic interaction was detected for 20 isolates (66.7%) at 4xMIC after 4 h. Consequently, VOR and CAS alone have been found effective, and high AmB MICs are remarkable against clinical C. krusei isolates in vitro. The combinations of CAS with VOR or AmB have exhibited promising results.

The evaluation of in vitro pharmacodynamic properties of amphotericin B, voriconazole and caspofungin against A. fumigatus isolates by the conventional and colorimetric time-kill assays.

Aspergillus fumigatus is an opportunistic pathogen responsible for life-threatening infections in immuncompromised patients. Data about the in vitro pharmacodynamics of antifungals against A. fumigatus are limited. In the present study, we investigated the fungicidal activities, at concentrations of 1, 4 and 16 times the minimum inhibitory concentration (MIC), of caspofungin (CAS), amphotericin B (AMB) and voriconazole (VORI) against eight A. fumigatus isolates through the use of time kill and 2,3-Bis [2-methoxy-4-nitro-5-(sulfenylamino) carbonyl-2H-tetrazolium-hydroxide] (XTT) reduction tests. By the conventional time kill test, AMB was fungicidal (≥99.9% reduction in colony forming units; CFU) for all isolates at 4-16 MICs after 48 h incubation. The fungicidal effect for VORI was determined at 4 × MIC for one isolate and at 16 × MIC for four isolates at 48 h of exposure. CAS was also fungicidal at 1 × MIC for one isolate and at 4-16 MICs for two isolates at 48 h. While the percentage of median killing of AMB was found by the time-kill method with XTT as 99% at 4 × MIC and 99.28% at 16 × MIC, that of VORI was 94.5% at 4 × MIC and 92.88% at 16 × MIC after 48 h of incubation. However, a significant increase was observed compared to initial inoculum size with CAS after 48 h. Since the XTT method measures all cellular viability in media, it may give more reliable results about pharmacodynamics of antifungal agents against Aspergillus spp. than the time kill test.