M.P. Arévalo

In vitro susceptibility of Candida dubliniensis to current and new antifungal agents.

Background: Candida dubliniensis is a recently described Candida species closely related to Candida albicans, which has been associated with oral candidiasis in HIV-infected patients. Fluconazole-resistant strains of C. dubliniensis are easily obtained in vitro and this fact could be a complication if this resistance develops during treatment with this drug. Methods: In the present study, the in vitro antifungal susceptibilities of 36 C. dubliniensis clinical isolates and culture strains to current and new antifungal agents, such as amphotericin B (AMB), amphotericin B lipid complex (ABLC), amphotericin B colloidal dispersion (ABCD), 5-fluorocytosine (5FC), fluconazole (FLC), itraconazole (ITC), ketoconazole (KTC), liposomal amphoteri- cin B (LAMB), liposomal nystatin (LNYT), LY303366 (LY), SCH56592 (SCH), and voriconazole (VRC), were determined according to the National Committee for Clinical Laboratory Standards M27-A broth microdilution method for yeasts. Results: Most isolates of C. dubliniensis were susceptible to both new and current antifungal drugs, with 75.9% isolates susceptible to KTC, 86.2% to FLC and to ITC, and ∼100% to the other antifungal agents tested. The cross-resistance phenotypes are detailed. Four isolates were resistant (MIC ≥64 μg/ml) to FLC. These 4 isolates were also resistant to KTC, and 3 of them were also resistant to ITC (MIC ≥1 μg/ml for both agents). However, these isolates were highly susceptible to 5FC and all polyene formulations (AMB, ABLC, ABCD, LAMB, and LNYT), triazole (SCH and VRC) and echinocandin (LY) antifungal agents. Conclusion: The new liposomal and lipidic formulations of AMB, LNYT, and the new triazoles and echinocandins may provide new alternatives to FLC for the treatment of infections by C. dubliniensis.

Biofilm formation by five species of Candida on three clinical materials

Most recalcitrant infections are associated with colonization and microbial biofilm development. These biofilms are difficult to eliminate by the immune response mechanisms and the current antimicrobial. Fungi can form biofilms on biomaterials commonly used in clinical practice (intravascular catheters, dentures, heart valves, implanted devices, contact lenses and other devices) and are associated with infections. A variety of in vitro models using different substrates/devices have been described. These models have been used to investigate the effect of different variables, including flow, growth time, nutrients and physiological conditions on fungal biofilm formation, morphology and architecture. The purpose of our study is to analyze biofilm formation capacity by 84 strains of Candida spp. (23C. albicans, 23C. parapsilosis, 16C. tropicalis, 17C. glabrata and 5C. krusei) on three materials used in medical devices and its quantification using a method based on viable cell count. Under the conditions of our study, all assayed Candida strains have been able to form biofilms. All species showed greater biofilm formation capacity on Teflon™, with the exception of C. glabrata which displayed higher biofilm formation capacity on PVC. Biofilm formation by Candida spp. varies depending on the type of material on which it grows and on the species and strain of Candida. The method we propose could be of great use to deepen scientific knowledge on this subject of remarkable clinical significance, considering the absence of standard biofilm formation and quantification techniques on the catheters and the level of difficulty associated to those available.

Antimicrobial-Resistant Salmonella enterica Serovars Isolated from Chickens in Spain

Abstract In order to analyze the antibiotic resistance of Salmonella enterica serovars, a total of 112 Salmonella strains were tested (54 S. enteritidis, 32 S. typhimurium, 11 S. heidelberg, 7 S. infantis, 4 S. virchow and 4 S. hadar). The bacteria were isolated from 691 samples of frozen and fresh chicken meat. Identification of microorganisms and antimicrobial sensitivity testing were undertaken by means of the automated MicroScan AutoScan 4 method (Baxter in Spain). 45.5% of 112 strains tested were susceptible to all antibiotics. The highest percentage of resistance was found to: chloramphenicol (44.6%), ampicillin (34.8%) and tetracycline (33.9%). Multiple resistance was observed in 49 strains (43.7%), whereas single resistance was seen in 12 isolates (10.7%). We found 12 different patterns of resistance in Salmonella enterica serovar enteriditis. Resistance to chloramphenicol was the most common single resistance. The most frequent patterns of multiresistant strains were ampicillin + amoxicillin/clavulanate + cefazolin + imipenem and chloramphenicol + impipenem. In this serotype, 49 isolates belonged to phagetype 4. Salmonella typhimurium showed the highest percentages of resistance to the tested drugs, with six different resistance patterns found. 25 strains out of 32 S. typhimurium isolates belonged to phagotype 120 and 13 of these showed the same resistance pattern: chloramphenicol + tetracycline + ampicillin. The high incidence of antibiotic resistant salmonellae found in chickens in our study suggests the need for public health interventions to decrease selective pressure on bacterial strains by antimicrobial agents.

Comparative in vitro Antifungal Activity of Amphotericin B Lipid Complex, Amphotericin B and Fluconazole

Amphotericin B (AMB) is considered the gold standard in the treatment of serious systemic mycoses in spite of its nephrotoxicity and adverse effects. Association with lipids enables larger doses of AMB to be given with a longer t½ and Cmax, without the toxic effects at lower concentrations. Liposome-encapsulated AMB shows a lower affinity for mammalian cells and improves Vd, thus decreasing toxicity. Amphotericin B lipid complex (ABLC) is an AMB formulation associated with a biodegradable phospholipid matrix (5% molar) from which the drug is released by cell phospholipases. ABLC is recommended for serious mycoses refractory to conventional antifungal therapy or when AMB is contraindicated. We compared the in vitro antifungal activity of ABLC, AMB and fluconazole (FLZ) against 328 strains of clinically significant opportunistic fungi using a microdilution method (NCCLS, M-27A). 64.9% of the yeasts were inhibited by MIC of ABLC ≤ AMB resulting in a similar or slightly superior efficacy compared to AMB when tested against Candida albicans, C. glabrata, C. guilliermondii, C. parapsilosis and C. tropicalis. Effectiveness against C. krusei was lower for ABLC (5.99 μg/ml for ABLC, 1.58 μg/ml for AMB). However, for Aspergillus fumigatus, the activities of AMB and ABLC were 1.62 and 2.46 μg/ml, respectively; A. niger 0.72 μg/ml, 0.76 μg/ml (ABLC and AMB, respectively); A. clavatus, A. candidus, A. tenuissima, A. corymbifera and Exophiala jeanselmei, Scedosporium spp. and Miceliophtora spp. showed a low susceptibility to both AMB formulations. ABLC is a useful alternative to AMB or FLZ for the treatment of severe fungal infections, due to the broad spectrum of antifungal actions observed in this study.

Comparison of Albicans ID2® agar plate with the germ tube for presumptive identification of Candida albicans

Albicans ID2 (bioMérieux, France) is a commercially available chromogenic medium that allows rapid and specific macroscopic identification of Candida albicans and facilitates the differentiation of species in mixed cultures. We compared it with the standard method for the identification of yeast species, the germ tube test (GT). This study involved 423 clinical isolates, including 163 C. albicans and 260 non-albicans yeasts. Sensitivity of Albicans ID2 agar plates regarding the identification of C. albicans were 98.2% after 48 h of incubation and specificity of 96.6%. This method using rapid enzymatic method shows the same similar sensitivity than the GT test The false negative rate (1.8%) for the GT test is consistent with that previously reported. None tests discriminated between C. albicans and C. dubliniensis isolates.

In vitro susceptibility of 545 isolates of Candida spp. to four antifungal agents

Summary. The in vitro susceptibility to amphotericin B, fluconazole, itraconazole and ketoconazole of 545 Candida strains from patients treated at the University Hospital of the Canaries was determined by means of a microdilution test. The distribution of the species was as follows: Candida albicans (342), Candida tropicalis (70), Candida glabrata (68), Candida parapsilosis (65). Of Candida albicans isolates, 8.5% and 7.6% showed resistance to itraconazole and fluconazole respectively. Of C. tropicalis isolates 34.3%, 27.1% and 2.9% were resistant to itraconazole, fluconazole and ketaconazole respectively. For C. glabrata, 10.3% and 4.4% of the isolates under study demonstrated resistance to fluconazole and itraconazole respectively. Only 4.6% and 1.5% of C. parapsilosis isolates demonstrated resistance to fluconazole and itraconazole respectively. C. tropicalis was the most resistant strain and C. parapsilosis the most sensitive. The greatest percentages of resistance in vitro were seen with the triazoles.

Candida glabrata: In vitro Susceptibility of 84 Isolates to Eight Antifungal Agents

The in vitro susceptibility of 84 isolates of Candida glabrata from patients treated at the University Hospital of the Canary islands to eight antifungal agents (amphotericin B, itraconazole, fluconazole, miconazole, clotrimazole, tioconazole and econazole) has been studied using the broth dilution micro-method. Among the eight antifungal agents tested, the smaller geometric mean corresponded to tioconazole, econazole, clotrimazole and miconazole. In contrast with fluconazole, a greater geometric mean has been achieved. All the C. glabrata isolates tested were sensitive to concentrations of 3.125 micrograms/ml of clotrimazole and miconazole, 6.25 micrograms/ml of amphotericin and ketoconazole. Concentrations of 12.5 micrograms/ml were needed to obtain 100% inhibition of isolates for econazole and tioconazole and concentrations of 25 and 50 micrograms/ml, respectively for itraconazole and fluconazole. Among our C. glabrata isolates, 2.4% were found to be resistant to amphotericin B. For fluconazole and itraconazole, 19.1% and 7.9% of isolates, respectively, were resistant. With reference to imidazoles, we obtained 2.4% and 3.6% resistance for tioconazole and econazole, respectively. No isolates were found to be resistant to ketoconazole, miconazole and clotrimazole. The results have shown a high activity of amphotericin B and itraconazole, observing a similar response with the five imidazole antifungals tested. The highest rate of resistance was found when fluconazole was used.

Fluconazole, itraconazole and ketoconazole in vitro activity against candida spp.

The in vitro activity of fluconazole, itraconazole and ketoconazole against 625 Candida yeast strains from patients treated at the University Hospital of the Canaries, by means of a micromethod of dilution in broth enriched with Yeast Nitrogen Base (YNB), and buffered to pH7, has been assessed. Species distribution was as follows: Candida albicans (388), Candida tropicalis (84), Candida glabrata (84), Candida parapsilosis (69). Our results show 10.0% and 8.8% of C. albicans resistant to itraconazole and fluconazole, respectively, and 1.8% resistant to ketoconazole; 39.5% of C. tropicalis were resistant to itraconazole, 34.5% to fluconazole and 2.4% to ketaconazole. 19.1% of C. glabrata were resistant to fluconazole and 13.1% to itraconazole; 4.4% of C. parapsilosis were resistant to fluconazole and 1.5% to itraconazole. In general C. tropicalis was the most resistant strain and C. parapsilosis the most sensitive. The greatest percentages of resistance in vitro were seen with the two triazols.