Interaction in a dual-species biofilm of Candida albicans and Candida glabrata co-isolated from intravascular catheter.

The use of catheters for vascular access may be associated with colonization by Candida species and their biofilm-forming ability. The latter can harbor two or more species of Candida yeast. In the sense, we conducted our study at the University Hospital of Tlemcen in west Algeria at the neuro-surgery unit, that aims (or which aims) to evaluate the ability to form mixed biofilm by dual-species Candida albicans/Candida glabrata co-isolated from intravascular catheters and their interaction in biofilm. That is the first report in Algeria. During this study, we took photographic images by scanning electron microscopy of 3 catheters implanted before 48 h and co-colonized by dual-species. From all taken samples, 34 catheters were altered by yeasts from which three were co-colonized by two Candida species and C. albicans established synergistic and competitive interactions with C. glabrata species in mixed biofilm tested.

In vitro activity of antifungal combinations against planktonic and sessile cells of Candida albicans isolated from medical devices in an intensive care department.

BACKGROUND: Invasive fungal infections are an emerging health problem worldwide. They are responsible for a significant rate of morbidity and mortality. Infections caused by Candida albicans involve proliferation of biofilms on biotic or abiotic surface. These adherent communities exhibit characteristics distinct from planktonic cells such as the ability to tolerate high concentrations of antifungal. OBJECTIVE: The object of our study was focused on the determination of the susceptibility to amphotericin B, caspofungin, voriconazole and two antifungal combinations (amphotericin B/caspofungin and amphotericin B/voriconazole) of both planktonic and sessile cells of C. albicans, which were isolated from catheters. MATERIAL AND METHODS: The susceptibility of C. albicans to antifungals was determined using the broth microdilution method according to Clinical Laboratory Standards Institute CLSI (2008). A Checkerboard assay was employed to evaluate the efficacy of drugs combinations. Biofilm susceptibility was determined using a metabolic [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] (XTT) reduction assay. RESULTS: The minimal inhibitory concentrations of individual antifungal drugs determined against C. albicans biofilms (SMICs) were significantly higher (P<0.05) than planktonic ones (MICs). They went from 2 to 64µg/mL for amphotericin B, from 1 to 64µg/mL for caspofungin and from 2 to 128µg/mL for voriconazole. The combination of amphotericin B to caspofungin or to voriconazole decreased significantly the MIC values for planctonic (P<0.0001) and sessile cells (P=0.0016). Based on Fractional Inhibitory Concentration Index (FICI), no antagonistic interaction was observed. CONCLUSION: The obtained results showed that the combination of amphotericin B with either caspofungin or voriconazole can be used as a new strategy for management of systemic mycoses associated to medical devices.

[Study of the infectivity of Candida on peripheral vascular catheters collected from the University Hospital of Tlemcen]. / Étude de l'infectivité des Candida sur cathéters vasculaires périphériques prélevés du Centre Hospitalier Universitaire de Tlemcen.

Yeasts can adhere to medical implants and cause infections responsible for high morbidity and mortality among hospitalized patients. The objective of this study is to investigate the infectivity on peripheral vascular catheters collected from general surgery and cardiology in University Hospital of Tlemcen. The results showed that from 29 samples altered by yeast, 35 Candida sp. strains were isolated. However, Candida albicans is the most isolated species in an infectious context. Risk factors that accompanied the infections are the duration of implantation, male gender, and bacterial presence.

Retrospective study of Candida sp. contaminations of endoscopes at the University Hospital of Tlemcen (Algeria).

BACKGROUND: Improper cleaning and disinfection of endoscopes has been responsible for multiple nosocomial outbreaks and sometimes serious life-threatening infections. OBJECTIVE: The aim of our study is, at first, to identify Candida species responsible for the contamination of endoscopes, and to determine the minimal inhibitory concentrations of planktonic (MIC) and sessile cells (SMIC) of amphotericin B (AmB) against our isolated strains. MATERIALS AND METHODS: The present study was performed on four endoscopes in the department of gastroenterology at the University Hospital of Tlemcen (Algeria). A total of 300 samples from endoscopes were examined over a period of 3years. RESULTS: Thirty-four strains of Candida sp. were isolated, representing 11.33% of the considered samples. The number of isolated strains dropped significantly in the second and the third year compared to the first year of our study. After testing the antifungal property of amphotericin B, we showed clearly that the sessile cells of Candida sp. were much more resistant than their planktonic counterparts (suspended cells). CONCLUSION: The methods of sterilization of the endoscopes are very important; drying by compressed air is a critical step that reduces significantly the number of yeasts contamination.

[Infectious risk related to the formation of multi-species biofilms (Candida - bacteria) on peripheral vascular catheters]. / Risque infectieux lié à la formation des biofilms multi-espèces (Candida ­ bactéries) sur cathéters vasculaires périphériques.

The Candida yeasts are the fourth leading cause of death from systemic infections, the risk may increase when the infection also involves bacteria. Yeasts and bacteria can adhere to medical implants, such as peripheral vascular catheters, and form a multicellular structures called "mixed biofilms" more resistant to antimicrobials agents. However, the formation of mixed biofilms on implants leads to long-term persistent infections because they can act as reservoirs of pathogens that have poorly understood interactions.

Study of strains of Candida spp. Isolated from catheters in UHC of Oran (Algeria): Identification and antifungal susceptibility.

OBJECTIVES: The increasing incidence of Candida spp., and the vital prognosis often compromise for patients with Candida species make urgent the exact knowledge of their distribution worldwide and exhaust action antifungals currently used in clinical. That why we carry out an epidemiological study of Candida species and testing their susceptibility against two antifungals: amphotericin B and caspofungin. MATERIALS AND METHODS: Samplings of peripheral venous catheters (PVC) were carried out from during 8months on the services of Internal medicine, Surgery A and Neonatology of Oran's University Hospital Center (UHC). The study of the susceptibility of Candida species to antifungal agents was performed according to the Clinical Laboratory Standards Institute (CLSI 2008). RESULTS: From 300 samples, 25 yeasts were isolated. The rate of colonization PVC was 8.33% by Candida spp. The most isolated strains were Candida parapsilosis with 64% of cases, followed by Candida albicans (12%) then 8% for Candida glabrata and Candida krusei. However, only 4% of isolates were Candida famata or Candida lusitaniae. Furthermore all isolated strains were susceptible to amphotericin B with Minimum Inhibitory Concentrations (MIC) ranging from 0.25 to 1µg/mL. MIC obtained with caspofungin vary from 0.0625 to 2µg/mL for all strains. Moreover, one strain of C. krusei is resistant to caspofungin with a MIC superior to 8µg/mL. CONCLUSION: All though caspofungin is at least as effective as amphotericin B, it is better tolerated for the treatment of invasive fungal infections.

[Fungal infectivities of implanted catheters due to Candida sp. Biofilms formation and resistance]. / Infectivités fongiques des cathéters implantés dues à Candida sp. Formation des biofilms et résistance.

BACKGROUND: Candidemia are the most common fungal infections in hospitals. However, the catheters are subject to be altered by Candida biofilms which increase the risk of invasive nosocomial infections due to the high resistance to antifungal agents. Therefore, the minimum inhibitory concentrations of planktonic (MIC) and sessile cells (CIMS) were evaluated. METHODS: To review the in vivo biofilms structures of Candida sp. formed on the inner and/or external surfaces of collected catheters, we used scanning electron microscopy (SEM). The level of biofilm resistance was assessed against two conventional antifungal agents: amphotericin B (AmB), which belongs to the class of polyenes, and fluconazole (FLZ) which is an azole. RESULTS: The SEM observation of biofilms of Candida sp. reveals complex structures. Compared to MICs, the calculation of CIMS showed an increase of 32 times with AmB and of 128 times with FLZ. CONCLUSION: Catheters offer an ideal surface to Candida sp. to form biofilms. This complex structure induces the increase of the resistance of sessile cells against two antifungal agents, AmB and FLZ.

[Evaluation of mixed biofilm formation between Candida albicans and a variety of bacterial species isolated from peripheral catheters at Tlemcen CHU. First study in Algeria]. / Évaluation du potentiel de formation de biofilms mixtes entre Candida albicans et quelques espèces bactériennes isolées de cathéters vasculaires périphériques au CHU de Tlemcen. Première étude en Algérie.

INTRODUCTION: Mixed-species biofilms constitute a reservoir of infection for a group of bacteria and yeasts that coexist on the same support. Peripheral venous catheters make up a good surface for the attachment of microorganisms that promote biofilm formation and this requires complex strategies for antimicrobial treatments. OBJECTIVE: No such studies on formation mixed biofilms have ever been conducted in Algeria. Therefore, we evaluated the potential for the formation of mixed-species biofilms by Candida albicans and some bacterial species isolated from peripheral vascular catheters at the University Hospital of Tlemcen, in Algeria. RESULTS: The results obtained showed that C. albicans have the potential to form mixed biofilms with three bacteria (Enterobacter cloacae, Bordetella spp. and Serratia liquefaciens) isolated from the same catheter as the yeasts. The amount of biofilms produced varies depending on the species and the composition of the growth medium. Observations by scanning electron microscopy showed that the structure of the mixed biofilm depends on the surface support the biofilm was formed on, and varies with the species. CONCLUSION: A competition was noted between bacteria and yeasts; it depends on the composition of the medium and its pH, which both play an important role in promoting the dominance of one over the other.

In vitro susceptibility of amphotericin-B, voriconazole and caspofungin against Candida guilliermondii biofilms, isolated from dentals units water pipes, under different growth phases.

OBJECTIVE: The dental units water pipes are a favorable medium for biofilms formation because of the small diameter of the pipe and the duration of water stagnation, but the question which arises is the nature of the biofilms which are formed inside? This article gives a progress report on the nature of this microbial contamination and precisely the fungal biofilms formation by examining their susceptibility to antifungal agents under different growth phases. METHODS: Sixteen samples of dental units water pipes were taken from public dental clinic and from stomatology unit at the university hospital of Tlemcen (Algeria). The isolated strains were identified by the conventional mycological methods and were analyzed to determine their minimal concentrations inhibiting their growth (planktonic and sessile forms) using three antifungal agents. RESULTS: Five strains type Candida guilliermondii were identified and analyzed for their resistance to antifungal agents. Antifungal susceptibility testing demonstrated the sensitivity of all planktonic Candida guilliermondii cells against amphotericin-B, voriconzole and caspofungin but the sessile cells of these strains revealed a less susceptibility to antifungal agents and even a resistance when the biofilm made mature. CONCLUSION: Several types of yeast contaminated the dental units water pipes and especially Candida guilliermondii that was the most founded. This specie was susceptible to antifungal agents under planctonic forms and resistance where the biofilm made mature.

Effects of ascorbic acid and α-tocopherol on the therapeutic index of amphotericin B.

Amphotericin B (AmB) remains the antifungal polyene of choice in deep fungal infections, but its high toxicity to mammalian cells limits its use. This toxicity is partly due to lipid peroxidation exerted by amphotericin B in cell membranes. The work we have undertaken focused on the one part the evaluation of the efficacy of amphotericin B in the presence of some antioxidants vitamins (vitamin C "ascorbic acid" and vitamin E "α-tocopherol") against the yeast Candida albicans ATCC 10231. Secondly, we have tested the cytotoxicity of these formulations on human red blood cells. The results showed a significant improvement in the efficiency of our formulations tested from 7% to 12% compared with amphotericin B alone at therapeutic concentrations. Furthermore, the addition of vitamin C and vitamin E protects human red blood cells against the cytotoxicity induced by amphotericin B with 17%. This is due may be to the antioxidant power of vitamins which confer protection against the autoxidation of the molecule of amphotericin B. On the other hand, it is noticed that the yeast regrows after 24h whatever in complex with vitamin C or vitamin E of the stock solution. On completion of this study, the incorporation of antioxidant vitamins that we propose to the reaction medium of antifungal improved the therapeutic index of amphotericin B.

[Catheterization and fungal infection risk in the University Hospital of Tlemcen: epidemiology and susceptibility to antifungals]. / Cathétérisme et risque infectieux fongique au centre hospitalo-universitaire de Tlemcen: épidémiologie et sensibilité aux antifongiques.

Fungal infections are a major cause of morbidity and mortality, and are frequently associated with the implantation of vascular catheters, especially in immune-compromised patients. Unfortunately, the therapeutic arsenal available for the treatment of these infections, caused generally by the yeasts of the genus Candida is still limited because of the toxicity and/or of the emergence of resistance against some antifungal agents. That is why we have undertaken this study, which is to determine the incidence and the degree of sensitivity of Candida spp., isolated from peripheral venous catheters at the University Hospital of Tlemcen (Algeria) to caspofungin and amphotericin B. The results show that the rate of colonization of vascular catheters was 19 % by yeasts of Candida spp., of which 60 % are Candida parapsilosis, 20 % Candida albicans, 14.3 % Candida glabrata and 5.7 % Candida famata. The minimum inhibitory concentrations (MIC) for amphotericin B are between 0.5 and 2 µg/mL and for caspofungin, they are between 0.125 and 2 µg/mL.

[Dormancy of Candida albicans ATCC10231 in the presence of amphotericin B. Investigation using the scanning electron microscope (SEM)]. / Dormance de Candida albicans ATCC10231 en présence d'amphotéricine B. Investigation au microscope électronique à balayage (MEB).

OBJECTIVES: The aim of this study was to visualize the morphology of Candida albicans ATCC10231 in the absence and in the presence of amphotericin B at 0.4 µg/mL, to better understand the phenomenon responsible for a large portion of cases of treatment failure called "dormancy phenomenon". The main objective was to determine the morphological changes adopted by C. albicans in the lag phase extended before resuming normal growth. MATERIALS AND METHODS: In order to define the morphological characteristics and surface properties of the cells in the absence and in the presence of amphotericin B at 0.4 µg/mL, cells were cultured in Sabouraud medium at 30°C, and the morphology index was determined by reference to the classification of forms in C. albicans determined by Merson-Davies and Odds. Then, the technique of scanning electron microscopy (SEM) coupled with image analysis was used to measure the surface and determined the morphological properties using the plugin analysis three-dimensional (3D) integrated into the ImageJ software. RESULTS: The dormant phase in C. albicans ATCC10231 grown in Sabouraud liquid at 30°C in the presence of amphotericin B at 0.4 µg/mL extends to 21 hours. The index morphology obtained for the two samples (in the absence and presence of amphotericin B) indicates that the cell even in the presence of amphotericin B retains its yeast form (Mi<1.5) The analysis of microphotography obtained by scanning electron microscopy (SEM) has shown that the cell in the presence of amphotericin B is partially deformed; the deformation is estimated at 33.25%, with various changes in the cell surface. CONCLUSION: This study showed that the morphology of C. albicans ATCC10231 in the presence of amphotericin B at 0.4 µg/mL changes with partial deformation of the cell. This rate is insufficient to induce cell death, which partly explains the phenomenon of dormancy adopted by C. albicans prior to cell repair and resume normal growth.

New strategy for enhancing the therapeutic index of Fungizone(®).

The aim of this study was to enhance the therapeutic index of Fungizone(®). For this reason we have prepared stock solutions of Fungizone(®) under different conditions of pHs (from 4.8 to 10.8) and temperatures (unheated solutions and heated ones during 20 min at 70 °C). Then we have evaluated the effect of those antifungal solutions on human red blood cells coming from one healthy donor, as well as on the dormancy of Candida albicans strain. The obtained results show that after exposure to antifungal stock solution prepared at pH 5.4 and heated during 20 min at 70 °C, the rate of hemolysis was largely reduced (only 5% of hemolysis). On the other hand, it is noticed the yeast regrowth after 26 h whatever is the temperature or the pH of the stock solution. However at pH 5.4, the growth of C. albicans is inhibited at 75% in comparison with the one obtained with the conventional Fungizone(®) after 30 h of culture where we observed 100% of growth. So, stock solution of Fungizone(®) prepared at pH 5.4 and heated 20 min at 70 °C seems to be a good strategy for enhancing the therapeutic index of Fungizone(®).

Antibacterial and antifungal activity of lawsone and novel naphthoquinone derivatives.

INTRODUCTION: Naphthoquinone derivatives are under investigation as potential therapeutic agents. The antibacterial and antifungal activity of lawsone and of some novel naphthoquinone derivatives was assessed in vitro. METHODS: The antimicrobial activity was determined using diffusion disk and the broth microdilution methods against seven bacteria and three Candida species, according to recommendations of the Clinical and Laboratory Standards Institute. RESULTS: Two compounds (P05 et P06) presented a good antibacterial effectiveness against two gram-positive bacteria. No antifungal potency was observed against the three Candida albicans strains used in the test. CONCLUSION: Our results prove that the introduction of substituents on ketone function position 4 decreased the antimicrobial properties of the synthetized compounds.

Candida albicans biofilms formed into catheters and probes and their resistance to amphotericin B.

In Algeria, many bacterial biofilms have been studied but those of fungal origin, particularly those due to the yeast Candida albicans remained unidentified. The present study was performed at the Chabane Hamdoune hospital in Maghnia (Algeria), where 51 strains of C. albicans representing 16.94% of all taken samples were isolated. They were collected from catheters and probes used in different hospital services with variable rates; the most concerned service was ICU (40.74%) followed by gynecology department (17.39%), while general surgery came third (15.79%). Testing the antifungal property of amphotericin B (AmB) we showed clearly that the sessile cells of C. albicans were much more resistant than their planktonic counterparts (suspended cells), especially when the resistance increased during the different phases of biofilm formation until it reached its threshold at the ripening stage (at 48h). Furthermore, scanning electron microscopy of the isolated strains in the laboratory revealed the formation of biofilms on catheters by C. albicans. Surprisingly, observations revealed the presence of a new structure in these biofilms: a chlamydospore?

Immobilized respiratory chain activities from Escherichia coli utilized to measure D- and L-lactate, succinate, L-malate, 3-glycerophosphate, pyruvate, or NAD(P)H.

The respiratory chain (membranous, multienzymatic system) from Escherichia coli, was coimmobilized with gelatin and insolubilized in film form by tanning with glutaraldehyde. The film was fixed onto an oxygen sensor. The enzyme electrode can be used for measuring NAD(P)H, D- and L-lactate, succinate, L-malate, 3-glycerophosphate, or pyruvate. The range of metabolites concentrations was from 1 to 50 mM. It was possible to discriminate between the different metabolites (if mixed): By inducing during bacterial growth the specific flavoproteins necessary for L-lactate, succinate, L-malate, and 3-glycerophosphate respirations. The constitutive activities are unaltered on glucose or glycerol, namely D-lactate, NAD(P)H, and pyruvate respiration. When intact bacteria were immobilized (with or without induction), D- and L-lactate, succinate, 3-glycerophosphate, and L-malate respiration were measured, no activities of pyruvate and NAD(P)H respiration were obtained. For these last activities, French press breakage (see section on Membrane Preparations) of bacteria prior to immobilization was necessary. Products of reactions can be used as enzyme inhibitors: Pyruvate inhibits D- and L-lactate; fumarate inhibits succinate, and oxaloacetate inhibits L-malate respirations. Heat denaturation of the bacteria at 55 degrees C for 1 h maintains full activity of succinate and pyruvate respiration. On the other hand, no activity of D- and L-lactate, L-malate, or NAD(P)H respiration was measurable. These enzyme electrodes have many applications in basic and applied research.