Marie-Hélène Rodier

In vitro efficacies of caspofungin or micafungin catheter lock solutions on Candida albicans biofilm growth

OBJECTIVES Caspofungin and micafungin belong to the echinocandins; the mechanism of action of echinocandins is based on the inhibition of (1,3)-beta-D-glucan synthase. The aim of this study was to investigate in vitro the optimal antifungal lock treatment details against a Candida albicans biofilm. METHODS An in vitro model of a C. albicans (ATCC 3153 or ATCC 66396) biofilm associated with 100% silicone catheters was used. The effectiveness of the antifungal treatment was assayed against biofilms aged 12 h or 5 days, after exposure to caspofungin (2 mg/L) or micafungin (5 mg/L) for 12 h. The durability of the reduction in the biofilm metabolic activity was investigated (1-3 days after echinocandin treatment). The efficacy of caspofungin and micafungin was determined by evaluating a significant decrease (P < 0.0001) in the metabolic activity of biofilm yeasts. RESULTS The results showed that the tested antifungal agents used as lock solution significantly (P < 0.0001) reduced the metabolic activity of C. albicans, whatever the biofilm maturation stage (12 h or 5 days old biofilms). The reduction in the metabolic activity of biofilm yeasts was maintained, even after 48 h. CONCLUSIONS These data suggest that caspofungin (2 mg/L) and micafungin (5 mg/L) could represent good candidates for the reduction or control of fungal biofilms associated with silicone medical devices, as part of an antifungal lock. They were able to induce a significant and persistent reduction in the yeast metabolic activity of intermediate and mature biofilms, 12 h and 5 days old, respectively, when used as catheter lock solutions.

Paranasal sinus fungus ball : Epidemiology, clinical features and diagnosis. A retrospective analysis of 173 cases from a single medical center in France, 1989-2002

Over the last decade, we have observed a high frequency of Aspergillus rhinosinusitis in french medical centers. The epidemiological data, clinical presentations, radiology, mycology and histology results of 173 consecutive patients with paranasal sinus fungus balls who were admitted from 1989 to 2002 have been reviewed. The most common symptoms included purulent nasal discharges and nasal obstructions, with the maxillary sinus being the most common site of infection (152 cases, 87.8%). Computed tomography scans (CT scan) were performed in 92% (159/173) of the cases and heterogeneous opacities were observed in 132 patients (83%). Histology examinations were performed in all cases and proved positive in 162 patients. Fungi were recovered, mainly Aspergillus fumigatus, from samples of 50 patients, while specimens from the remaining 123 patients were negative. Since no specific clinical sign could be found, a diagnosis of fungus ball is frequently made after a long term symptomatic period. CT scan findings of metallic or calcified densities within an opacified sinus cavity are highly suggestive of a fungus ball, but mycological and histological studies are essential to confirm the diagnosis. Treatment consisted of functional endonasal sinus surgery and was successful in 172 out of 173 cases.

Efficiency of water disinfectants against Legionella pneumophila and Acanthamoeba

Free-living amoebae might be pathogenic by themselves and be a reservoir for bacterial pathogens, such as Legionella pneumophila. Not only could amoebae protect intra-cellular Legionella but Legionella grown within amoebae could undergo physiological modifications and become more resistant and more virulent. Therefore, it is important to study the efficiency of treatments on amoebae and Legionella grown within these amoebae to improve their application and to limit their impact on the environment. With this aim, we compared various water disinfectants against trophozoites of three Acanthamoeba strains and L. pneumophila alone or in co-culture. Three oxidizing disinfectants (chlorine, monochloramine, and chlorine dioxide) were assessed. All the samples were treated with disinfectants for 1 h and the disinfectant concentration was followed to calculate disinfectant exposure (Ct). We noticed that there were significant differences of susceptibility among the Acanthamoeba strains. However no difference was observed between infected and non-infected amoebae. Also, the comparison between the three disinfectants indicates that monochloramine was efficient at the same level towards free or co-cultured L. pneumophila while chlorine and chlorine dioxide were less efficient on co-cultured L. pneumophila. It suggests that these disinfectants should have different modes of action. Finally, our results provide for the first time disinfectant exposure values for Acanthamoeba treatments that might be used as references for disinfection of water systems.

Free-living amoebae: what part do they play in healthcare-associated infections?

Free-living amoebae (FLA) are ubiquitous protozoa that do not require a host organism for survival. They are found in natural environments such as water or soil, and man-made environments including tap water or swimming pools, where they may interact with other micro-organisms, including bacteria, fungi and viruses. FLA can harbour micro-organisms including those found in hospital water systems, offering them protection against hostile conditions, providing a vehicle of dissemination, and enabling them to prepare for subsequent survival in macrophages. The interaction between Legionella pneumophila and FLA has been studied extensively; subsequent investigations have shown that FLA may serve as a reservoir for other bacteria including mycobacteria, Pseudomonas aeruginosa, Acinetobacter baumannii, or even fungi and viruses. Amoebae found in hospital water systems can serve as a reservoir of potential pathogens and thus be indirectly related to healthcare-associated infections.

Cellular, Biochemical, and Molecular Changes during Encystment of Free-Living Amoebae

ABSTRACT Free-living amoebae are protozoa found in soil and water. Among them, some are pathogenic and many have been described as potential reservoirs of pathogenic bacteria. Their cell cycle is divided into at least two forms, the trophozoite and the cyst, and the differentiation process is named encystment. As cysts are more resistant to disinfection treatments than trophozoites, many studies focused on encystment, but until recently, little was known about cellular, biochemical, and molecular modifications operating during this process. Important signals and signaling pathways at play during encystment, as well as cell responses at the molecular level, have been described. This review summarizes our knowledge and focuses on new findings.

Sensitivity of free-living amoeba trophozoites and cysts to water disinfectants

Free-living amoebae are naturally present in water. These protozoa could be pathogenic and could also shelter pathogenic bacteria. Thus, they are described as a potential hazard for health. Also, free-living amoebae have been described to be resistant to biocides, especially under their cyst resistant form. There are several studies on amoeba treatments but none of them compare sensitivity of trophozoites and cysts from different genus to various water disinfectants. In our study, we tested chlorine, monochloramine and chlorine dioxide on both cysts and trophozoites from three strains, belonging to the three main genera of free-living amoebae. The results show that, comparing cysts to trophozoites inactivation, only the Acanthamoeba cysts were highly more resistant to treatment than trophozoites. Comparison of the disinfectant efficiency led to conclude that chlorine dioxide was the most efficient treatment in our conditions and was particularly efficient against cysts. In conclusion, our results would help to adapt water treatments in order to target free-living amoebae in water networks.

Passage through Tetrahymena tropicalis enhances the resistance to stress and the infectivity of Legionella pneumophila

Legionella pneumophila is a gram-negative bacterium prevalent in fresh water which accidentally infects humans and is responsible for the disease called legionellosis. Intracellular growth of L. pneumophila in Tetrahymena is inconsistent; in the species Tetrahymena tropicalis stationary-phase forms (SPFs) of L. pneumophila differentiate into mature intracellular forms (MIFs) without apparent bacterial replication and are expelled from the ciliate as pellets containing numerous MIFS. In the present work, we tested the impact of L. pneumophila passage through T. tropicalis. We observed that MIFs released from T. tropicalis are more resistant to various stresses than SPFs. Under our conditions, MIFs harboured a higher gentamicin resistance, maintained even after 3 months as pellets. Long-term survival essays revealed that MIFs survived better in a nutrient-poor environment than SFPs, as a reduction of only about 3 logs was observed after 4 months in the MIF population, whereas no cultivable SPFs were detected after 3 months in the same medium, corresponding to a loss of about 7 logs. We have also observed that MIFs are significantly more infectious in human pneumocyte cells compared with SPFs. These results strongly suggest a potential role of ciliates in increasing the risk of legionellosis.

Update on infectious risks associated with dental unit waterlines.

Modern dental chair units consist of a network of interconnected narrow-bore plastic tubes called dental unit waterlines (DUWLs). The water delivered by these DUWLs acts as both a coolant for a range of instruments and an irrigant during dental treatments. The quality of water is of considerable importance because both patients and dental team are regularly exposed to water and aerosols generated by dental equipment. Studies have demonstrated that DUWLs provide a favourable environment for microbial proliferation and biofilm formation, and that water is consequently often contaminated with high densities of various microorganisms (bacteria, fungi, protozoa, viruses). The presence of high levels of microbial contamination may be a health problem for dentists and patients, especially those who are immunocompromised. The current status of knowledge on microbial contamination of DUWLs is presented, with an emphasis on the infectious risk associated with DUWLs and on the various approaches for disinfecting and protecting DUWLs.

Acanthamoeba castellanii: proteins involved in actin dynamics, glycolysis, and proteolysis are regulated during encystation.

Acanthamoeba castellanii is a pathogenic free-living amoeba. Cyst forms are particularly important in their pathogenicity, as they are more resistant to treatments and might protect pathogenic intracellular bacteria. However, encystation is poorly understood at the molecular level and global changes at the protein level have not been completely described. In this study, we performed two-dimensional gel electrophoresis to compare protein expression in trophozoite and cyst forms. Four proteins, specifically expressed in trophozoites, and four proteins, specifically expressed in cysts, were identified. Two proteins, enolase and fructose bisphosphate aldolase, are involved in the glycolytic pathway. Three proteins are likely actin-binding proteins, which is consistent with the dramatic morphological modifications of the cells during encystation. One protein belongs to the serine protease family and has been already linked to encystation in A. castellanii. In conclusion, this study found that the proteins whose expression was modified during encystation were likely involved in actin dynamics, glycolysis, and proteolysis.

Purification and characterisation of a metallopeptidase of Candida albicans.

A novel aminopeptidase was purified by high performance liquid chromatography from a cytosoluble 100,000 g extract of Candida albicans on the basis of its ability to cleave L-arginine 7-amino-4-methylcoumarin. The purification factor was 36 and the yield was 20%. The native enzyme had a mol. wt of 52 kDa as demonstrated by SDS-PAGE in the presence or absence of reducing conditions and exhibited an iso-electric point of 4.3. The aminopeptidase showed optimum activity at pH 7.2, a Michaelis constant of c. 50 microM and a Vmax at 19 mM AMC released/min/mg of protein for L-Arg-AMC. This enzyme was shown to cleave at low affinity L-leucine-7-amino-4-methylcoumarin as demonstrated by the spectrofluorimetric method. The enzyme was strongly inhibited by specific metallo-enzyme inhibitors-EDTA and o-phenanthroline. Furthermore, there is evidence that a similar or identical enzyme occurs in other C. albicans clinical isolates and other Candida spp.