Fungal biofilms formed on various types of medical implants represent a major problem for hospitalized patients. These biofilms and related infections are usually difficult to treat because of their ...resistance to the classical antifungal drugs. Animal models are indispensable for investigating host-pathogen interactions and for identifying new antifungal targets related to biofilm development. A limited number of animal models is available that can be used for testing novel antifungal drugs in vivo against C. albicans, one of the most common pathogens causing fungal biofilms. Fungal load in biofilms in these models is traditionally analyzed postmortem, requiring host sacrifice and enumeration of microorganisms from individual biofilms in order to evaluate the amount of colony forming units and the efficacy of antifungal treatment. Bioluminescence imaging (BLI) made compatible with small animal models for in vivo biofilm formation is a valuable noninvasive tool to follow-up biofilm development and its treatment longitudinally, reducing the number of animals needed for such studies. Due to the nondestructive and noninvasive nature of BLI, the imaging procedure can be repeated in the same animal, allowing follow-up of the biofilm growth in vivo without removing the implanted device or detaching the biofilm from its substrate. The method described here introduces BLI of C. albicans biofilm formation in vivo on subcutaneously implanted catheters in mice. One of the main challenges to overcome for BLI of fungi is the hampered intracellular substrate delivery through the fungal cell wall, which is managed by using extracellularly located Gaussia luciferase. Although detecting a quantifiable in vivo BLI signal from biofilms formed on the inside of implanted catheters is challenging, BLI proved to be a practical tool in the study of fungal biofilms. This method describing the use of BLI for in vivo follow-up of device-related fungal biofilm formation has the potential for efficient in vivo screening for interesting genes of the pathogen and the host involved in C. albicans biofilm formation as well as for testing novel antifungal therapies.
We here report on the in vitro activity of toremifene to inhibit biofilm formation of different fungal and bacterial pathogens, including Candida albicans, Candida glabrata, Candida dubliniensis, ...Candida krusei, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. We validated the in vivo efficacy of orally administered toremifene against C. albicans and S. aureus biofilm formation in a rat subcutaneous catheter model. Combined, our results demonstrate the potential of toremifene as a broad-spectrum oral antibiofilm compound.
The expansion of industry and the use of pesticides in agriculture represent one of the major causes of environmental contamination. Unfortunately, individuals and animals are exposed to these ...foreign and often toxic substances on a daily basis. Therefore, it is crucial to monitor the impact of such chemicals on human health. Several in vitro studies have addressed this issue, but it is difficult to explore the impact of these compounds on living organisms. A nematode Caenorhabditis elegans has become a useful alternative to animal models mainly because of its transparent body, fast growth, short life cycle, and easy cultivation. Furthermore, at the molecular level, there are significant similarities between humans and C. elegans. These unique features make it an excellent model to complement mammalian models in toxicology research. Heavy metals and pesticides, which are considered environmental contaminants, are known to have affected the locomotion, feeding behavior, brood size, growth, life span, and cell death of C. elegans. Today, there are increasing numbers of research articles dedicated to this topic, of which we summarized the most recent findings dedicated to the effect of heavy metals, heavy metal mixtures, and pesticides on the well-characterized nervous system of this nematode.
can attach to various medical implants and forms thick biofilms despite its inability to switch from yeast to hyphae. The current
biofilm models only provide limited information about colonization ...and infection and usually require animal sacrifice. To gain real-time information from individual BALB/c mice, we developed a noninvasive imaging technique to visualize
biofilms in catheter fragments that were subcutaneously implanted on the back of mice. Bioluminescent
reporter strains (
7/2/4 and
8/1/4), free of auxotrophic markers, expressing a codon-optimized firefly luciferase were generated. A murine subcutaneous model was used to follow real-time
biofilm formation in the presence and absence of fluconazole and caspofungin. The fungal load in biofilms was quantified by CFU counts and by bioluminescence imaging (BLI).
biofilms formed within the first 24 h, as documented by the increased number of device-associated cells and elevated bioluminescent signal compared with adhesion at the time of implant. The
model allowed monitoring of the antibiofilm activity of caspofungin against
biofilms through bioluminescent imaging from day four after the initiation of treatment. Contrarily, signals emitted from biofilms implanted in fluconazole-treated mice were similar to the light emitted from control-treated mice. This study gives insights into the real-time development of
biofilms under
conditions. BLI proved to be a dynamic, noninvasive, and sensitive tool to monitor continuous biofilm formation and activity of antifungal agents against
biofilms formed on abiotic surfaces
.
1 Department of Molecular Microbiology, VIB, K.U. Leuven,
Kasteelpark Arenberg 31, B-3001 Leuven, Belgium
2 Laboratory of Molecular Cell Biology, Institute of
Botany and Microbiology, K.U. Leuven, ...Kasteelpark Arenberg 31, B-3001 Leuven,
Belgium
3 Comenius University in Bratislava, Faculty of Natural
Sciences, Department of Microbiology and Virology, Mlynská dolina B-2,
842 15 Bratislava, Slovak Republic
4 Department of Chemistry, Laboratory of Biochemistry,
Molecular and Structural Biology, K.U. Leuven, Celestijnenlaan 200 G, B-3001
Heverlee, Belgium
5 Department of Medical Diagnostic Sciences, Laboratory
of Experimental Microbiology, K.U. Leuven, Herestraat 49, B-3000 Leuven, Belgium
Device-associated microbial growth, including Candida biofilms,
represents more than half of all human microbial infections and, despite a
relatively small risk of implant-associated diseases, this type of infection
usually leads to high morbidity, increased health-care costs and prolonged
antimicrobial therapy. Animal models are needed to elucidate the complex host–pathogen
interactions that occur during the development of attached and structured
biofilm populations. We describe here a new in vivo model to study Candida biofilm, based on the avascular implantation of small catheters
in rats. Polyurethane biomaterials challenged with Candida cells
were placed underneath the skin of immunosuppressed animals following only
minor surgery. The model allowed the study of up to ten biofilms at once,
and the recovery of mature biofilms from 2 days after implantation.
The adhering inoculum was adjusted to the standard threshold of positive diagnosis
of fungal infection in materials recovered from patients. Wild-type biofilms
were mainly formed of hyphal cells, and they were unevenly distributed across
the catheter length as observed in infected materials in clinical cases. The
hyphal multilayered structure of the biofilms of wild-type strains was observed
by confocal microscopy and compared to the monolayer of yeast or hyphal cells
of two well-known biofilm-deficient strains, efg1 /efg1 cph1 /cph1 and bcr1 /bcr1 , respectively. The subcutaneous Candida biofilm
model relies on the use of implanted catheters with accessible, fast and minor
surgery to the animals. This model can be used to characterize the ability
of antimicrobial agents to eliminate biofilms, and to evaluate the prophylactic
effect of antifungal drugs and biomaterial coatings.
Correspondence Patrick Van Dijck Patrick.vandijck{at}mmbio.vib-kuleuven.be
Abbreviations: CVC, central venous catheter
These authors contributed equally to this work.
We previously synthesized several series of compounds, based on the 5-aryl-2-aminoimidazole scaffold, that showed activity preventing the formation of Salmonella enterica serovar Typhimurium and ...Pseudomonas aeruginosa biofilms. Here, we further studied the activity spectrum of a number of the most active N1- and 2N-substituted 5-aryl-2-aminoimidazoles against a broad panel of biofilms formed by monospecies and mixed species of bacteria and fungi. An N1-substituted compound showed very strong activity against the biofilms formed by Gram-negative and Gram-positive bacteria and the fungus Candida albicans but was previously shown to be toxic against various eukaryotic cell lines. In contrast, 2N-substituted compounds were nontoxic and active against biofilms formed by Gram-negative bacteria and C. albicans but had reduced activity against biofilms formed by Gram-positive bacteria. In an attempt to develop nontoxic compounds with potent activity against biofilms formed by Gram-positive bacteria for application in antibiofilm coatings for medical implants, we synthesized novel compounds with substituents at both the N1 and 2N positions and tested these compounds for antibiofilm activity and toxicity. Interestingly, most of these N1-,2N-disubstituted 5-aryl-2-aminoimidazoles showed very strong activity against biofilms formed by Gram-positive bacteria and C. albicans in various setups with biofilms formed by monospecies and mixed species but lost activity against biofilms formed by Gram-negative bacteria. In light of application of these compounds as anti-infective coatings on orthopedic implants, toxicity against two bone cell lines and the functionality of these cells were tested. The N1-,2N-disubstituted 5-aryl-2-aminoimidazoles in general did not affect the viability of bone cells and even induced calcium deposition. This indicates that modulating the substitution pattern on positions N1 and 2N of the 5-aryl-2-aminoimidazole scaffold allows fine-tuning of both the antibiofilm activity spectrum and toxicity.
In this study, we demonstrated that in vitro Candida albicans biofilms grown in the presence of didofenac showed increased susceptibility to caspofungin. These findings were further confirmed using a ...catheterassociated biofilm model in rats. C. albicans-inoculated catheters retrieved from rats that were treated with both didofenac and caspofungin contained significantly fewer biofilm cells and showed no visible biofilms inside the catheter lumens, as documented by scanning electron microscopy, as compared to catheters retrieved from rats receiving only caspofungin or didofenac. This report indicates that didofenac could be useful in combination therapy with caspofungin to treat C. albicans biofilm-associated infections.
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