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Abstract
Biofilms represent the most prevalent type of microbial growth in
nature and are crucial to the development of clinical infections. They can
serve as a cause for disease and are often associated with high-level
antimicrobial resistance of the associated organisms.
The objectives of this work were to study the effect of different
antifungal drugs on the preformed C. albicans biofilm and to study the
antifungal penetration pattern as a mechanism of biofilm resistance.
A total of 152 isolates of Candida isolates were collected from
different clinical specimens from Egyptian hospitals. They were
identified as C. albicans (64); C. tropicalis (42); C. krusei (35); and C.
glabrata (11).
The antifungal sensitivity pattern and the antifungal`s minimum
inhibitory concentrations (MICs) of the tested planktonic C. albicans was
determined using the agar dilution method on RPMI-1640 medium. The
obtained data showed that nystatin showed activity against all tested
isolates followed by amphotericin B and clotrimazole which showed
resistance by only 3 isolates.
Determinations of antifungal susceptibilities and degree of
adherence of biofilm-grown Candida isolates were done in 96-well
microtiter plates using RPMI medium and using the semi-quantitative
XTT-reduction assay. For many years, the XTT assay has been the
mainstay for the estimation of biomass in yeast biofilms. The tetrazolium
salt XTT is intracellularly reduced to a water soluble formazan, which is
colorimetrically determined in the cell supernatant. The obtained results
showed that biofilm originated from vaginal isolates were more
complicated with high degree of adherence than those from catheters and
urinary tract infections. C. albicans cells in biofilm conditions display dramatically increased resistance to antifungal agents compared to that of
cells in planktonic conditions.
This work moved then to study the different stages and kinetic of
biofilm formation on the wells of microtiter plates over 48 hrs. The
results showed that the biofilms were highly metabolically active after the
first 12 hours but the complexity increased after (24 to 48 hrs). A marked
decrease in the complexity and the cellular density of the formed biofilm
when exposing C. albicans to planktonic subinhibitory levels of
antifungal agents and these results may point to approaches for preventive
or prophylactic treatment. A severe DROP in the finally formed biofilm
was obtained when adding nystatin at concentration equal to biofilm sub
SMIC50 after 3 and 6 hrs. While, there was a paradoxical rise in metabolic
activity of mature biofilms when adding nystatin after 12 or 24 hrs.
An investigation and evaluation of the penetration of antifungal
agents through 48-h biofilms (as a possible mechanism that may protect
microorganisms in biofilms from antibiotics) using polycarbonate
membrane filter were done. The results demonstrated that azole
antifungal agents permeated all Candida biofilms more rapidly than
terbinafine and polyene antifungal agents. Viability of biofilm cells after
exposure to antifungal agents for 24 h reveals the failure of penetrated
drug to produce complete killing of biofilm cells. Although, amphotericin
B was the least penetrant through biofilms but viable count observations
revealed that it caused the most damage to the biofilm cells in comparison
to fluconazole. These results indicate that poor drug penetration is not a
major resistance mechanism for Candida biofilms.
Scanning electron microscope (SEM) revealed that the fully mature
biofilms is produced after incubation for up to 48 hours and it is consisted
of a dense network of yeast cells. There was a wrinkled, ruptured, and
ballooning effect of the drug on yeast cells after applying nystatin in its
inhibitory concentration on 24-h biofilms.