Fernando César Bizerra

Biofilm production and evaluation of antifungal susceptibility amongst clinical Candida spp. isolates, including strains of the Candida parapsilosis complex

Candida cells can form biofilms that frequently are sources of infections and are less susceptible to antifungal drugs. Some authors have reported that Candida orthopsilosis and Candida metapsilosis isolates are not able to produce biofilms in vitro and there are no studies available on biofilm susceptibility for these species to antifungals. The aims of this study were to (i) quantify Candida spp. biofilms in vitro, and (ii) test the in vitro susceptibilities of Candida spp. biofilms to fluconazole (FLC) and amphotericin B (AMB). Isolates studied included four Candida albicans, six C. tropicalis, seven C. parapsilosis, eight C. orthopsilosis, and five C. metapsilosis. We compared two different methods to evaluate biofilm production, i.e., crystal violet (CV) staining and XTT-reduction assays (XTT). Scanning electron microscopy (SEM) was used to observe high, medium and low biofilm producing isolates screened by these two methods. To determine the minimum biofilm eradication concentration (MBEC) for FLC and AMB, XTT-reduction assay was used to measure cell metabolic activity. Biofilm quantification by CV and XTT showed that C. tropicalis isolates were the highest biofilm producer, followed by C. albicans, C. parapsilosis, C. orthopsilosis and C. metapsilosis. Examination of SEM images revealed that the extent of biofilms formed by high, medium, and low producers was highly correlated to the results generated by CV assay. Biofilm of all the isolates evaluated were resistant to FLC (MBEC(80) ≥ 256 ug/ml) but, in general, susceptible to AMB, except for six C. parapsilosis strains (MBEC(80) ≥ 8 ug/ml).

Changes in Cell Wall Synthesis and Ultrastructure during Paradoxical Growth Effect of Caspofungin on Four Different Candida Species

ABSTRACT Paradoxical growth (PG) has been described for echinocandins and is characterized by cell growth at drug concentrations above the MIC. In this study, two isolates each of Candida albicans, C. tropicalis, C. orthopsilosis, and C. parapsilosis, all of which displaying PG in response to caspofungin, were subjected to MIC, minimal fungicidal concentration (MFC), and time-kill curve assays to evaluate the levels of PG. Cell wall components and ultrastructural modifications of the PG cells were also investigated. The results showed that when cell growth and survival were evaluated by MFC or time-kill curve assays, high concentrations of caspofungin did not show fungicidal activity against PG cells. Furthermore, for C. parapsilosis and C. orthopsilosis, time-kill curves were more discriminatory than MFCs in detecting the PG effect. The four different Candida species studied demonstrated similar alterations in cell wall components and ultrastructure associated with PG. In PG cells, β-1,3-glucan content decreased from 2.7- to 7.8-fold, whereas chitin content increased from 4.0- to 6.6-fold. An electron microscopy study of the PG cells revealed morphological alterations, clumping of cells, enlarged cells, the absence of filamentation, abnormal septa, and accumulation of chitin in the cell wall. Also, PG cells basically exhibited a single dark high-density layer in the cell wall, indicating the loss of the β-1,3-glucan layer. Our results present novel details about the ultrastructural alterations that occur in C. albicans, C. parapsilosis, C. orthopsilosis, and C. tropicalis during PG and show that chitin is the major component of the cell walls of PG cells. Stimulation of chitin synthesis may represent a rescue mechanism against caspofungin activity.

Molecular Identification, Antifungal Susceptibility Profile, and Biofilm Formation of Clinical and Environmental Rhodotorula Species Isolates

ABSTRACT Rhodotorula species are emergent fungal pathogens capable of causing invasive infections, primarily fungemia. They are particularly problematic in immunosuppressed patients when using a central venous catheter. In this study, we evaluated the species distribution of 51 clinical and 8 environmental Rhodotorula species isolates using the ID32C system and internal transcribed spacer (ITS) sequencing. Antifungal susceptibility testing and biofilm formation capability using a crystal violet staining assay were performed. Using ITS sequencing as the gold standard, the clinical isolates were identified as follows: 44 R. mucilaginosa isolates, 2 R. glutinis isolates, 2 R. minuta isolates, 2 R. dairenensis isolates, and 1 Rhodosporidium fluviale isolate. The environmental isolates included 7 R. mucilaginosa isolates and 1 R. slooffiae isolate. Using the ID32C system, along with a nitrate assimilation test, only 90.3% of the isolates tested were correctly identified. In the biofilm formation assay, R. mucilaginosa and R. minuta exhibited greater biofilm formation ability compared to the other Rhodotorula species; the clinical isolates of R. mucilaginosa showed greater biofilm formation compared to the environmental isolates (P = 0.04). Amphotericin B showed good in vitro activity (MIC ≤ 1 μg/ml) against planktonic cells, whereas voriconazole and posaconazole showed poor activity (MIC50/MIC90, 2/4 μg/ml). Caspofungin and fluconazole MICs were consistently high for all isolates tested (≥64 μg/ml and ≥ 4 μg/ml, respectively). In this study, we emphasized the importance of molecular methods to correctly identify Rhodotorula species isolates and non-R. mucilaginosa species in particular. The antifungal susceptibility profile reinforces amphotericin B as the antifungal drug of choice for the treatment of Rhodotorula infections. To our knowledge, this is the first study evaluating putative differences in the ability of biofilm formation among different Rhodotorula species.

In Vitro Evaluation of Putative Virulence Attributes of Oral Isolates of Candida spp. Obtained from Elderly Healthy Individuals

Identification of Candida isolates obtained from oral cavity of elderly healthy individuals revealed the predominance of non-albicans Candida species (88.9%) compared to Candida albicans (11%). CHROMagar Candida differential medium and PCR revealed the presence of Candida tropicalis (33.3%), Candida glabrata (27.8%), and Candida krusei (16.7%). We investigated the presence of virulence attributes in a total of 18 isolates, including acid protease and phospholipase production, hemolytic activity, and biofilm production. Extracellular protease was found in five isolates (27.8%) whereas extracellular phospholipase was found in three isolates (17%). All isolates showed hemolytic activity. About 56% of the isolates were weakly positive for biofilm formation (score +) whereas a minority (5.6%) of them showed strong biofilm formation (score 4+). Susceptibility in vitro of the isolates to fluconazole was carried out by microdilution method. Fluconazole showed a strong inhibition against most buccal isolates. The resistant isolates were 2 C. tropicalis, 2 C. glabrata, and 1 C. krusei.

Unraveling the antifungal activity of a South American rattlesnake toxin crotamine

Crotamine is a highly basic peptide from the venom of Crotalus durissus terrificus rattlesnake. Its common gene ancestry and structural similarity with the β-defensins, mainly due to an identical disulfide bond pattern, stimulated us to assess the antimicrobial properties of native, recombinant, and chemically synthesized crotamine. Antimicrobial activities against standard strains and clinical isolates were analyzed by the colorimetric microdilution method showing a weak antibacterial activity against both Gram-positive and Gram-negative bacteria [MIC (Minimum Inhibitory Concentration) of 50->200 μg/mL], with the exception of Micrococcus luteus [MIC ranging from 1 to 2 μg/mL]. No detectable activity was observed for the filamentous fungus Aspergillus fumigatus and Trichophyton rubrum at concentrations up to 125 μg/mL. However, a pronounced antifungal activity against Candida spp., Trichosporon spp., and Cryptococcus neoformans [12.5-50.0 μg/mL] was observed. Chemically produced synthetic crotamine in general displayed MIC values similar to those observed for native crotamine, whereas recombinant crotamine was overridingly more potent in most assays. On the other hand, derived short linear peptides were not very effective apart from a few exceptions. Pronounced ultrastructure alteration in Candida albicans elicited by crotamine was observed by electron microscopy analyses. The peculiar specificity for highly proliferating cells was confirmed here showing potential low cytotoxic effect of crotamine against nontumoral mammal cell lines (HEK293, PC12, and primary culture astrocyte cells) compared to tumoral B16F10 cells, and no hemolytic activity was observed. Taken together these results suggest that, at low concentration, crotamine is a potentially valuable anti-yeast or candicidal agent, with low harmful effects on normal mammal cells, justifying further studies on its mechanisms of action aiming medical and industrial applications.

Breakthrough Candidemia Due to Multidrug-Resistant Candida glabrata during Prophylaxis with a Low Dose of Micafungin

ABSTRACT We identified a case of breakthrough candidemia in a 25-year-old patient receiving micafungin prophylaxis (50 mg/day). Five Candida glabrata isolates were obtained from blood cultures and were classified as multidrug-resistant isolates, since all of them exhibited high MICs for echinocandin and azole drugs. A mutation (S663F) in hot spot 1 of the FKS2 gene was found in all five isolates. This mutation yielded a 1,3-β-d-glucan synthase enzyme with highly reduced sensitivities to echinocandin drugs.

Multiple species of Trichosporon produce biofilms highly resistant to triazoles and amphotericin B.

Invasive infections caused by Trichosporon spp. have increased considerably in recent years, especially in neutropenic and critically ill patients using catheters and antibiotics. The genus presents limited sensitivity to different antifungal agents, but triazoles are the first choice for treatment. Here, we investigated the biofilm production and antifungal susceptibility to triazoles and amphotericin B of 54 Trichosporon spp. isolates obtained from blood samples (19), urine (20) and superficial mycosis (15). All isolates and 7 reference strains were identified by sequence analysis and phylogenetic inferences of the IGS1 region of the rDNA. Biofilms were grown on 96-well plates and quantitation was performed using crystal violet staining, complemented with Scanning Electron Microscopy (SEM). Susceptibility tests for fluconazole, itraconazole, voriconazole and amphotericin B were processed using the microdilution broth method (CLSI) for planktonic cells and XTT reduction assay for biofilm-forming cells. Our results showed that T. asahii was the most frequent species identified (66.7%), followed by T. faecale (11.1%), T. asteroides (9.3%), T. inkin (7.4%), T. dermatis (3.7%) and one T. coremiiforme (1.8%). We identified 4 genotypes within T. asahii isolates (G1, G3, G4 and G5) and 2 genotypes within T. faecale (G1 and G3). All species exhibited high adhesion and biofilm formation capabilities, mainly T. inkin, T. asteroides and T. faecale. Microscopy images of high biofilm-producing isolates showed that T. asahii presented mainly hyphae and arthroconidia, whereas T. asteroides exhibited mainly short arthroconidia and few filaments. Voriconazole exhibited the best in vitro activity against all species tested. Biofilm-forming cells of isolates and reference strains were highly resistant to all antifungals tested. We concluded that levels of biofilm formation by Trichosporon spp. were similar or even greater than those described for the Candida genus. Biofilm-forming cells were at least 1,000 times more resistant to antifungals than planktonic cells, especially to voriconazole.

Accurate Identification of Candida parapsilosis (Sensu Lato) by Use of Mitochondrial DNA and Real-Time PCR

ABSTRACT Candida parapsilosis is the Candida species isolated the second most frequently from blood cultures in South America and some European countries, such as Spain. Since 2005, this species has been considered a complex of 3 closely related species: C. parapsilosis, Candida metapsilosis, and Candida orthopsilosis. Here, we describe a real-time TaqMan-MGB PCR assay, using mitochondrial DNA (mtDNA) as the target, which readily distinguishes these 3 species. We first used comparative genomics to locate syntenic regions between these 3 mitochondrial genomes and then selected NADH5 as the target for the real-time PCR assay. Probes were designed to include a combination of different single-nucleotide polymorphisms that are able to differentiate each species within the C. parapsilosis complex. This new methodology was first tested using mtDNA and then genomic DNA from 4 reference and 5 clinical strains. For assay validation, a total of 96 clinical isolates and 4 American Type Culture Collection (ATCC) isolates previously identified by internal transcribed spacer (ITS) ribosomal DNA (rDNA) sequencing were tested. Real-time PCR using genomic DNA was able to differentiate the 3 species with 100% accuracy. No amplification was observed when DNA from other species was used as the template. We observed 100% congruence with ITS rDNA sequencing identification, including for 30 strains used in blind testing. This novel method allows a quick and accurate intracomplex identification of C. parapsilosis and saves time compared with sequencing, which so far has been considered the “gold standard” for Candida yeast identification. In addition, this assay provides a useful tool for epidemiological and clinical studies of these emergent species.

In Vitro Susceptibility of a Large Collection of Candida Strains Against Fluconazole and Voriconazole by Using the CLSI Disk Diffusion Assay

We evaluated all Candida spp. isolates obtained from patients admitted to two tertiary care hospitals between 1999 and 2003 in the city of São Paulo, Brazil. The in vitro activities of fluconazole (FCZ) and voriconazole were determined by the agar disk diffusion test using the Clinical and Laboratory Standards Institute M44-A guidelines. The inhibition zone diameters were read and interpreted automatically by the BIOMIC® image-analysis plate reader system. We tested a total of 4,625 strains, including 2,393 strains of C. albicans (51.7%), 658 of C. tropicalis (14.2%), 503 of C. glabrata (10.9%), 495 of C. parapsilosis (10.7%), 292 of C. rugosa (6.3%), 195 of C. guilliermondii (4.2%) and 89 of other Candida species (2.0%). Only 2.0% of the strains tested were classified as dose-dependent susceptible (DDS), and 5.8% of them were resistant to FCZ. The resistance or DDS to fluconazole was verified mainly among C. glabrata (7.8%), C. krusei (67.9%) and C. rugosa (65.1%). Voriconazole exhibited better activity in vitro than fluconazole, even in isolates fluconazole resistant. The resistance of fluconazole and voriconazole did not increase in the isolates of Candida spp. during the evaluated period.

Antimicrobial compounds from natural sources

Infectious diseases are one of the main causes of morbidity and mortality worldwide. Nowadays many infections are often caused by multi-resistant microorganisms resulting in difficult to treat diseases and, consequently, substantial increases in healthcare costs. The relative easy access to the antimicrobials and also the massive employment of these compounds for industrial purposes, including food production, have both strongly contributed to the progressive increase of resistant microorganisms. As a result, these multi-resistant microorganisms are reasserting themselves as worldwide threats. Research into natural products has demonstrated significant progress in the discovery of new compounds with antimicrobial activity. In fact, nature is a generous source of compounds with the potential to treat diseases, including infectious diseases. Among the known sources of natural compounds with valuable antimicrobial activity, we highlighted the medicinal plants and marine and terrestrial organisms, including fungi and bacteria. Nevertheless, there is still a vast fauna and flora that once systematically explored, could provide additional antimicrobial leads and new drugs. Thousands of natural products with the potential to act as antimicrobial compounds or as a structural lead compound still await further investigation. In this Research Topic Ebook, we present several scientific studies mainly focused on natural products with antimicrobial activity, which are the case of the natural antimicrobial peptides (AMPs) and host defense peptides (HDPs). This topic also includes recent studies on the roles of honey hydrogen peroxide in antimicrobial activity against resistant microbial strains, as well as the use of essential oils for food preservation. Such a wide and interesting topic also gave us an opportunity to include diverse sources, including plants, terrestrial and sea animals. Not to mention the interesting and unusual sources such as coal or lignite, which may provide future antimicrobial compounds candidates. The recent development of a patented process to GMP standards (PA107470/GB), rendering the obtainment of carbohydrate derived fulvic acid (CHD-FA), stimulated Sherry et al. (2012) to study and describe for the first time a highly effective novel antiseptic effect of fulvic acid with exquisite biofilm activity that acts by disrupting cell membranes. The antifungic peptide from Amazonian Pink Toe spider Juruin, described by Ayroza et al. (2012) is another outstanding example of the potential contribution of a systematic exploration of nature aiming to provide additional antimicrobial leads and drugs. In other words, nature is a generous source of compounds, with the potential to treat diseases, including infectious diseases. Studies exploiting the mechanism of action and the structure-activity aspects of these natural compounds may provide both additional antimicrobial leads and drugs, and also significant insight into potential possibilities to overcome the antimicrobial resistance.