Anti-Bacterial and Anti-Biofilm Activities of Anandamide against the Cariogenic Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium in the oral cavity involved in plaque formation and dental caries. The endocannabinoid anandamide (AEA), a naturally occurring bioactive lipid, has been shown to have anti-bacterial and anti-biofilm activities against Staphylococcus aureus. We aimed here to study its effects on S. mutans viability, biofilm formation and extracellular polysaccharide substance (EPS) production. S. mutans were cultivated in the absence or presence of various concentrations of AEA, and the planktonic growth was followed by changes in optical density (OD) and colony-forming units (CFU). The resulting biofilms were examined by MTT metabolic assay, Crystal Violet (CV) staining, spinning disk confocal microscopy (SDCM) and high-resolution scanning electron microscopy (HR-SEM). The EPS production was determined by Congo Red and fluorescent dextran staining. Membrane potential and membrane permeability were determined by diethyloxacarbocyanine iodide (DiOC2(3)) and SYTO 9/propidium iodide (PI) staining, respectively, using flow cytometry. We observed that AEA was bactericidal to S. mutans at 12.5 µg/mL and prevented biofilm formation at the same concentration. AEA reduced the biofilm thickness and biomass with concomitant reduction in total EPS production, although there was a net increase in EPS per bacterium. Preformed biofilms were significantly affected at 50 µg/mL AEA. We further show that AEA increased the membrane permeability and induced membrane hyperpolarization of these bacteria. AEA caused S. mutans to become elongated at the minimum inhibitory concentration (MIC). Gene expression studies showed a significant increase in the cell division gene ftsZ. The concentrations of AEA needed for the anti-bacterial effects were below the cytotoxic concentration for normal Vero epithelial cells. Altogether, our data show that AEA has anti-bacterial and anti-biofilm activities against S. mutans and may have a potential role in preventing biofilms as a therapeutic measure.

The Distinction between Dematiaceous Molds and Non-Dematiaceous Fungi in Clinical and Spiked Samples Treated with Hydrogen Peroxide Using Direct Fluorescence Microscopy.

Dematiaceous fungi are pigmented molds with a high content of melanin in their cell walls that can cause fatal infections in immunocompromised hosts. Direct microscopy is the main method for the rapid diagnosis of dematiaceous fungi in clinical specimens. However, it is often difficult to distinguish their hyphae from non-dematiaceous hyphae and yeast pseudohyphae. Our aim was to develop a fluorescence staining method that targets melanin for the detection of dematiaceous molds in clinical specimens. Glass slide smears of clinical samples and sterile bronchoalveolar lavage spiked with dematiaceous and non-dematiaceous fungi were treated with hydrogen peroxide, and digital images were recorded using direct microscopy with different fluorescent filters. The images of fungi were compared for their fluorescence intensity using the NIS-Elements software. The fluorescent signal between dematiaceous and non-dematiaceous fungi demonstrated a markedly increased mean intensity for dematiaceous molds following hydrogen peroxide treatment (7510.3 ± 10,427.6 vs. 0.3 ± 3.1, respectively, p < 0.0001). No fluorescent signal was detected in the absence of hydrogen peroxide. "Staining" fungal clinical specimens with hydrogen peroxide, followed by fluorescence microscopy examination, can differentiate between dematiaceous and non-dematiaceous fungi. This finding can be used for the detection of dematiaceous molds in clinical specimens and enables the early and appropriate treatment of infections.

Genotypes and population genetics of cryptococcus neoformans and cryptococcus gattii species complexes in Europe and the mediterranean area.

A total of 476 European isolates (310 Cryptococcus neoformans var. grubii, 150 C. neoformans var. neoformans, and 16 C. gattii species complex) from both clinical and environmental sources were analyzed by multi-locus sequence typing. Phylogenetic and population genetic analyses were performed. Sequence analysis identified 74 sequence types among C. neoformans var. neoformans (VNIV), 65 among C. neoformans var. grubii (56 VNI, 8 VNII, 1 VNB), and 5 among the C. gattii species complex (4 VGI and 1 VGIV) isolates. ST23 was the most frequent genotype (22%) among VNI isolates which were mostly grouped in a large clonal cluster including 50% of isolates. Among VNIV isolates, a predominant genotype was not identified. A high percentage of autochthonous STs were identified in both VNI (71%) and VNIV (96%) group of isolates. The 16 European C. gattii species complex isolates analyzed in the present study originated all from the environment and all belonged to a large cluster endemic in the Mediterranean area. Population genetic analysis confirmed that VNI group of isolates were characterized by low variability and clonal expansion while VNIV by a higher variability and a number of recombination events. However, when VNI and VNIV environmental isolates were compared, they showed a similar population structure with a high percentage of shared mutations and the absence of fixed mutations. Also linkage disequilibrium analysis reveals differences between clinical and environmental isolates showing a key role of PLB1 allele combinations in host infection as well as the key role of LAC1 allele combinations for survival of the fungus in the environment. The present study shows that genetic comparison of clinical and environmental isolates represents a first step to understand the genetic characteristics that cause the shift of some genotypes from a saprophytic to a parasitic life style.

The prevalence and clinical significance of microcolonies when tested according to contemporary interpretive breakpoints for fluconazole against Candida species using E-test.

Changes in the interpretive-breakpoints for antifungals against various Candida species have raised the need to examine the significance of the phenomenon of the growth of microcolonies in agar diffusion inhibition zones, which has generally been considered negligible. The objective was to determine the incidence of cases in which microcolonies demonstrate fluconazole resistance according to current interpretive-breakpoints and whether their growth is associated with therapeutic failure. The fluconazole minimum inhibitory concentrations (MICs) of 100 blood culture isolates of Candida were performed by E-test on Roswell Park Memorial Institute (RPMI) agar and examined for the appearance of microcolonies. Fluconazole MICs of microcolonies were then determined over three generations. The significance of the phenomenon of microcolonies was determined according to clinical data retrieved from electronic files. Microcolonies were a common phenomenon among Candida isolates following incubation on RPMI agar, with a higher frequency among C. albicans isolates as compared to non-albicans Candida across generations (57-93% vs 31-93%, respectively) and a similar fluconazole susceptibility rate over three generations. The rate of microcolonies was similar in both patients with successful and unsuccessful outcome (41% vs 42%, respectively). Microcolonies are a common phenomenon. No increase in MIC was demonstrated throughout three generations of microcolony inoculation on RPMI, and no difference in clinical outcome was observed.

Importance of Resolving Fungal Nomenclature: the Case of Multiple Pathogenic Species in the Cryptococcus Genus.

Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. The two varieties within C. neoformans were raised to species level, and the same was done for five genotypes within C. gattii. In a recent perspective (K. J. Kwon-Chung et al., mSphere 2:e00357-16, 2017, https://doi.org/10.1128/mSphere.00357-16), it was argued that this taxonomic proposal was premature and without consensus in the community. Although the authors of the perspective recognized the existence of genetic diversity, they preferred the use of the informal nomenclature "C. neoformans species complex" and "C. gattii species complex." Here we highlight the advantage of recognizing these seven species, as ignoring these species will impede deciphering further biologically and clinically relevant differences between them, which may in turn delay future clinical advances.

Fundamental niche prediction of the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii in Europe.

Fundamental niche prediction of Cryptococcus neoformans and Cryptococcus gattii in Europe is an important tool to understand where these pathogenic yeasts have a high probability to survive in the environment and therefore to identify the areas with high risk of infection. In this study, occurrence data for C. neoformans and C. gattii were compared by MaxEnt software with several bioclimatic conditions as well as with soil characteristics and land use. The results showed that C. gattii distribution can be predicted with high probability along the Mediterranean coast. The analysis of variables showed that its distribution is limited by low temperatures during the coldest season, and by heavy precipitations in the driest season. C. neoformans var. grubii is able to colonize the same areas of C. gattii but is more tolerant to cold winter temperatures and summer precipitations. In contrast, the C. neoformans var. neoformans map was completely different. The best conditions for its survival were displayed in sub-continental areas and not along the Mediterranean coasts. In conclusion, we produced for the first time detailed prediction maps of the species and varieties of the C. neoformans and C. gattii species complex in Europe and Mediterranean area.

Unique aggregation of conjugated amphotericin B and its interaction with lipid membranes.

The purpose of this paper was to investigate the aggregation of amphotericin B (AMB) and AMB-arabinogalactan conjugate (AMB-AGC), and the interactions of these drugs with free and membrane-embedded sterols. Aggregation of AMB and AMB-AGC was studied by circular dichroic (CD) and UV absorbance spectroscopic techniques. The effect of liposomes on the spectra was utilized to investigate the interactions of aggregates with membrane-embedded sterols. Interaction with free sterols was studied by measuring sterols' effect on AMB/AMB-AGC susceptibility test. The results demonstrated that AMB-AGC forms unique aggregates in aqueous solution which differ from those formed by free AMB. Ergosterol and cholesterol embedded in liposomes, affected the CD spectra obtained for both AMB and AMB-AGC, indicating interactions of these sterols with both drugs. Interaction with both cholesterol and ergosterol resulted in an increase of AMB-AGC's minimal inhibitory concentration (MIC) in Candida albicans. In conclusion, AMB-AGC forms unique aggregates in aqueous solution; these aggregates interact with membrane-embedded cholesterol and ergosterol and with free sterols. These results indicate that the selectivity of AMB-AGC to fungal cells may not occur due to inability to bind cholesterol but probably as a result of this unique aggregation. Understanding this mechanism may help to develop a safer AMB formulation for therapy.

The Effect of Novel Heterocyclic Compounds on Cryptococcal Biofilm.

Biofilm formation by microorganisms depends on their communication by quorum sensing, which is mediated by small diffusible signaling molecules that accumulate in the extracellular environment. During human infection, the pathogenic yeast Cryptococcus neoformans can form biofilm on medical devices, which protects the organism and increases its resistance to antifungal agents. The aim of this study was to test two novel heterocyclic compounds, S-8 (thiazolidinedione derivative, TZD) and NA-8 (succinimide derivative, SI), for their anti-biofilm activity against strains of Cryptococcus neoformans and Cryptococcus gattii. Biofilms were formed in a defined medium in 96-well polystyrene plates and 8-well micro-slides. The effect of sub-inhibitory concentrations of S-8 and NA-8 on biofilm formation was measured after 48 h by a metabolic reduction assay and by confocal laser microscopy analysis using fluorescent staining. The formation and development of cryptococcal biofilms was inhibited significantly by these compounds in concentrations below the minimum inhibitory concentration (MIC) values. These compounds may have a potential role in preventing fungal biofilm development on indwelling medical devices or even as a therapeutic measure after the establishment of biofilm.

Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean basin.

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

Association of chronic Candida albicans respiratory infection with a more severe lung disease in patients with cystic fibrosis.

BACKGROUND: Despite the increase in fungal isolates, the significance of chronic Candida albicans airway colonization in CF is unclear. AIM: To investigate the impact of C. albicans airway colonization on CF disease severity. METHODS: Longitudinal analysis of clinical data from CF patients followed during 2003-2009 at our CF center. Patients were stratified based on their C. albicans colonization status--chronic, intermittent, and none. RESULTS: A total of 4,244 cultures were obtained from 91 patients (mean age 19.7 years, range 5-68). The three colonization groups were similar in age, gender,and body mass index (BMI). Compared to the non-colonized group (n = 27, 30%), the chronic C. albicans colonization group (n = 34, 37%), had a significantly lower FEV1 percent predicted (74.3 ± 23.1% vs. 93.9% ± 22.2) with a higher annual rate of FEV1 decline (-1.9 ± 4.2% vs. 0.7 ± 4.5%). The patients who were intermittently colonized with C. albicans had intermediate values. CONCLUSIONS: Chronic respiratory colonization of C. albicans is associated with worsening of FEV1 in CF. Prospective studies are needed to confirm this finding and to corroborate whether indeed C. albicans drives a deleterious lung phenotype.

Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex.

Phylogenetic analysis of 11 genetic loci and results from many genotyping studies revealed significant genetic diversity with the pathogenic Cryptococcus gattii/Cryptococcus neoformans species complex. Genealogical concordance, coalescence-based, and species tree approaches supported the presence of distinct and concordant lineages within the complex. Consequently, we propose to recognize the current C. neoformans var. grubii and C. neoformans var. neoformans as separate species, and five species within C. gattii. The type strain of C. neoformans CBS132 represents a serotype AD hybrid and is replaced. The newly delimited species differ in aspects of pathogenicity, prevalence for patient groups, as well as biochemical and physiological aspects, such as susceptibility to antifungals. MALDI-TOF mass spectrometry readily distinguishes the newly recognized species.

Unraveling the biology of a fungal meningitis pathogen using chemical genetics.

The fungal meningitis pathogen Cryptococcus neoformans is a central driver of mortality in HIV/AIDS. We report a genome-scale chemical genetic data map for this pathogen that quantifies the impact of 439 small-molecule challenges on 1,448 gene knockouts. We identified chemical phenotypes for 83% of mutants screened and at least one genetic response for each compound. C. neoformans chemical-genetic responses are largely distinct from orthologous published profiles of Saccharomyces cerevisiae, demonstrating the importance of pathogen-centered studies. We used the chemical-genetic matrix to predict novel pathogenicity genes, infer compound mode of action, and to develop an algorithm, O2M, that predicts antifungal synergies. These predictions were experimentally validated, thereby identifying virulence genes, a molecule that triggers G2/M arrest and inhibits the Cdc25 phosphatase, and many compounds that synergize with the antifungal drug fluconazole. Our work establishes a chemical-genetic foundation for approaching an infection responsible for greater than one-third of AIDS-related deaths.

Activity, reduced toxicity, and scale-up synthesis of amphotericin B-conjugated polysaccharide.

Amphotericin B (AMB) arabinogalactan (AG) conjugate was synthesized by the conjugation of AMB to oxidized AG by reductive amination. The conjugate was evaluated for in vitro antifungal activity and in vivo toxicity. Optimization of the conjugation process was investigated using large batches of 100 g, which are 20 times larger than previously reported for AMB-AG conjugation. The efficacy of AMB-AG conjugates was studied as a function of reaction conditions and time, aldehyde/reducing agent mole ratio, and purification procedure. The most potent AMB-AG conjugate having low minimal inhibitory concentration (MIC) and high maximal tolerated dose (MTD) was obtained following reduction with NaBH4 at 1:2 mol ratio (AG units/NaBH4) at 25 °C for 24 h. AMB-AG conjugate prepared under these conditions demonstrated MIC of 0.5 mg/L (equiv of AMB) in Candida albicans, and an MTD of 60 mg/kg (equiv of AMB) in mice, while AMB clinical formulation (Fungizone) demonstrated high toxicity (MTD = 3 mg/kg). These findings confirm the simplicity and reproducibility of the conjugation allowing this method to be applied on larger scale production.

Anti-Candida albicans biofilm effect of novel heterocyclic compounds.

OBJECTIVES: The aims of this study were to develop new anti-biofilm drugs, examine their activity against Candida albicans biofilm and investigate their structure-activity relationship and mechanism of action. METHODS: A series of thiazolidinedione and succinimide derivatives were synthesized and their ability to inhibit C. albicans biofilm formation and destroy pre-formed biofilm was tested. The biofilms' structure, metabolic activity and viability were determined by XTT assay and propidium iodide and SYTO 9 live/dead stains combined with confocal microscopic analysis. The effect of the most active compounds on cell morphology, sterol distribution and cell wall morphology and composition was then determined by specific fluorescent stains and transmission electron microscopy. RESULTS: Most of the compounds were active at sub-MICs. Elongation of the aliphatic side chain resulted in reduced anti-biofilm activity and the sulphur atom contributed to biofilm killing, indicating a structure-activity relationship. The compounds differed in their effects on biofilm viability, yeast-to-hyphal form transition, hyphal morphology, cell wall morphology and composition, and sterol distribution. The most effective anti-biofilm compounds were the thiazolidinedione S8H and the succinimide NA8. CONCLUSIONS: We developed novel anti-biofilm agents that both inhibited and destroyed C. albicans biofilm. With some further development, these agents might be suitable for therapeutic purposes.

Ancient dispersal of the human fungal pathogen Cryptococcus gattii from the Amazon rainforest.

Over the past two decades, several fungal outbreaks have occurred, including the high-profile 'Vancouver Island' and 'Pacific Northwest' outbreaks, caused by Cryptococcus gattii, which has affected hundreds of otherwise healthy humans and animals. Over the same time period, C. gattii was the cause of several additional case clusters at localities outside of the tropical and subtropical climate zones where the species normally occurs. In every case, the causative agent belongs to a previously rare genotype of C. gattii called AFLP6/VGII, but the origin of the outbreak clades remains enigmatic. Here we used phylogenetic and recombination analyses, based on AFLP and multiple MLST datasets, and coalescence gene genealogy to demonstrate that these outbreaks have arisen from a highly-recombining C. gattii population in the native rainforest of Northern Brazil. Thus the modern virulent C. gattii AFLP6/VGII outbreak lineages derived from mating events in South America and then dispersed to temperate regions where they cause serious infections in humans and animals.

Mechanism of activity and toxicity of Nystatin-Intralipid.

A novel lipid formulation of Nystatin (NYT), Nystatin-Intralipid (NYT-IL), which was found to be more active and less toxic in vitro and in vivo, was developed in our laboratory. The aim of the present study was to explore the possible mechanisms underlying its biological activity. To assess mechanisms affecting fungal cells we conducted the following experiments: killing kinetics, scanning and transmission electron microscopy (EM), measurements of potassium ion leakage and susceptibility in the presence of ergosterol. To study mechanisms affecting mammalian cells, we evaluated the effect of NYT-IL on a kidney cell line, with respect to viability, metabolic activity, potassium leakage and internalization of FITC-labeled human transferrin. NYT-IL exhibited killing kinetics patterns against Candida albicans similar to those of NYT and caused disruption of fungal cells and potassium ion leakage. Susceptibility tests showed that NYT-IL had lower antifungal activity in the presence of ergosterol. Thus, NYT-IL acts apparently by damaging fungal membrane, possibly through interaction with ergosterol, and maybe by additional modes of action. NYT-IL did not cause potassium leakage from mammalian kidney cells at any tested concentration and was not cytotoxic, whereas NYT, at high concentrations, led to K(+) leakage and was cytotoxic. Furthermore, the high NYT concentration interfered in the internalization process of human transferrin receptor (hTfnR) while NYT-IL did not. In summary, the Intralipid formulation of NYT diminishes the mechanisms responsible for toxicity to mammalian cells but preserves mechanisms of action against fungi, thereby suggesting superiority of NYT-IL as compared to NYT as an antifungal agent.

Toxicity mechanisms of amphotericin B and its neutralization by conjugation with arabinogalactan.

Amphotericin B (AMB) is an effective antifungal agent. However, its therapeutic use is hampered by its toxicity, mainly due to channel formation across kidney cell membranes and the disruption of postendocytic trafficking. We previously described a safe injectable AMB-arabinogalactan (AG) conjugate with neutralized toxicity. Here we studied the mechanism of the toxicity of free AMB and its neutralization by conjugation with AG. AMB treatment of a kidney cell line modulated the trafficking of three receptors (C-X-C chemokine receptor type 4 [CXCR4], M1 receptor, and human transferrin receptor [hTfnR]) due to an increase in endosomal pH. Similar data were also obtained in yeast but with an increase in vacuolar pH and the perturbation of Hxt2-green fluorescent protein (GFP) trafficking. The conjugation of AMB with AG neutralized all elements of the toxic activity of AMB in mammalian but not in fungal cells. Based on these results, we provide an explanation of how the conjugation of AMB with AG neutralizes its toxicity in mammalian cells and add to the knowledge of the mechanism of action of free AMB in both fungal and mammalian cells.