MICROBIO 4.1 - MYCOSES INTRODUCTION & ANTIFUNGALS

• Antifungal resistant fungi are a growing threat
• Knowing specific antifungals is vital to counter fungal growth

Class Objectives

• Defining fungi and related vocabulary is key
• Distinguishing fungi from viruses and bacteria will aid understanding
• Illustrating the anatomy, role, and function of fungi is essential
• Comparing and contrasting fungal morphology clarifies traits
• Understanding how antifungals disrupt fungal growth is critical
• Knowing effective antifungals for specific fungal species is necessary
• Being aware of potential side effects and contraindications of antifungals is important

Phylogenetic Tree of Life

• Bacteria and Archaea are distinct from Eukaryota in the phylogenetic tree of life
• Fungi fall under the Eukaryota domain

Prokaryotes vs Eukaryotes

• Prokaryotes like Bacteria and Archaea differ from Eukaryotes like Animals, Plants, and Fungi
• Prokaryote DNA is naked and circular, while Eukaryote DNA is bound to protein and linear
• Prokaryotes lack a nucleus, while Eukaryotes have a nucleus
• Prokaryotes have 70S ribosomes, while Eukaryotes have 80S ribosomes
• Prokaryotes reproduce via binary fission, while Eukaryotes use mitosis and meiosis
• Prokaryotes are generally smaller (~1-5 μm) than Eukaryotes (~10-100 μm)

What are Fungi?

• Fungi are eukaryotes
• Fungi consist of a separate kingdom called Fungi
• Fungi can be multicellular filamentous structures (molds) or unicellular forms (yeasts)
• Some species are dimorphic, existing in either form depending on environmental conditions
• Fungi cause superficial, cutaneous, subcutaneous, and systemic infections
• Fungi are typically free-living in nature and acquired from environmental sources
• Some fungi are part of normal human flora
• Fungi cause disease by eliciting an inflammatory response or through direct invasion/destruction of tissues
• Endemic mycoses are infections caused by geographically restricted pathogens, causing serious systemic infection in healthy individuals
• Opportunistic mycoses are caused by fungi that cause life-threatening systemic disease, mainly in immunosuppressed patients
• Subcutaneous mycoses involve the skin, subcutaneous tissue, and lymphatics
• Superficial and cutaneous mycoses are common fungal infections limited to the skin

Fungal Cell Anatomy

• Cell walls are present
• Cell membranes are present
• Contains Endoplasmic reticulum, Nucleus, Mitochondria and Golgi

Basics of Fungi

• Fungi are eukaryotic with a nucleus, mitochondria, Golgi, and endoplasmic reticulum
• Rigid cell walls are made of chitin & glucan
• Cell membranes contain sterol, specifically ergosterol, which can be targeted by antifungals
• Fungi can be Unicellular (e.g., yeast) or Multicellular (e.g., mold)
• Dimorphic species alternate between yeast and mold forms depending on the environment (temp, pH, CO2)
• Fungi reproduction can be sexual, asexual, or both and produce spores/budding etc.

Fungi Morphology - Yeasts & Molds

• Unicellular fungi (aka yeast) are ovoid or round-shaped
• They replicate via budding or fission to form discrete, separate cells
• Pseudohyphae are chains of cells with constriction at septal junctions
• Pseudomycelium refers to the mass of pseudohyphae
• (Uni)Cellular-to-Pseudohyphae transition involves germ tubes becoming chains of cells that remain attached at septa
• This happens in response to environmental conditions
• Filamentous fungi are Hyphae
• Hyphae appear as smooth branching filaments
• The filaments may be septated (divided by partitions or cross-walls) or coenocytic (hollow and multinucleate)
• Mycelium refers to the mass of hyphae, commonly known as mold

Fungi Reproduction & Nomenclature

• Reproduction can be asexual or sexual
• Asexual reproduction involves reproductive structures called conidia, which vary in appearance
• Sexual reproduction occurs through the development of spores, often formed into complex structures
• Nomenclature uses a dual naming system
• Classification is based on structures associated with sexual reproduction
• Anamorph is the form of a species that produces asexual spores, given one name
• Teleomorph is the form of a species that produces sexual spores, given a different name
• Members of Ascomycota & Basidiomycota have dual nomenclature because they produce both asexual & sexual spores
• Aspergillus fischerianus is the anamorph of Neosartorya fischeri

Mycoses - Fungal Disease

• Most fungi are opportunistic pathogens
• Some cause primary respiratory infections that can disseminate
• Mycotoxins are toxins produced by fungi
• They can cause mycotoxicosis
• They can be used to create antibiotics
• Mycoses:
• Superficial- infect very superficial surfaces of skin & hair (non-destructive & cosmetic; e.g. pityriasis versicolor)
• Cutaneous- infect keratinized layer of hair, skin, and nails (symptomatic; e.g. ringworm)
• Subcutaneous- localized, chronic infections (e.g. produce abscesses, ulcers, or sinuses with drainage tracts)
• Endemic (systemic)- begin as primary infections in the lungs that disseminate (e.g. histoplasmosis)
• Opportunistic- caused by fungi found in human microbiome or environment, typically limited to immunosuppressed, prosthetic devices, or underlying disease (e.g. HIV/AIDS)

What is a Fungi and How Does it Differ from Viruses and Bacteria?

• Fungi are a kingdom of mostly multicellular eukaryotic organisms that are heterotrophs and reproduce sexually and asexually
• Fungi have important roles in nutrient cycling in ecosystems

Medically Important Fungi

• Phylum: Glomeromycota (Mucormycetes); Order: Mucorales; Genera: Rhizopus, Mucor, Lichtheimia, Saksenaea; Human Disease: Mucormycosis
• Phylum: Basidiomycota (Basidiomycetes); Genera: Cryptococcus, Malassezia, and Trichosporon species; Human Disease: Cryptococcosis
• Class: Pneumocystidomycetes; Genus: Pneumocystis jirovecii; Human Disease: Pneumocystis pneumonia
• Teleomorphs of Candida Species are medically important fungi causing numerous mycoses

Antifungal Terminology

• Fungistatic: inhibits fungal growth
• Fungistatic activity: level of antifungal activity that inhibits growth of an organism
• Minimum inhibitory concentrations (MICs)- Lowest concentration of drug that inhibits growth of organism
• Fungicidal: kills the fungal cells
• Fungicidal activity: ability of an antifungal agent to kill an organism in vitro or in vivo
  • Minimum fungicidal concentration (MFC) = lowest concentration of drug that kills 99.9% of the test population
• Antifungal resistance is when fungi alter antifungal target sites and/or reduce access to them, & when the fungal cells produce efflux pumps that prevent accumulation of antifungal agents
• Antifungal spectrum: range of activity of antifungal against fungi
  • Broad-spectrum: inhibits a wide variety of fungi (yeast like fungi & molds)
  • Narrow-spectrum: active only against a limited number of fungi

Antifungals Basic

• Antifungal combinations enhance efficacy for a refractory fungal infection
• Antifungal combinations broaden the spectrum of empiric antifungal therapy
• Antifungal combinations prevent the emergence of resistant organisms
• They achieve a synergistic killing effect
• Antifungal synergism includes combinations of antifungal agents that enhance antifungal activity when used together compared with activity alone
• Antifungal antagonism includes combinations of antifungal agents where the activity of one interferes with another
• Efflux pumps are families of drug transporters that actively pump antifungal agents out of fungal cells
• This decreases the amount of intracellular drug available to bind to its target

Polyenes

• Polyenes act by binding ergosterol in the cell membrane, leading to cytoplasmic leakage and cell death
• Examples of Polyenes are Amphotericin B IV ("ampho-terrible"), Amphotericin B deoxycholate, lipid, and Nystatin (oral, topical)
• Polyenes are used to treat mycoses like cryptococcal meningitis, invasive aspergillosis, mucormycosis, candidiasis, fungal cystitis, and oropharyngeal & vaginal candidiasis, and diaper rash
• Side effects include being Nephrotoxic (especially deoxy), QT prolongation, electrolyte wasting (K & Mg), acute infusion reactions, GI rash, Stevens-Johnson syndrome (rare)

Azoles

• Azoles block ergosterol synthesis by binding Cyp-450, making them fungistatic with a slow fungicidal activity
• Examples of Azoles include Imidazoles (oral, topical), Triazoles (Oral, IV), fluconazole, itraconazole, voriconazole, and Isavuconazole (newest addition)
• Azoles treat dermatophytoses, vaginal & oropharyngeal candidiasis (topical imidazoles), and systemic candidiasis (keto)
• Azoles treat Vaginal and oropharyngeal Candidiasis, Cryptococcosis, and Coccidioidomycosis (fluconazole)
• Side effects includes interactions with other Cyp450 drugs, hepatic toxicity possible, QT prolongation, BUT GI effects, lowers testosterone, fatal liver injury/death when prescribed incorrectly (i.e. pills for topical infection) (ketoconazole)
• *note that isavuconazole shortens QT interval

Allylamines

• Allylamines Block ergosterol synthesis by inhibiting squalene expoxidase
• Naftifine (topical) and Terbinafine (oral or topical) are Allylamines
• Treat Dermatophytoses, Aspergillosis, and Chromoblastomycosis
• Side effects are rare and pretty well tolerated
• Terbinafine treats dermatophyte infections of the skin that have not responded to application of creams and for fungal nail infections
• It is not indicated for systemic fungal infections

George Washington Carver - Also a Scientist

• born to slave parents around 1864
• interested in microfungi & sent samples to University of Wisconsin to catalog and the USDA
• several species named after him, including Cercospora carveriana, Metasphaeria carveri, Pestalotia carveri, and Taphrina carveri

Elizabeth Lee Hazen & Rachel Fuller Brown - Also Scientists

• Inspired by Alexander Fleming
• Discovered Nystatin (Mycostatin) for NY State from Streptomyces noursei
• Hazen cultured organisms found in soil samples and tested them in vitro for activity against two fungi, Candida albicans and Cryptococcus neoformans
• Brown employed painstaking solvent extractions to isolate the active agent in the culture before mailing them back to Hazen to test in animals