Fungi: Identification and Classification in Mycology 1

 

Classification

Kingdom: Mycetae (Fungi) 3 domain system: Eukarya (eukaryotes): yeasts & Molds Eu: means true, karyon = nucleus Cell walls:made up of chitin (polysaccharide) Chitin is made up of NAG (N-acetylglucosamine), modified glucose Recent methods for fungi identification uses molecular tools & basic morphology This lecture aims to provide basic & traditional morphology of fungi & still described especially in identification. 2

Microbial Growth rate comparison Typical bacteria: 18 – 24 hrs in Media (nutrient or blood agar) turbidity ( broth) or colonies ( solid medium) bacterial multiplication (logarithmic phase): 4 hrs ( 1 to 2 cells) Mycobacterium tuberculosis: 3 to 6 weeks/ 2 months = obvious + growth in Lowenstein-Jensen media ( inspissated egg media) ( kills AIDS patients in Philippines) Algae: 2 months ( 1 cell to 2) in constituted media Fungi: (microbial succession) Yeasts: 48 hrs ( turbidity & colony) Molds ( 5 – 7 days) ( zygomycota:5 days) Deuteromycota ( 7 days ) ascomycota members ( 10 – 14 days) Basidiomycota: 3 weeks in plants (natural substrate) ( ex: mushroom)

BENEFITS OF FUNGI Edible Mushroom (basidiomycota) Saccharomyces cerevisiae yeast for Beer, wine, alcohol (fermentation in ENCLOSED System of facultative anaerobes), bread (leavening of yeast) Trichoderma: produce cellulase, for clear fruit juice Penicillin during world war 2 by Penicillium rubens (mold discovered by Alexander Fleming, Nobel Prize winner) Mycorrhizae: symbiotic fungi, help plants’ roots absorb minerals & water from soil. Nearly all plants have it. Taxol (anticancer drug) from plants = produced also by Taxomyces fungi

Parasitic Lifestyle Fungi are categorized by lifestyle into: 1.saprophytes/necrotrophs – feeding on dead material like dead leaves - decomposers .In soil,they breakdown complex compounds into simpler forms so nutrients will still be available for plants to absorb. 2.biotrophs – feeding on living host Example: Trichoderma spp. feeding on other fungi 3. hemibiotrophs – feeds on both living & dead material Example: Colletotrichum gloeosporioides : versatile plant pathogen, infects wide range of plants worldwide. - adaptibility, an advantage for survival under harsh conditions. 5

Role of fungi as etiological agent of infection

Humans become an accidental host by -Inhalation of spores & by trauma (wounds) Economic losses thru damage to plant crops ( phytopathogens) Most fungal infections: nosocomial and/or occur in babies, immunocompromised individuals (opportunistic infections). (Nosocomial: hospital acquired) Humans are resistant to fungal infection except for dimorphic fungi Provoke allergic response to host ( molds growing in homes, spores on atopic allergies) Major factor for infection: alteration of immune system

  • serious disease (HIV infection, diabetes)
  • immune system suppressive drugs, long term antimicrobial therapy, organ transplant
  • side effect of vaccines ( dry mouth in oral cavity favors yeast growth)   6

Virulence Factors: Fungi Capsules Adhesion factors Hydrolytic Enzymes Inflammatory stimulants Toxin-like substances

Allergies & Asthma Farmer’s lung Teapicker’s lung Bark stripper’s disease Mycotoxicoses – ingesting or inhaling fungal toxins Aflatoxins by Aspergillus flavus. Toxic.carcinogenic.on grains, peanuts (peanut butter) poultry & livestock. Stachybotrys chartarum – Sick building syndrome.Hematologic & neurological damage. Mycotoxins by mushroom – potent neurotoxins, result in death Phalloidin & Amanitin by Amanita phalloides (death cap)

Ingestion causes mushroom poisoning in Amanita 8

Normal X-ray

Farmer’s lung X-ray

Fungal Toxins & Enzymes Trichothecenes – fungal toxin, inhibit protein synthesis in eukaryotic cells by Fusarium & Stachybotrys Ingestion causes: Headaches, chills, severe nausea, vomitting & visual disturbances Virulence Factors: Candida albicans & Trichophyton secrete proteases, modify cell membrane to allow attachment of fungi Cryptococcus neoformans – causes meningitis, produces capsule to resist phagocytosis Claviceps purpurea – on grains.Disease: ergotism (Europe, Middle Ages) Ergot toxin – alkaloid, cause hallucinations like LSD.Constricts capillaries, gangrene of limbs, preventing blood circulation. Ergot toxin is in sclerotia ( resistant part of mycelium, detachable)

Taxonomy

Old Divisions of Fungi Deuteromycota: removed in new Classification (2017) due to molecular technology, asexual forms (imperfect fungi) Penicillium chrysogenum for Penicillin 2.Ascomycota: yeasts 3. Basidiomycota: mushrooms 4. Zygomycota: no septa in hyphae Zygomycota hyphae

No Septa: Easier Identification for Zygomycota Division members No Septa/ coenocytic hyphae Faster growth (5 days) Grows faster in high sugar content conjugation fungi & Saprophytes Rhizopus stolonifer ( common black bread mold) Rhizopus & Mucor Opportunistic

Rhizopus stolonifer

No Septa: Easier Identification for Zygomycota Division members Spores are inside sporangium

Major Division of fungi

For Basidiomycota and Ascomycota members: asexual form called anamorph sexual form called teleomorph Some identified deuteromycota members were eventually identified as the asexual forms of Basidiomycota or Ascomycota This of course bring about confusion in identifying Fungi like molds all over the world.

Mushrooms are under Basidiomycota: teleomorphs

Molds in Petridish: usually anamorphs 15

Major Phyla To avoid confusion, scientists therefore mentions the scientific name of anamorph & teleomorph Example: Colletotrichum gloeosporioides is the anamorph.(asexual stage) teleomorph is Glomerella sp. (sexual stage) classified under Ascomycota A lot of recognized human and plant pathogens are in asexual forms

Acervulus (fruiting body) is a distinct feature of Colletotrichum Glomerella teleomorph form of fruiting body 16

IMPORTANT DIVISIONS OF FUNGI Ascomycota (Sac Fungi) Molds with septate hyphae and some yeasts. Asexual Reproduction: Conidiospores not enclosed in a sac. Become airborne easily. Form chains (broom-like structures). Sexual Reproduction: Ascospores enclosed in a sac-like structure (ascus). Include common antibiotic producing fungi and yeasts, and several human pathogens. Saccharomyces cerevisiae (Brewer’s yeast) Ajellomyces (sexual stage of Histoplasma capsulatum) Arthroderma (sexual stage of Trichophyton spp. ) Claviceps purpurea: Produces ergot toxin in wheat and rye 17

IMPORTANT DIVISIONS OF FUNGI Basidiomycota (Club Fungi): Have septate hyphae. Include mushrooms, toadstools, rusts, and smuts and bracket fungi Sexual Reproduction: Produce basidiospores: Spores formed externally on a club shaped sexual structure or base called basidium.Asexual Reproduction: Through hyphae. Filobasidiella spp.(sexual stage of Cryptococcus neoformans) Causes opportunistic respiratory and CNS infections in AIDS patients. Cryptococcus neoformans: asexual stage Amanita: Mushroom produces lethal toxins to humans.

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Rapid Identification: Yeast Germ tube test: conventional/traditional Recent Rapid Test: API system for Yeasts (Biomeriux is a brand), 48 hrs growth & reading time computer program will identify, just type numerical value of API reading (Genus & species epithet) Miniturized media & sugars where yeast broth culture is placed thru micropipette Turbidity means sugar is utilized Computer reveals the scientific name Rapid kit test still relies its tests from traditional sugar utilization schemes (derived from media & biochemical test)

Classical Identification: Molds

NO API kits for Molds Molds classified thru morphology (classical method) & molecular tools Molds morphology use taxonomic keys to identify until species level (scientific journal or book) still updating its classification till now due to molecular inf Penicillium rubens: source of Penicillin by Fleming Classification Identification: Mold

Media for Fungi : culture remains definitive for medical diagnosis Non-selective Synthetic media for molds and yeasts: PDA (potato dextrose agar).best recommended for plant pathogenic molds. PDB ( potato dextrose Broth) for stock culture. Other media: SDA (Sabouraud Dextrose Agar), Czapek Dox Agar Mounting medium Lactophenol cotton blue: commonly used for plant pathogenic molds Lactophenol cotton blue is also used in human pathogens like members of Trichosporon, Geotrichum Staining methods Periodic acid schiff stain: fungi stain is bright red or purplish red Gram-stain: all are gram-positive Acid-fast stain for Nocardia Wright stain for Histoplasma capsulatum 21 21

Other Methods for Fungi Identification 1.Reverse plate method ( from Dr Dogma) Turn petri dish medium upside down ( ex: PDA/Potato Dextrose Agar) View the plate directly under the microscope If fungi: Yeasts: pseudohypha outside round colonies Molds: hypha is evident 2. Wet mount 3. Agar Block 4. Petri dish Moist Chamber

Moist chamber (plant part with fungal inoculum) Agar block stabbed by Fungal inoculum

Mold & Yeasts Appearance, Hyphae & Spores

Fungi forms based on colony appearance 1.Molds Multicellular Fluffy, Cottony, Wooly ( 5 to 7 days in media) Take note of fluffy colony color Powdery colonies filamentous fungi Identified by:physical appearance, colony characteristics, and reproductive spores Conidiospore or conidium: spore produced by mitosis (asexual reproduction) Plural: conidiospores or conidia

Axenic culture of Penicillium chrysogenum produces penicillin 26

Hypha Types

  1. Vegetative Hypha – obtains nutrients
  2. Reproductive Hypha –    Bears reproductive spores    Projects above surface of medium    9/5/12

Characteristics of Fungal Hyphae: Septate versus Coenocytic 28

Molds Thallus: Body of a mold or fleshy fungus Hyphae : Long filaments of cells joined together. Plural: hypha Septate hyphae: Cells are divided by cross-walls (septa) Septum:singular Coenocytic (Aseptate) hyphae: Long, continuous cells, no septa

CHARACTERISTICS OF FUNGI 29

Molds Mycelium: loose network, Large, visible, filamentous mass made up of many hyphae. Hyphae grow by elongating at the tips. Each part of a hypha is capable of growth. For the case of the septate hyphae, fungi are classified in terms of pigmentation If hyphae is septate and pigmented: it is called dematiaceous fungi If hyphae is septate but non-pigmented: it is called hyaline fungi

CHARACTERISTICS OF FUNGI septate hypha 30

LIFECYCLE OF FUNGI Asexual spores: Formed by the aerial reproductive hyphae.genetically identical to the parent (commonly found) Clinical identification is based on microscopic examination of asexual spores/ conidia. Produced by mitosis

Conidiophores: specialized hyphae that produces conidia Macroconidia: big spores Microconidia: small spores Arthroconidia/Arthrospores: conidia from fragmentation of hypha Blastoconidia/blastospores: conidia formed thru budding Chlamydospore: Resistant spores.Thick-walled spore formed within a hyphal segment.When conditions are not favorable. Phialoconidia – produced by phialide (vase-shaped) in Aspergillus fumigatus 31

Arthrospores Blastospores in Candida Chlamydospore

True Fungal Reproductive Spores 2. Sexual spores = fusion of nuclei of mating cells of same species . New organisms are different from both parents. From 3 phases

  1. Plasmogamy = union of cytoplasm
  2. Karyogamy = union of nuclei to form diploid zygote    3.Meioisis = diploid to haploid sexual spores

Example: Sexual spores in Zygospongium in Zygomycota: zygospores

Fungi forms based on colony appearance 2.yeasts Moist, creamy Opaque or pasty colonies on media (48 hrs) Unicellular fungi Nonfilamentous oval or spherical cells Reproduce by mitosis Facultative anaerobes Fission yeasts: Divide evenly to produce two new cells (Schizosaccharomyces).

Saccharomyces cerevisiae The brewer`s yeast 35

CHARACTERISTICS OF FUNGI Yeasts Budding yeasts: Divide unevenly by budding (Saccharomyces cerevisiae) Budding yeasts can form pseudohypha- a short chain of undetached cells. Candida albicans: normal flora of skin, mucous membranes,GIT Opportunistic C.albicans, attaches to epithelial cells but invades deeper tissues through pseudohyphae. Yeasts without Oxygen: go to fermentation of carbohydrates producing ethanol & CO2 Left:Candida albicans colonies Right: Candida albicans pseudohypha 36

Opportunistic Infection by Candida albicans in an AIDS Patient disease is called oral thrush 37

Candidiasis: Caused mainly by Candida albicans. Part of normal mouth, esophagus, and vaginal flora. 70% of nosocomial fungal infections (USA) by C. albicans Superficial to overwhelming fatal systemic diseases. Vulvivaginal or oral thrush, occurs in newborns, AIDS patients, people under broad spectrum antibiotics Vulvoginal yeast infection: painful inflammatory condition, ulceration & discharge Cutaneous candidiasis – chronically moist areas of skin & burn patients 38

Candida For detection of Candida albicans, pseudohypha,production of germ tubes or chlamydospores

Germ tubes: hyphal-like extension of yeast cell.

Chlamydioconidia on Cornmeal Agar with Tween 80

Rapid Urease test: Hydrolysis of urea by urease to produce ammonia.Ammonia wil change the medium from yellow to red (separates Candida from Cryptococcus)

C.albicans is Urease test negative: no color change

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Germ Tube Test for Candida

  1. Put 0.5 ml of sheep serum into a small tube.
  2. Using a Pasteur pipette, touch a colony of yeast and gently emulsify it in the serum.(Too much of inoculum will inhibit germ tube formation)
  3. Incubated tube at 37°C for 2 to 4 hours.    4.Transfer a drop of serum to a slide.
  4. Place Coverslip & examine microscopically under LPO & HPO

Dimorphism: switching forms

Dimorphic fungi Can exist as both multicellular fungi (molds) and yeasts depending on growth conditions (dimorphism) Many pathogenic species to humans, an adaptation to survival to human host Dimorphism in pathogenic fungi typically depends on temperature: At 35- 37 ºC: Yeast form (parasitic) At 25-30 ºC: Mold form (free-living) ( Tortora et al.) CHARACTERISTICS OF FUNGI:DIMORPHISM Mold Yeast 42

Mold form produces aerial and vegetative hyphae. Yeast form reproduces by budding. Dimorphism in nonpathogenic fungi may depend on other factors Ex: Carbon dioxide concentration. Stachybotrys grows on cellulose (dead plants), water damaged walls of homes. Spores cause fatal pulmonary hemorrhage in infants CHARACTERISTICS OF FUNGI: DIMORPHISM 43

CHARACTERISTICS OF FUNGI: DIMORPHISM

CHARACTERISTICS OF FUNGI Dimorphic Fungi Disease Histoplasma capsulatum histoplasmosis Sporothrix schenckii Sporotrichosis: ulcers in skin, lymph node Coccidioides immitis coccidioidomycosis Blastomyces dermatitidis Blastomycosis Paracoccidioides brasiliensis Paracoccidioidomycosis (mucosal lesions by eating vegetation with spores) Penicillium marneffei From bamboo rats in China Penicillium marneffei is recently added as dimorphic fungi. Coccidioides immitis is dimorphic but temperature is not the factor for its dimorphism

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Coccidioides life cycle: dimorphism

Primary pathogens: dimorphic types

FUNGI NUTRITION Food acquisition method: absorptive Chemoheterotroph: need organic compounds for energy & carbon like humans Oxygen Requirement: Aerobic (molds) or facultative anaerobic/(fermenters) for yeasts

Yeasts have higher water activity ( needs more moisture) Molds can grow in dry areas ( wood types, plastic, clothes, leather, paper), in human body where skin tends to be dark & moist Mold skin infection can also occur on dry skin during hot weather

FUNGI NUTRITION Mold: Typical Lab contaminant of bacteria Penicillin mold, lab contaminant, discovered by serendipity by Fleming Resistant to osmotic pressure than bacteria Yeasts & molds can grow in high sugar ( ex: syrup) or high salt Can grow in very low moisture content ( ex: molds on powder & biscuits) ( low water activity compared to bacteria) Can grow better at pH = 5 ( Tortora et al.) Capable of breaking down complex carbohydrates ( example lignin from plants)