Subject: Microbiology and Parasitology Topic: Introduction to Mycology Lecturer: Dr. Eleanor P.

Padla Date of Lecture:July 26, 2011 Transcriptionists: JC Luna, Fluffy Editor: JC Luna Pages: 7

Mycology study of fungi Medical mycology deals with fungi that cause human diseases 3 Morphologic Forms: 1. Yeasts 2. Filamentous fungi (molds) 3. Fleshy fungi (e.g. mushrooms, most common) Fungi VS Bacteria
Characteristics Size Fungi 3 (yeasts) - 3 ft. (molds; due to longer hyphae) Unicellular (yeasts) to Multicellular (molds and mushrooms) Eukaryotic Cell wall is mainly Chitin; sterols (primarily ergosterol) are usually present on cell membranes Sexual and Asexual (binary fission) For Reproduction; are not Thermoresistant Bacteria 1-1.5

Mesophilic can thrive at temperature below that of the body (<37 C) with the exception of true pathogens.

Cellular Differentiation

Unicellular

Nuclear & Cellular Structure Chemical Composition

Prokaryotic Cell wall consists of peptidoglycans; Cell membranes have no sterols (except mycoplasma) Asexual; binary fission For Thermoresistance and survival during periods of Desiccation (survival in adverse conditions) Simple (binary fission) Nucleolus and Ribosome

STRUCTURE y Cell Wall 1. Chitin: structure of cell wall 2. Glucans: forms fibrils that increase strength of cell wall 3. Mannans: together with some proteins, are responsible for the serologic/antigenic activity of the cell wall y Cell membrane 1. Sterols (Ergosterol), glycoproteins, lipids 2. Most available antifungals act on the sterols of the fungal cell membrane y Cytoplasmic Contents 1. True nucleus, membrane-bound organelles y Capsule 1. Ex. Cryptococcus neoformans only pathogenic yeast with a capsule MORPHOLOGY 2 morphologic forms of importance: 1. YEAST y Unicellular y Facultatively anaerobic y Reproduction: 1. ASEXUAL: budding, binary fission (fissionists) 2. SEXUAL: production of ascospores, basidiospores

Reproduction Spore Formation

Life Cycle Cellular Structure

Simple (yeasts) to Complex (molds) With Membranebound Organelles

GENERAL CHARACTERSITICS OF FUNGI y Eukaryotic y Unicellular (yeasts) to multicellular (molds) y Microscopic to macroscopic in size y Non-photosynthetic (they are achlorophyllous<do not contain chlorophyll> as opposed to plants. This is one of the reasons they are re-classified years ago as non-plants) y Chemoheterotrophic; absorb nutrients, do not digest y Saprophytes / parasites y Nutritionally adaptable can thrive with little amount of organic substrate and moisture y Aerobic (molds <although there are not strictly aerobic molds> to facultatively anaerobic (yeasts, that is why they can be used for fermentation)

Diagram: Budding of Yeast Budding: Process 1. Formation of protuberance on the mother cell 1

SY 2011-2012

2. Nucleus undergoes mitosis 3. One of the subsequently formed nuclei will go to the daughter cell 4. Septum formation 5. Pinching off of the bud from the mother cell

True budding y When daughter cell is pinched off, a scar will be left. The presence of the scar concludes the formation of a new bud. y Usual limit to the number of buds produced = 24 30 1. Once an area already has a scar, a bud can no longer form on it 2. FILAMENTOUS FUNGI (MOLDS) y Multicellular y Generally Aerobic y Grows by apical elongation of the hypha y Hyphae / mycelia 1. Hypha microscopic form; tubular form 2. Mycelia macroscopic; mass of intertwining hyphae 3. Thallus colony mass 4. Classification:  In terms of segmentation y Septate hyphae divided into small units by cross-wall or septa o Each portion enclosed by 2 septa has its own nucleus, organelles and everything that it needs to survive. Hence when it is broken off, it will live. y Non-septate/Aseptate or coenocytic; there are no cross walls; hyphae appears as long continuous multinucleated structure. o If a segment of the hypha is accidentally broken off, that segment will die. o Ex. Zygomycota 5. In terms of pigmentation:  Hyaline no pigment  Dematiaceous pigmented 6. In terms of function  Reproductive also known as aerial hyphae; produced from the vegetative hyphae and grows vertically. It produces the reproductive spores. (Thousands of) Spores are enclosed in a Sporangium  Vegetative embedded into the substrate; used by the fungi to absorb nutrients and water. DIMORPHISM y Fungi are dimorphic: able to exist in 2 different forms: yeast and mold y Thermally dimorphic: conversion of mold to a yeast is induced by a temperature change y Many human pathogens are thermally dimorphic

1. At room temperature fungi will be in the mold form.Hence, in culture if you want to produce the form with hyphae/mycelia (mold form), fungi must be incubated at room temperature (25 degrees). Additionally, this is called the Saprobic Phase of the fungi. 2. The parasitic form (yeast form, or Spherules<are multinucleate>), requires body temperature (37 degrees). This means at 37 C, either in an incubator or the body, the mold form of the fungi will be converted to the yeast form. 3. To determine whether a thermally dimorphic species (e.g. Histoplasmacapsulatum) has been isolated, both forms, the moldand theyeast, should be demonstrated in culture. Dimorphic Fungi y Sporothrixschenckii y Histoplasmacapsulatum y Blastomycesdermatitidis y Coccidioidesimmitis y Paracoccidioidesbrasiliensis y Penicilliummarneffei REPRODUCTION y Fungi are usually classified by the mode of reproduction. The divisions are generally based on the type of sexual spores y Sexual Reproduction (Teleomorphs) Steps: 1. Plasmogamy 2. Karyogamy 3. Genetic Recombination 4. Meiosis reduction-division; formation of the haploid sexual spores y Asexual Reproduction (Anamorphs) Steps: 1. Binary fission 2. Budding 3. Fragmentation 4. Sporangiospore or Conidia Formation * these fungi undergo mitosis all the cells are genetically identical Sexual Reproduction 1. Sexual Spores a. Zygospores (phylum: zygomycota) b. Ascospores (phylum: Ascomycota) c. Basidiospores (phylum: Basidiomycota) *** These 3 are perfect fungi *** *Deuteromycota imperfect fungi; do not produce sexual spores (note: she said that this is the 4th phylum. However an internet source says, once considered a formal phylum of the kingdom Fungi. The term is now used only informally, to denote species of fungi that are asexually reproducing members of the fungal phyla Ascomycota and Basidiomycota. Also

on Jawetz p. 629 the 4th phylum mentioned was Chytridiomycota) Zygospores y Containing structure is the zygosporandium which contains thousands of zygospores Ascospores y Contained within a sac: ascus y Inside the ascus are the ascospores (8/ascus) Basidiospores y Terminal cells arising from the Basidium Asexual Reproduction 2. Asexual Spores a. Sporangiospores i. inside sporangium<containing sac> ii. typical of members of phylum Zygomycota iii. produced by aseptate hyphae iv. can germinate and disseminate b. Conidia i. devoid of sporangium, or the containing sac ii. typical of members belonging tothe phyla Ascomycota,Basidiomycota and Deuteromycota iii. produced by septate hyphae

2. Poroconidia  Bud growing out of small pores

3. Phialoconidia  Growing out from a small vase like vessels called phialides

4. Anneloconidia  Grows out from a vase like structure (annelids)  When the conidia grows out from the annelid, it leaves a remnant on the parental cell wall (annelations)

A. Sporangiospores B. Types of Conidia Formation: BlasticConidiogeny y formation by budding/blowing off from the parent cell 1. Blastoconidia (budding)  Bud

B. Types of Conidia Formation: Thallic conidiogeny y grows from the differentiation of the thallushyphae 1. Microconidia and Macroconidia  Produced when the whole length of the hyphae differentiated into a conidia

Basidiomycete(Zygomycete not considered because it is aspetate) 3. It can be said that this is just a holding category phylum until such time that sexual reproduction can be observed. 1. Zygomycota y Sexual reproduction by gametangial fusion producing zygospores y Asexual reproduction by sporangiospores w/n sporangia y Hyphae aseptate (coenocytic) y Includes: 1. Rhizopus 2. Mucor 3. Absidia 2. Ascomycota y Sexual reproduction by ascospores within an ascus(8 ascospores/ascus) y Asexual reproduction by conidia y Hyphae septate y Includes: 1. Saccharomyces 2. Peidraia Imperfect State 3. Arthroconidia  Rectangular thick walled cells  From the hyphael fragmental segment  You can see them without spaces. Sometimes contain separator spaces called disjunctor cells Blastomyces Histoplasma Penicillium Aspergillus Perfect State (depending on species) Ajellomyces Emmonisella, Gymnoascus Talaromyces Sartorya, Eurotium

2. Chlamydoconidia  Circular thick walled cells  Grows by the thickening of the thallus

3. Basidiomycota y Sexual reproduction by basidiospores on basidium y Asexual reproduction by conidia y Hyphae septate with specialized connections contains small port that allows cytoplasmic migration y Includes: 1. Cryptoccousneoformans (anamorphic form)  Filobasidiella perfect form Dolipore Septum y Dolipore with very small pore allowing cytoplasmic leakage from one portion to another portion. Enclosed by parenthosome (leaf like structure)

CLASSIFICATION 4 phyla (Kingdom Eumycota): y Zygomycota (conjugation fungi) y Ascomycota (sac fungi) y Basidiomycota (club fungi) y Deuteromycota (imperfect fungi) 1. Imperfect b/c don t have asexual reproduction 2. Once a sexual form has been observed, organism will be reclassified either as an Ascomycete or 4. Deuteromycota (Imperfect Fungi) 4

y y y y y

No sexual reproduction Asexual reproduction by conidia Hyphae septate Classified on basis of morphological structures of vegetative phase, types of asexual spore Includes: 1. Sporotrhix 2. Coccidioides 3. Candida albicans 4. Epidermophyton

y Significance and Application A. Desirable Effects y Used in biotechnology (bread and wine production) y Used as protein supplements y Production of cellulase (digests plant walls and clears fruit juices) and herbicides y Decomposers / recyclers y Ushered age of chemotherapy B. Undesirable Effects y Spoilage of food, grains y Plant pathogens y Cause infectious disease in humans: 1. Hypersensitivity reactions 2. Toxicosis  Mycotoxicosis ingestion of food contaminated with toxins producing fungi y Ex. Ingestion of moldy peanuts - aflatoxicosis  Mycetismus ingestion of the fungi itself producing the toxin y Ex. Mushroom poisoning  Mycoses human infection with fungi y Etiology o About 50 fungal species are known to produce disease y Epidemiology: o Not reportable o Not communicable from person to person (except dermatophytosi s&pityriasisvers icolor o Acquired through contact with environmental source / patient s own flora (candida spp.) y Portal of Entry

o Skin, puncture wounds o Hair o Nails o Respiratory tract Mode of Entry o Directly on the skin (cutaneous) o By implantation via an injury (subcutaneous) o By inhalation (respiratory) o From previous focus of infection (dissemination) Pathogenesis o Adherence skin and mucosal surfaces o Invasiveness o Resist phagocytic killing o Cause tissue injury Determinants of Pathogenicity o Ability to produce toxicant (toxin production) o Ability to cause physiological alterations used by fungi to spread o Thermotolerance o Dimorphism  Man s temperature protects us from most fungi (only thermally dimorphic can affect man). Additionally, oxygen concentration is most tissues are lower than what is required by fungi (which are mostly aerobic) o Nature of infectious particles o Infectious Dose Infectious Particles:Part of the fungi that will contaminate you o Yeast cells o Hyphae can be spread or be injected into the skin o Sporangiospores - When a sporangium is disrupted it can release thousands of sporangiospores o Conidia *** There are more asexual spores than sexual spores. Ergo, infectious particles usually are asexual spores *** Immunity: o Humoral immunity  Not active in most mycoses o Cellular Immunity  Of primary importance Classification of Mycoses: (according to the tissue invaded) o Superficial mycoses  Pitriasisversicolor  TineaNigra  Black Piedra  White Piedra o Cutaneous mycoses(Dermatophytoses)  Tineacapitis (Kerion<inflammatory>, Favus)  Tineabarbae  Tineacorporis ring worm on the body 5

 Tinaeimbricata  Tinaecruris  Tinaepedis  Tinaemanuum  Tineaungium Subcutaneous Mycoses  Sporotrichosis  Mycetoma  Chromomycosis  Phaeohyphomycoses Systemic Mycoses  Endemic Respiratory  Histoplamosis  Blastomycosis  Coccidioidomycosis  Paracoccidioidomycosis  Penicilliosismarneffei  Opportunistic  Candidiasis  Aspergillosis  Cryptococcosis  Zygomycosis

are harder to handle compared to tube preparations Tube cannot isolate the colonies

Micro-culture y Done to identify the species of fungi, type of spore, and sporulation pattern) y Undisturbed sporulation to be able to see the formation and the bearing structures y Very tedious but good for identifying the species Examination of cultures y Gross morphology analyzed Microscopic Morphology (with LPCB) y Scotch tape preparation y Tease / wet mount y Do this if you don t want to do a micro-culture y Can see the spores, but not used for identification C. Identification of Cultures y Yeasts 1. Biochemical/ physiologic tests; morphologic features y Molds 1. Morphology of spore, spore-bearing structures y Newer methods: 1. Exo-antigen test 2. Nucleic acid probes D. Histopathologic Examination y Stains PAS, GMS, Wright s / GIEMSA y Tissue Forms 1. Yeast cells  Intracellular (histopalasmosis)  Broad-based (blastomycosis) y Parent cell attached to daughter through a broad base  Multiple buds (paracoccidioidomycosis)  Encapsulated (cryptococcosis) y White capsule  With transverse septum (penicillosismarneffei)  Cigar-shaped (sporotrichosis) 2. Hyphae  Coenocytic (zygomycosis)  Dichotomously branched (aspergillosis)  Hyaline, septate (dermatophytosis)  Dematiaceous, septate (pheohyphomycosis, tineanigra) 3. Yeast cells & hyphae  Candidiasis 4. Granules  Mycetoma 5. Spherules

Laboratory Diagnosis Specimen Selection or Clinical Specimens y Respiratory tract secretions y CSF y Blood y Hair, skin, nail scrapings y Urine y Tissue, bone, sterile body fluids A. Direct Microscopic Exam (rapid method to see of fungi is involved) y KOH (10% - 20%) preparation 1. Demonstrate the hyphael elements, hyaline, septate, dimatatiocious, yeast cells y India ink preparation 1. For capsulated species of fungi 2. Looking at streptococcus y Calcifluor white staining 1. Utilizes a die that reacts with the cell wall of the fungi, when you view under UV light will fluoresce easy to see the fungal cell wall B. Cultural Procedures Media y Sabouraud s Dextrose Agar y Mycobiotic agar (Mycosel) y Brain Heart Infusion (BHI) 1. Convert the mould into a yeast to determine the thermal dimorphism y Potato Dextrose Agar 1. This is used when you want to enhance the correlations y Chlamydospore (corn meal) agar 1. Used for Candida Plate / Tube Cultures y When you do a plate culture, can get isolated colonies, they are more of aerobic type these

 Coccidioidomycosis 6. Fission (schlerotic) bodies  Chromomycosis E. Immunologic Methods y Skin testing y Antibody detection 1. Latex agglutination 2. Complement fixation 3. Immunodiffusion Therapy Targets of Antifungals y Allylamnes y Polyenes y Azoles y Griseofolyn y Flucytosine

1. Nucleus y Benzofurans (Griseofulvin) 1. Block intracellular microtubules y Pyrimidines (5-FC) 1. Block DNA/RNA synthesis 2. Cytoplasmic Membrane y Polyenes (Amphotericin B, Nystatin) 1. Bind to and disrupt cell membranes y Azoles (Imidazoles, triazoles) 1. Block ergosterol formation y Allymines (Terbinafine) 1. Block ergosterol formation 3. Cell wall y Echnocandins (Caspofungin, Micafungin) 1. Inhibit glucan (part of the fungal cell wall) synthesis ============= END TRANSCRIPTION ============== NOTE: I was only able to edit the parts before laboratory diagnosis . Mag-aaral pa kongpatho. jcluna Hello batch 2014. Just wishing everyone luck! We can do it!! Hiya Argent Babies Happy Anniversary! Hahahaha Rest if you must, but don t you quit. There is no point in doing something if you won t give your best 7