MICROBIOLOGY LECTURE M1 – Generalities, Principles and Proper Collection of SpecimensNotes from Murray (97) and Fisher (98)USTMED ’07 Sec C – AsM

MYCOLOGY = is the study of fungi!!!

fungi reside in nature and are essential in breaking down and recycling organic matter (a saprobe – organism that brings about decay)

used in production of food and spirits in medicine, they provide useful bioactive

secondary metabolites such as antibiotics and immunosuppressive drugs

used in model systems for the investigation of a variety of eukaryotic processes

are phytopathogens that cause huge agricultural losses (major pathogens of plants)

only a few hundred species of fungi have been implicated in human disease

90% of human infections can be attributed to a few dozen fungi

FUNGI

a group of nonmotile eukaryotic organisms that have definite cell walls, devoid of chlorophyll and reproduce by means of spores (or conidia)

each fungal cell has at least one nucleus and nuclear membrane, ER, mitochondria and secretory apparatus

cell walls contain chitin or cellulose, or both are heterotrophs – uses many different organic

compounds as nutrients are obligate or facultative aerobes are chemotrophic, secreting enzymes that

degrade a wide variety of organic substrates into soluble nutrients, which are then passively absorbed or taken into the cell by active transport

often reside on body surfaces as transient environmental colonizers but obtain no obvious benefit

DEFINITIONS

hyphae – long strand of cells with or without crosswalls (septa) ; filamentous tubular structures which grow by elongation at the tips or by branching

septa – crosswalls; hyphae may be septate or aseptate

mycelium – masses of hyphae which comprises the colony of the fungus (also called thallus)

yeasts – single round to oval cell that usually buds to form daughter cells; unicellular form

thermally dimorphic fungi – develop mould-form colonies at RT and another form at human body temperature

Conidium – refers to any reproductive structure moulds – fungi that form hyphae (as opposed to

yeast) Mycosis (mycoses) are any disease caused by a

fungus

3 types of mycelia (generally cannot be distinguished from one another)

1. Vegetative mycelia- grow in or on the medium

- absorbs nutrients from the medium2. Aerial- grow above the surface of the agar- forms most of the visible part of the colony3. Fertile or reproductive mycelia - from which the reproductive structures arise

MACROSCOPIC EXAMINATION

Colony texture (the way the colony looks)

a. Glabrous leathery or waxy little if any aerial mycelium

b. velvety resembles plush or velvet fabric

or suede have short aerial hyphae, few

conidia or sopresc. yeastlike

resembles colonies of coagulase-negative staphylococci

“bacteria like” yeasts appear dryer and duller no aerial mycelia

d. cottony develop when colonies produce

long aerial hyphaee. granular

fungi that conidiate or sporulate heavily

Colony topography (the way the colony surface is arragned)

a. Flat

b. Folded

c. Rugose

d. Crateriform

e. Cerebriform

f. Verrucose

MYCOTOXICOSES

fungi are metabolically versatile organisms and sources of innumerable secondary metabolites such as alkaloids and other toxic compounds

mycotoxicoses are most often the result of the accidental or recreational ingestion of fungi that produce these compounds

source of toxin is determined by obtaining the history of the patient

when fungi are ingested, emesis (vomiting) should be induced, and supportive measures should be instituted consistent with the physiological signs exhibited by the patient

1. Ergot Alkaloids- produced when grain is infected with

Claviceps purpurea - history of epidemics (St. Anthony’s fire) –

associated with consumption of bread and other bakery products made with contaminated rye

- symptomso inflammation of the infected tissues

(cellular response to injury)o necrosis (cell death)o gangrene (death of large masses of

tissue)- pharmacologically – they produce alpha-

adrenergic blockade, w/c inhibits responses to epinephrine and 5-hydroxytryptamine

o create marked peripheral vasoconstriction

restricts blood flow necrosis and gangrene

- directly stimulates smooth muscle contraction- used as oxytocic agents to induce labor

(increases force and frequency of uterine contractions)

- affects CNS by stimulating the hypothalamus and other sympathetic portions of MB

2. Psychotrophic Agents- used by primitive tribes- recreation use of agents such as psilocybin

and psilocin as well as the semisynthetic derivative, lysergic acid diethylamide (LSD)

3. Aflatoxins- contamination with Aspergillus flavus- resulted in outbreak of Turkey X disease in

England- Turkey developed symptoms:

o Lethargyo Anorexiao Muscle weaknesso Spasmso Eventually death

- postmortem studies revealed gross hemorrhage and necrosis of the liver

- histophath = parenchymal cell degeneration and extensive proliferation of the bile duct epithelial cells

- etiological agents were A. flavus toxins that are bisfuranocoumarin metabolites

- are potent carcinogens but have not been shown to play specific role in human carcinogenesis

4. Others- yellow rice toxicosis (Japan)- alimentary toxic aleukia (Soviet Union)

HYPERSENSITIVITY DISEASES

to measure degree of air pollution, fungal spore counts are conducted because they are ubiquitous in nature

airborne spores and other fungal elements can be an antigenic stimulus and may induce (depending on an individual’s immunological status),

hypersensitivity from the production of immunoglobulins or sensitized lymphocytes

clinical manifestation of hypersensitive pneumonitis:

o rhinitiso bronchial asthmao alveolitiso forms of atopy

growth of the fungus in the tissues is not required for the development of hypersensitivity; clinical manifestations are seen only in sensitized persons, after subsequent exposure to the fungus, its metabolites, or other cross-reactive materials

FUNGAL INFECTIONS

most pathogenic fungi are exogenous natural habitats

o watero soil o organic debris

Candidiasis and dermatophytosis (mycoses with the highest incidence) are caused by fungi that are part of the normal microbial flora or highly adapted to survival on the human host

Mycoses may be classified as: (refer to table1)o Superficialo Cutaneouso Subcutaneouso Systemico Opportunistic

categories reflects their usual portal of entry and initial site of involvement

overlaps do occur, since systemic mycoses can have subcutaneous manifestations and vice versa

patients who develop opportunistic infections have serious underlying diseases and compromised host defenses

during infections, most patients develop significant cellular and humural immune responses to the fungal antigens

COLONIZATION AND DISEASES

in general people have a high level of innate immunity to fungi

most fungal infections are mild and self-limiting skin – primary barrier to any infection caused by

fungi that primarily colonize the superficial, cutaneous and subcutaneous layers of skin

mucosal surfaces – discourage colonization by organisms that cause pulmonary infections

Fatty acid content, pH, epithelial turnover and normal bacterial flora of the skin contribute to host resistance

Transferrin (humoral factors) – restrict the growth of several fungi by limiting the amount of available iron

Some fungi have gained significance with their association with acquired immunodeficiency syndrome

o Result of low pathogenic potentialo Produce disease only under unusual

circumstances involving host debilitation Some circumstances that lead to infection by once

innocuous saprobes are:o Change in normal intestinal flora (use of

broad-spectrum drugs)o Debilitation of the host by the use of

therapeutic measures (cytotoxins, xray, steroids)

o Alteration of host’s immune system by underlying endocrine disorders (DM)

GENERAL PROPERTIES AND CLASSIFICATION OF FUNGI

Fungi grow in 2 basic forms:1. yeasts

- unicellular, spherical to ellipsoid fungal cells that usually reproduce by budding

- some species produce buds that characteristically fail to detach and become elongated

o continuation of the budding process then produces a chain of elongated yeast cells called pseudohyphae

- colonies are usually soft, opaque and cream-colored

- yeast species are identified on the basis of physiologic tests and few key morphologic differences

- some species of fungi are dimorphic and are capable of growth as yeast or mold depending on environmental conditions

2. molds- growth is by production of multicellular

filamentous colonies- colonies consist of branching cylindric tubules

called hyphae- the mass of intertwined hyphae that

accumulates during active growth is a mycelium

- some hyphae are divided into cells by cross-walls called septa, forming at regular intervals during hyphal growth

- substrate hyphae – hyphae that penetrate the supporting medium and absorb nutrients

- aerial hypahe – project above the surface of the mycelium and usually bear the reproductive structures of the mold

- molds produce colonies with characteristic features such as rates of growth, texture, and pigmentation

- the genus of most clinical molds can be determined by microscopic examination of the ontogeny and morphology of their asexual reproductive spores, or conidia

TYPES OF SEXUAL REPRODUCTION

1. a single zygosporangium containing zygospore is produced by mating of two compatible hyphal branches

2. multiple basidiospores form on denticles at the tips of the club-shaped basidia within a basidium

3. Ascospores form w/in asci inside the protective ascocarp (a perithecium is shown)

FUNGAL CELL WALL

are rigid and determines the shape of the fungi cell walls are composed:

o largely of carbohydrate layerso long chains of polysaccharideso glycoproteinso lipids

during infectiono surface components of the cell wall

mediate attachment of the fungus to host cells

o cell wall polysaccharides may activate the complement cascade and provoke an inflammatory response

are poorly detected by the host and can be detected by special stains

o release immunodominant antigens that may elicit cellular immune responses and diagnostic antibodies

dematiaceous fungi – yeasts or molds that have

melanized cell walls, imparting a brown or black pigment

SPORES

chances of survival are increased when fungi produce spores

characteristics:o readily dispersedo more resistant to adverse conditionso can germinate when conditions of growth

are favorableo can be derived from asexual

(anamorphic state) or sexual (teleomorphic state) reproduction

Asexual sporeso Are mitotic progeny (ie. Mitospores)o Genetically identicalo Fungi produce 2 major types of asexual

spores Conidia Sporangiospores

(zygomycetes) Features include

o Ontogeny Conidiogenic structures

o Morphology Size Shape Texture Color Uni or multicellularity

vegetative cells may transform into conidia (eg. Arthroconidia, chlamydospores)

conidia are sometimes produced by conidiogenous cell such as a phialide, which itself may be attached to a specialized hypha called a conidiophore

In zygomycetes, sporangiospores result from mitotic replication and spore production w/in a sac-like structure called a sporangium, which is supported by a sporangiophore

TAXONOMIC CLASSFIICATION (refer to table1)

based on the mechanism and spores that result from sexual reproduction (involves mating, nuclear fusion, meiosis, and the exchange of genetic information)

a species may be recognized and defined on the basis of its asexual state, but its telemorph, or sexual identity, may have a different name

GROWTH AND ISOLATION OF FUNGI

most fungi occur in nature and grow readily on simple sources of nitrogen and carbohydrate

Traditionally, Sabouraud’s agar (contains glucose and modified peptone pH7.0) has been used because it does not readily support the growth of bacteria

o Used to exhibit morphologic characteristics of fungi

Inhibitory mold agar – used to recover fungi from clinical specimens

Antibiotics (gentamicin, chloramphenicol) and cycloheximide are added to media to inhibt bacteria and saprophytic molds respectively in recovery of fungi from clinical specimens

MEDICAL CATEGORIES OF FUNGAL IMPORTANCE

1. Mycotoxicoses2. Hypersensitivity diseases3. Colonization of the host and resultant disease

TABLE 1 MAJOR TAXONOMIC GROUPS

GROUP CHARACTERISTICS EXAMPLEZygomycetes Sexual reproduction

results in a zygospore

Asexual reproduction occurs via sporangia

Vegetative hyphae are sparsely septate

Rhizopus

Absidia

Mucor

Pilobolus

Ascomycetes Sexual reproduction involves a sac or ascus

Ajellomyces (anamorphic

in which karyogamy and meiosis occur producing ascospores

Asexual reproduction is via conidia

Molds have septate hyphae

genera, blastomyces, histoplasma)

Arthroderma (anamorphic genera, microsporum, trichophyton)

Yeast genera (saccharomyces)

Basidiomycetes

Sexual reproduction results in four progeny basidiospores supported by a club-shaped basidium

Hyphae have complex septa

Mushrooms

Filobasidiella neoformans (anamorph, Crytococcus neoformans)

Deuteromycetes

An artificial grouping of the imperfect fungi for which a teleomorph or sexual reproduction has not been discovered

Anamorphic state is characterized by asexual conidia

Coccidioides immitis

Paracoccidioides brasiliensis

Candida albicans

TABLE 2 MAJOR MYCOSES AND CAUSATIVE FUNGI

TYPE OF MYCOSES

CAUSATIVE FUNGAL AGENTS

MYCOSIS

Superficial

infections limited to the outermost “dead” layers of skin and hair

Malassezia furfur

Hortaea weneckii

Trichosporon species

Peidraia hortae

Pityriasis versicolor

Tinea nigra

White piedra

Black piedra

Cutaneous

Infections that extend deeper into the epidermis as well as invasive hair and nail disease (keratinized portions)

Microsporum species, trchophyton species, and Epidermophyton floccosum

Candida albicans and other candida species

Dermatophytosis

Candidiasis of skin, mucosa, or nails

Subcutaneous

Infections involving the dermis, subcutaneous tissues, muscles and fascia

Sporothrix schenckii

Phialophora verrucosa, Fonsecaea pedrosoi, others

Pseudallescheria boydii, Madurella mycetomatis, others

Exophiala, bipolaris, exserohilum, and others

Sporotrichosis

Chromoblastomycosis

Mycetoma

Phaeohyphomycosis

Endemic (primary, systemic)

Infections that originate primarily in the lung but may spread to many organ systems (lymphatic, circulatory)

Coccidioides immitis

Histoplasma capsulatum

Blastomycoses dermatitidis

Paracoccidioides brasiliensis

Coccidioidomycosis

Histoplasmosis

Blastomycosis

Paracoccidioidomycosis

Opportunistic

Candida albicans and other candida

Systemic candidiasis

Infections caused by fungi that infect because of compromising situations

species

Cryptococcus neoformans

Aspergillus fumigatus and other aspergillus species

Species of rhizopus, absidia, mucor, and other zygomycetes

Penicillium marneffei

Crytococcosis

Aspergillosis

Mucormycosis (zygomycosis)

Penicilliosis

MODE OF ACTION OF ANTIFUNGAL DRUGS

A/N: guys, gawa ko lang ‘to. Kasi walang available na slides or pictures from the lecture. Of course, I was too busy dozing off to actually listen to the lecture kaya I had no idea what to put in this handout. I think I placed too much information… oh well, mabuti na kaysa bitin!

This is my Last lecture transcription for SY 2004-2005…

Sorry for the lack of availability of the files, slow circulation, dozens and dozens of typographical errors, grammatical

errors, blurred pics, and wrong information… tao lang po!

Thanks to all the people who helped me in the transcriptions… marc ostrea, jayveeh Navarro, Luver Lei Manzon, Kat Manibog, Ate Mea Mendoza, Pons, Anne and Boom. (Thanks also to the profs and doctors for their hardwork in preparing the slides! Siyempre kung wala silang lecture, wala ring trans!)

For the support – Roxy, cocoy, c3 and section C! *BIG HUG*

Section A, B and D – thanks for the powerpoints!

Lastly I’d like to share this with the batch… It’s what I always (well, not really always…hehe!) tell myself pag tinotopak ako and pag gusto ko na mag-quit…it’s just a short prayer, super daling i-memorize!

“Give me grace, O my Father, to persevere in the work to which you have called me, neither leaving it half-done nor giving up when the first enthusiasm has faded and other interests attract.”

…yun lang… MORE POWER TO USTMED BATCH 2007!!!

- Auds Martinez -

audsmartinez@gmail.comustmedc3@yahoogroups.com