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MICR570/ZMR/F12 6-1

BIOLOGY OF ATYPICAL BACTERIA & INTRODUCTION TO FUNGILecture 6

LEARNING OBJECTIVES At the end of these lectures, you should be able to:

1. Distinguish “atypical” bacteria from the “norm” by being able to describe the structuraldifferences and/or metabolic capabilities of the atypical bacteria.

2. Identify the differences between Yeasts & Moulds and be able to differentiate between

the two by selecting the key characteristic features.

3. Describe the significance of fungal dimorphism, by recognizing its relevance to human

disease and be able to identify the triggers.

INTRODUCTION

We shall examine the four groups of “atypical” bacteria (Mycoplasmataceae, Rickettsiaeceae, Anaplasmataceae & Coxiella and the Chlamydiaceae) in terms of what separates them fromthe “norm”. Basically all have unique features that differentiate them from the “typical” bacteriacell, described in Lectures 4 & 5.

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i. MYCOPLASMATACEAE Family includes 2 Genera: Mycoplasma sp. & Ureaplasma sp. with 4 human pathogenicspecies: M. pneumoniae (URT); M. genitalium & M. hominis; U. urealyticum (Genitourinary).

SIZE & SHAPE

Smallest known free-living organisms (0.15-0.3μm dia - approx. size of poxvirus). Contain minimal set of organelles required for growth & replication.Shape varies: pleomorphic (coccoid - long filaments) relates to cellular division.

UNIQUE FEATURE: POSSESS NO CELL WALL - entire life Classified in Class Mollicutes (“soft skin”). DO NOT synthesize peptidoglycan (BUT will stain pink on Gram staining – WHY?).

∴resistant to certain antimicrobial agents (i.e., penicillin & lysozyme).

CYTOPLASMIC MEMBRANE is the external surface layer A typical phospholipid bilayer structure

UNIQUE FEATURE: contains STEROLS (Cholesterol in some species), provide integrity &strength

Membrane proteins provide structural, catalytic, immunological support (used in classification& identification).



MICR570/ZMR/F12 6-2

CYTOPLASM Cytoplasmic division lags behind genome replication, giving rise to filamentous shape.

UNIQUE FEATURE: ATTACHMENT ORGANELLE Allows attachment to epithelial cells lining respiratory & urogenital tract. Has a tapered tip, coated with P1 adhesin (for adherence), attaches to receptors at base of

cilia. Result : tissue destruction due to H2O2 & superoxide anion? Absence of organelle leads to avirulence.

NUCLEOID Small Genome: M. genitalium 0.5 - 1x109 Daltons (compare to “norm”) Smallest capable of self reproduction. Encodes for few proteins: requires supplemental media

ii . RICKETTSIAECEAE Family includes 2 Genera: Rickettsia & Orientia (previously also included Ehrlichia & Anaplasma).

SIZE & SHAPE

Small (0.3-0.5 x 0.8-1.0μm)Shape: bacilli – coccoidal (pleomorphic)Closely related to Gram -ve’s (do not stain well): Use Giemsa or Gimenez stain.

UNIQUE FEATURE: OBLIGATE INTRACELLULAR PATHOGENS

Unable to produce sufficient energy to replicate extracellularly CAN NOT BE CULTIVATEDON AGAR. Only grow in/on cell culture, embryonated eggs, susceptible animals. Reason: Possess NO Coenzyme A, NAD or ATP – all obtained from host (essential for adequate growth).

Genus Rickettsia: grow in cytoplasm of cells

ZOONOTIC (Refer to Murray et al. p450. Fig. 45-1: Epidemiology of Common Rickettsia &Orientia infections) Transmitted via arthropod vectors such as ticks, lice, fleas, mites (See Zoonosis & BiodefenseLectures). E.g., R. prowazekii – human body louse; R. rickettsiae – wook tick

iii. ANAPLASMATACEAE & COXIELLA Family Anaplasmataceae now contains four genera: Ehrlichia, Anaplasma, Neorickettsia, andWolbachia.

Medically important: E. chaffeensis, E. ewingii, A. phagocytophilum (previous name: HGEagent, E. equi, E. phagocytophilum) causing Ehrlichiosis; C. burnetti causes Q fever.

Small: 0.2-2.0 μm OBLIGATE INTRACELLULAR PATHOGENSZOONOTIC: Anaplasmataceae

Arthropod vectors: ticks, (See Zoonosis & Biodefense Lectures)



MICR570/ZMR/F12 6-3

iv . CHLAMYDIACEAE Family contains 2 Genera: Chlamydia (C. trachomatis) & Chlamydophila (C. psittaci, C.pneumoniae)

DIVIDED IN TO 2 Ecological GroupsHuman infections ONLY: Trachoma, inclusion conjunctivitis & lymphogranuloma venereum

(See UTI & STI Lectures).

Zoonotic infections (birds → man): Parrots, finches, pigeons, chickens, ducks, turkeys &seabirds (See Zoonosis & Biodefense Lectures).

SIZE & SHAPE

Small: 0.2-0.7μm dia (< largest virus)Non-motile, coccoidal

UNIQUE FEATURE: OBLIGATE INTRACELLULAR PATHOGENS

Grow in cytoplasmic vacuoles in limited host cell types

Depend upon host for ATP & NAD+

Can not synthesize ATP or reoxidise NADP No detectable flavoproteins or cytochromes

NO Peptidoglycan detected in 1 stage of life cycle

COMPLEX LIFE CYCLE PRODUCING 2 DEVELOPMENTAL FORMSDetected by Gimenez & Giemsa stains

ELEMENTARY BODY (EB) RETICULATE (RB) or INITIAL BODY (IB)

Non-replicating Replicating

Infectious Non-infectious

Small 0.25-0.3μm dia Larger 0.5-0.7μm dia Adapted for extracellular survival Adapted for intracellular growth

Induce endocytosis Does not induce endocytosis

Metabolically Inactive Metabolically active

DEVELOPMENTAL CYCLE

Refer to: Murray et al. p465. Fig 47-1: Growth Cycle of C. trachomatis.a. EB taken into host cell by phagocytosis

b. Next 8 hours EB reorganises→ RB (IB)c. RB grows & divides by binary fission

d. 24-48hr RB reorganises→ EBe. Completed: host cell liberates EB’s

This developmental cycle is unique to the Chlamydia

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MICR570/ZMR/F12 6-4

INTRODUCTION TO THE FUNGIThe fungi are widespread and are involved in the decomposition of organic matter and play animportant role in the recycling of organic compounds in nature. Although more than 100,000species have been identified most fungal diseases of humans are caused by only 10-15different fungi.

N.B: Eukaryotic: nucleus/nuclear membrane BUT NO chlorophyll (differ from plants)

Macroforms: mushrooms, puffballs, gill fungi (NOT DISCUSSED)Microforms: molds & yeast

ALL FUNGI ARE SAPROBES (live on dead or decaying organic matter)TYPES OF RELATIONSHIPS

Symbionts: association with mutual advantageCommensals: one benefits, other neither benefits nor is harmedParasites: live on or within a host, harmful to host

MORPHOLOGICAL FORMS OF MICRO-FUNGI YEASTS: Unicellular

Typically round/oval single cells. 4-5μm dia (up to 24μm dia). Reproduce Asexually by budding (Refer to Murray et al. p69. Fig 7-2A: Yeast cell reproducing)Form elongated tubes termed Pseudohyphae.

MOLDS: Multicellular Long filamentous/tubular structures (HYPHAE) which intertwine to form the MYCELIUM.

Hypha range from 5-50μm length; 2-4μm dia.Reproduce Sexual &/or Asexual by producing spores (See Microbial Growth & ReplicationLectures).

HYPHAE Refer to: Murray et al. p69. Fig 7-2B: Types of Hyphae. Thread-like branching filaments - nucleated tubes of cytoplasm.Characterised by presence/absence of wall partitions.

SEPTATE: partitioned by cross walls ASEPTATE or COENOCYTIC: not partitioned

DIFFERENCES BETWEEN HYPHAE vs YEAST PSEUDOHYPHAE Feature Present in:

Permanent True HyphaeDifferentiation True HyphaeBranching True Hyphae

Arthrospores & Chlamydospores True HyphaeNOTE: True hyphae can be easily differentiated from Pseudohyphae (See handout p 5).



MICR570/ZMR/F12 6-5

MYCELIUM An intertwined mat of hyphaeComposed of 2 portions

VEGETATIVE: attached to substrate/penetrates to obtain substrateREPRODUCTIVE: - represented by aerial structures

Asexual reproduction propagules (conidia)NOTE: presence of mycelium gives rise to a cotton wool appearance on agar

PLEASE NOTE: Fungi can reproduce Sexually or AsexuallyRefer to: Murray et al. p69. Fig 7-3A & B: Examples of Asexual spore formation

DIMORPHIC FUNGI (DIMORPHISM)Morphological & physiological conversion of certain fungi from one phenotype to another i.e.,SAME organism can exhibit 2 different formsDEPENDS UPON CHEMICAL & PHYSICAL FACTORS (Temperature)IMPORTANCE: Pathogenicity (See Pathogenesis Lectures)

E.g., Candida albicans, Coccidioides immitis, Histoplasma capsulatum, Ajellomyces(previously known as Blastomyces) dermatitidis, Paracoccidiodes brasiliensis,Paracoccidoides marneffei & Spirothrix schenckii

Refer to http://gsbs.utmb.edu/microbook/ ch075.htm

WHAT ARE THE DIMORPHIC FORMS?FREE-LIVING STATE: MYCELIAL or HYPHAL FORMSOccurs at sub-physiological temperature (25oC); Distinct sexual forms are displayed.

PARASITIC STATE: YEAST FORMDue mainly to physiologic temperature (37oC); Oval morphology (Nothing distinct).

PLEASE NOTE: MOST, BUT NOT ALL fungi are dimorphic Aspergillus sp. ALWAYS in mycelial phase (even in deep tissue infections) Torula sp. Exist ONLY as yeast

Candida albicans Yeast & Mycelial form in human tissue; Mycelial form (in culture) producesPseudohyphae (in biofilm: superficial cutaneous & mucus membrane infections)

FUNGAL STAINING PROCEDURESKOH PREPARATION/PROCEDURE

Take scraping from margin (not centre) of lesion

Place on clean slide Add 2-3 drops 10% KOH in water Warm slide (DO NOT BOIL)OPTIONAL: Add 1 drop Lactophenol Cotton Blue

Add cover slipExamine immediately under high dry magnification

USE OF STAININGDetermine Fungal Morphology i.e., yeast or mould If mould identify hyphae (septate/non septate); spores (thallospores or sporangium)



MICR570/ZMR/F12 6-6

FUNGAL ARCHITECTURERefer to: Murray et al. p68. Fig 7-1: Diagram of a Fungal Cell.

Cell surface - Glycocalyx (Capsule in only 1 species)Cell wallCytoplasmic membrane

Eukaryotic cell Nucleus - Nuclear envelope, Nucleolous, Chromosome

Cytoplasm - Organelles - ER, Golgi, Mitochondria, VacuolesCytoskelton - Microtubules, MicrofilamentsRibosomes - 80S

ABSENCE of locomotion structures ABSENCE of capsules EXCEPT Cryptococcus neoformans (Important for diagnosis)

CELL WALL

Contains NO PEPTIDOGLYCAN (differs from bacteria) Thick & Rigid. Complex Composition of 75% Polysaccharide & 25% Protein CHITIN (Polymer of N-acetyl-D-glucosamine)

Additional Polysacc’s such as Glucans (1,3-β- & 1,6- β-glucan); MannanProteinsMay contain lipids

CYTOPLASMIC MEMBRANE

Typical phospholipid bilayer but differs from bacteriaContains sterols: ERGOSTEROL not Cholesterol

Site of antifungal drugs (See Control of Microbial Populations Lecture)Functions the same as all membranes

CYTOPLAMSIC CONTENTS Differs from Bacteria More Complex: Golgi Body (GB); Endoplasmic Reticulum (ER); CytoskeletonRibosomes: 80S (large 60S subunit & small 40S subunit)Microtubules (spindle fibers): Tubulin - site of antifungal drug (See Control of MicrobialPopulations Lecture).

NUCLEUS (Eukaryotic)Differs from BacteriaNucelar membrane bound (Nuclear envelope)Most Haploid

Contains the chromosome (larger than bacteria)



MICR570/ZMR/F12 6-7

FUNGAL CLASSIFICATIONKINGDOM: FUNGI2 DIVISIONS: Myxomycota & Eumycota 5 subdivisions (4 are pathogenic to humans)Refer to: Murray et al. p70. Table 7-3: Medically Important Fungi.

Class Zygomycetes

Most primitive; Filamentous; Non-septate (separates them from other subdivisions)Reproduce sexually (zygospores) & asexually (sporangiospores)NOT Primary Pathogens (OPPORTUNISTIC)E.g., Rhizopus sp.; Mucor sp., water molds

Class AscomycetesSeptate; Produce sexual (ascospores) & asexual (conidia) sporesMembers Include: yeasts & moulds - Dermatophytes (skin infections); some Aspergillus sp.

Class BasidiomycetesSeptate; Produce sexual spores (basidiospores)Members Include: Mushrooms & Puffballs

Only Human Pathogen: Filobasidiella neoformans (Sexually reproductive form of C.neoformans)

Class Deuteromycetes Septate; Reproduce asexually; septate hyphae, Sexual reproductive structures UNKNOWNIncludes yeasts & moldsE.g., Candida, Coccidioides sp., Epidermophyton sp., Malassezia, Microsporium sp.,Trichophyton sp.

Class Arch iascomycetes Reproduce sexually (fusion) & asexually (binary fission)Only member: Pneumocystis jirovecii (previously known as P. carnii)

FUNGAL DISEASES: MYCOSESRefer to: Murray et al. p72. Table 7-5: Classification of Human Mycoses.

Systemic: by inhalation, initially pulmonary → disseminated

Subcutaneous: infections of deep layers most by wound puncture, remain localised

Cutaneous: invade keratinized and cutaneous tissue

Superficial: infections of limited to surfaces of skin & hair, innocuous, reappear

Opportunistic infections: debilitated & traumatized patients, immuno-suppresed

Mycotoxins: Mycetismus ( Amantia mushroom) Afloatoxin ( Aspergillus flavus)

Each wil l be discussed in the relevant sections wi thin the course



MICR570/ZMR/F12 6-8

IN SUMMARYFUNGAL FEATURES SHARED WITH BACTERIAMicroorganisms (forms not seen with the naked eye) ALL fungi grow axenically (without others) - on artificial media ALL fungi are aerobic or facultativeMOST fungi are NOT capable of invading living human tissue

FUNGAL FEATURES DIFFERENT FROM BACTERIA ALL fungi EUKARYOTIC (no exceptions)Different internal organelles: membrane bound nucleus; chromosomes that sort by mitosis;80S RibosomesLarger than bacteriaBiochemically different (See Diagnosis of Infections Lectures)Fungi grow more slowly (See Microbial Growth & Multiplication Lectures)Fungi have multiple reproductive patterns - Usually but not always (See Microbial Growth &Multiplication Lectures)

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Review Questions1. What makes each of the bacteria discussed here atypical (compared to the “norm”)?2. Why are the penicillin group of antibiotics ineffective against Mycoplasma species?3. What links the Rickettsiae, Anaplasmataceae, Coxiella & Chlamydial bacteria?4. What differentiates the Chlamydial bacteria from the others?5. What is the difference between true hyphae & pseudohyphae?6. What is the importance of fungal dimorphism?

References in Murray, P.R., Rosenthal, K. S. & Pfaller, M.A. (2009) Medical Microbiology, 6 th Edition (Recommended Course Text)

For the Atypical Bacteria: Chapter 43: Mycoplasma & Ureaplasma p421-426Chapter 44: Rickettsia & Orientia p427-434Chapter 45: Ehrilichia, Anaplasma & Coxiella p435Chapter 46: Chlamydiaceae p441-450

For the Fungi: Chapter 5: Fungal Classification, Structure & Replication p67-64

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