Institute of Medical Microbiology Semmelweis University Budapest

http://mikrobiologia.sote.hu

Dr. Dóra Szabó MD, DSci

Head

Dr. Ágoston Ghidán, PhD

Tutor

email:ghidan.agoston@med.semmelweis-univ.hu

Medical Microbiology

Microbes and vectors swim in the evolutionary

stream, and they swim faster than we do.

Bacteria reproduce every 30 minutes. For them, a

millennium is compressed into a fortnight.

They are fleet afoot, and the pace of our research

must keep up with them, or they will overtake us.

Microbes were here on earth 2 billion years before

humans arrived, learning every trick for

survival, and it is likely that they will be here 2

billion years after we depart …

(Krause 1998).

Sahara Yellowstone National Park

Magadi-lake, Kenya Anktarktisz, Vosztok-lake

photosyntesis Nitrogen fixation

Decomposition or biodegradationOil degradation

Production of Foods and Fuels

• Agricultural microbiology

• Aquatic microbiology

• Industrial microbiology

• Medical microbiology

• Space microbiology

• Environmental microbiology

Medical Microbiology1) Bacteriology: bacteria, 0,5-2μm

2) Virology: virus: protein + nucleic acid, 20nm-400nm

3) Micology: fungi: 3-5 μm size eukaryota

4) Parazitology 5) protozoons: eukariota1-2 μm 60-80 μm

trophozoid, cyst

6) helmintology: worms,

eggs, 1-3 μm

7) Prion: protein only

The size of the microorganisms

Largest Smallest (nm)

Fungi Bacteria Virus

Bacteria colony

Fungi colony

- Antibiotics

- Antibiotics are produced

in nature by molds such

as Penicillium and

bacteria such as

Streptomyces and

Bacillus.

- Vaccines

Medical, Pharmaceutical and

Biotechnological Applications

• recombinant DNA technology and genetic engineering. Important tools of biotechnology are microbial cells, microbial genes and microbial enzymes.

• Genetically engineering them to produce a substance or conduct a process. The genes source: human, animal, plant or microbial. – microbial production of foods, fuels,

enzymes, hormones, diagnostic agents, medicines, antibiotics, vaccines, antibodies, natural insecticides and fertilizers,

Biotechnology

History of Microbiology

Paralyticus poliomyelitis

1500 bc Egypt

Lepra

– XIV. century - plague Europe 20-45 % inhabitant 20-45% †

– 1831 - cholera Kairó 13% inhabitant †

– 1854-56 - Krím war dysenteria 10X több † , more than in the war

– 1899-1902 - Bor war háború – dysenteria 5X more † ,

Quarantine 1350

The plaque in Europe

History of Biological Warfare• 15th Century – Pizarrio’s army presented South

American natives clothing laden with the variola virus

• 1710 – Russian troops hurl the corpses of plague victims over the city wall (Russian – Sweden war)

Anton van Leeuwenhoek (1632 - 1723) was the

first to report microorganisms in 1673 (Royal

Society) (Animalcules)

– 50-300X magnification

Discoveries in Microbiology

Ignaz Semmelweis

•Demonstrated that childbed fever

(puerperal fever), caused by

streptococcal infections, was

transmitted to patients by doctor’s

hands

– Pioneer of antisepsis in

obstetrics

– Women giving birth in

hospitals by medical students

and physicians were 4x more

likely to contract puerperal

fever compared to those by

midwives

Handwashing with leach powder

– developed a system of surgery

designed to prevent

microorganisms from entering

wounds – phenol sprayed in air

around surgical incision

– Decreased number of post-

operative infections in patients

– his published findings (1867)

transformed the practice of

surgery

Joseph Lister

1827-1912

E. JENNER 1796 - L. PASTEUR 1885

Louis Pasteur 1922-

1895Pasteurization – heat killed

bacteria

Germ theory

Developed other vaccines including

those for chicken cholera,

anthrax, and rabies

• Vaccination against rabies

• Pasteur Institute in Budapest

Hőgyes Endre

(1847-1906)

Challanges in Mircobiology

Drug-resistance timeline

The Bacterial Cell Wall

Essential components: cell wall, cytoplasmic membrane, ribosome,

nucleoid, mesosome, periplasm

The bacterial cell wall is a unique structure which surrounds the cell

membrane. Although not present in every bacterial species, the cell wall is very

important as a cellular component. Structurally, the wall is necessary for:

* Maintaining the cell's characteristic shape- the rigid wall compensates for

the flexibility of the phospholipid membrane and keeps the cell from assuming a

spherical shape

* Countering the effects of osmotic pressure- the strength of the wall is

responsible for keeping the cell from bursting when the intracellular osmolarity

is much greater than the extracellular osmolarity

* Providing attachment sites for bacteriophages- teichoic acids attached to the

outer surface of the wall are like landing pads for viruses that infect bacteria

* Providing a rigid platform for surface appendages- flagella, fimbriae, and

pili all emanate from the wall and extend beyond it.

The structure, chemical composition, and thickness of the cell wall differ

in gram-negative and gram-positive bacteria.

Cell wall of Gram-positive

bacteria

-the peptidoglycan layer is much

thicker; multilayered

PG: sugar backbone with

peptide side chains that are

cross-linked

Gives rigid support, protects

against osmotic pressure; is

the site of action of penicillins

and cephalosporins and is

degraded by lysozyme

- have fibers of teichoic acid, that

protrude outside the peptidoglycan

major surface antigen

- no LPS

Cell wall of Gram-

negative bacteria

- the peptidoglycan

layer is thinner; single

layered

- no teichoic acids

- lypopolysaccharid

(endotoxin)

LPS layer is attached

to the outer membrane

of gram negative

bacteria.

1. Repeating units are

used to identify

different gram negative

bacteria.

2. Lipid A portion is

responsible for toxic

properties of LPS

Cytoplasma

Constituents

* Proteins including enzymes

* Vitamins, Ions

* Nucleic acids and their precursors

* Amino acids and their precursors

* Sugars, carbohydrates and their derivatives

* Fatty acids and their derivatives

The nucleoid is one long, single molecule of double stranded,

helical, super coiled DNA.

The two ends of the double-stranded DNA are covalently bond

together to form both a physical and genetic circle.

The chromosome is generally around 1000 µm long and may

contain as many as 3500 genes.

E. coli, is 2-3 µm in length but has a chromosome approximately

1400 µm long.

Ribosomes

Ribosomes are cytoplasmic organelles found in prokaryotes and

eukaryotes.

They are large complexes of proteins and three (prokaryotes 50S

and 30S ) or four (eukaryotes) rRNA (ribosomal ribonucleic acid)

molecules called subunits made in the nucleolus.

- serve as the site of mRNA translation ( = protein synthesis, the

assembly of amino acids into proteins); These are the site of action

of aminoglycosides, erythromycin, tetracyclines, and

chloramphenicol

Periplasmic space

• between the inner and outer membrane of gram negative organisms

• numerous enzymes

– metabolism of large substrates (hydrolytic enzymes), those that would be toxic if inside

of the cell (DNase) and molecules such as toxins that are destined for excretion to the

environment.

– For most substrates (and agents that act inside of the cell), passage through the

periplasmic space is essential.

– The activity of b-lactam antibiotics (the penicillins) for example is dependent upon

their getting access to the cell wall.

Flagella

- Flagella are long, whiplike appendages.

- Composed of a protein called flagellin.

- Flagellated bacteria have a characteristic number and location of

flagella: some bacteria have one, and others have many (a tuft at

one pole, or multiple flagella covering the entire surface).

-

Flagella-- Flagella move the bacteria towards nutritients and other

attractants (chemotaxis)

- Flagella are medically important for two reasons:

- May play a role in pathogenesis / e.g. urinary tract

infection: E.coli, P.mirabilis

- Identification in the clinical laboratory by the use of

specific antibodies against flagellar components

/Spirochetes move by using a flagellumlike structure called the

axial filament/

Fimbriae (Pili)

- Thin, hairlike appendages

- 1 to 20 microns in length

- Often occurring in large numbers, present on the cells of gram-

negative bacteria, (particularly enterobacteriaceae and neisseria)

- They do not possess motility, but being protein (pilin) in nature,

they possess antigenic and haemagglutinating (agglutination of

erythrocytes) properties.

- They are of medical importance because some fimbriae mediate

the attachment of bacteria to cells via adhesins (adhesins,

bacterial).

- Bacterial fimbriae refer to common pili, to be distinguished

from the preferred use of "pili", which is confined to sex pili (pili,

sex).

The capsule is important for four reasons

- it is determinant of virulence of many bacteria, since it limits

the ability of phagocytes to engulf the bacteria

- specific identification of an organism can be made by using

antiserum against the capsular polysaccharide (quelling reaction:

in the presence of the homologous antibody, the capsule will swell

greatly)

- capsular polysaccharides are used as the antigens in certain

vaccines

- the capsule may play a role in the adherence of bacteria to

human tissues (this is an important initial step in causing

infection

Capsule

The capsule is a gelatinous layer covering the entire bacterium.

- It is composed of polysaccharide, except in the anthrax bacillus, which has a capsule

of polymerized D-glutamic acid.

- The sugar components of the polysaccharide vary from one species of bacteria to

another and frequently determine the serologic type within species. (e.g. >90 different

serologic types of Streptococcus pneumoniae)

-

Glycocalix (slime layer)

The glycocalix is a polysaccharide coating that is secreted by many

bacteria.

- allows the bacteria to adhere firmly to various structures: skin,

catheters, heart valves

- Adherence of certain bacteria, such as Streptococcus mutans , to

the surface of teeth ( This plays an important role in the formation

of plaque, the precursor of dental caries.)

Granules

They serve as storage areas for nutrients and stain

characteristically with certain dyes.

For example, volutin is a reserve of high energy stored in

the form of polymerized metaphosphate –

“metachromatic” granule (Neisser stain).

Spores

These highly resistant structures are formed in response to adverse

conditions (clostridia, bacillaceae).

- Sporulation occurs when nutrients (such as sources of carbon

and nitrogen), are depleted.

- The spore forms inside the cell

- Contains bacterial DNA, a small amount of cytoplasm, cell

membrane, peptidoglycan, very little water, thick, keratinlike coat

(dipicolinic acid)

- The spore has no metabolic activity and can remain dormant for

many years

- Upon exposure to water and the appropriate nutrients -

germination into a metabolizing, reproducing bacterial cell occurs

(one!)

The medical importance of spores

1. Highly resistant to heating (sterilization!)

2. Highly resistant to many chemicals, including most

disinfectant

3. They can survive for many years, especially in the soil

(wounds contaminated with soil can be infected with spores and

cause diseases such as tetanus and gas gangrene

4. They exhibit no measurable metabolic activity (antibiotics are

ineffective)

5. Spores are not often found at the site of infections

(germination)

6. Infections transmitted by spores are caused by species of either

Bacillus or Clostridium

1.Crystal violet 2. Lugol iodine sol. 3.alcohol 4.Safranin

Before

staining

1

2

3

4

After the Lugol iodine is added there will be a purple complex , called iodine para rosanilin

in the bacteria.

This complex can not be washed out from the Gram positives during differentiation, which

therefore stained blue-purple.

In contrast the complex disappears from the Gram negatives which stain red.

Gr+ Gr-

Morphology of the bacteria

Gram-positive cocci in “stiffly-like”

arrangement (Staphylococcus)

Gram-positive cocci in long chain arrangement

(Streptococcus)

Gram-positive bacilli, in the centre non-stained endospore, that

didn’t deform the shape of the bacterial cell (Bacillus

cereus)

Gram negative cocci

(Neisseriaceae)

Gram negative bacilli (Escherichia coli)

Gram negative bacilli (Pseudomonas

aeruginosa)

Gram positive cocci in blood

sample (Enterococcus faecalis)

Gram positive cocci in blood sample

(Staphylococcus aureus)

Gram positive diplococci in sputum sample (Streptococcus

pneumoniae)

Gram positive pseudohyphae in vaginal discharge (Candida albicans)

Gram positive „bubbles” in vaginal discharge (Candida albicans)

Neisseria gonorrhoeae in urethral discharge

N.gonorrhoeae

Haemophilus ducrey in pus

Thank you for your attention!