Bacterial cell

Bacterial cell

Jan Tkadlec

Crucial impact of microbiology on medicine

Surgery almost imposible

Major types of advanced medical procedures are amputation or dissection

Surgery (cardio, neuro, plastic, …) Transplantation (heart, lungs, …)

Oncologic treatment (chemotherapy, radiotherapy…) Immunosupresion

Modern medicine will be imposible without knowledge of antibiotics,

antiseptics, disinfection and in general microbes as a cause of diseases

What if we never learn about the microbes ….?

Size of human population: 6 bilions in 2000

Projection based on previous trend: 3 bilions

Understanding microbes: rise of human population

Efect of antibiotics, improved hygiene etc.

Is there any other scientific discipline that could claim to save so many lifes?

Penicilin itself saved more than 200 milions lifes

Understanding microbes: increasing life expectansy

1908 – water chlorination

Twenties- vaccination campaings 30. – 40ies. Antibiotics

Deaths by infectious diseases Mean life expectancy

Cause of death per year :

Infectious diseases Cancer

Cardiovasvular diseases

Total Violence/injuries

Chronic lung diseases

Pregnancy related Other Git disorders

Neuro-psychologic disorders

Still a lot of work for microbiologists, infectionists and epidemiologists.

You could make a career!

MICROBIOLOGY

Dark prophecies…

„The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant.“

Sir Alexander Fleming, Nobel prize speech.

„"If we are not careful, we will soon be in a post-antibiotic era. And for some patients and for some microbes, we are already there.".“

Tom Frieden; CDC

„Drug-resistant infections - or “superbugs” – could claim 10 million lives a year and could cost a cumulative $100 trillion of economic output by 2050 .“

Jim O´Neil; Review on Antimicrobial Resistance.

Simon

• Healthy 1,5 year boy

• Died after rapid progression of respiratory tract infection by community MRSA unsuccesfully treated by wide spectrum antibiotics

Rebeca

17 years, student, swimmer

Seemingly common cold symptoms developed into serious MRSA pneumonia requiring intensive care hospitalisation and extracorporeal oxygenation(ECMO) After 4 months of hospitalization she died

David

19 years, travel to India as volunteer

Returned after train accident to USA, his wounds were infected by multiresistant bacteria (P.

aeruginosa, Klebsiella pneumoniae, Morganella morgani, Enterococcus), including NDM-1 producing bacteria. More than one year it takes to treat the infections

Meredith

• 19 years, treated with leukemia

• Died after succesfull bone marrow transplantation due to multiresistant Pseudomonas aeruginosa infection

Not only sick and old…..

Bacterial cell

What is interesting on bacterial cell? Medical point of view

1. Features/parts of bacterial cell that could be used for detection or

identification of bacterial patogens

2. What gives them ability to cause harm to human body

What is interesting on bacterial cell? Medical point of view

3. How to kill bacteria without (to much) harm to the host

What is interesting on bacterial cell? Medical point of

view

Size of human patogens

Light microscope Direct observation of stained bacteria

Bacteria are define by their small size!

They are microbes

Size in micrometers (µm)

Not so many shapes of bacterial cell

Coccus

Diplococci Streptococci

(chain) Staphylococci

(cluster)

Rod (bacillus)

Vibrio

Spirocheates Spirillum

Corynebacterium

Eu vs prokaryotes

Eu

Chromosome separated by membrane

Nucleus Organelles:

• Mitochondria

• Golgi

• Endoplasm. reticulum

• Lysosome Eu. flagellum Eu. ribosome

Pro

Circular chromosome (one copy=haploid)

Plasmids

Cell wall of peptidoglycan Outer membrane

Bacterial flagellum Bact. ribosome

Both

Cytoplasm

Plasmatic membrane

Structure of cell wall

Brown et al 2015 Nat Rev Microbiol

• Two basic variants

Thick peptidoglycan layer Simple

Terestric bacteria

Physical resistance (desication, irradiation) Thin peptidoglycan layer

Outer membrane Periplasmic space Requires humidity

Chemical resistance (antibiotics, enzymes, toxines)

Peptidoglycan

• Only in bacteria

• Rigid but permeable

• Integrity and shape of bacterial cell

• Protects from

• irradiation, desiccation, mechanical damage, inner pressure

• Polysacharide:

• N - acetylglucosamine

• N - acetylmuramic acid

• Polymer fibers croslinked via peptide side

chain into net like structure

Peptidoglycan synthesis – target of antibiotics

PBP – penicilin binding proteins - transpeptidases cross-link of peptidoglycan fibers – main target of antibiotics

• Beta-lactams (penicilins, carbapenems) and cefalosporins binds and inhibits PBP

• Glycopeptides (vancomycin, teicoplanin) binds aminoacid side chains and prevents PBP binding PBP

Inhibited action of PBP cause lysis of the cell when growing.

Outer membrane

Outer membrane is target of polymyxins (colistin)

Lipopolysacharide (LPS)

Endotoxin = part of the cell

• O-antigen

• Highly variable – e.g. serotyping E. coli O157:H7

• Core polysaccharide

• Lipid A – toxic part of LPS

LPS is PAMP (Pathogen-Associated Molecular Pattern)

Molecule specific for bacteria recogised by innate immunity (complement, macrophages) Septic shock

Only Gram negatives

Gram staining – most importatnt staining in microbiology

Procedure

V L A K

Violet Lugol Alcohol Karbolfuchsin

Allow visualise and distinguish bacteria in clinical sample

Vlak = train

Gram staining principle

• v

1. Crystal violet (CV) penetrates into the cell

2. Iodine solution (mordant) – formatio of precipitates – binding to the cell wall

3. Alcohol wash– decolorizing gram negatives – thin cell wall could not keep the stain. Grampositive keep its color (purple)

4. Counter staining of gram negatives with carbolfuchsin (pink)

Wilhelm et al 2015 ACS Chem. Biol.

Mycobacteria – Gram resistant bacteria

High content of wax-likes compounds - mycolic acids Repulse stain and alcohol

Cause of high physical and chemical resistance of mycobacteria

 Antibiotic resistance

Fast acid or Ziehl-Nielsen staining

Mycobacterium tuberculosis M. leprae

Capsule

• Additional protective layer – polysacharide

• Important for adhesion

• Protection from desication and immune system

• Hides surface antigens (PAMPs) from phagocytes, complement and antibodies

• Antigenic variation

• Frequently found in serious patogens

• Neisseria meningitidis, Haemophilus influenzae, Streptococcus pneumoniae, Bacillus anthracis etc.

• Essential for full virulence

Halo around bacterial cells when stained by bury stain

Electron microscopy

Example S. pneumoniae capsule

• S. pneumoniae (pneumococcus) – pneumonia, meningitidis, otitis media

• Main virulence factor, but not toxic itself

• Noncapsulated strains are avirulent

• Protects from phygocytes and opsonisation

• Variable (80-100 antigenic variants)

• Capsule is a target of S. pneumoniae vaccine

• Polyvalent vaccines:

• Covers multiple variants but not all

Neisseria meningitidis and Haemophillus influenzae type B vaccines targets also capsule antigens

Bacterial ribosom

Function – translation= protein syntesis

Size - 70S (S =Svedberg unit of sedimentation)

vs. Eukaryotic 80S

• large (50S) and small (30S) subunit vs eukaryotic 60S a 40S

16S rRNA (18S Eu) – part of small subunit

• In all living organisms

• Sequence – taxonomy, diagnostics

Antibiotics targeting ribosom

Macrolides and linkosamides

• Large subunit

• Block the syntesis of protein

Aminoglycosides

• Small subunit

• Mistranslated peptides - toxic

Tetracyclines

• Small subunit

• Prevents tRBA recognition Mostly bacteriostatic effect

Great when production of toxin is goal of the treatment

Bacterial flagellum

• Different from eukaryotic one

• Anchored in the membrane

• screw-propeller like mechanism

• Energy from proton gradient

• Some bacteria lacks flagella (streptococci, staphylococci)

• Spirochetes flagellum (=axial filament) corkscrew like motion

• Highly antigenic

• Serotyping of enterobacteria: H antigen

Different arrangement

Monotrichal

Lophotrichal

Peritrichal Amphitrichal

Chemotaxis – where are you going?

Two modes of movement based on orientation of flagella rotation

• Forward

• Tumbling

Movement on the solid surface

Swarming e.g. Proteus

• CLED agar – inhibition of swarming

• Otherwise Proteus will overgrow other

bacteria

Movement of non-motile bacteria

• Intermicrobial Hitchhiking

Staphylococcus aureus and Pseudomonas aeruginosa

• S. aureus and Candida

• Mechanical inoculation of

S. aureus by growing of the

yeast

Fimbria

• syn. pili (pilus)

• Shorter than flagella, around the surface of the cell

• Made of proteins

• Main function is adhesion

• Top of pili – adhesins

• adhesin bind sacharide on the surface of human cells

• Tissue specific

Fimbrie

• Escherichia coli urinary tract infection

• PAP fimbria (Pyelonephritis-Associated Pili)

• Home made medicine: cranberries

• Sacharides from cranberries goes into urine and blocks

adhesins on fimbria

Targets of antibiotics in bacterial cell

Mostly processes associated with cell growth

Biofilm

• Bacteria dont form a tissue

• But they could form a biofilm!!!

• Biofilm is attached structured consorcium of bacteria enveloped by extracelular matrix from polysacharides, proteins and extracelular DNA secreted by these bacteria

• Base of the biofilm is protected from

• antibodies

• complement

• phagocytes

• antibiotics

• Regeneration from the base

Biofilm – Clinical impact

• Majority of patogenic bacteria forms biofilm

• Protects from antibiotics and immune system

• Infection of foreign bodies:

• implants

• prostetics

• dental implants

• catheters

• vascular

• urinary

• Dental plaque

• Biofilm is impossible to eradicate

Growth and division of bacterial cell

Growth rate of bacteria is limited by speed of genome replication (cca 40 min.) But E. coli is able to divide each 20 minutes. How is ti possible?

Binary fission

Growth and division of bacterial cell

Solution: replication of more genomes et once Binary fission

Growth of bacteria

Light source

Lambert-Beer law

Turbidity corelates with amount of bacteria

Growth curve

1. lag phase – adaptation to the new environment 2. Exponential - rapid growth

3. Stationary – Limited nutrients availability 4. Death – accumulation of toxic products of

metabolism

Sporulation: special type of fission

McKenney et al, Nature Reviews Microbiology 2013 In unfavourable conditions

Resulting in formation of durable spore

Endospore

Mother cell dies

Bacillus and Clostridium

Germination

Spora

McKenney et al, Nature Reviews Microbiology 2013 B. subtilis B. anthracis

Durable, non-metabolising and dormant form Multiple protective layers

High chemical (alcohol) and physical resistance (boiling, UV irradiation)

Highly toxigenic bacteria

• Clostridium tetanii

• Clostridium botulinum

• Bacillus anthracis

Bacteria and the oxygen

a) Aerobic (e.g. Mycobacterium)

• Requires oxygen

• respiratory metabolism

• Oxygen is final electron acceptor b) Anaerobic (e.g. Clostridium)

• Hates the oxygen (fermentation), oxygen makes them sick

c) facultative anaerobic (most of the bacteria)

• Prefer oxygen (respiration), but are OK without it (fermentation)

d) Microearophilic (e.g. Campylobacter)

• Requires oxygen bur not too much (cca 2 %) e) Aerotolerant - (e.g.. Streptococcus)

• Dont care about oxygen (fermentation)

• Reactive oxygen species are toxic

• Result of metabolism processes

• Damage to the proteins, DNA/RNA and lipids

• Detoxifying enzymes

• Superoxid dismutase

• Peroxidase

• catalasa (

• Diferentiation staphylococci from streptococci

Oxygen as a poison

Anaerobic bacteria

Lacks detoxyfying enzymes that deals with reactive oxygen species

Dying in oxygen presence – transport of the sample Anaerobiosis

• Chemical reaction

• Supplementation with nitrogen

Patogenic bacteria

Relation to the host

Symbiotic bacteria

Comensal bacteria

Patogenic bacteria

• Primary (obligatory) patogen – cause disease of the healthy person

• Streptococcus pyogenes, Treponema pallidum, Bordetella pertussis, Salmonella typhi, Neisseria gonorrhoeae, Bacillus anthracis,

Mycobacterium tuberculosis, Yersinia pestis

• Facultative (oportunistic) patogen – requires decrease immunity

• E. coli, S. aureus, etc

Patogenic bacteria

• Patogen - biological factor (microorganism) able to cause disease

• Patogenity = ability to cause disease

• qualitative: patogenic vs non-patogenic

• Associated with species

• Virulence = quantitative measure of patogenity

• Associated with bacterial strain

• Letal dose LD

– dead of 50 % of tested subjects in 24 hours from exposition – measured as amount of inoculated bacteria

• Infective dose ID

– dose able to cause disease of 50% tested subjects

• Depends on the bacterial strain and host condition

Staphylococcus commander (Once Upon a Time... Life) Purple nose =It is Gram-positive!!!

Amphibiosis: patogen or symbiont

Helicobacter pylori only bacterium known to be living in stomach.

Associated with peptic ulcers and stomach cancer BUT – lower chance of esophageal cancer in H.

pylori colonised people

Helicobacter pylori

Virulence factors

• Allows bacteria to cause a disease Could be divided

• From the perspective of the host

• Invasivity – adhesion and colonisation

• Toxicity – direct damage to host tissues

• Immune dependent factors – the damage is mediated by the reaction of the immune

system

Invasivity

• Adhesion – attachment to the epithelia (adhesins, lipoteichoic acid, capsule)

• Invasion – pentration of the epithelium or into the host cell

• Enzymes – destruction of extracell matrix – hyaluronidase, colagenase, elastase

• Invasins – phagocytosis by non profesional phagocytes (e.g. epitelia)

• Flagellum – penetration through the mucus or epitelia

Toxicity

• Damage by direct action or through immune reaction

Endotoxins

• Part of bacterial cell – release after its disintegration

• Pathogen-Associated Molecular Pattern (PAMP)

• Molecules recognised by immune system (complement and macrophages= innate immunity) as foreign

• Lipopolysacharide

• Peptidoglycan

• Teichoic and lipoteichoic acid

• flagellum

• porins

 Cause massive immune response (cytokin storm) – septic shock

Exotoxins

• = „true“ toxins

• Extracelullar protein moleculles (compare to endotoxin – outer membrane G-)

• Direct and serious damage to host

• Diferentiation – variable criteria:

• chemical structure (single molecule or macromolecullar complex)

• Targett structure (cell surface or intracellular)

• Mechanism (neurotoxic, enterotoxic, cytotoxic)

Exoenzymes

• enzymatic destruction of host cells and tisues

• Penetration of mechanical barriers (epitelia, mucous membranes) – extracelular matrix

• hyaluronidase

• kolagenase, gelatinase

• elastase

• streptokinase, staphylokinase

• cytolytic enzymes

• (phospho)lipases

• Degradation of phospholipids in membranes

• Cell lysis

Cytotoxins

Pore forming toxins Mechanism of action

1. Subunits recognise receptor 2. Polymerisation of subunits 3. Insertion of the pore into the

mebrane 4. Ion leakage 5. Cell lysis

Cytotoxins of S. aureus

lukED, luk AB, PVL - leukocidins

Intracelular A-B toxins

• Two subunits A (action) and B (binding)

• Toxins inhibiting proteosyntesis – diphteric toxin, shiga toxin

• Hyperactivating toxins – choleratoxin, anthrax toxin, pertusis toxin

• Neurotoxins

Neurotoxins

• Extremely low lethal dose

• Produced by clostridia

• sporulating

• anaerobic

1. botulotoxin

2. tetanospasmin

Neurotoxins

• Mechanism of action: inhibition of fusion of synaptic vesiculles with presynaptic membrane

• botulotoxin: inhibits acetylcholin release → muscle relaxation

• tetanospasmin: inhibits GABA and glycine release→ muscle contraction

• Disruption of respiration

Enteric toxins

• Many patogenic bacteria

• E. coli, Shigella, Salmonella, Vibrio cholerae, Campylobacter, Clostridium difficile, S. aureus

• Poissoning of the intestinal epithelium→ diarrhoea

• Typical symptom of intestinal infection

• Diarrhoea

• patogen: rapid spread in host population

• Lots of liquid stool filled with bacteria

• host: cleaning of the intestines

Superantigens

• superantigens

• Direct cross connection between MHC II and TCR

• Without specific antigen

• Masive activation of T-

lymphocytes

Superantigens

• Staphylococcus aureus: 23 superantigens

• Streptococcus pyogenes: 11 superantigens

• For example:

• Pyrogenic exotoxins of Streptococcus pyogenes

• Toxin shock syndrome toxin of S. aureus

• Staphylococal enterotoxins

• Food poissoning

Toxic shock

• Caused by superantigens

• TSST-1 of S. aureus (less frequently caused by S. pyogenes)

• Similar to septic shock

• Leads to massive cytokine production by activated T-lymfocytes

• fever

• Colaps of immune and regulatory homeostasis

• Systemic patological changes

• CZ 1983-2011 - 159 cases, (47 menstrual form).

letality 11 %(staphylococci), 50% (streptococci)

biofilm on tampon fibres reservoir of staphylococci producing toxic shock toxin

Toxin detection

• Detection of toxin moleculles (protein)

• Specific antibodies

• latex bead agglutination

• ELISA

• Rapid antigen tests

• Detection of toxin genes(DNA)

• PCR specific for selected genes (primers)

Non-textbook literature

Fine