1. PATHOGENICITY AND VIRULENCE FACTORS OF PATHOGENICITY AND VIRULENCE – I
Institute for Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospital in Brno
Miroslav Votava
PATHOGENICITY AND VIRULENCE
FACTORS OF PATHOGENICITY AND VIRULENCE – I
The 7th + 8th lecture for 2nd-year students
April 8, 2013

2. Resistance of biofilm towards toxic substances – revision
MICROBES IN THE BIOFILM FORM ARE ALWAYS
MORE RESISTANT THAN IN THE PLANKTONIC FORM
Higher resistance applies also to disinfectants and antibiotics
Differences in sensitivity sometimes amount up to 3 orders
General mechanism of the higher resistance is not known
In each microbe-antimicrobial combination the mechanism can be different

3. Possible causes of higher resistance of biofilm – revision
More difficult penetration of toxic matter through the biofilm
Character of environment in the biofilm is altered
Also the microbial population in the biofilm is altered

4. Biofilm and disease 1 – revision
Biofilm takes part in the pathogenesis of
chronic infections in general
infections of implanted devices
the progress of these infections is slow
they are without distinctive symptoms
acute exacerbations occur occasionally
the effect of antibiotic therapy is transitory only
after stopping antibiotics infections recur (even if
bacteria grown from them appear sensitive in vitro)

5. Biofilm and disease 2 – revision
Chronic infections of natural bodily surfaces
dental caries (oral streptococci, mainly Streptococcus mutans)
periodontitis (Gram-negative oral anaerobes)
otitis media (Haemophilus influenzae)
osteomyelitis (Staphylococcus aureus)
cholecystitis and cholangoitis (enterobacteriae)
prostatitis (Escherichia coli)
subacute bacterial endocarditis (oral streptococci)
pneumonia in cystic fibrosis (Pseudomonas aeruginosa)

6. Biofilm and disease 3 – revision
Chronic infections of artificial surfaces
central venous catheters (coagul. neg. staphylococci, candidae)
prosthetic heart valves (Staph. aureus, Staph. epidermidis)
joint prostheses (Staphylococcus aureus, Staph. epidermidis)
surgical sutures (Staphylococcus aureus, Staph. epidermidis)
vascular grafts (Gram-positive cocci)
endotracheal tubes (various bacteria and yeasts)
intrauterine contraceptive devices (Actinomyces israelii)
urinary catheters (E. coli or others, mainly Gram-negative rods)
contact lenses (Pseudomonas aeruginosa, Gram-positive cocci)

7. Pathogenicity
Pathogenicity = ability of a microbe to be harmful to health and to cause disease
× Infectiousness = ability to cause infection
Infection – broader term than disease
In the disease the symptoms of disease are present (the infection is manifest)
But the infection may proceed without symptoms (inapparent infection)
Apart from infections microbes can cause food poisoning, as well

8. Infection
The definition of infection is not easy
Infection = situation when the etiological agent of infection invades an organism and multiplies in it; or it settles on bodily surfaces and acts adversely there
× Colonization = settlement of bodily surface by a nonpathogenic microbe (or by a pathogen that does not cause pathological symptoms there)

9. Relationship between the microbe and the host
The relationship is dynamic and influenced by the environment:
microbe host
environment
Illness is not a rule – peaceful coexistence is usually better for the parasite
In spite of that the host tries to get rid of the parasite – to destroy, remove or at least to localize it

10. Pathogenicity
Pathogenicity = the ability to cause a disease It depends on both microbial and host species
Particular microbial species is pathogenic for a specific host species only, for another species it may be non-pathogenic
This host species is susceptible to the relevant microbial species, to a different microbial species it can be resistant

11. Primary and opportune pathogens
Primary (obligate) pathogens → cause disease even in otherwise healthy individuals = chiefly agents of classical infections (diphtheria, typhoid fever, plague, gonorrhea, tetanus, influenza, morbilli etc.)
Opportunistic (facultative) pathogens → cause disease under certain conditions or at a certain disposition only = usually members of normal flora
when they reach another site in the body
or when the immunity of the individual is lowered

12. Natural and experimental pathogenicity – examples
Microbes naturally pathogenic for man & animals:
Staph. aureus, Francisella tularensis, Clostridium botulinum, rabies v., tick-borne encephalitis v.
Microbes pathogenic for animals experimentally:
Bacillus anthracis, Streptococcus pneumoniae, Clostridium tetani – mouse
Mycobact. tuberculosis, rickettsiae – guinea pig
Treponema pallidum, herpes simplex v. – rabbit
Microbes pathogenic for man only:
Neisseria gonorrhoeae, Haemophilus ducreyi
Microbes non-pathogenic for man:
Majority of soil and water microorganisms

13. Opportunistic pathogens – I
Typical opportunistic pathogen:
Escherichia coli
A part of normal colonic flora (but <1 % only)
Outside the large intestine = pathogen
cystitis, pyelonephritis, urosepsis
cholecystitis, peritonitis
wound infections
At lowered immunity (newborns):
meningitis
diarrhea (EPEC – serotypes O55, O111)

14. Opportunistic pathogens – II
Another opportunistic pathogen:
Staphylococcus epidermidis
Part of normal skin and mucosal flora
Outside the skin and mucosae = pathogen
wound infections (also surgical: sternum, eye)
cystitis
At lowered immunity:
above all blood stream infections in individuals with i.v. catheters, infections of implants and other devices
sepsis in newborns and neutropenic individuals

15. Virulence Virulence = degree (measure) of pathogenicity
Virulence = property of certain strain of the microbe – a pathogenic species can incorporate highly virulent strains as well as almost avirulent ones
Indicator of strain virulence: ability to kill
LD50 = 50% lethal dose (the amount of microbe that is able to kill exactly ½ of experimental animals)
Increasing virulence: repeated passages of the strain (be cautious with the strains from dissection material)
Attenuation = artificial weakening of virulence (attenuated strains serve for the preparation of vaccines)

16. Attenuation – an example
BCG-vaccine against TBC (bacille Calmette-Guérin)
Original strain – Mycobacterium bovis – is less pathogenic for man than Mycob. tuberculosis
The selected strain was „tormented“ 12 years on potato with bile until it lost most of its virulence (it is almost avirulent)
In a normal newborn BCG causes only a local process in the site of injection or in a regional lymph node
Very rarely in an immunodeficient newborn it can cause the generalized infection

17. MICROBE obligately opportunistically Species: pathogenic pathogenic non-pathogenic Strain: virulent avirulent
Individual: sensitive nonspecifically unresponsive
or specifically immune
Species: susceptible resistant
HOST

18. FACTORS OF PATHOGENICITY AND VIRULENCE – I

19. Three elements of pathogenicity and virulence
1. Transmissibility (communicability) = ability to be transmitted between hosts
2. Invasiveness = ability to:
- enter the host ability to
- multiply within = overcome
- spread within the defence
3. Toxicity = ability to do harm to the host

20. Transmissibility – I It depends on
the way of transmission – especially on
- the way in which microbes leave the body the amount of excreted microbes the portal of entry into other host
the microbe tenacity – the degree of resistance to the external environment
the minimum infectious dose – the number of microbes required for the start of infection
the behaviour of the host – the abuse of the host‘s defensive reflexes for the transmission

21. Transmissibility – II
Way of elimination from the body: not only respiratory secretions and diarrhoeic stool are infectious but every biological substance, as well
Typhoid fever – Salm. Typhi is more dangerous in kidney than in gallbladder (urine × stool)
Amount of eliminated microbes: 102 new infectious virions per 1 infected respiratory epithelial cell → 109 virions in 1 ml of respiratory secretion
Portal of entry: in general, the infection penetrates better through mucosae than through skin (e.g. respiratory infection with tick-borne encephalitis virus acquired in a laboratory is more dangerous and usually ends fatally: )

22. Transmissibility – III
Microbe tenacity (stability in environment)
Resistant: bacterial spores (Clostridium tetani), protozoal cysts (Giardia lamblia), helminth eggs (Taenia saginata)
Delicate microbes rely on:
- direct transmission (sexual contact in particular – gonococci, treponemae)
- biological vectors (ticks, mosquitoes – borreliae, arboviruses)
- transmission by water (leptospirae, shigellae)

23. Transmissibility – IV Infectious dose (ID)
high: V. cholerae, salmonellae – roughly 108 cells
(high ID: always in an immune individual!)
low: shigellae cells
gonococci, Mycob. tuberculosis
Coxiella burnetii (Q fever) (!)
Host behaviour
Abuse of defence reflexes such as cough, sneezing, diarrhoea
Goal-directed change of behaviour: rats infected with Toxoplasma gondii lose fear of cats (so as the parasite can finish its life cycle in cat bowel)

24. Invasiveness – entering the host
Most often through mucosae
Sometimes the entering is preceded by the colonization = overcoming the concurrence of commensals
Prerequisite of successful entry: ability to
- adhere to epithelium by means of adherence factors
- penetrate through epithelium by means of penetration factors

25. Adherence factors of bacteria
Fimbriae (pili)
Size ca. 5100 nm
Their end reacts specifically with a receptor on epithelial surface P-fimbriae of urinary E. coli strains CFA I & CFA II of enteropathogenic E. coli fimbriae of gonococci
Nonfimbrial adhesines
- hemagglutinins of yersinae, bordetellae F protein of Streptococcus pyogenes

26. Adherence factors of other microbes
Viruses
envelope projections (hemagglutinin) of influenza virus
glycoprotein gp120 of HIV
Parasites
lamblial suction disc
suckers or rostellum of taeniae
Micromycetes
glucans and mannans of yeasts keratophilia of dermatophytes (skin moulds)

27. Penetration into internal environment – I
A. Direct penetration by means of
small cracks in skin (S. aureus, Str. pyogenes, B. anthracis, Francisella tularensis, wart viruses)
small cracks in mucosa (T. pallidum, HBV, HIV)
animal bite (rabies virus, Pasteurella multocida)
arthropod bite (arboviruses, borreliae, plasmodia)
enzymes (penetration factors)
lecithinase (C. perfringens): surface membrane
hyaluronidase (S. pyogenes): intercellular cement

28. Penetration into internal environment – II
B. Forced phagocytosis by normally non-phagocyting cells by means of
- invasines, which change cellular framework
Ipa (shigellae) internalin (L. monocytogenes) YadA (Y. enterocolitica)
- ruffling of epithelial surface
(salmonellae)
- unknown mechanism as yet
(legionellae, chlamydiae)

29. Ability to multiply in vivo
Intracellular multiplication is better → a lot of available nutrients, defence against immunity Intracellular parasites: mycobacteria, rickettsiae, chlamydiae, listeriae, salmonellae etc.
Extracellular multiplication – not so easy → it is obstructed by
- antibacterial substances in blood (complement, lysozym, antibodies)
- high temperature (M. leprae, M. haemophilum)
- but above all by shortage of free Fe (Fe is bound to lactoferin and transferin in serum)
To get Fe bacteria produce siderofores and hemolysins

30. Ability to spread through the macroorganism
According the ability to spread different infections evolve:
- localized infections (common cold, salmonellosis, gonorrhoea)
- systemic infections (influenza, meningitis)
- generalized infections (morbilli, typhoid fever, exceptionally even localized and systemic infections)
Way of spreading:
by means of lymph
by means of blood
per continuitatem (into immediate neighbourhood)
along nerves
(more details will be dealt with later)

31. Defense against infection
Two tightly linked defense systems:
1. Innate immunity (nonspecific one)
2. Acquired (specific, adoptive) immunity
Both systems hand in hand
a) prevent microbes from colonizing bodily surfaces
b) bar the penetration of microbes into tissues
c) inhibit their spread through the body
d) neutralize their toxins
e) aim for their liquidation and removal of their remains

32. Innate immunity – properties
- acts nonspecifically against whole microbial groups (bacteria, viruses etc.)
- is inherited, therefore it exists from the birth
- is present in all members of the given species
- is no match for obligate pathogens
- works instantly – which is extremely important!
- acts uniformly even during repeated contact

33. Innate immunity – tools
Barriers of colonization and penetration – skin, mucosae, normal microflora, protective reflexes
Barriers of spread and tools liquidating microbes
- cellular – mainly phagocytosis
- humoral – complement, lysozyme, cytokins (interferon) etc.
Fever
Inflammation – calor, dolor, rubor, tumor, functio laesa

34. Acquired immunity – properties
- affects specifically only a particular microbe
- forms only during the lifetime after the contact with the agent in question
- develops in only a particular individual
- protects also against virulent strains of obligate pathogens
- starts to operate relatively late, after immune reaction has developed
- has a memory, so after repeated contact with the same microbe it acts more quickly and efficiently

35. Acquired immunity – tools
Antigen-presenting cells (phagocytes; antigen = foreign structure on the microbial surface)
T cells (lymphocytes) and activated macrophages (cell-mediated immunity)
B cells (lymphocytes) = producers of antibodies (humoral immunity)

36. Protection by cell-mediated immunity – I
Indispensable in the defense against intracellular parasites (mycobacteria, listeriae, brucellae, francisellae, viruses, some fungi, protozoa)
In a non-immune macroorganism:
Microbes remain alive and multiply in phagocytes, which disseminate them through the body
In an immune macroorganism:
immune lymphocytes Th1 react with microbial antigens and produce cytokines (IFN-γ, TNFα etc.), which attract and activate macrophages

37. Protection by cell-mediated immunity – II
Activated macrophages go on the rampage:
- they release enzymes and damage microbes as well as the neighboring tissue (delayed hypersensitivity)
- they phagocyte more vividly
- they reliably kill engulfed microbes
In virus infections and tumors the afflicted cells are recognized and killed by cytotoxic Tc lymphocytes

38. Protection by antibodies
Bacterial infections:
support of phagocytosis – opsonization of encapsulated bacteria (IgG)
inhibition of adherence to epithelium – mucosal antibodies IgA
neutralization of bacterial toxins (IgG)
bacteriolysis by complement (IgM, IgG)
transfer of immunity across the placenta (IgG)
Parasitic infections:
expulsion of helminths (IgE)
Viral infections:
neutralization of virus infectivity (IgG, IgA) …

39. Recommended reading material
Paul de Kruif: Microbe Hunters
Paul de Kruif: Men against Death
Axel Munthe: The Story of San Michele
Sinclair Lewis: Arrowsmith
André Maurois: La vie de Sir Alexander Fleming
Hans Zinsser: Rats, Lice, and History
Michael Crichton: Andromeda Strain
Albert Camus: Peste (= The Plague)
Thank you for your attention