1. 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 + part of the 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 antibioticsHigher resistance applies also to disinfectants and antibiotics Differences in sensitivity sometimes amount up to 3 ordersDifferences in sensitivity sometimes amount up to 3 orders General mechanism of the higher resistance is not knownGeneral mechanism of the higher resistance is not known In each microbe-antimicrobial combination the mechanism can be differentIn each microbe-antimicrobial combination the mechanism can be different

3. Possible causes of higher resistance of biofilm – revision 1.More difficult penetration of toxic matter through the biofilm 2.Character of environment in the biofilm is altered 3.Also the microbial population in the biofilm is altered

4. Biofilm and disease 1 – revision Biofilm takes part in the pathogenesis of 1. chronic infections in general 2. infections of implanted devices the progress of these infections is slow the progress of these infections is slow they are without distinctive symptoms they are without distinctive symptoms acute exacerbations occur occasionally acute exacerbations occur occasionally the effect of antibiotic therapy is transitory only the effect of antibiotic therapy is transitory only after stopping antibiotics infections recur (even if after stopping antibiotics infections recur (even if bacteria grown from them appear sensitive in vitro) 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. Possibilities of affecting the biofilm I – revision Prevention of the biofilm development Now:modifying the surface of biomaterials (change of charge) Now: modifying the surface of biomaterials (change of charge) impregnation of biomaterials with antimicrobials (antibiotics, antiseptics) impregnation of biomaterials with antimicrobials (antibiotics, antiseptics) In future:interference with quorum-sensing signals In future: interference with quorum-sensing signals inhibition of extracellular matrix production inhibition of extracellular matrix production inhibition of highly resistant persistors development inhibition of highly resistant persistors development ce se signály typu quorum-sensing inhibice tvorby mimobuněčné hmoty inhibice tvorby mimobuněčné hmoty inhibice vzniku vysoce odolných perzistorů inhibice vzniku vysoce odolných perzistorů

8. Possibilities of affecting the biofilm II – revision Disrupting the already present biofilm Now:high concentration of an antimicrobial – so-called antibiotic plug in a venous catheter Now: high concentration of an antimicrobial – so-called antibiotic plug in a venous catheter combination of antimicrobials with different mechanisms of action combination of antimicrobials with different mechanisms of action disruption of extracellular matrix – e.g. with enzymes (polysaccharide lyases) disruption of extracellular matrix – e.g. with enzymes (polysaccharide lyases) In future: use of molecules causing the autodestruction of biofilm

9. Detection of biofilm 1 – revision Phenotypic methods staining of biofilm on the inner wall of a vessel (test tube, well in microplate) staining of biofilm on the inner wall of a vessel (test tube, well in microplate) = Christensen method universal for most microbes character of colonies on agar with Congo red character of colonies on agar with Congo red for staphylococci only negative – colonies red, glossy positive – colonies black, rough

10. Inoculum: 0.5 McFarland scale; culture: Sabouraud broth with 8 % glucose, 48 hrs, 37 °C PS = polystyrene, S = glass PS S S Biofilm + Biofilm─ Biofilm ─ Biofilm production on glass and on hardened polystyrene

11. Positive production of slime on agar with Congo red Black colonies of a biofilm-positive staphylococcus strain

12. Detection of biofilm 2 – revision Genotypic methods e.g. proof of a gene set called ica- operon responsible for the production of intercellular adhesin in Staphylococcus epidermidis e.g. proof of a gene set called ica- operon responsible for the production of intercellular adhesin in Staphylococcus epidermidis●●●

13. 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

14. Ecological remark Ecology = science on mutual relations among organisms and relations between organisms and their environment Symbiosis = close association of two different organisms Three forms of symbiosis: Mutualism – both partners benefit from the association and are unable to survive without it Commensalism – the association is beneficial for one partner and indifferent to the other Parasitism – the association benefits one partner and harms the other (the host) → consequence = pathogenicity

15. 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 thereInfection = 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)× Colonization = settlement of bodily surface by a nonpathogenic microbe (or by a pathogen that does not cause pathological symptoms there)

16. History of infectious diseases Physiological thesis of Hippocrates: The disease (incl. the infectious one) = consequence of certain inadequacy of organism Microbial antithesis of Pasteur and Koch: The cause of the infectious disease is a microbe Ecological synthesis: = synthesis of physiological thesis and microbial antithesis – for the occurence of the infectious disease 1. the microbe, 2. the host and 3. their environment are responsible

17. Relationship between the microbe and the host The relationship is dynamic and influenced by the environment: microbe host microbe host environment 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

18. 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

19. 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 bodywhen they reach another site in the body or when the immunity of the individual is loweredor when the immunity of the individual is lowered

20. 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, mnohé viry (VZV, CMV aj.) Microbes non-pathogenic for man: Majority of soil and water microorganisms

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

22. 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)wound infections (also surgical: sternum, eye) cystitiscystitis At lowered immunity: above all blood stream infections in individuals with i.v. catheters, infections of implants and other devicesabove all blood stream infections in individuals with i.v. catheters, infections of implants and other devices sepsis in newborns and neutropenic individualssepsis in newborns and neutropenic individuals

23. 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 LD 50 = 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)

24. 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

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

26. FACTORS OF PATHOGENICITY AND VIRULENCE – I

27. 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

28. Transmissibility – I It depends on the way of transmission – especially onthe 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 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 environmentthe microbe tenacity – the degree of resistance to the external environment the minimum infectious dose – the number of microbes required for the start of infectionthe 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 transmissionthe behaviour of the host – the abuse of the host‘s defensive reflexes for the transmission

29. 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: 10 2 new infectious virions per 1 infected respiratory epithelial cell → 10 9 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: )

30. 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)

31. Transmissibility – IV Infectious dose (ID) high: V. cholerae, salmonellae – roughly 10 8 cells (high ID: always in an immune individual!) (high ID: always in an immune individual!) low: shigellae 10 2 cells gonococci, Mycob. tuberculosis 10 1 gonococci, Mycob. tuberculosis 10 1 Coxiella burnetii (Q fever) 10 0 (!) Coxiella burnetii (Q fever) 10 0 (!) 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)

32. 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) mvotava@med.muni.cz Thank you for your attention