Miroslav Votava PATHOGENICITY AND VIRULENCE FACTORS OF PATHOGENICITY AND VIRULENCE – I

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 + 8th lecture for 2nd-year students April 8, 2013

2. Resistance of biofilm towards toxic substances – revision MICROBES IN THEBIOFILM FORMAREALWAYS MORE RESISTANTTHAN 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 difficultpenetration of toxic matter through the biofilm • Character of environment in the biofilmisaltered • Also the microbial population in the biofilmisaltered

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, mainlyStreptococcus 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(Staphylococcusaureus, Staph. epidermidis) surgical sutures(Staphylococcusaureus, 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 andto 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 withoutsymptoms (inapparentinfection) 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 dynamicand 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 onboth microbial and host species Particularmicrobial 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 agentsofclassical 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 ofnormal 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 strainof 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-vaccineagainst 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) Ina normal newborn BCGcauses 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. MICROBEobligately opportunisticallySpecies:pathogenic pathogenic non-pathogenicStrain: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 withinthe 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– thedegree 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 eliminationfrom 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: 102new 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 102 cells gonococci, Mycob. tuberculosis 101 Coxiella burnetii (Q fever) 100 (!) Host behaviour Abuse of defence reflexes such ascough, 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 throughmucosae Sometimes the entering is preceded by the colonization = overcoming the concurrence of commensals Prerequisiteof successful entry: ability to - adheretoepithelium by means of adherence factors - penetratethrough 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 lamblialsuction 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 smallcracks in skin (S. aureus, Str. pyogenes, B. anthracis, Francisella tularensis, wartviruses) smallcracks 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 phagocytosisby normally non-phagocyting cells by means of - invasines,whichchange cellular framework Ipa (shigellae) internalin (L. monocytogenes)YadA (Y. enterocolitica) - rufflingof 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 Febacteria produce sideroforesandhemolysins

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 oflymph by means of blood per continuitatem(into immediate neighbourhood) along nerves (more details will be dealt with later)

31. Defense against infection Two tightly linkeddefense 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 Innate immunity - acts nonspecifically against whole microbial groups (bacteria, viruses etc.) -isinherited, therefore it exists from the birth -is present in all members ofthe given species -is no match for obligate pathogens -works instantly – which is extremely important! -actsuniformly even during repeated contact

33. Innate immunity – tools • Barriers of colonizationand 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 Acquired immunity - affects specifically only a particular microbe - forms only during the lifetimeafter 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 samemicrobe 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-mediatedimmunity) • 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 Th1react with microbial antigens and producecytokines (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) mvotava@med.muni.cz Thank you for your attention