Institute for Microbiology, Medical Faculty of Masaryk University and St. Anna Faculty Hospital in Brno Miroslav Votava PATHOGENICITY AND VIRULENCE The 7th lecture for 2nd-year students of Dentistry April 4, 2012

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

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

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)

Biofilm and disease 2 – revision Chronic infections of natural bodily surfaces dental caries (Streptococcus mutans, Str. sobrinus, actinomycetes, lactobacilli, veillonellae & oth.) periodontitis (Porphyromona gingivalis, Tannerella forsythia, Treponema denticola & oth.) otitis media (Haemophilus influenzae) osteomyelitis (Staphylococcus aureus) cholecystitis and cholangoitis (enterobacteria) prostatitis (Escherichia coli) subacute bacterial endocarditis (oral streptococci) pneumonia in cystic fibrosis (Pseudomonas aeruginosa)

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)

Dental plaque – revision = adhering microbial layer, composed of live and dead bacteria and their products, together with host components Microbial composition: differs with time and localization Supragingival plaque: mutans & mitis group streptococci, then lactobacilli and actinomycetes then lactobacilli and actinomycetes Subgingival plaque: two layers thin adhering plaque (G+ cocci, rods and filaments) non-adherent bacteria (G – anaerobes: prevotellae, porphyromonads, fusobacteria, treponemae)

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

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

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

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

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

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 - - -

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 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

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

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)

History of infectious diseases – I Leviticus (3rd book of Moses) – ban of pork Quarantine (= 40 days) – plague Fracastoro ( ): „De contagione“ 1676 van Leeuwehoek – observed bacteria 1838 Schönlein – mould in hair during favus 1850 Davaine – bacteria in sheep with anthrax 1857 Pasteur – microbes → spoil wine and beer 1865 Villemin – microscopically: bacteria in TBC 1869 Pasteur – parasites in silkworm 1876 Lister – antisepsis 1876 Robert Koch and Louis Pasteur – anthrax

History of infectious diseases – II 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 the microbe, the host and their environment are responsible

Koch´s postulates A particular microbe is the etiological agent, if 1.it is present in every case of the disease and its localization in the body corresponds to the damages observed; 2.it is isolated from the host and kept in pure culture for several generations; 3.by means of the microbe grown in this way it is possible to imitate the disease in another host; 4.it is again isolated from the experimentally infected host.

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

Pathogenicity Pathogenicity (= the ability to cause a disease) 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

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

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

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)

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

Virulence Virulence = degree (measure) of pathogenicity Virulence = property of certain microbial strain – 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 on the host (be cautious with the strains from dissection material) Attenuation = artificial weakening of virulence by many passages on other host (viruses) or in vitro (bacteria) Attenuated strains serve for the development of vaccines Attenuated strains serve for the development of vaccines

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“ (attenuated) 12 years on potato with bile until it lost most of its virulence (in normal host 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

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

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 Thank you for your attention