Practical 7 - Pseudomonas, Bordetella, Bacillus

1. Practical 7- Pseudomonas, Bordetella, Bacillus • Hospital infections • Whooping cought • Antrax a bioterorismus • Microscopy - sc. Gram - Ps. aeruginosa, Bacillus anthracis, Bacillus cereus, sc.Wirtz Coonklin Bac. anthracis • Cultivation Ps. aeruginosa on blood agar and Endo, Bordetella pertussis on Bordet Gengou, B. anthracis a B. cereus on blood agar • Biochemical properties of Ps. aeruginosa • Ascoli thermoprecipitation • Cultivation of B. pertussis • ATB therapy of Ps. aeruginosa

2. Hospital - nosocomial infections • Hospital infection - infection, that arises in connection to hospitalisation or to diagnostical, therapeutic or preventive processes. I does not necessary have to present during the hospitalisation and not every infection arising during hospitalisation is nosocomial • Risk factors - age,accompanying diseases, surgical processes therapy - ATB, imunosupression, irradiation, not vital reservoire - indwelling catheters) • Microbes • Ways of transmission - in direct (inhalation, ingescion, inoculation) , direct (contact of infected skin or mucous membrane with healthy) • Prevetion - organisation of health process, construction, food supply, health process technics, asepsis and antisepsis, nursery approches, isolation, monitoring, surveillance, role of microbiologickal laboratory) • ATB therapy

3. Role of microbes in hospital infections • Staphylococcus aureus - problems of per secundam healing wounds (70 ies). Virulence, colonisation capacitiy, resistence - MRSA - methicilin resistent staphylococcus aureus (80 ies) • G-rods (60% of HI) - urinary tract infections, respiratory infections, wound infection, GIT • Opportunistic pathogens - Ps. aeruginosa (Hospital environmente – food, cut flowers, water, toels, mops, respiration devices, desinfection solutions. Persistent carriage in less than 6% helathy, 38% in hospitalised, 78% imunocompromised) and other non fermenting G- rods - Acinetobacter, Stenotrophomonas maltophilia, Burkholderia cepacia… - present in environmentí (Legionella pneumophila - climatisation) • PK negative staphylococci - colonisation of plastic material of indwelling catheters • Viruses - blood borne infection agenses HIV, VHB, VHC, CMV….

4. ATB therapy in hospital infections • Overuse of ATBe - resistence and multiresistence (selection pressure of ATB and transmission in hospital environment), toxic side effects, economic burden, deterioration of physiological microflora • Racional indication - preventive in spread of HI • Prophylaxis - oriented to anaerobe infectione - perioperative preparation for GIT and UGT surgery, in instrumental examination of patients with bacteriuria • ATB surveys - monitoring of ATB susceptibility • Restrictive policy - time restricted contraindication of some ATB • Rotation of atb - periodical changes of used - decreasing of selection pressure • Combination of atb - agains possible resistent mutnants, broader antimicrobial spectrum

5. Whooping cought • Clinical signs Inhalation of infectious droplets Patogenesis – exposition to bacteria and attachment to cylindric epitelium of bronchial stroma, production of toxinu and of tissu destruction + systemic toxicity – catharral phase (1-2 weeks) - sy influenza disease – paroxysmal phase (2- 4 weeks) - destruction of epitel – paroxysmus of cought – restriction of respiratory ways, vomiting, lymfocytosis – reconvalescence – decrease of paroxysmi - secundary complication Prevention Whool cell vaccine - neurological? ( combination with diphtheric, tetanic and Hib or VHB vakccine). Acellular vaccine – imunogenicity ? Therapy - supporting, nursing, survey, ATB do not necessary have to ameliorate the state – intoxication and destruction of epithel - ERY -eradiction of bacteriae, reduction of infectiousity

6. Anthrax a bioterorismus • Very virulent (toxin) • Inhalation, ingescion and contact - spread by air - aerosol, bombes, water • Resistent - broad thermal interval (14-42*C). Spores - resistent to drying

7. Mikroscopy • Mikroscopy - sc. Gram - Ps. aeruginosa,: G- huge rod • G+ huge rod , long chains of rods with centrally located spores: Bacillus anthracis. In smear from tissue - not spores. • Bacillus cereus: G+ rod without capsule, motile • - sc. Wirtz Coonklin (practicals 4 ST) - detection of spores of Bacillus anthracis

8. Cultivation • Ps. aeruginosa on blood agar - mucous gray colonies with methal lood and pigment and characteristic smell Production of diffusibile pigment - pyocyanin and of capsule • B. anthracis on blood agar- aerobe, facult. anaerobe rod, t.: 14- 45*C, small graish adherent colonies lining in paralel way - growing in picture of caput medusae, colonies with wave edges. Encapsulated colonies are soft, non encapsulated are rough, Older colonies growing in aerobe environment contain spores - dry wrinkled look. Growth on medium with PNC - chain of pearls • B. cereus on blood agar - not producing capsule, gray colonies

9. Cultivation of Bordetella pertussis • Very difficult, aerobe, prolonged cultivation, production of toxic metabolites, must be absorbed with active charcoal, high concentration of blood and ATB - Bodette Gengou medium • Plates with high level of agar, ensured agains drying • Transparent colonies (B. parapertussis - brown pigment)

10. Biochemical properties of Ps.aeruginosa • Minimal nutrition requirements, thermotolerant 4*- 42* C, rezistent to ATB and desinection Nonfermenting – cytochromoxidase – dif.dg. (COX test), Hajn tube medium - without change - red - detection of pigment and smell.On transparent media - green pigment • Oportunistic: factors of virulence: Pilli - adherence Polysacharid capsule – antifagocytosis, attachment, Endotoxin – LPS sepsis Exotoxin A – most important, blocs proteosynthesis of eukaryotic cells Alkalic protease – destruction of tissu Phospholipase C – destruction of lipids and lecithin, destruction of tissue

11. Ascoli termoprecipitation reaction • Detection of antigens extracted by heat directly from biological material by antibodies in agar • Factors of pathogenicity - capsule and toxins - both have antifagocytic properties, toxin - effect on CNS, leu. Fagocyted spores are not destroyed • Toxin - 3 subunits : EF - oedematogenous factor, LF - lethal factor, protective antigen PA • EF+LF - not toxic effect • LF+PA - lethal, not oedematogenous • EF+PA - only len oedem • EF+PA+LF - maximal toxicity • PA - most immunogenic • EF, LF solely not immunogenic • EF+PA, EF+LF, LT+PA, EF+LF+PA - immunogenic

12. B.pertussis - sampling and cultivation • Sample from nasopharynx - on bound wire, humidity. Coughing plates - overgroth of contaminating flora - (Blood agar + active charcoal +ATB, or Bordette Gengou) Bordetella pertussis – nutritionally very requiring, virulent. Pertussic toxin – 2 subunits: A (active) multiple biological effects, B(binding) Dermonecrotic toxin – vasoconstriction, tissue destructione Filamentous haemaglutinin – attachement, hemagglutination -protective Ab, Adenyl cyclase - toxin – interference with immunity cells (inhibition), Tracheal cytotoxin – cilliostasis, LPS - endotoxin Laboratory diagnosis Sensitiveto drying, fat acid in cotton of sampling devices are toxic, not living in common transport media, direct innoculation on Bordet Gengou plate. Humid chamber - prolonged incubation – 7 days Serology -Agglutination: patient serum + Ag B. pertussis - 2 samples in 14 days interval, 4 fold increase of titer, conversion from negat to pozit

13. ATB susceptibility of pseudomonas • Therapy - Frustrating – immunocompromised defence of patient, typical ATB rezistence induction of ATB inactivating enzymes resistence transmission via plasmids Isolation without signs of infection is not indication for ATB therapeutic intervention • Aminoglycosides – useless in the place of infection, acid environment in abscess Combination of ATB – beta lactams + aminoglycosides, Meronem, Imipenem, Sulperason, Cefoperason • Preventive approaches - Interruption of contamination of steril materials and cross contamination. Decontamination of fits and and tubes and catheters and environment - nosocomial strains in intensive care units. Broad spectrum ATB use - very carefully - suppression of normal flora and overgrowth of resistent bacteria and mutants