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

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

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….

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

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

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

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

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

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)

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

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

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

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