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 (14-42C).
  Spores - resistent to drying

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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

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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- 45C, 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

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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)

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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

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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
• EFLF - not toxic effect
• LFPA - lethal, not oedematogenous
• EFPA - only len oedem
• EFPALF - maximal toxicity
• PA - most immunogenic
• EF, LF solely not immunogenic
• EFPA, EFLF, LTPA, EFLFPA - immunogenic

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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

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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