4/15/20131BordettelaSharq Elneil CollegeSchool of Medical LaboratorySciencesDepartment of MicrobiologyMedical Bacteriology course U.Mahadi Hassan MahmoudBsc, Msc, MIBMS Microbiology
4/15/20132In 1906, Jules Bordet and OctaveGengou (another famous Belgianbacteriologist : 1875-1957) discoveredthe microbe that causes whoopingcough (Bordet-Gengou bacillus orBordetella pertussis).Medically Important spp the genus contains threemedially important speciesB. pertussisB. parapertussisB. bronchoseptica
4/15/20133General propertiesMorphology and culturalcharacteristicsSmall gram negative coccobacilliB. parapertussis and B.bronchoseptica both grow onsheep BA (SBA) in 1-2 daysBordetella pertussis
4/15/20134B. pertussis for initial isolation(The best clinical specimen is anasopharyngeal swab.) theorganism requires special mediawith additional nutrients forgrowth and absorbents to removetoxic substances found incomplex media such as fatty acidsand sulfides.Borget-Gengou media – containsglycerol, potato infusion, albumin(binds fatty acids), and up to 50%defibrinated SRBCsCharcoal agar supplemented with10% horse blood with or withoutcephalexin.May take 3-7 days for growth andcolonies are smooth, raised, andglistening (phase 1 colonies).They are also hemolytic and producetoxin.
4/15/20135Upon extensive subculturing, the coloniesbecome rough (they progress throughphases 2, 3, and finally 4) and can now begrown on SBA.They are now less virulent due to loss ofcapsule, hemolytic activity, and toxinproduction.These changes, however, are reversible.The organisms are strict aerobes andgrow best at 35-370 C. No growth on Mac for B. pertussis,others are variableOxidase test is variableVirulence factors (B. pertussis)Pili for attachmentPertactin, an outer membrane proteinalso acts as an adhesionFilamentous hemagglutinin – is foundon the cell surface of and is alsosecreted. It attaches to cilia by binding to exposedlactose receptors.
4/15/20136Pertussis toxinHas one A subunit (toxic part),plus four different kinds of Bsubunits (involved in binding).Structure of pertussis toxinA subunitB subunits
4/15/20137Activation of pertussis toxinTrachael cytotoxin – is related to theB.pertussis peptidoglycan.When this is incubated with cells inculture, the cells are destroyed, so itmight contribute to the killing andsloughing off of ciliated cells in therespiratory tract.Lipooligosaccharide associated with thesurface of the bacteria and has potentendotoxin activity.
4/15/20138PathogenesisRespiratory droplet exposureEnter respiratory tractAttach to ciliated epithelial cellsEndotoxin inhibits cilia clearanceReplication on outside of respiratorycellsCells eventually die and release toxinThree stages of WhoopingCoughCatarrhal stageFirst stage as bacteria just start to die andrelease toxinMild cold symptoms, coughing, sneezingChild is not that sick so parent thinks theyhave a common cold and don’t isolate fromother childrenThis is the MOST contagious stage sincemany bacteria still alive in respiratory
4/15/20139Paroxysmal stageMaximum cell death and toxin releaseSevere Cough40 – 50 cough spells/day, 20-30 coughs ina row with no chance to breathCoughing causes stomach upset and vomitingMucous build-up in LungsAir blockage can in rare cases lead to deathSecondary pneumonia is biggest threat Caused by other bacterial pathogensH. influenzae, S. aureus, S. pneumoniaeConvalescent stageCoughing spells diminishslowlydecrease in number of spellsand severityPossible CNS complications insome children. Thepathogenesis is not clear
4/15/201310Clinical significanceB. pertussis – causes whooping coughAcquired by inhalation of dropletscontaining the organismThe organism attaches to the ciliated cellsof the respiratory tract.During an incubation period of 1-2 weeks,the organism multiplies and starts to liberateits toxins.Next the catarrhal stage occurs - thepatient has a mild cough and sneezingwhereby large numbers of organisms arespread through the respiratory secretions. This last ~ 2 weeks.Next is the paroxysmal stage thatlasts 4-6 weeks.The patient has rapid, consecutivecoughs with a rapid intake of airbetween the coughs (has a whoopingsound).The ciliary action of the respiratorytract has been compromised, mucoushas accumulated, and the patient istrying to cough up the mucousaccumulations.The coughs are strong enough to breakribs!
4/15/201311Other symptoms due to the activity of thereleased toxins include:Increased peripheral lymphocytes due toa blocking of homing of lymphocytes tothe spleen and lymph nodes.Metabolic alteration such as increasedinsulin release and the resultinghypoglycemiaIncreased capillary permeability andincreased susceptibility to histamine,serotonin, and endotoxin shockFinally there is a convalescent stageduring which symptoms graduallysubside.This can last for months.B. pertussis rarely spreads to other sites,but a lot of damage may occur, such asCNS dysfunction which occurs in ~10 %of the cases and is due to an unknowncause.Secondary infections such as pneumoniaand otitis media are common.
4/15/201312B. pertussis pathogenesisB. parapertussis – causesa mild form of whoopingcoughB. bronchosepticaWidespread in animals whereit causes kennel cough.Occasionally causesrespiratory or woundinfections in humans.
4/15/20131325Specimens: Pernasal swab from Naso pharyngealsecretion.Microscope: B-Pertusis issmall-Non Motile –capsulated G-VeCoccobacilli – Non sporing.Lab Diagnosis:
4/15/20131427Culture: B-Pertusis, when culture oncharcoal cephatexin blood Agar(CCBA) or Bordet – Gengoupencillin. It is strike O2 incubation (35 – 37Co)for 3 days produce mucoid – grayish– white colonies with shiny surfaceand high convex shape (bisectedpearl) or (mercury drop)appearance28Biochemical: we can differential betweenspecies of B-Pertusis by manytest e.g (Motility – Urea –Oxidase – catalase – pigment– Producing – Growth onblood agar.
4/15/20131529B.Pertusis: Non Motile – catalase +ve –oxidase +ve urea –ve. can not grow on blood agar. not produce pigment. It differ from Haemophilusinfluenzae in its continuedviability at low temp. (00 – 10C0).30Serology:B.Pertusis has three major Ags(Serotype)Type 1,2/type 1,2,3/ type 1,3B. Pertusis can be detected by:Slide agglutinating.Immunofluorescentmicroscopes.Complement Fixation.ELISA.
4/15/20131631B. Parapertusis:Oxidase –ve - grow rapidy on bloodAgarUrea +ve - Produce brown Pigment inN. AgarNon Motile - it can grow on blood AgarB. Bronchiseptica:Motile - it can grow in blood Agar.Urea +ve - oxidase +veBordetella bronchisepticaLeifson flagella stain
4/15/20131733Antimicrobial Sensitivity:Erythromycin –only effective inearly stages of the disease beforethe toxin(s) have been released chloramphenicol.Tetracycline.Vaccination Vaccination (DPT – diphtheria, pertussis,tetanus) CNS toxicity was major stumbling block Blamed on whole cell pertussis prep in the DPT vaccine Many parents avoided vaccine and apathy led to widespread outbreaks New genetic engineered noncellular preparationshave helped to alleviate fear in parents However, only effective in 80-85% of children Therefore, we still need to give antibiotics tocontacts