PSEUDOMONASPSEUDOMONAS

IntroductioIntroductionn

Large group of aerobic Large group of aerobic non sporingnon sporing gram negativegram negative motile by polar motile by polar

flagellaflagella ubiquitousubiquitous oppurtunistic infectionsoppurtunistic infections newer genera-Burkholderianewer genera-Burkholderia StenotrophomonasStenotrophomonas

SpeciesSpecies Pseuomonas aeruginosaPseuomonas aeruginosa Stenotrophomonas maltophilaStenotrophomonas maltophila Burkholderia cepaciaBurkholderia cepacia Burkholderia malleiBurkholderia mallei Burkholderia pseudomalleiBurkholderia pseudomallei

Pseudomonas Pseudomonas aeruginosaaeruginosa

MorphologyMorphology slender,Gram negative bacillusslender,Gram negative bacillus size-1.5 microns-3*1.5micronssize-1.5 microns-3*1.5microns motile by polar flagellamotile by polar flagella non capsulated though some mucoid non capsulated though some mucoid

strains may sometimes occurstrains may sometimes occur some are pilatedsome are pilated

Cultural Cultural characteristicscharacteristics Obligate aerobeObligate aerobe

Wide range of temperature 5Wide range of temperature 5°c°c-42-42°c°c optimum 37optimum 37°c°c Ordinary mediaOrdinary media – – large,opaque,irregular,with large,opaque,irregular,with

distinctive musty,mawkish,earthy distinctive musty,mawkish,earthy smellsmell

Cont..Cont..

Nutrient agar-Nutrient agar- Colonies are Colonies are

smooth,large,translucent,low smooth,large,translucent,low convex,2-4mm in diameter.convex,2-4mm in diameter.

Produce sweetish aromatic odourProduce sweetish aromatic odour Greenish blue pigment diffuses Greenish blue pigment diffuses

Cont..Cont..

Blood agarBlood agar Similar to nutrient agar Similar to nutrient agar Many are haemolyticMany are haemolytic Mac conkeyMac conkey agaragar Colourless,non lactose fermentersColourless,non lactose fermenters Cetrimide agarCetrimide agar selective mediaselective media

Pigment Pigment productionproduction

PyocyaninPyocyanin Bluish green phenazine pigment Bluish green phenazine pigment Soluble in chloroform and waterSoluble in chloroform and water Not produced by other speciesNot produced by other species Pyoverdin(fluorescin)Pyoverdin(fluorescin) It is a greenish yellow pigment It is a greenish yellow pigment Insoluble in chloroform but soluble in Insoluble in chloroform but soluble in

waterwater Produced by many other speciesProduced by many other species

Pyocyanin Pyocyanin Pyoverdin Pyoverdin

Cont..Cont..

PyorubinPyorubin Reddish brown pigment Reddish brown pigment Insoluble in chloroform but soluble Insoluble in chloroform but soluble

in waterin water pyomelaninpyomelanin Brown to black pigmentBrown to black pigment Production is uncommonProduction is uncommon

Biochemical Biochemical reactionsreactions

O/F test-oxidativeO/F test-oxidative Catalase-Catalase-positivepositive Oxidase-positiveOxidase-positive Nitrate reduction-positiveNitrate reduction-positive

Cont..Cont..

Indole test-negativeIndole test-negative Methyl red test-negativeMethyl red test-negative Vp test-negativeVp test-negative Citrate test-positiveCitrate test-positive Urease test-negativeUrease test-negative

Cont…Cont…

Sugar fermentation testsSugar fermentation tests

Glucose-only acid Glucose-only acid

Lactose-negativeLactose-negative

Sucrose-negativeSucrose-negative

Mannitol-negativeMannitol-negative

ResistanceResistance Susceptible to Susceptible to

heat,killed at 55heat,killed at 55°c °c in one hourin one hour

acidsacids

beta glutarldehydebeta glutarldehyde

Silver saltsSilver salts

strong phenolic disinfectantsstrong phenolic disinfectants So,silver sulphonamide as a topical cream in So,silver sulphonamide as a topical cream in

burns is used burns is used

Cont…Cont…

Resistant toResistant to

chemical agentschemical agents

common antisepticscommon antiseptics

disinfectants -ammonium disinfectants -ammonium compoundscompounds chlroxylenolchlroxylenol

hexachlorophanehexachlorophane

antibiotics -aminoglycosidesantibiotics -aminoglycosides

cephalosporinscephalosporins

pencillinspencillins

Antigenic Antigenic structurestructure

O antigensO antigens Possesses 19 distinct,group specific Possesses 19 distinct,group specific

O antigensO antigens Heat labileHeat labileH antigens H antigens Two heat labile antigens recognised Two heat labile antigens recognised

in Ps.aeruginosain Ps.aeruginosa

Toxins and Toxins and enzymesenzymes

Extracellular productsExtracellular products Pyocyanin inhibits mitochondrial enzymes Pyocyanin inhibits mitochondrial enzymes

and causes disruption and cessation of and causes disruption and cessation of ciliary beat on ciliated nasal eptheliumciliary beat on ciliated nasal epthelium

Extracellular enzymes and haemolysinsExtracellular enzymes and haemolysins Produces proteases,haemolysins and Produces proteases,haemolysins and

lipaseslipases ExotoxinsExotoxins Produces two exotoxns A and SProduces two exotoxns A and S EndotoxinsEndotoxins It is a lipopolysaccharide have pyrogenic It is a lipopolysaccharide have pyrogenic

actionaction

Typing methodsTyping methods Bacteriocin typingBacteriocin typing Three types of bacteriocins are produced-Three types of bacteriocins are produced-

R,F,SR,F,S Pyocin produced by test strain is employed Pyocin produced by test strain is employed

to assess the growth inhibition of 13to assess the growth inhibition of 13 Depending upon the growth inhibition of Depending upon the growth inhibition of

13 indicators strains,105 types are 13 indicators strains,105 types are recognisedrecognised

Most popular method used Most popular method used

Phage typingPhage typing SerotypingSerotyping

based on O and H,17 based on O and H,17 serotypes of Ps.aeruginosa are serotypes of Ps.aeruginosa are recognisedrecognised

Molecular MethodMolecular Method

Restriction endonuclease Restriction endonuclease typing with pulsed-field gel typing with pulsed-field gel electrophoresis(PAGE) is most reliableelectrophoresis(PAGE) is most reliable

Cont..Cont..

PATHOGENESISPATHOGENESIS Important agent in causing nosocomial Important agent in causing nosocomial

infectionsinfections Most common infections are Most common infections are Urinary tract infections following Urinary tract infections following

catheterisationcatheterisation Acute purulent meningitis following Acute purulent meningitis following

lumbar puncturelumbar puncture Post-tracheostomy pulmonary infectionPost-tracheostomy pulmonary infection Septicaemia in debilitated patientsSepticaemia in debilitated patients

cont..cont..

Wound and burn infectionsWound and burn infections Chronic otitis media and otitis Chronic otitis media and otitis

externaexterna Eye infectionsEye infections Acute necrotising vasculitis Acute necrotising vasculitis Infantile diarrhoeaInfantile diarrhoea

Mechanism Of Mechanism Of PathogenesisPathogenesis

Caused by Caused by

exotoxins,proteases,elastases,haemolyexotoxins,proteases,elastases,haemolysins,lipases and enterotoxinssins,lipases and enterotoxins

Exotoxin A-lethal toxinExotoxin A-lethal toxin Elastases-haemorrhagic lesions Elastases-haemorrhagic lesions Enterotoxins-diarrhoeal diseaseEnterotoxins-diarrhoeal disease Slime layer acts as a capsule and Slime layer acts as a capsule and

enhances virulenceenhances virulence

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HAEMOPHILUSHAEMOPHILUS

INTRODUCTIONINTRODUCTION

HAEMOPHILUS HAEMOPHILUS INFLUENZAEINFLUENZAE

MORPHOLOGYMORPHOLOGY SMALL [ 1.0 X 0.3 MICRONS]SMALL [ 1.0 X 0.3 MICRONS] GRAM NEGATIVEGRAM NEGATIVE NON MOTILENON MOTILE NON SPORINGNON SPORING EXHIBIT PLEOMORPHISMEXHIBIT PLEOMORPHISM

CULTURAL CULTURAL CHARACTERISTICSCHARACTERISTICS

FACTOR XFACTOR X HEAT STABLEHEAT STABLE

HEMIN HEMIN

AEROBIC AEROBIC RESPIRATIONRESPIRATION

FACTOR VFACTOR V HEAT LABILEHEAT LABILE

BACTERIAL BACTERIAL VITAMINVITAMIN

ANEROBIC ANEROBIC RESPIRATIONRESPIRATION

CONT…CONT…

AEROBICAEROBIC GROWS ANEROBICALLY ALSOGROWS ANEROBICALLY ALSO OPTIMUM TEMPERATURE-37COPTIMUM TEMPERATURE-37C SOME STRAINS REQUIRE 10% SOME STRAINS REQUIRE 10%

CO2CO2 BLOOD AGAR-SCANTY GROWTHBLOOD AGAR-SCANTY GROWTH

SATELLITISMSATELLITISM

Staph.aureus is streaked across a Staph.aureus is streaked across a plate of blood agar on which a plate of blood agar on which a specimen containing H.influenzae has specimen containing H.influenzae has inoculatedinoculated

After overnight incubation-After overnight incubation- ColoniesColonies

large -along the streak of large -along the streak of Staph.aureusStaph.aureus

small –farther awaysmall –farther away

satellitismsatellitism

CONT…CONT… LEVINTHAL`S MEDIUMLEVINTHAL`S MEDIUM Boiling and filtering a mixture of Boiling and filtering a mixture of

blood and nutrient brothblood and nutrient broth Capsulated strains produce Capsulated strains produce

translucent coloniestranslucent colonies FILDES AGARFILDES AGAR By adding a peptic digest of blood By adding a peptic digest of blood to to

nutrient agarnutrient agar Best for primary isolationBest for primary isolation Copious growthCopious growth

BIOCHEMICAL BIOCHEMICAL REACTIONSREACTIONS

Catalase-positiveCatalase-positive

Oxidase-positiveOxidase-positive

Glucose,Xylose-fermented with Glucose,Xylose-fermented with acid productionacid production

Lactose,Sucrose,Mannitol-not Lactose,Sucrose,Mannitol-not fermentedfermented

RESISTANCERESISTANCE Destroyed by heatingDestroyed by heating

refrigerationrefrigeration

dryingdrying

disinfectantsdisinfectants Cultures may be preserved for Cultures may be preserved for

about a month on choclate agar about a month on choclate agar slopes in screw capped bottlesslopes in screw capped bottles

Long term preservation –culture Long term preservation –culture may be lyophilisedmay be lyophilised

ANTIGENIC ANTIGENIC PROPERTIESPROPERTIES

3 Major surface antigens3 Major surface antigens

Capsular polysaccharideCapsular polysaccharide

Outer membrane protienOuter membrane protien

Lipo oligosaccharideLipo oligosaccharide

Major antigenic determinant of capsulated Major antigenic determinant of capsulated strains Capsular polysaccharidestrains Capsular polysaccharide

Pittman classified into 6 capsular strains Pittman classified into 6 capsular strains i.e type a to fi.e type a to f

•Meningitis belong to type b strain•Type b strain contains pentose sugars ribose &ribitol instead of hexoses & hexosamines in other 5 serotypes

•Capsular polyribosyl ribitol phosphate antigen of Hib induces IgG IgM IgA antibodies which are bactericidal and protective

CONT….CONT….

Strains lacking a capsule cannot Strains lacking a capsule cannot be typed and are called NON be typed and are called NON TYPABLE STRAINSTYPABLE STRAINS

Outer membrane protein antigens Outer membrane protein antigens of H influenzae type b have been of H influenzae type b have been classified into 13 sub typesclassified into 13 sub types

Lipo oligosaccharides are Lipo oligosaccharides are antigenically complexantigenically complex

PATHOGENECITYPATHOGENECITY

Exclusively human pathogen Exclusively human pathogen Not pathogenic for animals but Not pathogenic for animals but

intra peritoneal inoculation of intra peritoneal inoculation of large doses is fatal in large doses is fatal in mice,guinea pigs &rabbitsmice,guinea pigs &rabbits

Diseases due to H influnzae Diseases due to H influnzae considered under 2 groups –considered under 2 groups –invasive and non invasiveinvasive and non invasive

INVASIVE•Spread through Blood

•Meningitis,arthritis, endocardits,conjuctivitis,pancarditis

•Seen in children

•Caused by capsulated strains

NON INVASIVE •Spread through local invasion

•Otitis media,sinusitis

•Seen in adults

•Caused by non capsulated strains

CLINICAL CLINICAL MANIFESTATIONSMANIFESTATIONSMENINGITISMENINGITIS

Bacilli reach meninges from Bacilli reach meninges from nasopharynx through blood streamnasopharynx through blood stream

Common in childrenCommon in children

LARYNGO EPIGLOTTISLARYNGO EPIGLOTTIS Acute inflammaton of epiglottis with Acute inflammaton of epiglottis with

obstructive laryngitisobstructive laryngitis Children above 2 yearsChildren above 2 years

CONT….CONT….

PNEUMONIAPNEUMONIA Infants accompanied by empyemaInfants accompanied by empyema Older children & adults by lobar Older children & adults by lobar

pneumonia pneumonia These are primary infections due to These are primary infections due to

capsulated strainscapsulated strains Broncho pneumonia may occur as Broncho pneumonia may occur as

secondary infection due to non secondary infection due to non capsulated strainscapsulated strains

CONT…CONT…SUPPURATIVE LESIONSSUPPURATIVE LESIONS Arthritis,endocarditis& pericarditis Arthritis,endocarditis& pericarditis

may result from hematogenous may result from hematogenous disseminationdissemination

Otitis media occurs by direct spread Otitis media occurs by direct spread from from nasopharynxnasopharynx

BRONCHITISBRONCHITIS Associated with pneumococci in Associated with pneumococci in

acute exacerbationof chronic acute exacerbationof chronic bronchitis &bronchiectasisbronchitis &bronchiectasis

LAB DIAGNOSISLAB DIAGNOSIS

1.1.SPECIMENSSPECIMENSCSFCSFBlood Blood Throat swabThroat swabSputumSputumPus Pus Aspirates from joints ,middle Aspirates from joints ,middle

earsears

2.COLLECTION 2.COLLECTION &TRANSPORT&TRANSPORT

Collected in sterile containers & Collected in sterile containers & under aseptic conditionsunder aseptic conditions

Very sensitive to low temp so Very sensitive to low temp so should not be refrigeratedshould not be refrigerated

Transported to laboratory with Transported to laboratory with out delayout delay

Inoculated on culture media Inoculated on culture media immediatelyimmediately

CONT.....CONT.....3.DIRECT MICROSCOPY3.DIRECT MICROSCOPY

Gram stainingGram staining Immunoflouroscence and Immunoflouroscence and

quellung reactionquellung reaction Antigen detectionAntigen detection

Latex agglutinationLatex agglutinationCo-agglutinationCo-agglutinationCounter immuno Counter immuno electrophoresiselectrophoresis

CONT…CONT… 4.CULTURE4.CULTURE

CSF cultureCSF cultureBlood cultureBlood cultureSputum cultureSputum culture

55.COLONY MORPHOLOGY .COLONY MORPHOLOGY AND STAININGAND STAINING

6.SEROTYPING6.SEROTYPING

TREATMENTTREATMENT

Cefotaxime & Ceftazidime is the Cefotaxime & Ceftazidime is the drug of choice for meningitisdrug of choice for meningitis

Ampicillin &Cotrimoxazole –Ampicillin &Cotrimoxazole –respiratory infectionsrespiratory infections

Amoxycillin - clavulanate or Amoxycillin - clavulanate or clarithromycin is more effectiveclarithromycin is more effective

EPIDEMIOLOGY & EPIDEMIOLOGY & PREVENTIONPREVENTION

Infection transmitted by Infection transmitted by respiratory routerespiratory route

Immunity is type specific Immunity is type specific Active immunization with Hib Active immunization with Hib

PRP vaccine is indicatedPRP vaccine is indicated Conjugate Hib PRP is used for Conjugate Hib PRP is used for

younger childrenyounger children Rifampicin -4 days prevents Rifampicin -4 days prevents

secondary infection in contacts & secondary infection in contacts & also eradicates carrier statealso eradicates carrier state

Haemophilus aegyptiusHaemophilus aegyptius

KOCH –WEEKS BACILLUSKOCH –WEEKS BACILLUS Non capsulatedNon capsulated Causes highly contagious form Causes highly contagious form

of conjuctivitis [ pink eye ]of conjuctivitis [ pink eye ] Causative agent of Brazilian Causative agent of Brazilian

purpuric fever [BPF] purpuric fever [BPF] Responds to local Responds to local

sulphanamides & gentamicinsulphanamides & gentamicin

Haemophilus ducreyiHaemophilus ducreyi

Ducrey demonstrated in chancroid lesionsDucrey demonstrated in chancroid lesions Short ,ovoid bacillus Short ,ovoid bacillus Gram nagative often may appear gram Gram nagative often may appear gram

positive & frequently show bipolar positive & frequently show bipolar stainingstaining

May be arranged in small groups or May be arranged in small groups or whorls or in parallel chains giving a whorls or in parallel chains giving a SCHOOL OF FISH SCHOOL OF FISH or or

RAIL ROAD TRACK RAIL ROAD TRACK appearanceappearance

CONT…CONT… Can be grown on fresh clotted rabbit Can be grown on fresh clotted rabbit

bloodblood May also grown on chorio allontioc May also grown on chorio allontioc

membrane of chick embryomembrane of chick embryo On choclate agar enriched with On choclate agar enriched with

isovitalex and fetal calf serum & isovitalex and fetal calf serum & containing vancomycin as a selective containing vancomycin as a selective agentagent

Forms small ,grey ,translucent colonies Forms small ,grey ,translucent colonies after incubation at 35 c under 10 % co2 after incubation at 35 c under 10 % co2 & high humidity in 2 – 8 days& high humidity in 2 – 8 days

CONT…CONT…

Antigenically homogenous Antigenically homogenous Cultures may be identified by Cultures may be identified by

agglutinatoin with anti serumagglutinatoin with anti serum Susceptible to sulphonamides & Susceptible to sulphonamides &

many antibioticsmany antibiotics Erythromycin ,Cotrimoxazole ,CipErythromycin ,Cotrimoxazole ,Cip

rofloxacin may be used for rofloxacin may be used for treatmenttreatment

Choclate agarChoclate agar

Haemophilus Haemophilus parainfluenzaeparainfluenzae

Requires factor V & not the Requires factor V & not the factor Xfactor X

Commensel in the upper Commensel in the upper respiratory tract respiratory tract

Causes bacterial Causes bacterial endocarditis ,urethritis ,acute endocarditis ,urethritis ,acute pharyngitis pharyngitis

Haemophilus Haemophilus haemolyticushaemolyticus

Requires both factor X & VRequires both factor X & V Commensal of upper respiratory Commensal of upper respiratory

tracttract Strains that donot require factor Strains that donot require factor

X have been designated as H X have been designated as H parahaemolyticusparahaemolyticus

Haemophilus Haemophilus aphrophilusaphrophilus

Requires factor X but not factor Requires factor X but not factor V V

Cause bacterial Cause bacterial endocarditis ,brain endocarditis ,brain abscess ,sinuisitis pneumoniaabscess ,sinuisitis pneumonia

Strains requiring factor V but not Strains requiring factor V but not factor X have been termed as H factor X have been termed as H paraaphrophilusparaaphrophilus

HACEK group bacteriaHACEK group bacteria Fastidious slow growing bacteriaFastidious slow growing bacteria

HH -- Haemophilus speciesHaemophilus species

A A - - Actinobacillus Actinobacillus

C C - - Cardiobacterium Cardiobacterium hominishominis

EE - - Eikenella corrodensEikenella corrodens

K - K - Kingella kingaeKingella kingae

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