Fastidious Gram-Negative Organisms PDF

Fastidious gram negative rods Haemophilus, pasteurella, Bordetella, Brucella, francisella, legionella, bartonella & hacek group Haemophilus species  Facultatively anaerobic  Small, pleomorphic, Gram-negative rods  Requirement for factors present in blood  Oxidase and catalase-variable Haemop...

Fastidious gram negative rods Haemophilus, pasteurella, Bordetella, Brucella, francisella, legionella, bartonella & hacek group Haemophilus species  Facultatively anaerobic  Small, pleomorphic, Gram-negative rods  Requirement for factors present in blood  Oxidase and catalase-variable Haemophilus Pathogens Normal flora (usually)  H. influenza  H. aphrophilus  H. parainfluenzae  H. paraphophilus  H. ducreyi  H. hemolyticus  H. aegypticus  H. parahemolyticus Pleomorphic bacilli of Haemophilius influenzae in a Gram stain from in vitro culture Gram stain of Haemophilus influenzae  CSF Gram stain  Thin, pleomorphic forms from a 1-yr-old, unvaccinated child in Africa w/ overwhelming meningitis Biochemical ID  May be identified by their nutritional requirements for X and V factor and their ability to grow and hemolyze rabbit or horse blood agar  Other tests performed in commercial ID systems:  Indole  Urease  Fermentation of various carbohydrates  Porphyrin Test Porphyrin test (ALA)  Tests for the presence of enzyme which convert aminolevulanic acid (ALA) into porphyrins  A much more accurate means of determining X factor requirement compared to using X & V factor disks  Provides no information on the V factor requirement ALA Test procedure  Place one ALA disk for each organism to be tested on the inside of a Petri dish using forceps.  Moisten each disk with one drop of sterile water. 3. Rub a loopful of the test organism onto the moistened disk holding it in place with sterile forceps.  Saturate gauze with water, squeeze out any excess and place it in the petri dish as far away from the disks as possible.  Incubate at 35 C.  Examine at hourly intervals for 6 hours by removing the top of the petri dish and exposing the disks to UV light in a darkened room. NB: Wear UV safety goggles when using the UV light.  Positive: Orange-red fluorescence  Negative: No fluorescence observed Growth Requirements of Haemophilus spp.  Fastidious, require either hemin or a precursor (factor X) or NAD (factor V), or both for growth on media lacking blood products  Grow in CO2 enriched environment on CHOC agar  See satellite colonies adjacent to Staphylococcus aureus on BAP for spp. requiring V factor  Use paper strips impregnated with each growth factor for aid in species identification Haemophilus = "blood loving"  Best growth is on CHOC agar  Sheep blood agar heated to 80ºC to rupture RBCs and inactivate an enzyme that degrades NAD  Rabbit blood & horse blood lack the NADase that is present in sheep & human blood; thus these BAPs will support growth, but CHOC is still best  Mousy odor to growth of Haemophilus influenzae on CHOC agar Satellitism of Haemophilus on sBAP via "Staph streak" test  Inoculate entire surface of plate w/ suspected Haemophilus isolate  Streak Staphylococcus aureus on plate; it liberates NAD into the agar as it grows due to its hemolysin  Haemophilus spp. requiring V factor (NAD) appear as small, dewdrop-like satellite colonies X & V factors  X factor = heme, hemin, or hematin  Hemin is a component of RBCs in blood  V factor = NAD  Nicotinamide adenine dinucleotide, a coenzyme and electron carrier.  Found in chocolate agar but not blood agar.  Chocolate agar is blood agar that has been heated. Contents of RBC are released into the media so that bacteria can use them.  Organisms with “para” in their name do not require X factor X & V factor procedure  Prepare a heavy suspension of cells of the organism in saline.  Note it is important not to carry over any X-factor or V factor contained in the medium that the organism is taken from  Dip a sterile swab into the organism suspension and roll the swab over the entire surface of a trypticase soy-agar plate  Place the X, V , and XV factor disks on the agar surface.  If using separate disks, place them at least 4 to 5 cm apart.  Carefully invert the plate and incubate overnight at 35°C in place it in a CO2-incubator or candle extinction jar X and V factor Controls  Positive:  Haemophilus influenzae will show a halo of growth around XV disk; the rest of the agar surface will show no growth.  Haemophilus parainfluenzae will show a halo of growth around XV and V disks.  Negative:  Haemophilus aphrophilus will grow over the entire surface of the plate. Neither X, nor V, nor XV factors are necessary for growth. X &/or V factor requirements Aid Speciation of an Isolate Growth factor X V requirement: heme NAD+ Catalase ALA H. influenzae differentiate on basis of ability to use xylose + + + - H. aegyptius differentiate on basis of ability to use xylose + + + - H. ducreyi + - - - H. parainfluenzae - + +/- + Haemophilus influenzae  Most common pathogen isolated  Does NOT cause influenza “the flu” but originally was thought to  Often a secondary invader in individuals infected with influenza  Can be capsulated or uncapsulated nontypeable  Most Haemophilus influenzae of the normal flora are nontypeable  Several serotypes based on their capsules with Type b (HIB) being the most virulent  HIB causes otitis media, acute epiglottitis, meningitis and septicemia particularly in children under the age of 2  There is now a vaccine routinely given for HIB  Other strains cause pneumonia, sinusitis, bronchitis, otitis media, conjunctivitis, rarely causes sepsis Haemophilus influenzae Virulence Factors  Pili and nonpilus adhesions facilitate colonization of the oropharynx  Cell wall components (LPS & a low MW glycopeptide) impair ciliary action, leading to damage to respiratory epithelium & entry of organism into blood  IgA protease produced by both capsulated and unencapsulated forms  Polysaccharide capsule is major virulence factor for type b (polyribitol phosphate is anti-phagocytic)  Haemophilus influenzae type b (Hib)  Before immunization with an effective vaccine was introduced in the 1980s, 95% of H. influenzae diseases were caused by type b  It was the most common cause of meningitis in children 3-18 mo  Caused a life-threatening epiglottitis in children