Presentation on theme: "MLAB 2434: Microbiology Keri Brophy-Martinez"— Presentation transcript:
1MLAB 2434: Microbiology Keri Brophy-Martinez Haemophilus and Other Fastidious Gram-Negative Rods
2Haemophilus and Other Fastidious Gram-negative Rods The fastidious group of gram-negative bacilli include:HaemophilusHACEK( Haemophilus, Actinobacillus, Cardiobacteria, Eikenella & Kingella)LegionellaBordetellaPasteurellaBrucellaFrancisellaBartonella
3Haemophilus Species Haemophilus = “blood loving” Require either heme (X factor) or NAD (V factor)Haemophilus is facultative and can grow anaerobicallyOrganism is sensitive to drying and extremes in temperatureDistinctive “mousy” or “bleach-like” odor
4Haemophilus Influenzae Misnamed – originally thought to cause the “flu”Now know that flu is caused by virusesIn some cases of flu, H. influenzae is secondary infection
5Haemophilus Influenzae: Virulence Factors CapsuleAntiphagocyticIgA ProteaseCleaves IgA on mucosal surfacesLipid AEffects ciliated respiratory epitheliumPiliAttachment
6Haemophilus Influenzae: Clinical Infections: Typable strains Acute epiglottitis or laryngotracheal infection in small childrenCan cause airway obstruction needing immediate tracheostomyCellulitis/arthritischeek and upper extremitiesMeningitisChildren under 6 yearsContagious, vaccine has decreased incidencePneumonia/septicemiaIn childrenConjunctivitis “pink eye”very contagious
7Haemophilus Influenzae: Clinical Infections: nontypable strains Otitis mediaChildren 6 months- 2 yearsSinusitisPneumonia, bronchitisIn adultsThese sites are all in proximity to respiratory tract
8Haemophilus Species Haemophilus species require growth factors: X-factor ( hemin)Heat-stable substancePresent in RBC and released with degradation of hemoglobinV-factor (NAD: nicotinamide adenine dinucleotide)Heat- labileFound in blood or secreted by certain organisms
9Haemophilus SpeciesH. influenzae satellitism around and between the large, white, hemolytic staphylococci.This occurs when another organism produces V factor as a bi-product.
10Haemophilus Species Gram Stain Morphology Usually very small pleomorphic gram negative cb or rodMay be able to observe a halo around the organismGram stain can be enhanced by extending time for safranin to 2 minutes OR substitute carbolfuschin for safranin
11Haemophilus SpeciesDirect smear of H. influenzae in CSF in a case of meningitis. Note the TINY intracellular and extracellular pleomorphic gram-negative bacilli.Remember to look for capsules surrounding the rod.
12Haemophilus Species Colony Morphology No growth on BAP or MAC On CA: semi-opaque, gray-white, convex, mucoid.
13Haemophilus Species: Identification Gram stainGram negative cocco-baccillusCatalase +Oxidase +X and V factor strips or disksQuad platesRapid ID PanelsNHI cards- automated
14Haemophilus Species: Identification This organism would be identified as H. influenzae because it is using both X and V factors.
15Haemophilus Species: Identification This organism would be identified as H. parainfluenzae because it is using V factor only.
16Haemophilus Species: Identification Quad platesContain X and Vfactors & sheep bloodagar
17Haemophilus ducreyiCausative agent of chancroid or soft chancre (STD), highly contagiousSpecimens should be collected from base of lesion, inoculated directly to enriched media and held for 5 daysGram stain appears as groups of coccbacilli that resemble a ‘school of fish” or “railroad tracks”Requires only X factor to grow
19Haemophilus Antibiotic therapy Historically ampicillin was the drug of choice. However, resistance has developed due to production of beta-lactamase or altered penicillin binding proteins and cell wall permeabilitySusceptibility testing can be performed by disk diffusion, broth dilution or E-testPrimary antibiotics include cefotaxime or ceftriaxone
21HACEK GroupHACEK is an acronym of the first initial of each genus that belong in the group:Haemophilus aphrophilus:NAME ALERT: Now called Aggregatibacter aphrophilusNot a true Haemophilus because does not need X nor VActinobacillus actinomycetemcomitansCardiobacterium hominisEikenella corrodensKingella speciesHabitatCommensals of oral cavityClinical SignificanceInfective endocarditisPeridontal diseaseDental cariesInfections following dental procedures
22HACEK Group: General Characteristics Gram-negative bacilliRequire an increased CO2 (5%-10%) environmentSlow/poor growersUsual flora of the oralpharyngeal cavityOpportunists in immunocompromised hosts
23Capnocytophaga sp. Capnophilic Facultative anaerobe Part of the normal oralpharygeal floraCause periodontal disease, sepsis
24Pasteurella species General characteristics Colonizes mucous membranes of the upper respiratory tract and gastrointestinal tracts of mammals and birdsHuman infections occur from bites and scratches inflicted by animals, primarily felinesResults in a localized, pus- producing infectionCan cause life-threatening systemic diseaseMost common isolated species is Pasteurella multocida
29Brucella species Causes infection in cattle (zoonosis) Acquired through aerosol, percutaneous and oral routes of exposureBrucellosisPrimarily seen with animal handlers and those who handle animal productsAlso known as Malta or undulant feverType 3 biohazard – can be transmitted through unbroken skinCategory B Biological agent- easy to disseminate and cause moderate morbidity, but low mortality.
31Francisella tularensis Highly infectious Type 3 biohazard – can be transmitted through unbroken skin, bite from an insect, direct contact with infected animals or inhalation of aerosolsCategory A Biological agent-it can be spread from person to person or disseminated, high mortality ratesInfection in rabbits, sheep, squirrels and ticksZoonotic infection in humansTularemia
32Francisella tularensis: Identification Colony MorphologyBAP = No growthMAC = No growthChoc = Small, smooth, gray gncb at 2-5 daysRequires special media (BCYE or MTM)Oxidase: negativeCatalase: negative- weak positiveFerments glucoseX and V negativeNOTE: Usually identified by DFA or direct agglutination tests due to risk of lab acquired infection
33Legionella Species General characteristics Habitat Aquatic sourcesCooling towers, condensersUbiquitous gram-negative rodsAcquired by humans primarily through inhalation of aerosols
34Legionella Species: Clinical Infections Legionnaire’s diseaseDisease with pneumonia and extrapulmonary involvementMalaise, rapid onset of dry cough and feverIllness is fatal in 15-30% of cases not treatedPontiac feverInfluenza-likeFever, headache, malaiseNot fatal- short lived (2-5 days)
35Legionella Species Specimen Handling & Processing BAL, bronchial washings, lung biopsy and pleural fluid are appropriate specimensAvoid aerosolization & transport ambient temperatureBuffered Charcoal Yeast Extract (BCYE) most widely usedOrganism requires cysteine & iron salts for growthIncubate at 35o C in 5-10% CO2 with increased humidity for 10 daysSlow growth (2-4 days)
36Legionella pneumophila B(A) Nonselective buffered charcoal yeast extract (BCYE) plate inoculated with sputum specimen. Colonies appear blue-green or gray-white and glistening(B) Selective BCYE ( has added antibiotics) inoculated with the same specimen but treated before inoculation. Legionella colonies are the smallest visible colonies. Colonies are grayish-white and glistening at 2-4 days.
37Legionella Species: Identification Oxidase positiveCatalase PositiveMotile by polar flagellaShort, thin GNR, may be faint staining
38Legionella pneumophila Misc. Identification methodsRapid Methods for IdentificationUrine Antigen testDirect Fluorescent Antibody test (DFA)DNA DetectionSerological tests (IFA)
39Legionella spp.: Treatment Susceptibility testing not routinely performedErythromycin alone or Rifampin used to treat
40Bordetella spp. B. pertussis and B. parapertussis Cause pertussis “Whooping cough”Highly communicable disease of childrenStrict human pathogen, spread by airborne dropletsLives in ciliated epithelium of URTProduces toxins and virulence factorsRequired vaccination (DTaP)
41bordetella spp: Specimen collection, transport and processing Nasopharyngeal swab or aspirate is the specimen of choice.Swabs should be calcium alginate or dacron polyesterSpecimen should be plated at the bedside and a smear made OR placed in casamino acid for transportRegan-Lowe is recommended for transport
42Bordetella spp: Identification Requires Bordet-Gengou agarCough plateAppears slightly beta hemolytic smooth, shiny, resembling a mercury dropletRegan-Lowe agarDomed and shiny with a white mother of pearl opalescenceBAP & MAC: no growthOrganism is a fastidious obligate aerobeGram stain: small faint staining GN coccobacilliCan increase counterstain of safranin to 2 minutes for improved visibilityOxidase positiveNonmotile
44Bartonella Spp. Facultative Intracellular gram negative cocco-bacillus Transmitted by direct contact or blood-sucking arthropodsInfect RBCs and vascular endothelial cells in the host leading to circulatory system infectionsClinical InfectionsCat Scratch diseaseOthersCarrion’s diseaseTrench fever
45ReferencesEngelkirk, P. G., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams & Willkins.Kiser, K. M., Payne, W. C., & Taff, T. (2011). Clinical Laboratory Microbiology: A Practical Approach . Upper Saddle River, NJ: Pearson Education, Inc.Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.