Chapter 19 Haemophilus and Other Fastidious Gram-Negative Rods

1. Chapter 19Haemophilus and Other Fastidious Gram-Negative Rods MLAB 2434: Microbiology

3. Haemophilus Influenzae Misnamed � originally thought to cause the �flu� Now know that flu is caused by viruses In some cases of flu, H. influenzae is secondary infection

4. Haemophilus Influenzae: Virulence Factors Capsule Antiphagocytic IgA Protease Cleaves IgA on mucosal surfaces Lipid A Found in cell wall Effects ciliated respiratory epithelium Pili Attachment

5. Haemophilus Species Haemophilus = �blood loving� Haemophilus is facultative and can grow anaerobically Organism is sensitive to drying and extremes in temperature Distinctive �mousy� or �bleach-like� odor Sheep blood agar contains NADase that destroys NADSheep blood agar contains NADase that destroys NAD

6. Haemophilus Influenzae: Clinical Infections Infections caused by encapsulated typable strains (H. influenzae type b most common � Hib vaccination) Acute epiglottis or laryngotracheal infection in small children � can cause airway obstruction needing immediate tracheostomy Cellulitis/arthritis (cheek and upper extremities) Meningitis children under 6 years, contagious, vaccine has decreased incidence Pneumonia/septicemia (in children) Conjunctivitis �pink eye� very contagious

7. Haemophilus Influenzae: Clinical Infections Infections caused by nontypable strains Otitis media: children 6 months- 2 years Sinusitis Pneumonia, bronchitis (in adults) These sites are all in proximity to respiratory tract

8. Haemophilus Species Haemophilus species require hemoglobin for growth: X-factor ( hemin) Heat-stable substance Present in RBC and released with degradation of hemoglobin V-factor (NAD: nicotinamide adenine dinucleotide) Heat- labile, coenzyme I, found in blood or secreted by certain organisms

9. Haemophilus Species Staph aureus provides NAD for Haem. To growStaph aureus provides NAD for Haem. To grow

10. Haemophilus Species Gram Stain Morphology Usually very small pleomorphic gram negative cb or rod Gram stain can be enhanced by extending time for safranin to 2 minutes OR substitute carbolfuschin for safranin

11. Haemophilus Species

12. Haemophilus Species Colony Morphology No growth on BAP or MAC On CA: semi-opaque, gray-white, convex, mucoid.

13. Haemophilus species

14. Haemophilus Species: Identification Catalase + Oxidase + X and V factors

15. Haemophilus Species: Identification

16. Haemophilus Species: Identification

17. Haemophilus Species: Identification Although BAP contains both X and V factors, only X is directly available V is not available on BAP because sheep blood contains NADase that hydrolyzes V Quad plates contain X and V factors & sheep blood agar

18. Haemophilus ducreyi Causative agent of chancroid or soft chancre (STD), highly contagious Specimens should be collected from base of lesion, inoculated directly to enriched media and held for 5 days Gram stain appears as groups of coccbacilli that resemble a �school of fish� or �railroad tracks� Requires only X factor to grow

19. Haemophilus Species: Identification

20. Haemophilus 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 permeability Susceptibility testing can be performed by disk diffusion, broth dilution or E-test Primary antibiotics include cefotaxime or ceftriaxone

21. Case Study A 2-year-old unvaccinated child was seen in the emergency room because of complaints of headache and fever A cerebrospinal fluid(CSF) sample was obtained and sent to the laboratory for culture The Gram stain showed many white blood cells and many gram-negative, small bacilli

22. Points to Consider What clinical findings led the physician to request a CSF culture? Based on the patient�s age, which organism would be suspected? What growth requirements must be met to recover the suspected agent? Which other fastidious organisms would be considered? Other points to consider?

23. HACEK Group and Capnocytophaga HACEK is an acronym of the first initial of each genus that belong in the group: Haemophilus aphrophilus (not a true Haemophilus because does not need X nor V) Actinobacillus actinomycetemcomitans Cardiobacterium hominis Eikenella corrodens (often associated with human bite infection) Kingella species Capnocytophaga sp. Has similar requirements as the HACEK group

24. HACEK Group Clinical Significance Infective endocarditis Diagnosis from blood culture Must hold blood cultures for extended periods, making blind subcultures to enriched media

25. HACEK Group and Capnocytophaga: General Characteristics Gram-negative bacilli Require an increased CO2 (5%-10%) environment Slow/poor growers Usual flora of the oralpharyngeal cavity Opportunists in immunocompromised hosts

26. HACEK Group

27. HACEK Group

28. HACEK Group

29. HACEK Group

30. HACEK Group and Capnocytophaga

31. HACEK group

32. Pasteurella species General characteristics Colonizes mucous membranes of the upper respiratory tract and gastrointestinal tracts of mammals and birds Human infections occur from bites and scratches inflicted by animals, primarily felines Most common isolated species is Pasteurella multocida

33. Pasteurella species P. multocida: Clinical manifestations Localized infection after a bite or scratch, usually purulent Life-threatening systemic diseases (e.g., meningitis, bacteremia)

34. Pasteurella multocida

35. Pasteurella multocida Culture characteristics Growth on 5% blood or chocolate shows small, smooth, grayish,convex colonies Non-hemolytic �Musty� or mushroom odor No growth on MacConkey agar

36. Pasteurella multocida Microscopic examination Very small gram-negative rods Bipolar staining with Giemsa or methylene blue �Safety-pin� appearance

37. Pasteurella multocida: Identification oxidase positive indole positive Nonmotile Catalase positive Glucose fermenter

38. Pasteurella multocida Treatment Sensitive to penicillin

39. Brucella species Causes infection in cattle (zoonosis) B. abortus causes abortions in cattle (Brucellosis) Acquired through aerosol, percutaneous and oral routes of exposure Primarily seen with animal handlers and those who handle animal products Type 3 biohazard � can be transmitted through unbroken skin Category B Biological agent- easy to disseminate and cause moderate morbidity, but low mortality.

40. Brucella species: Clinical Infections In humans, causes �undulant fever� Named because fever fluctuates regularly Can become chronic, presenting 1 year post exposure

41. Brucella species: Identification Smooth, raised, translucent colonies Browns with age

42. Brucella species: Identification Gram Stain Morphology Small gram-negative coccobaccilli Nonmotile Aerobic Oxidase positive Catalase positive Lab diagnosis is difficult Hold cultures at least 21 days Serologic tests Patient history Disease status

43. Francisella tularensis Highly infectious Type 3 biohazard � can be transmitted through unbroken skin, bite from an insect, direct contact with infected animals or inhalation of aerosols Category A Biological agent-it can be spread from person to person or disseminated, high mortality rates Infection in rabbits, sheep, squirrels and ticks Zoonotic infection in humans Requires special media (BCYE or MTM) Treatment: Streptomycin is drug of choice

44. Francisella tularensis: Clinical Infections Ulceroglandular Ulcer forms at site of inoculation and lymph node enlargement follows

45. Francisella tularensis: Identification Colony Morphology BAP = No growth MAC = No growth Choc = Small, smooth, gray gncb at 2-5 days Oxidase: negative Catalase: negative- weak positive Ferments glucose X and V negative NOTE: Usually identified by DFA or direct agglutination tests due to risk of lab acquired infection

46. Legionella Species General characteristics Habitat Aquatic sources Cooling towers, condensers Ubiquitous gram-negative rods Acquired by humans primarily through inhalation of aerosols

47. Legionella Species: Clinical Infections Legionnaire�s disease disease with pneumonia and extrapulmonary involvement Malaise, rapid onset of dry cough and fever Illness is fatal in 15-30% of cases not treated Pontiac fever without pulmonary involvement Fever, headache,malaise Not fatal- short lived (2-5 days) Asymptomatic infection

48. Legionella Species Specimen Handling & Processing BAL, bronchial washings, lung biopsy and pleural fluid are appropriate specimens Avoid aerosolization & transport ambient temperature Organism requires cysteine & iron salts for growth Buffered Charcoal Yeast Extract (BCYE) most widely used Incubate at 35o C in 5-10% CO2 with increased humidity for 10 days Organisms usually take 2-4 days to grow

49. Legionella Species: Identification Oxidase positive Catalase Positive Motile by polar flagella Short, thin GNR, may be faint staining BAP & MAC= no growth CHOC= may grow very slowly

50. Legionella pneumophila

51. Legionella pneumophila

52. Legionella pneumophila Misc. Identification methods Conventional Gram stain, DFA, L-cysteine Urine Antigen test DNA Detection Serological tests (IFA)

53. Legionella sp. Treatment Susceptibility testing not routinely performed Erythromycin alone or Rifampin used to treat

54. Case Study Ten members of a group of 2 dozen retirees from the tobacco industry became acutely ill with pneumonia during a 2-week cruise The group was staying in a block of rooms together and spent time socializing over drinks and cigarettes in the cabins and saunas and at the poolside

55. Case Study On the fifth day of the cruise, several members went to see the ship�s doctor because of a worsening cough Chest x-rays revealed patchy lobar pneumonia in all affected individuals The condition improved with erythromycin therapy

56. Bordetella sp. B. pertussis and B. parapertussis Cause pertussis �Whooping cough� Highly communicable disease of children Strict human pathogen, spread by airborne droplets Lives in ciliated epithelium of URT Required vaccination (DPT) Produces an exotoxin (pertussis toxin) and has a cell wall endotoxin

57. Bordetella sp Specimen collection, transport and procesing Nasopharyngeal swab or aspirate is the specimen of choice. Specimen should be plated at the bedside and a smear made OR placed in casamino acid for transport Regan-Lowe is recommended for transport

58. Bordetella sp: Identification Requires Bordet-Gengou agar Cough plate Appears slightly beta hemolytic smooth, shiny, resembling a mercury droplet Regan-Lowe agar Domed and shiny with a white mother of pearl opalescence BAP & MAC: no growth Organism is a fastidious obligate aerobe Gram stain: small faint staining GN coccobacilli Can increase counterstain of safranin to 2 minutes for improved visibility Oxidase positive Nonmotile

59. Bordetella sp Serologic Identification Identified by direct fluorescent antibody, nucleic acid probe, or sero-conversion Treatment Erythromycin is the drug of choice for treatment Vaccination is the best protection

61. Points to Remember Clinical manifestations presented by the patient The types of infections these organisms produce The risk factors that predispose susceptible individuals

62. Points to Remember What special growth supplements are required for isolation Where these groups of organisms are usually found Characteristic features of the organisms for identification and differentiation among closely-related species