1. Haemophilus, Pasteurella, Gardnerella and Pseudomonas
Microbiology Department KUMS
Dr. Mohajeri

2. The Haemophilus Species
G-ve, pleomorphic bacteria that require enriched media, usually containing blood or its derivatives
- H. influenzae type b is an important human pathogen
- H. ducreyi, a sexually transmitted pathogen (STD) causes chancroid
- other haemophilus species are among the normal flora of mucous membranes and only occasionally cause disease.

3. Haemophilus influenzae
is found on the mucous membranes of the upper respiratory tract (URT) in humans
is an important cause of meningitis in children and occasionally causes respiratory tract infections in children and adults
In specimens, the organisms are short coccoid bacilli, sometimes occurring in pairs or short chains. (pleomorphic forms)

4. The capsule is the antigen used for "typing" H influenzae.
On chocolate agar, tiny colonies
does not grow on sheep blood agar except around colonies of staphylococci ("satellite phenomenon")

5. IsoVitaleX in media enhances growth.
H haemolyticus and H parahaemolyticus are hemolytic variants of H influenzae and H parainfluenzae, respectively.

6. Identification depends in the need for certain growth factors called X and V
Factor X acts physiologically as hemin
Factor V can be replaced by nicotinamide adenine nucleotide (NAD) or other coenzymes
Colonies of staphylococci on sheep blood agar cause the release of NAD, yielding the satellite growth phenomenon.

8. Variation
- In addition to morphologic variation, H influenzae has a marked tendency to lose its capsule and the associated type specificity.
Transformation
- Resistance to ampicillin and chloramphenicol is controlled by genes on transmissible plasmids.

9. Antigenic Structure
contains capsular polysaccharides of one of six types (a–f)
The capsular antigen of type b is a polyribose-ribitol phosphate (PRP).
Encapsulated H influenzae can be typed by slide agglutination, coagglutination with staphylococci, or agglutination of latex particles coated with type-specific antibodies.

10. A capsule swelling test with specific antiserum is analogous to the quellung test for pneumococci.
Most H. influenzae organisms in the normal flora of the upper respiratory tract are not encapsulated.
The somatic antigens of H influenzae consist of OMPs.
LPS share many structures with those of neisseriae.

11. Pathogenesis
H influenzae produces no exotoxin.
The capsule is antiphagocytic in the absence of specific anticapsular antibodies.
The polyribose phosphate capsule of type b H influenzae is the major virulence factor.
The carrier rate in the upper respiratory tract for H influenzae type b is 2–4%.

12. The carrier rate for non-typeable H influenzae is 50–80% or higher.
Type b H influenzae causes meningitis, pneumonia , empyema, epiglottitis, cellulitis, septic arthritis
Nontypeable H influenzae causes chronic bronchitis, otitis media, sinusitis, and conjunctivitis following breakdown of normal host defense mechanisms.

13. The carrier rate for the encapsulated types a and c–f is low (1–2%), and these capsular types rarely cause disease.
Similarly, nontypeable H influenzae only occasionally causes invasive disease (about 5% of cases).
The blood of many persons over age 3–5 years is bactericidal for H influenzae, and clinical infections are less frequent in such individuals.

14. Clinical Findings
type b enters by way of the respiratory tract
There may be local extension with involvement of the sinuses or the middle ear.
H influenzae type b and pneumococci are two of the most common etiologic agents of bacterial otitis media and acute sinusitis.

15. The organisms may reach the bloodstream and be carried to the meninges or, less frequently, may establish themselves in the joints to produce septic arthritis.
Prior to the use of the conjugate vaccine, H influenzae was the most common cause of bacterial meningitis in children age 5 months to 5 years in the United States.

16. H influenzae types of disease
Type b
Unencapsulated
Meningitis
CSF 50%-95% culture positive
Blood 50%-95% culture positive
Otitis Media
Tymphanocentesis
50% - 70% culture positive
Sinusitis
Sinus aspirate
50% - 75% culture positive
Conjunctivitis
Eye 50%-75% culture positive
Blood < 10% culture positive
Cellulitis
Skin 75% - 90% culture positive
Blood 50% - 75% culture positive
Pneumonia, bronchitis
Sputum
20% - 75% culture positive
Blood
10% - 30% culture positive
Epiglottitis
Blood 90% - 95% culture positive
Epiglottitis culture
contraindicated
Arthritis
Synovial fluid
70% - 90% culture positive
Blood
50% - 80% culture positive

17. Diagnostic Laboratory Tests
Specimens consist of nasopharyngeal swabs, pus, blood, and spinal fluid for smears and cultures.
Commercial kits are available for immunologic detection of H influenzae antigens in spinal fluid.
Specimens are grown on IsoVitaleX-enriched chocolate agar until typical colonies appear.
H. influenzae is differentiated from related gram-negative bacilli by its requirements for X and V factors and by its lack of hemolysis on blood agar.

18. V factor is heat-labile.
Growth of haemophilus in the area between the strips indicates requirement for both factors.
Haemophilus species that synthesize porphyrins (and thus heme) are not H influenzae.

19. Sputum chains of H influenzae

21. Immunity
Infants under age 3 months may have serum antibodies transmitted from the mother.
H. influenzae has been the most common cause of bacterial meningitis in children from 5 months to 5 years of age.

22. Treatment
The mortality rate of untreated H influenzae meningitis may be >90%.
Many strains of H influenzae type b are susceptible to ampicillin, but up to 25% produce β-lactamase under control of a transmissible plasmid and are resistant.
Essentially all strains are susceptible to the newer cephalosporins. (Cefotaxime)

23. Epidemiology, Prevention, & Control
Encapsulated H influenzae type b is transmitted from person to person by the respiratory route.
Children aged 2 months or older can be immunized with H influenzae type b vaccine conjugated with one of two carriers (HbOC with protein carrier CRM197 mutant Corynebacterium diphtheriae toxin protein; or Neisseria meningitidis outer membrane complex) with appropriate booster doses according to standard recommendations.

24. Children aged 15 months or older can receive H influenzae type b vaccine conjugated with diphtheria toxoid (which is not immunogenic in younger children).
The vaccine reduces the carrier rates for H influenzae type b.
Prophylaxis with rifampin is recommended for such children.

25. H influenzae Type b Conjugate Vaccines
Abbreviation
Protein Carrier
HbOC
CRM 197 ( a nontoxic mutant of diphtheria toxin)
PRP - OMP *
OMP (an outer membrane protein complex of Neisseria meningitidis)
PRP - T †
Tetanus toxoid
* PRP-OMP is also available as a combination vaccine with hepatitis B vaccine (Comvax). This should
not be used for hepatitis B immunization at birth.
† PRP-T can be reconstituted with Connaught DTaP vaccine (Tripedia), to produce a combination marketed
as TriHIBit, which is acceptable only for the booster (4th) dose in infants≥ 15 mo of age.
‡ Marketed by GlaxoSmithKline in the United States

26. Haemophilus aegyptius
This organism was formerly called the Koch-Weeks bacillus
it is sometimes called H influenzae biotype III.
It resembles H influenzae closely and has been associated with a highly communicable form of conjunctivitis.
H aegyptius is the cause of Brazilian purpuric fever (BPF), a disease of children characterized by fever, purpura, shock, and death.

27. Haemophilus aphrophilus
This organism is sometimes encountered in infective endocarditis and pneumonia.
It is present in the normal oral and respiratory tract flora.
It is related to Actinobacillus (Haemophilus) actinomycetemcomitans.

28. The regional lymph nodes are enlarged and painful.
Haemophilus ducreyi
causes chancroid (soft chancre), a sexually transmitted disease (STD)
The regional lymph nodes are enlarged and painful.
The disease must be differentiated from syphilis, herpes simplex infection, and lymphogranuloma venereum (LGV).

29. requires X factor but not V factor
It is grown best from scrapings of the ulcer base on chocolate agar containing 1% IsoVitaleX and vancomycin, 3 g/mL, and incubated in 10% CO2 at 33 °C.
There is no permanent immunity following chancroid infection.
Treatment with intramuscular ceftriaxone, oral trimethoprim-sulfamethoxazole, or oral erythromycin.

30. Other Haemophilus Species
H. haemoglobinophilus
- requires X factor but not V factor and has been found in dogs but not in human disease.
H. haemolyticus
- is the most markedly hemolytic organism of the group in vitro
- it occurs both in the normal nasopharynx and in association with rare upper respiratory tract infections of moderate severity in childhood.

31. H. parainfluenzae
- resembles H influenzae and is a normal inhabitant of the human respiratory tract
- it has been encountered occasionally in infective endocarditis and in urethritis
H. suis
- resembles H. influenzae bacteriologically and acts synergistically with swine influenza virus to produce the disease in hogs.

32. Pasteurella
are primarily animal pathogens, but they can produce a range of human diseases
The generic term pasteurellae formerly included all yersiniae, francisellae, pasteurellae
Pasteurellae are nonmotile G-ve coccobacilli with a bipolar appearance on stained smears.
They are aerobes or facultative anaerobes that grow readily on ordinary bacteriologic media.

33. They are all oxidase-positive and catalase-positive but diverge in other biochemical reactions.
Pasteurella multocida occurs worldwide in the respiratory and gastrointestinal tracts of many domestic and wild animals.
It is perhaps the most common organism in human wounds inflicted by bites from cats and dogs.
can also produce human infections in many systems. (may be part of normal human flora).

34. Pasteurella haemolytica occurs in the upper respiratory tract of cattle, sheep, swine, horses, and fowl.
Human infection appears to be rare.

35. Pasteurella pneumotropica is a normal inhabitant of the respiratory tract and gut of mice and rats and can cause pneumonia or sepsis when the host-parasite balance is disturbed.
A few human infections have followed animal bites.
Pasteurella ureae has rarely been found in animals but occurs as part of a mixed flora in human chronic respiratory disease or other suppurative infections.

36. Clinical Findings
The most common presentation is a history of animal bite followed within hours by an acute onset of redness, swelling, and pain.
Regional lymphadenopathy is variable, and fever is often low-grade.
Pasteurella infections sometimes present as bacteremia or chronic respiratory infection without an evident connection with animals.

37. Pasteurella multocida is susceptible to most antibiotics.
Penicillin G is considered the drug of choice for P multocida infections resulting from animal bites.
Tetracyclines and fluoroquinolones are alternative drugs.

38. معیارهای Amsel’s : گاردنرلا گاردنرلا واژینالیس
باسیل گرم منفی، بدون اسپور، بدون حرکت، ساکن واژن
یکی از عوامل باکتریال واژینوزیس
معیارهای Amsel’s :
1- Clue cell
2- تست آمین (KOH 10%)
3- pH پایین
4- ترشح چسبنده و هموژن
عفونت داخل رحمی، PID، UTI

39. Noramal vaginal smaer

40. Clue cells

41. The Pseudomonad Group
The pseudomonads and acinetobacters are widely distributed in soil and water.
The pseudomonads are gram-negative, motile, aerobic rods.
Pseudomonads occur widely in soil, water, plants, and animals.

43. Pseudomonas aeruginosa
is the major human pathogen of the group.
is an important nosocomial pathogen.
is frequently present in small numbers in the normal intestinal flora and on the skin of humans .
is commonly present in moist environments in hospitals.
It can colonize normal humans, in whom it is a saprophyte.

44. It causes disease in humans with abnormal host defenses.
Identification
is motile and rod-shaped, gram-negative
is an obligate aerobe
grows readily on many types of culture media
produce a sweet or grape-like odor

45. Some strains hemolyze blood.
forms smooth round colonies with a fluorescent greenish color.
It often produces the nonfluorescent bluish pigment pyocyanin.
Other Pseudomonas species do not produce pyocyanin.
Many strains of P aeruginosa also produce the fluorescent pigment pyoverdin, which gives a greenish color to the agar.
Some strains produce the dark red pigment pyorubin or the black pigment pyomelanin.

46. P aeruginosa in a culture can produce multiple colony types.
P aeruginosa from different colony types may also have different biochemical and enzymatic activities and different antimicrobial susceptibility patterns.
Cultures from patients with cystic fibrosis (CF) often yield P aeruginosa organisms that form mucoid colonies as a result of overproduction of alginate, an exopolysaccharide.
the exopolysaccharide appears to provide the matrix for the organisms to live in a biofilm.

47. Growth Characteristics
P aeruginosa grows well at 37–42 °C;
its growth at 42 °C helps differentiate it from other Pseudomonas species in the fluorescent group.
It is oxidase-positive.
It does not ferment carbohydrates (Non-Fermentative Bacteria=NFB)

48. Antigenic Structure & Toxins
Pili (fimbriae) extend from the cell surface and promote attachment to host epithelial cells.
The exopolysaccharide is responsible for the mucoid colonies seen in cultures from patients with cystic fibrosis.
LPS
P aeruginosa can be typed by lipopolysaccharide immunotype and by pyocin (bacteriocin) susceptibility.

49. produce elastases, proteases, and two hemolysins: a heat-labile phospholipase C and a heat-stable glycolipid.
produce exotoxin A, which causes tissue necrosis and is lethal for animals when injected in purified form.
Exotoxin A blocks protein synthesis by a mechanism of action identical to that of diphtheria toxin, though the structures of the two toxins are not identical.

50. Pathogenesis
P aeruginosa is pathogenic only when introduced into areas devoid of normal defenses, eg, when mucous membranes and skin are disrupted by direct tissue damage; when intravenous or urinary catheters are used; or when neutropenia is present, as in cancer chemotherapy.
Lipopolysaccharide plays a direct role in causing fever, shock, oliguria, leukocytosis and leukopenia, disseminated intravascular coagulation, and adult respiratory distress syndrome.

51. P aeruginosa and other pseudomonads are resistant to many antimicrobial agents.
Clinical Findings
P aeruginosa produces infection of:
- wounds
- burns (giving rise to blue-green pus)
- meningitis
- urinary tract infection (by catheters)
- respiratory tract (contaminated respirators)
- mild otitis externa in swimmers

53. - invasive (malignant) otitis externa in diabetic patients
- Infection of the eye (eye pathologist), which may lead to rapid destruction of the eye, occurs most commonly after injury or surgical procedures.
- In infants or debilitated persons, may invade the bloodstream and result in fatal sepsis;
Hemorrhagic necrosis of skin occurs often in sepsis due to P aeruginosa; the lesions, called ecthyma gangrenosum.

55. Diagnostic Laboratory Tests
Specimens from skin lesions, pus, urine, blood, spinal fluid, sputum, and other material should be obtained as indicated by the type of infection.
Gram-negative rods are often seen in smears.
Specimens are plated on blood agar.
P aeruginosa does not ferment and is easily differentiated from the lactose-fermenting bacteria.
Culture is the specific test for diagnosis of P aeruginosa infection.

57. Treatment
should not be treated with single-drug therapy, because the success rate is low with such therapy and because the bacteria can rapidly develop resistance when single drugs are employed.
A penicillin active against P aeruginosa—ticarcillin or piperacillin—is used in combination with an aminoglycoside, usually tobramycin.
Other drugs active against P aeruginosa include aztreonam, imipenem, and the newer quinolones, including ciprofloxacin.

58. Of the newer cephalosporins, ceftazidime and cefoperazone are active against P aeruginosa; ceftazidime is used in primary therapy of P aeruginosa infections.
The susceptibility patterns of P aeruginosa vary geographically.

59. Epidemiology & Control
P aeruginosa is primarily a nosocomial pathogen (Nosocomial Infection)
pseudomonas thrives in moist environments, special attention should be paid to sinks, water baths, showers, hot tubs, and other wet areas.