Haemophilus and other

fastidious GNB
CHAPTER 18
 Haemophilus general facts
 General characteristics
 Family Pasteurellaceae
 13 species: 8 associated with humans
e.g. Haemophilus influenzae, H. aegypytius, H. ducreyi, H.
parainfluenzae
 Small pleomorphic Gram negative coccobacilli or bacilli
 Facultative anaerobe
 Non motile
 Oxidase positive, except H. ducreyi
 Catalase positive
 NF of upper respiratory tract
 H. influenzae virulence factors
 Capsule – purpose?
 Utilized for further typing
 Six antigenic types: a, b, c, d, e, f
Hib primary serotype in invasive infection in unvaccinated population
Has unique anti-phagocytic properties
Non encapsulated strains are untypeable (NTHi)
 IgA protease: Allows for colonization
 Only produced by H. influenzae
 Adherence mechanisms
 NTHi strains adhere well – more localized infections?
 Hib strains don’t adhere – systemic infections?
 Clinical infections of H. influenzae
 Localized
 Non-typable H. influenzae (non encapsulated strains)
 Invasive
 Caused by encapsulated strains
 General risk factors
Asplenic

Sickle cell disease

Complement deficiencies
Other immunocompromised states

 Transmission from colonizing strains or via respiratory droplets

Clinical infections of H. influenzae
 Meningitis: usually cause by HiB
 Children age 6 months- 2 years
 Mortality 2-5% (cdc.gov)
 Symptoms: Headache, sore neck, light sensitivity
 15-30% survivors suffer from sequelae: hearing damage, mental retardation,
paralysis, hydrocephalus, ataxia
 Decreased Incidence with vaccine
 Acute epiglottitis: usually caused by HiB
 Children 2-5 y.o.
 Symptoms: Fever, sore throat, hoarse, “barking” cough
 Rapid disease progresseion
 Pneumonia
 Reduction of mucosal clearing of organism
Smoking, COPD, viral infection,
 Bacteremia
H. Influenzae cases decreasing (Taiwan
cases )

cdc.gov
Non-invasive clinical infections of H.
influenzae
 Usually caused by non-encapsulated strains
 Otitis media
 Sinusitis

 Bronchitis
 Treatment for Haemophilus influenzae

 Invasive
 3rd generation cephalosporin
 Non-invasive
 ~25% produce beta-lactamase
 Beta-lactam/beta-lactamase inhibitor
 Other Haemophilus species
 H. aegyptius
 Closely related to H. influenzae
 Acute, contagious conjunctivitis
“pink eye”
 H. ducreyi Fig 18.2

 Causative agent of chancroid

STD – genital ulcer disease
 Rare in U.S.A but common in Africa, Asia, Latin
America
 H. parainfluenzae
 Normal flora of mouth and throat
 Rarely associated with bacterial endocarditis
Haemophilus spp. Growth Requirements
 Require growth factors
X factor: Hemin
Directly available in blood agar Fig 18.1
 V factor: Nicotinamide adenine dinucleotide (NAD)
Within intact red blood cells
V factor dependent organisms will not grow on blood agar
 Chocolate agar: Lysed red cells releases V factor
 Satellitism

Small colonies around colony of other organism, S.

aureus
Organism produces V factor as by product alpha-toxin
Laboratory Diagnosis of Haemophilus species

 Specimen collection
 Blood, CSF, swabs (ears and eyes)
 Organisms die rapidly : Prompt processing essential
 Culture requirements
 Chocolate agar is media of choice
 35 ℃ + Increase CO2 content
 Colony morphology
 Chocolate agar: Grayish/tan, smooth, moist (always?)
 Blood agar: No growth, or satellite colonies
Laboratory Diagnosis of Haemophilus species

 Microscopic morphology
 Small, pleomorphic, Gram-negative coccobacilli
 May see capsule in primary smear
 Faint staining
 Often need to observe edge of smear to visualize
 Growth pattern
 X and V factor requirements
 Disk test
 Quad plate
 Requires overnight incubation
X or V . . . What is your preference?
Laboratory Diagnosis of Haemophilus species

 Porphyrin
 Detectsability of organism to convert ALA into
porphyrins
 Occurs in organisms that DO NOT require hemin to grow
 Porphyrins detected via UV light
 Advantages: Quicker (4 hour test)
 Disadvantage: Identification of H. influenzae based
off a negative results
H. ducreyi + - +/- -
 HACEK group
 Group of fastidious organisms
 Haemophilus (H. paraphrophilus)
 Aggregatibacter spp.
 Cardiobacterium hominis: capnophilic
 Eikenella corrodens
 Kingella spp.

 Most commonly associated with bacterial endocarditis

 Part of normal oral flora
 Opportunistic pathogens
HACEK group
 Aggregatibacter aphrophilus
 Most prevelent HACEK organism
 Found in dental plaque
 Does not require CO2, but grows better with it
 Endocarditis

 Aggregatibacter actinomycetemcomitans
 Oral flora
 Does not require CO2, but grows better with it
 Slow growing, >24 hours to observe Fig 18.13
 Star shaped in center of colony after 48 hours
 Typical animal pathogen
 peridontitis
 HACEK group
 Cardiobacterium hominis
 Normal flora in nose, throat, mouth
 Grow slowing on blood and choc
 “Rosettes” on Gram stain
 Eikenella corrodens
 Normal flora of oral and bowel
 Wound infections from human bites or fights
 Endocarditis associated with poor dental hygiene or oral surgery
 Cellulitis in IV drug users
 Growth

Colonies “pit” agar

Greening around colonies

HACEK group (KNOW Table 18.4)
 Kingella
 Colonize upper respiratory tract/tonsils
 Poor dental hygiene or oral surgery associate with infection
 Bone and joint infections in children (<3 y.o.)
 Gram stain morphology
Rod with square ends
Resist decolorization, may stain purple
 Capnocytophaga spp.
 General characteristics
 Nine species
 Normal oral flora
 Facultative anaerobe
 Require increased CO2 for growth
 Long thin Gram negative bacilli
 Bacteremia

Neutropenic patients
 Pasteurella spp. general
 General characteristics
 21 species
P. multocida is most common human pathogen
 Normal oral flora of birds and mammals
Infection usually result of animal bite
 Growth on blood and chocolate
Do not grow on MacConkey
 Small Gram negative coccobacilli
 Oxidase positive
 Brucella spp.
 Normal flora in animals
 Four common species are human pathogens
B. melitensis – sheep and goats
B. abortus – cattle
B. suis – swine
B. canis – dogs
 Transmission

Direct via work with animals or animal products

Indirect via consumption of contaminated food
Laboratory acquired
 Disease

Brucellosis

7-21 days post exposure

Malaise, fever, chills
Body aches, headache
Brucella spp. laboratory diagnosis
 Blood most common specimen
 Slow growing
 Growth on blood and chocolate
 Small Gram-negative coccbacillus (similar to Haem)
Faint staining, may require longer counterstain time
 Considered to be agent of bioterrorism
Rule out testing
Oxidase positive
Catalase positive
Urea positive
 Francisella spp.
 Normal flora in wild animals (rabbits most common)
 F. tularensis is most common human pathogens
 Transmission

Direct via work with animals or animal products

Bite or scratch from infected animal
Tick bites
Deer flies
Laboratory acquired
 Disease

Tularemia

Clinical presentation depends on route of infection

Most common form is ulceroglandular
Ulcer at site of inoculation
Followed by swelling of regional lymph nodes
 2001-2010: CDC

https://www.frontiersin.org/articles/10.3389/fmic
b.2011.00034/full
Francisella spp. laboratory diagnosis
 Blood most common specimen
 Slow growing
 Growth on chocolate
 Small pleomorphic Gram-negative coccobacilli
 Considered to be agent of bioterrorism
 Rule out testing
Oxidase negative
Catalase weakly positive
Urea negative
 Bordetella spp.
 Two clinically significant human isolates
 B. pertussis
 B. parapertussis
 Obligate aerobe
 Small Gram negative bacilli or coccobacilli
 Bordetella pertussis virulence factors

 Virulence factors
 Anatomical

Capsules

Pili

Filamentous hemagglutinin: attachment

 Exotoxins

Pertussis toxin: Antibodies to PT confer immunity

Tracheal cytotoxin: Stops movement of cilia
Adenylatecyclase toxin: overproduces cyclic adenosine
monophosphate
 Bordetella pertussis clinical diseases
 Pertussis - Whooping cough
 Human disease
 7-10 day incubation
 Catarrhal phase
Initial symptoms similar to common cold
1-2 weeks
Highly communicable
 Paroxysmal phase
Sudden onset of severe repetitive cough cdc.gov
Followed by “whooping” sound as gasp for air
2-4 weeks
 Convalescent phase
B. parapertussis
Decrease in coughing spells Similar disease course, but milder
May take weeks to months to fully recover
 Risk of secondary bacterial pneumonia
Bordetella pertussis laboratory diagnosis
 External nares swab (one through each): dacron or flocked swabs
 Culture
 Special media: Bordet-Gengou or Regan-Lowe
 Direct plating ideal
 Incubation

35 °C, ambient air Regan-Lowe

Hold MINIMUM of 7 days
Colonies resemble tiny mercury drops :Smooth, glistening, silver
 Gram stain
Small Gram-negative coccobacilli
 Confirmation via DFA or slide agglutination tests
 Non culture
 Molecular assays - PCR
 Legionella spp.
 L. pneumophila is primary human pathogen
 Habitat: Water
Resist chlorine water treatment
Often colonize water systems
 Transmission

Environment via aerosolized water particles

No human to human transmission
 Legionella pneumophila clinical diseases

 Legionnaire’s disease
 Acute pneumonia
Cough, chest pains, shortness of breath
 Opportunistic: Immunocompromised, smokers, COPD
 Can become systemic
 15-30% mortality rate
 Pontiac fever
 Previously healthy patients
 Acute flu-like symptoms
 Self limiting (2-5 days)
Legionella pneumophila laboratory diagnosis

 Gram: Gram (-) bacilli

 Culture
 Difficult to cultivate: Require 5-7 days
 Requires acid treat specimen to reduce normal
flora
 Needs L-cysteine
 Require special media
Buffered charcoal yeast extract (BCYE) agar
Small blue/green colonies
 Definitive identification referred to public
health labs
 Legionella pneumophila laboratory
DFA
diagnosis
 Non-culture
 Urine antigen assays
Detects soluble antigen excreted in
urine within 3 days of infection
 Direct Fluorescent Antibody test
 Indirect Fluorescent Anitbody – fix
bacteria, then add patient serum
 Molecular assays - PCR
Current test of choice