HAEMOPHILUS AND OTHER FASTIDIOUS
GRAM-NEGATIVE BACILLI
General Characteristics
Family Pasteurellaceae
• Haemophilus and Pasteurella
-Gram-negative coccoid to rod shaped
-Nonmotile
-Aerobic to facultatively anaerobic
-Convert nitrate to nitrite
-Oxidase positive and catalase positive
HAEMOPHILUS SPECIES
• Gram-negative pleomorphic coccobacilli
or rods
-Coccobacilli in direct smears
-Rods with occasionally long filamentous
rods from colony growth
• Biochemical reactions
-Nonmotile, oxidase positive, catalase
positive, nitrate reduction
• Obligate parasites of the mucous
membranes
• 10 species involved in human infection
-Emphasis on H. influenzae, H.
haemolyticus, H. aegyptius, H. influenzae
biotype aegyptius, and H. ducreyi
• Haemophilus: blood loving
Require X and V factors
-X factor is hemin, hematin
-V factor is nicotinamide-adenine
dinucleotide (NAD)
• Para: require only V factor
-H. parainfluenzae
Produces X factor, requires V factor
• Hemolysis of 5% horse or rabbit blood
agar
-H. hemolyticus species and sometimes H.
ducreyi
-Sheep blood agar (SBA) only contains X
factor and not V factor.
*Thus, some will not grow without V factor
added because NADases present in blood
agar plate (BAP) destroy V factor.
*Chocolate agar releases X and V factor and
deactivates NADases.
GROWTH PATTERNS
• Satellitism
-Growth of fastidious organisms around
other bacteria that release the necessary
growth factors or break down toxic
products
✓S. aureus, S. pneumoniae, and Neisseria
species that cause hemolysis release the
factors or naturally produce V factor.
-Exceptions: H. aphrophilus and H. ducreyi
EXAMPLE OF SATELLITISM
• 10% of normal flora of the upper
respiratory tract in adults
-2% to 6% in children from birth through
childhood, with a higher percentage
colonization in day care centers
✓As they mature they convert from
encapsulated to nonencapsulated strains
H. influenzae: Historical Perspective
• H. influenzae was isolated from
pandemic influenza victims.
-Was not cause of disease
-Was frequently isolated
(Secondary infections)
Virulence Factors
• Capsule
-Serotypes a-f
• Immunoglobulin A (IgA) protease
-Cleaves IgA on mucous membranes
• Outer membrane proteins and
lipopolysaccharide (LPS)
-Not well defined
-Antibody to these proteins is somewhat
protective.
-LPS paralyzes the cilia.
(Cannot clear the lungs)
• Adherence
-Pili and other structures
Capsule • H. influenzae biogroup aegyptius
• Classification based on capsule serotype Brazilian purpuric fever (BPF)
• Contains ribose, ribitol, and phosphate -Occurs in warm tropical climates
-Antiphagocytic and anticomplement -Recurrent conjunctivitis, high fever,
activity vomiting, petechiae, purpura, septicemia,
• H. influenzae serotype b (Hib) and shock
-Primarily causes disease in children -High mortality, as high as 70%
-Causes bacteremia and spread to other
tissues H.DUCREYI
-Hib vaccine is useful in reducing incidence • Sexually transmitted infection
of disease. -Chancroid
• Nontypable strains (no capsule) • Incubates 4 to 14 days
-Invade the respiratory tract and tissues ✓Painful lesion with an irregular edge
located around the same area ✓Genital and perianal areas
-Localized infections -Penis
-Pneumonia, otitis media, sinusitis -Labia or vagina
✓Enlarged and draining lymph nodes
Clinical Manifestations of H. influenzae -Buboes
Diseases of Hib strains
• Meningitis CHANCROID LESIONS
-Invasion of the respiratory tract
-Bacteremia allows spread to the meninges
and cerebrospinal fluid (CSF).
-Infants and children 3 months to 6 years
old
• Epiglottitis
Acute inflammation and swelling, causing
airway obstruction Miscellaneous Haemophilus Species
-Affects children 2 to 4 years old • H. parainfluenzae
-Emergency tracheostomy -Endocarditis
• Bacterial tracheitis •H. aphrophilus
-Life-threatening disease in young children -Bite wound infections
• Cellulitis -Endocarditis
Cheek
-Causing swelling, pain, and reddish blue Specimen Processing
color of the inflamed area •Haemophilus species
-Children younger than 2 years old Die rapidly
• Other infections -Plated within 10 minutes for maximum
-Acute pharyngitis and pneumonia recovery
-Children about 1 year old •H. ducreyi
• Diseases of nonencapsulated strains -Clean specimen site with sterile saline and
-Otitis media, bronchitis, sinusitis, sterile gauze
pneumonia in the elderly, genital tract -Swab base of ulcer with cotton swab
infections moistened with sterile saline, and plate
immediately
H.AEGYPTIUS -Can also aspirate pus from buboes
• H. aegyptius and H. influenzae biogroup •H. influenzae
aegyptius can cause conjunctivitis Chocolate agar with bacitracin (300mg/L)
Pinkeye -Helps inhibit other organisms, especially
-Acute, contagious, purulent conjunctivitis respiratory flora
-Particularly in children
•H. aegyptius
-Chocolate agar supplemented with 1%
isovitaleX
•H. ducreyi
-GC agar (hemoglobin, fetal bovine serum
[FBS], vancomycin to reduce genital flora),
enriched chocolate, or Mueller-Hinton agar
with 5% lysed horse blood can be used as
well
•Grow at 33°C at 5% to 10% carbon
dioxide (CO2) for 7 days
•Humidity chamber
-Prevent drying
•Plate inoculated nutrient broth on a
nutrient agar plate or a Mueller-Hinton agar
plate
-Add X and V strips to the media
-Incubate at 35°to 37°C in 5% to 10% CO2
18 to 24 hours
•Read plates
ORGANISM REQUIRING X AND V FACTORS

Culturing Haemophilus
•Colony morphology
-Translucent, moist, smooth, convex
colonies
-Mousy or bleach-like odor
-Encapsulated strains appear more mucoid.

Haemophilus Microscopic Morphology

•H. influenzae
-Gram: coccobacilli or small rods
•H. ducreyi
-Gram: coccobacilli arranged in groups
resembling “school of fish,” “railroad
tracks,” or “fingerprints” PORPHYRIN TEST
•If they do not require X factor
GRAM STAINS OF H. INFLUENZAE -Porphyrin positive (make X)
•Δ-aminolevulinic acid (ALA) gets converted
to porphyrins or protoporphyrins.
✓ Kovacs reagent
-Red color produced in lower part of the
tube
✓ Wood ultraviolet (UV) lamp
-Fluoresce reddish-orange under UV light
Laboratory Identification
•Take colonies from an initial isolation of EXAMPLE OF PORPHYRIN TEST
Haemophilus
-Do not pick up the growth media
•Place the colonies in nutrient broth and
mix
PORPHYRIN TEST
•Clinical and Laboratory Standards Institute
(CLSI) guidelines
•Respiratory tract or CSF specimens
-Gram-negative bacilli or coccobacilli that
exhibit colonies greater than 1 mm on
chocolate agar
-No growth on SBA (excluding satellitism)
-Negative porphyrin
•Can identify as H. influenzae
Treatment
• Cefotaxime or ceftriaxone
-Recommended
•Trimethoprim sulfamethoxazole,
imipenem, ciprofloxacin, and
chloramphenicol with ampicillin
-Alternative treatments
•H. ducreyi
-Azithromycin, ceftriaxone, ciprofloxacin, or
erythromycin
Chromogenic Cephalosporin Test
•Disk impregnated with nitrocefin is
moistened.
Smear several colonies onto disk
-If β-lactam ring is broken, a red color
develops on the disk.
•Acidometric test
-Test strip contains benzylpenicillin and
bromocresol purple.
-If β-lactam ring is broken, acid develops,
causing a color change from purple to
yellow.
HACEK
•H – Haemophilus spp.
-Now Aggregatibacter aphrophilus
•A – Aggregatibacter
actinomycetemcomitans
-Formerly Actinobacillus
actinomycetemcomitans
•C – Cardiobacterium hominis
•E – Eikenella corrodens
•K – Kingella spp.
HACEK Group General Characteristics
•Gram-negative bacilli
•Require an increased CO2 (5%-10%)
environment
•Significant cause of endocarditis
•Usual flora of the oral cavity
-Thus are pathogens associated with human
bite wounds, causing septicemia and
subacute endocarditis
•Opportunists in immunocompromised
hosts
A. aphrophilus
•Aggregatibacter aphrophilus
From the Greek word for foam loving
-Desiring high concentrations of CO2
•Chocolate agar
-Yellow, granular, convex colonies with
opaque centers
A. actinomycetemcomitans
•Primarily an animal pathogen but has
been found in human oral cavity
-Nonmotile gram-negative coccobacilli
-6 serotypes a-f
✓a, b, c are most common
-24 to 48 hours to grow
Distinctive star formation in the center of
the colonies
✓Under ×100 magnification of the colony
•Ferments carbohydrates in the presence
of serum
-Not lactose or sucrose
•Catalase positive, oxidase variable
•No growth on MacConkey (MAC) plates
•Negative for X and V growth factors
•Negative for urease, indole, esculin, and
citrate
COLONY OF A. •Biochemical tests
ACTINOMYCETEMCOMITANS -Nonmotile
-Nonfermenter
-Oxidase positive and catalase negative
-Lysine decarboxylase positive
-Ornithine decarboxylase positive
-Arginine decarboxylation negative

C. hominis
•Pleomorphic, nonmotile, gram-negative
bacillus
-Sometimes appear false positive on Gram
stain
-Show rosette, swellings, or long filaments Kingella Species
•Normal flora of the nose, mouth, and •Coccobacilli or short bacilli with square
throat ends in pairs or chains
-Can be present in gastrointestinal (GI) tract -Resist decolorization in the Gram stain
•Involved in endocarditis •Nonmotile
-Grows on the heart valve and is resistant •Oxidase positive, catalase negative; sugar
to antibiotics, so valve must be replaced fermenters without gas production
•Grow on SBA, chocolate agar
-Not MAC plates •K. kingae
-Growth requires 5% CO2 -May grow on Thayer-Martin medium and
•Pitting may be produced on agar plates. resemble N. gonorrhoeae
•Ferments sucrose ✓Colonies do not always pit.
-Gram-negative, short, coccoid bacilli have
square ends.
-β-hemolytic colonies on SBA
-No growth on MAC
-Catalase and oxidase negative
•Biochemical tests
E. corrodens -Glucose and maltose weak positive
•Normal flora of oral and bowel cavities -Sucrose negative
-Fight and bite wounds -Isolates of children less than 3 years old
•Infections ✓Affecting bone and joints
-Meningitis -Other patients present with endocarditis
-Pneumonia
-Osteomyelitis • K. dentrificans
-Arthritis -Catalase and superoxol negative
-Postoperative tissue infections usually •Biochemical tests
from bacteremia associated with wounds -Glucose positive
-Cellulitis -Reduces nitrate (positive)
(Inoculation with needles “licked clean”) -Urease negative
•Grow in capnophilic environment -Indole negative
•Require hemin (X factor) and CO2 -Esculin negative
•Gram-negative coccobacilli -Gelatin negative
-Bleach-like odor with yellow colonies -Citrate negative
pitting the agar (about 45% of isolates)
-Hence the name corrodens
Capnocytophaga
•Previously called dysgonic fermenters 1
and 2 (DF-1 and DF-2)
-Gam-negative bacilli, often fusiform
-May resemble Fusobacterium
•Facultatively anaerobic
-Require CO2
•More common in septicemia than
endocarditis
•Biochemicals
Ferments
-Sucrose, glucose, maltose, and lactose
-Negative indole
-Hydrolyzes esculin
-May have negative triple sugar iron (TSI)
without enrichment
P. multocida
•Five serogroups A, B, D, E, F
•Gram-negative pleomorphic coccobacilli
with bipolar staining
-Oval shaped, short rods, longer filaments
•Grow on SBA and chocolate agar
-Nonhemolytic mucoid colonies with a
narrow green or brown halo
-Generally does not grow on MAC plates
-Bipolar staining
✓Safety pin appearance when the poles of
the cells are more intensely stained
•Zoonosis
-Colonize the upper respiratory tract (URT)
and GI tract of mammals and birds
-Exposure from dog and cat bites or
scratches
•Biochemical tests
-Nonmotile
-Catalase positive
-Oxidase positive (most)
-Glucose fermentation
✓Weak acid (sick acid) production
PLATE GROWTH OF P. MULTOCIDA
Brucella Species
Brucellosis or undulant fever
•Category B select biologic agents
-Centers for Disease Control and Prevention
(CDC) reportable disease
•Reoccurring fever at regular intervals that
persist for days, months, or years
•Zoonotic infection acquired from animals
or animal products, and laboratory workers
-Exposure through aerosol, percutaneous,
and oral routes
Clinical Stages of Brucellosis
•Acute
-Fever, malaise, headache, anorexia,
myalgia, and back pain
Within 1 to 4 weeks after exposure
•Subchronic (undulant)
-Low temperature in morning followed by
rising temps in the afternoon and evening
-Arthritis
-Epididymo-orchitis
•Chronic
-Depression, arthritis, and chronic fatigue
syndrome
•Most common species for human
brucellosis
-B. melitensis (most common isolate), B.
abortus, B. canis, B. suis
•Handle under biosafety level 3 (BSL3)
conditions
-Due to aerosol transmission
Characteristics of Brucella Species
-Strict aerobes (some require CO2)
-Non–spore-forming, gram-negative
coccobacilli
-Nonmotile, nonencapsulated, intracellular
parasites
•Biochemical tests
-Oxidase positive
-Catalase positive
-Hydrogen sulfide (H2S) production and
urease help differentiate
•Blood and bone marrow cultures
-Acute and convalescent sera
Category B select biologic agent
✓Serology test are safer.
COLONIES OF B. MELITENSIS
Francisella Species
•Facultative intracellular parasite
•Nonmotile, small gram-negative
coccobacilli
•Strict aerobe
•Zoonotic infection
✓ Causes tularemia
-Acute febrile, granulomatous disease with
rapid onset and flulike symptoms
✓Category A select agent
-Handle in BSL3
✓Diagnosis from serology
-Culture is less safe.
•Also known as rabbit, deerfly, and
lemming fever
•Four subspecies
•F. tularensis subsp. tularensis (type A),
more virulent
-Transmitted by rabbits, sheep, ticks
•F. tularensis subsp. holarctica (type B)
-Transmitted by rodents and mosquito
•F. tularensis subsp. mediasiatica
•F. tularensis subsp. novicida
-Similar to F. tularensis but less lethal
Colony Growth on Chocolate Agar for F.
tularensis subsp. Tularensis
General Characteristics of Legionella
•Ubiquitous gram-negative bacilli
-Primarily acquired through inhalation
-Patients present with wide range of
conditions.
•Methods of isolation
-Isolation on special media
-Urine antigen detection
-Direct fluorescent antibody detection
-Serology
Clinical Significance
•Cause 2% to 15% of community-acquired
pneumonia
-Incidence is likely higher than reported.
-Also associated with nosocomial infections
•Most are L. pneumophila
Other important species
-L. micdadei
-L. longbeachae
-L. dumoff
•Virulence factors
-Ability to invade and survive in
macrophages
•Infection
-Disease has a large range from
asymptomatic to deadly.
-Results from the inhalation of aerosols
Note: Person to person via aerosol
transmission has not been documented.
•Detection
-Culture
-Antigen in the urine
-Direct fluorescent antibody staining
-Serology
•L. pneumophila
-Legionnaires’ disease
-Pontiac fever
-14 serogroups
•Sources
-Present in environmental water sources
-Air conditioning systems
-Respiratory secretions on objects used by
patient
Legionnaires’ Disease
•Sporadic cases: community acquired
•Epidemic outbreaks
-Usually from air conditioners
•Nosocomial infections in
immunocompromised
-Respiratory equipment and aerosols
•One of the top four pathogens in
community-acquired pneumonia
-Streptococcus pneumoniae, Mycoplasma
pneumoniae, Chlamydia pneumoniae
•Incubation period is 2 to 10 days
Symptoms
-Nonproductive cough, fever, headache,
and myalgia
-Progressing to bloody or purulent sputum,
rales (crackling), difficulty breathing, and
shaking chills
•Dissemination via circulatory system can
occur
Invades the kidneys, liver, heart, CNS, and
lymphatic system
-Tissue destruction leads to death;
generally pneumonia does not.
•Mortality rate from 15% to 30%,
sometimes approaching 50%
•Most cases are serogroup 1 (85%)
-Also serogroup 4 and 6
-Sometimes L. longbeachae or L. micdadei
Pontiac Fever
•Incubation period of 2 days
Flulike symptoms
-Fever, headache, myalgia
-Lasting 2 to 5 days
•Spontaneous recovery
-Does not generally lead to mortality
-Mild form of Legionella
Epidemiology
•aquatic sources
•Lake, river, springs, man-made water
treatment systems
-Can survive in chlorinated water up to 3
mg/L
•L. longbeachae: associated with gardening
materials such as compost and potting soil
-Significant source of infection in New
Zealand and Australia
•Specialized sources
-Heating and cooling towers of buildings
-Fountains
•Can colonize a variety of sources due to
•Ability to multiply at a large range of
temps
-20°to 43°C
•Survive in temps
-40°to 60°C
-Adherence to pipes, rubber, and plastics
-Can live intracellularly in protozoa,
bacteria, and algae
•Transmitted by aerosolized water particles
from environmental sources
Clinical Infections of Legionella
• Intracellular parasite of cells
-Enter and multiply within host cells
-Particularly bronchoalveolar macrophages
• Predisposition for disease
-Immunocompromised
-Patients with chronic lung disease
-Alcoholics and heavy smokers
Identifying Legionella
•Specimens
•Predominantly respiratory specimens
✓Bronchoalveolar lavage (BAL), sputum,
bronchial washings
-Culture and direct detection
✓Urine specimens for antigen test
-Soluble antigen found in the urine for
serogroup 1
-Stays positive for a long time, thus only
confirms infection happened
✓Serology
-Used for Pontiac fever
•Refrigerate specimens if more than 2-hour
delay in processing
-Freeze at -70°C if delayed for several days
•Microscopic examination
-Pleomorphic, thin, weakly staining, gram-
negative rods
-Found within macrophages and
neutrophils as well as extracellularly
•Urine antigen test
•Direct antigen tests available
-Fluoresce if positive for Legionella
GRAM STAIN OF L. PNEUMOPHILA
Isolation Methods for Legionella Antimicrobial Susceptibility
•Aerobic bacteria •Susceptibility not routinely performed
•Require L-cysteine for growth, so do not -Use erythromycin or in combination with
grow on routine media rifampin
•Alternatives
•When suspected -Doxycycline, trimethoprim-
Grow on buffered charcoal yeast extract sulfamethoxazole, newer macrolides, or
(BCYE) fluoroquinolones
-Grow as small pinpoint colonies
-Acid wash specimens to reduce Bordetella Species
contaminants •Eight major species
✓ Dilute sample 1:10 in 0.2N potassium •Major pathogens
chloride-hydrochloric acid (KCl-HCl) solution B. pertussis
for 5 minutes B. parapertussis
-Both of these are agents of
•Semiselective BCYE whooping cough.
Contains polymyxin B, anisomycin, and -B. parapertussis is usually milder
vancomycin or cefamandole
-Can reduce contaminants but also inhibits •Minor pathogens
Legionella B. bronchiseptica
B. avium
•Grow plates in normal atmosphere at 35°C B. hinzii
to 37°C for at least 7 days B. holmesii
•Grayish-white or blue-green convex, wet- B. petrii
looking colonies B. trematum
Confirmation
-Requires proving L-cysteine is required for Virulence Factors
growth •Filamentous hemagglutinin (FHA)
-Can use molecular methods -Facilitates attachment to epithelial cells
•Pertussis toxin (PT)
BCYE Agar with L. pneumophila -Interferes with signal transduction
•Adenylate cyclase toxin
-Increases cyclic adenosine monophosphate
(cAMP) levels
•Tracheal cytotoxin
-Ciliostasis: cilia do not clear the airway

Infection of B. pertussis and parapertussis

EXAMPLE OF DIRECT FLUORESCENT
ANTIBODY (DFA) TEST FOR LEGIONELLA
Rapid Methods
•Urine antigen tests
•Direct fluorescent antibody test
•DNA detection
•Serologic testing
-Indirect fluorescent antibody assay
• Breathing in aerosols
-Bacteria adhere to and grow on ciliated
respiratory epithelial cells.
•Highly contagious disease
-90% of exposed contacts get disease
✓Vaccine widely used; however,
effectiveness drops rapidly after
administered
-Adults can become transient carriers.
✓With and without symptoms
Clinical Infections and Disease Course •Smooth silver pinpoint colonies
•Whooping cough resembling mercury droplets
•Exposure and incubation period of 1 to 3 •Direct fluorescent antibody tests are used
weeks for confirmation.
•Initially cold or flulike symptoms, sneezing, Agglutination tests
runny nose -Require larger amount of organism
-Catarrhal phase 1 to 2 weeks (highly •Biochemicals to differentiate species
contagious)
•Sudden severe repetitious coughing COLONIES OF B. PERTUSSIS
Followed by “whoop”
-Rapid gasp for air
-Can be followed by vomiting

•Paroxysmal phase
•Serious infection in young children
-Cyanosis from lack of oxygen
-Sometimes need assistance in keeping
airway open
•Convalescent phase Serologic Testing
-Begin recovery about 4 weeks after initial •Used to study outbreaks and document
symptoms seroconversion
-Decrease in bouts of coughing but may Antibody levels do not correlate well to
take weeks or months for complete immunity.
resolution -Not approved for diagnostic use
•Paired sera and detection of multiple
Diagnosis and Isolation immunoglobulin classes are best for
•Specimens are best from the nasopharynx. diagnostic sensitivity.
-Aspirates of fluid
-Calcium alginate or Dacron swabs up each Antimicrobial Susceptibility
nasal passage as deep as possible •Erythromycin
•Inoculate a transport system -Most common
-Regan-Lowe transport medium -Must be started during catarrhal phase of
-Direct to plate media disease
•Nucleic acid detection •Azithromycin
•Trimethoprim-sulfamethoxazole
Isolation and Identification -Can be used as an alternative
•Culture •Routine antimicrobial testing on B.
-Bordet-Gengou potato infusion agar with pertussis or B. parapertussis is not
glycerol and sheep blood necessary.
-Alternative is charcoal agar with 10% horse -B. bronchiseptica must be tested but is
blood and 40 mg/L cephalexin generally susceptible to aminoglycosides.
✓Also plate to BAP and MAC agar
✓Chocolate agar if you need to rule out
Haemophilus
•Incubate plates
-35°C in aerobic conditions
-Detected in ~3 to 5 days
-Adequate moisture to prevent plate drying
•Tiny gram-negative rods or coccobacilli
•Obligate aerobic bacteria