Haemophilus, HACEK,

Legionella,and Other
Fastidious
Gram-Negative Bacilli
Rochelle D. Darlucio, RMT, MPH
College of Medical Laboratory Science
Our Lady of Fatima University
 HACEK Group
Haemophilus spp., e.g., H. paraphrophilus
Aggregatibacter actinomycetemcomitans, formerly
Actinobacillus actinomycetemcomitans and A.
aphrophilus, formerly H. aphrophilus
Cardiobacterium hominis
Eikenella corrodens
Kingella spp.
 Haemophilus
GENERAL CHARACTERISTICS
• gram-negative, pleomorphic • Haemophilus - “________
coccobacilli or rods ______________”
• nonmotile • require preformed growth
• facultatively anaerobic factors present in blood:
• ferment carbohydrates 1. _______________________
• oxidase and catalase positive 2. _______________________
• reduce nitrates to nitrites
 Haemophilus influenzae
VIRULENCE FACTORS Clinical Manifestations
• Capsule
• Immunoglobulin A (IgA)
proteases
• Adherence by fimbriae and
other structures
• Outer membrane proteins
and lipopolysaccharide (LPS)
 Infections Associated with Other Haemophilus Species

Haemophilus influenzae
Haemophilus aegyptius biogroup aegyptius
• ____________________ • conjunctivitis primarily in
pediatric populations.
• associated with an • non-encapsulated
acute, contagious
• first caused a severe
conjunctivitis, systemic disease known as
commonly referred to _______________________
as “_______________”. _______________________
in Brazil in 1984.
 Infections Associated with Other Haemophilus Species
Haemophilus ducreyi
• strictly human pathogen H. parainfluenzae
• causative agent of ____________ • found in the oral cavity
• commonly referred to as _________
___________________
• causes a few cases of
• not part of the normal microbiota otitis media and acute
sinusitis
• rarely implicated as a
causative agent of
endocarditis.
 Laboratory Diagnosis
Specimen Processing and Isolation
• Genital sites first should be cleaned
with sterile gauze moistened with
sterile saline before specimens are
collected for the isolation of this
organism.
• Next, a swab premoistened with sterile
phosphate-buffered saline should be
used to collect material from the base
of the ulcer.
• As an alternative, pus can be aspirated
from buboes if they are present.
Culture Media
• ___________________________is
a commonly used medium
incubated between 33° and 37° C
in an atmosphere of 5% to 10%
carbon dioxide (CO2)
• CHOC agar supplemented with
_________________________ -
Haemophilus spp. from respiratory
specimens
• enriched CHOC medium, or
_____________________________
– H. ducreyi
 Laboratory Diagnosis
Colony Morphology
• translucent, tannish, moist, smooth,
and convex, with a distinct
________________ or ______________
Microscopic Morphology
• varies from small, gram-
negative coccobacilli to long
filaments.
• _______________________-
gram-negative coccobacilli
arranged singly or in groups
(clusters),
• commonly referred to as
“_______________________________”
or “______________________________”
• loosely coiled clusters of organisms lined
up in parallel or appearing as
“_________________________________”
 Laboratory Diagnosis
X Factor and V Factor
Requirements
- using impregnated strips or disks

Porphyrin Test
- based on the ability of the
organism to convert the substrate
______________________________
into porphyrins or porphobilinogen,
which are intermediates in the
synthesis of X factor
 Aggregatibacter aphrophilus
• A. aphrophilus
• Greek aphros and philia:
____________________________
or desiring high concentration of
CO2)
• one of the most prevalent
species in the HACEK group
involved in endocarditis.
• linked to bone and joint
infections.
• found in dental plaque and
gingival scrapings.
 Aggregatibacter actinomycetemcomitans
• animal pathogens or animal
endogenous biota that in general do
not routinely cause infections in
humans
• grows better with increased CO2.
• a distinctive “_____________________
with four to six points” in the center of
the colonies is often seen at 48 hours
• catalase positive and oxidase variable
• do not grow on MAC agar
• negative for X and V growth factor
requirements, urease, indole, esculin,
and citrate.
 Cardiobacterium hominis
• Pleomorphic
• Nonmotile
• Fastidious
• gram-negative bacilli
• found as normal microbiota of the
nose, mouth, and throat and may be
present in the gastrointestinal tract.
• organisms tend to form rosettes,
swellings, long filaments, or sticklike
structures in yeast extract
• On agar, “_______________________”
can be produced
 Eikenella corrodens
• a member of the normal biota of the
oral and bowel cavities
• fastidious, gram-negative coccobacilli
that grow best under conditions of
increased CO2 with hemin.
• Tnonmotile, oxidase positive, and
asaccharolytic.
• catalase negative and often produce a
yellow pigment.
• “pit” (make a depression) or corrode
the surface of the agar
 Kingella
• coccobacillary to short bacilli with
squared ends that occur in pairs or
short chains .
• resist decolorization in Gram stains.
• nonmotile.
• fastidious
• oxidase-positive
• catalase-negative fermenters of
glucose and other sugars but with no
gas.
 Capnocytophaga
• belongs to the family • C. ochracea
Flavobacteriaceae
• Fastidious
• facultatively anaerobic
• gram-negative bacilli and require
increased CO2 for growth and isolation
from blood cultures.
• thin and often fusiform (pointed ends)
resembling Fusobacterium spp.;
spindle-shaped, coccoid, and curved
filaments may be also seen.
• “_______________________________”
-most common clinical isolate.
• C. canimorsus and C.
cynodegmi
-normal inhabitants of the oral
cavity of dogs and cats.
• C. canimorsus
-cause a fulminant, lifethreatening
septicemia in humans, particularly in
patients with asplenia or alcoholism, after a
dog or cat bite or through continuous
contact.
 Pasteurella
• Zoonosis • grow on SBA and CHOC agar,
• animal bites- most common presentation producing grayish colonies
• gram-negative, nonmotile, facultative, • P. multocida
anaerobic coccobacilli that appear ovoid,
filamentous, or as bacilli. -nonhemolytic colonies on SBA
• _________________________________ -appear mucoid after 24 hours
• catalase and oxidase (most isolates) of incubation at 37° C
positive -production of a ____________
• ferment glucose with weak to moderate _____________________________
acid production without gas. around the colony after 48 hours.
 Brucella
• Brucellosis, infection with bacteria from
the genus Brucella • facultative intracellular
• . Characterized by chronic and recurring pathogens
fever with weight loss and anorexia
____________________________ • small gram-negative, appear as
____________________________ coccobacilli or bacilli
____________________________ • aerobic, nonmotile,
____________________________ unencapsulated bacteria
____________________________
• smooth, raised, and translucent
colonies
• - REQUIRES BIOHAZARD LEVEL III in
handling specimens suspected with
Brucella
 Francisella
• Tularemia
• small, nonmotile, gram-negative bacilli
or coccoid bacteria and are strictly
aerobic.
• facultative intracellular parasites.
• fastidious and require supplementation
with cysteine, cystine, or thiosulfate
• CHOC, MTM, and buffered charcoal
yeast extract (BCYE) agars and
thioglycollate broth may be used.
• Broth cultures are not recommended.
- zoonotic disease and has many other
names, including :
- _______________________________
- _______________________________
- _______________________________
- _______________________________
- _____________________________
can be contracted through ingestion,
inhalation, arthropod bite (e.g., ticks,
biting flies), or contact with infected
tissues.
 Francisella
• Common species:
-Francisella tularensis subsp. tularensis – TYPE A – most severe; all forms of tularemia
-Francisella tularensis subsp. holartica – TYPE B – least severe; all forms of tularemia
-Francisella tularensis subsp. mediasiatica - severe
 Francisella
• IDEAL MEDIA and METHODS of IDENTIFICATION:
Culture Media of Choice:
Blood-Cystine-Glucose Agar with thiamine
Modified/Buffered Charcoal Yeast Extract Agar (BCYE)
Chocolate Agar with ISOVITALEX

-Direct Fluorescent Antibody (DFA) Stain

-Antibody titers as low as 1:40 in the absence of previous disease is
diagnostic and may rise up to 1:640 or greater within the 1st 3 weeks

-______________________________– susceptibility test for tularemia

 Legionella
• ubiquitous gram-negative bacilli
• acquired primarily through inhalation
from environmental sources
• Slow growth (3–5 days)
• Characteristic
“____________________________”
colony morphology
• Lightly staining, gram-negative bacillus
• Requires L-cysteine for primary
isolation
• No growth on unsupplemented sheep
blood agar
• Asaccharolytic
• Catalase or oxidase:
________________________________
CLINICAL INFECTIONS
• Legionnaires’ disease
- febrile disease with pneumonia
- Incubation Period: 2 to 10
days
• Pontiac fever
- influenza-like febrile disease
- nonpneumonic form of legionellosis
- Incubation Period; 2 days
- L. pneumophila is responsible for
most cases of this illness
 Laboratory Diagnosis
• pleomorphic, weakly staining, gram-
negative bacilli that are approximately
1 to 2 µm × 0.5 µm in size.
• Extending the safranin counterstaining
time to at least 10 minutes can enhance
the staining intensity of the organisms.
• _________________________________
is weakly acid fast in tissue and stains
best with the modified Kinyoun
procedure
• Will not grow on standard Sheep
Blood Agar
• Buffered Charcoal Yeast Extract Agar
(BCYE)
1. Cysteine is essential for growth
2. Iron is essential for growth
On BCYE agar, colonies appear
as grayish white or blue-green, convex,
and glistening, measuring
approximately 2 to 4 mm in diameter
• “ground-glass” appearance
 Bordetella
• small, gram-negative bacilli or
coccobacilli
• obligate aerobic bacteria, grow best at
35° to 37° C
• do not ferment carbohydrates
• B. pertussis is inhibited by fatty acids,
metal ions, sulfides, and peroxides,
constituents found in many media.
• Media for the isolation of B. pertussis
require protective substances, such as
charcoal, blood, or starch, to bind and
neutralize inhibitory substances.
• Bordetella pertussis and
Bordetella parapertussis
-primary human pathogens of
the respiratory tract, causing
whooping cough or pertussis
VIRULENCE FACTORS
• Filamentous hemagglutinin (FHA).
• Pertussis toxin (PT)
• adenylate cyclase toxin
• Tracheal cytotoxin
 Bordetella
CLINICAL MANIFESTATIONS
• PERTUSSIS
1. CATARRHAL PHASE
- Symptoms are insidious and
nonspecific and include sneezing, mild
cough, runny nose, and perhaps
conjunctivitis
- infants can develop apnea or
respiratory distress or both
- highly communicable
2. PAROXYSMAL PHASE
- hallmark: sudden onset of severe,
repetitive coughing followed by the
characteristic “whoop” at the end of the
coughing spell.
- B. parapertussis- causes a similar
disease with milder symptoms.
3. CONVALESCENT PHASE
-begins within 4 weeks of onset with a
decrease in frequency and severity of
the coughing spells.
 Laboratory Diagnosis
Specimen Collection and Handling
Isolation Methods
• Specimen of choice: • _______________________
Nasopharyngeal aspirates or _______________________
swabs of Dacron polyester with glycerol and horse or
sheep blood

• Transport media:
• _______________________-
contains charcoal, starch,
• casamino acid (1% casein horse blood, cephalexin,
hydrolysate) and amphotericin B
• Amies with charcoal
• Regan-Lowe transport medium
• ______________________-
contains casamino acids
 Laboratory Diagnosis
Colony Morphology
• Young cultures - smooth, glistening,
and silver, resembling mercury droplets Identification Methods
• turn whitish gray as they age • Gram stain: tiny gram-negative
coccobacilli

• Because of the fastidious nature

of these organisms,
identification by biochemical
testing is difficult.
Anaerobes of Clinical
Importance
Rochelle D. Darlucio, RMT, MPH
College of Medical Laboratory Science
Our Lady of Fatima University
 Anaerobe

• a bacterium able to replicate in the absence of oxygen

 Gram-Positive, Spore-Forming
Anaerobic Bacilli
Clostridium spp.

• Gram positive large _____________________________

• oval subterminally located spores
• round terminally located – C. tetani
• drumstick, tack head, lollipop, tennis racket bacillus
• No spores – box or car shaped
 Clostridium spp.
• Food Botulism – ingestion of
CLINICAL INFECTIONS:
improperly canned goods
1. Clostridium perfringens Food Poisoning
• type A- mild and self-limited GI illness
• type C (enteritis necroticans )- more serious but
• Wound Botulism – severe
rarely seen disease. wound infection
2. Botulism
• ingestion of preformed botulinum toxin, • Infant Botulism – ingestion of
produced in food by C. botulinum.
contaminated home-made
• _________________________________________
is also used medically to treat strabismus honeys
(wandering and chronic migraines, and as a
beauty enhancer by temporarily improving facial
wrinkles.
 Clostridium spp.
CLINICAL INFECTIONS: 5. Bacteremia
Clostridium perfringens- most common
• 3. Tetanus.
• attributed to the neurotoxin tetanospasmin C. septicum (present in the bloodstream)
produced by Clostridium tetani.
- marker organism for a malignancy in
the GI tract
• 4. Myonecrosis or gas gangrene.
• usually occurs when organisms contaminate
wounds, through trauma or surgery. 6. Clostridioides difficile Associated
• C. perfringens – most common cause Disease.
• C. histolyticum C. difficile
• C. septicum - the most common but not the sole
• C. novyi cause of antibiotic-associated diarrhea
and pseudomembranous colitis.
• C. bifermentans
 Gram-Positive, Non–Spore-Forming
Anaerobic Bacilli
• Actinomyces spp.
• straight to slightly curved bacilli of
differing lengths, from short rods to long
filaments.
• some are fastidious, requiring special
vitamins, amino acids, and hemin for
adequate growth.
• Young Actinomyces colonies are
frequently _________________________
• older colonies of A. israelii usually have a
___________________________________
 Gram-Positive, Non–Spore-Forming
Anaerobic Bacilli
• Bifidobacterium spp.
• variable in shape, ranging from
coccobacilli to long branching rods. • Lactobacillus spp.
• ends of the cells may be pointed,
bent, club-shaped, spatulated, or
• pinpoint α-hemolytic colonies
bifurcated (forked). on SBA.
• singly or in chains and as starlike • medium in size, with a rough
aggregates, V arrangements, or appearance and gray color.
palisade clusters.
• Colonies of are convex, entire, and • catalase negative
cream to white, smooth, glistening,
and soft.
 Gram-Positive, Non–Spore-Forming
Anaerobic Bacilli
• Propionibacterium and Cutibacterium spp.
• pleomorphic rods with a diphtheroid
appearance.
• _____________________________ is a
common member of the skin microbiota, it is
frequently isolated from blood culture
bottles as a contaminant
• __________________________________can
cause actinomycosis.
• ___________________is a major metabolic
end product of Propionibacterium spp.
 Anaerobic Gram-Negative Bacilli
• Bacteroides spp.
 Anaerobic Gram-Negative Bacilli

• Bilophila spp.
• __________________________
is a bile-resistant anaerobe
• grow on BBE agar with a
characteristic fish-eye
appearance
• also grow on KVLB agar.
• The organism is strongly
catalase positive and nitrate
positive
 Anaerobic Gram-Negative Bacilli
• Prevotella spp.
• gram-negative coccobacilli or
bacilli
• will grow on KVLB agar but not
on BBE agar.
• resistant to vancomycin and
kanamycin but are variable in
colistin
• produce ____________________,
a dark pigment that causes their
colonies to become brown to
black with age
• Porphyromonas spp.
• produce brick red
fluorescence under UV light,
similarly to Prevotella, but
some species do not
fluoresce.
• not grow on KVLB agar
• resistant to colistin
• Most are spot indole positive.
 Anaerobic Gram-Negative Bacilli
• Campylobacter ureolyticus
Group
• pitting anaerobes of the C.
ureolyticus group:
• C. ureolyticus
• C. gracili
• C. curvus
• C. rectus
• Sutterella wadsworthensis
•microaerophiles rather
than obligate anaerobes
• Fusobacterium
• long, thin, tapered rods, a
morphology characteristically
referred to as fusiform
• ____________________________-
has cells that are consistently
fusiform in shape
• ________________________-
appear pleomorphic and
exhibit globular forms,
swellings, and other bizarre
shapes