ALCANTARA S.

Specimen Collection and Processing

▪ Upper and lower respiratory tract

specimens; bronchial washing (for
culture)
Chapter 18: Haemophilus and other Fastidious ▪ Swabs from conjuctiva (for suspected
Gram-Negative Bacilli conjuctivitis)
▪ Vaginal swabs
▪ Abscess drainage
General Characteristics Haemophilus spp. die rapidly in clinical specimens;
prompt transportation and processing are vital for
their isolation
Haemophilus is derived from the the Greek words
"aima" and "philia" meaning blood-lover Haemophilus spp. do not grow on MAC agar

○ Gram- negative CHOC agar w/ bacitracin is an excellent medium

○ Pleomorphic coccobacilli or rods to for the islation of Haemophilus spp. from
filamentous respiratory specimens.
○ Fastidious
○ Facultatively anaerobic Haemophilus influenzae
○ Non-motile
○ Carbohydrate fermenters
○ Catalase (+) ; Oxidase (+)
○ Nitrate reduction (+)

Most species are normal URT flora ○ Pfeiffer bacillus

Require growth factors present in blood:
• X factor = Hemin or hematin ("X for
unknown")
• V factor = NAD ("V for vitamin") ; heat-
labile
Haemophilus spp. wit the prefix para- require only
V factor for growth.
○ Haemophilus parainfluenzae
○ Haemophilus parahaemolyticus
○ Haemophilus paraphrohaemolyticus
CHOC agar is used for the recovery for
Haemophilus spp. from clinical specimens
Satellitism - phenomenon that helps in the
recognition of Haemophilus spp. that require V
factor; occurs when organisms, like
Staphylococcus areus, Streptococcus
pneumoniae, or Neisseria spp., produces NAD as
a by-product of metabolism
○ 6 capsular serotypes: A-F
○ 8 biotypes: I-VIII
Causative agent of Influenzae
Leading cause of Meningitis in children
VIRULENCE FACTORS:
• Capsule
• IgA protease
• Fimbriae
• Outer membrane protein and LPS
Serotype - encapsulated strains of H. influenzae
NTHi - non-encapsulated strains of H. influenzae
CLINICAL MANIFESTATION
• Meningtis - headaches, stiffnecks, mild
respiratory disease
• Epiglottis - intense edema of epiglottis;
tracheostomy
• Bacterial Tracheitis - thick secretions
occluding trachea; antimicrobial agents

Sources of Specimens: MICROSCOPIC MORPHOLOGY:

▪ Blood • "Halos" = capsules of haemophilus
▪ CSF (for suspected meningitis) influenzae may be observed in Gram-
▪ Middle ear exudate (for suspected otitis stained direct smears as clear, non-
media) staining areas.
▪ Joint fluids

COLONY MORPHOLOGY
Preferred culture media: CHOC Agar, or Horse
blood bacitracin agar
Incubation temperature: 33 - 37 C
Duration of incubation: 18-24 hours
Incubation condition: Capnophilic 5%-10% CO2
 CHOC agar: Non-hemolytic, translucent,
tannish, moist, smooth, convex
 "Mousy or bleachlike" odor
 Encapsulated strains grow larger and
more mucoid than the NTHi
Haemophilus aegyptius
○ Koch-Weeks bacillus
○ Causative agent of Pink eye
○ Genetically related to H. influenzae
○ Difficult to differentiate H. influenzae from
H. aegyptius and H. influenzae biogroup
aegyptius in the clinical laboratory
○ Preferred culture media: Encriched
CHOC agar supplemented with 1%
IsoVitaleX
○ Duration of Incubation: 4 days
Haemophilus aegyptius biogrup aegyptius
○ Non-encapsulated
○ Conjuctivitis, primarily in pediatric
populations
○ First caused a severe systemic disease
known as Brazillian Purpuric Fever in
Brazilin 1984
Haemophilus influenzae biogroup
aegyptius
○ Strict human pathogen
○ Not part of the normal biota
○ Extremely fastidious
Chancroid/Soft chancre: highly communicable
sexually transmitted genital ulcer disease (GUD)
caused by H. ducreyi; nonindurated, painful lesion
with irregular edge; penis, labia or within the
vagina
Buboes; suppurative (pus-forming), enlarged,
draining, inguinal lymph nodes
ALCANTARA S.Y
SPECIMEN PROCESSING AND ISOLATION:
• Genital sites cleansed with sterile gauze
moistened with sterile saline
• A swab, premoistened with sterile
phosphate-buffered saline, should be used
to colllect material from the base of the
ulcer
• As an alternative, pus can be apirated
from buboes if they are present
• Direct plating on selective media at the
bedside is preferred instead of using
transport media/ specimen processing in
the laboratory should occur soon after
collection for maximum recovery
MICROSCOPIC MORPHOLOGY:
 Pale staining gram-negative coccobacilli
arranged singly or in groups
 "Railroad tracks"
 "School of fish"; "fingerprints"
COLONY MORPHOLOGY
Preferred culture media: Enriched CHOC medium,
Nairobi plate
Incubation temperature: 33C
Duration of incubation: 7 days
Nairobi plate medium components:
○ GC agar base
○ 2% bovine hemoglobin + 5% fetal calf
serum + Mueller Hinton agar on one side
○ 5% chocolatized hourse blood on the
other
○ 3 mg/L of vancomycin on both sides
CHOC agar: Small, flat, smooth, nonmucoid,
transparent to opaque, tan or yellow
Haemophilus parainfluenzae
○ Found in the oral cavity
○ Very low incidence of pathogenicity
○ Disease associated: Endocarditis
COLONY MORPHOLOGY:
 Tannish and drier
 Medium to large size compared with H.
influenzae
Haemophilus parahaemolyticus
 ALCANTARA S.Y

○ Indigenous microbiot of the URT of adults ▪ Media with X and V Factors with horse red
○ Diease associated: Pharyngitis blood cells

COLONY MORPHOLOGY
○ resembles H. parainfluenzae
○ on horse or rabbit blood agar, it is -
hemolytic

Laboratory Identification

 Growth of gram-negative pleomorphic

coccobacilli on CHOC agar
 No growth on SBA and MAC

X and V Factor Requirement

▪ Traditional approach
▪ Impregnated strips or disks
Porphyrin Test
▪ Method for differentiating the heme-
producing species of Haemophilus
▪ Can be performed in agar, in broth, or on
a disk

Test Principle:
 Abiility of the organism to convert the
substrate aminolevulinic acid into
porphyrins or porphobilinogen
▪ Both X and V factors are found within red (intermediates in synthesis of X factor)
blood cells; however only X factor is  Incubation temperature: 35C
directly available.  Duration of incubation: 4 hours
▪ Haemophilus spp. that are V factor  Porphobilinogen is detected by the
dependent do not grow on SBA because addition of p-
the red blood cells are still intact, and the dimethylaminobenzaldehyde (Kovac's)
sheep red blood cells contain enzymes red color forms if porphobilinogen is
(NADases) that hydrolyze V factor, present
▪ The lysing of the red blood cells by heat in  Porphyrins can be detected using an
the preparation of CHOC agar releases ultraviolet light with a wavelenght of
both the X factor and the V factor and about 360 nm (Wood's lamp)
inactivates NADases  Porphyrins fluoresce reddish orange under
ultraviolet light
Haemophilus spp X Factor V Factor
H. influenzae + + • Species that cannot synthesize heme/ X
H. haemolyticus + + Factor dependent = Porphyrin (-)
• Species that can synthesize hem/ V Factor
H. ducreyi + - dependent = Porphyrin (+)
H. parainfluenzae - +
H. parahaemolyticus - + Haemophilus spp. ALA
H. paraphrohaemolyticus - + H. influenzae -
H. haemolyticus -
Haemophilus Quad Plate H. ducreyi -
▪ Media with X factor only
H. parainfluenzae +
▪ Media with V Factor only H. parahaemolyticus +
▪ Media with X and V Factors H. paraphrohaemolyticus +
 ALCANTARA S.Y

• Grow better with increased CO2

HACEK Group concentration
• Duration of Incubation: 24-48 hours
• "Star shape with four to six points"
H ⎯ Haemophilus • Do not grow on MAC agar
A ⎯ Aggregatibacter 2 species:
1. Cardiobacterium hominis
C ⎯ Cardiobacterium
E ⎯ Eikenella Cardiobacterium hominis
K ⎯ Kingella
2. Cardiobacterium valvarum
Aggregatibacter aphrophilus
 Gram-negative bacillus
 Pleomorphic
▪ Greek aphros and philia" "foam loving"  Nonmotile
▪ H. aphrophilus and H. paraphrophilus have  Fastidious
been reclassified into the single species,
Aggregatibacter aphrophilus Found in nose, mouth, throat, gastrointestinal tract
▪ Does not require CO2, but grow better in
high concentrations of CO2 Disease Associated:
▪ Most prevalent species in the HACEK • Endocarditis
group involved in endocarditis • Meningitis
▪ Found in dental plaque and gingival
scracpings MICROSCOPIC MORPHOLOGY:
• Forms rosette
CLINICAL MANIFESTATIONS: • Swellings
▪ Fever • Long filaments or stick-like structures in
▪ Heart murmur, congestive heart failure yeast extract
▪ embolisn
COLONY MORPHOLOGY:
COLONY MORPHOLOGY: • Grow slowly o SBA and CHOC; do not
grow at all on MAC
CHOC agar: convex, granular, yellow, opague • Capnophilic = requires 5% CO2
zone near the center of the agar • "Pitting" may be produce on the agar

Aggregatibacter actinomycetemcomitans Eikenella corrodens

▪ Formerly in the genus Actinobacillus

▪ Normal oral microbiota in humans GENERAL CHARACTERISTICS
 Fastidious
▪ Disease associated:  Gram-negative
 periodonitis  Coccobacilli
 subacute bacterial endocarditis  Grow best with increased CO2 and hemin
 Nonmotile
▪ Six serotypes (A-F) based on a  Assacharolytic
surface polysaccharide
• Normal biota of the oraland bowel cavities
MICROSCOPIC MORPHOLOGY:
• Small bacilli to coccoid Disease associated:
• Gram-negative ○ IInfection from human bites
• Nonmotile ○ "Clenched fist wound"
○ Endocarditis
VIRULENCE FACTOR: ○ 45% of the isolates of E. corrodens "pit"
• Collagenase leukotoxin = toxic to (make a depressio) or corrode the
polymorphonuclear cells & monocytes surface of the agar
 ALCANTARA S.Y

COLONY MORPHOLOGY • 2 species are part of normal microbiota of

 Non-hemolytic on SBA the oral cavity of dogs and cats:
 "Chlorine bleach-like" odor Catalase/Oxidase (+)
 Do not grow on MAC and EMB agar
 Broth media: Produce granules 1. C. canimorsus
2. C. cyanodegmi
Kingella spp.
GENERAL CHARACTERISTICS:
 Fastidious
4 Species:  Facultatively anaerobic
 Gram-negative bacilli
1. Kingella kingae
2. Kingella denitrificans Disease Associated
3. Kingella oralis • Septicemia in patients with neutropenia
4. Kingella potus • Endocarditis (rarely)

GENERAL CHARACTERISTICS: COLONY MORPHOLOGY:

 Nonmotile • Flagella are usually absent, gliding on solid
 Fastidious surfaces
 Catalase (-); Oxidase (+) • Yellow-orange pigment
 Colonize the upper respiratory tract, • Most are nonhemolytic except for
especially the tonsils Capnocytophaga haemolytica (-
 Poor dental hygiene or oral surgery is hemolytic)
associated with infection
 Can grow on Neisseria selective agar (e.g., MICROSCOPIC MORPHOLOGY:
modified Thayer-Martin medium) and • Thin and often fusiform (pointed ends)
can resemble Neisseria gonnorrhea resembling Fusobacterium
(catalase +) • Coccoid, and curved filaments may be
also seen
Disease Associated:
○ K. denitrificans = bacteria, abscesses
Pasteurella spp.
○ K. kingae + pediatric, predilection for
bones and joints, endocarditis

MICROSCOPIC MORPHOLOGY:
• Resist decolorization in the Gram stain
• Coccobacilliary to short bacilli
• Squared ends
• In pairs or short chains
• Normal flora of respiratoy tract and oral
cavity of birds abd nannaks
• Pasteurellosis - zoonotic disease

Species:
Capnicytophaga spp.
▪ Pasteurella multocida; most common
isolate
• Family: Flavobacteriaceae
• 5 species are part of normal microbiota of 5 serogenous (A-F) defined by capsular antigens 3
the oral cavity of humans: subspecies:
Catalase/Oxidase (-)  Multocida
 Septica
1. C. ochracea  Gallicida
2. C. gingivalis
3. C. sputigena • Pasteurella canis
4. C. haemolyticus • Pasteurella stomatis
5. C. granulosa • Pasteurella dogmatis
• Pasteurella bettyae
 ALCANTARA S.Y

GENERAL CHARACTERISTICS:
 Fastidious Culture media: SBA, CHOC, Modifies Thayer-
 Gram-negative Martin, Matin-Lewis
 Nonmotile Duration of incubation: 18 hours
 Facultative anaerobic
 Coccobacilli that appear ovoid, Note: Plates should be kept for 4 days before
filamentous reporting as negative

Bipolar staining: "Safety pin" appearance = when

the poles of the cells are more intensely stained
Francisella tularensis
COLONY MORPHOLOGY
• On CHOC & SBA
 grayish colonies • Three subspecies/boivars:
• Do not grow on MAC agar  subsp. tlarensis (Type A)
• on SBA only, P. multocida  subsp. holartica (Type B)
 nonhemolytic  subsp. mediastica
 mucoid after 24 hours at 37C
 production of a narrow green to brown • Causes tularensis (rabbit fever, deerfly
halo around the colony after 48 hours fever/ lemming fever/ water rat trappers'
disease
• Ulceroglandular ulcer forms at the site of
Brucella spp. inoculation; enlargemnt of the regional
lymph nodes
• Bang's bacillus • Potential bioterrorism agent
• Causes brucellosis, also known as Undulant • Category A select biological agent
fever/ Malta fever • Biosafety level 3 conditions
• Normal flora of respiratory tract and oral
cavity of birds and mammals GENERAL CHARACTERISTICS:
• Potential bioterrorism agent  Small
• Category B select biological agents  Nonmotile
 Non-spore forming
4 species associated with humans:  Gram-negative
1. Brucella melitensis - goat/sheep  Bacilli or coccoid
2. Brucella abortus - cattle  Strict aerobes
3. Brucella suis - swine  Fastidious; requires the ff. supplements
4. Brucella canis - dogs • Cysteine
• Cystine
2 additional species isolated from marine animals: • Thiosulfate
1. Brucella ovis
2. Brucella neotomae Culture media: CHOC, Modified Thayer-Martin,
Buffered Charcoal Yeast Extract (BYCE). Mueller-
GENERAL CHARACTERISTICS: Hinton, Tryptic Soy Broth (TSB)
 Small
 Gram-negative • Do not grow on MAC and EMB
 Aerobic • CHOC agar: gray-white, raised colonies
 Nonmotile with smooth appearance (slow
 Unencapsulated growing, after 72 hours)
 Non-spore former
 Appear as coccobacilli or bacilli
 Facultative intracellular pathogens
 Must be handled under biosafety level 3
conditions
 Isolated from bone and bone marrow
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• Acid treatment must be first done on

specimens contaminated by other
Legionella pneumophila
bacteria = enhances isolation of
Legionella spp.
• Fastidious, aerobic, do not grow on SBA
• Ranging from mild upper respiratory tract • Require L-cysteine
infections to pneumonia
• Acquired primarily through inhalation Culture Media:
• Found on natural water sources and water • CHOC agar with L-cysteine
supplies • Most preferred culture media: Buffered
• Assacharolytic Charcoal Yeast Extract agar with L-
cysteine.
VIRULENCE FACTOR
 Ability to enter, survive and multiply within COLONY MORPHOLOGY:
the host's cells, especially  BYCE agar: grayish white or blue-green,
bronchoalveolar macrophages convex and glistening
 Production of proteolytic enzymes  When viewed on dissecting microscope:
"Ground glass" appearance
Disease Associated:
• Legionnaire's disease = febrile disease + Rapid methods:
pneumonia  Urine antigen - L- pneumophila serogroup
• Pontiac fever = febrile disease only, no 1
pulmonary involvement  DFA -LRT specimen; bright yellow to green,
short or coccobacillary bacilli with
SPECIMEN COLLECTION AND HANDLING intense peripheral staining

• Source of Specimens: Bordetella spp.

 Sputum
 Bronchoalveolar lavage
 Bronchial washings
 Environmental sources
 Urine
2 species implicated in human infections:
1. Bordetella pertussis
2. Bordetella parapertussis

• Methods: 6 other species:

 Direct examination 1. Bordetella bronchiseptica - opportunistic
 Culture - most important pathogen
 Serology - IFA 2. Bordetella avium - wild & domesticted
animal pathogen
Legionnaires' disease - culture + urine antigen 3. Bordetella hinzii - avian commensal
Pontaic fever - serology 4. Bordetella holmesii - immunocompromised
bacteremia
• Delays of > 2 hours - specimen 5. Bordetella petrii
refrigerated 6. Bordetella trematum- wound & ear
• Delays take several days - freeze at -70C infections

MICROSCOPIC MORPHOLOGY: • Inhabits mucous membranes of respiratory

 Pleomorphic tract
 Weakly staining, gram (-) bacilli
 Extend safranin counterstain at least 10 GENERAL CHARACTERISTICS
mins
 Intracellular and exracellular pathogen  Small, gram-negative bacilli or
coccobacilli
ISOLATION AND IDENTIFICATION:  Obligate aerobic bacteria
 Optimum growth temperature: 35C - 37C
 Non-carbohydrate fermenter
 ALCANTARA S.Y

MICROSCOPIC MORPHOLOGY:
Bordetella pertussis
• DFA Staining - small, fat bacilli or
coccobacilli with intense peripheral
 Oxidize amino acids yellow green fluorescence and darker
centers
• Inhibited by fatty acids, metal ions, sulfides, • Gram stain - tiny gram (-) coccobacilli;
and peroxides, constituents found in many increase safranin counterstain = n time to
media 2 minutes
• Require protective substances, charcoal,
blood, starch Culture media:
• Bordet-Gengou potato with glycerol and
VIRULENCE FACTORS: horse or sheep blood
• Filamentous hemagglutinin (FHA) and • Regan Lowe selective agar with full
pertactin = attachment to ciliated strenght charcoal sugar supplemented
epithelial cells with 10% horse blood and 40 mg/L
• Pertussis toxin = protein exotoxin cephalexin
• Adenylate cyclase toxin • Incubation temperature: 35C
• Tracheal cytotoxin • Duration of incubation: 7 days; adequate
moisture
Pertussis (Whooping cough) = via respiratory
droplets or direct contact with infectious  B. pertussis colonies are detected in 3-
secretions 5days;
 B. parapertussis colonies are detected in
3 Stages of Pertussis: 1 day or sooner
1. Catarrhal - general flulike symptoms
2. Paroxysmal - repetitive coughing (whoop COLONY MORPHOLOGY
sound)  Small and shiny
3. Convalescent - Recovery  "mercury droplets"

SPECIMEN COLLECTION AND HANDLING: ○ Bordotella parapertussis causes mild

respiratory infections in humans
• Sources of specimens: Nasopharyngeal ○ Bordotella bronchiseptica causes kennel in
aspirate/swabs dogs and infrequent cause of respiratory
• Acceptable swab material: calcium cough infections in humans
alginate or Darcon polyester + flexible
wireshaft
• Two swabs are collected, one through
each of the external nares; the swabs
should bbe inserted as far back as possible
into nasopharynx, rotated, held a few
seconds, and then gently withdrawn
• Transit time <2hours / DFA test - swab put
into a solution of 1% casein hydrolysate
(casamino acids) broth
• Amies transport medium - up to 24 hours
• Regan-Lowe transport medium - overnight
or days

Methods:

• Culture
• DFA
• PCR - more sensitive

Note: DFA should always be with culture.

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