LESSON 12.

SMALL,
MEDT 14 | CLIN. BACTERIOLOGY (LEC)
Jonnel P. Andaya || 3rd Year PLEMORPHIC, GRAM-
Transcribed by: Ybanez, Trisha Mae C. | De los Reyes,
John Dominic M.
NEGATIVE BACILLI

 Normally inhabits the urinary tract of humans except

OUTLINE: only to Haemophilus ducreyi.
I. Introduction  Oxidase and catalase positive except to Haemophilus
II. Haemophilus spp. segnis
A. H. influenzae  They are facultative aerobes, non-motile, capnophilic
B. H. ducreyi and fastidious bacteria
C. Differential characteristics of Haemophilus  Requires several growth factors: X (hemin factor), V
spp. (nicotinamide adenine dinucleotide factor)
D. Laboratory diagnosis  They are very susceptible to drying and very high
III. HACEK (AACEK) group temperature
A. Characteristics  They are “blood lover” but most species cannot grow at
pure Blood agar plate
B. Aggregatibacter aphrophilus
 Under microscopy, they are gram negative, small and
C. Aggregatibacter actinomycetemcomitans
pleomorphic coccobacilli or rods.
D. Cardiobacterium hominis
 Human pathogenic species are Haemophilus
E. Eikenella corrodens
influenzae, Haemophilus ducreyi, Haemophilus
F. Kingella spp.
parainfluenzae, Haemophilus paraphrophilus,
G. Differential characteristics of the HACEK Haemophilus parahaemolyticus, Haemophilus
group pittmaniae, Haemophilus aegypticus, and Haemophilus
IV. Brucella spp. segnis
A. Laboratory diagnosis
B. Differential characteristics of Brucella spp. HAEMOPHILUS INFLUENZAE
V. Bordetella spp.  Main etiologic agent of meningitis among children
A. General characteristics  Spread starts form the nasopharynx and the regional
B. Bordetella pertussis lymph nodes going to blood and finally to the meninges.
C. Laboratory diagnosis  Can also cause septic arthritis and osteomyelitis
VI. Francisella tularensis
A. Laboratory diagnosis
VII. Pasteurella spp.
A. General Characteristics
VIII. Legionella spp.
A. General Characteristics
B. Distinguishing Characteristics
C. Primary Clinical Manifestations  Fastidious organisms and can be immediately killed by
D. Laboratory diagnosis the phagocytes
 This is the only genus that can produce IgA protease
INTRODUCTION  They do not produce endotoxin
 Five small gram-negative coccobacilli are part of the  Primary virulence factor: Polysaccharide capsule
normal oral flora and are associated occasionally with (serotype A and F)
bacterial endocarditis and rarely with other infections.  Other virulence factors are fimbriae (for attachment),
They are opportunists that enter the bloodstream, settle IgA protease (to degrade immunoglobulin A in
on damaged heart valves, and cause a relatively slowly secretions), and lipopolysaccharide
progressive, indolent form of endocarditis. They typically  Mode of acquisition: From person to person -
require an additional 1–2 days before they are isolated exposure and inhalation of contaminated respiratory
from blood cultures, and they are uniformly susceptible droplets)
to many antimicrobial agents. The word HACEK is an  Biochemical test: porphyrin negative
acronym for thebacteria responsible for this disease:  In Chocolate agar their colonies are- translucent,
Haemophilus spp. (influenzae, parainfluenzae), convex, tan-colored, and mucoid with a “mousy” of
Aggregatibacter (Haemophilus) aphrophilus (most “bleach-like” odor
commonly, Aggregatibacter [Actinobacillus]
actinomycetemcomitans), Cardiobacterium hominis, 2 CATEGORIES OF H. INFLUENZAE
Eikenella corrodens, and Kingella spp. Some taxonomic  Haemophilus influenzae is catergorized base on the
changes have beennoted more recently, and some of characteristics of their capsule.
the HACEK members have been reassigned to the 1. Typeable form
genus Aggregatibacter (Norskov-Lauritsen, 2006). o Encapsulated strains are type A, B, C, D,
E and F- capsular serotypes
o Haemophilus influenza type B: causative
HAEMOPHILUS SPP.
agent of serious infections in humans
 Came from the Greek word “haima” and “philos” which and the leading cause of meningitis in
means “blood lover”. unvaccinated children.
 Obligate parasites of human’s mucous membranes o The composition of this vaccine is H.
o Obligate- requires suitable host to influenzae type B capsular
complete their life cycle. polysaccharide conjugated with
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diptheriae toxi, and isadministered in 2- DIFFERENTIAL CHARACTERISTICS OF HAEMOPHILUS
18 months of age. SPP.
o Other associated infections are
septicemia, arthritis, epiglottitis, Species Distinguishin Growt Associated
tracheitis, osteomyelitis, and pneumonia g h infection/
o Invasive type characteristic factor disease
s
2. Non-typeable form H. influenzae Mousy/bleach X,V Meningitis,
(Pfeiffer’s like odor; non- epiglottitis,
o Non-capsulated strains of Haemophilus bacillus) hemolytic arthritis
influenzae H. aegypticus Genetically X,V Pink eye
o Indigenous microbiota of urinary tract (Koch-weeks related to H. conjunctiviti
and may adhere at human epithelial bacillus) influenzae s
cells. H. influenzae Non-typeable X,V Brazilian
o Second to the prevalent cause of otitis biogroup purpuric
media with effusions (middle ear aegypticus fever
infection) H. haemolyticus Beta-hemolytic X,V
o Localized infections are: Conjunctivitis H. ducreyi School of fish X Chancroid
and sinusitis or soft
o Less invasive chancre
o They can produce biofilms so they can H. Tan and dry V Pharyngitis
secrete exopolysaccharide to hide parahaemolyticu colonies, B-
themselves from our immune system. s hemolytic
o They have the capacity to change their H. Fructose and V Endocarditis
structure especially their antigenic parainfluenzae maltose
structure such as oligosaccharide. fermentation
o We can acquire reinfection from H.
influenza because of its ability to LABORATORY DIAGNOSIS
change structure whereas our body
 Specimens: CSF, sputum, genital lesions or ulcer, joint
does not recognize it.
fluid, vaginal swab, abscess drainage, conjunctival
HAEMOPHILUS DUCREYI swab, bronchial washing, and blood
 Not a part of Human normal flora o Sample to be collected depends on the
 Etiologic agents of chancroid or painful “soft chancre” infection caused by specific Haemophilus spp.
which is highly contagious, sexually transmitted, genital  CSF samples should be centrifuge
ulcer disease  Haemophilus species are very susceptible to drying and
 Hallmark of chancroid: Buboes or suppurative, enlarge, extreme temperature, hence the need for an immediate
draining, inguinal, lymph nodes. transport and processing for their isolation.
 It infects the mucosal epithelium, genital and non- o Bedside plating is preferred instead of
genital skin, and regional lymph nodes. transport medium.
o For example, for H. ducreyi that causes ulcer.
 It enters the body through skin and it have mayvirulence
The ulcerated area should be pre-moistened
factors that could attack and invade host cells. Then
with phosphate buffered saline to prevent
after invasion of the tissue, they can release toxins that
drying.
could arrest the cell cycle leaving it big and may cause
cell death.  For the recovery of Haemophilus ducreyi, the ulcer
should be cleansed with sterile gauze that is pre-
 Can secrete cytotoxic hemolysin. It destroys ephitelial
moistened with sterile phosphate- buffered saline
cells and fibroblasts.
 They can activate the secretion of inflammatory GRAM STAINING
chemicals which can activate the T-helper cell and also
the polymorphonuclear cells (neutrophil).  Haemophilus species resemble an “amorphous
o These cells will move to the site of infection serous material” because of their pleomorphic
and then excrete their oxidative radicals (ex.
appearances
Superoxide) to kill H. ducreyi.
 H. ducreyi cells have a “school of fish” arrangement
o But H. ducreyi is smart and secrete its own
enzymes to degrade the superoxide and that  resembling like a bunch of long chains gram (-) bacilli
is the copper-zinc superoxide dismutase.
 Since it destroys epithelial cells and fibroblasts,
disorganization occurs in the epidermis and then forms
erythematosus in the papules. Then it may form into
pustules and then forms painful ulcer with soft irregular
margins.
 It causes chancroid which is a sexually transmitted
disease characterized by painful and tender genital
lesions that advance to ulcers with satellite lesions.
 Culture: CAP- colonies are transparent, small, non-
mucoid and tan or yellow. CULTURE
 *Haemophiluss parainfluenzae – common indigenous  Culture media: CAP, BAP, BHI and thioglycollate
microbiota or normal flora of the urinary tract of adults.  CAP is the preferred medium for haemophilus because
it contains the X and V
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o V factor is found in the RBC and we have to
lyse RBC to release V factor; NOTE that CAP
is composed of lysed RBC
o because CAP contains blood, it automatically
contains X factor or hemin factor
Factors
 Most strains cannot grow on Pure BAP because the
medium only contains hemin
o BAP do not compose of lysed RBC so it does
not contain V factor so we can oly cultivate
haemophilus that grows in X factor (also the
blood in BAP kasi is not lysed) therefore only
the spp that require X factor can grow in this
medium
 ex. H.ducreyi
o Provide BAP then sample na H.influenzae that
will be inoculated in the whole surface BAP
and then vertically streak at the middle.
Incubate at appropriate condition for their
growth. After incubation the growth pf
H.influenzae is found around the growth of
Staphy bec staphy is capable of producing the
hemolysin factor which could lyse the RBC.
Lysis is found aroud the colonies of
Staphylococcus. Once the RBC si lysed,
hemolysisn will be released. That is why their
growth is found around the colonies if Staphy
and this is called SATELLITISM GROWTH.
 Rabbit’s or Horse’s blood agar is preferred for
observing hemolysis over sheep’s blood.
 All clinically significant Haemophilus species, except for
Haemophilus ducreyi require nicotinamide adenine
dinucleotide
 Importance of CAP: The lysing of the RBCs by heat in
the preparation of CAP releases
 both the X and V factors and inactivates NADases
The selective media for isolation of Haemophilus species:
a. Haemophilus influenzae - horse’s blood-bacitracin
agar for respiratory secretions of patients with cystic
fibrosis
b. Haemophilus aegypticus- CAP with 1% IsovalateX or
Vitox
c. Haemophilus ducreyi – selective Nairobi biplate
medium (combination of gonococcal agar and MHA
with horse’s blood and vancomycin)
PORPHYRIN TEST (δ-aminolevulinic acid)
 It detects the presence of enzymes that converts δ-
aminolevulinic acid (ALA) into porphyrins
 It is a test for identifying the heme-producing species of
Haemophilus.
 Reagent: Kovac’s reagent (p-
dimethylaminobenzaldehyde)
 Endproducts:
o Porphobilinogen: red color
o Porphyrins: reddish- orange color (UVL-300
nm)
 (+) Result: Exhibit a red color :
o Haemophilus parainfluenzae
o Haemophilus parahaemolyticus
o Haemophilus paraphrophilus
o Haemophilus aphrophilus
 (-) Result:
o Haemophilus influenzae
o Haemophylus haemolyticus
o Haemophilus aegypticus
o Haemophilus ducreyi
 Interpretation of results:
o Haemophilus species that need the X factor are
unable to synthesize porphyrin from δ –ALA

SEROLOGIC TEST
 Serotype can be determined through the identification
of the distinct capsular antigen by latex agglutination,
capsular swelling, or immunofluorescence test.
The growth patterns of Haemophilus species are as follows:  Neufeld- Quellung reaction is a rapid direct
a. Haemophilus species grow best at 35 degrees Celsius identification test of the capsular antigen of H.
to 37 degrees Celsius (except H. ducreyi which grows influenzae
at 33 degrees Celsius) and in an environment with 5%
to 10 % Carbon dioxide HACEK (AACEK) GROUP
b. V-factor: dependent Haemophilus species, like H.  HACEK (Haemophilus aphrophilus OR ,
influenzae, grow as “satellite” on BAP around bacterial Aggregatibacter aphrophilus, Aggregatibacter
colonies and produce NAD, like S. aureus actinomycetemcomitans, Cardiobacterium hominis,
c. When Haemophilus species are grown anaerobically, Eikenella corrodens, and Kingella species)
only NAD, and not Hemin, is required  It includes the following organisms:
d. Haemophilus aegypticus requires four days of a. Aggregatibacter aphrophilus (formerly H.
incubation while H. ducreyi requires seven. aphrophilus)
e. No growth occurs on a Mac Conkey agar. b. Aggregatibacter actinomycetemcomitans
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c. Cardiobacterium hominis
d. Eikenella corrodens AGGREGATIBACTER ACTINOMYCETEMCOMITANS
e. Kingella species  Formerly known as Actinobacillus
actinomycetemcomitans
CHARACTERISTICS OF THE HACEK GROUP  This organism is the common cause of periodontitis
 These organisms are considered human oral
(gum infection/inflammation esp after tooth extraction
indigenous microbiota and are also opportunistic
or if and when there is a formation of plaques in the
pathogens
teeth)
 They are small gram negative bacilli that are non- motile
 It has been isolated with Actinomyces in a
and cannot grow at Mac Conkey
polymicrobial infection
 They are fastidious bacteria and most species are slow
 It is the only member of HACEK that is catalase
grower at Blood Agar Plate and Chocolate Agar Plate.
positive
 Since they are included to small pleomorphic gram (-)
 Virulence factor: Collagenase and leukotoxin
bacilli, so they are coccobacillary to bacillary in form
 Serotype: A, B, C, D, E, and F
under the microscope
 Culture: Colonies exhibit a “star-shaped” appearance
 They require 7-14 days of incubation
after 48 hour of incubation
 since they are the predilection of heart valve attachment,
 Biochemical test: (-) urease
they are the etiologic agent of slow and progressive
bacterial endocarditis (vegetation of fibrinous clots in
heart valves)
 Bacterial group that utilize δ - aminolevulinic acid

AGGREGATIBACTER APHROPHILUS
(“FOAM-LOVING BACTERIUM”)
 "H" in HACEK group
 This organism is formerly known as Haemophilus
aphrophilus
 “aprohilus” is came from the Greek works “aphros” and
“philos” collectively means
“foam- loving”
 Most common cause of endocarditis among HACEK CARDIOBACTERIUM HOMINIS
group species.  It infects the aortic valves more frequently than the
 Can be isolated form dental plaques and gingival other HACEK species.
scrapings

 Culture: CAP- colonies are raised, convex, granular,

and yellowish  It shows “false Gram-negative” reactions in some parts
of the cells
 It is only indole-positive HACEK member
 Culture:
a. BAP – Colonies are capnophilic and may exhibit
“pitting”
b. Yeast extract – Colonies exhibit a “rosette”
formation and appear filamentous
 Biochemical test: (+) Oxidase and indole
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EIKENELLA CORRODENS (CORRODING BACILLI)  K. kingae is the major Gram negative bacterium that is
 It is least common isolate of the HACEK group in isolated from degenerative joint and bone infections
adult infections endocarditis. (osteoarthritis) in children younger than three years old
 It is assacharolytic like the species of the genus
Moraxella. DIFFERENTIAL CHARACTERISTICS OF THE HACEK
 It causes various infections from human bites or GROUP
clenched fist injuries
Species Oxid Gluc Lact Malt Sucr
ase ose ose ose ose
A. aphrophilus v + + + +
A.actinomycetem v + - + -
comitans
C. hominis + + - + +
E. corrodens + - - - -
K. kingae + + - + -

BRUCELLA (BANG’S BACILLUS)

 the higher risk of acquiring infection is among
immunocompromised individuals
 Culture: BAP- Colonies exhibit a yellow color; pit or
corrode the agar; and have a “sharp bleach” odor.
 a zoonotic infection
 The species of this genus are important human and animal
pathogens
 They are intracellular parasites (they could live inside the
hosts cell ex. in macrophage of humans) and strictly
aerobic bacteria
 They are non-capsulated, non-motile, and assacharolytic
 Some species require an increased supply of CO2 for
growth
 They are localized in tissue that are rich in erythritol
(placental tissue) and induces spontaneous abortion
among animals
 Preferred specimen: Blood and bone marrow for isolation
 Microscopy: Small coccobacilli that are arranged singly, in
pairs, or in short chains, and
which have a “sandy appearance”

 Biochemical test:
(+) lysine decarboxylase and
(-) arginine dihydrolase

KINGELLA
 It has a tendency to resist decolorization.
 Microscopy: Plump, square-ended, Gram negative
rods to coccobacilli that are arranged in pairs or short
chains

 Culture: BAP-Colonies are small,convex, translucent,

yellowish and non-hemolytic; they
are become brownish in color with age.
 since they are slow grower bacteria they require longer time
of incubation for the exhibition of their colonies in the
medium (2-4 weeks)

 Culture:
o BAP – Colonies exhibit white to yellowish-
brown pigmentations
o TMA – K. denitrificans resembles the growth
of N. gonorrhoeae
How to identify K.dentrificans from Neisseria?
 K. dentrificans is Superexol (-)
 Neisseria is Superexol (+)
 Biochemical test:
 Species: K. kingae, K oralis, and K denitrificans (+) catalase and oxidase; rapid urease producer
 It the most virulence species in K. kingae (causative  Diseases: Malta/Crimean/Mediterranean fever or
agent of endocarditis). undulant fever (brucelliosis)
 Superoxol test: Nagative ( K. denitrificans)
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 Brucella species should be handled as a biosafety level 3
agent in a class III cabinet (since Brucella belong to risk
group 3 agent) due to their aerosol mode of transmission.

GRAM STAINING
 Carbol fuchsin should be substituted for safranin O to
improve the gram stain.

CULTURE
 Culture media: BAP, trypticase soy agar (TSA), and
Castaneda’s medium
 B. abortus requires niacin or nicotinic acid for growth but
can be inhibited by adding thionine dye
 Species:  Isolates can be recovered after seven days but may
o B. abortus require prolonged incubation up to 30 days.
o B. canis  Turbidity of the specimen is a normal occurrence in positive
o B. suis
culture bottles.
o B. melintensis
 B. melintensis is the common isolate SEROLOGIC TEST
 The most virulent species are B. melintensis and B. suis  Serum agglutination test (SAT):
 B. suis, and B. melintensis can cause severe infection o Positive with > 1:160 titer
o ex. B suis can cause long course destructive  B. suis is not detected by SAT
lesions
 B. abortus, B. canis could cause mild diseases and rare DIFFERENTIAL CHARACTERISTICS OF
complication BRUCELLA SPP
 CLINICAL SIGNS AND SYMPTOMS: non-specific (which
Specie Natu Gro H2S Urea Inhibit Thion
means like those in common diseases) s ral wth produc se ion by ine
 ex. flu, fever chills, weakness, lethargy, muscle and joint Host in tion Test dyes
aches, headaches. Most importantly, remember the Night 5%- (Lead Fuchs
sweats wherein their sweat is mold-like odor 10% Acetat in
 they are intracellular parasites so they could live inside the CO2 e
Metho
host cell esp macrophage. If you ingested brucella, the
d)
bacteria is phagocytized by the macrophage, so they could
B. Cattl +/- + + - +
live inside the macrophage. This macrophage carrying abortus es
brucella goes into the lymphatic tissue, into the lymph B. Dogs - - + + -
nodes, there will be infiltration in the lymph nodes causing canis
its enlargement resulting to lymphadenopathy (kulani) B. Goat - - v - -
 it could also infect the spleen and liver, there will be melinte s
infiltration that will lead to hepatosplenomegaly nsis and
Shee
 even in our body it is slow growing so its incubation period ps
is longer B. suis Swin - + + + -
 it takes 1-3 weeks before the manifestation of signs and es
symptoms
 BRUCELLIOSIS- the most common zoonoic infection in BORDETELLA
the world affecting all ages, group, and gender GENERAL CHARACTERISTICS
● Obligate aerobic, fastidious gram negative coccobacilli
Undulant fever: ● Non-carbohydrate fermenters and are non-motile except for
B. bronchiseptica
 It is characterized by normal temperature in the morning
● Replicates on ciliated respiratory epithelial cells of humans
and then followed by high temperature in the afternoon and ● Are mostly inactive in biochemical test systems
evening. ● Culture: Bordet-Gengou agar – Colonies are smooth,
 Primary route of infection: glistening, and have a silver color
o Ingestion of unpasteurized and contaminated milk
or cheese from infected animals
o Inhalation of air around animal carcasses (aerosol
infection)
o Penetration of ocular or oral mucosa
o Direct inoculation into the bloodstream through
abrasions in the skin or needle stick injuries

LABORATORY DIAGNOSIS
 Specimens: Blood, bone marrow, skin lesion, and placental
tissues. ● Biochemical test:
o (+) catalase
o (-) indole
● Growth factors: Nicotinic acid, cysteine, and methionine
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Species: B. pertussis, B. parapertussis, B. brochiseptica, and B.
avium
BORDETELLA PERTUSSIS
● Usually affects the upper respiratory, and is not associated
to pneumonia but can increase the risk of acquiring the said
infection
● Does not survive well outside the host
● Only infects and causes diseases in humans; meaning a
Human pathogen
● Is the etiologic agent of whooping cough.
● Culture: Bordet-Gengou agar – Colonies are small and shiny
and resemble “mercury drops”
● Growth inhibitors: Fatty acids, metal ions, sulfides and
peroxides
● Are fastidious bacteria requiring growth protectors:
Charcoal, blood, and starch
● Principal virulence factor:
o Pertussis toxin (protein toxin)
⮚ Inhibits chemokine receptors which
causes cells unable to receive any signal
to proceed to the site of infection
⮚ Stimulates WBC apoptosis
● Other virulence factors:
o Filamentous hemagglutinin
⮚ used for attachment to the ciliated
epithelial cells of the airways; once
attached, proliferates and causes
localized damage
o Adenylate cyclase toxin (CyaA)
⮚ Increases the mucous and respiratory
secretion triggering violent cough reflex
o Tracheal toxin
⮚ Inhibits the cilia from moving/causes
paralysis, and interrupts the normal
clearing mechanism of the respiratory
restricting the release of the bacteria
o Pertactin (adherence factor)
● Preferred specimen for isolation: Nasopharyngeal swab
Related infection: Whooping Cough
● Highly contagious, acute infection of the upper respiratory
tract (URT) and is disease that primarily affects children
● Mode of acquisition: Inhalation of infected droplets
● Incubation period: 7- 14 days
Three stages of Whooping Cough
● Catarrhal Stage
o When B. pertussis is inhaled, it will bind to the lining of
the upper respiratory tract and shall proliferate/replicate
- time when the infected will show clinical signs and
symptoms
o 1-2 weeks after bacteria exposure; long period of
incubation for the bacterium is a slow-grower
o The infected is highly communicable and contagious;
main reason is that the bacteria are already replicated
in the system
o Characterized by mucous membrane inflammation,
rhinorrhea, malaise, fever, sneezing, anorexia, and may
seem like a usual rhinovirus infection but is not
o It is highly communicable stage that is characterized by
mucous membrane inflammation and mild coughing
with runny nose (from handout)
● Paroxysmal stage:
o It is associated with repetitive coughing, vomiting and
“whooping” or hurried, deep respiration that may last for
six weeks
o Infected individuals experience severe coughing for
atleast 15 to 25 times in 24 hours (reason is that the
epithelial cells are damaged)
● Convalescent stage:
o Stage in which the symptoms slowly decline;
diminished paroxysmal cough, and may developed
secondary complications. Since epithelial cells are
damaged, this is an opportunity for opportunistic
pathogen to get in the lower respiratory. Hence, one
secondary complication is pneumonia. Other
complications include seizure and encephalopathy
This period may last for six months after infection
LABORATORY DIAGNOSIS
● Specimens: Nasopharyngeal swab and bronchoalveolar
lavage
● A Nasopharyngeal swab is preferred for B. pertussis.
● Nasopharyngeal swabs should be inoculated directly into
the culture media or transferred into a transport medium as
the bedside.
● Calcium alginate or a Dacron swab is used for the collection
of specimens.
Gram stain
● The use of a two-minute safranin or 0.2% basic fuchsin as
counterstain enhances its visibility.
Culture
● Culture media: Regan-Lowe agar, Bordet-Gengou potato
infusion agar, modified Jones-Kendrick charcoal agar, and
Casamino acid broth
● The commonly used medium for B. pertussis is the Bordet-
Gengou potato infusion agar
o composed of potato infusion, sheep’s blood, and
cephalexin
● Regan-Lowe agar is selective agar which contains charcoal,
horse blood and cephalexin
o Favorable transport and enrichment medium
● Modified Jones-Kendrick charcoal agar contains yeast
extract and cephalexin antibiotic
● B. pertussis and B. parapertussis have hemolytic reactions
in a Bordet-Gengou potato infusion agar
● Plates are incubated for seven days at 35 degrees Celsius
without an increase CO2
● Casamino broth is used for transporting swab specimens.
● Species cannot grow on MAC except for B. bronchiseptica
because it is less fastidious.
Serologic Tests
● Greater amount of bacteria is required for an agglutination
test; thus it is sometimes necessary to perform subculture
from the primary isolation plate.
● Clinical specimens can be microscopically examined for the
presence of Bordetella species using direct fluorescent
antibody (DFA) stains.
Nucleic acid test
● The polymerase chain reaction (PCR) Assay is a rapid test
that can be used to identify Bordetella species in clinical
specimens
● The PCR assay is considered as more sensitive test than
cultures and DFA assays in identifying Bordetella species.
FRANCISELLA TULARENSIS
General Characteristics:
 Gram-negative small non-motile coccobacillus
 Processed according to biosafety level 3 condition
because they are included in risk group 3 agent.
 Infection can be acquired via inhalation
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 It is included in category A bioterrorism agent meaning
it is one of the highest concerns in bioterrorism.
 Low infective dose but high mortality
 Facultative intracellular
 Zoonotic, infection can be passed from animals to
humans
Microscopy:
 Faint bipolar staining
Cultivation:
 CAP supplemented with cysteine
 Buffered charcoal yeast extract agar with L-cysteine
 Fructose cysteine agar
 Colonies are round, smooth, bluish gray to white, and
slightly mucoid.
 Growth factors are cysteine and thiosulfate
Biochemical Tests:
 Weak positive catalase (+)
 Oxidase (-)
Serologic Test:
 An agglutination titer of 1:40 is diagnostic
Virulence Factors:
 Capsule – antiphagocytic
 Inactive (LPS) Liposaccharides – it is used to bind in
(TLR4) protein Toll-like receptor 4 protein that is found
in macrophage.
 Acid Phosphatase 4 Enzyme – inhibit the formation of
phagolysosome inside the macrophage.
Diseases:
 Rabbit fever / Tularemia
o 6 Forms of Tularemia infections
 Ulceroglandular – lesions in skin
and lymphadenopathy
 Glandular – no skin lesion but there
is lymphadenopathy
 Occuloglandular – lymphadenopathy
accompanied with eye infection
 Oropharygeal – ingestion of
contaminated food or water that
leads to the infection of throat and
mouth. It can cause sore throat and
difficulty in swallowing
 Pneumonic – directly inhalation of
the bacteria
 Typhoidal – when the bacteria
access the blood it can cause acute
sepsis and chronic febrile illness
LABORATORY DIAGNOSIS
 Specimens: scrapings from infected ulcers, lymph
nodes, and sputum (best specimen)
 Gram stain: It requires an acridine orange stain to
visualize organisms that are obtained from a blood
culture bottle
 Culture:
o Culture media: CAP supplemented with
cysteine, MTM, non-selective buffered
charcoal yeast extract agar with L-cysteine
(BCYE), MHA, and TSB
o Growth is not enhanced by incubation at an
increased carbon dioxide
o Slowly growing organisms require two to four
days for colony formation
PASTEURELLA SPP. (ZOONOTIC BACTERIA)
GENERAL CHARACTERISTICS
● Facultatively anaerobic and non-motile
● Mode of transmission: Isolated from animal bites (mainly
from cats) or scratch wounds
● Normal commensal bacteria of healthy animals such as
dogs and cats
● Grow well on BAP and CAP but most species cannot grow
on MAC
● Culture: BAP and CAP - colonies are gray and non-
hemolytic
● Virulence factor: Endotoxin and capsule
● Microscopy: Small, straight, Gram negative bacilli with a
“safety pin” appearance
● Biochemical test:
o (+) oxidase, catalase, and indole
o Weak glucose fermenter
● Species: P. multocida, P. stomatis, P. dagmatis, P. bettyae,
and P. canis
Pasteurella multocida
● Most commonly isolated species in humans
● Disease associated: Skin and soft tissue infections (e.g.
Cellulitis for general populations; Blood infection and
systemic inflammation for immunocompromised individuals)
● Commonly isolated in dog and cat bite infections
● Culture has characteristics “mushroom smell”
● Grows only BAP and susceptible to penicillin
● Biochemical test:
o (+) oxidase, OD, indole, and urease
o (-) ONPG
Pasteurella bettyae
● Isolated from amniotic fluid, blood and urogenital specimens
from humans
● Can be sexually transmitted
● Both glucose and fructose fermenter
● Can grow on MAC
● Biochemical Test:
o (+) catalase
o Variable indole
o Production of oxidase
LEGIONELLA SPP.
GENERAL CHARACTERISTICS
● Only genus in the family Legionnellaceae.
● Fastidious, aerobic, motile, and non-carbohydrate-
fermenting
● Species are primarily acquired through inhalation.
● Microscopy: Faintly staining, thin, gram negative, bacillary
or coccobacillary in form (Silver stain can be used for better
visualization)
● Culture: BCYE (Buffer Charcoal Yeast Extract Agar w/ L-
cysteine) - colonies appear sticky and exhibit a “rainbow”
color
● Biochemical Test:
o (+) catalase, gelatinase
o Weak positive in oxidase
● Major reservoirs: Hot water system, cooling towers, and
evaporative condensers
● Species: L. pneumophila, L. micdadei (Pittsburg pneumonia
agent), L. bozemanii (WIGA agent), and L. dumoffii
DISTINGUISHING CHARACTERISTICS
● Can infect and multiply within some free-living amoeba
(species of Hartmanella, Acanthamoeba and Naegleria),
ciliated protozoa (Tetrahymena) and biofilms.
● Can be isolated from lakes, rivers, hot springs and mud.
● Can be tolerated up to 3 mg/L of chlorine, and thus resist
water disinfection and treatments.
● Cannot grow on routine primary plated media like BAP
 PAGE
MEDT\* 14 CLINICAL BACTERIOLOGY (LECTURE) | LESSON TITLE
MERGEFORMA
T2
Legionella pneumophila ● Selective medium: BCYE with L-cysteine, ferric salt, and
● Most commonly isolated human pathogen in the genus alpha-ketoglutarate
Legionella ● Legionella species can only grow in media with L-cysteine.
● Etiologic agent of Legionellosis which has two forms: ● The application of acid treatment (KCL-HCL) on
Legionnaire’s Disease and Pontiac fever contaminated specimens enhances the isolation of
● Invades the bronchoalveolar macrophage, which is a Legionella species
facultative intracellular pathogen - can live either inside or ● Saline or Buffer should not be used in processing or
outside the host cell transporting the specimens because of the inhibitory effect
● Isolated in air-conditioned units, cooling towers humidifiers of sodium to Legionella.
and nebulizers
● Serogroups: 1 to 7 For early diagnosis of infection:
● Serogroups associated to the Legionnaire’s disease: 1, 4
and 6 Serologic Test
● Preferred medium: BCYE with L-cysteine that is buffered to ● Indirect fluorescent antibody (IFA) test - is the most common
pH 6.9 method used for the serologic diagnosis of Legionnaire’s
● Culture: BCYE - colonies are blue green and glistening and disease
have a convex elevation. ● Direct fluorescent antibody test - is used for detecting
common Legionella species in the lower respiratory tract. A
PRIMARY CLINICAL MANIFESTATIONS positive result exhibits yellow-or-green colored bacilli.
Legionnaire’s disease:
● Also known as Legionellosis which is febrile and pneumonic Rapid methods
illness ● DNA test (PCR)
● Direct physical contact does not spread the infection ● Urine antigen test - detects L. pneumophila as early as three
● Mode of transmission: Airborne spread or inhalation of days from infection.
infectious aerosols
● Symptoms: High fever, non-productive cough, headache,
neurological, and severe bronchopneumonia
● Diagnostic test: Antigen testing in urine and antibody titer
● Administration of antibiotics can be done - Azithromycin,
Fluoroquinolones
Bacteria are inhaled, and will enter the lower respiratory
(lungs). These will be engulfed by macrophages and shall
form phagosomes. To destroy L. pneumophila inside the
macrophage, phagosomes must be fused with lysosomes.
However, bacteria have Type IV secretory system, secreting
effector protein to avoid fusion of lysosomes and
phagosomes, in other words an interruption in lysosome-
phagosome formation. L. pneumophila stays alive inside the
macrophage and shall replicate repetitively leading to the
macrophage bursting. Because of this, bacteria shall be
released to the extracellular space and are ready to infect
other cells. On one hand, the macrophages will produce
chemotactic factors causing leukocytosis in the peripheral
blood. As a result, formation of fibrin happens in the alveoli
leading to destructive pneumonia.

Pontiac fever
● Non-fatal respiratory infection that resembles an allergic
disease, but exhibits the symptoms of pneumonia
● Milder form of infection; without pneumonia
● Flu-like symptoms
● Self - limiting; antibiotics are not necessary

Wound abscess & Encephalitis

LABORATORY DIAGNOSIS
● Preferred specimens: Sputum and bronchoalveolar lavage
● Other specimens: Urine, pleural fluid, blood, and
transbronchial lung biopsy materials
● Urine is an important specimen for antigen detection.

Staining
● Microscopy: Faint staining and usually undetectable through
Gram staining
● Prolonging the contact of smear with safranin for 10 minutes
enhances the staining of the cells
● Legionella is intracellularly and extracellularly located in the
phagocytes.
● L. micdadei is weakly acid fast when using the modified
Kinyoun method

Culture
● Culture is the most important test for the Legionella species