CHAPTER 18 Caused invasive & Caused localized

HACEK Group systemic infection infection

Haemophilus Septicemia, Septic
Blood-lovers arthritis, Otitis media,
Gram(-) pleomorphic coccobacilli or rods Meningitis, Conjunctivitis,
Oxidase(+), Catalase (+) Osteomyelitis, Sinusitis, Bacteremia,
Nonmotile Cellulitis, Pneumonia(lower
Facultative anaerobic Pneumonia, respiratory tract
Pericarditis, infection)
Ferment CHO
Epiglottis
Reduce nitrate to nitrite
Obligate parasite Haemophilus aegyptius
Fastidious=dysgenic(slow grower) Koch-weeks bacillus
Normal biota of oral cavity Contagious/Purulent conjunctivitis
SBA & MAC (-) (pink-eye) [in children]
capnophilic Nonencapsulated
Require growth factors (found in RBC) BPF (Brazilian purpuric fever)
X (unknown)factor: hemin or Severe systemic disease
hematin
V(vitamin) factor: NAD Haemophilus ducreyi
(niclotinamide Strict human pathogen
adenine Cause GUD (genital ulcer disease)
dinucleotide) Not part of normal microbiota
Chancroid
Haemophilus influenzae
Soft chancre
Pfeiffers Bacillus
Ragged ulcer, painful lesion
Capsule
Differentiated to:
Have serotype a-f
LGV: lympho granuloma
Serotype B (Hib) [capsule]
venereum
Unique polymer composed of HSV: Herpes Simplex virus
ribose, ribitol & PRP Syphilis: hard chancre
(polyribitol phosphate)
Site of infection: penis, or labia w/in
Caused the most invasive
vagina
infection w/c occurs normally
in children
Laboratory Diagnosis:
If unvaccinated, it is the
Collected by premoistened swab:
leading cause of meningitis
sterile phosphate-buffered saline
Immunoglobulin A (IgA) protease
Specimen: blood, CSF, middle-ear exudates,
Adherence by fimbrae & other joint fluids,
structures vaginal swabs, abscess
Most nonencapsulated strains drainage, upper &
are adherent to human lower respiratory tract
epithelial cells, whereas specimens
serotype b are not Culture Media:
OMP & LPS CHOC AGAR
Influenza (flu): viral disease H. H.
H. ducreyi
characterized by influenzae aegyptius
Acute inflammation of Used Supplemente Nairobi
upper airways medium bet. d w/ 1% biplate
330 C & 370C IsoVitalex or medium
Encapsulated NonEncapsulated in 5-10% CO2 Vitox for at Gonococc
Lacks adherent May be carried for 18-24 least 4days al agar
capability asymptomatically hours (GC) w/
2% bovine
 hemoglobi nonhomogene
n ,5% fetal ous
calf serum appearance in
1% saline
hemoglobi
n, 1% X & V Factor Requirement
IsoVitalex X Factor V Factor
& 3mg/L H. influenza + +
vancomyci H. + +
n aegyptius
Oxida Catala Gluco Sucro Lacto
Mueller- ONPG H. + Nitra Escul
+
se se
Hinton se se se te in
haemoliticu
X(+), V(+), agar w/ s
Porphyrin (-) + + -
5% H.+ducreyi- - + + --
H. influenzae chocolatiz
H. haemoliticus + + - + - - + -
ed horse
X(-), V(+), blood
Porphyrin (+) + V
Incubated in V + + - + -
H. parainfluenzae + + CO2 -
5-10% + + - + -
H. for at least 7
parahaemoliticus - V 330C
days - Weak Weak - + -
A.Supplemented
segnis w/ bacitracin(300mg/L) for
X(+), V(-), isolation of
Haemophilus
Porphyrin (-) from respiratory specimens
- - - - - - + -
H. ducreyi
X(-), V(-),
Porphyrin (+)
+/- - + + + + + -
H. aphrophilus
Acidometric Test
Microscopic Morphology pH indicator: Bromcresol purple
Gram-stain -lactam ring of benzylpenicillinis
H. influenzae H. ducreyi broken by -lactamase = penicilloic
Capsules are clear, Pale-stain, tiny acid
nonstaining areas gram(-) coccobacilli (+): yellow
(halo) surrounding arranged singly or (-): purple
the organism clusters.
Stains pink (serum- School of fish Hemolysis
like or proteinaceous Railroad H.
background material) tracks H.
parainfluenza
fingerprints influenzae
e
Horse/Rab
Colony Morphology: bit blood -hemolytic -hemolytic
CHOC AGAR agar
H. H.
influenza H. ducreyi parainfluenza Porphyrin Test
e e ability of the organism to convert the
Translucen Small, flat, Tannish & drier substrate delta-aminolevulinic acid
t, tannish, Smooth, w/ a medium (ALA) into porphyrins or
moist & nonmucoid, to large size porphobilinogen
convex transparent to incubation at 350C for 4 hours
Mousy or opaque porphobilinogen is detected using
beachlike colonies or KOVACS REAGENT (p-
odor appears tan or dimethylaminobenzaldehyde)
yellow porphyrins can be detected using UV
Clumpy
light (360nm, wood lamp)
 porphyrins fluoresce reddish-
orange
RESULTS:
(+) reddish-orange
Ex. H. parainfluenzae
(-) no fluoresce
Ex. H. Influenzae
Staphylococcus Streak
Haemophilus is streaked w/ S.aureus,
S.pneumoniae, Neisseria or yeast
(+): Satellitism w/ S. Aureus
Treatment:
H. influenzae
A.) life-threatening
cefotaxime or ceftriaxone
B.) nonlife-threatening
amoxicillin-clavulanate
H. ducreyi: erythromycin
Aggregatibacter aphrophilus
foam loving/ CO2lover
Most prevalent spp.
Found in dental plaque and gingival
scrapings.
Gram (-) coccobacilli
V Factor dependent
CHOC agar:
o Convex
o granular and yellow
o opaque zone near the center
(CHOC)
Aggregatibacter
actinomycetemcomitans
formerly in the genus Actinobacillus
produce small bacilli to coccoid G(-)
bacilli
divide into 6 serotypes (a f) based on
SURFACE POLYSACCHARIDES
a, b and c most common
normal oral microbiota
fastidious
capnophilic
isolated from blood causative agent
of subacute bacterial endocarditis
(SBE)
Isolated from lung tissue,
Isolated from abscesses of mouth and
brain, and sinuses
Etiologic pathogen in the devt of
destructive PERIODONTITIS
(inflammation of gingiva 2/ plaque of
bacteria)
Virulence Factors:
o Collagenase
o Leukotoxin (toxic to PMN and
Monocytes)
Fermenter (needs additional serum)
Bipolar stain morse code
appearance
24hours
Pinpoint colonies
48 hrs
star shape with 4 6 points
(100x light microscope)
Granular and may adhere to the sides
of TT (in broth)
Catalase (+) and oxidase variable
MAC(-)
Negative X and V growth factors,
urease, indole, esculin,and citrate
Glucose fermentation is positive (w/ or
w/o gas)
Isolates dont ferment Lactose/
sucrose
Susceptible to Penicillin,
AMINOGLYCOSIDES. 3RD Generation
cephalosporins, quinolones,
chloramphenicol, an tetracycline
Resistant to vancomycin and
erythromycin
Cardiobacterium hominis
pleomorphic,
Nonmotile
Fastidious
G(-) Bacillus
Normal microbiota of the nose, mouth
and throat (maybe present in GI tract)
Disease: Meningitis, SABE & oral
infection
Form rosettes, swellings, long
filaments or in yeast extract,
sticklike structures.
Grow slowly in SBA and CHOC
Dont grow on MAC
Incubation (aerobic/anaerobic)in 5%
CO2 enhances growth
 Fermenter(dysgonic) glucose, Colonize Upper Respiratory tract esp.
mannitol, sucrose Tonsils
Microscopic morphology: Dysgonic; pits agar
Pitting May grow on Nesseria selective agar
Oxidase(+) (Thayer Martin medium)
Catalase (-) 3 species:
Indole (+)
Urease (-), Nitrate(-), Gelatin(-), Esculin(-) Kingell
Kingella Kingella
a
kingae denitrificans
Eikenella corrodens oralis
Normal biota of the oral and bowel Weakly
cavities ferments
Result trauma in human bites/ fights glucose &
Opportunistic pathogen maltose but Glucose
Least common isolate sucrose (-)
fermentation (+),
Fastidious Produce yellow- nitrate reduction,
brown pigment grow on 420C
G(-) coccobacilli
2 colonies indole, urease,
Non Fermenter
Smooth, esculin, gelatine,
Capnophilic (CO2 with hemin)
convexor citrate, MAC,
Nonmotile, oxidase(+), Asaccharolytic Spreading catalase &
Catalase(-) produce yellow pigment corroding superoxol (-)
Microscopic Characteristics - -hemolytic
pit/Depression hemolytic 2 colonies
Nonhemolytic (SBA) with slight colony Smooth,
greening effect Obtained
convex
Chlorine bleachlikeodor clinically from
Spreading
Dont grow on MAC/ EMB blood, bone,
corroding
Adhere to sides of TT and produce joint fluid,
granules(Broth medium) urine,and
Lysine(+);Ornithine decarboxylase(+); wounds
Arginine dehydrolase(-)

Kingella
Coccobacillary to short bacilli with
squared ends
Occurs in pairs/short chains
Resist decolorization in gram stain
Nonmotile
Nutritionally fastidious
Oxidase (+)
Other Gram (-) Bacilli
Catalase (-)
1.)Capnocytophaga
Fermenters of glucose and other
Req. CO2 for enhanced growth
sugars (no gas)
Fastidious
 Facultative anaerobe
Gram(-) bacilli
Thin & fusiform (pointed ends),
spindle-shaped, coccoid & curved
filaments 3.)Brucella
Flagella is absent Small gram(-), aerobic, nonmotile,
Haze (Gliding motility) on solid unencapsulated bacteria w/c do not
surface form spores & may appear
Opaque, shiny; pale beige or yellow- coccobacilli or bacilli
orange Smooth, raised & translucent colony
-hemolytic except C. haemolytica Facultative intracellular pathogens
ferment sucrose, lactose, glucose & Oxidase(+), Catalase(+), Urease(+)
maltose Require CO2
indole(-) Using lead acetate=H2S(+)
reduce nitrate & hydrolyze esculin Biosafety level 3
C. ochracea most clinical isolate Category B select biological agents
Brucellosis/Undulant Fever
2.)Pasteurella Acquired through aerosol,
Gram(-) percutaneous & oral routes of
Cat scratch exposure
Animal or feline bite/animal licking Transmitted by unpasteurized
Nonmotile milk products
Facultative anaerobe coccobacilli 3 stages:
appearing ovoid, filamentous or Acute
bacilli Subchronic
Bipolar staining (safety pin chronic
appearance)
Catalase (+); Oxidase (+)
Weak glucose fermentation
Will grow on SBA & CHOC agar w/c
will producing grayish colonies.
P. multocida:
Most frequent isolate
-hemolytic (SBA), mucoid,
narrow green-brown halo
around the colony after 24-
48hours of incubation at 370C
P. bettyae
solated from placenta,
amniotic fluid, blood, rectal
sites, abscess & urogenital
specimens
fastidious, capnophilic
coccobacilli & bacilli w/c are
facultative anaerobe
ferment glucose & fructose
catalase(+), oxidase(V), indole
(+) nonmotile
 Growth in
Serum
Natural Dyes H2S (lead
Agglutina Urease CO2
Host Thioni Fuch acetate)
tion
ne sin
B. Goat or
+ V - - - -
melitensis Sheep
Cattle or +<2hou
B. abortus + + - + +/-
Cow rs
Swine or +<0.5h
B. suis + - + + -
Pig our
+<0.5h
B. canis Dog - - - - -
our
Legionella
Legionella
4.)Francisella
ubiquitous gram (-) bacilli
Small, nonmotile, non-sporeforming,
gram(-) bacilli or coccoid bacteria & acquired through inhalation
strictly aerobic multiply in free-living amoebas &
Facultative intracellular pathogen coexist w/ them in biofilms
Fastidious & require supplementation Found on bodies of water, also in air-
w/ cysteine, cystine or thiosulfate for con
growth
3.1 Virulence factors
MAC/EMB (-)
- ability to enter, surive,and multiply
Gray-white, raised colonies w/
within hosts cells esp.
smooth appearance bronchoalveolar macrophages
Negative: oxidase, urease, satellite - ability to produce proteolytic enzymes
or X & V - survive in an extracellular
Weakly positive for catalase & - environment
lactamase activity
Virulence Factor: Endotoxin 3.2. Infections
Causative agent: rabbit 1. Legionnaires disease
Category A; biosafety level 3 febrile disease w/ pneumonia
Tularemia flu-like symptoms
Rabbit fever usually produces lobar,
Deerfly fever segmental or patchy
Lemming fever pulmonary infiltration
Water rat trappers disease incubation of 2-10 days
Focal ulcer (ulceroglandular) 3 major patterns:
at site of entry of organism & Sporadic cases
enlargement or regional lymph most common and
nodes usually occur in
community
2) Epidemic outbreaks
short duration and low
attack rates
3) Nosocomial clusters
occurring in
compromised patient
population
Serogroup 1 = L.
Pneumophila
2. Pontiac fever
febrile disease w/o
pulmonary involvement
 doesnt develop & infection
does not spread beyond
lungs
incubation of 2 days
caused by L. pnemuophila
3. Pneumonia
predominant manifestation of
legionellosis.
a. S. pneumonia most common
cause of bacterial pneumonia.
4. Atypical Pneumonia
disease caused by Mycoplasma
pneumoniae, Chlamydophila
pneumoniae and Legionella spp
3.4 Epidemiology
naturally occurring in aquatic sources
(lakes,rivers,hot springs, and mud)
can tolerate chlorine conc. Up to
3mg/L
resist water treatment
Hot water systems, cooling
towers and evaporative
condensers major reservoirs
Factors that contribute to
colonizing ability to these
sources:
ability to multiply over the tempt
range of 20 degrees to 43
degrees and survive for varying
periods at 40 degrees to 60
degrees
capacity to adhere to pipes,
rubber, plastics, and sediment
and persist in piped water
systems even when flushed
ability to survive and multiply
w/in free living protozoa and in
the presence of commensal
bacteria and algae
LABORATORY DIAGNOSIS
1. Combination of Urine Antigen
Detection and Culture best
approached in diagnosis of
Legionnaires disease
2. Serology diagnosis of Pontiac fever
4.1 Specimen Collection and Handling
Specimens: sputum,
bronchoalveolar lavage, and
bronchial washings.
Respiratory secretions (acid
treatment) and body fluids (lysis
centrifugation) are submitted in
sterile, leak proof containers.
Saline and buffer should not be used
because of the inhibitory effects of
sodium.
When transport of specimens is
prolonged, specimens should be
refrigerated if more than 2 hours
pass between collection and
processing.
If processing will be delayed for
several days, 700C
For blood cultures, the Isolator
system w/c uses the lysis
centrifugation method is preferred.
Urine is an impt specimen to be
collected for antigen detection.
Specimens are collected in sterile,
leak proof containers and assayed
within 24 hours of collection
If testing is delayed, specimens should
be stored at 2 degrees to 8 degrees or
frozen at 200 C
4.2 Microscopic Examination
Legionella spp. are pleomorphic,
weakly staining, gram(-) bacilli
L. micdadei is weakly acid fast in
tissue and stains best with the
modified Kinyoun procedure.
Giemsa, Diertle
4.3 Isolation and Identificaiton
4.3.1 Isolation Methods
i. Acid treatment of specimens
Aliquot of specimen is first
diluted 1:10 w/ 0.2N KCL
HCL and allowed to stand
for 5 mins.
ii. Inoculated medium is
incubated at 35 to 37 degrees
in air for at least 7 days.(3 to
5 days), Legionella spp colonies
are visible
iii. Legionella spp are fastidious,
aerobic bacteria that will not
grow on sheep blood agar and
require L cysteine for growth
 iv. buffered charcoal
extract agar w/ L cysteine is
best for Legionella isolation
charcoal: acts as
detoxifying agent
yeast avian commensal
B. holmesii and B. Trematum
respective agents of immune
compromised bacteremia and
wound or ear infection

v. Semiselective BYCE contains GENERAL CHARACTERISTICS

polymyxin B, anisomysin, and small gram(-) bacilli or coccobacilli
either vancomycin (PAV) or obligate aerobe
cefamandole(PAC)
grows best in 35-370C
do not ferment CHO
4.3.2 Colony Morphology
BCYE: colonies appear as grayish oxidize amino acids
white or blue green, convex and catalase + (variable in B. pertussis)
glistening
central portion of young colonies has Bordetella pertussis
a ground glass appearance, Fastidous, requires special collection
light gray and granular, whereas and culture media
the periphery of the colony has pink inhibited by fatty acids, meal ions,
and/or light blue or bottle green sulfides, and peroxides
bands with a furrowed pinpoint mercury droplets
appearance colony
specimen: nasopharygeal swab
(cough)
fluorescent antibody technique more
rapid
B. holmesii, B. parapertussis, B.
trematum
oxidase negative

B. avium, B. bronchisepica, B.
Henzii
oxidase positive

BORDTELLA
Bordetella pertusis and Bordetella
parapertussis are primary human
pathogen of respiratory tract
causing whooping cough or
pertussis.

Other Species
B. bronchiseptica
non fastidious, RT pathogens of
wild, domestic birds, and
mammals. (Opportunistic
human pathogen causing RT
and wound infection.)
B. avium Virulence factors
non fastidious, RT pathogens of Filamentous hemaglutinin (FHA)
wild, domestic birds, and and peractin (69 kDa ouermembrane
mammals proein)
B. hinzii
 o Provides adherence to ciliated
epithelial cells
Pertussis toxin (PT)
o A Protein exotoxin
o Modifies host proteins by ADP
ribosyl transferase which
interferes in signal transduction
o Produe wide variety of response
in vivo
Adenylate cyclase toxin
o Inhibits host epithelial and immune
effector cells by inducing cAMP
Tracheal cytotoxin
o Causes ciliostasis (inhibits DNA
synthesis and promotes cell
death)
Clinical manifestations
Pertussis or whooping cough
( 1-3 weeks incubation usually
7-10 days)
Human disease
Infections are acquired through
respiratory droplets or direct
contact with infectious
secretions.
One of most highly
communicable diseases of
childhood
1. Catarrhal phase
(highly communicable stage)
symptoms are insidious and
non specific (sneezing, mild
cough, runny nose,
conjunctivitis. Infants can
develop apnea or
respiratory distress)
2. Paroxysmal phase
the onset of severe (repetitive
cough with whoof at end of
coughing spell)
Whooping sound is caused by
rapid gasp for air
3. Convalescent phase
begins within 4 weeks of
onset with decrease of
frequency and severity of
coughing spells.
Vaccination
Single dose vaccines, administered with
diphtheria and tetanus toxoids are use
in both adolescents and adults.
Miscellaneous Species
B. bronchiseptica is a Respiratory
Tract pathogen of number of animals
which causes kennel cough.
Specimen : Nasopharyngeal aspirates or
Swabs
(Calcium alginate or Dacron
polyester w/ a
flexible wire shaft)
Transport system
If transmit time is less than 2 hours or
DFA is the desired test:
o the swab can be expressed into a
solution of 1% casein hydrolysate
broth
o Amies transport medium with
charcoal is appropriate for up to
24 hours
Media:
Charcoal Horse Blood
Regan-lowe (1-2 days @ 350C)
Bordet Gengou potato infusion agar
Incubate @ 350C in ambient air(moist
chamber) for >7 days
Microscopic Examination
Organism appear as small, fat bacilli or
coccobacilli with intense peripheral
yellow green fluorescence & darker
centers
Clinical specimens can ONLY be examined
for Bordetella spp. microspically using DFA
staining.
Slides for DFA testing may be
prepared:
directly from the swab specimen
 or expression of swab to a solution of
1% casein hydrolysate
At least two slides should be
prepared.
Slides are dried, heat fixed & stained
on the same day of collection or
stored at 70 degrees Celcius & heat
fixed immediately before staining.

Colony Morphology
Charcoal-horse blood & Regan-
Lowe media
young colonies are smooth,
glistening, and silver (like
mercury droplets)
Colonies turn whistish gray as
they age.
Bordet Gengou agar
colonies of B. pertussis & B.
parapertussis are hemolytic

Identification Methods
On Gram stain:
appear as tiny gram negative
coccobacilli
maybe elongated if recovered from
media containing cephalexin
Necessary to increase the safranin
counterstaining time to 2 minutes
to see typical morphology.

B. pertussis B. parapertussis B. bronchiseptica

Charcoal-horse
+ (3-5 days) + (2-3 days) + (1-2 days)
blood
Blood agar - + +
MAC agar - - +
Catalase + + +
Oxidase + - +
Urease - +(24 hours) +(4 hours)
Nitrate reduction - - +
Motility - - +
Antimicrobial Susceptibility alternative for treatment or
prophylaxis
Erythromycin
drug of choice for treatment &
prophylaxis of pertussis
for eradication & preventation of
secondary cases but has clinical
efficacy ONLY if treatment is started
during the catarrhal phase
Azithromycin
fewer & milder side effects
has longer half life
requires fewer daily doses

Trimethroprim sulfamethoxazole

Zoonotic Diseases
Infected by tick
bite, direct contact
w/ infected animal
Tularemia (Rabbit fever, tissue, inhalation
Gram (-) coccobacilli
Francisella Lemming Fever,Water of aerosols or
Pinpoint colonies
tularensis rat trappers disease, ingestion of
(mercury droplet)
Deerfly fever) contaminated food
or water
High risk to lab.
Personnel (BSL-3)
Most common organism
Pasteurell
Gram (-) bacilli in human wounds
a Mousy odor
Bipolar staining inflicted by Dog & Cat
multocida bites
Characterized by
Streptobac Long filamentous gram(-) recovering fever,
bacilli w/ swellings arthritis-like
illus
(pleomorphic); Rat bite fever (Haverhill symptoms
monolifor Puffball or string of pearls Fever) (inflammation
ms or colonies on Thioglycolate pain, stiffness) &
Spirillum broth infection of the
lymphatic vessel