Chapter 18: Haemophilus and Other Fastidious Gram-Negative Bacilli Flashcards
(189 cards)
1
Q
Staining Characteristic of Haemophilus spp.
A
Gram Negative
2
Q
Appearance of Haemophilus spp. (3)
A
- Pleomorphic
- Small coccobacili from direct smears of clinical samples
- Long filaments from colony growth
3
Q
Motility of Haemophilus spp
A
Non-motile
4
Q
Mode of Respiration and Sugar for Haemophilus spp.
A
Facultative Anaerobic, Carbohydrate Fermenter
5
Q
Kind of Parasite on the mucous membranes of Humans and Animals
A
Obligate Parasites
6
Q
Kind of Parasite on the mucous membranes of Humans and Animals
A
Obligate Parasites
7
Q
8 Species of Haemophilus species associated with humans
A
Haemophilus influenzae - “Pfeiffer’s Bacillus” Haemophilus parainfluenzae Haemophilus haemolyticus Haemophilus parahaemolyticus Haemophilus paraphrohaemolyticus Haemophilus pittmaniae Haemophilus aegyptius - “Koch-Weeks Bacillus” Haemophilus ducreyi
8
Q
Three Pathogenic Species of Haemophilus
A
Haemophilus influenzae - Meningitis, epiglottitis
Haemophilus aegyptius - Pink Eye Conjunctivitis
Haemophilus ducreyi - Chancroid / Soft Chancre
9
Q
General Characteristics of Haemophilus spp.
A
- Indigenous microbiota of the healthy upper respiratory tract
- Non-encapsulated strains of Haemophilus influenzae in healthy children average 2% of the normal bacterial biota.
- Approximately 10% of normal bacterial biota in adults consists of Haemophilus spp.
- Haemophilus parainfluenzae
- Non-encapsulated Haemophilus influenzae
10
Q
Haemophilus is derived from the Greek words ____
A
“aima” and “philia”
11
Q
X Factor
A
hemin or hematin (“X for unknown”)
12
Q
V factor
A
nicotinamide adenine dinucleotide [NAD] = (“V for vitamin”)
13
Q
Haemophilus spp. with the prefix para require only ______
A
V factor for growth.
14
Q
Both X and V factors are found within red blood cells; however, only _____ is directly available.
A
X factor
15
Q
Haemophilus spp. that are V factor dependent do not grow on SBA because the red blood cells are still intact, and the sheep red blood cells contain ______
A
Enzymes (NADases) that hydrolyze V factor
16
Q
Clinical laboratories use _____ for the recovery of Haemophilus spp. from clinical specimens.
A
CHOC agar
17
Q
The lysing of the red blood cells by heat in the preparation of CHOC agar releases both _______
A
the X factor and the V factor and inactivates NADases
18
Q
- Phenomenon that helps in the recognition of Haemophilus spp. that require V factor is satellitism
- occurs when organisms, like Staphylococcus aureus, Streptococcus pneumoniae, or Neisseria spp., produces V factor as a by-product of metabolism
A
Satellitism
19
Q
Pfeiffer’s Bacillus
A
Haemophilus influenzae
20
Q
Most significant of all virulence factors in H. influenzae
A
Capsule
21
Q
Most significant of all virulence factors in H. influenzae
A
Capsule
22
Q
Capsular polysaccharide serve as a basis for serologic grouping
What are the 6 serotypes?
A
A, B, C, D, E, and F
23
Q
Serotype b capsule is a unique polymer composed of ______
A
ribose, ribitol, and phosphate (polyribitol phosphate)
24
Q
___________ of the type b capsule are important factors in virulence
A
antiphagocytic property and anticomplementary activity
24
___________ of the type b capsule are important factors in virulence
antiphagocytic property and anticomplementary activity
25
Most invasive infections were caused by encapsulated strains of H. influenzae belonging to _______
Serotype b (Hib)
26
In unvaccinated children, type b is a leading cause of ______
Meningitis
27
Not all strains of H. influenzae are encapsulated, referred to as _________
Non-typable H. influenzae (NTHi)
28
Haemophilus influenzae is the only member of the genus that produces ________
This enzyme has the ability to cleave secretory IgA
Immunoglobulin A Proteases
29
Adherence mechanisms of H. influenzae
Studies indicate that most NTHi strains are adherent to human epithelial cells; most encapsulated serotype b strains are not adherent
30
Antibody directed against these antigens may play a significant role in human immunity
Have a paralyzing effect on the sweeping motion of ciliated respiratory epithelium
Outer Membrane Proteins & Lipopolysaccharides
31
Infections by encapsulated, serotype B strains (Hib) : systemic
```
Septicemia
Meningitis
Arthritis
Epiglottitis
Tracheitis
Pneumonia
```
32
Infections by non-encapsulated, non-typable Haemophilus influenzae (NTHi) : localized
Conjunctivitis
Sinusitis
Otitis media with effusion / middle ear infections
33
* Widespread before the use of Hib vaccine
* were common among children between the ages of 3 months and 6 years
* Bloodstream invasion and bacteremic spread follow colonization of this organism in the respiratory mucous membranes
Meningitis
34
Symptoms of Meningitis
mild respiratory disease
headache
stiff neck
other meningeal signs
35
Occurs in children between 2 and 4 years old
Rapid onset, acute inflammation, and intense edema of the epiglottis
complete airway obstruction
Epiglottitis
36
* life-threatening disease in young children
* arise after an acute, viral respiratory infection; mild to moderate illness for approximately 2 to 7 days progresses rapidly
* thick secretions can occlude the trachea
Bacterial Tracheitis
37
Koch-Weeks Bacillus
Haemophilus aegyptius
38
Disease associated with Haemophilus aegyptius
“Pink Eye” = Acute Purulent Conjunctivitis
39
Historical Background of Haemophilus aegyptius
observed in conjunctivitis exudates from Egyptians by Koch in 1883
40
* Non-encapsulated
* caused a severe systemic disease known as Brazilian Purpuric Fever (BPF) in Brazil in 1984
* Disease associated :
- conjunctivitis, primarily in pediatric populations
- Brazilian Purpuric Fever
Haemophilus influenzae biogroup aegyptius
41
Characterized by:
* recurrent or concurrent conjunctivitis
* High fever
* Vomiting
* Petechial / purpural rash
* Septicemia, shock, and vascular collapse
* Mortality Rate : 70% within 48 hours after onset
Brazilian Purpuric Fever (BPF)
42
Strict human pathogen
Not part of the normal microbiota
* Extremely fastidious
* Disease associated : Chancroid / “Soft Chancre”
highly communicable sexually transmitted genital ulcer disease (GUD)
facilitates the transmission of other STDs
Incubation Period : 4 – 14 days
Haemophilus ducreyi
43
Clinical Manifestations of Chancroid / “Soft Chancre”:
1. nonindurated, painful lesion with irregular edge
2. generally on the genitalia or perianal areas
3. Buboes = suppurative (pus-forming), enlarged, draining, inguinal lymph nodes
4. Men have symptoms related to the inguinal tenderness and genital lesions
5. Most women are asymptomatic
44
1. found in the oral cavity
2. very low incidence of pathogenicity
Disease associated : endocarditis
3. Primary Site of Infection : mitral valve
symptoms appear approximately 1 month after routine dental procedures
Haemophilus parainfluenzae
45
* indigenous microbiota of the URT of adults
| * Disease associated : pharyngitis
Haemophilus parahaemolyticus
46
Specimen Processing & Isolation
sources of specimen :
* Blood
* CSF (for suspected meningitis)
* middle ear exudate (for suspected otitis media)
* joint fluids
* upper and lower respiratory tract specimens; bronchial washing (for culture)
* swabs from conjunctiva (for suspected conjunctivitis)
* vaginal swabs
* abscess drainage
47
_________, nasal and nasopharyngeal swab specimens have no clinical value in evaluation for respiratory tract infections caused by Haemophilus influenzae
Except in the case of patients with cystic fibrosis
48
Haemophilus spp. are ______ in nature
Fastidious
49
Most conventional media ___________ the growth of Haemophilus spp.
Do not support
49
Haemophilus spp. do not grow on
MAC agar
50
Haemophilus spp. die __________
rapidly in clinical specimens; prompt transportation and processing are vital for their isolation
51
* genital sites first should be cleaned with sterile gauze moistened with sterile saline
* a swab, premoistened with sterile phosphate-buffered saline, should be used to collect material __________
from the base of the ulcer
51
* genital sites first should be cleaned with sterile gauze moistened with sterile saline
* a swab, premoistened with sterile phosphate-buffered saline, should be used to collect material __________
from the base of the ulcer
52
as an alternative, pus can be aspirated from _______
buboes if they are present
53
___________ is preferred instead of using transport media / specimen processing in the laboratory should occur soon after collection for maximum recovery
direct plating on selective media at the bedside
54
Haemophilus ducreyi Preferred culture media:
Enriched CHOC medium, Nairobi biplate
55
Haemophilus ducreyi Optimum growth temperature
33°C
56
Haemophilus ducreyi Duration of incubation
7 days
57
Nairobi biplate medium components
GC agar base
2% bovine hemoglobin + 5% fetal calf serum + Mueller Hinton agar on one side
5% chocolatized horse blood on the other
3 mg/L of vancomycin on both sides
58
Colony Morpholoy for H. ducreyi on CHOC agar
```
Small
Flat
Smooth
Nonmucoid
transparent to opaque
tan or yellow
Individual colonies can be pushed intact using a loop across the agar plate surface
```
59
Haemophilus influenzae preferred culture media:
CHOC Agar, or Horse Blood Bacitracin Agar
60
H. influenzae Incubation Temperature
33° - 37°C
61
H. influenzae duration of incubation
18 - 24 hrs
62
H. influenzae incubation condition :
CAPNOPHILIC = 5% - 10% carbon dioxide
63
H. influenzae incubation condition :
CAPNOPHILIC = 5% - 10% carbon dioxide
64
CHOC agar supplemented with ______ is an excellent medium for the isolation of Haemophilus spp. from respiratory specimens
bacitracin (300 mg/L)
65
H. aegyptius Preferred Culture Media
enriched CHOC agar supplemented with 1% Iso-VitaleX
66
H. aegyptius Duration of Incubation
4 days
67
Colony Morphology of H. influenzae on CHOC agar
NOTE : the same colony morphology may be observed from Haemophilus influenzae biogroup aegyptius
```
Non-hemolytic
Translucent
Tannish
Moist
Smooth
Convex
“mousy” or bleach-like odor
```
68
For Haemophilus influenzae, encapsulated strains grow ____________
larger and more mucoid than the NTHi strains
69
Haemophilus parainfluenzae Colony Morphology
tannish and drier
| medium to large size compared with H. influenzae
70
Haemophilus parahaemolyticus Colony Morphology
resembles H. parainfluenzae
| on horse or rabbit blood agar, it is β-hemolytic
71
Microscopic Morphology of Haemophilus spp.
* small, gram-negative bacilli
* pleomorphic : coccobacilli to long filaments
* “halos” = capsules of Haemophilus influenzae may be observed in Gram-stained direct smears as clear, non-staining areas
* other stains that may be used :
Acridine orange
Methylene blue
72
Microscopic Morphology of H. ducreyi
“railroad tracks” / “school of fish” = Haemophilus ducreyi appearing as pale staining gram-negative coccobacilli arranged singly or in groups
73
traditional approach : using impregnated strips
X & V Factor Requirement
74
The Haemophilus Quad Plate contains four zones
media with X factor only
with V factor only
with X and V factors
with X and V factors with horse red blood cells
75
X & V Factor Requirement: H. influenzae
X, V
76
X & V Factor Requirement: H. aegyptius
X, V
77
X & V Factor Requirement: H. influenzae biogroup aegyptius
X, V
78
X & V Factor Requirement: H. ducreyi
X only
79
X & V Factor Requirement: H. parahaemolyticus
V only
80
X & V Factor Requirement: H. parainfluenzae
V only
81
X & V Factor Requirement: H. haemolyticus
X, V
82
* method for differentiating the heme-producing species of Haemophilus
* can be performed in agar, in broth, or on a disk
Porphyrin Test
83
Principle of Porphyrin Test
ability of the organism to convert the substrate δ-aminolevulinic acid (ALA) into porphyrins or porphobilinogen (intermediates in synthesis of X factor)
incubation temperature : 35°C
duration of incubation : 4 hrs
porphobilinogen is detected by the addition of p-dimethylaminobenzaldehyde (Kovacs’ reagent)
red color forms if porphobilinogen is present
porphyrins can be detected using an ultraviolet light with a wavelength of about 360 nm (Wood’s lamp). Porphyrins fluoresce reddish orange under ultraviolet light.
84
Species that are porphyrin negative __________
cannot synthesize heme and are X factor-positive (require hemin) when the impregnated strip is used
85
Haemophilus spp. that can synthesize heme ________
are porphyrin-positive (X strip negative, do not require hemin)
86
Haemophilus influenzae Drugs of choice
cefotaxime
| ceftriaxone
87
Haemophilus influenzae Alternative Drugs
```
trimethoprim-sulfamethoxazole
imipenem
ciprofloxacin
chloramphenicol
ampicillin
```
88
For non–life-threatening H. influenzae infection :
amoxicillin clavulanate
oral second-generation
third-generation cephalosporin
trimethoprim-sulfamethoxazole
89
Treatment : Haemophilus ducreyi
azithromycin
ceftriaxone
ciprofloxacin
erythromycin
90
Greek aphros and philia : “foam loving
does not require, but grow better in high concentrations of CO2
most prevalent species in the HACEK group involved in endocarditis
found in dental plaque and gingival scrapings
Aggregatibacter aphrophilus
91
__________ have been reclassified into the single species Aggregatibacter aphrophilus
H. aphrophilus and H. paraphrophilus
92
Clinical Manifestations Aggregatibacter aphrophilus
fever
heart murmur, congestive heart failure
embolism
93
Colony Morphology Aggregatibacter aphrophilus
```
convex
granular
yellow
opaque zone near the center
can either be X factor-dependent or independent
```
94
formerly in the genus Actinobacillus
normal oral microbiota in humans
General Characteristics :
small bacilli to coccoid
gram-negative
nonmotile
Aggregatibacter actinomycetemcomitans
95
Disease associated :
periodontitis
subacute bacterial endocarditis
divided into six serotypes (a through f) based on a surface polysaccharides; most common serotypes : a, b, c
Aggregatibacter actinomycetemcomitans
96
grow better with increased CO2 concentration
duration of incubation : 24 – 48 hrs
colony morphology : “star shape with four to six points”
Collagenase as a virulence factor
Aggregatibacter actinomycetemcomitans
97
toxic to polymorphonuclear cells & monocytes
Collagenase
98
A HACEK microbe that can be isolated from
```
blood
lung tissue
abscesses of the mouth
brain
sinuses
```
Aggregatibacter actinomycetemcomitans
99
```
General characteristics :
gram-negative bacillus
pleomorphic
nonmotile
fastidious
```
Cardiobacterium hominis
100
```
normal microbiota of the :
nose
mouth
throat
gastrointestinal tract
```
Cardiobacterium hominis
101
Disease associated :
endocarditis, primary site of infection : aortic valve
meningitis
Cardiobacterium hominis
102
* grow slowly on SBA and CHOC; do not grow at all on MAC
* Capnophilic = requires 5% CO2
* Colony morphology :
“pitting” may be produced on the agar
* Microscopic morphology :
form rosettes
swellings
long filaments or sticklike structures in yeast extract
Cardiobacterium hominis
102
* grow slowly on SBA and CHOC; do not grow at all on MAC
* Capnophilic = requires 5% CO2
* Colony morphology :
“pitting” may be produced on the agar
* Microscopic morphology :
form rosettes
swellings
long filaments or sticklike structures in yeast extract
Cardiobacterium hominis
103
```
General Characteristics :
fastidious
gram-negative
coccobacilli
grow best with increased CO2 and hemin
nonmotile
asaccharolytic
```
Eikenella corrodens
104
```
General Characteristics :
fastidious
gram-negative
coccobacilli
grow best with increased CO2 and hemin
nonmotile
asaccharolytic
```
Eikenella corrodens
105
_______ of the isolates of E. corrodens “pit” (make a depression) or corrode the surface of the agar
45%
106
normal biota of the oral and bowel cavities
poor dental hygiene or oral surgery has also been associated with infections
Disease associated :
infection from human bites
“Clenched-fist Wounds”
endocarditis
Eikenella corrodens
107
```
for immunocompromised individuals :
periodontitis
meningitis
empyema
pneumonia
osteomyelitis
arthritis
postoperative tissue infections
```
for drug abusers :
cellulitis
Eikenella corrodens
108
colonize the upper respiratory tract, especially the tonsils
viral infections can be a precursor to infections by these organisms
poor dental hygiene or oral surgery is associated with infection.
Kingella spp.
109
can grow on Neisseria selective agar (e.g., modified Thayer-Martin medium) and can resemble Neisseria gonorrhoeae
Kingella spp.
110
Four species of Kingella spp.
Kingella kingae
Kingella denitrificans
Kingella oralis
Kingella potus
111
bacteremia, abscesses of Kingella spp
K. denitrificans
112
predilection for bones and joints, endocarditis of Kingella spp
K. kingae
113
Microscopic morphology Kingella spp.
resist decolorization in the Gram stain
coccobacillary to short bacilli
squared ends
in pairs or short chains
114
family : Flavobacteriaceae
General characteristics :
fastidious, facultatively anaerobic, gram-negative bacilli
require increased CO2 for growth & isolation from blood cultures
Disease associated :
septicemia in patients with neutropenia
endocarditis (rarely)
Capnocytophaga spp.
115
5 species are part of normal microbiota of the oral cavity of humans (Capnocytophaga spp.)
```
C. ochracea
C. gingivalis
C. sputigena
C. haemolyticus
C. granulosa
```
116
Capnocytophaga spp. Colony Morphology
Although flagella are usually absent, gliding motility can be seen on solid surfaces.
adherent and produce a yellow-orange pigment
resemble colonies of Eikenella corrodens
most are nonhemolytic except for Capnocytophaga haemolytica (β-hemolytic)
117
Microscopic morphology Capnocytophaga spp.
thin and often fusiform (pointed ends) resembling Fusobacterium
coccoid, and curved filaments may be also seen
118
General Characteristics of Pasteurella spp.
normal flora of respiratory tract and oral cavity of birds and mammals
fastidious
gram-negative
nonmotile
facultative anaerobic
coccobacilli that appear ovoid, filamentous, or as bacilli
Bipolar staining / “safety pin” appearance = when the poles of the cells are more intensely stained is frequently observed
119
Most common isolate in Pasteurella spp.
Pasteurella multocida
120
5 serogroups of Pasteurella spp
A, B, D, E, and F
121
3 Subspecies of Pasteurella spp.
multocida
septica
gallicida
122
4 other clinical species of Pasteurella
Pasteurella canis
Pasteurella stomatis
Pasteurella dagmatis
Pasteurella bettyae
123
```
Disease associated :
* Pasteurellosis = zoonotic disease; acquired from exposure to infected animals or products made from infected animals
* most common : cutaneous infection
* systemic infections :
Septicemia
Arthritis
Endocarditis
Osteomyelitis
Meningitis
```
Pasteurella spp.
124
Biochemical Test Results Pasteurella spp.
```
catalase positive
oxidase positive
indole positive
glucose fermenter
fructose fermenter
weak to moderate acid production
non-gas producer
```
125
Colony morphology Pasteurella spp.:
| on CHOC & SBA
grayish colonies
126
Pasteurella spp. on SBA only
optimum temperature : 37°C
mucoid after 24 hrs
production of a narrow green to brown halo around the colony after 48 hrs
127
“Bang’s Bacillus”
Brucella
128
considered potential bioterrorism agent / category B select biological agents by the CDC
normal flora of respiratory tract and oral cavity of birds and mammals
Brucella spp.
129
populations at risk :
people handling animals & animal products
laboratory workers
Disease associated :
zoonotic disease that is found throughout the world
Undulant Fever / “Malta Fever”
Brucella spp.
130
Brucella spp. Modes of Transmission
aerosol
percutaneous
oral routes
131
Rare documented modes of transmission (Brucella spp.)
sexual contact
| breast feeding
132
three clinical stages of Brucella spp:
acute
subchronic
chronic
133
Identify the Disease Phase:
```
1 – 4 weeks after exposure
fever
malaise
headache
anorexia
myalgia
```
Acute Phase of Brucella spp
134
Identify the Disease Phase:
within a year
undulating fevers = normal temperatures in the morning followed by high temperatures in the afternoon / evening
arthritis
epididymoorchitis
Subchronic / Undulant Form of Brucella spp
135
Identify the Disease Phase:
1 year after exposure
depression
arthritis
chronic fatigue syndrome
Chronic Phase of Brucella spp
136
four species associated with humans (Brucella spp.)
Brucella melitensis
Brucella abortus
Brucella suis
Brucella canis
137
two additional species isolated from marine animals (Brucella spp)
Brucella ovis
| Brucella neotomae
138
General characteristics of Brucella spp:
```
small
gram-negative
aerobic
nonmotile
unencapsulated
non-spore former
appear as coccobacilli or bacilli
facultative intracellular pathogens; can reside within phagocytic cells
must be handled under biosafety level 3 conditions
```
139
Sources of specimen of Brucella spp
blood
| bone marrow
140
Appropriate culture media for Brucella spp
SBA
CHOC
Modified Thayer-Martin
Martin-Lewis
141
duration of incubation for Brucella:
18 hrs
142
plates should be kept for _______ before reporting as negative (Brucella spp)
4 days
143
Biochemical Test Results for Brucella spp:
oxidase positive
catalase positive
urease positive
144
Three subspecies of Francisella tularensis
subsp. tularensis (type A)
subsp. holarctica (type B)
subsp. mediasiatica
145
Disease associated with Francisella tularensis
Tularemia
zoonotic disease
rabbit fever
deerfly fever / lemming fever / water rat trappers’ disease
146
modes of transmission for Francisella tularensis
```
ingestion
inhalation
arthropod bite
contact with infected tissues
person-to-person
```
147
most common form of tularemia
Ulceroglandular
ulcer forms at the site of inoculation
enlargement of the regional lymph nodes
148
other forms of tularemia
```
pulmonary
glandular
oropharyngeal
oculoglandular
typhoidal forms
```
149
Francisella tularensis is a CDC ________
category A select biological agent
150
General Characteristics Francisella tularensis
```
small
nonmotile
non–sporeforming
gram-negative
bacilli or coccoid
strict aerobes
fastidious; requires the following supplements :
cysteine
cystine
thiosulfate
```
151
appropriate culture media Francisella tularensis
```
CHOC
Modified Thayer-Martin
Buffered Charcoal Yeast Extract (BCYE)
Mueller-Hinton
Tryptic Soy Broth (TSB)
```
NOTE : MAC and EMB agars do not support Francisella tularensis growth
152
Francisella tularensis Colony Conditions and Colony Morphology
optimum temperature : 37°C
duration of incubation : 48 hrs
Plates should be checked daily for 14 days.
Colony Morphology :
gray-white
smooth
raised
153
Virulence factor of Legionella pneumophila
ability to enter, survive, and multiply within the host’s cells, especially bronchoalveolar macrophages
production of proteolytic enzymes
154
Disease associated with Legionella pneumophila
Legionnaires’ Disease = febrile disease + pneumonia
| Pontiac Fever = febrile disease only, no pulmonary involvement
155
Legionnaires’ Disease / Legionellosis
```
incubation period : 2 – 10 days
predominant manifestation : pneumonia
“atypical pneumonia”
most cases are brought by serogroup 1
also implicated in human infections :
Legionella pneumophila serogroups 4 and 6
Legionella longbeachae
Legionella micdadei
```
156
Clinical Manifestations Legionella pneumophila:
```
nonproductive cough
fever
headache
myalgia
pulmonary infiltrates develop
bloody sputum
dissemination via the circulatory system = extrapulmonary infections
```
157
Clinical Manifestations Legionella pneumophila:
```
nonproductive cough
fever
headache
myalgia
pulmonary infiltrates develop
bloody sputum
dissemination via the circulatory system = extrapulmonary infections
```
158
incubation period : 2 days
flu-like symptoms : fever, headache, myalgia
last 2 to 5 days
subside without medical intervention
Pontiac Fever
159
factors that allow Legionella spp. to colonize water supplies:
can tolerate chlorine concentrations of 3 mg/L
can multiply over the temperature range of 20° C to 43° C
can survive for varying periods at 40° C to 60° C
can adhere to pipes, rubber, plastics, and sediment
persists in piped water systems even when flushed
ability to survive and multiply within free-living protozoa and in the presence of commensal bacteria and algae
160
Specimen Collection & Handling for Legionella
```
sources of specimen :
sputum
bronchoalveolar lavage
bronchial washings
environmental sources
```
When delays of more than 2 hours between collection and processing are unavoidable, specimens should be refrigerated.
if delays take several days : freeze @ −70° C
161
Microscopic Examination of Legionella
pleomorphic, weakly staining, gram-negative bacilli
162
Isolation & Identification of Legionella
Acid treatment must first be done on specimens contaminated by other bacteria = enhances isolation of Legionella spp.
aliquot of the specimen is first diluted 1 : 10 with 0.2 N KCl-HCl
allowed to stand for 5 minutes
proceed with inoculation to culture media
optimum incubation temperature : 35° C to 37° C
duration of incubation : at least 7 days;
colonies usually visible in 3 - 5 days
fastidious, aerobic, do not grow on SBA
require L-cysteine; most preferred culture media : Buffered Charcoal Yeast Extract (BCYE) with L-cycteine
163
Colony Morphology of Legionella
```
grayish white or blue-green
convex
glistening
approx. 2 to 4 mm in diameter
“ground-glass” appearance
```
164
Identification Methods for Legionella
L-cysteine requirement
Gram stain
Direct Fluorescence Antibody (DFA)
165
two species implicated in human infections in Bordetella spp
Bordetella pertussis
| Bordetella parapertussis
166
six other species of Bordetella spp
Bordetella bronchiseptica
opportunistic pathogen
Bordetella avium
wild & domesticated animal pathogen
Bordetella hinzii
avian commensal
Bordetella holmesii immunocompromised bacteremia
Bordetella petrii
Bordetella trematum
wound & ear infections
167
General Characteristics of Bordetella spp.
small gram-negative bacilli or coccobacilli
obligate aerobic bacteria
optimum growth temperature : 35° C to 37° C
non-carbohydrate fermenter
oxidize amino acids
catalase producer (variable for Bordetella pertussis)
relatively inactive in biochemical test systems
168
inhibited by fatty acids, metal ions, sulfides, and peroxides, constituents found in many media
require protective substances; charcoal, blood, starch
Bordetella pertussis
169
Virulence Factors of Bordetella pertussis
Filamentous hemagglutinin (FHA) and pertactin
Pertussis toxin (PT)
Adenylate cyclase toxin
Tracheal cytotoxin
170
facilitate attachment to ciliated epithelial cells
Filamentous hemagglutinin (FHA) and pertactin
171
protein exotoxin; modifies host proteins by adenosine diphosphate–ribosyl transferase
Pertussis toxin (PT)
172
inhibits host epithelial & immune effector cells by inducing supraphysiologic concentrations of cyclic adenosine monophosphate
Adenylate cyclase toxin
173
causing ciliostasis, inhibiting DNA synthesis, and promoting cell death
Tracheal cytotoxin
174
incubation period : 7 – 10 days or 1 – 3 weeks
| three phases : catarrhal, paroxysmal, convalescent
Pertussis / “Whooping Cough”
175
sneezing, mild cough, runny nose, conjunctivitis; apnea & respiratory distress for infants; highly infectious stage
Catarrhal
176
severe, repetitive coughing + characteristic “whoop” sound at the end due to rapid gasping for air. Young children might experience apnea, pneumonia, or both and require aid in maintaining a patent airway.
Paroxysmal
177
within 4 weeks of onset with a decrease in frequency and severity of the coughing spells
Convalescent
178
contain the structural gene for PT but do not express the complete operon.
generally causes a similar disease as pertussis with milder symptoms.
Bordetella parapertussis
179
Specimen Collection & Handling for Bordetella
sources of specimen : nasopharyngeal aspirate / swabs
acceptable swab material : calcium alginate or Dacron polyester + flexible wire shaft
two swabs are collected, one through each of the external nares; the swabs should be inserted as far back as possible into the nasopharynx, rotated, held a few seconds, and then gently withdrawn
180
Methods for Bordetella
culture
DFA
PCR
181
Staining Procedure for Bordetella
two-minute safranin or 0.2% basic fuchsin as counterstain enhances visibility
DFA staining may also be used:
should be used only along with culture because the lack of sensitivity diminishes the clinical utility of a negative result, and false-positive results can occur
DFA should not be used as a replacement for culture
182
preferred culture media for Bordetella spp
Bordet-Gengou Potato Infusion Agar with glycerol and horse or sheep blood
Regan-Lowe selective agar with charcoal agar supplemented with 10% horse blood and 40 mg/L cephalexin
183
optimum incubation temperature for Bordetella spp
35° C
184
duration of incubation for Bordetella spp
7 days; B. pertussis colonies are detected in 3 – 5 days; B. parapertussis colonies are detected in 1 day or sooner
185
Colony Morphology of Bordetella spp.
small & shiny
| “mercury droplets”
