Test 2: Anaerobes Flashcards
1
Q
List four obligate anaerobes and what percent oxygen
A
Prevotella sp.
Fusobacterium sp.
Bacteroides spp.
Clostridium perfringens
-less than 0.5% oxygen
2
Q
What is the normal habitats of anaerobes?
A
Mucosal surfaces, bowel, skin
3
Q
Where are anaerobes part of the normal microbiota?
A
Feces, skin, vagina, mouth, upper respiratory.
4
Q
Anaerobes outnumber aerobes ______ to 1 in feces,
____ to 1 in in skin, mouth and vagina.
A
1000
10
5
Q
Indigenous microflora of the colon:
Majority of bacteria are ___________ with a small amount of facultative organisms.
A
obligate anaerobes
6
Q
Clues to anaerobic infections?
A
*A foul odor emanating from the specimen
*A lesion close to a mucosal surface
*The presence of an underlying disease:
tissue necrosis, impaired blood supply
*Previous antibiotic therapy
*Infection following a bite wound
*Sulfur granules exudating from the lesion
7
Q
Vincent’s angina is now called…
A
Anaerobic tonsillitis
8
Q
Types of infections of the head, throat, mouth, and repiratory?
A
-Anaerobic tonsillitis
-Ludwig’s angina
-Otitis media and sinusitis
-Aspiration pneumonia
-Cervico-facial actinomyces
9
Q
What bacteria causes acne?
A
Propionibacterium acnes
10
Q
Ture or false,
anaerobic infections are common in bite wounds.
A
True
11
Q
Anaerobic infections are commonly seen following…
A
trauma or surgery
12
Q
Two soft tissue infections…
A
-cellulitis
-gas gangrene
other infections include food poisoning, tetanus, Pseudomembranous colitis
13
Q
What specimens are used?
A
-Body fluids (not urine)
-Deep abscess exudates
-Transtracheal
-specimens/lung aspirates
-Tissue biopsies
-CSF (only in shunt infections)
14
Q
What are inappropriate specimens?
A
-Sputum, bronchial washings or BAL, gingival
-Gastric, feces, rectal
-Surface material
-Urine
-Vaginal
15
Q
Anaerobic culture media…
A
-Primary: CDC anaerobic agar, Brucella agar
-Thioglycollate broth
-Chopped meat broth
16
Q
Selective media
A
-PEA: Phenylethyl Alcohol
-LHV: Laked Kanamycin Vacomycin
-BBE: bacteroides Bile Esculin
17
Q
Selective media that Inhibits enteric GNR and swarming by some Clostridia
A
PEA: Phenylethyl Alcohol
18
Q
Selective media that selects for Prevotella and Bacteroides
A
LKV: Laked Kanamycin Vancomycin
19
Q
What is PRAS media?
A
Pre-reduced Anaerobically Sterilized media
-reduces oxygen presence
20
Q
Anaerobic incubation at ______ temp.
Incubate _____hours before initial check of plates.
A
35-37
48
21
Q
Anaerobic incubation finalize culture at _____ days.
A
5-7
Exception—joints may be held for 14 days, for Propionibacterium
22
Q
anaerobic systems:
Pouch, jars and boxes use CO2 or CO2and _________ generators
A
Hydrogen
23
Q
Anoxomat II:
Evacuation method- vacuum removes air and then replaces with the following 3 gases…
A
-80% to 90% nitrogen
-5% to 10% hydrogen
-5% to 10% CO2
24
Q
Anaerobic Glove Box:
5% CO2, 10% hydrogen, and 85% nitrogen, plus a __________ catalyst (reacts with water to produce hydrogen and carbon dioxide), maintain the anaerobic environment inside the chamber.
A
palladium
25
When subculturing for aerotolerance, what agar is used?
Chocolate agar in CO2
26
After incubation, what is examined?
-Colony morphology
-Hemolysis
-Pigments/fluoresce
27
Aerotolerance test?
?
28
What is Ludwig's angina?
infection of oral cavity
29
Chopped meat broth:
most use ______ as source of energy.
protein
30
What plate can provide anaerobic conditions?
chocolate agar
31
What percentage of oxygen is in the candle jar method?
10%
32
Identification tests?
1. Catalase
2. Indole (p-dimethylaminocinnamaldehyde – DMCA)
3. Special Potency Antibiotic Disks
33
What are the special potency antibiotic disks? And what is SPS used for
-Kanamycin
-Vancomycin
-Colistin
-SPS (for GPC only)
34
Esculin hydrolysis is used to differentiate...
Bacteroides sp. (causes abscess)
-Dark brown or black complex
35
What is in Bacteroides Bile Esculin Agar (BBE) and what is it used for?
-gentamicin and oxgall-inhibit facultative anaerobes and most GN anaerobes
36
What is a positive result when using Bile Esculin Agar (BBE)?
dark brown or black complex
37
What type of media is Egg Yolk Agar?
Nonselective and differential
38
What is in Egg Yolk Agar?
-Lecithinase (Nagler test)
-Lipase
-Proteolytic activity
39
Egg Yolk Agar:
Degradation of lecithin =
opaque precipitate surrounding area of growth
40
Egg Yolk Agar:
Lipase positive...
iridescent sheen on colony surface (breakdown of free fats)
41
Egg Yolk Agar:
Proteolytic activity=
clearing around colony (proteins)
42
What agar is used for differentiation of Clostridium sp.?
Egg Yolk Agar
43
What is the reverse CAMP test used to differentiate?
Clostridium perfringens from other Clostridium sp.
44
The alpha toxin produced by Clostridium perfringens interacts with CAMP factor from ____________ and produce synergistic hemolysis.
Streptococcus agalactiae
45
What indicates a positive reaction for the reverse CAMP test?
hemolysis
46
What SPS susceptibility test used for?
For P. anaerobius (susceptible)
Sodium Polyanetholsulfonate disk
47
What are commercial ID kits usually used for?
Blood and cultures and other sites
48
An identification system used by many hospitals. It uses spectrophotometry.
MALDI-TOF
49
EXAMINATION OF ANAEROBIC
CULTURES
always use what
Chocolate agar in CO2
50
Anaerobic glove box
5% CO2, 10% hydrogen, and
85% nitrogen, plus a palladium
catalyst (reacts with water to
produce hydrogen and carbon
dioxide), maintain the anaerobic
environment inside the chamber.
51
Anoxomat 2
Evacuation method- vacuum removes air and then
replaces with the following 3 gases -80% to 90%
nitrogen, 5% to 10% hydrogen, and 5% to 10% CO2
52
Anaerobic systems
Pouch, jars and boxes use CO2 or
CO2and hydrogen generators
53
Anaerobic incubation
*Incubated at 35-37°C
*Incubate 48 hours before initial check of
plates
*Finalize culture 5-7 days
*Exception—joints may be held for 14
days, for Propionibacterium
54
Candle jar
Flame uses oxygen.
Increased CO2 extinguishes flame
55
PRAS media
Pre-reduced anaerobically sterilized media
56
Identification systemss are
commercial ID kits – usually used for blood
cultures and other sites
57
Gas liquid chromatography
* Separates components
* All use mobile phase & stationary (separating )
phase
* Anaerobes:
– Short chain volatile end-products or
– Fatty acid cellular components
Gas: mobile phase, Liquid: stationary phase
Sample: volatized into column
Acids are recovered in specific order based on
chemical/physical properties
Compared to standard
58
Steps for anaerobic infection
1. Gram stain
2. Aerotolerance test
3. Decide which test to set up
for identification
59
ID pathway for Gram - rod
* Indole
* Kanamycin, Colistin,
Vancomycin disks
* Bile esculin
* LK agar, optional
60
ID pathways for gram + rod
Large
*EYA
*Reverse CAMP test,
optional
Small:
*Catalase
*Indole
61
ID pathway for gram positive cocci
* disk
* Vancomycin disk
* Colistin disk
* Indole
62
ID testing for gram-negative cocci
No further testing
63
Anaerobic gram postive cocci
* Peptostreptococcus
* Peptococcus
* Finegoldia
* Staphylococcus saccharolyticus
* Gallicola
* Parvimonas
* Peptoniphilus
* Murdochiella
* Anaerococcus
*** 9 species and some not studied
64
Anerobic gram positve cocci
normal microbiota
* Oral cavity, upper respiratory tract, GI tract,
female genitourinary tract and skin
65
Anerobic gram positve cocci
Clinical manifestations
* A great variety of infections including abscesses,
gangrene, cellulitis, bacteremia, pneumonia,
peritonitis, bite wounds, and pelvic inflammatory
disease.
* Brain abscesses: 40% mortality
66
Gram +anaerobes cocci
Difficult to speciate
P. anaerobius: susceptible to sodium polyanethol
sulfonate (SPS)
P. asaccharolytic: indole positive
P. micros: very small cocci
Peptococcus niger: black colony
Finegoldia magna: large cocci; look like
staphylococci
67
Peptostreptococcus
Anaerobic GPC can form tetrads, chains, clusters, pairs
or clumps and can vary in size of the cocci
68
Gram - cocci
Vellonella
* Veillonella sp. (historically the only one in
human infections)
– Part of the normal microbiota-oral cavity,
genitourinary, respiratory, and GI tract
– Usually in mixed infections; rarely significant
– Biochemically inert
69
Gram negaive cocci
other less common genera
– Megasphaera
– Anaeroglobus
– Negativicoccus
– Acidaminococcus
70
Anaerobic gram negative rods
* Bacteroides
* Prevotella
* Porphyromonas
* Fusobacterium
71
Baceroides fragilis group are the most of any what
· They are the anaerobic bacteria most often
isolated from human infection
· They are the most common of all anaerobes in
producing life-threatening illness
72
Bacteroides fragilis group
All are
Catalase positive
73
B fragilis is indole
Indole -
74
B.thetaiotamicron is
the second most common species
Indole +
75
B.ovatus and B.uniformins is
Indole +
76
B.vulgatus and B.distasonis is
Indole -
77
Other species of bacteroides are
B. eggerrthii, B. merdae, B.
stercoris, and B. caccae.
* B. ureolyticus
78
Bacteroides spp-
beneficial normal flora
Produce
butyrate (SCFA) which provides
energy to enterocytes
79
Bacteroides spp-
beneficial normal flora
Play a role in
bile acid recirculation and
biotransformation/help ferment carbs
80
Bacteroides spp-
beneficial normal flora
Compete with
pathogenic microorganisms
81
Bacteroides spp-
beneficial normal flora
synergy
with other coliforms
82
Bacteroides spp-
beneficial normal flora
Note
30-50% of fecal matter may be
composed of B. frag
83
Bacteroides are not always beneficial
*Capsule confers resistance to host
mechanisms
*Fibrin deposition and abscess formation can
be stimulated by capsular material
*Most common infections are abscesses
which may lead to septicemia
84
diseases of Bacteriodes
Intra-abdominal infection, skin & soft
tissue
infection, bacteremia
85
Laboratory diagnosis and culture of Bacteroides
Gram stain: GNR with rounded ends,
safety pin appearance (containing vacuoles)
Culture: B. fragilis exhibit
Concentric whirls or ring-like structures
(under stereoscope)
86
ID tests of Bacteroides
· Growth in the presence of 20% bile: positive
· Penicillin: resistant
· Kanamycin: resistant
· Vancomycin: resistant
· Colistin resistant
87
Prevotella and porphyromonas genera
they are the 2nd most
common group of anaerobic bacteria
88
Prevotella and porphyromonas genera
they are normal inhabitant of the
oropharynx,
nose, GIT, GUT
89
Prevotella and porphyromonas genera
most of them develop
pigmented colonies
(brown or black) and demonstrate florescence
under UV light (brick red)
90
Prevotella and porphyromonas genera
They are mostly associated with what infection
orofacial and
pleuropulmonary infections
91
Fusobacterium genus
important member
* F. nucleatum
* F. necrophorum
* F. mortiferum
* F. varium
92
Fusobacterium genus
diseases
pleuropulmonary infection, intra-abdominal
infection, abscess, systemic infection , Vincent’s disease
(anaerobic tonsillitis)
93
Fusobacterium genus
gram stain
slender, spindle-shaped
GNR with tapered end
Others -- pleomorphic GNR with spherical swelling
94
Fusobacterium genus
culture
· F. nucleatum: speckled opalescent appearance,
bread-crumb colonies
· F. necrophorum: convex with irregular margins
· F. mortiferum, varium: fried egg appearance
95
ID test of Fusobacterium
-Fusobacterium sp. are susceptible to
Kanamycin and colistin-
no growth on KV BAP
-F. mortiferum is positive for esculin
hydrolysis and lactose fermentation,
-F. varium is negative for both
GLC: butyric acid peak
96
Vincents disease description in detail
· It is a necrotizing gingivostomatitis
·It is caused by Treponemes, P.
melanogenica,
other anaerobic GNR and anaerobic cocci
·Fusobacterium necrophorum is involved
with the severe form of the disease, which is
accompanied by sepsis
97
Vancomycin(5 ug)
* Majority of Gram positive organisms are
susceptible
* Exception-some species of Lactobacillus sp.
* Majority of Gram negative organisms are
resistant
* Useful when organism is Gram variable or falsely
staining negative (some Clostridia)
98
kanamycin
1000ug
99
Colistin
10ug
Most gram postive are resistant
100
Prevotella
disk suceptible or R and testing
Flourescence= +
Pigment= may be + or -
BBE= negative
Kanamycin=R
Vanvomycin=R
Colistin= R or S
Growth on LKV= growth
Glucose fermentation= +
101
Porphyromonas
Disk su or Re and testing
Fluorescence +
Pigmentation +
BBE= neg
Kanamycin= R/S
Vancomycin= S
Colistin= R
growth on LKV= negative
glucose fermentation= negative
102
B.fragilis group resistance or susceptibility
Fluorescence -
Pigmentation -
BBE= growth
Kanamycin= R
Vancomycin= R
Colistin= R
growth on LKV= growth
glucose fermentation= Postive
103
Fusobacterium
Fluorescence -
Pigmentation -
BBE= neg
Kanamycin= S
Vancomycin= R
Colistin= S
growth on LKV= negative
glucose fermentation= NA
104
Anaerobic spore-forming rods
clostridium, botulism, Tetani, perfringes, difficile
105
4 types of botulism
1. Classical or food-borne: ingestion of improperly canned food
2. Infant botulism: ingestion of botulinal spores from soil, dust, honey
3. Wound botulism: contamination of traumatic wound with botulinal spores
4. Undetermined: unknown source
106
C.Botulinum symptoms
weakness, dizziness, blurred vision, muscle weakness,
respiratory and flaccid paralysis
107
C.Botulinum virulence factors
Exotoxin that blocks the release of acetylcholine
108
C.botulinum Lab diagnosis
Demonstration of C. botulinum and/or its toxin
in specimens
109
Clostrium tetani
disease
Tetanus
110
Clostridium tetani
symptoms
muscular spasm, lockjaw, respiratory distress
111
C.Tetani virulence factors
tetanospasmin (neurotoxin) that
inhibits inhibitory neurotransmitters of motor neurons
112
C.Tetani diagnosis
it is usually based on
clinical symptoms
113
C.tetani gram stain and what is availble
drumstick or tennis-racket
appearing GPR
Vaccine available
114
Clostridium perfringens diseases
(Soft tissue):
1. Wounds
2. Cellulitis
3. Myonecrosis or gas gangrene: caused by 12 toxins
(lecithinase or a-toxin)
4. Food poisoning: caused by enterotoxin
115
Gas gangrene may be caused by
other Clostridia like C.
novyi, C. septicum, C. sordellii, C. histolyticum
116
Gas gangrene most common cause
C. perfringens is the most common agent
117
Laboratory diagnosis of Clostridium per-fringes
Gram stain and culture
Gram stain: large, box-car shaped spore-forming
GPB
Culture: double zone of b hemolysis
118
Laboratory diagnosis of Clostridium per-fringes
ID tests
· Positive Nagler test (lecithinase test): opaque
precipitation
· Positive reverse CAMP tes
119
Reversed CAMP test
The alpha toxin
produced by
Clostridium
perfringens interacts
with CAMP factor
produced by Group B
Streptococcus and
produce synergistic
hemolysis.
120
C.difficile is a
Gram postive spore forming bacilli
121
C difficile diseases
Antibiotic associated diarrhea or
colitis, pseudomembranous colitis
122
C difficile
antibiotic therapy
Antibiotic therapy will diminish the normal
bowel flora, which may be followed by
intestinal colonization with Clostridioides
difficile
123
C.difficile
CDC facts
* It’s estimated to cause almost half a million
illnesses in the United States each year.
* About 1 in 5 patients who get C. diff will get
it again.
* Within a month of diagnosis, 1 in 11 people
over age 65 died of a healthcare-associated
C. diff infection.
124
C.difficile risk factors
* Age - elderly
* Hospitalizations or long-term care
* Antibiotic treatment
* Previous infection
* Recent exposure to organism
125
C.difficile colonization
0-6 months
0-37% carriage rates
126
C.difficile colonization
6-12 months
14% carriage rates
127
C.difficile colonization
by 3 years of age
~3% = non-
hospitalized adults
128
C.difficile testing is not
* Testing is not recommended in <1
years of age and other causes should be
ruled out before testing in ages 1-3
years
129
C.difficile testing
many PCR tests
* **Many PCR tests are not validated in
children 2 years of age and younger**
130
C.difficile symptoms
* Loose (watery) stools
* Fever
* Tender stomach or pain
* Fever
* Loss of appetite
* Nausea
131
C.difficile complications
* Pseudomembranous Colitis
* Recurrence – up to 30% of patients will
* relapse with the same strain or a new strain
132
C.difficile virulence factors
Toxin A, toxin B test by
NAP1/B1/027 (North American pulsed-field gel EF type
1, RE type B1, PCR ribotype 027) – For hypervirulent
strain
133
C.difficile Lab diagnosis
tissue culture cytotoxin assay
demonstration of toxin B
134
C.diff Lab diagnosis
Culture isolation agar type
Cycloserine-cefoxitin egg yolk fructose
agar (CCFA)
135
C.diff Lab Diagnosis
GDH
(Glutamate dehydrogenase
136
C.diff Lab Diagnosis
toxin testing
(EIA)
137
C.diff Lab Diagnosis
PCR for
toxin A and or B gene detection
138
C.diff Lab Diagnosis
specificity and sensitivity in
the high 90-100% range depending on the manufacturer
139
C.diff Lab Diagnosis
detecting gene does not
Mean it is producing the gene
140
CCFA
(Cycloserine-cefoxitin egg yolk
fructose)
141
Cycloserine-cefoxitin egg yolk
fructose
* Enriched, selective, and differential
* Consists of animal peptones and fructose, and
supplemented with cefoxitin and cycloserine at to
inhibit growth of most normal fecal flora.
* Cycloserine -inhibits Gram-negative bacteria
* Cefoxitin will inhibit both Gram-positive and Gram-
negative organisms
142
Cycloserine-cefoxitin egg yolk
fructose.
C.difficile is not
makes characteristic
turns neutral PH
* C. difficile is not inhibited
* Makes characteristic yellow, ground-glass colonial
morphology.
* It turns the neutral red pH indicator from pink/orange
to yellow (change in pH from utilization of the amino
acids)
143
C.difficile algorithm
One testing algorithm
A One test algorithm may be used if the bacteria is GDH positive and Toxin positive then the final report is positive. If the bacteria are GDH positive and Toxin negative, then a Genexpert is used to determine if the organism is positive. If both are negative, then the clinical report is negative
144
Treatment for C.difficile
* Metronidazole
* Vancomycin
* Severe or Recurrent infection:
* Both metronidazole and vancomycin
* Fidaxomicin
* FMT (fecal microbiota transplant)
* Monoclonal antibodies
145
Transmission/prevention for C.difficile
* Fecal oral route
* Spread by spores
* Spores can survive on surfaces for months to
years
* Frequent HAI (Hospital Acquired Infection)
* Patients must be isolated (contact isolation)
* Bleach or other sporicidal cleaners must be
used
146
Anaerobic non-spore-forming Bacilli
Members:
· Propionibacterium
· Eubacterium
· Actinomyces
· Bifidobacterium
147
Propionibacterium acnes
Is responsible for
Human acne
148
Propionibacterium acnes
can be found as
· It can be found as normal flora in the mouth, skin, GIT, GUT,
nasopharynx
149
Propionibacterium acnes
It is a frequent
· It is a frequent contaminant of blood cultures
150
Propionibacterium acnes
Can cause
· Can cause joint infections and CSF shunt infections
151
Propionibacterium acnes
gram stain and culture
· Gram stain: diphtheroid-like or club-shaped GPR with Chinese
letter formation
· Catalase positive and indole positive
152
Eubacterium lentum ( now Eggerthella lenta)
· It is a true agent of endocarditis (normal oral
flora)
· It is recovered from mixed culture from wound
or abscess
· Gram stain: diphtheroid-like GPR
153
Actinomyces species
disease and infection is initiated
* Disease: actinomycosis (cervicofacial or lumpy jaw, thoracic,
abdominal)
Note: Infection is initiated when the endogenous organism is inoculated into damaged tissue
after another infection, trauma, or surgical manipulation
154
Actinomyces species symptoms
formation of draining sinus tract containing "sulfur
granules"
155
Actinomyces species Lab Diagnosis
Gram stain and culture
* Gram stain: Branching, beaded Gram-positive rods
* Culture: CDC-BAP young colonies of A. israelii look like spider
colonies (slow-grower)
156
Actinomyces species Lab Diagnosis
older colonies
* Older colonies are referred to as molar tooth (heaped or raised up)
157
Actinomyces species Lab Diagnosis
The most Frequent species
The most frequent species involved in actinomycosis is
Actinomyces israelii
158
Bifidobacterium dentium
Inhabitant and gram stain
* It is a normal inhabitant of the oral cavity and
the GI tract
* Gram stain: Y or bifid shaped Gram-positive
rods
159
Bifidobacterium dentium
Are what
* “Friendly” Probiotic Along with
Lactobacillus,
* inhibits harmful microorganisms
* (Claimed to promote good digestion, boost the
* immune system.)
160
Mobiluncus
* Strict anaerobe
* Gram variable curved rods with tapered
ends
* Normal flora but believed to be involved in
Bacterial Vaginosis.
* Usually not cultured in Clinical lab because
found in samples unacceptable for
anaerobic culture
