Anaerobes!!! MICROM442 Deck 21 Flashcards
1
Q
ambient air=
A
About 21% O2, 0.03% CO2
2
Q
Obligate anaerobes can only handle up to 0.5% O2, what is an example?
A
clostridium species
3
Q
Facultative anaerobes: use fermentation to grow in places without oxygen,
but use aerobic respiration in places with oxygen, what is an example?
A
ecoli and staph. aureus
4
Q
Aerotolerant anaerobes: do not use oxygen to live, but can exist in its
presence for a period of time, what is an example?
A
cutibacterium acnes
5
Q
Microaerophiles: Prefer lower oxygen (5%), increased CO2 (8-10%), what is an example?
A
camphylobacter
6
Q
Capnophiles (=carbon dioxide “loving”): Require increased CO2 (5-10%), what is an example?
A
Neissseria gonorrhoea
7
Q
obligate anaerobe growth in thioglycate media
A
very bottom of the tube only
8
Q
obligate aerobe growth in thioglycate media
A
only at the top of the tube
9
Q
facultative anaerobe growth in thioglycate media
A
half and half mix, but microbes growing in the top half
10
Q
aerotolerant anaerobe
A
grows throughout the tube
11
Q
aerotolerant + facultative anerobes grow in larger amounts of the media beccause
A
aerobic respiration produces more ATP
12
Q
aerobic respiraton
A
- O2 = final electron acceptor
- Yields lots of ATP
- Oxygen can grab electrons and
form reactive oxygen species
(ROS) that damage cells - Bacterial antioxidant enzymes
help detoxify these products
13
Q
Bacterial antioxidant enzymes
help detoxify these products
A
- Catalase
- Peroxidase
- Superoxide dismutase
14
Q
Anaerobes
A
- Can use inorganic compounds as
alternative electron acceptors:
NO3- NO2− SO₄²- - Some use fermentation
- Overall yield less ATP
- Some are prone to forming ROS
- Some lack antioxidant enzymes
15
Q
oxygen tolerance is a?
A
SPECTRUM
16
Q
what factors typically influence a bacterias sensitivity to oxygen?
A
- Protective antioxidant enzymes
-How active?
-How abundant?
-Where in the cell they’re located - How quickly the bacteria take up oxygen
- If they possess enzymes that promote internal formation of ROS
- How sensitive their overall systems are to ROS
-Enzymes with iron cofactors are particularly sensitive
-DNA damage
17
Q
what percentage of oral bacteria are anaerobes?
A
90
18
Q
what percentage of colon bacteria are anaerobes
A
99%
19
Q
where do anaerobes live on the body?
A
-upper respiratory tract
-colon
-oral cavity
-skin
-vagina
20
Q
upper respiratory tract infx
A
-oral/neck anscesses
-pnemonia
21
Q
colon infx
A
-intra-abdominal infx
-abscesses
-appendicitis
22
Q
more infx :’(
A
-bloodstream
-endocarditis
23
Q
oral cavity infx
A
-dental infx
-sinusitis
-brain abscess
-pneumonia
24
Q
skin infx
A
-acne
-would infx
-surgical site infx
25
vagina infx
-bacterial vaginosis
-adverse pregnancy outcomes
-pelvic inflammatory disease
26
internal displacement cause of infx
-trauma, burns, wounds
-surgery
-aspiration pneumonia
-bowel or intestinal perforation
-appendicitis
-bloodstream infx
-endocarditis
27
host factors
-abx therapy
-chemotherapy injures mucosa
-immunosuppression
-diabetes
28
external sources
-environmental contamination
-food-borne illness
-water exposure
-injection drug use
-animal bites
29
toxin treatments
-antitoxin
-immune globulin
-vax
30
source control treatment
drain abscess, deride/remove necrotic tissue
31
antibiotics
-anaerobic infx often mixed, often need multiple
-metronidazole, clindamycin
-Piperacilllin/tazobactam, Ampicillin/sulbactam
-carbapenems
32
blood culture enrihment
-patient with possible bloodstream infx
-always use BOTH aerobic and anaerobic bottles
-put into fancy machine
-nutrient broth + pH colorimetric indicator
33
anaerobic bottle contains both?
CO2 and NO2
34
what signifies growth in the blood culture instrument?
CO2 production and pH change
35
Anaerobic blood agar plates
Hemolysis patterns with sheep’s blood
36
Enrichment broths
* Chopped meat/Cooked meat broth
* Thioglycolate broth
-Differential: where the organisms grow
* Reducing agents deplete oxygen
37
Selective media
Inhibitors like antibiotics or bile
38
Differential media
* Pigment production
* Esculin hydrolysis = dark brown colonies
* pH indicators
* Egg yolk to visualize toxin activity
39
anaerobic indicator
white= anaerobic
pink=oxygen present
40
palladium catalyst
2H2 + O2 -> 2H2O
41
For other specimens
Tissue, abscesses, normally sterile body fluids (joint, pleural, CSF, etc.)
42
Incubation conditions without oxygen
* Specialized airtight containers
* A way to remove oxygen
* A way to generate an anaerobic gas
mixture
* An indicator to show if it’s working
43
Example: Vacuum system
* Evacuates air with vacuum pump
* Replaces with anaerobic gas mixture
* Add anaerobic indicator to jars
* Add PALLADIUM catalyst to jars to take care of remaining oxygen
44
Example: Anaerobic chamber
* Passbox/vacuum airlock to transfer materials
* Armholes to reach plates on the inside
* Palladium catalyst box
* 5% CO2, 5% H2, 90% N2 mixture pumped in
* Temperature control to incubate
45
ID with gram stain
-Fusobacterium nucleatum
pointy Gram-negative rods
-Clostridium spp.
Boxy Gram-positive rods
Can chain, can have spores
46
ID with biochemical tests
OLD NEWS: Indole, catalase, urease, susceptibility to special antibiotic
MORE COMMON: Long-wave UV fluorescence
47
Long-wave UV fluorescence
* C. difficile, Fusobacterium: chartreuse, yellow-green
* Some Prevotella, Porphyromonas: red
48
ID with MALDI-TOF MS (Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry)
* Mix bacteria with an organic matrix, hit with a laser to generate protonated ions
* Accelerate through a vacuum to get a peptide profile based on time to flight
* Compare pattern to a database to match with known organisms
* Instrument $$$, but consumables cheaper and hands-on time is minimal
* Becoming the standard in larger laboratories
49
ID with sequencing
* After isolation on plates or directly from patient specimen
* PCR + Sanger sequencing
* Next generation sequencing
-Can resolve templates if multiple species are present
-Many anaerobic infections are polymicrobial
50
Sanger cannot anwser the
cause of infx
51
NGS better if there is a
polymicrobial infx
52
NGS can be used for either
growth on plates or directly from patient ssample
53
Gm+ rods we are concerned with (CCCA)
* Clostridium
* Clostridioides
* Cutibacterium
* Actinomyces
54
GM- rods we are concerned with (BF)
* Bacteroides
* Fusobacterium
55
Gram-negative rods with pleomorphic morphology, gut
commensals
bacteroides
56
what are bacteroides often isolated from?
blood, intra-abdominal
abscesses, appendicitis
57
* B. fragilis
* B. thetaiotaomicron
* Others
Bacteroides fragilis group
58
Baceroides abx resistance B. fragilis
beta-lactamase
production → treatment with BL/BLIs metronidazole
59
Baceroides abx resistance nimA-H genes
nitroimidazole reductase decreases efficacy of metronidazole, a common antibiotic for
anaerobic coverage
60
Bacteroides Bile Esculin Agar
-esculin hydrolysis = dark colonies (black)
-abx + bile inhibit most bacteria other than fragilies group
61
what type of rods are fusobacterium
gram neagtive rods
62
* Often found in polymicrobial brain
abscesses, especially with preceding
sinusitis or dental treatment
* Long and tapered appearance
Fusobacterium nucleatum
63
fusobacterium necophorum
* Associated with Lemierre’s syndrome
* Rare complication in post-antibiotic era
* Oropharyngeal infection first
* Infected blood clots in jugular vein
* Septic emboli spread to other organs
* Gram stain morphology variable
64
"spidery" on gram stain
cutibacterium acnes
65
Cutibacterium acnes
* Aerotolerant anaerobe
* Often found on skin, causes acne
* Can form biofilm, concern with implant
infection after orthopedic surgeries
66
* Gram-positive, filamentous, beaded
looking, grow very slowly
* Facultative anaerobes
* Part of oral & GI flora
* Slow and chronic infections
* “Lumpy jaw”
* Yellow “sulfur granules” containing
organism in abscesses
Actinomyces species
67
* Gram-positive rods
* Some form spores
* Some make toxins
* Most are obligate anaerobes
Clostridium and Clostridioides
68
Gas gangrene, botulism, tetanus, food poisoning can be caused by different
Clostridium species
69
causes diarrhea/colitis in association with abx use and transferred from genus clostridium to clostridioles
C. dif
70
what triggers sporulation
starvation/stress
71
what triggers germination
nutrients
72
why can clostridium be mistaken as GM- rods if they should stain GM+?
overdecolorize easily (often taken from blood culture)
73
source of inx for clostridium gas gangrene
trauma-associated injury or spontaneous
74
trauma-associated injury for gas gangrene
war, natural disasters, post-operative
75
spontaneous (GI tract commonly) gas gangrene
* Most often C. septicum
* Underlying malignancy, diabetes, immunosuppression
76
gas gangrene mechanism and clinical picture
* Spreads via blood to invade muscles
* Vegetative cells or spores enter deep tissue, quickly
spread and destroy several inches per hour
* Shock, organ failure, high mortality rate
* Best treatment: amputation :'(
77
what two toxins are in gas gangrenee promoted by C.perfringens?
alpha and theta
78
Alpha toxin (phospholipase C)
* Damages cell membranes
* Promotes blood clots = anaerobic tissue environment
79
theta toxin (perfringolysin O)
* Pore-forming toxin
* Synergy with alpha toxin
80
X-ray shows gas in tissue
Case of gas gangrene after
intramuscular saline injection
associated with IV drug use
81
C. perfringens on Egg Yolk Agar
* Made with egg yolk suspension
* Phospholipase C/alpha toxin forms opaque
precipitate
82
C. perfringens on brucella blood agar
* Double zone of hemolysis
* Narrow zone: theta toxin
* Wider zone: alpha toxin
83
* Caused by C. botulinum, also C. butyricum, C. baratii
* Neuroparalytic illness caused by toxin
BOTULISM
84
infant botulism
baby consumes spores, they germinate in
intestine and vegetative bacteria produce toxin
85
Wound botulism
bacteria enter a wound through injury, surgery,
or illicit drug injection
86
Foodborne botulism
homemade, improperly
canned/preserved/fermented food with toxin in it
87
Iatrogenic botulism
too much is injected during a medical or
cosmetic procedure (iatrogenic botox poisoning)
88
toxin that binds at nerve ending synapses, prevents release of neurotransmitters at neuromuscular junctions and results in LACK OF STIMULUS TO MUSCLE FIBERS
botulinum toxin
89
what are the symptoms of botulism?
slurred speech, trouble breathing, weak muscles, droopy eyelids, blurry vision, trouble swallowing
90
Dx of botulism
DO NOT WAIT FOR LAB CONFIRMATION TREAT IMMEDIATELY, PCR, Mouse bioassay, culture
91
treatment botulism
*Heptavalent antitoxin targets 7 serotypes
* Arrests progression of illness, but does not reverse it
* Long term recovery requires regeneration of new neuromuscular
connections
92
Tetnaus caused by clostridium tetani leads to spastic paralysis caused by toxin. How is it transmitted?
* Spores in the environment contaminate
wounds
* Crush injury, puncture wounds
* Injection drug use
* Spores germinate into vegetative cells
which then produce toxin
93
* Blocks inhibitory signals to motor neurons at
CNS system
* Similar mechanism, but different binding site
from botulinum toxin which is at
neuromuscular junctions
* Painful, spastic muscle contractions
tetani toxin
94
symptoms/treatment of tetanus
* Jaw cramping/lockjaw, muscle spasms, jerking and
stiffness, seizures
* Can progress to respiratory paralysis and death
* Human tetanus immune globulin (TIG) – antibodies
to toxin
* CDC recommends routine tetanus vaccination with
boosters throughout life
95
DTaP vax for
young children
96
Tdap for
young teens
97
Td/Tdap for
adults
98
* Spores survive in improperly cooked/heated meat and then germinate
* Humans ingest vegetative cells in the food
* Form spores and enterotoxins (damage intestinal epithelia) in the small intestine
* Diarrhea
* Self-limiting but can be severe in young, elderly, immunocompromised
C. perfringens
99
The most frequently reported nosocomial
clostridioides difficile
100
C. diff transmission
* Spores are found in the environment and are difficult to clean from hospital surfaces
* Fecal-oral transmission, spores germinate in the intestine
101
C. diff risk factors
Healthcare-associated infection
* Previous C. diff infection
* Older age (65+)
* Recent stay at hospital or nursing home
* Immunocompromised patients
* Antibiotic exposure wipes out competing intestinal bacteria and allows C. diff to take over
Community-acquired infection
* Usually younger and healthier patients, less severe infection
102
what organ do c.diff toxins act on
large intestine (colon)
103
internalized by epithelial cells and disrupts tight junctions
Toxin A (TcdA)
104
promote collapse of the actin cytoskeleton = cell rounding and death
* Strong host inflammatory response
* Pseudomembranous colitis: plaques on mucosal surface with inflammatory exudate
* Hypervirulent strain (ribotype 027): hyperexpression of toxins
Toxin A and Toxin B (TcdB)
105
* Watery diarrhea
* Fever
* Abdominal pain
* Loss of appetite
C. diff infx
106
C. diff diagnosis
* Complicated and controversial to diagnose
* False positives: asymptomatic colonization, particularly in infants and children
* People get diarrhea for other reasons: laxatives, antibiotics, other infections
* PCR to detect genes that encode toxin
* Toxin testing (tissue culture cytotoxicity, enzyme immunoassay) – less sensitive
107
C. diff treatment/prevention
* C. diff can be treated with 10 days vancomycin or fidaxomicin
* Use isolation and contact precautions to prevent spread
* Wear gloves and a gown
* Wash hands between seeing patients (soap and water is best)
* Keep infected patients in separate rooms
* Prescribing more targeted, appropriate antibiotics in general (vs broad-spectrum)
108
C. diff fecal transplant
* Goal: reestablish a healthy gut microbiota in patients with recurrent C. diff infection
* Method:
* Screen for healthy donors (clinical history, test stool for pathogens)
* Oral capsules (poop pills), colonoscopy to inject stool, nasoenteric tubes
* Concerns:
* Long-term risks, unknown health issues with donors
* One case in 2019: drug resistant E. coli from stool donor cause bloodstream infection and
death in an immunocompromised patient and clinical trial was halted
