Test 4 Flashcards
Anaerobes usually fail to grow under what conditions?
usually fail to grow in the presence of oxygen thus carry out fermentation
Anaerobes can be either gram positive or gram negative
T/F
True
aerotolerant
(5% O2) anaerobic bacteria not killed by small amounts of oxygen
1-Clostridium
2-Actinomyces spp.
*grow best under anaerobic conditions
Most anaerobes are _______________.
obligat (strict) anaerobes requiring 0% O2.
How long does it take for obligate anaerobes begin to die with brief exposure to O2
~10 minutes
Anaerobic organisms lack…..
1-superoxide dismutase
2-catalase
enzymes required to break down reactive oxygen species during aerobic metabolism, harmful to bacteria
Obligate Aerobes
(21% O2, and 0.3% CO2, ambient air AKA room temp) cannot grow anaerobically
-Have no fermentative pathways, typically produce superoxide dismutase
Obligate Anaerobes
( 5-10 % hydrogen and CO2 both, 80-90% N) grow only in or near 0% O2 levels
-Lack superoxide dismutase, generally lack catalase, are fermenters that cannot use oxygen as a terminal electron acceptor
Microaerophiles
(5-10% O2) CO2 (8-10%) only grow under reduced O2 levels
Facultative anaerobe
can grow under aerobic or anaerobic conditions thus some can be normal flora-skin
*will respire aerobically until oxygen is depleted and then
ferment
Capnophiles
like increased CO2(5-10%) and 15% O2.
*Candle jars achieve about 3-5% CO2 and 8-10% O2
Anaerobes are the most abundant bacteria in …..
the human gastrointestinal tract.
- the number of bacteria increase as you move distally
ex. . colon has more than the duodenum
What are the most abundant bacteria (normal flora)
in the human colon?
Bacterioides spp.
Why are bacteria like Bacterioides important for us to have?
- these bacteria are important in the breakdown of food products into forms our body can absorb.
- they also break down carcinogens.
Clostridial spp- normal flora?
many Clostridial app are part of normal flora-GI and skin
additional anaerobes as normal flora of skin and/or
upper respiratory tract:
1-Propionibacterium
2-Prevotella
3-Fusobacterium
Microaerophilic gram positive thin rods with spiral forms,
normal flora of female genital tract
Lactobacillus
Lactobacillus
1-colony morphology on sheep blood
2-hemolytic pattern
3-catalase
1-pinpoint
2-alpha hemolytic-resembles streptococci
3-negative
Endogenous infections
opportunistic infections, pathogens gain access to sterile sites (trauma)
Exogenous infection sources
rusty nails
skin puncture
botulism
ingestion of improperly canned foods
Human to human spread
C. difficile
Disease Transmission of Anaerobes (4)
1-Endogenous infections
2-Exogenous sources
3-Ingestion of improperly canned foods (botulism)
4-Human to human spread (C. difficile)
Infection sites (9)
1-Brain/lung/liver/intra-abdominal abscess
2-Chronic otitis media
3-Dental infections (abscess formation)
4-Aspiration pneumonia
5-Peritonitis/appendicitis/cellulitis
6-Trauma/surgical wounds
7-Post-abortion sepsis-gynecological infections
8-Myonecrosis
9-very rarely urine (only 1% isolated from urines are anaerobes)
-Voided or catheterized urine not acceptable (normal flora contaminants)
-Suprapubic bladder aspirate is acceptable
Specimens that limit the amount of normal flora contamination? (3)
1-aspirates
2-tissue
3-blood
Acceptable specimens for anaerobes?
aspirated material or tissue biopsy
which is preferred, needle aspiration or swab?
needle aspiration
Disadvantages of swabs?
- small amounts
- drying
- normal flora contamination-is normally taken from from skin or mucosal surfaces
- toxic exposure to air
Blood specimens should always be collected utilizing…
both aerobic and anaerobic blood culture bottles
How are blood cultures collected?
-sanitize the collection site (skin) with iodine or clorhexidine gluconate in combo with 70% isopropyl alcohol.
Specimens to accept?
- skin-aspirates of wounds are acceptable
- Swabs not preferred-they may contain anaerobes associated with normal flora that normally inhabit hair follicles and sebaceous glands(i.e. more easily contaminated with normal flora, also O2 is present on skin surface
skin aspirates
- aspirations typically contain less contamination with normal flora and are thus preferred over swabs
- once in the syringe, the extra needs to be expelled
- the aspirate should then be injected into an oxygen free transport tube or vial preferably containing PRAS media.
When should swabs be used?
when aspirated material is not possible or tissue is unavailable
The steps for processing sputum specimens
1-digestion-liquify mucus with mucolytic agents to release bacteria from mucin
2-decontamination-eliminate normal flora, high lipid content of Mycobacteria cell walls makes them resistant to this step
3-neutralization-with phosphate buffer or water
4-concentration
Nonphotochromagens (organisms)
Mycobacteria avium/intracellular complex
- M. shimoidae
- M. genavense
- M. xenopi
Mycobacteria xenopi
Nonphotochromagen that is found in hot water taps in hospitals, causes pulmonary infections and disseminated disease
What is MAC?
Mycobacteria avian complex
Important pathogen in AIDS patients
Pulmonary infections and lymphadenitis
exhibit mucus production, if long standing can cause mechanical stress on bronchioles and result in bronchiectasis
Difference between MAC and TB?
MAC is not spread person to person, rarely presents with granulomas or tissue death
TB does
ID scheme for TB
Culture growth within 4-8 weeks that is rough, dry, buff, non-pigmented on Lowenstein-Jensen media
- Acid-fast: strong + with slightly curved, beaded rods
- niacin: +
- Nitrate reduction: +
- 68 degree catalase test: -
Rapid Growers
organisms
Mycobacteria fortuitum
M. chelonae
Can grow on MacConkey with CV omitted
All Mycobacteria produce…
Niacin
M. tuberculosis and simile are “positive” due to high accumulation of niacin in the media due to lacking the enzyme to convert niacin to niacin ribonucleotide
Percentage of O2 and CO2 for….
Obligate aerobes Obligate anaerobes Microaerophiles Facultative anaerobes Capnophile
Obligate aerobes- 21% O2, 0.3% CO2 Obligate anaerobes- 0% O2, 5-10% H and CO2 Microaerophiles-5-10% O2, 8-10% CO2 Facultative anaerobes- doesn't matter Capnophiles- 15% O2, 5-10% CO2
What type of bacteria are most abundant in the human gastrointestinal tract
Anaerobes
As you move more distally, the number of bacteria increases or decreases?
Increases
Cell wall deficient bacteria
Mycoplasma and Ureaplasma
2 major groups of Leptospira app.
1-interrogans-causes human leptospirosis
2-L. biflexa- environmental strains
2 groups of Mycobacteria
Mycobacterium tuberculosis complex-humans are natural reservoirs
*All are slow growers and have non-pigmented colonies!
(NTM) Nontuberculosis Mycobacterium-found in the environment and can transiently colonize skin, GI, and RT
Causative agent of cervical identities in children
Mycobacteria scrofulaceum
Which organism colonizes with a “fried egg” appearance on agar?
Mycoplasma hominis
Clostridium botulinum
Spores are: Virulence Factors: Group 1 and 2: Group 3: Group 4:
Spores are: subterminal (racket shape) Virulence Factors: neurotoxin causing flaccid paralysis Group 1 and 2: human botulism Group 3: animal botulism Group 4: not disease associated
Mycoplasma and Ureaplasma
1-Media
2-ID
3-Treatment
1-Media-SP4 media
2-ID-molecular method (off of specimen) or serodiagnosis
3-Treatment- tetracycline-resistant to beta-lactams due to lacking cell wall
Most definitive test to ID Mycobacteria
Niacin
Safety precautions for Mycobacterium
AST Levels 1-3
AST 1-grow organism and stain, but not ID
AST 2- ID TB only
AST 3-ID all Mycobacteria and perform susceptibility testing
What allows an organism to be acid fast and which actinomycetes are acid-fast, which ones are not?
mycelia acid content in the cell wall
Acid fast: Nocardia and Rhodococcus
Non-Acid fast: Streptomyces
Nonculticatable Nontuberculosis Mycobacterium
M. leprae
- Obligate intracellular bacteria that cannot be grown in vitro
- found in armadillos and mice
- Causes leprosy
Mycobacteria specimen sources
virtually any tissue or organ
- sputum (collected early morning for 3 consecutive days)
- Tissue granuloma
- Urine
- Bone
- CSF
- Blood
- GI
- Stool
Mycobacteria specimen processing
- Specimens from sterile sources can be directly plated to media
- digestion/decontamination, neutralization, and concentration of sputum cultures
TB Pathogenesis
post established infection
After ~3 weeks, TH1 cells release cytokines that cause macrophages to mount a bacterial response and granuloma formation and recruit monocytes, which differentiate into “epithelioid” like cells that wall off the bacteria
TB Epidemiology
Not every person exposed will become infected
Contagious-inhalation of a single bacilli
Prevalent in elderly, chronic malnourished, alcoholics, homeless, crowded living conditions
1/3 of the world’s population is infected
Mycoplasma and Ureaplasma
1-Lack
2-Normal flora of
3-Pathogenesis
1-Lack cell walls
2-Normal flora of the mouth, URT, upper GI
3-Mycoplasma pneumonias-causes walking pneumonia, primary atypical pneumonia
-Mycoplasma hominis-systemic infection in neonates, urogenital infection in adults
-Ureaplasma urealyticum-isolated from stillborn infants, controversial cause of chorioamnionitis
BSL 3 requires….
- directional air flow
- labs and hoods equipped with air pressure gauges
- respirators
- disposable clothing to be autoclaved
- Nonpermeable walls and surfaces in rooms
TB
1-Primary:
2-Secondary:
3-Miliary:
1-Primary: initial infection in a previously exposed individual with 2 potential outcomes:
-Latent TB: exposure, develop antibodies, and test positive skin test, but no active disease not contagious
-Active infection/Disease
2-Secondary: (Reactivation) person with latent TB manifests later with disease
3-Miliary: spreads to other organs hematogenously
TB Pathogenesis
establishing the infection
Respiratory droplets from infected individual get suspended in air and inhaled and the droplets make their way to alveoli.
Macrophage migrate to the alveoli and take up the bacteria via endocytosis, but are unable to kill the bacteria due to mycobacteria blocking the fusion of the lysosome with the phagosome through protein inhibition
Scotochromagens
organisms
(Mycobacteria)
- M. scrofulaceum
- M. szulgai
- M. gordonae
Photochromagens
organisms
(Mycobacteria)
- M. kansasii
- M. marinum
- M. asiaticum
What are Runyon groups
Nontuberculosis Mycobacteria
Photochromagens-require light to form pigment following
incubation in the dark
Scotochromagens-produce pigment in dark or light
conditions
Nonphotochromagens-non-pigmented regardless of
incubation conditions
Rapid Growers-take <7 days to grow on solid media
Organisms part of the Mycobacterium tuberculosis complex
- M. tuberculosis
- M. bovis
- M. africanum
Lowenstein-Jensen media
The medium most often used for isolation and cultivation of Mycobacterium spp.
Mycobacteria spp characteristics
non-spore forming slender, gram +/variable bacilli slow growing (2-60 days) (due to high lipid content in the cell wall causing them to clump making nutrient uptake more difficult) Obligate aerobes (CO2 enhances some) Acid-fast
ID for Mycobacteria
Biochemical/Growth characteristics
High Performance Liquid Chromatography
T-SPOT-TB blood test
Molecular methods
Incubation of Mycobacteria spp
If skin specimen-25-30 degrees C
(M. marium, M. ulcerans)
35 degrees C in the dark, 5-10% CO2 and high humidity
TB takes 2-4 weeks to grow and susceptibility testing takes an additional 2-4 weeks for a total of 4-8 weeks on average
What should be done for optimal recovery of Mycobacteria
-uses and least one solid media: Middlebrook 7H10 ( non-selective) egg-potato base (Lowenstein-Jensen and Petragnant) -AND at least one liquid media: BACTEC 12B Middlebrook 7H9 broth
Non cultivatable Mycobacteria
Mycobacteria leprae
Causes Leprosy (Hansen’s Disease)
-chronic disease of the skin, mucous membranes,nerve tissue
obligate intracellular bacteria-never cultured in vitro
-Humans are the primary reservoir
Transmission via contact with infected skin/nasal secretions
Stain methods for Mycobacteria
1-Acid-fast: high lipid content binds fuchsin dye and resists destaining
-(Ziehl-Melson (hot), Kinyoun (cold)-higher concentration of phenol to accelerate staining process)
2-Fluorochrome: Mycobacteria have affinity to flurochrome (Auramine O with/without rhodamine)
BSL 3
Recommended BSL when working with Mycobacterium
Pigment production of:
1-Scotochromagens
2-Photochromagens
3-Nonphotochromagens
1-Scotochromagens-deep yellow-orange in both dark and light
2-Photochromagens-light exposure
3-Nonphotochromagens- non-pigmented in either dark or light (can produce pale yellow, buff,tan, but do not intensify upon light exposure)
Mycobacteria that have positive nitrate reduction
M. tuberculosis (strong positive)
M. kansasii
Clinical Screening for TB
QuantiFERON-TB Gold In-Tube test
-test for interferon-gamma release from the T-cells after stimulation via 3 proteins derived from M. tuberculosis
Purified Protein Derivative (PPD)- Intradermally
- delayed appearance of indurated reaction with or without erythema
- positive test does not distinguish between current or past infection/exposure
General Characteristics of Anaerobes
Cannot grow in presence of Oxygen (some are aerotolerant-5% O2), thus can only ferment
Can be both gram+ and gram-
Lack superoxide dismutase and catalase, which are used to break down reactive oxygen radicals
Anaerobe specimens of choice
aspirates, tissue, blood (where limited normal flora)
Disadvantage of swabs: organism is toxic if exposed to air, dry, small amount, normal flora contamination
Anaerobic PEA
inhibits proteus swarming and facultative enterobacteriaceae, but allows growth of gram+ bacteria
A major cause of pelvic inflammatory disease and ectopic pregnancy
Chlamydia trachomatis