Week 11 - Anaerobes Flashcards
What do aerobic and faculatative anerobes have to protect them from superoxide anions and their toxic derivatives
superoxide dimutase and/or catalase where are anaerobes have neither
where are anerobes found
-environment soil and water
-NF - places where oxygen exposure is minimal most in like GI tract, genital tract or skin
-when other facultative orgs are in the same environment it helps to reduce O2 availability
What are the characteristics of a anerobic infection
-polymicrobic where both aerobes and anaerobes work together to cause disease
-smells bad
- necrotic tissue blocks blood flow and allows the anaerobes to grow
-caused by both endogenous and exogenous bacteria
Obligate and strict = dies upon O2 exposure
aerotolerant - what we use in the labs for testing **
Clostridium novei - control org for ana gas pak
What are endogenous infections
-grow from own flora = GI tract, gingival crevices around teeth , skin and glands
-the infection will occur close to where the bacteria is found = deep wound infection occurring after surgery or septicemia after dental work
-reduced blood flow = reduced oxygen tension perfect anerobe growing conditions
-ABotic treatment for aerobes can also allow anaerobe growth
What are exogenous infections
-from environment = soil
- caused by spore bearers Clostridia = contamination of trauma or puncture wound
tentanus , animal bites, septic abortion and gangrene
What are some predisposed conditions that can cause anaerobe associated infections
-bites
-vomit in lungs
-oral cavity puncture - dental
-GI tract puncture
how will we know an infection in close to the mucosal surface or area of NF
-odor
-large amount of gas (gas gangrene due to clostridium)
-black color or red fluorescence
-sulfur granules
-gram will show PMN leukocytes but if you dont see them that okay too because C perfringens produce enzymes that destroy neutrophils and macrophages
What is clostridium ?
found?
Anaerobic Gram Positive Bacilli
-large GPB
-pathogenic due to exotoxins
-produces spores -terminal (swelling in the end) and subterminal (swelling in middle)
found in
-soil, dust, water
-GI tract and genital tract
Clostridium tentani
ANA GPB
found :
-soil, manure
-spores can survive many years
-not invasive and must be inoculated aka puncture wound
-produces exotoxin at site which spreads through the body
-the exotoxin is the one that causes the symptoms its a neurotoxin not the bacteria
C . tetani – Clinical Significance
- convulsive muscle contractions
-head and legs back = risus sardonicus with lock jaw
-death from resp failure
C. tetani: Laboratory Diagnosis
- grows on BA anaerobic conditions as fine film over the agar and looks like its not inoculated
-spready and thin
-motile
-detect with biochem kits, molecular or GLC
box car GBP= round terminal spores = drumstick
Clostridium tetani
prevention and treatment
prevention
-tetanus TOXID vaccine containing inactivated tetanus toxin stimulating the body to make AB to toxin
-ARTIFICIAL ACTIVE LONG LASTING
Treatment
-antitoxin injection = pre formed AB to neutralized the toxin
ARTIFICAL PASSIVE SHORT
Clostridium botulinum
Clinical Significance:
-food intoxication
-poorly canned foods or stored food - toxin is produced - baby food, bar clams
-meat fish, beans
-acidic foods will not support growth
-infant instances after ingesting honey with spores = hypotonia no muscles
-in botox
-flaccid paralysis - relives muscle opposite to c tetani
most lethal bacterial poison known
-odorless, tasteless
-attacks CNS causing paralysis
-affects respiratory muscles like heart - cardiac arrest
-destroyed by heat - after boiling 15 mins
-possible bioterrorism
-three types of effects : food borne, wound and infant botulism
Laboratory Diagnosis and treatment
Clostridium botulinum
-oval subterminal spores
-motile
-GLC
-in Ba ANA large grey irregular, grainy and filamentous border
treat
Botulinum antitoxin neutralizes the toxin
Clostridium perfringens
Clinical Significance:
-found in soil
-readily contaminates wounds and food
-produces exotoxin that causes the death of local tissue producing alpha and beta toxin = double zone
-food intoxication and mild necrosis (gas gangrene)
-production of gangrene - bubble because muscle sugars start fermenting under the skin
-if there is food poisoning its mild and self resolving
-not invasive its the toxin and necrosis . once the bacteria in the area and system is cleared the infection leaves too
-infection cant jump from one area to the next
Clostridial myonecrosis caused by
Clostridium perfringens
especially in those with restricted blood flow to limbs like diabetics
-needs debridement in the area if not cleaned then you need to amputate
-better prognosis if the infection is away from organs aka lower extremities
-toxins dont get far unless its gotten into the blood stream
Actinomyces
significance
-actis = ray beam and mykes = mucus
-relationship with long term IUD users
-causing draining sinuses with ppt yellow colonies = sulphur granules surrounded by PMNs which you crush and gram
-occurs from tooth abscess or extraction where the endogenous infection causes a suppurative infection - actinomycosis
-lumpy jaw - secondary to trauma or dental procedure
-abcesses are not kept at the jaw they can spread to the abdomen and thoracic area
-treatment with penicillin after lesions are drained
-can travel to areas nearby causing neck abcess
Actinomyces
lab diagnosis
-short branching GPB- rod
-NF in mouth and intestinal tract
-lab must be told specifically to culture
-like a starburst
-colonies look like molar tooth and are slow growing
-culture for 7 days
Laboratory Diagnosis
Clostridium difficile
-isolated from stool
-large flat colonies
-barnyard odor
-chartreuse fluorescence
-Toxin in stool is most important
-ELISA - similar to pregnancy test look for the AG on the org and the tox (need two tests to detect pathogenic clostridium because it can be NF)
-if pos for AG and neg for TOX = org is not the problem
- if pos for both AG and TOX then it the issue
-PCR also done
-Gram = thin large GPB spore forming
-anaerobe
-no hemolysis
Treatment
-Vanc or Metronidazole (treat anaerobes)
Lab Diagnosis
Clostridium perfringens
-target
-lots of spore
-anaerobe
-double zone inner due to 0 (theta) toxin and outer due to alpha toxin
-non motile
-reverse CAMP positive - this is the differentiating test from other Clostridium
-GLC
-BOX CAR LOOK
- semi opaque , shiny
-lecithinase pos on egg yolk agar
Clostridium difficile
-can occur after using broad spectrum AB as it kills all NF in the bowel allowing pseudomembranous colitis to grow
-cytotoxin overgrowth produces the strain C diff
-causes severe diarrhea and tissue
necrosis
-nocosomial
-infection in large intestine
-high mortality
Propionibacterium
found
gram?
-called cutibacterium
NF on skin
-most common ANA now called Cutibacterium acnes
-contaminant in cultures or spinal fluids
-causes acne
-causes brain abscess
Gram: Pleomorphic GPB - curved; coryneform bacilli
-on direct sample there will a variation - cocci or bacilli
On anaerobic BA: small opaque, white, circular
Cat + (we dont do catalase on anaerobes because they need air but if you do you need to leave it for 10-15 mins before you do the test need to expose to air and has the catalase enzyme),
-indole POSITIVE (P.acnes)
Bacteroides fragilis group
significance
anaerobe
-NF in oral cavity, GI tract , found in poop
-most common genus that causes human infection
-can be mixed with other infections
-intra-abdominal and pelvic abscess, liver abscess, brain abscess
Laboratory Diagnosis
Bacteroides fragilis group
-Small GNB or GNCB with rounded ends; look vacuolated - not spores the bubble at the end is a vacule
-Anaerobic
-pleomorphic
-grows in presence of 20% bile any inhibition is sensitive**
-hydrolyses esculin **
-catalase positive
-grows on kanamycin-Vancomycin-laked blood selective agar
-turns BBE medium brown to black - diff from enterococci
resistant to Kanamycin and Vancomycin
shiny or dull, brown black entire edge only young colonies will fluoresce under woods
Prevotella species
-NF of oropharynx, nose, GIT and urogential tract
-associated with disease in head, neck and oral cavity
-Species associated with human disease: P. melaninogenica, P. intermedia, P. bivia, P. nigrescens, and P. disiens
what does a mixed infection with prevotella occur
-necrotizing fascitis can occur in those that used drugs
-when mixed with Strep milleri (part of anginosis group)
-can occur when pt greases or licks the needle before injection
-abscess formation with mixed anaerobes
Prevotella spp - Lab Diagnosis
-growth on BA and KVL
-Anaerobic
-slow grower - days to weeks
-saccharolytic
-fluoresces brick red under UV light
-pigmented -black or brown
-catalase negative
-NG in 20% bile
-Eschulin - NEGATIVE
-GNB
-anaerobe
Porphyromonas
clinical significance
used to be called bacteroides
-NF in oropharynx, URT, GIT and Urogental tract
-causes pulmonary head, neck and oral cavity
-associated with human periodontal disease
Porphyromonas – Lab Diagnosis
-slow grower
-similar to provetella
-Anaerobic
-fluoresces brick red under UV light
-older culture look black
-ASACCHAROLYTIC - does not use sugars
-bile esculin negative - diff test
-NG in 20% bile -diff test
-Inhibited by penicillin, kanamycin, rifampin and vancomycin
Comparing Porphyromonas and Prevotella, what differentiates them in the lab
Porphyromonas is asaccharolytic while Prevotella is saccharolytic
Fusobacterium sp.
significance
Fusobacterium nucleatum
-anaerobe
-NF in URT, GIT, urinary tract
-causes sinus and dental infections, brain abscesses, pulmonary infections and blood infection
Fusobacterium species
lab diagnosis
-Anaerobic
-Long spindle-shaped GNB with pointed ends
-growth on BA incubated Ana look like BREAD CRUMBS
-tiny zone AH - green
-if every exposed to air itll die
-slow grower
-convex , raised opaque, dry bread crumb
Mobiluncus species
clinical significance
-Anaerobic
-NF in female genital tract
-found in synergy with other orgs in bacterial vaginosis
-small, curved, Gram-negative (Gram-variable) bacilli
-hard to isolate
-usually in mixture seen through a stain
Gram Positive Anaerobic
Cocci - GPAC
significance
-NF of GIT, female genital tract and oral cavity
-Genera include Peptostreptococcus, Finegoldia, Parvimonas
-most common cause of anerobic infections
-brain abscesses, intra abdominal wound infections , lung and pelvic infections
-POLYMICROBIAL INFECTIONS
Finegoldia magnus
found where
type of grower
GPAC
- Anaerobic
-most pathogenic of anaerobic cocci and most frequently isolated
-NF of skin microbiota
-Opportunistic - degrades collagen in skin
-isolated from wound, chronic wound (diabetic or pressure ulcers), soft tissue abscess and prosthetic joint infection
-slow growing 2-5 days
-PPT on ANA blood
-slow grower 2-5 days
-MALDI
Parvimonas micra
-predominant GPAC
-ORAL microdata
-increased with periodontal trauma
-small zone in SPS disc no ZOI
Anaerobic
Peptostreptococcus anaerobius
GPAC
-BPC long Chains
-Anaerobic
-obligate anaerobe
-slow growing; needs 48 hours for recognizable
-presumptive = Sensitive to sodium polyanethol sulfonate (SPS) with ZOI >12mm
-small ppt on ana BA
Veillonella
-Anaerobic small GNC
-NF in GIT, oral cavity and female genital tract
-rarely implicated in disease
-isolation and ID not attempted often
Culturing Specimens for
Anaerobes
COLLECTION AND TRANSPORT
- anaerobes are found in URT, GIT and female genital tract and skin
-doing anaerobic culture in these areas are not appropriate
-try not to contaminate
-swabs of these areas are discouraged but cant be avoided so transport them in media
-needle aspirates with cap
-collect aseptically from deep wounds and abscess nothing superficial
-curetting or tissue
-gauze from draining sinus if Actinomyces - sulfur granules
-send to lab in 20 mins
-dont refrigerate as anaerobes can be killed off in the cold
Macroscopic Examination of anaerobic specimen
-before culture look for:
-odor
-gas
-discharge
-pus or blood
-necrotic tissue
Microscopic Examination anaerobic specimen
-use carbol fuchsin rather that safranin in negatives - makes the bacteria brighter
-methanol fixing is better than heat fixing
-methanol preserves cell integrity
what type of plate would be best suited for anaerobes
-fresh plate of a prereduced plate so the presence of oxygen is reduced
-anaerobic media can have Vit K, hemin, laked blood, yeast extract and 1-cystine
What is anaerobic selective media
PEA (phenyl ethyl alcohol) added to BA and inhibits facultative gram neg - like a CNA
-always have a series of plates to determine what the org is
-Kanamycin and Vancomycin in KVL are added to anaerobic basal media selecting for bacteroides
-make sure to use BA and MAC to detect facultative or aerobic bacteria in the sample
What is Brucella agar
- enriched agar with hemin and Vit K which supports bacteroides and Gram Pos growth
-Whole blood is used to determine hemolysis
Culture Media – Tube for anaerobes
-fluid or broth media
-Thioglycolate broth - a reducing agent that maintains lowered oxygen tension
-shows what the preferred environment for the org
Resazurin - redox indicator that tells if there is O2 (pink) or no (no pink)
Vit K and hemin - reduces inhibitory effect of thioglycolate on some anaerobes
Cooked Meat (or Chopped Meat) glucose
-contains reducing agent glutathione and glucose
-supplemented with Vit K and hemin
-turbid if inoculated
-used specifically for C perfringens
-good for anaerobes
how will anaerobes be incubated
gas pak
- GasPak that produces hydrogen gas that mixes with O2 to form water
-methylene blue indicator ; blue when oxygenated and colorless under ANO2
-the whole tube can be placed in O2 incubator
-anaerobic atmospher will be produced in hours
-sachet has inorganic carbon, activated carbon, ascorbic acid and water which all becomes activated when exposed to air
how will the Identification flow work
-Gram
-**test for aerotolerance for every org that grows on an ANA plate.
-Subculture suspect colonies from Brucella plate onto CHOC (co2) (helps with fastidious) and BA for (ANA) -see what it grows on PROVES ANA
-next test is discs - gram stain helps to choose the disc if small GNB a potential ana use the 20% bile disk (can do with the aerotolerance disk but put the bile disk on the primary quad of the blood agar)
-with ANA GPC same use an SPS disk with aerotolerance on primary quad
-for large GPB NO DISK - GRAM, AEROT TEST and REVERSE CAMP (if neg then grouped as OTHER ANA GNB)
-for ANA GNC gram , aerot test and prove its ana
-Haemophilus flu will grow on anaerobically incubated Brucella agar but can be differentiated from an anaerobe by growth on CHOC plate from CO2 incubator
-ID disks: Potency discs/MicroRing or Anident, SPS, Bile
-Catalase
-Spot indole
-Reverse CAMP test
-Fluorescence
how to use the Identification Discs – Special Potency Antimicrobial discs
FOR ANIDENT discs
-using known susceptibility patterns to identify orgs
-streak no incubation
-GNB mainly
-streak for lawn of growth on BA, put discs on plate and incubate anaerobically
-B fragilis is S to rifampin and R to Kanamycin, Penicillin, Vanc, and Colistin
GP are Vanc-S and Colistin - R
GN are Vanc -R and Colistin -S
SPS disc
-place in primary quad of AEROT
-streak with 2 quad method and look for ZOI >12mm is S (anaerobius)
-all other GPC ANA are R
What is the reverse CAMP
-when alpha toxin of C prefingens interacts with the CAMP factor of Group B strep to enhance hemolysis
-other Clostridium spp dont show this
-streak strep agalactiae through the middle and streak perpendicular but dont touch IT
-alpha factor interacts with the CAMP factor and diffuses into the agar and creates an arrowhead hemolysis
Susceptibility testing of anaerobe
-not usually done on slow growers
-MICE has been used
-follow CSLI for broth dilution adn micro dilution
-B. fragilis needs brucella broth with lysed horse blood
-penicillin not effective against Bacteroides
-Erythromycin not effective against Fusobacterium
-Vancomycin effective against gram positive
Preferred Antibiotics For Suspected Anaerobic Infections
Above the Diaphragm
Below the Diaphragm
Everywhere
Above the Diaphragm
Clindamycin
Below the Diaphragm
Metronidazole
Everywhere
B-Lactam/combos
Carbapenems
Moxifloxacin
