Clinical microbio I: Anaerobes Flashcards

Varying degrees of oxygen intolerance

• strict anaerobe

• aerotolerant anaerobe

• facultative anaerobes

Strict (or obligate) anaerobe

• use alternate electron acceptor for respiration such as sulfate, nitrate, iron, manganese, mercury, and CO2

• tolerate <0.5% O2

• are killed after exposure to air for only a few minutes

• Treponema denticola, Selenomonas ruminatium, Clostridium novyi type B, Peptostreptococcus spp.

Aerotolerant anaerobe

• majority of anaerobic organisms

• tolerate 2-8% O2

• survive exposure to air for several hours on an agar plate without loss of viability

• require anaerobic environment to multiply

• Bacteriodes fragilis, Porphyromonas melaninogenicus and Clostridium tertium

Oxygen tolerance

• correlated with concentration of super oxide dismutase (SOD), catalase, or peroxidases

• gives ability to breakdown toxic O2 radicals

• no enzymes = toxicity

oxidation reduction (redox) potential (Eh)

• O2 tolerance also linked to redox potential

• Eh +150mV = normal tissue

• Eh = -420mV = lowest in nature

• large bowel = -250 mV

• any decrease in BF results in decrease in the Eh to that tissue

• anaerobic bacteria don’t survive is Eh is above -100mV

• some organisms need both low O2 & low redox potential

Growth in thioglycolate broth

• thioglycolate is a reducing agent that takes away O2 from tube (more O2 on top vs less on bottom)

• gradient of O2 is created

• semi-solid medium

Anaerobic rod-shaped bacteria

• GBP spore forming

  • Clostridium spp

• GPB non spore forming

  • actinomyces

  • bifidobacterium

  • propionibacterium

  • anaerobe lactobacillus

• GNB

  • bacteroides fragilis

  • prevotella

  • porphyromonas

Anaerobic cocci

• GNC

  • Veilonella spp

• GPC

  • Peptostreptococcus spp

  • Peptococcus spp

Specimen collection

• use needle + syringe

• tissue samples or biopsies

• if swab is used, sample should be transported using special transport medium (e.g. Amies)

Specimen transport

• depends on nature of specimen

  • large volumes of material keep viability of anaerobes for longer periods

  • small volumes, small biopsies require anaerobic transport device

• avoid extreme heat or cold

• never transport material for culture in a syringe with needle

Specimen processing

• visual examination

  • purulent, bloody, necrotic tissue, foul odor sulfur, granules, etc.

• specimen prep

  • vortex purulent material, grind bone or tissue, centrifuge non-purulent material and use sediment

Specimen inoculation

• inoculate specimen in aerobic and anaerobic media, and liquid medium

• place inoculated plates immediately into an anaerobic atmosphere

• incubate the rest of the plates in 5% CO2 or aerobic incubator

Media for anaerobic culture

• Pre-reduced anaerobically sterilized media (PRAS)

  • made in anaerobic conditions

  • no O2 contamination

PRAS media - non-selective

• Trypticase soy agar (TSA)

• Brain-heart infusion (BHI)

• Columbia, Brucella, or Schaedler’s

  • they all have 5-10% sheep RBC, a nutrient base and added enrichments

PRAS media - selective

• Kanamycin and Vancomycin (KV)

  • inhibits GP organisms

  • selective for GN (like MAC)

  • no color differential

Phenyl ethyl alcohol agar (PEA)

• aka rose agar = smells like roses

• selective for GP

• inhibits most GN, facultative-anaerobic bacteria and prevents swarming

  • Bacteroides and Prevotella (GNB) grow in PEA

Bacteroides bile esculin agar (BBE)

• selective for certain Bacteroides species - bile tolerance

• Differential based on esculin hydrolysis - dark brown color

Laked blood agar

• lysis of sheep RBC by freeze-thaw method

• produces clear red lysate

• similar nutritional value to chocolate agar

• everything grows

Laked blood agar with antibiotics

• with kanamycin and vancomycin (CDC medium)

• enriched, but also selective for GN anaerobes

• however, not all GN grow on LKV

Cycloserin-cefoxitin fructose agar (CCFA)

• selective for Clostridium difficile

• growth in this medium is not definitive diagnosis

Glove box chamber

mixture of gases such as nitrogen or argon and H is flushed through the glove box to assure that the interior of the chamber is maintained anoxic

Anaerobic jars

• tablets in pouch

  • citric acid with Na bicarbonate

  • Na borohydrate with cobalt chloride

  • add water

• generates CO2 + H2 which combines with ambient O2 → H2O

anaerobic indicators

• mechanism to detect O2 in system and media

• dyes act as electron receptors or donors

  • colorless = reduced

  • color = oxidized

• methylene blue

• resazurin (pink if O2)

Microscopic examination

• large GPB box car shape

  • Clostridium perfringens

• Thin GNB with tapered ends

  • Fusobacterium nucleatum

• Pleomorphic palely stained GNB

  • Bacteroides

Identification of anaerobe organisms

• RapID ANA strips or Vitek ANI cards with a set of biochemical reactions

• always correlate with:

  • microscopy

  • colonial morphology

  • source of specimen

• If biochemicals give equivocal results, use antibiotic identification disks

  • Colistin 10ug

  • Vancomycin 5ug

  • Kanamycin 1ug

• typical susceptibility pattern

Additional tests for identificaiton

Stormy clot formation

Gas liquid chromatography

• heats liquids to vaporize

• temp and time to vaporize is distinct for each product

• detects acid or alcohol products from glucose or amino acid fermentations

• patterns give primarily genus identification

  • Bacteroides, Prevotella, Porphyromonas

  • Clostridium

  • Fusobacterium

Polymicrobic anaerobic infection

• many species in human flora

• many grow simultaneously

  • opportunistic growth

    • injured tissue *limited blood/O2

  • No growth

    • healthy tissues *high O2 content

• facultative anerobe

  • diminishes O2 supply further

  • aids growth of obligate anaerobes

Clostridium

• C. tetani

• C. botulinum

• C. perfringens

• C. difficile

Clostridium tetani

• GPB

• produces terminal spherical swollen spores (drumstick, tennis racquet, lollipop)

• slow grower

• swarming growth on lab media

• rarely isolated

C. tetani virulence factors

• produces toxin = tetanospasmin

  • responsible for pathological effects

  • heavy chain binds receptors on motor neuron

  • lighter chain taken up through endocytosis

• tetanospasmin blocks inhibition of motor neurons, causing paralysis

  • muscle contraction is uncontrolled

  • muscles don’t relax

  • paralysis usually begins in the jaw (locked jaw)

Tetanus

• spastic paralysis

• aka “lockjaw”

• frequently fatal

• bacterial spores prevalent in dust and soil

  • difficult to avoid exposure

Tetanus epdemiology/treatment/prevention

• found in dirt/dust and GI tract of humans and other animals

• nearly half of infections result from puncture wounds including

  • body piercing, tattoo, animal bites, injected drug abuse

• 30-60 cases in US annually with 25% mortality rate

• immunization has decreased incidences in economically advanced countries

Clostridium botulinum

• causes botulism, rare but serious paralytic illness caused by Botulinum neurotoxin

• Foodborne botulism

• Wound botulism

  • injected drug users are at increased risk

• infant botulism

  • caused by consuming the spores, which then grow in the intestines and release toxin

Botulinum toxin: neurotoxin

• BTX is the most potent toxin known

• 1ug is deadly for humans

• metalloproteinase that acts as on presynaptic membranes at the neuromuscular junctions

• it cleaves SNAREs, proteins involved in release of acetylcholine at the synapse

• w/o release of acetylcholine, paralysis of the motor system occurs

Identification of C. botulinum

• the definitive diagnosis is ID of toxin in serum, stool, or gastric contents (bioassay) only done in reference labs such as CDC

• identifying it alone in food is not enough

• cells are gram variable bacilli that show profuse sub-terminal and free spores

• Hemolysis is variable, but odor is strong and reminiscent of rotten eggs due to production of H2S

• Lipase + Indole/urease -

• can be isolated from clinical samples such as - feces, wounds, tissues, and pus as well as from foods

Clostridium perfringens

• large rectangular GPB

• subterminal spores, seldom seen

• non motile

• produces several toxins

  • alpha (lecithinase)

  • beta (necrotizing enteritis)

  • Enterotoxin

• double zone of hemolysis

where is C, perfringens found

• feces of humans and animals

• vaginal tract

  • established in vaginal tract of 1-9% of healthy women

• gas gangrene of uterus

  • fairly common after self-induced abortion

  • rarely seen after miscarriage and childbirth

C. perfringens diseases

• bacteremia

• myonecrosis

• food poisoning

• enteritis necrotica (pig bel)

Clostridial myonecrosis or Gas gangrene

• caused by

  • presence of dirt and dead tissue in wound

  • long delays in treatment

• primarily disease of wartime

Clostridial myonecrosis or Gas gangrene pathogenesis

• Produces α toxin

  • Toxin attacks host cell membrane

    • diffuses and kills tissue cells

  • Other enzymes breakdown macromolecules of dead tissues

• C. perfringens unable to grow in healthy tissue

  • Survives well in dead or poorly oxygenated tissue

  • Releases toxin in tissue

• Bacteria produces gas through fermentation

  • Gas accumulates in tissue, contributing to spread

Clostridium difficile

• large, straight, thin GPB

• produces oval, subterminal spores rarely seen

• motile

• circular or rhizoid colonies

  • 2-5mm

• characteristic odor: manure, stable

• in BAP fluoresce chartreuse under UV light

• grows in cycloserine, cefoxitin-fructose agar (CCFA)

  • yellow colonies w ground appearance

C. difficile pathogenesis

• found in human GI tract in small #s

• associated with antibiotic use

  • clindamycin, ampicillin, cephalosporins

  • kill NF, increase number of C. diff in GI tract

• Toxin A-enterotoxin & toxin B -cytotoxin

C. difficile diagnosis

• detection of toxins in stools (EIA, cell culture, NAAT)

• culture of organism

• Clinical - AAC (ATB associated colitis) or Pseudomembranous colitis (PMC)

• treatment: omit antibiotic if possible/oral vancomycin, or fecal transplant

Actinomyces

• strict anaerobic GP bacilli typically arranged in hyphae-like structures which fragment into short bacilli

• NF of upper respiratory tract, GI tract, and female genital tract

• low virulence

• produce disease when mucosal barrier is breached (dental trauma or surgery)

• establishes chronic infection that spreads through normal anatomical barriers

Actinomycosis

• Chronic suppurative disease

• Cervicofacial, abdominal, and thoracic

  • progresses slowly; painful swelling under the skin

  • swollen regions open and drain pus

    • chronic condition

    • openings usually heal

      • lesions reappear at the same or nearby region within days or weeks

• most cases involve jaw or neck

  • recurrent lesions can develop on chest and abdominal wall or genital tract of women

• scars and swelling give rise to name “lumpy jaw”

Actinomycosis diagnosis

• Gram stain of ‘sulphur’ granules

• culture

• treatment - surgery and long term penicillin

Actinomycosis pathogenesis

• A. israeli cannot penetrate healthy mucosa

• infection is characterized by cycles

  • abscess formation → scarring → formation of sinus tracts

    • passageways underneath the skin that can extend in any direction through soft tissue and allows for spread

• disease progresses to skin and can penetrate bone or CNS

• in tissue, culture grows as dense yellow colonies

• nearly 50% cases originate in mouth

Actinomyces israelii

• GP

• filamentous

• branching

• anaerobic

• slow growing

Cutibacterium acnes

• previously known as Propionibacterium acnes

• GP pleomorphic rod

• inhabits human skin, sebaceous glands, nasopharynx, GI/GU tracts

• most common, non-spore forming anaerobic rod found in clinical specimens

• ACNE

• frequent blood culture contaminant

• most frequent serious infection: CNS shunt infection

Lactobacillus

• GPB

• major part of the lactic acid bacteria group

  • convert lactose and other sugars to lactic acid

• NF of vagina and GI tract

• symbiotic and make up small portion of gut flora

• production of lactic acid makes the vaginal environment acidic, which inhibits the growth of some harmful bacteria and yeast

Bifidobacterium

• GPB non motile

• NF of the vagina and GI tract

• make up big portion of the gut flora in colon

• aid in digestion, associated with lower incidence of allergies

• used as probiotics

Mobiluncus

• GPB

• part of polymicrobial bacterial vaginitis

• crescent-shaped or curved rods

• motile with polar flagella

• inconsistently GP

Peptostreptococci

• GPC, non-spore forming

• slow grower

• NF of muco-cutaneous surfaces

  • mouth, intestinal tract, vagina, urethra, skin

• P. magnus is the most commonly isolated anaerobic cocci

• includes species within the genus formerly known as peptococcus

Anaerobic GN bacilli

• Bacteroides, Prevotella, Porphyromonas and Fusobacterium

• large component of NF of GI tract

• >80% infections associated with B. fragilis

• grow in leaked blood agar KV

  • stimulates pigmentation of porphyromonas

• Clinical- Endogenous infections

  • intra-abdominal pyogenic infections

  • pleuro-pulmonary infections

  • genital infection

Bacteroides

• slender rods or cocobacilli

• Prevotella and Porphyromonas were previously classified as Bacteroides

• infection cause by injury of the colon (peritonitis)

• ID: grow in laked blood agar KV

  • colonial characteristic

  • short chain fatty acid by LGC

  • biochemical features

Bacteroides fragilis

• rapid ID is important bc is more resistant to ATB than other anaerobes

• bacteroides bile esculin agar is selective for Bacteroides (contains gentamicin and bile salts)

• differential for hydrolysis of esculin

  • other bacteroides grow but don’t turn medium brown

Prevotella

• GNB similar to Bacteroides

• mostly in upper respiratory tract (mouth)

  • P. melaninogenica

    • tooth gum infection

• also in female GT

  • P. bivia and P. disiens

• brain and lung abscess

• PID and tubo-ovarian abscesses

• associated with other ANA

Porphyromonas

• GNB

• produce porphyrin pigments

  • dark brown/black pigments

• red brick fluorescence under UV

• also breast and axillaries(armpit) infections

• male GT and perianal infections

• found in gingival or tooth infection

• P. gingivalis

Fusobacterium

• GNB pleomorphic

  • fusiform, tapered ends

• produce butyric acid and propionic acid

• mix bacterial infection

• also neck, breast and axillaries infections

• male GT and perianal infections

• F. necrophorum, F. nucleatum, F. novum

Veillonella

• GN coccus

• NF of the mouth and nasopharynx

• implicated in cases of osteomyelitis and endocarditis

• polymicrobic infection

Antibiotic disks for ID of GN anaerobes