Introduction to anaerobes

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Introduction to anaerobes
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Introduction

• Believe it or not oxygen is a highly toxic
  substance. Some forms are more toxic than
  others.
• Use of oxygen, or in some cases merely surviving
  in its presence requires mechanisms to deal with
  oxygen toxicity.
• Charged, electron withdrawing oxygen free
  radicals are referred to as reactive oxygen
  species (ROS). ROS are potent oxidants that can
  at least break bonds and cause damaging mutations
  (sometimes carcinogenic).
• All organisms capable of aerobic respiration
  depend on 2 protective mechanisms 1) oxygen
  reducing cytochrome oxidase proteins (vs
  cytochrome oxidases involved in anaerobic
  respiration) that terminate their electron
  transport systems, and 2) two enzymes that work
  together to detoxify a particular ROS by-product
  of aerobic respiration superoxide (O-2)

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Introduction

• 2 H 2 O-2 ? Superoxide dismutase (SOD) ? H2O2
• 2 H2O2 ? Catalase ? 2 H2O O2
• Together these enzymes convert the ROS superoxide
  into harmless water and molecular oxygen.
• Obligate anaerobes produce neither of these
  enzymes.
• The term obligate anaerobe does NOT necessarily
  imply that oxygen is bacteriacidal, although this
  is the case for some unusual strict obligate
  anaerobes such as the Methanogens.
• Medically important obligate anaerobes can
  survive the presence of oxygen but cannot grow in
  it (ie. oxygen is bacteriastatic to them).
  Actually, they can grow in O2 less than 0.5.

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Introduction

• Strict obligate anaerobes could not function as
  pathogens due to limiting concentrations of
  oxygen in every body tissue. Consider the
  difficulty of manipulating samples and culturing
  strict obligate anaerobes!
• Obligate anaerobes are also sensitive to positive
  redox potential values (redox is short for
  oxidation-reduction).
• Positive redox values (Eh) indicate a tendency
  to oxidize (withdraw electrons) as does oxygen
  (aerobic env.). Negative redox values (-Eh) tend
  to reduce (donate electrons) which is indicative
  of anaerobic environments, and favorable to
  obligate anaerobes.
• They prefer Eh lt -50mV. Reducing conditions are
  maintained in media using reducing agents such
  as thioglycolate or the amino acid cysteine.
  Reducing agents are synonymous with the idea of
  anti-oxidants.

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Introduction

• Obligate anaerobes (I will just call them
  anaerobes from now on) fall into 2 metabolic
  groups. The fermenters do not conduct
  respiration at all and require no terminal
  electron acceptor. They are slow growing as
  fermentation is a relatively inefficient means of
  acquiring energy. Among the anaerobes,
  fermenters are relatively oxygen tollerant
  (generalization). Medically important anaerobes
  are fermentors.
• Other anaerobes conduct anaerobic respiration.
  Here a non-oxygen terminal electron acceptor is
  used. They inhabit sub-surface environments
  (soil, benthose, other) and generate reduced
  compounds such as sulfide, ammonia, methane, and
  reduced metals such as ferrous iron. They are
  cool but not medically important so who cares,
  right?

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Introduction

• Anaerobes can be found in or on virtually any
  body location, but are most prevalent in the oral
  cavity, GI tract, genitourinary tract, all mucous
  membranes and even skin.
• These guys utilize your body chemistry and other
  microbes to shelter them from oxygen or scrub
  oxygen from their surroundings.
• Mucous membranes possess natural reducing agents
  such as vitamins, poysaccharides, lipids and
  proteins. Anaerobes can submerge in the mucous
  as a O2 shelter
• Anaerobes often exist in a microbial community
  which includes aerobic organisms that consume or
  scrub oxygen from the surroundings. These
  biofilms are stratified with anaerobes being in
  a lower layer of the film.

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Introduction

• Anaerobes relationship with the human host is
  varied.
• Some anaerobic species have important human
  mutualistic function, including normal colon
  flora that aid in digestion (Bacteroides), and
  acidification of the female genitorurinary tract
  (Lactobacillus). Others are now considered
  commensal, but all of the above at least control
  growth of opportunistic pathogens.
• Types of human pathology associated with
  anaerobic bacteria include intra-abdominal
  infections, pulmonary infections, pelvic
  infections, brain abscesses, skin and soft tissue
  infections, oral infections, and bacteremia and
  endocarditis. With the exception of the
  Clostridia, the mechanism by which the anaerobes
  cause human pathology is not well understood

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Methods

• An anaerobic environment can be provided in
  several ways. These include pre-reduced
  anaerobically sterilized media (PRAS), Gas Pak
  Jar, anaerobic pouch, anaerobic glove box, and
  roll tubes
• PRAS media are commercially available in agar
  plates, agar slants, or broths. They are
  manufactured in an oxygen free environment and
  packaged individually in air-tight sealed pouches
  or bags.
• Regular commercial media can be pre-reduced by
  placing it an anaerobic jar or glove box for a
  few hours prior to inoculation with clinical
  specimens
• The Roll tubes technique was developed at
  Virginia Techs Anaerobe Lab, and is excellent
  for maintaining a completely anaerobic
  environment. However, the technique is very
  tedious and rarely used in the clinical laboratory

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Methods Gas Pak

• The anaerobic jar method (e.g. Gas Pak Jar) was
  introduced into the clinical laboratory over 30
  years ago and was the most commonly used method
  until introduction of the anaerobic pouch
• Inoculated anaerobic culture media are placed in
  the jar
• An envelope (i.e. Pak) containing sodium
• borohydrite and sodium carbonate is placed
• in the jar to generate hydrogen and CO2
• Water is added to the envelope which is
• also placed in the jar. A methylene blue test
• strip is added, and the jar is sealed.

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continued

• Hydrogen generated when water combines with the
  borohydrite chemically binds to oxygen trapped in
  the sealed jar to form water an anaerobic
  environment
• A small wire basket attached under the lid
  contains aluminum pellets coated with palladium.
  This serves to rapidly catalyze the reaction.
• If the lid of the jar is not warm to the touch
  within 40 minutes after it is sealed, or if
  condensation does not form within the jar within
  25 minutes, the jar should be opened and entire
  process be repeated
• Likewise, if the methylene blue indicator strip
  in the jar or pouch does not turn white within
  one hour, the process should be repeated

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continued

• The sodium bicarbonate in the envelope releases
  carbon dioxide. This is unrelated to the
  production of the anaerobic environment, however,
  growth of most anaerobes in culture is enhanced
  by the presence of carbon dioxide
• Hydrogen sulfide and other gases produced by
  bacterial growth can poison the palladium
  catalysts when used over and over. To prevent
  this, the palladium catalyst should be
  periodically heated in a dry oven at 160-170oC to
  remove the contaminating gasses and thus
  regenerate the palladiums catalytic abilities.
  This is referred to as re-charging the
  palladium.

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Gas Pak Jar
Clamp
Lid
Palladium catalyst in wire basket
H2 CO2
H2O
Sodium bicarbonate
Foil Envelope Gas Pak
Sodium borohydrite
Culture Plates
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Anaerobic Pouch

• Anaerobic pouches work similarly to gas pak jars
  and are currently most often used in the clinical
  laboratory
• They are sealable bags made of oxygen impermeable
  see-through plastic material
• They hold one to two agar plates or a few tubes
• The major advantage of anaerobic pouch is that
  plates and tubes can be examined without opening
  to pouch thus avoiding exposing them to oxygen if
  there is no growth
• They are also more convenient.
• See Figure 14-7 on page 729.

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Anaerobic Chambers and Holding Jars

• Anaerobic glove boxes or chambers and holding
  jars are self-contained anaerobic chambers.
• Air is removed with a vacuum pump and replaced by
  oxygen free gas (usually 85 nitrogen, 10
  hydrogen and 5 carbon dioxide) from compressed
  gas cylinders
• The anaerobic glove box has a sufficiently large
  volume to allow culture and media manipulation.
  Specimens and media can be passed in and out of
  an automatic entry lock
• Technicians place their hands in the gloves
  protruding into the chamber
• The main purpose of the anaerobic holding jar is
  to pre-reduce media and hold it under these
  conditions until there are enough specimens to
  fill a gas generating jar

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Glove box
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Culture of Anaerobes

• Specimens must be placed in a transport system
  that excludes oxygen (reducing agent, etc.), and
  be processed with a minimum exposure to oxygen
• Media must be less that 1 week old or be
  pre-reduced, in either case to reduce ROS
  concentration
• Tom Pre-reduced media can be placed in a sealed
  cellophane bag in a refrigerator (???) for a few
  days or it can be used immediately.
• Anaerobe recovery media should include
  non-selective, selective and enrichment types.
• Vitamin K and hemin are commonly added to
  anaerobe culture media as these are absolute
  growth requirements for some clinically
  significant anaerobes
• For a non-selective medium, CDC suggest a
  modified blood agar (AnBAP) with the above as a
  general purpose medium. It will recover
  anaerobes but other pathogens as well, which is
  important. BHI is also good, among others

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Culture of Anaerobes

• Thioglycollate enriched with vitamin K and hemin
  or chopped meat glucose are usually included as a
  back-up broth in case the anaerobic technique
  fails due to technical problems. Thioglycolate
  (S) is a good reducing agent and anaerobes such
  as Clostridia like meat.
• For selective media, anaerobic phenyl ethyl
  alcohol (PEA) agar can grow most anaerobic
  bacteria, as well as aerobic or facultatively
  anaerobic bacteria. The PEA inhibits the
  swarming of Proteus and growth of most other
  potentially contaminating enteric gram negative
  rods
• Kanamycin/Vancomycin is a good selective medium
  when Gram negative anaerobes such as Bacteroides,
  Prevotella and Fusobacterium are suspected.
  Kanamycin inhibits the enteric rods (but these
  anaerobic genera are resistant), and vancomycin
  inhibits Gram positive cells.

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Aerotolerance Tests

• Anaerobic growth is no guarantee that the
  organism is an obligate anaerobe could be
  facultative or aerotollerant.
• Colonies growing on agar plates incubated
  anaerobically must be subcultured to two
  different media to see if it is an obligate
  anaerobe
• One of the sub-cultured plates is incubated in
  air and the other is incubated in an obligately
  anaerobic environment

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Example Aerotolerance Tests
A
B
A
B
C
D
D
C
Four different morphotypes grew on the original
an- aerobic plate. Each was subcultured to two
non-selective enriched blood agar plates. The
plate on the left was incu- bated anaerobically
and the plate on the right was incubated in air.
Organisms A and C are obligate anaerobes.
Organ- ism B is a facultative anaerobe. Organism
D is either an very strict obligate anaerobe or
requires longer incubation (further testing is
required)
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Identification

• Identifying anaerobic bacteria to species is
  technically challenging in most cases.
• Fortunately species identification is often not
  necessary. A mixture of anaerobes in a clinical
  specimen is often reported as such with no
  further testing.
• With a few exceptions presumptive ID via
  morphology and a few simple tests is sufficient
  information to allow appropriate antimicrobial
  therapy
• Most anaerobes have a predictable antimicrobial
  sensitivity pattern antimicrobial susceptibility
  testing of anaerobes is therefore rarely required
• Standard drugs such as penicillin, clindamycin or
  metronidazole are almost always effective against
  obligate anaerobes

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Species Identification

• There are many commercially available
  identification kits for identifying obligate
  anaerobic bacteria.
• Some require standard inoculation and extended
  anaerobic incubation prior to reading results.
  Others utilizing heavy inocula give quick results
  from pre-existing enzymes. These therefore do
  not require anaerobic incubation.
• Reference labs ID anaerobes chromotographically
  on the basis of fatty acid methyl ester (FAME)
  profiles
• Other macromolecule probes or homology can be
  used as well