Unit VIII
Anaerobes
Anaerobe Terminology
Terminology:
• 1. Aerotolerant: ability of an anaerobic microorganism to grow in air; usually poorly,
especially after initial anaerobic isolation.
• 2. Glove/Gloveless Box: a device with flexible, semi-rigid or complete rigid plastic
walls used for the cultivation of anaerobic bacteria. The bacteriologist must work by
way of glove ports and materials that are brought into and out of the anaerobic work
area via an interchange (air lock-port hole) that can be evacuated and refilled with an
anaerobic atmosphere repeatedly until it is anaerobic. The interchange has one door leading
into the glove box and another door to the outside. Some glove boxes have incubators within
them . Also, called “anaerobic chamber” or “anaerobic cabinet”.
• 3. Laked Blood: a.k.a. “hemolyzed blood” – hemolysis of red blood cells used in agar may be
effected in various ways but alternate freezing and thawing is a simple method.
• 4. Meconium: pasty, greenish mass in the intestines of fetuses; made up of mucus, desquamated
cells and bile and such.
• 5. Gas Liquid Chromatography (GLC): a method for separating substances by allowing their
volatile phase to flow through a heated column with a carrier gas (usually helium) and
measuring the time required to detect their presence at the distal end of the column.
• 6. Pseudomembrane: necrosis of mucosal surface simulating a membrane
• 7. Pseudomembranous Colitis (PMC): syndrome in the large bowel characterized by a layer of
necrotic tissue and dead inflammatory cells often caused by the toxin of Clostridium difficile.
• 8. Sulfur granules: small colonies of organisms with surrounding club-like material; yellowbrown; resembles grain of sulfur.
ANAEROBES
• Obligate anaerobe - an organism that can not grow in the
presence of oxygen. The anaerobic bacteria are subdivided
according to their requirement for O2. Obligate anaerobes are
killed by exposure to atmospheric O2 for 10 minutes or longer.
• Facultative anaerobe - an organism that can use aerobic or
anaerobic respiration or anaerobic fermentation system,
depending on the availability of oxygen.
• Microaerophilic – organisms that will not grow in an aerobic
incubator on solid media, and only minimally under anaerobic
conditions. However, they will grow in minimal oxygen (5%
O2). Superoxide dismutase (SOD) is produced by many
anaerobes, which catalyzes the conversion of superoxide radicals
to less toxic H2O2 and molecular O2.
Eh POTENTIAL: (oxidation-reduction potential)
• Eh is a measure of the tendency of a system to give up electrons
• expressed as millivolts
• as Eh potential is lowered by 60 mv, the pH increases by 1
• pH affects the oxidation-reduction potential (Eh) of media for anaerobic
bacteria more than oxygen
• ** Eh is most affected by pH and is expressed at pH 7.0. In cultivating
anaerobic bacteria, reducing agents such as thioglycollate and L-cysteine are
added to anaerobic transport and culture media in order to maintain a low
Eh. Certain anaerobes will not grow in the media above a specific critical
Eh level
• most anaerobic media is at - 150 mv
• actively growing cultures are Eh = -250 mv
• if Eh is low enough, anaerobes may grow in the presence of oxygen
concentrations which would ordinarily be inhibitory
• when surfaces are damaged or necrosis occurs then the Eh potential is
lowered
CLINICAL SIGNS OF ANAEROBIC INFECTIONS:
• foul smelling discharge
• infection in proximity to a mucosal surface
• necrotic tissue, gangrene; pseudomembrane formation
• gas in tissues or discharge or presence of “sulfur
granules” in discharges
• endocarditis with negative blood cultures
• infection associated with malignancy or tissue destruction
• infection related to the use of aminoglycoside drug
therapy (oral, parenteral or topical)
• infection following human bites, septic abortion or
gastrointestinal surgery
Anaerobic Specimens
UNACCEPTABLE Specimens for Anaerobic Culture
• - throat, ear, NP swabs, sputum, tracheostomy
• - feces or rectal swabs
• - voided or cath urine
• - vaginal or cervical swabs
• - urethra
• - superficial wounds/abscesses
• - GI or abdominal wounds contaminated with feces
ACCEPTABLE Specimens for Anaerobic Culture
• - transtracheal aspirate or direct lung puncture
• - feces only requested for Clostridium difficile
• - suprapubic bladder tap
• - culdocentesis aspirate, placenta, Bartholin’s gland
• - prostatic or seminal fluid
• - abscess aspirates or deep wounds collected to avoid skin flora
Specimen Collection and Handling
SPECIMENS
• Proper Collection:
• 1. Specimens for anaerobe culture must be sent in the proper anaerobic transport tubes
which may include: “Port-a-Cult” semi-solid agar tubes, COPAN tubes, a closed syringe
(best) or a gassed out tube.
• 2. Tissue may be cultured anaerobically but it should be ground inside the chamber to minimize
the amount of oxygen forced into the tissue during the grinding process.
• 3. When anaerobic culture on CSF is requested, the culture may be set up from the regular
CSF collection tube as long as it is done immediately upon receipt of the specimen.
• 4. If a valid specimen for anaerobic culture is received in an inadequate transport device and
cannot be recollected, consult with the laboratory supervisor or the Dept. Head.
• 5. Certain specimens are unsuitable for anaerobic culture. These include specimens coming from
body sites normally inhabited with normal anaerobic flora. (See acceptable versus unacceptable
specimens).
• 6. On vaginal and cervical cultures (from surgery which the doctor is persistent about culturing)
which yield a light or moderate growth of anaerobes mixed with aerobic flora, note the
presence of the anaerobe on the worksheet, but do not identify. Send the report out as "No
Significant Anaerobes Isolated" or “Mixed Anaerobic Flora”. Identify (either by site or Rapid
ID ANA) only a heavy or pure growth. NO SENSI.
Specimen Collection and Handling:
Specimens must be collected and transported anaerobically
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** Needle aspiration - is the best method of collection when possible. The specimen should not sit in the syringe for more than
30 minutes due to the passage of air through
the plastic
** Gassed-Out tubes - are appropriate (gassed with CO2);
Resazurin is often used as the indicator for anaerobic systems.
colorless = Eh -43 mv
pink = increased oxygen
** Inoculating loop should be platinum or plastic NOT nichrome due to agar surface tension breakdown
Inoculation and Incubation:
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1. CDC Anaerobic Blood Plate containing Vitamin K1
Allows for the growth of all facultative organisms (aerobic and anaerobic) including gram positive and gram negative rods,
gram positive and gram negative cocci.
2. Laked Blood plate with Kanamycin and Vancomycin
For selective isolation of obligate anaerobic gram negative bacteria (Bacteroides, Prevotella, Fusobacterium and Veillonella species).
Essentially inhibits all other bacteria encountered in clinical specimens including anaerobic gram positive bacteria.
3. Anaerobic Blood plate with Phenyl Ethyl Alcohol
For isolating obligate gram negative and gram positive anaerobic bacteria. The anaerobic formulation (CDC blood agar with
PEA) inhibits facultative gram negative bacteria such as Proteus species and other members of the Family Enterobacteriaceae but will
support gram positive aerobes and anaerobes and gram negative obligate anaerobes.
4. 5% TSA (tryptic soy agar) Sheep Blood Agar plate
Supports the growth of most aerobic gram negative and gram positive organisms. This plate is incubated aerobically at 35OC
and is used for comparison with the anaerobic BAP.
5. Gram stain slides (2) Usually performed and reported by the Exudate Bench technologist.
All media are inoculated and incubated inside the anaerobe chamber at 35-37oC with the exception of the TSA blood plate. This
is incubated aerobically in CO2 for comparison.
Any organisms recovered from the aerobic plate that were not seen on the corresponding exudate culture are then sent back to
the exudate bench for complete work up and reporting.
All cultures are held for a minimum of 5 days. Any specimen with a request for isolation of Actinomyces species will be held for 7
days. Currently, specimens of vaginal or cervical swabs will be held for 3 days.
Other Media:
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1. Egg Yolk Agar - recommended for determining the production of lecithinase and lipase that aids in the
identification of the Clostridium species
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Lecithinase + : colonies with opaque zone around colony (essentially a surface phenomenon); reaction
occurs within the agar
Lecithinase
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Lipase
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Lipase + : colonies are covered with an iridescent, multicolored sheen, sometimes described as resembling
the appearance of “gasoline on water” or “mother of pearl”. This sheen may also appear on the surface
of the agar in a narrow zone around the colony
** Modified McClung or Neomycin egg yolk agar is used to determine the presence of lecithinase activity,
which causes an insoluble, opaque, whitish precipitate within the agar. Lipase activity is indicated by an
iridescent sheen or pearly layer on the surface of the colony or agar.
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2. Bacteroides Bile Esculin Agar - used for the determination of Bacteroides fragilis which is tolerant of
increased bile concentrations
3. Litmus milk - a differential medium on the basis of lactose fermentation caseolysis and casein coagulating
properties. Skim milk and litmus are used in preparation. Litmus has the advantage of being readily
reduced by certain bacteria
Reactions:
1. Acid: pink color indicates an acid reaction caused by fermentation of lactose.
2. Alkaline: a purple/blue color indicates no fermentation of lactose.
3. Reduction: a white color results when litmus serves as an electron acceptor and is reduced to its leuco base.
4. Coagulation: Clotting is caused by precipitation of the casein by the acid produced from lactose. It may
also be caused by the conversion of casein to paracasein by the enzyme rennin.
5. Peptonization: Dissolution of the clot indicates digestion of the curd or milk proteins by proteolytic
enzymes.
Anaerobic Respiration
Anaerobic Respiration:
• The mechanism of electron transport and oxidative phosphorylation is basically the same in
anaerobes as in aerobes with minor variations:
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The final electron acceptor is not oxygen but is some other inorganic molecule, such as nitrate
(NO3), sulfate (SO4), nitrite (NO2), or an organic molecule, such as fumarate.
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The terminal enzyme component in electron transport in anaerobes is not cytochrome oxidase
but enzymes such as reductases that catalyze the reduction of nitrate to nitrite, sulfate to hydrogen
sulfide and nitrite to molecular nitrogen. One of the most frequently used inorganic electron
acceptors is nitrate, where as fumarate is a more common organic electron acceptor. The
synthesis of terminal reductases is repressed and their activities inhibited by oxygen. This
mechanism is especially important in facultative anaerobes that switch from anaerobic to aerobic
growth when oxygen becomes available.
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Less energy is realized during electron transport under anaerobic conditions. The difference in
the oxidation-reduction potential between the donor and acceptor (for example, between NADH
and nitrate) is not sufficient to generate three molecules of ATP. Depending on the donor and
acceptor, the number of ATP molecules generated during electron transport under anaerobic
conditions is usually between one and two. The rate of growth of anaerobes is therefore,
considerably slower than that of aerobes. A facultative anaerobe, for example, under anaerobic
conditions might require 48 hours of optimal growth conditions to produce a population of cells
that under aerobic conditions should be established in 16 hours or less.
Oxygen and Anaerobic Bacteria:
• 1. Obligate anaerobes are not able to grow in the presence of air, but they do
show different sensitivities to oxygen. Some anaerobic bacterial populations
are almost completely killed when exposed to oxygen for 3 minutes, whereas
others remain viable even though they cannot grow. Indifference to oxygen
by some anaerobes is the result of their inability to reduce oxygen; therefore
toxic intermediates are not produced. In other anaerobes the concentration
of oxygen is the critical factor. Obligate anaerobes are often found associated
with facultative anaerobes in areas where oxygen is present. Facultative
anaerobes can remove oxygen from the environment by using it in aerobic
respiration, thus making the environment suitable for the growth of obligate
anaerobes. Studies with oxygen-tolerant anaerobes have demonstrated that a
positive correlation exists between oxygen tolerance and the content of
superoxide dismutase in the cell. It is now clear that microbial cells whether
aerobes or anaerobes rely on scavenger systems such as catalase, peroxidase,
and superoxide dismutase to remove toxic intermediates produced by the
reduction of oxygen.
Work Up Protocol
WORK-UP PROTOCOL
• A. Identification
• Since the type of specimen accepted for anaerobic culture is carefully defined, any
amount of any anaerobe should be identified. The technologist must remember that
anaerobic infections are rarely caused by one organism; the usual situation is isolation
of at least two or three different anaerobes. Blood cultures and CSF may however,
yield only one organism.
• B. Susceptibility testing:
• Susceptibility testing will be performed on any number of pathogens in any amount
from any normally sterile body site, bone specimens and brain abscesses. (See E Test
Procedure). Occasionally, if a pure or predominant anaerobic organism is recovered
from a non-sterile body site, then discretion is used by the MLS reading the plates.
• C. Direct Gram Stains:
• Gram stains prepared from the specimens will be performed on all cultures. The
results will be reported for both the aerobic culture (sent to Exudate bench) and the
anaerobic culture by the Exudate bench technologist. Occasionally there are instances
when the gram stain shows organisms and the culture is negative.
• D. Beta Lactamase Testing:
• A beta-lactamase test will be performed on all anaerobic gram negative rods and
anaerobic spore-forming gram positive rods. The results of the test will be reported.
(Positive versus Negative).
Gram Stain Procedure for Anaerobes
GRAM STAIN PROCEDURE FOR ANAEROBES
• Procedure:
• 1. Fix the slide with methanol for about one minute. Drain.
• 2. Flood slide with Crystal Violet for one minute.
• 3. Using a freshly mixed bottle of Gram's Iodine (if using unstabilized iodine), wash off the
Crystal Violet and flood the slide with Iodine for one minute.
• 4. Decolorize quickly and gently with 95% alcohol.
• 5. Wash with water.
• 6. Counterstain with Basic-fuchsin for one minute.
• 7. Wash with water.
• 8. Air dry.
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The cell wall of anaerobic organisms is much more fragile than that of aerobic organisms,
and may be destroyed by heat fixing. Fixing the slide with methanol is more protective of the
cell wall. The mordant ability of Gram's Iodine declines rapidly once the solution is prepared.
The gram positive characteristic of anaerobes is often difficult to demonstrate. For these two
reasons, the anaerobe Gram stain must be performed with a freshly mixed bottle of Gram's
Iodine. The decolorizer used for routine gram stains is too harsh and easily over decolorizes
most anaerobes thus using just 95% alcohol yields more accurate results. The interpretation of
Gram stains on anaerobes is often difficult to interpret. As a general rule, an organism which
shows even small areas which stain Gram positive should be considered to be gram positive, as it
is unlikely that the smear has been under-decolorized.
The Gram Positive, Spore-Forming Rods
CLOSTRIDIUM
• - large, gram positive rods
• - obligate anaerobes
• - spores cause cell walls to swell
• - found mainly in soil and intestines of man and
animals
• - discovered in 1877 by Pasteur
CLOSTRIDIUM PERFRINGENS - a. k. a. "WELCH'S BACILLUS"
• isolated by Welch in the early 1900's
• most common cause of gas gangrene (myonecrosis)
• most common isolate of the clostridium
• encapsulated and non-motile; box-car shaped bacilli seen
on gram stain; may stain gram-negative
• spore production is not usually seen; if present, centrally located
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only Clostridium to have a double zone of hemolysis
on anaerobic blood agar (an inner zone of complete beta
hemolysis and an outer zone of partial hemolysis)
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causes stormy fermentation of litmus milk: media changes
from blue to pink, forms a clot, the clot explodes and is
digested to a whey color
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“Reverse camp test" positive
“Unknown” Clostridium
Postive result shows
“arrow-head” hemolysis
www.microbelibrary.org
www.asm.org
Known ATCC Strep agalactiae
www.microbelibrary.org
Clostridium perfringens continued:
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Nagler reaction positive which is recommended for presumptive rapid ID: This test employs
an EYA plate (inoculated at set up of specimen) and a reagent known as C. perfringens type A
antitoxin. This reagent inhibits the lecithinase reaction produced by C. perfringens thus
presumptively identifying C. perfringens. (This test is also positive for 3 other Clostridia as well)..
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on egg yolk agar it is lecithinase positive, lipase negative
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Clostridium perfringens is normal in GI tract; gets in through perforation in GI tract or wound.
Evidence of toxemia precedes gas development. Anemia occurs due to the hemolytic action of
the toxin. Organism ferments sugars in muscle tissue to form gas. The enzyme hyaluronidase
helps spread the gangrene. Jaundice results from RBC destruction.
TREATMENT:
• - antibiotics sprinkled directly into the wound
• - hyperbaric oxygen chamber
• - remove the necrotic tissue (amputation)
CLOSTRIDIUM TETANI - causes "lock jaw"
• grows well on anaerobic blood agar
• colonies have a compact grayish center which flattens out to be a fine, gray filamentous network
described as "maple leaf or fern-like"
• small zone of hemolysis; they are motile organisms
• litmus milk negative, lecithinase and lipase negative
• spores are terminal and appear "racket-shaped"
or "drumstick" shaped
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the organisms are introduced by spore into deep,
lacerated wounds in which there has been considerable
tissue destruction (compound fractures, gunshot
wounds, etc..) The organism has limited invasive power
and there is only a localized infection with no systemic
results until there has been sufficient time (5 days to a
www.textbookofbacteriology.net
week) for the vegetative forms to produce the exotoxin. The toxin "tetanospasmin" becomes
bound to gangliosides within the central nervous system acting like strychnine, suppressing motor
neuron activity (disrupts nerve impulses to muscles). This results in a spastic type of paralysis with
continuous muscular spasms leading to trismus (stiffness of the muscles of the neck and
jaw.a.k.a.lockjaw), and difficulty in swallowing. The damage to the tissues and nerve endings is
irreversible resulting in brain damage. The prognosis depends upon incubation time with an 80%
mortality rate. If left untreated, spasms continue to be triggered by even minor stimuli, leading to
exhaustion and, eventually death due to asphyxiation (respiratory failure) during a muscle spasm.
Clostridium tetani continued:
TREATMENT:
• antitoxin usually coupled with one or more antibiotics.
The antitoxin is prepared from horses. Recovery from
the disease does not give lasting immunity
• excellent immunity is gained from the toxoid vaccine,
though revaccination is recommended every 10
years. ** However, if one acquires a major wound
infection with Clostridium tetani 5 years out from their
vaccination, then they should receive a “booster” of the
tetanus toxoid as a precautionary measure.
• Penicillin is the drug of choice for the vegetative cell.
CLOSTRIDIUM BOTULINUM - causes "food poisoning"
• motile, forms subterminal spores
• grows best at 25 C, slightly hemolytic
• produces the most powerful toxin known to man
• this exotoxin is not destroyed by the enzymes in the GI tract
• this organism is most commonly found in home canned vegetables, home-cured meats such as ham,
fermented fish and other preserved foods (protein). The exotoxin is heat labile.
• Symptoms may occur as soon as 2 hours due to toxin absorbed in the small intestine. (There are 6
antigenic types with A and B being the most common. The tuna fish scare of the 1960's was type E).
• The condition is caused by ingestion of the preformed toxin.
• the botulinal toxin attaches to individual motor nerve terminals preventing acetylcholine release at
the nerve endings . Death occurs due to a flaccid type of paralysis of the respiratory muscles.
• ** persons who have eaten food suspected of containing botulinum toxin should be purged with
cathartics (laxatives), have their stomach pumped AND be given enemas***
• The antitoxin is available from CDC.
• on egg yolk agar it is lecithinase - and lipase +
• recovery gives lasting immunity
• the toxin is destroyed at 1000C for 10 minutes
• confirmation of botulism is made by demonstration of the toxin in serum, gastric, or stool
specimens
• ** Infant botulism is the most frequent form occurring in the United States. Epidemiological
studies have demonstrated that infant botulism results from the ingestion of spores via breastfeeding or exposure to honey. Preformed toxin has not been detected in food or liquids taken by
the infants. C. botulinum spores germinate, colonize in the gut (colon) of the infant and produces
the neurotoxin in situ.
Clostridium species continued:
CLOSTRIDIUM RAMOSUM
• second most commonly encountered
Clostridium in clinical infection
• often appears gram negative in smears made
from culture
• very resistant to antibiotics
CLOSTRIDIUM SEPTICUM
• strong association between bacteremia with this
organism and malignancy.
Clostridium species continued:
CLOSTRIDIUM DIFFICILE:
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Clostridium difficile has become well recognized as a major nosocomial pathogen associated with antibiotic use
within hospitals and communities. After treatment with antibiotics, many patients develop gastrointestinal
problems ranging from mild diarrhea to severe pseudomembranous colitis. Many cases of the milder forms of
GI illness and most cases of pseudomembranous colitis are caused by Clostridium difficile. This organism is an
opportunistic anaerobic bacterium that grows in the intestine once the normal flora has been altered by the
antibiotic. The disease results from the toxins that the organism produces. C. difficile disease is responsible for
substantial strain on the hospital system by increasing patients’ length of stay and increasing costs. Diagnosis
should be considered if patients have diarrhea and a history of antibiotic exposure. Toxin detection assays
remain the first step in proving infection; however, sigmoidoscopy should be considered if patients have
possible alternative diagnoses or if assay results are negative but clinical suspicion is high. Prevention of
infection, by prudent restriction of antibiotic use, strict adherence to hand washing, disinfection, and infection
control precautions for infected patients, is crucial. Antibiotic therapy for first-episode infections is well
established; however, treatment of relapsed cases is less well defined and multiple relapses could require lengthy
therapy.
www.nlm.nih.gov
Clostridium difficile continued:
Risk Factors
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Antibiotic exposure is the single most important risk factor, contributing to altered gut flora
and allowing overgrowth of C. difficile. Disease can occur after a few days of antibiotic therapy or
up to 2 months after use. Clindamycin has been identified as the main culprit. Other antibiotics
commonly found to predispose patients to infection include the second- and third-generation
cephalosporins and penicillins. Less common agents include the quinolones, sulfonamides, and
metronidazole. In addition to antibiotics, cancer chemotherapy is also a risk factor as well as age
and severity of underlying illness.
• Research has shown the organism can be carried
asymptomatically or acquired nosocomially. Patients
treated with antibiotics are then at risk for developing
Clostridium difficile associated diarrheal disease
(CDAD) which, if left untreated may progress into
pseudomembranous colitis (PMC), toxic megacolon
and death. The rate of nosocomial infections will vary
with the institution, ward, housekeeping procedures,
recent epidemiology and patient population. Facilities
with large in-patient population, chemotherapy wards
or long term patient care facilities need to be particularly
aware of the problems related to nosocomial acquisition
of Clostridium difficile. Two distinct groups which can
harbor toxigenic Clostridium difficile asymptomatically
are infants and cystic fibrosis patients.
A picture of pseudomembranes
and lesions within the colon.
www.cfpc.ca
Clostridium species continued:
Diagnosis
• The clinical symptoms associated with the disease are believed to be primarily
due to toxin A which is a tissue-damaging enterotoxin. Clostridium difficile also
produces a second toxin, designated toxin B. Essentially all strains either
produce both toxins or neither toxin.
• Clostridium difficile strains that produce high levels of toxin A also produces
high levels of toxin B. Likewise, strains that produce low levels of toxin A
produce low levels of toxin B, indicating that the toxin production may be
regulated similarly. Tests that detect either or both toxins are best used as
diagnostic aids. There is some evidence suggesting that toxins A and B act
synergistically and that the initial tissue damage caused by toxin A allows
toxin B to exert its toxicity. Therefore, although toxin A is believed to cause
most of the clinical signs, toxin B also may play an important role in the
disease.
Clostridium species continued:
Tests for Detection
• The C. difficile TOX A/B TEST that we use is an enzyme immunoassay (EIA) for the
detection of toxins A and B produced by toxigenic strains of Clostridium difficile. It
can be used to detect toxins A and B in fecal specimens from persons suspected of
having C. difficile disease. The diagnosis of AAPMC is dependent upon the
identification of the characteristic lesions by sigmoidoscopy or colonoscopy exam ,
recovery of the organism by culture or detection of its toxins present in the stool. This
test that we perform is to be used as an aid in the diagnosis of C. difficile disease and
results should be considered in conjunction with the patient history.
• If test results are reported negative, sending one or two additional stools on
subsequent days could improve sensitivity. In addition, if only toxin A alone is tested,
diarrhea secondary to toxin A–negative—toxin B–positive strains will be missed.
Principle of the Test:
• In the assay, an aliquot of a fecal specimen is emulsified in the diluent and the diluted
specimen is then transferred to the microtiter well containing the detecting antibody.
If toxins A and B are present in the specimen, they will bind to the detecting antibody
and the immobilized polyclonal antibody during the incubation phase. Any unbound
material is removed during the washing steps. Following the addition of substrate, a
color is detected due to the enzyme -antibody-antigen complexes that form in the
presence of toxin. Any shade of yellow is a positive test result indicating toxin A
and/or toxin B are present in the stool.
Clostridium species continued:
Other Tests for Detection:
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1. Cytotoxicity assay/Tissue Culture: This test is positive when stool filtrate inoculated onto cell culture
monolayer demonstrates cytopathic effect due to the presence of C difficile toxin. It remains the
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criterion standard of detection. Sensitivity and specificity are 67% to 100% and 85% to 100%,
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respectively. The test, however, is relatively expensive; it requires 24 to 48 hours for completion; and
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inactivation of toxins during transport, through dilution of sample, and through age of the cell line
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gives rise to false-negative results.
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2. Latex agglutination: based assays recognize the enzyme glutamate dehydrogenase but lack sensitivity.
These latex agglutination tests detect the antigens of C. difficile in the supernatant of stool specimens.
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The antigens and toxins are soluble and are found in the supernatant solution. Agglutination is
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observed for a positive test.
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3. Stool culture: for C difficile is sensitive, but will also detect nontoxigenic strains. A special media is
used to grow C. difficile that contains cycloserie, cefoxitin, fructose and egg yolk (CCFYA). The
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colonies have a “mustard-yellow” color and will fluoresce under a UV light.
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4. Genetic analysis: via polymerase chain reaction is available, but confined to research settings.
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5. Endoscopy: should be reserved for when a patient’s condition necessitates rapid diagnosis or when
other diagnoses are being considered. Colonoscopy can detect cases not apparent during sigmoidoscopy
because C difficile infection can involve the transverse colon or ascending colon cecum without evidence of
involvement of the more distal segments of the colon.
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False-negative results can occur in any of these assays, so if the clinical suspicion is high and the assay is
negative, either the test should be repeated or a sigmoidoscopy or colonoscopy should be considered. This is
critical, for we have seen several cases where the initial assay is negative and empiric C difficile therapy is
stopped or the diagnostic possibility of C difficile is abandoned, resulting in delayed treatment and unfortunately
serious adverse outcomes for some patients.
Clostridium species continued:
Treatment:
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Once C. difficile diarrhea is diagnosed, inciting antibiotic therapy must be discontinued (if
possible). Anti-diarrheal agents should be avoided and narcotics minimized in order to prevent
colonic stasis and decreased toxin clearance. Volume resuscitation is necessary for dehydrated
patients. Specific treatment involves antibiotic regimens directed at C. difficile.
• Metronidazole. Metronidazole is recommended as first-line therapy for .C difficile diarrhea in
non-pregnant adults. It is inexpensive and generally well tolerated; side effects may include a
metallic taste, nausea, peripheral neuropathy, and disulfiram effect.
• Vancomycin. Oral Vancomycin is not well absorbed from the gastrointestinal tract and has an
intracolic effect. Vancomycin is second-line therapy and, because of its cost and the need to
prevent development of vancomycin resistance in species such as Enterococcus, is reserved for
patients whose treatment with metronidazole fails to provide benefit.
Fluid Replacement:
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Electrolyte replacement such as Pedialyte (Each liter of Pedialyte © provides sodium 45
mEq, potassium 20 mEq, chloride 35 mEq, citrate 30 mEq, and dextrose 25 g)
Complications:
Some major complications of pseudomembranous colitis are perforation of the colon,
dehydration with electrolyte imbalance, and toxic megacolon, which can be a life threatening
complication of other intestinal conditions. It is characterized by a very inflated colon, abdominal
distention, and sometimes fever, abdominal pain, or shock.
Non-Spore Forming Gram Positive Rods
ACTINOMYCES - causative agent of “Actinomycosis”
• Actinomycosis: a chronic suppurative or granulomatous infection
characterized by the
• formation of abscesses, multiple draining sinuses, and the appearance of
tangled mycelial masses, or
• "granules" (sulfur granules: small colonies of bacteria)
in the discharges (pus) or in tissue sections.
• most common species is A. israelii
• gram positive rods often branching and beaded
• normal flora in the upper respiratory tract,
oral cavity, bowel and the vagina;
can be an opportunistic pathogen
• can be isolated from carious teeth
and tonsillar crypts in the absence of infection.
Gram stain of branching, gram positive rods
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Actinomyces continued:
1.
Cervicofacial actinomycosis is an infection of the jaw and tissues of the face and neck, characterized by swelling and hardness
(“lumpy jaw”) with the formation of multiple abscesses which eventually break down to form draining sinuses. The
sanguinopurulent drainage from the sinuses usually contains macroscopic granules of A. israelii. Such infections follow tooth
extractions or other operative procedures in the mouth, and the disease progresses by slow and direct extension of the infection
through the tissues.
“Lumpy jaw”
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www.articleroad.com
2. Thoracic actinomycosis is an infection of the lungs and thoracic cage. Until extension through the thoracic skin results in
multiple draining sinuses, the diagnosis is often not suspected. Infection usually is confined to the hilar region and the base of
the lungs. Direct extension through the tissue to the lung surface and pleura may result in an inflammatory process, with pleural
thickening, empyema, and osteomyelitis of the ribs. Hematologic spread from a primary lung infection can result in the formation
of foci in tissues, such as the liver, kidneys, and brain.
3. Abdominal actinomycosis originates in the region of the cecum and may stimulate acute or subacute appendicitis. Infection
by A. israelii should be expected when an apendectomy wound fails to heal and irregular tender masses appear in the abdomen.
- typically produces a characteristic white, heaped, rough lobate colony resembling a "molar tooth"
- a large number of cases of Actinomycosis is associated with the use of intrauterine devices (IUD)
- pus from lesions often contain "sulfur granules"
- slow grower; hold all specimens for 7 days; may grow micro-aerophilic but better anaerobically
- thio broth has "bread crumb" appearance or tight aggregates
- produces major acetic, succinic and lactic acid in GLC
- most Actinomyces are nitrate positive and all are indole negative
- Penicillin G is the drug of choice; Metronidazole is relatively inactive
Actinomyces continued:
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3. Abdominal actinomycosis originates in the region of the cecum and may stimulate acute or
subacute appendicitis. Infection by A. israelii should be expected when an apendectomy wound
fails to heal and irregular tender masses appear in the abdomen.
- typically produces a characteristic white, heaped, rough lobate colony resembling a "molar tooth"
- a large number of cases of Actinomycosis is associated with the use of intrauterine devices (IUD)
- pus from lesions often contain "sulfur granules"
- slow grower; hold all specimens for 7 days; may grow micro-aerophilic but better anaerobically
- thio broth has "bread crumb" appearance or tight aggregates
- produces major acetic, succinic and lactic acid in GLC
- most Actinomyces are nitrate positive and all are indole negative
- Penicillin G is the drug of choice; Metronidazole is relatively inactive
“molar tooth colonies”
www.microblog.me.uk
Abdominal actinomycotic abscess
www.carene.org
www.carene.org
Non-Spore Forming, Gram Positive Rods
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BIFIDOBACTERIUM
- normal flora in the GI tract and oral cavity
- rarely isolated from clinical material
- most diagnostic feature is their various shapes, with pronounced clubbing at both ends (“dog bones”)
or bifurcated ends
- B. dentium, species of significance
- like Lactobacillus, they are acidophilic and grow best on low pH agar
- produce major amounts of acetic and lactic acid
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EUBACTERIUM
- normal flora in the GI tract and oral cavity, dental caries or plaque
- E. lentum now classified as Eggerthella lenta
- E. aerofaciens now classified as Collinsella aerofaciens
- not particularly pathogenic
- gram stain appearance varies; mostly diphtheroid like
- colonies are non-descriptive; raised to convex and transparent to translucent
- ID by default; any anaerobic gram positive rod that does not fit any of the other genera is classified as
Eubacterium
- catalase negative; indole negative, nitrate positive; most are non-motile
- can be presumptively identified on the basis of arginine stimulation in broth culture and nitrate
reduction
- produce acetic and butyric acids
Non-Spore Forming, Gram Positive Rods
PROPIONIBACTERIUM
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- referred to as the "anaerobic diphtheroid"
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- colonies are small and white to gray-white with older colonies often yellow
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- aerotolerant but grow best anaerobically
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- P. acnes, most often recovered
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- presumptive ID can be made by catalase positive, spot indole positive and diphtheroid like GPR see on
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gram stain
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- normal flora of the skin; sometimes GI tract, upper respiratory tract and uro-genital tract
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- often contaminants of blood cultures and sterile body fluids due to improper decontamination of the
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skin prior to obtaining specimen
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- P. acnes has been implicated in infections related to heart valves and prosthetic devices which may
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lead to osteomyelitis, bacteremia, endocarditis and meningitis
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- P. acnes produces a lipase that frees long chain fatty acids in the skin that may be irritating enough to
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contribute to the formation of acne pustules
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- produces major amounts of propionic and acetic acids
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- very sensitive to Penicillin and like most aerotolerant anaerobic gram positive bacilli, resistant to
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metronidazole
www.microbewiki.kenyon.edu
Non-Spore Forming, Gram Positive Rods
• LACTOBACILLUS ACIDOPHILUS
• - also known as "Döderlein’s Bacillus"
• - prominent as normal flora in the vagina and colon and found
in small numbers in the mouth
• - straight and uniform rods with rounded ends and may form
chains
• - microaerophilic but grow best anaerobically; we see this
organism aerobically as small pinpoint colonies (especially
from urine cultures)
• - grow best on a low pH medium
• - produces major amounts of propionic and acetic acids
• - very sensitive to Penicillin; resistant to Metronidazole
Anaerobic Cocci
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VEILONELLA - anaerobic gram negative diplococci
- although several genera of anaerobic gram-negative cocci are found in the
indigenous microflora, only Veillonella (especially species parvula) are implicated as
pathogens
- small, convex translucent to transparent colonies
- nitrate positive, catalase positive, red fluorescence under long wave UV light
- strict anaerobic cocci that occurs in pairs, short chains and irregular clumps
- normal mouth flora, normal upper respiratory, GI and vaginal flora
- causes disease usually associated with fractured jaws, gums or lung infections
PEPTOCOCCUS - except for Peptococcus niger all pathogenic species in genus
Peptococcus have been transferred to the genus Peptostreptococcus.
Peptococcus niger – gram positive cocci occurring in pairs, tetrads and irregular masses
- colony morphology reveals tiny, black pigmented, convex, shiny, smooth, circular,
entire colonies
Anaerobic Cocci continued:
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PEPTOSTREPTOCOCCUS
- normal flora in bowel, female genital tract, oral cavity and skin
- most prevalent in respiratory tract and related infections
- all species are catalase negative and produce acetic acid from PYG broth on GLC
- all species are susceptible to Metronidazole (Flagyl), if not susceptible then probable
anaerobic Streptococcus or Staphylococcus
- may act synergistically with Bacteroides or Staph aureus to produce tissue necrosis
Most commonly encountered species in infections are:
Ps. magnus: now called Finegoldia magna (resembles Staphylococci on gram stain)
Ps. asaccharolyticus – one of two species that is indole positive
Ps. indolicus – one of two species that is indole positive
Ps. anaerobius – glucose positive and only species that produces isocaproic acid
Ps. micros: now called Micromonas micros (GS reveals tiny, little cocci in short chains)
Anaerobic Cocci
STREPTOCOCCUS SPECIES
• the Streptococcus species are facultative
anaerobes
• Streptococcus intermedius is found occasionally in our
lab
• Usually treat with Penicillin G, Cephalothin or
Metronidazole
• *** Definitive ID of the anaerobic cocci
depends on gas chromatographic analysis of end
products or enzymatic substrate productivity.
Anaerobic Gram Negative Rods
Family Bacteroideaceae
• - obligate anaerobic, non-spore forming bacilli
• - indigenous flora of humans primarily on mucosal surfaces
• - constitute one third of the total anaerobic isolates from clinical
specimens
• - infection with anaerobic gram negative rod is characterized by
abscess formation and tissue destruction
• - most common infections include pleuropulmonary,
intra-abdominal and female genital tract
• - produce many enzymes that aid in the pathogenicity
(collagenase, heparinase, DNase )
• - the Bacteroides Group grows well in 20% bile and are usually
resistant to penicillin (thus beta-lactamase positive)
Anaerobic Gram Negative Rods:
Bacteroides species
BACTEROIDES:
• normal flora of the oropharynx, GI tract and genitourinary tract
• many species are catalase positive, produce acetic and succinic acids from PYG broth on GLC
• Bacteroides fragilis group forms the largest part of the gut flora
• the pigmented Bacteroides reside in the oropharynx, upper respiratory tract, genitourinary and GI
tract
• Clindamycin, Metronidazole and Chloramphenicol are active against the Bacteroides groups
Bacteroides fragilis group:
• Bacteroides fragilis: is the most commonly encountered pathogen of all the anaerobic bacteria; pale
staining, pleomorphic rods with rounded ends; resembles a “safety pin”
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Bacteroides thetaiotaomicron: is the second most commonly encountered species of this group;
- catalase positive and indole positive
- usually causing infections below the diaphragm
- non-pigmented and growth enhanced by bile
- resistant to penicillin (and kanamycin).
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- other members include: B. distasonis, B. caccae, B. uniformis, B. vulgatus, and B. ovatus
Bacteroides Pigmented Saccharolytic Species:
• placed in the genus Prevotella
• have been implicated in head and neck infections,
aspiration pneumoniae and lung abscesses; some from
urogenital tract
• typically produce protoporphyrin, a dark pigment that
causes colonies to turn color (brown to black) with age
(best seen on KVLB agar); also fluoresces brick-red on
long wave UV light
• ferment glucose and bile sensitive
• members include: most common P. melaninogenica,(black
pigment), P.intermedia & P. corporis,(dark brown to black,
dry), P. bivia, P. loescheii and P. denticola
• resistant to Penicillin
Bacteroides Pigmented Asaccharolytic Species:
• - placed in the genus Porphyromonas
• - Porphyromonas is primarily isolated from dental structures and
may play a major role in gingivitis and periodontitis
• - mostly isolated from the oral cavity (infected root canals)
• - forms tan to buff colonies that fluoresce brick-red under UV
light (long wave)
• - does not ferment glucose
• - fails to grow on KV agar
• - inhibited by bile, produces indole
• - sensitive to penicillin and rifampin not kanamycin
• *NOTE: The pigmented species are often fastidious and slow
growing, taking 2 days to 3 weeks to form a pigment. They are
the second most common group of anaerobic bacteria
encountered in human infections.
Other Bacteroides species:
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normal flora in the oral cavity, GI and genitourinary tract
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Bilophila wadsworthia
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Bacteroides ureolyticus
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Bacteroides gracilis
grows in bile, catalase positive, nitrate positive, urea positive, H2S positive
causes pitting of the agar; colonies are usually thin, flat, spreading, irregular translucent
that extend out from a slightly raised central area
urease positive; actually an anaerobic Eikenella corrodens
most frequently isolated from superficial soft tissue or bone infections
only species that is susceptible to penicillin
moved to a new genus Campylobacter and renamed Campylobacter gracilis due to its
microaerophilic nature (not a true anaerobe after all)
urea negative
isolated from more serious deep-seated infections, including head and neck, pulmonary
and sites within the abdomen and pelvis
more resistant to antibiotics and more virulent than B. ureolyticus.
Fusobacterium species
FUSOBACTERIUM
• - long, slender, spindle-shaped rods with tapered ends (fusiform)
• - normal flora in the upper respiratory tract, GI tract and genitourinary tract
• - most commonly encountered species in human infections are F. nucleatum and F.
necrophorum
• - produces large amounts of butyric acid , fluoresce chartreuse
• - resistant to Vancomycin and susceptible to Kanamycin and Colistin
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Fusobacterium nucleatum:
- important pathogen in head, neck and
lower respiratory tract; can cause serious
infections/abscesses in the brain and lung,
intra-abdominal, pleuropulmonary and blood
3 colony types:
a) white, bread-crumb like
b) speckled
www.microbewiki.kenyon.edu
c) smooth
- gram stain has “pick up sticks” appearance with long, thin tapered rods
- colonies may be convex, glistening , alpha-hemolytic colony with flecked internal structure and
grows in balls in broth culture
- Fusobacterium nucleatum when combined with Borrelia vincentii (spirochete) causes
“Vincent's Angina” or “Trench mouth” an acute, necrotizing, ulcerative gingivitis
(See Unit VII)
Fusobacterium species continued:
Fusobacterium necrophorum:
• - more virulent
• - known to invade the bloodstream and cause a widespread
metastatic infection that often includes lungs, liver and bones
• - produces an umbonate colony on media but grows in balls in
broth culture
• - indole and lipase positive
Other Fusobacterium species:
• - F. mortiferum and F. varium have a distinct fried-egg appearance
with raised, opaque centers and a flat, translucent margin
• - F. mortiferum is Esculin hydrolysis positive
Synergistic Gangrene
Causative agents of Synergistic gangrene:
• 1. Peptostreptococcus with a gram negative rod
• 2. Peptostreptococcus and Staphylococcus aureus
• 3. Eikenella with Alpha strep - often found in
cultures around the face and mouth as both
are normal respiratory flora
Identification Methods
IDENTIFICATION OF ANAEROBES
• Several methodologies are available for use:
• 1. PRAS biochemicals
• 2. Miniaturized biochemical systems (API 20A)
• 3. Rapid, preformed enzyme detection panels (RapID ANA)
• 4. Gas Liquid Chromatography (GLC) - for end products of glucose fermentation
• 5. High Resolution GLC - for cellular fatty acid analysis
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Probably the 2 most common methods are:
1. Preformed Enzymes
2. Gas Liquid Chromatography
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Preformed Enzymes:
- Several rapid ID systems are available that do not require growth of the organism as much as
relying on chromogenic and conventional substrate breakdown by preformed enzymes. A pure
culture of the organism grown on an individual non- selective agar plate medium is harvested and
suspended into inoculating fluid to a specific turbidity. Panels are inoculated, incubated
(aerobically 350 C) for 4 hours. Factors that can affect the ID results are the culture plate medium,
inoculum density, purity, correct filling of wells, incubation time and atmosphere.
ID Methods continued:
Gas Liquid Chromatography:
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- Some cells can not oxidize carbohydrates and other substrates to carbon dioxide and
water without oxygen. Instead they produce partially oxidized organic acids and gases
that are characteristic for certain groups of anaerobic bacteria. With Gas Liquid
Chromatography (GLC) it has become practical to identify these anaerobic bacteria on
the basis of their metabolic pathways.
- Anaerobic metabolism produces volatile and non-volatile fatty acids and alcohols.
Nutrient broths such as a) Pre-reduced Peptone-Yeast extract-Glucose (PYG) and b)
chopped meat with carbohydrates support the growth of anaerobic bacteria. The end
products from the metabolism of the peptone and carbohydrates are volatile fatty acids
that help to identify the bacteria. After incubation, the broth (peptone yeast or
chopped meat) is centrifuged and the supernatant is used on the chromatograph to
analyze the metabolic pathways. By-products are extracted (with ethyl-ether) and
injected into a column (either glass or tubular metal) where helium gases pass through
at a controlled rate. The by-products are eluted off the column and produce an
electrical displacement on a thermal conductivity detector, thus displacing the needle
of the graph to make a peak. These peaks are measured, compared to a known
standard, which in turn identifies the anaerobic bacteria in question. GLC is a valuable
procedure for the analysis of metabolic products of anaerobic bacteria. It is also a
practical, inexpensive procedure that provides both speed and accuracy. It is a
valuable diagnostic and research tool for large institutions or reference laboratories.
** To ensure accurate identification one should use a combination of either PRAS
biochemicals and GLC or HR-GLC.
References
Most of the lecture notes produced in this power point presentation were acquired from:
• Required Textbook:
Mahon, Connie R., Lehman, Donald C., Manuselis, George,(2015). Textbook of
diagnostic microbiology (5th edition). Saint Louis, Missouri: Saunders-Elsevier
•
Journals/Publications: Notes have also been ascertained using the following:
a) Laboratory Medicine
b) Advance for Medical Laboratory Professionals
•
Laboratory Procedures: The procedures used in the following notes with and without
pictures were obtained with permission from Carolinas Medical Center-Main,
Microbiology Laboratory Procedure Manuals, to be used for educational purposes only
by the faculty of Carolinas College of Health Sciences, School of Clinical Laboratory
Sciences, Medical Laboratory Science Program.
•
Hand-outs and Lecture Notes from various members and lecturers of the South
Eastern Association for Clinical Microbiologists (SEACM) Annual Meeting Conference
Any Questions???
Write them down or highlight them in your
notes and bring them with you to Test Review.