Transcript Laboratory Methods for Diagnosis of Non0fermenting Gram

Laboratory Methods for
Diagnosis of Non-fermenting
Gram-Negative Bacilli
Dr Mohammad Rahbar
General Characteristics of Nonfermenters
• Nonfermenting gram-negative bacilli are
grouped together because they fail to
acidify oxidative-fermentative (OF) media
overlaid with mineral oil or triple sugar
iron agar (TSIA) butts .They prefer and
grow much better in an aerobic
environment ;some group
members
oxidize carbohydrates to derive energy
for their metabolism ;they are referred to
as oxidizers.
General Characteristics of Nonfomenters
• Others do not break down carbohydrates
at all and are inert or biochemically
inactive; they are
referred
to as
nonoxidizer or asaccharolytic .Additional
characteristics can differentiate this group
of nonfernenters from other gram-negative
bacilli: motility ,pigmentation and their
ability
or lack of ability to grow on
selective gram-negative media such as
MacConkey agar.
General Characteristics of Nonfermenters
• Most
nonfermentative
gram-negative
bacilli are oxidase positive, a feature that
differentiate
them
from
the
Enterobacteriaceae (except plesiomonas
witch is oxidase positive.
General Characteristics of Nonfermenters
• In general nonfermentative gram-negative
bcilli are ubiquitous and found in most
environments: in soil and water .on plants
and decaying vegetation and in many
foodstuffs. They prefer moist environment
,and in hospitals that can be isolated from
nebulizers, dialysate,fluide saline and on
catheters and other devices.
General Characteristics of Nonfermenters
• Nonfermenters may withstand treatment
with
chlohexidine
and
quaternary
ammonium compounds .They are rarely ,if
ever part of the normal host flora but can
easily colonize hospitalized patients,
especially
those
who
are
immunocompromised
.Nonfermentative
gram-negative bacilli tend to be resistant
to several Antimicrobial agents
TAXONOMY, BIOCHEMICAL
CHARACTERISTICS, AND
CLINICAL SIGNIFICANCE OF
MEDICALL Y IMPORTANT
GENERA OF
NONFERMENTERS
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• Unlike
the
Enterobacteriaceae
the
nonfermenting
gram-negative
bacilli do not fit conveniently into a single
family of well-characterized genera, and
the correct taxonomic placement of many
nonfermentative, gram-negative bacilli
(NFBs) remains unresolved.
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Consequently, the study of nonfermenters
is often confusing for the beginning
microbiologist. The major genera of
nonfermenting, gram-negative bacilli
have been classified into at least 15
families.
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• One
approach
to
studying
the
nonfermenters is to group them on the
basis of the presence or absence of
motility and on the type of flagella present
in strains that are motile.
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• Organisms That Are Motile With Polar
Flagella
Pseudomonads
Fluorescent Group.
• The species within this group are all by
the production of a water-soluble white to
blue-green
under
long
wavelength
pyoverdin pigment that fluoresces (400nm) ultraviolet light. Production of
fluorescent pigments is particularly
enhanced in media with a high
phosphate concentration.
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• Although all three members of this group
produce pyoverdin, only one species. P.
aeruginosa ,produces the distinctive blue,
water-soluble pigment pyocyanin
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• Pseudomonas aeruginosa produces a
characteristic appearance when grown on
BAP. It appears as large gray colonies
with a spreading periphery and exhibits
hemolysis. Colonies often have an
alligator skin appearance and exhibit
a metallic sheen.
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• Rapid identification of P. aeruginosa in
culture can be made whenever the
characteristics are observed: typical
colony morphology production of diffusible
pigments the presence of a fruity odor,
and oxidase positivity .
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We have occasionally observed strains
that produce a pungent, "rotten-potato"
odor. There has been at least one report
of a nosocomial outbreak caused by
strains of malodorous P. aeruginosa .
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• Pseudomonas aeruginsa is the
most
frequently
recovered
from
clinical
specimens. p, aeruoginosa infection is
especially prevalent among patients with
burn wounds, cystic fibrosis, acute
leukemia, organ transplants, and drug
addiction.
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• Infections commonly occur at any site
where moisture tends to accumulate
tracheostomies,
indwelling
catheters,
burns, the external ear ("swimmer's ear"),
and weeping cutaneous wounds. The
exudation of bluish pus, with a grape-like
odor from the production of pyocyanin, is
characteristic.
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• P aerugnosa also causes urinary tract
and lower respiratory tract infections;
the latter can be severe and even lifethreatening in immunocompromised hosts.
The organism can also cause
devastating infections of the eye
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• Pseudomonas keratitis. Infection of corneal
ulcers, and endophthalmitis must be approached
as a medical emergency that can be fulminant
and threaten permanent loss of vision. Individual
cases of endocarditis,meningitis, brain abscess,
and infections of bones and joints from
hematogenous spread appear with regular
frequency in the literature.
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• Most cases of endocarditis require valve
replacement because the infection is difficult to
eradicate. P. aeruginosa dermatitis and otitis
externa outbreaks associated with swimmingpool and hot-tub use are well described. The
CDC reported at least 75 cases during six
outbreaks occurring between 1997 and 1998.
Sporadic P. aeruginosa infections following ear
piercing have also been reported.
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• P .aerugillosa produces several substances that
are thought to enhance the colonization and
infection of host tissue. These substances,
together with a variety of virulence factors,
including lipopolysaccharide (LPS), exotoxinA,
leukocidin, extracellular slime, proteases,
phospholipase,and several other enzymes (Box
7-5), make P. aerugillo.l'a the most clinically
significant bacteria among the NFB.
Virulence Factors
• An unusual mucoid morphotype of P.
aeruginosa is frequently recovered from
respiratory secretions of patients with cystic
fibrosis who are chronically infected with P.
aeruginosa The mucoid morphotype is due to
the production of large amounts of a (called
alginate)that surrounds the cell. The production
of alginate is ultimately responsible for the poor
prognosis and high mortality rates among
patients with cystic fibrosis.
Virulence Factors of Pseudomonas
aeruginosa
•
Alginate:
Capsular polysaccharide that allows
infecting bacteria to adhere to lung
epithelial cell surfaces and form biofilms
which, in turn, protect the bacteria from
antibiotics and the body's immune
system
Virulence factors
• PIlli
Surface appendages that allow adherence
of organism to GM-I ganglioside receptors
on host epithelial cell surfaces
Neuraminidase
Removes sialic acid residues from GM-I
ganglioside receptors. Facilitating binding
pili
Viurlence factors
• Exotoxin A :
• Tissue destruction, inhibition of protein
synthesis; interrupts cell activity .
Enterotoxin
• Interrupts normal gastrointestinal
activity. leading to diarrhea
Virulence Factors
• Exoenzyme S:
Inhibits protein synthesis
Phospholipase C:
Destroys cytoplasmic pulmonary
surfactant; inactivates opsonins
Virulence Factors
• Elastase:
Cleaves immunoglobulins and , disrupts
neutrophil activity
• Leukocidin:
• Inhibits neutrophil and lymphocyte function
Virulence Factors
• Pyocyanins:
• Suppress other bacteria and disrupt ciliary
activity; cause oxidative damage to
tissues, particularly oxygenated tissues
such as lung
Summary Key Tests for
Identification P. aeruginosa
• Minimum Requirements for Definitive Identification
of P. aeruginosa
Identification based on all of the following:
• I. Gram-negative rod
• 2. Oxidase-positive
• 3. Typical smell (fruity grape-like odor or corn tortilla)
• 4. Recognizable colony morphology
• a. On blood or chocolate agar appear as large colonies
with
• metallic sheen, mucoid, rough. or pigmented (pyocyanin)
• and often p-hemolytic
•
Summary Key Tests for
Identification P. aeruginosa
• b. On MacConkey, appear as lactose-negative with
greenpigmentation, or metallic sheen
• Limitations:
• I. Rare Aeromonas isolates may resemble P. aerugirrosa
(lacking the typical smell) but will be spot indole-positive
(P. aeruginosq are indole-negative).
• 2. Some Burklto/der;a cepac;a isolates from patients with
cystic fibrosis may exhibit morphotypes that resemble
P.aeruginosa.
Colonies of Pseudomonas aeruginosa typically display beta
hemolysis, a metallic sheen, and blue or green pigment
.
Pseudomonas aeruginosa (beta hemolysis and metallic
sheen
Pseudomonas aeruginosa (beta hemolysis with transmitted
light)
. Pseudomonas aeruginosa (beta hemolysis with
transmitted light
FIG. 5. Pseudomonas aeruginosa (beta hemolysis and
pigment with transmitted light
encapsulated strain of Pseudomonas aeruginosa
recovered from a cystic fibrosis patient at 24 hours.
Same plate as FIG. 23 at 48 hours, this strain of Pseudomonas
aeruginosa make abundant, mucoid capsular material.
Acinetobacter
• The genus Acinetobacter ,now a member
of the family Moraxellaceae ,cosist of 25
ِDNA homology groups or
genomospeecies .Only 10 species have
been officially named:the two species
most commonly seen in clinical specimens
are : A.baumannii and A. lwoffii
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• Acinetobacter spp are unique in the
environment in soil, water and foodstuffs in
the hospital environment
they have
been associated with ventilator ,humidifies
catheter and other devices. About 25% of
adults carry the organism in their phrynx.If
not harboring Acinetobacter spp ,already
hospitalized patients may become easily
colonized,
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• As many as 45% of patents with a
trachetomy may be colonized. When
Acinetobacter spp isolated from urine,
feces ,vaginal secretion ,and many
different type of respiratory specimens,
they are often considered insignificant
colonizer or contaminants.
Acinetobacter baumannii
• A. baumannii is the second most frequent
nonfermenter encountered in clinical
laboratories, but with only about one tenth
the frequency of P. aerugi1losa. The
following are the characteristics by which a
presumptive identification can be made.
Clinical Infections
• Acinetobacter spp are opportunistic
accounting 1% to 3% of all nosocomial
infections< they are second only to
P.aeruginosa in frequency of isolation of
all
nonfermenters
in
the
clinical
microbiology laboratory.
Disease in particular with
A.baumannii
•
•
•
•
•
UTI
Pneumonia, Tracheobronchitis,or both
Endocarditis with up 25% mortality
Meningitides
Septicemia
Truman infections, Burn infections,
• Eye infections.
• A.lwoffii is much less virulent
Laboratory Diagnosis
• Appear as cocci or coccobacilli on Gram
stain .
• Grow well on MacConkey agar (colonies
may have slightly pinkish tint ,a helpful
characteristic when present
• Exhibit rapid utilization of glucose, with
production of acid
• Are non- motile
• Are penicilin resistant
Lab Diagnosis
• The initial clue is the observation of tiny
diplococci on Gram stains prepared
directly from clinical materials. When
Gram stains are prepared from agar or
broth cultures, the cells may appear larger
and more like coccobacilli
Lab Diagnosis
• Acinetobacter species are not pigmented
when grown on blood agar, a helpful
characteristic in differentiating them from
certain other nonfermenters, such as
occasional oxidase-negative, nonmotile
strains of Burkholderia cepacia.
Lab Diagnosis
• However, colonies growing on MacConkey agar may produce a faint pink tint
or a deeper cornflower blue when
observed on eosin methylene blue agar
Resistance to penicillin helps distinguish
• A. baumannii from the highly penicillinsensitive Moraxel/ a species, which also
usually appear as coccobacilli on Gram
stain.
Lab Diagnosis
• Most strains of Moraxel/a species are also
cytochrome oxidase-positive. A. lwoffii is
nonsaccharolytic and can be differentiated
from A. baumannii because it produces
no acid when grown in media that contain
carbohydrates.
Summary for Diagnoses of
Acinetobacter Spp
•
•
•
•
•
Obligate Aerobe
Nonmotile
Oxidase Negative
Nonhemolytic
Saccharolytic: acidifies most OF
carbohydrates ,including glucose and
xylose.
• Produce acid from lactose
Summary for Diagnoses of
Acinetobacter Spp
• Grows well on MacConkey agar
• Resistant to penicillin
Although Acinetobacter baumanii is incapable of
fermentation, its very strong lactose oxidation leads to
weakly acid/purple colonies on MacConkey agar
Stenotrophomonas ma/tophi/ia
• S. maltophilia is the third most frequently
encountered nonfermenter in clinical
laboratories. Before 1983 it was a member
of genus Pseudomonas. It was later
classified as a member of the plant
pathogen Xanthomonas .Following DNA
homology andsequencing analysis it was
classified
as
a
member
of
Stenotrophomonas
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• Isolates are ubiquitous in the envir• ointment ,being found in water, sewage,
and plant materials ; they are very
common to the hospital environment
,where they can be found contaminating
blood
–drawing
equipment
,disinfectant,tranducer,
and
other
equipment.
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• Clinically ,when S.maltophilia is isolated
from clinical specimens ,it is initially
regarded as saprophyte or colonize .
Although not considered part of normal
flora ,S.maltophilia cab quickly colonize
the reparatory tracts hospitalized patients
,in
particular
those
exposed
to
antimicrobial agents to
which
S.
maltophilia may be inherently resistant
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• These antimicrobial include cephalosporins
,penicillins, carbapenems .and aminoglycosides.
With increased use of agents to which it is
innately resistant ,there have been more reports
of disease attributed to this organism. Reported
disease include endocarditis, especially in a
setting of prior intravenous drug abuse or heart
surgery ,wound infections, including cellulitis and
ecthyma gangrenosum,bactermia and rarely
meningitides.
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• With rare exceptions ,infections have
occurred in a nosocomial setting
.S.maltophilia is rarely associated with
lower respiratory tract infections, although
it has been isolated from 6.4 to 10.2 of
patients with CF. Pseudoinfections have
also occurred a result contaminated
collection tubes or cups. (e.g Blood
collection tubes)
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• The single most important risk factor in
affected individuals was the presence of a
venous chatter. Most patients with
bactermia responded well to therapy
unless they had concomitant pneumonia
or shock.
Important Reactions for Diagnosis
of S.maltophilia
•
•
•
•
•
•
•
Yellow –tan pigment on tryptycase Soy agar.
Lavender-green pigment on sheep blood agar
Growth at 42C: positive
Oxidase =negative
Catalase :positive
Oxidize glucose in OF medium weakly positive
Oxidize maltose in OF medium strongly positive.
Important Reactions for Diagnosis
of S.maltophilia
•
•
•
•
•
•
•
Pyoverdin=negative
ONPG = positive
DNASe = positive
Nitrate not reduced to nitrogen gas
Lysin decarboxylase=slovely positve
Arginine dehydrolase :negative
Ornithine decarboxylase : negative
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•
•
•
•
Esculin hydrolysis :positive
Gelatin hydrlysis: positive
Susceptibility to SXT: positive
Susceptibility to colisitin:positive
Important Reactions for Diagnosis
of S.maltophilia
• The antibiotic susceptibility pattern can
also be a clue to the identification of S.
maltophilia, which is typically resistant
• to most antibiotics, , butis susceptible to
trimethoprim- and colistin.
Figure 8
Stenotrophomonas maltophilia on
EMB
. Same plate as FIG. 19 at 48 hours, Stenotrophomonas
has distinct non-lactose fermenting colonies. The indicator
has turned an alkaline tan color
Gram Stain of S.maltophilia
Burkholderia
• B.cepacia
• Burkholderia (Pseudomonas ) cepacia is a
complex of nine distinct genomic species
(geneomvars) that has in the past been called
P.multivorans,P.kngiiand and EQ-1.Clinically
B.cepacia is a lowgrade ,nosocomial pathogen
that has most often been associate with
pneumonia inpatients with CF or chronic
granulomatous disease( CGD).
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• It has been reported to cause endocarditis
( especially in drug addicts) pneumonia
,UTIs ,osteomylitis , dermatitis, and other
wound infections resulting from use of
contaminated water. It has been isolated
from
irrigation
fluids,
anesthetics
,nebulizers, detergents, and disinfections.
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• Research supports the association of B,cepacia
and increased severity of disease and death in
patients with CF and CGD.In the United State
about 3%of CF population is infected with
B.cepacia ,but rats up to 30%in some adult CF
patients populations have been repotted.
Outside these population morbidity and mortality
rates remain low and consideration needs to be
given to the possibility of contamination rather
than infection when isolated.
Laboratory Diagnosis
• The organism grow well on most laboratory
media but may also viability on Sheep blood
agar in 3 to 4 days without appropriate transfers
.
• B.cepacia
grow on MacConkey agar, but
selective media containing antimicrobial to
reduce the growth of P.aeruginosa ,as well as
other gram-negative bacilli ,are available to
increase the recovery of B.cepacia.
Lab Diagnosis
• These media include PC( pseudomonas
cepacia), OFPBL (oxidative fermentative
base
,polymyxinB, bacitracin ,lactose
)and BCSA ( B.cepacia selective agar).
Studies have suggested that BCSA is
most effective in reducing overgrowth
while maintaining good recovery of
B.cepacia.
Lab diagnosis
• B.cepacia complex often produce a week
oxidase reaction. Nearly all strains oxidize
glucose, and many will oxidize maltose,
lactose and manitol.
• Most strains are LDC positive
• Most strains are ONPG positive
• Most strains are ODC negative.
Lab diagnosis
• Nitrate positive
• Are motile by means of polar tuftsof
flagella
• They do not fluoresce like P .aeruginosa,
but they can produce a nonfluorescing
yellow or green pigment that may diffuse
into media.
Lab Diagnosis
• Colonies of B.cepacia are nonwrinkled
,and this may be used to differentiate
isolates from P.stutzeri ,which also
produce a yellow pigment.
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• B.cepacia is usually susceptible to
chloramphenicol, cetazidim, piperacillin,
and SXT, but resistant to most other
agents. Susceptibility to the carbapenems
is variable. resistance can develop quite
rapidly. The CLSI recommended that if
disk diffusion method of susceptibility
testing
,then
only
ceftazidime
,meropenem, minocycline and SXT should
be reported.
. Same plate as FIG. 17 at 48 hours, Burkholderia cepacia
displays small non-lactose fermenting colonies. Some
strains appear somewhat purple due to strong lactose
oxidation
Oxidation-fermentation HughLeifson - Uninoculated
OF glucose oxidative metabolism
OFglucose fermentative
metabolism (Enlarged view)
Amino acid decarboxylase
Figure 7
Nitrate reduction test (Labeled
view)
Phenol red broth