Laboratory%20Methods%20for%20Diagnosis%20of%20Non-fermenting%20Gram-Negative%20Bacilli

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Laboratory Methods for Diagnosis of
Non-fermenting Gram-Negative Bacilli

• Dr Mohammad Rahbar

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General Characteristics of Non-fermenters

• 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.

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General Characteristics of Non-fomenters

• 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.

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General Characteristics of Non-fermenters

• Most nonfermentative gram-negative bacilli are
  oxidase positive, a feature that differentiate
  them from the Enterobacteriaceae (except
  plesiomonas witch is oxidase positive.

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General Characteristics of Non-fermenters

• 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.

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General Characteristics of Non-fermenters

• 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

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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

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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 (400-nm) 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
  life-threatening 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 swimming-pool
  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.

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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.

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Virulence Factors of Pseudomonasaeruginosa

• 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

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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

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Viurlence factors

• Exotoxin A
• Tissue destruction, inhibition of protein
• synthesis interrupts cell activity .
  Enterotoxin
• Interrupts normal gastrointestinal
• activity. leading to diarrhea

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Virulence Factors

• Exoenzyme S
• Inhibits protein synthesis
• Phospholipase C
• Destroys cytoplasmic pulmonary surfactant
  inactivates opsonins

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Virulence Factors

• Elastase
• Cleaves immunoglobulins and , disrupts
• neutrophil activity
• Leukocidin
• Inhibits neutrophil and lymphocyte function

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Virulence Factors

• Pyocyanins
• Suppress other bacteria and disrupt ciliary
  activity cause oxidative damage to tissues,
  particularly oxygenated tissues such as lung

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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

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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/dera cepaca isolates from
  patients with
• cystic fibrosis may exhibit morphotypes that
  resemble P.aeruginosa.

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Colonies of Pseudomonas aeruginosa typically
display beta hemolysis, a metallic sheen, and
blue or green pigment.
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Pseudomonas aeruginosa (beta hemolysis and
metallic sheen
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Pseudomonas aeruginosa (beta hemolysis with
transmitted light)
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. Pseudomonas aeruginosa (beta hemolysis with
transmitted light
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FIG. 5. Pseudomonas aeruginosa (beta hemolysis
and pigment with transmitted light
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encapsulated strain of Pseudomonas aeruginosa
recovered from a cystic fibrosis patient at 24
hours.
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Same plate as FIG. 23 at 48 hours, this strain of
Pseudomonas aeruginosa make abundant, mucoid
capsular material.
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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 namedthe 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.

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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.

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Clinical Infections

• Acinetobacter spp are opportunistic accounting
  1 to 3 of all nosocomial infectionslt they are
  second only to P.aeruginosa in frequency of
  isolation of all nonfermenters in the clinical
  microbiology laboratory.

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Disease in particular with A.baumannii

• UTI
• Pneumonia, Tracheobronchitis,or both
• Endocarditis with up 25 mortality
• Meningitides
• SepticemiaTruman infections, Burn infections,
• Eye infections.
• A.lwoffii is much less virulent

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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

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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

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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.

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Lab Diagnosis

• However, colonies growing on Mac- Conkey 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 penicillin-sensitive
  Moraxel/ a species, which also usually appear as
  coccobacilli on Gram stain.

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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.

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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

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Summary for Diagnoses of Acinetobacter Spp

• Grows well on MacConkey agar
• Resistant to penicillin

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Although Acinetobacter baumanii is incapable of
fermentation, its very strong lactose oxidation
leads to weakly acid/purple colonies on MacConkey
agar
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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.

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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.

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Important Reactions for Diagnosis of S.maltophilia

• Pyoverdinnegative
• ONPG positive
• DNASe positive
• Nitrate not reduced to nitrogen gas
• Lysin decarboxylaseslovely positve
• Arginine dehydrolase negative
• Ornithine decarboxylase negative

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• Esculin hydrolysis positive
• Gelatin hydrlysis positive
• Susceptibility to SXT positive
• Susceptibility to colisitinpositive

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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.

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Figure 8Stenotrophomonas maltophilia on EMB
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. Same plate as FIG. 19 at 48 hours,
Stenotrophomonas has distinct non-lactose
fermenting colonies. The indicator has turned an
alkaline tan color
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Gram Stain of S.maltophilia
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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 3of CF population is infected with
  B.cepacia ,but rats up to 30in 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.

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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.

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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.

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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.

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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.

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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.

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. 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
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Oxidation-fermentation Hugh-Leifson - Uninoculated
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OF glucose oxidative metabolism
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OFglucose fermentative metabolism (Enlarged view)
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Amino acid decarboxylase
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Figure 7Nitrate reduction test (Labeled view)
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Phenol red broth