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Laboratory Methods for Diagnosis of Non-fermenting

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Laboratory Methods for Diagnosis of Non-fermenting Gram-Negative Bacilli Dr Mohammad Rahbar Gram Stain of S.maltophilia Burkholderia B.cepacia Burkholderia ... – PowerPoint PPT presentation

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Title: Laboratory Methods for Diagnosis of Non-fermenting


1
Laboratory Methods for Diagnosis of
Non-fermenting Gram-Negative Bacilli
  • Dr Mohammad Rahbar

2
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.

3
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.

4
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.

5
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.

6
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

7
TAXONOMY, BIOCHEMICAL CHARACTERISTICS,
AND CLINICAL SIGNIFICANCE OF MEDICALL Y
IMPORTANT GENERA OF NONFERMENTERS

8
Continue..
  • 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.

9
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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.

10
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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.

11
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  • Organisms That Are Motile With Polar Flagella
  • Pseudomonads

12
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.

13
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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

14
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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.

15
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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 .

16
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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 .

17
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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.

18
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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.

19
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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

20
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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.

21
Continue..
  • 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.

22
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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.

23
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.

24
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

25
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

26
Viurlence factors
  • Exotoxin A
  • Tissue destruction, inhibition of protein
  • synthesis interrupts cell activity .
    Enterotoxin
  • Interrupts normal gastrointestinal
  • activity. leading to diarrhea

27
Virulence Factors
  • Exoenzyme S
  • Inhibits protein synthesis
  • Phospholipase C
  • Destroys cytoplasmic pulmonary surfactant
    inactivates opsonins

28
Virulence Factors
  • Elastase
  • Cleaves immunoglobulins and , disrupts
  • neutrophil activity
  • Leukocidin
  • Inhibits neutrophil and lymphocyte function

29
Virulence Factors
  • Pyocyanins
  • Suppress other bacteria and disrupt ciliary
    activity cause oxidative damage to tissues,
    particularly oxygenated tissues such as lung

30
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

31
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.

32
Colonies of Pseudomonas aeruginosa typically
display beta hemolysis, a metallic sheen, and
blue or green pigment.
33
Pseudomonas aeruginosa (beta hemolysis and
metallic sheen
34
Pseudomonas aeruginosa (beta hemolysis with
transmitted light)
35
. Pseudomonas aeruginosa (beta hemolysis with
transmitted light
36
FIG. 5. Pseudomonas aeruginosa (beta hemolysis
and pigment with transmitted light
37
encapsulated strain of Pseudomonas aeruginosa
recovered from a cystic fibrosis patient at 24
hours.
38
Same plate as FIG. 23 at 48 hours, this strain of
Pseudomonas aeruginosa make abundant, mucoid
capsular material.
39
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40
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

41
Continue..
  • 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,

42
Continue..
  • 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.

43
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.

44
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.

45
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

46
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

47
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

48
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.

49
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.

50
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.

51
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

52
Summary for Diagnoses of Acinetobacter Spp
  • Grows well on MacConkey agar
  • Resistant to penicillin

53
Although Acinetobacter baumanii is incapable of
fermentation, its very strong lactose oxidation
leads to weakly acid/purple colonies on MacConkey
agar
54
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

55
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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.

56
Continue..
  • 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

57
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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.

58
Continue..
  • 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)

59
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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.

60
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.

61
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

62
Continue.
  • Esculin hydrolysis positive
  • Gelatin hydrlysis positive
  • Susceptibility to SXT positive
  • Susceptibility to colisitinpositive

63
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.

64
Figure 8Stenotrophomonas maltophilia on EMB
65
. Same plate as FIG. 19 at 48 hours,
Stenotrophomonas has distinct non-lactose
fermenting colonies. The indicator has turned an
alkaline tan color
66
Gram Stain of S.maltophilia
67
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).

68
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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.

69
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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.

70
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.

71
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.

72
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.

73
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.

74
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.

75
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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.

76
. 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
77
Oxidation-fermentation Hugh-Leifson - Uninoculated
78
OF glucose oxidative metabolism
79
OFglucose fermentative metabolism (Enlarged view)
80
Amino acid decarboxylase
81
Figure 7Nitrate reduction test (Labeled view)
82
Phenol red broth
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