Nonfermenting Gram Negative Rods

1
Nonfermenting Gram Negative Rods

• GNNFs Tom calls them GNFBs

2
GNNF Characteristics

• Non-fermenting Gram negative rods (GNNFs) are
  mostly non-fastidious obligate aerobes that grow
  well on SBA or even TSA
• GNNFs either generally do not utilize carbs (at
  least compared to enterics) or do so by
  biochemical pathways other than fermentation (see
  later)
• Almost all grow on selective media such as Mac,
  but growth may be slow and scant. Since they are
  non-fermentors the colonies are colorless and do
  not alter media pH no color change
• Most GNNFs are oxidase positive, but there are
  exceptions
• Some GNNFs are more fastidious than those
  included here and are discussed in other groups
  in this series
• Many GNNFs like low-nutrient waters, so they grow
  in faucets, aerators, respiratory therapy
  equipment, sinks and water baths
• They contaminate medications and sterile
  solutions intended for intravenous therapy

3
continued

• A small percentage of GNNFs are pathogens, and
  those that are generally have lower virulence
  than the enterics only 15 of Gram-negative rod
  infections are caused by GNNFs as you will see,
  Pseudomonas aeruginosa is the bad boy of the
  group
• GNNFs usually only infect patients with
  predisposing conditions therefore many are
  nosocomial infections
• Infections include UTI, septicemia, meningitis,
  osteomyelitis, and wound infections following
  trauma or surgery
• GNNFs tend to be resistant to the many of the
  antibiotics used to treat enteric infections, as
  well as antiseptics disinfectants especially
  true of Pseudomonas aeruginosa
• Treatment of severe infections may require
  combinations of antibiotics

4
P. aeruginosa

• Most GNNF infections are caused by Pseudomonas
  aeruginosa. P. aeruginosa constitutes 80 to 90
  of GNNFs isolated from patients in the U.S.
• P. aeruginosa hardly ever infects uncompromised
  tissues, but there is basically no tissue that it
  cannot infect a big problem
• P. aeruginosa is the 4th most commonly isolated
  nosocomial pathogen causing gt10 of nosocomial
  infections, especially in patients compromised by
  burns, cancer or cystic fibrosis
• Cells of P. aeruginosa and most other GNNFs are
  thinner and slightly longer than enterics
• A few GNNFs appear more like coccobacilli in
  stationary phase on agar but tend to be longer in
  log phase in broth, while others produce short
  fat rods, often appearing in pairs
  (diplococcobacilli)
• Colonies of P. aeruginosa are larger than those
  of other GNNFs (less fastidious) and they have a
  unique morphology .
• Irregular, spreading, rough-edged, pitted,
  ground glass or beaten copper appearance and
  often with a metallic sheen.

5
P. aeruginosa

• Most produce a blue-green tint (pyocyanin), but
  some produce red (pyorubin), brown or brown-black
  (pyomelanin) pigments.
• P. aeruginosa and two other Pseudomonas species
  produce a yellow pigment (pyoverdin) that
  fluoresces when exposed to UV light.
• They produce a grape-like odor grape Cool-Aid
• Only about 5 of P. aeruginosa strains do not
  produce obvious pigments on primary isolation
  media
• 25-30oC incubation favors the production of these
  pigments
• Of the three fluorescent pseudomonads, P.
  aeruginosa is the only one to grow at 42oC
• P. aeruginosa usually produces weakly beta
  hemolytic colonies on SBA that tends to be more
  obvious after 48h incubation

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P. aeruginosa pathology

• urinary tract infections (mainly nosocomial)
• respiratory system infections
• dermatitis
• otitis media swimmers ear
• wound infections - various soft tissue infections
• bacteremia 6 of all bacteremias and up to 74
  of nosocomial bacteremias
• bone and joint infections
• gastrointestinal infections
• a variety of systemic infections, particularly in
  patients with severe burns, in cancer, cystic
  fibrosis and AIDS patients (immuno-compromised)

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Virulence factors of P. aeruginosa

• Lipopolysaccharide endotoxin, pyrogen
• Capsule anti-phagocytosis, inhibits complement
  fnc.
• Proteases damage host proteins such as
  complement and IgA
• Hemolysins
• Exotoxin A a diptheria-like toxin - toxic for
  macrophages
• Pyocyanin impairs ciliary activity
• Fimbriae adherence factor
• Other non-pilus adhesins
• Others..

8
Acinetobacter

• Acinetobacter baumanii is the second most
  commonly isolated GNNF in the clinical lab, and
  is associated with nosocomial UTIs, pneumonia,
  URT infection, endocarditis, septicemia,
  meningitis and cellulitis from contaminated
  indwelling catheters
• Since the organism is oxidase negative, it can be
  confused with enterics so is often included in
  commercial enteric ID systems
• Fortunately A. baumanii is more biochemically
  active than most GNNFs and yields a good ID
  with most of these systems
• Three clues might suggest one is working with A.
  baumanii
• Coccoid cells seen microscopically
• Reddish-purple colonies on Mac after 48hr
  incubation rather than brick red or pink colonies
  produced by lactose enterics
• Strong O reaction in Hugh and Leifsons O/F
  glucose test

9
Stenotrophomonas

• S. maltophilia (formerly Pseudomonas (until 1993)
  then Xanthomonas (until 1995) reclassified due
  to nucleic acid homology) is the third most
  frequently isolated GNNF
• S maltophilia is not usually found as an
  indigenous microbe in the general population but
  it can cause nosocomial infections
• Infections include pneumonia, endocarditis and
  wound infections

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Differentiating Common NFBs and Enterics
Flagellar Arrangement Peri ( None)
Monopolar Lophopolar None
HL Glucose F O O/ or K O
Organism Enteric GNR P. aeruginosa S.
maltophilia A. baumanii
KIA KorA/A K/K K/K K/K
Oxidase Neg Pos Neg Neg
HL,Hugh and Leifson KIA, Kliglers Flagellar
arrangements Peri, peritrichous Monopolar,
one flagellum on the end of the cell,
Lophopolar, tuft of flagella at the end A.
baumanii is nonmotile
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Differentiating Common NFBs
Nitrate reduction -/ -
Organism P. aeruginosa S. maltophilia A.
baumanii
ONPG - -
LDC - -
42oC 1 /-
Pigment 2 3 -
1 Differentiates P. aeruginosa from other
fluorescent pseudomonads 2 Yellow fluorescent and
blue green or other diffusable pigments 3 Yellow
or green non-fluorescent usually non-diffusable
pigment
12
Other Less Frequent NFBs
Notable Infection Severe lung infections in
cystic fibrosis melliodosis (Vietnamese time
bomb) Glanders (systemic disease of horses and
rarely man) Nosocomial wound and
UTI infection Neonatal septicemia and meningitis
Organism Burkholderia cepacia B.
pseudomallei B. mallei Alcaligenes
faecalis Chryseomonas meningosepticum
13
Definitive identification

• With the exception of the florescent pseudomonads
  and possibly A. baumanii, GNNFs may require a
  large battery of biochemical and physiological
  tests for identification
• This may not be cost effective or appropriate in
  many hospital labs for all isolates, and even
  commercially available ID kits may not
  satisfactorily identify many of them to the
  species level
• If an isolate is deemed to be clinically
  significant and species ID cannot be attained
  with a kit, it can be sent to a reference lab
• Reference labs can identify GNNFs in a cost
  effective manner

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Detection of Acid Produced by GNNFs

• Accurate ID via biochemical means depends on
  knowledge of an organisms carbohydrate
  utilization characteristics. Media that indicate
  acid from fermentation from pH change are
  commonly used for this, as for enterics. Most
  GNNFs do not produce acid from carb fermentation,
  or too little to be detectable in this way
• GNNFs produce more alkaline products from peptone
  than do enterics. The pH increase from amino
  acid deamination can neutralize any acid produced
  from carb fermentation false (-)
• Eventhough carb fermentation media such as KIA
  have a carb peptone ratio of 12, enterics
  result in sufficient acid from carb fermentation
  to be readily detectable. Most fermenters
  produce acid in the butt of KIA but GNNFs produce
  an alkaline butt
• For these reasons, carb fermentation media for
  GNNFs must have a higher carbpeptone ratio

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Test for Fermentation of Glucose
Kliglers or Triple Sugar Iron
Stab the butt once and streak the slant
Incubate 37oC 12-48h
Alkaline butt indicates nonfermenter
16
Detection of Acid Produced by GNNFs

• Lack of acid in KIA or TSI is often one of the
  first clues that the isolate is a GNNF
• The next step is to determine if and how the
  organism produces acid from sugars.
• Hugh and Leifson O/F media contains 0.2 peptones
  and 1 sugar. The low peptone, sensitive pH
  indicator and pH buffer is perfect for the
  situation that we have described for GNNFs the
  slight acidification caused by GNNFs the media
  turns yellow
• Organisms that do not produce acid usually cause
  the medium to turn dark blue in the presence of
  oxygen (due to alkali from peptones) and no
  change in the absence of oxygen
• Recall the information on O/F media from the
  enteric section

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Test for Fermentation of Glucose
Hugh and Leifsons Oxidation/Fermentation test
Stab inoculate
Incubate 30-35oC 35oC 12-24h
Acid below the oil indicates Fermentation
No oil
Oil
18
Test for Fermentation of Glucose
Hugh and Leifsons Oxidation/Fermentation test
Stab inoculate
Incubate 30-35oC 12-48h
Acid in top of open tube only indicates oxidation
No oil
Oil
19
Test for Fermentation of Glucose
Hugh and Leifsons Oxidation/Fermentation test
Stab inoculate
Incubate 30-35oC 12-48h
No change or Alkali in top of the open
tube indicates lack of oxidation or fermentation
No oil
Oil
20
Culture media

• No special media or incubation conditions are
  required for the initial growth of GNNFs from
  clinical specimens
• The media used for enterics will suffice
• Since GNNFs tend to grow slower than enterics
  incubation times should be no less than 48h
• Incubators should have a high humidity and
  include carbon dioxide