Nonfermenting Gram-negative rods

􀻨 Pseudomonas
Pseudomonas (Migula, Walter 1894)

Gk. pseudo = “false” + monas = “unit”. “Cells with polar organs of motility. Formation of spores occurs in some
species, but it is rare”— Migula2

Facultative anaerobic, non-spore forming, Gram-negative rods which are straight or

slightly curved and are 0.5-1.0 by 1.5-5.0 µm depending on the growth phase

Motile with one or more polar flagellum (mono-, peritrichous)

Possess aerobic respiratory metabolism with oxygen as the terminal electron acceptor.

Most species are oxidase positive except P. luteola, and P. oryzihabitans

Catalase positive and are chemolithotrophs.

Oxidase positive

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Nonfermenting Gram-negative rods

􀻨 Pseudomonas
Two distinct groups:

1. Fluorescent group (6)

• Produces pyoverdin (water soluble) yellow-green or yellow-brown pigment that

fluoresces blue-green when exposed to UV light

• P. aeruginosa, P. fluorescens, P. putida, P. veronii, P. monteilii, P. mosselii

• Many P. aeruginosa also produce the blue water-soluble phenazine pigment

pyocyanin. When pyoverdin combines with the blue pigment pyocyanin, the bright
green color of P. aeruginosa is created. Other diffusible pigments include,
pyomelanin (brown-black) and pyorubrin (red)

2. Nonfluorescent group (6)

• P. stutzeri, P. mendocina, P. alcaligenes, P. pseudoalcaligenes, P. luteola, P.

oryzihabitans
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 Nonfermenting Gram-negative rods
􀻨 Pseudomonas
Characteristics:

1. Compose of species with both clinical and environmental implications.

2. Many species are saprophytic and pathogenic for plants.

3. Extremely versatile, survives in a broad range of habitats— organism’s cell grow as

biofilms. It can be found in sinks, showers, respirator equipment, and the water of
dental water-cooled drilling equipment.

NOTE: Pseudomonas spp. may be colonizers— isolation does not always link with clinical
disease but their presence intracellularly in polymorphonuclear cells is clinically significant.

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Nonfermenting Gram-negative rods

􀻨 Pseudomonas
Endogenous vs. Exogenous Infection
Endogenous, typically occurs in individuals whose colonization resistance has been
perturbed (disturbed).

Example: bacteremia 2º GI colonization in neutropenic hosts and pneumonia

individuals who have required endotracheal intubation (ventilator-associated
pneumonia, VAP)2

Exogenous infection likely occurs in Cystic fibrosis patient (CF), since their initial infecting
isolates usually resemble environmental morphotypes.

_____

Other infections are acquired (eg., otitis externa, folliculitis, chronic wound infections, and
osteomyelitis of the foot in diabetic patients.

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 Nonfermenting Gram-negative rods
􀻨 Pseudomonas

Some species prefer incubation temperatures lower than 25ºC, while Pseudomonas
aeruginosa (Schroeter, 1872) can grow at temperatures up to 42ºC

Alkalinazation of acetamide

Denitrification of nitrates and nitrites

_____

Organisms are easily recovered for clinical specimens (eg., sputum, discharges, etc.)

Samples containing Pseudomonas spp. can be refrigerated at 2 to 8ºC for up to 4 weeks.

Non-aeruginosa pseudomonads may grow better at lower temperatures (RT, 28 to 30ºC)

P. fluorescens

P. putida

P. oryzihabitans

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Nonfermenting Gram-negative rods

􀻨 Pseudomonas

Microscopy
Gram-negative bacilli (thinner than Enterobacteriaceae).
Some appear elongated (P. putida) and pleomorphic (older
cultures)

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 Nonfermenting Gram-negative rods
􀻨 Pseudomonas

Colonial Morphology
Mucoid, nonmucoid phenotype (revertants frequently due
to algT gene mutations)

๏ Mucoid forms represents biofilms in the lungs —

should be documented

Small-colony variants (SCV)

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Nonfermenting Gram-negative rods

􀻨 Pseudomonas
Colonial morphology

Simple nutritional requirements. Grow well on standard broth media and solid media (eg., TSA +5%
sheep’s RC- β-hemolytic, CA, and McC- NLF)— recommended media.

Mucoid strains may appear in McC especially from CF patients.

Good growth can be attained after 24- to 48h of incubation, but plates may be incubated for 48- to
72h since some isolates grow slowly.
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 Nonfermenting Gram-negative rods
􀻨 Pseudomonas
Identification
Easily recognizable based on colonial morphology, production of diffusible pigments, a grape-like odor or
“corn tortilla-like” odor, and a positive oxidase reaction.

Colonies are usu. flat, spreading, and have a serrated edge and a metallic sheen (often assoc. with autolysis
of the colonies)

Smooth, mucoid, and dwarf (SCV) may be present.

Grow at 42ºC

TSI: K / NC, Arginine +, reduced nitrate to nitrite,

NOTE 1: Mucoid isolates from CF patients may produced slow growth, Non-motile, and loss of pigment
production

NOTE 2: SCV may require prolonged incubation, Non-motile, adherence to agar.

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Nonfermenting Gram-negative rods

􀻨 Pseudomonas
Fluorescent group
Produce pyoverdin— fluoresces under short wavelength UV light.

King’s medium B detects pyoverdin

King’s medium A detects pyocyanin (blue pigment resulting from extraction with
chloroform)— exhibits by most strains except form CF patient with chronic lung infection.

Other pigments (water-soluble)

1. Pyorubrin (red)

2. Pyomelanin (brown-black)

NOTE 1: Conditions of iron limitations enhance pigment production, because these

pigments acts as siderophores in iron uptake system of the bacteria

NOTE 2: Use of non-dye-containing media enhance the visualization of pigments.

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 Nonfermenting Gram-negative rods
􀻨 Pseudomonas
Identification

Urease (V)
TSI, LIA:
King A
King B
OF:
TSB
TSI: K/NC
Gray Metallic Pyocyanin:
Fluorescein:
Open:
(Peptone):
LIA: -/+
sheen Blue-green Green Glu+; Mal -
42ºC

CIT: +
fluorescence
Closed:
Positive
SIM: - - +
(UV light) Glu+; Mal - growth:
turbid

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Nonfermenting Gram-negative rods

􀻨 Pseudomonas
Pseudomonas fluorescens and P. putida
Does not posses a distinctive colony morphology or odor.

Cannot reduce nitrate to nitrogen gas, acid production from xylose — distinguish these two
from other fluorescent pseudomonads

P. fluorescens grow at 4ºC and hydrolyze gelatin

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 Nonfermenting Gram-negative rods
􀻨 Pseudomonas
Pseudomonas veronii and P. monteilii, P. mosselii
P. veronii reduce nitrates to nitrogen gas, acetanimide hydrolysis negative. P. veronii (LMG
17761 strain) does not produce acid from lactose and maltose negative and does not grow
at 36ºC

P. monteilii unable to reduce nitrates either to nitrites or nitrogen gas, gelatin hydrolysis
negative, and no acid production from xylose.

P. mosselii cannot reduce nitrates to nitrites nor nitrogen gas, no gas production from
xylose, but most isolate hydrolyze gelatin

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Nonfermenting Gram-negative rods

􀻨 Pseudomonas

Nonfluorescent Group

P. stutzeri, P. mendocina
P. stutzeri produce dry, wrinkled colony that can pit or adhere to the agar and are buff to
brown — similar to Burkholderia pseudomallei.

Arginine dihydrolase negative, no acid production from lactose, hydrolyze starch

(unique reaction of this species)

P. mendocina colonies are smooth, nonwrinkled, and flat producing a brownish yellow
pigment

Key characteristics: reduce nitrates to nitrogen gas, positive for arginine dihydrolase,
acetamide and starch hydrolysis negative

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 Nonfermenting Gram-negative rods
􀻨 Pseudomonas

Nonfluorescent Group

P. stutzeri

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Nonfermenting Gram-negative rods

􀻨 Pseudomonas
Nonfluorescent Group

P. alcaligenes, P. pseudoalcaligenes
Rarely encountered from clinical samples and biochemically inert Gram negative rods

P. alcaligenes differ from P. pseudoalcaligenes by its inability to oxidize fructose.

P. luteola and P. oryzihabitans

Oxidase negative, and production of an intracellular, nondiffusible yellow pigment.

Exhibit rough, wrinkled, adherent colonies or, more rarely smooth colonies

P. luteola hydrolyze o-nitrophenyl-β-D-galactopyranoside and esculin.

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