This document provides an overview of the bacterium Pseudomonas, including its morphology, natural habitats, pathogenicity, diseases caused, laboratory diagnosis, isolation procedures, cultural characteristics, identification, and biochemical characteristics. Pseudomonas is a common gram-negative rod found in soil and water that can cause opportunistic infections. Key points discussed include that it is oxidase-positive, produces pigments like pyocyanin, and diseases include pneumonia and wound infections. Identification involves culture characteristics, biochemical tests like arginine dihydrolase, and morphology under gram stain.

1. Advanced Bacteriology and mycology assignment
Pseudomonad
Presented by Tamirat Tekilegiorgis
Bahir Dar Jan 2019 GC

2. Out line
o Introduction
o Morphology
o Natural habitat
o Pathogenesis and pathogencity
o Disease caused by pseudomonad
o Laboratory diagnosis
o Isolation procedure
o Cultural characteristics
o Identification
o Biochemical characteristics

3. PSEUDOMONADS
INTRODUCTION
Genus Pseudomonas and Burkholderia
Pseudomonas is a bacteria mostly saprophytic in nature, is found
in soil, water and other moist environment.
o It has emerged as an important cause of Health Care Associated
and Opportunistic Infections.
o Most of the clinical isolates of Pseudomonas are resistant to many
antibiotics.
o The family comprises of about eight groups and 191 species, the
type species is Pseudomonas aeruginosa.

4. Pseudomonads morphology
o rod shaped, slender (0.5 to 0.8 μm by 1.5 to 5.0 μm)
o Strict aerobic
o Non spore forming
o glucose non fermenting: cannot catabolize glucose, and are thus
unable to ferment
o gram-negative rods
o They are catalase positive and split sugars by oxidation

5. Pseudo.morph, con’t
o all have polar flagella except Burkholderia mallei, which is non
motile
o Some strains of Pseudomonas particularly those isolated from
cases of cystic fibrosis are very mucoid and have kind of pseudo
capsule (glycocalyx) made of polysaccharides this protects
pseudomonas from host defense
o grow on macConkey agar as pale colonies (NLF) except B.mallei b/c
it needs 1% glycerol in media for optimal growth.

6. Natural habitat
Pseudomonas aeruginosa: This important potential pathogen
occurs widely in nature, especially on mucous membranes.
B. pseudomallei: This potential pathogen is found in soil and
water in Southeast Asia, Australia, and central Africa.
B. mallei: This species differs from the others in that it is an
obligate pathogen that does not occur in nature
p.fluorescens: present in soil and water associated with food
spoilage and can cause lesions in reptiles and fish.

7. Pathogenesis and Pathogenicity
Pseudomonas aeruginosa
P. aeruginosa relative resistance to drugs, it may persist in
infectious processes from which other more susceptible organisms
have been eliminated by treatment.
o Produces a number of protein exotoxins, an entero toxin that is
responsible for diarrhea during initial infection an endotoxin and
numerous extracellular products such as protease and haemolysins
that may play a role in the pathogensis.
o Posses pili which facilitate adherence to epethelial cells and some
strains have a capsule that is anti phagocytic.

8. Pathog Con’t
Burkholderia pseudomallei
o The disease in humans is referred to as melioidosis or pseudoglanders. It
may assume a benign, chronic, or septicemic form in humans and animals.
o The toxins include a lethal factor with anticoagulant activity and a skin
proteolytic agent.
o Infections have been reported in humans, primates, cattle, sheep, goats,
pigs, horses, dogs, cats, and rodents.
o In the more common chronic form, nodules and abscesses occur in the
lungs, liver, spleen, lymph nodes, and subcutis.

9. Burkholderia mallei
B. mallei is the cause of glanders, a disease principally of the Equidae. It has
been eradicated from North America and from central and western
Europe, but it still occurs in eastern Europe and Asia.
o Glander in horse, mule, and donkey has three forms:
1. Pulmonary: Lung lesions in pulmonary glanders commence as small light-
coloured nodules surrounded by a haemorrhagic zone
2. Nasal : Ulceration in nasal glanders may spread within upper respiratory
passages; perforation of the nasal septum has been observed
3. Cutaneous or farcy form: farcy, which is a lympangitis with ulcers along
lymphatic vessels of the limbs and chest The ulcers eventually heal leaving
‘star-shaped’ scars

10. Con’t
o Infections are characterized by the formation of encapsulated nodules
that contain yellow caseous pus.
o Carnivores and humans become infected as a result of contact with
infectious materials. Swine, cattle, rats and birds are considered to be
resistant.
o Psedomonus fluorescens and P. putidu occasionally infect fresh water
fish
o Glanders is an infectious disease that is caused by the bacterium
Burkholderia mallei. While people can get the disease through contact
with tissues or body fluids of infected animals, It also affects donkeys
and mules and can be naturally contracted by other mammals such as
goats, dogs and cats.

11. Other Pseudomonades
o There are numerous free-living pseudomonades. They occur
occasionally as contaminants in clinical materials.
o Several species other than P. aeruginosa have been incriminated in
infrequent infections, mostly in humans.
o The most common of these, P. maltophilia, P. stutzeri, P.
fluorescens, P. acidovorans, and P. putida, are usually contaminants
in clinical specimens.

12. Disease caused by pseudomonads
Species Host(s) Disease
p. aeruginosa Cattle Mastitis, uterine infection, skin infections, abscesses, enteritis and arthritis
Sheep and goat Mastitis, pneumonia, lung abscesses and ‘green wool’ (a skin infection in sheep)
Pigs Enteritis, respiratory infections and otitis
Horse Metritis, lung abscesses and eye infection
Dogs and cat Otitis externa, cystitis, endocarditis, dermatitis, wound infection and conjuctivities
Many animal spp Infection of burns and other wounds, diarrhea, genital and nosocomial infections.
B. pseudomallei Many animal spp Melidosis (pseudoglanders)
Horses The disease can mimic glanders
cattle Acute and chronic forms with localization of lesion in lungs, joints and uterus.
Sheep Arthritis and lymphangitis predominate
Goats Loss of condition, respiratory and central nervous disturbances, arthritis and mastitis.
pigs As for goats but in addition diarrhea and abortion
Dogs Febrile disease with localizing suppurative foci
B. mallei Horse and other equade Glanders:
Human, cats and other animals Acute, septicemia disease

13. LABORATORY DIGNOSIS
o Collect the appropriate sample. This will be according to the tissue/system
affected. Specimen can be pus, urine, blood, tissue, etc.;
o Carry out the Gram staining- Gram negative bacilli will be seen;
o Culture the specimen on Blood agar and MacConkey agar plates. Incubate for
24-48 hrs at 37° C;
o Examine the bacterial growth : Examine type of colonies, pigment production,
odour and do oxidase test from MacConkey agar.
o Pale colonies on MaConkeyAgar, fruity odour, pigment (greenish, brownish)
and oxidase positive test means the growth is probably Pseudomonads; TSI
agar unchanged

14. Gram staining- Gram negative bacilli
Figure 1 Gram staining of pseudomonas: Gram
negative, rod shaped bacteria seen

15. Lab diagnosis Con’t
o Confirm by Arginine dihydrolase test
o Carry out the antibiotic susceptibility
testing by Kirby Bauer disc diffusion
method.
o Read the result-measure the inhibition
zones and label as sensitive , resistant or
intermediate sensitivity taking into
account the zone size. Figure 1: Kirby-Bauer Test to Measure
Antibiotic Sensitivity.

16. Isolation Procedures
 Pseudomonas spp. grow well on blood and on less complex media.
o They may be recovered on various enteric media. Glycerine
stimulates the growth of B. mallei, and for this reason, a glycerol
agar is sometimes used.
o B. mallei grows slowly on blood agar
o The culture of P. aeruginosa, B.pseudomallei and B.mallei are
incubated aerobically at 370c for 24-48 hr but P.floursence grow
extermly poorly, or not at all, at 370c as 300c is often the upper
temperature limit of their growth range.

17. Con’t
Animal Inoculation
o Hamsters and guinea pigs are highly susceptible to B. mallei.
o The guinea pig is favored because of the well-known Straus's
phenomenon.
o Cultures are inoculated into male guinea pigs intraperitoneally, and
pathological material is inoculated subcutaneously.
o The glanders organisms produce a septic orchitis, Straus's
phenomenon, in 3-4 days.
o The organism is readily recovered from the testicle.
o Lesions are also found in the spleen, liver, and other visceral organs.

18. Cultural Characteristics
 Pseudomonas aeroginosa
o Colonies are large (3-4mm) and grayish-blue grap- like odor of
aminoacetophenone and irregular spreading margins.
o Some cultures are markedly mucoid.
o On blood agar, colonies are frequently β-hemolytic.
o Greenish and/or yellowish-green pigments may diffuse throughout clear
media.
o On MacConkey colonies are colorless
o while on brilliant green agar, the metallic sheen displayed by this strain a
feature of some isolate

19. Con’t

20. P. aeruginosa produces two pigments: pyocyanin, which is green or blue-green and
pyoverdin, yellow to green in color.
A similar pigment is produced by P. fluorescens, a nonpathogenic organism that does not
usually grow at 37°C. Some strains of P. aeruginosa produce the pigments pyorubin (red)
and pyomelanin (brown-black).

21.  P. maltophilia
o Colonies are round, smooth, non-
pigmented, and glistening, with
regular margins.
o They are not hemolytic but may
display a greenish discoloration of
blood agar.

22.  B.pseudomallei
o Colonies are evident after 24-hr
incubation on blood and other simple
media.
o After 48-hr incubation, colonies obtain a
size of 1-2 mm in diameter and are
smooth, umbonate, and cream colored.
o Colonies enlarge markedly when left at
room temperature for about a week.
Cultures have an earthy or ammoniacal
odor.

23.  B. mallei
o Grow slower than P. aueroginesa
and B.pseudomallei
o Colonies are shallow, round
convex, opaque becoming
yellowish green or brown on aging.
o Growth is slow on blood agar un
able to grow on macconkey agar
o The colonies have a tendency to be
slimy and tenacious in consistency.

24. Identification
The species referred to above, with the exception of B. mallei, are
motile, and stained smears disclose gram-negative, straight or
slightly curved rods.
 P. aeruginosa
o Characteristic colonies, sometimes with a metallic sheen, are
usually β-hemolytic.
o A blue-green or yellowish-green pigment is often produced in clear
media.
o Other characteristics include alkaline reactions on TSI with no gas or
H2S produced, positive oxidase reaction.

25. Con’t
Other reactions include the following:
o Nitrates are reduced to nitrites.
o Indole is not produced.
o Gelatin is liquefied.
o A selective medium, Cetrimide agare base, is available for the
isolation of P. aeruginosa; other pseudomonads are generally
inhibited.

27. B. mallei
Identification is based on the features listed below.
o Non motile
o Growth is enhanced by glycerin.
o Growth on blood agar is relatively slow.
o Carbohydrates for the most part are not broken down, although
several may be split by oxidation.
o Indole is not produced;
o catalase and ammonia are produced.
o Nitrate is reduced.
o Gelatin is not liquefied.

28. BIOCHEMICAL CHARACTERISTICS
o Pseudomonads has oxidative metabolism. Since organism is non
fermentative the acid is not produced from peptone water sugars.
o Oxidase test positive;
o Catalase test positive
o Nitrates are reduced to nitrites;
o Glucose is utilized oxidatively ⇒ Oxidative reaction in of media
o Indole, Methyl red (MR), Vogues Prauskar (VP) and H2S production
test are negative.
o Arginine dihydrolase test positive;
o Commonest screening diagnostic biochemical test used in lab is
the oxidase test.

29. References
Brokopp, C. Dv and Farmer, J.: Typing methods for Pseudomonas aeruginosa. In Doggett, R. G. (Ed.):
Pseudomonas aeruginosa: Clinical Manifestations of Infection and Current Therapy. New York, Academic
Press, 1979.
Gilardi, G. L.: Pseudomonas. In Lennette, Ε. H. (Ed-in-chief): Manual of Clinical Microbiology, 4th ed.
Washington, D.C., American Society for Microbiology, 1985.
Govan, J. R. W.: Pyocin typing of Pseudomonas aeruginosa. In Bergan, T., and Nonis, J. R. (Eds.): Methods in
Microbiology, Vol. 10. London, Academic Press, 1978, pp. 6 1 -91.
Miller, W. R., Pannell, L., Cravitz, L., Tanner, W. Α., and Ingalls, M. S.: / Bacteriol, 55:115, 1948.
P.J.Quinn, Peter J. Quinn, M.E. Carter, Bryan Markey, G.R. Carter : Clinical Veterinary Microbiology; pp 237-242
Wolfe 1994