Pseudomonas is a gram negative, non-spore forming bacteria that is commonly found in the environment. It produces several pigments including pyocyanin and pyoverdin. It can cause various infections, especially in hospitalized patients. Some key infections include ventilator-associated pneumonia, surgical site infections, and bloodstream infections from central lines. Laboratory identification involves culturing on selective media, observing its morphology and growth characteristics, and conducting biochemical tests to confirm its identity.

1. Pseudomonas
Dr. Rakesh Prasad Sah
Associate Professor, Microbiology
Gram Negative Non Fermenters (GNNF)

2. Introduction
GNNF do not ferment any sugars but utilize them oxidatively
GNNF are a group of unrelated bacteria
Most are environmental commensals
causes various infections in hospitalized patients
are resistant to majority of antibiotics

3. • Non-fermenters include
– Pseudomonas
– Stenotrophomonas
– Burkholderia
– Acinetobacter etc.

4. Pseudomonas

5. Gram Negative
Pseudomonas
Non-sporing
Pigment production
Motile (Polar flagella)
Oxidase Positive
Non-fermentive

6. Pseudomonas aeruginosa
Morphology
• Gram negative
• Non-capsulated
• Non-sporing
• Motile (Polar flagella)

7. Culture
• Strict aerobe
• Grows well on ordinary media.
• Opt temperature 370C. (5-420C)
• Nutrient Agar
– Smooth, large, translucent.
– Sweetish aromatic odour (2-aminoacetophenone).
– Greenish blue pigment. (diffuse into the medium).

8. Culture
• Blood Agar
– Many strains are hemolytic in nature.
• MacConkey agar
– NLF colonies
• Cetrimide agar
– Selective media

9. Pigment Production
Pyocyanin
Pyoverdin
Pyorubin
Pyomelanin

10. Biochemical Reactions

11. Toxin and Enzymes
• Extracellular products
– Pyocyanin  inhibits mitochondrial enzymes in mammalian tissue
and causes disruption and cessation of ciliary beat on ciliated nasal
epithelium  favours colonization of these organisms in the nasal
mucosa.
• Extracellular enzymes and hemolysins
– Proteases
– Hemolysins
– Lipase
Local Lesions

12. • Exotoxin
– Two exotoxins A and S.
– Exotoxin A
• Most important virulence factor of P. Aeruginosa
• Inhibits protein synthesis by inhibiting elongation factor-2. (similar to
diptheria toxin).
• Endotoxin
– LPS in nature.
– Pyrogenic in nature.

13. • Pigment production: Produces a number of pigments -
diffuse freely into the surroundings, inhibit other bacteria and
mediate tissue injury
 Pyocyanin: Blue green pigment.
 Fluorescein (or pyoverdin): Greenish-yellow
 Pyorubin (imparts red color)
 Pyomelanin (imparts brown-black color).

14. Clinical Manifestations
• Can cause infections at almost all sites, most common being
– Lungs, Skin and Soft Tissues
• Most infections are Hospital Acquired Infections.
• Risk factors
– Burn wounds,
– Immunosuppression &
– Post surgeries

15. Healthcare-associated infections
 Ventilator
associated
pneumonia (VAP)
 Central-line
associated
bloodstream
infection (CLABSI)
 Catheter-associated
urinary tract
infection (CAUTI)

16.  Surgical site infection (SSI)
 Post-tracheostmy pulmonary
infection.
 Septicaemia  malignancy or
immunosuppressive therapy.
 Wound or burn infections

17.  Chronic Otitis
media and Otitis
externa
 Eye infections
 Acute necrotising
vasculitis
(hemorrhagic
infection of skin &
internal organs)
 Infantile diarrhoea.

18. • Shanghai fever - Mild febrile illness resembling typhoid fever
• Skin and soft tissue infections –
– Burns wound infection,
– Green nail syndrome and
– Cellulitis with blue-green pus.

19. • Other infections
 Bone and joint infections - osteomyelitis and septic arthritis
 Meningitis (in postoperative or post-traumatic patients).

20. Laboratory diagnosis
• Sample collection: Pus, wound swab, urine, etc.
• Direct smear: Gram-negative bacilli, and pus cells

21. Culture
 Nutrient agar: Opaque, irregular colonies with metallic sheen
(iridescence) and blue green diffusible pigments.

22.  Blood agar: β-hemolytic grey moist colonies
 MacConkey agar: NLF colonies
 Selective media: e.g. cetrimide agar

23. • Culture smear and motility: Motile, gram-negative bacilli
• Identification:
 Catalase positive and oxidase positive
 ICUT tests: Indole (–), Citrate (+), Urease (–), TSI:K/K, gas (–),
H2S(–)
 Automated identification such as MALDI-TOF or VITEK
• Antimicrobial susceptibility testing

24. • Useful during outbreaks.
• For epidemiological studies, various typing methods are used such
as—
 (i) bacteriocin (pyocin) typing,
 (ii) antibiogram typing,
 (iii) serotyping,
 (iv) molecular typing methods - pulse-field gel electrophoresis
(PFGE) and sequence-based typing method.

25. • Intrinsically resistant to ceftriaxone, amoxicillin-clavulanate,
ampicillin-sulbactam, ertapenem, tetracyclines, tigecycline,
cotrimoxazole and chloramphenicol.

27. ACINETOBACTER
• Saprophytic bacilli, present in environment
• Emerged as nosocomial pathogen.
• Genomospecies:
 A.baumannii – most pathogenic species.
 A.calcoaceticus and A. Lwoffii – rarely pathogenic to man

28. • Sources
 Hospital environment, Hospital staff
 Commensals in skin, oral cavity and intestine
• Promote colonization:
 Unhygienic practices in hospitals - Contaminated hands of staff
 Patients with underlying diseases or immunosuppression -
predisposed to invasion and pathogenesis

29. Pathogenesis:
• Multidrug resistance
• Virulence factors
 Outer membrane protein A (OmpA)- adhesion, invasion and
cytotoxicity through mitochondrial damage
 LPS - inflammatory responses  tissue injury
 Ability to form biofilm

30. Clinical manifestations
• Ventilator associated pneumonia
• Central line associated bloodstream infection
• Post-neurosurgical meningitis
• Catheter- associated UTI
• Wound and soft tissue infections
• Infections in burn patients.

31. Laboratory diagnosis
• Obligate aerobe, not fastidious
• Blood agar - Non-hemolytic colonies
• MacConkey agar - Lactose non-
fermenting colonies with faint pink
tint
Lactose non-fermenting colonies
(with faint pink tint)

32. • Gram staining: Gram-negative
coccobacilli
• Oxidase negative and catalase positive
• Non-fermenter of sugars and non-motile
• ICUT tests: Indole test (negative), citrate
test (positive), urease test (negative) and
TSI (triple sugar iron agar) test shows
alkaline slant/alkaline butt with no gas and
no H2S