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1. ARYA.J
MSc MICROBIOLOGY

2. GENERAL PROPERTIES
• Gram negative , areobic , non sporing , non capsulated bacilli.
• Motile :polar flagella
• Non fermentative and oxidase positive
• Sprophytes ;mostly in soil water and decomposing matter
• Major pathogen in hospitalized and cystic fibrosis patients
• Pathogenic member :Pseudomonas aeruginosa

3. PSEUDOMONAS AERUGINOSA
Morphology
• Slender , gram negative bacilli ,non sporing, non capsulated ,motile.
• Most strain possess pili
• Occasionally strains have 2 or 3 polar flagella

4. VIRULENCE FACTORS AND PATHOGENESIS
• Pathogenesis mainly :develop widespread resistance to multiple
antibiotics and disinfectants
 Colonization :organism adhere and colonize the host surface
• polar flagellum to reach the host’s surface and pili ,fimbria to attach.
 Toxins
• Non diffusible toxins :exotoxin S,U,T &Y
Colonized organism via type 111 immune system inject the toxin to host cells
bacteria invade phagocytic cells cytotoxic activity tissue injury

5. • Diffusible toxins: exotoxin A by organism’s type 11 secretion system
act freely and cause tissue injury
most important virulence factor
inhibits protein synthesis by inhibiting elongation factor 2
 Enzymes :elastases ,proteases ,phospholipases ,hamolysins e.t.c…
 Host’s inflammatory response :as a defence mechanism
against bacilli components like endotoxin and flagellin mediated via
TLR 4&5 produce tissue injury and septic shock

6.  Pigment production :diffuse freely , inhibits other bacteria and
mediate tissue injury
• Pyocyanin :blue green phenazine by Ps.aeruginosa ;soluble in
chloroform and water
• Fluorescein (pyoverdin) :greenish yellow insoluble in chloroform and
soluble in water
• Pyorubin :reddish brown pigment ;soluble in water
• Pyomelanin :brown to black pigment

7. pyoverdin
pyorubin
pyomelanin

8.  Alginate coat :some mucoid strains have slime layer /alginate layer
and produce biofilm ,helps in adhesion to host cell ,cause infection
 Capsule :preventbacteria from phagocytosis
 Multidrug resistance
 Biofilm formation :prevent entry of antibiotics into bacterial cell
 Multi disinfectant resistance
 Wide temperature range :survives in extremes of temperature 5-45°C

9. CLINICAL MANIFESTATIONS
• Cause infection in almost all sites ;common in lungs ,skin , and soft t/s
• Mostly in hospitalized patients colonized with organisms
from contaminated hospital environment /hospital staff.
• Colonized patients :risk factors burnwounds,immunosupressed
and post surgical patients.
VAP : in patients on ventillator in ICU
Chronic respiratory tract infctions :in patients with cystic fibrosis,
bronchiectasis / chronic panbroncholithiasis.

10. o mucoid strains of pseudomonas cause this infection
o These strains adhere the mucous initiates infection
Bacteremia :to sepsis and septic shock
Infective endocarditis : IV drug abusers
Ear infections :mild ; Swimmer’s ear ( children ) & malignanat
otitis externa (elderly diabetic patients )
Eye infections :corneal ulcers and endopthalmitis
Shanghai fever :mild febrile fever resmbling typhoid fever
Skin and soft tissue infection

11. • Burn patients :infect burn wounds
• Ecthyma gangrenosum :acute necrotising condition in AIDS
febrile neutropenia patients
• Dermatitis :folliculitis and papular/vesicular lesions
• Toe –web infections
• Green nail syndrome :inflammation of t/s adjacent to nail with green
pus formation
• Cellulitis :blue green pus
Other infections like osteomyelitis,septic arthritis.meningitis,UTI

12. LABORATORY DIAGNOSIS
Specimen :pus, wound swab, urine, sputum, blood or CSF
Direct smear :Gram negative slender bacilli and pus cells
[occasionally capsulated no spores]
Culture :non fastidious ,obligate aerobe
 Peptone water :uniform turbidity with surface pellicle
 Nutrient agar :large ,opaque ,irregular with metallic sheen
(iridescence) with blue green diffusible pigment
Pigment production enhanced : King’s media

13. Smell : most have sweet ether / alcohol like fruity odor
• After aerobic incubation on NA for 24 hrs at 37°C six distinct
colonial types are observed
 Type 1: large , low convex ,rough , often oval
 Type 2: small , smooth domed
 Type 3 & 4 :small , rough and rugose
 Type 5 : mucoid , alginate producing
 Type 6: dwarf and slightly mucoid

14.  Blood agar :β hemolytic grey moist colonies
 Macconkey agar : pale NLF colonies
 Selective media cetrimide agar :to isolate organism from mixed
growth in purulent specimen
 Culture smear and Motility :Gram negative bacilli and actively motile
by single polar flagellum

16. Biochemical reaction
 Oxidase and catalase :positive
 Non fermenter but utilize sugars oxidatively
 OF : Oxidative
 Indole test : negative
 Citrate : utilized
 Urease :hydrolysed
 TSI : k/no change no gas &no H2S

17. Typing Methods
 Various typing for epidemiological studies are
• Bacteriocin (pyocin) typing :ability of strain producing distinct
bacteriocin that inhibits certain indicator bacterial strains.
• Antibiogram typing : is based on the antimicrobial resistance pattern
of strain.Easiest and most commonly used method.
• Serotyping : based on O and H antigens ,17 serotypes of P.aeruginosa
have been recognized
• Molecular methods : pulse field gel electrophoresis (PFGE) and
sequence based typing method

18. TREATMENT
Pseudomonas species are resistant to most of the antibiotics
• Penicillins : piperacillin , mezlocillin , ticarcillin
• Cephalosporins : ceftazidime , cefoperazone , ceftolozane ,and
cefepime
• β –lactamase inhibitor : combinations ( piperacillin – tazobactam and
cefoperazone – sulbactam)
• Carbapenems :imipenem , meropenem ,doripenem
• Monobactam :aztreonam
• Aminoglycosides :tobramycin , gentamicin , amikacin
• Quinolones : ciprofloxacin , levofloxacin
• Polymyxins : polymyxin B , colistin

19. DRUG RESISTANCE
Possesses a number of drug resistant plasmids
Many strains are producers of β lactamases such as ESBL ,
carbapenemases and AmpC β lactamases .
Many strains are resistant to aminoglycosides and quinolones
PREVENTION
Hand hygiene is important to limit the spread of infection

20. OTHER NON FERMENTERS

21. BURKHOLDERIA
• Oxidase positive , non fermenters
• Bipolar staining (safety pin appearance )
• Resistant to polymyxin B
BURKHOLDERIA PSEUDOMALLEI
• the causative agent of melioidosis
• Habitat: B. pseudomallei is a saprophyte of soil and water.
Melioidosis also occurs in rats, rabbits and guinea pigs.
• Mode of transmission: Humans and animals are infected by inoculation, inhalation or
ingestion.
Man to man transmission is very rare.
• Virulence factors: B. pseudomallei is perhaps the most virulent among the non-
fermenters. Several virulence factors such as polysaccharide capsule, type 1V secretion
system, LPS, toxins and enzymes.

22. Risk factors
• Diabetes
• Renal failure in adults
• Traumatic inolcutaion in children
• Rainy season
• Occupation [rice farmers]
 Incubation period
• From 2 days to many years
• Some cases have long latency ;presesented long time after exposure so
mellioidosis also called “Vietnam time bomb disease”

23. Clinical features
• Can present with array of manifestations so called great mimicker
 Acute , localized infection : nodule , fever , general muscle aches , & progress
rapidly to blood
 Sub acute / pulmonary : mild bronchitis to severe tuberculosis like pneumonia
 Acute blood stream infection : seen in HIV , renal failure and diabetes patients :;
ptresent as septicemia with metastatic pus – filled skin lesions
 Chronic suppurative infection : form abscesses in joints , viscera , lymoh nodes ,
skin . Brain , liver , lungs , bones , spleen .

24. Bioterrorism
Used as a potential agent of biological warfare
Laboratory diagnosis
• Specimen: depends on site of infection,
sputum, purulent discharge from lesion, etc
• Direct microscopy : gram-negative bacilli , typically exhibit a bipolar or safety pin
appearance which is better seen when stained with methylene blue.
• Culture :obligate aerobe, grows in various media, e.g. nutrient agar, blood agar
and MacConkey agar. colonies are typically rough and corrugated
 Ashdown's medium is used as a selective medium, where it produces wrinkled
purple colonies.

26. Biochemical reactions:
Important properties that differentiate it from Pseudomonas stutzeri include:
• Gelatin liquefaction positive
• Utilizes arginine
• Positive for intracellular poly β hydroxy butyrate
 Latexagglutination test:
Cultures can be confirmed by latex agglutination test using specific antisera
Treatment of melioidosis
Intensive phase (2 weeks): Ceftazidime or acarbapenem is given followed by
Maintenance phase (12 weeks): Oral cotrimoxazole is given to eradicate the bacilli