This document provides information about Pseudomonas aeruginosa and other non-fermenting gram-negative bacilli. P. aeruginosa is a common opportunistic pathogen able to infect many sites in hospitalized and immunocompromised patients. It has many virulence factors that aid colonization and pathogenesis, including pili, flagella, and various toxins and enzymes. Treatment can be challenging due to its high intrinsic and acquired antibiotic resistance. Other non-fermenters described include Burkholderia pseudomallei, the causative agent of melioidosis. It is a serious disease endemic in Southeast Asia that can have varied clinical manifestations.

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