This is a detailed overview of the elusive but increasingly important group of Oxidase Positive Non-fermenters.

1. Non Fermenter GNB
Oxidase positive

2. Definition
In Microbiology lab. Non fermenters are defined as all aerobic gram- negative
rods/coccobacilli that show abundant growth within 24hrs on the surface of KIA or
TSI media, but neither grow in nor acidify the butt of media

4. Initial clues to a NFGNB
1. Lack of evidence for glucose fermentation in TSI/KIA
2. Positive cytochrome oxidase reaction*
3. Failure to grow on MacConkey agar**
The two most widely used identification schemes based on phenotypic biochemical reactions are those of
Weyant and associates (CDC) and Schreckenberger * (Practical approach to identification of NFGNB)

5. Key tests for identification
1. Hugh Leifson OF medium
2. Oxidase reaction
3. Motility & flagellar stains
4. Pigment production
5. Polymyxin B
6. Urea hydrolysis
7. Nitrate-Nitrite reduction
8. Indole
9. Decarboxylation of amino acids
10.Esculin hydrolysis

6. Clinically important NFO*GNB
Family
Pseudomonadaceae
Family
Burkholderiaceae
Family
Sphingobacterium
Family
Sphingomonadaceae
Genus:
-Pseudomonas
Genus:
-Burkholderia
-Ralstonia
Genus:
-Sphingobacterium
Genus:
-Sphingomonas
Family
Alcaligenceae
Family
Shewanellaceae
Family
Moraxellaceae
Family
Flavobacteriaceae
Genus:
-Bordatella
-Alcaligenes
Genus:
-Shewanella
Genus:
-Moraxella
Genus:
-Elizabethkingia
-Chryseobacterium

7. Pseudomonas

8. Non – fluorescent pigmented Pseudomonas
Pseudomonas luteola Pseudomonas oryzihabitans

9. Most common & most important NFO*GNB
Pseudomonas

10. Pseudomonas

11. Pseudomonas
For practical purposes, if an isolate is encountered that produces fluorescein (pyoverdin) pigment and does not produce
pyocyanin or grow at 42°C, laboratories may wish to report the isolate as “P. fluorescens/putida complex,” with no further
testing, since most laboratory isolates are not considered clinically significant.

12. Pseudomonas

13. NFO*GNB other than Pseudomonas - Approach
• Oxidase
• Pigment
• Sensitivity to Colistin, Polymyxin B & Cotrimoxazole
• Motility
• Morphology – Bacilli/Coccobacilli
• Indole*
• TSI* H2S

14. New & increasingly common NFO*GNB
Burkholderia

15. Burkholderia cepacia
Left: Colonies of B. cepacia on oxidative-
fermentative base, polymyxin B, bacitracin,
lactose (OFPBL) agar are yellow.
Right: Colonies of B. cepacia on Burkholderia
cepacian selective agar.
Chromagar by Biomeriux for B. cepacia

16. Burkholderia contaminans

17. Burkholderia

18. Burkholderia
The genus was named after Walter H. Burkholder, plant pathologist at Cornell University.
Phytopathogen - onion bulb rot in plants and foot rot in humans, opportunistic human
infections, particularly in patients with chronic granulomatous disease and cystic fibrosis (CF)
Most frequently recovered from CF: B. contaminans followed by B. cenocepacia B. cepacia &
B. multivorans – epidemic spread & “Cepacia syndrome”
Burkholderia species are resistant to polymyxins, aminoglycosides and beta lactams - can also
use Penicillin G as the sole source of carbon!
Typical pattern: Cot sensitive – PB & Colistin resistant *

19. Burkholderia
Hospital outbreaks of B. cepacia infection are usually due to a single contaminated source such
as anesthetics, disinfectants, intravenous solutions, nebulizer solutions, mouthwash and
medical devices, including respiratory therapy equipment
Disinfectants in which B. cepacia will grow include povidone–iodine, quaternary ammonium
compounds, and chlorhexidine.
Pseudobacteremias (false-positive blood cultures) have been reported following the use of B.
cepacia-contaminated disinfectant solutions
B. cepacia can also grow in distilled water with a nitrogen source owing to the ability of this
organism to fix CO2 from air

20. B. Mallei causes Glanders – which is a highly contagious and often fatal zoonotic disease
primarily of solipeds such as horses, mules, and donkeys – also transmitted to humans
B. mallei was one of the first biological warfare agents used in the 20th century, specifically
during World War I
Glanders is transmitted by direct invasion of abraded or lacerated skin; inhalation with deep
lung deposition; and by bacterial invasion of the nasal, oral, and conjunctival mucous
membranes - chronic, disseminated, pulmonary, and septicemic presentation can be seen
The “mallein test” is used for veterinary diagnostic purposes
In humans - definitive diagnosis of glanders requires isolation of the organism and positive
identification
Burkholderia mallei

21. B. pseudomallei causes Meliodosis - can range from asymptomatic to acute melioidosis or
chronic meliodosis (with reactivation potential – “Vietnamese time-bomb”)
People with diabetes mellitus or regular exposure to the bacteria are at increased risk -
Disease should be considered in anyone staying in endemic areas who develops fever,
pneumonia, or abscesses in their liver, spleen, prostate, or parotid gland.
Symptoms of sepsis (predominantly fever) with or without pneumonia, or localized abscess or
other focus of infection.
Non-specific signs and symptoms - "the great mimicker“
Chronic - symptoms lasting greater than two months - loosely mimics tuberculosis and has
sometimes been called "Vietnamese tuberculosis“
Burkholderia pseudomallei

22. Burkholderia mallei & pseudomallei
Burkholderia pseudomallei
Burkholderia mallei

23. Burkholderia mallei & pseudomallei - key tests

24. Important Yellow pigmented non-fermenters

25. Yellow pigment
Myroides odoratus

26. Sphingomonas
S. paucimobilis has been isolated from a variety of clinical specimens, including blood, CSF,
urine, wounds, vagina, and cervix and from the hospital environment, community-acquired
bacteremia and peritonitis have also been reported in
patients receiving long-term ambulatory peritoneal dialysis.
There have been a few reports of nosocomially acquired S. paucimobilis infections from
contamination of hemodialysis fluids, contamination of a hospital water system,
contamination during in vitro processing of bone marrow for transplantation & catheter-
related sepsis.
Motility* Few cells are actively motile in broth culture, thus making motility a difficult
characteristic to demonstrate. Motility occurs at 18°C to 22°C, but not at 37°C.
Oxidase reaction is weak positive or negative.

28. Elizabethkingia
E. meningoseptica (formerly Flavobacterium meningosepticum and CDC group IIa) is the
species most often associated with significant disease in humans.
Adults - causes pneumonia, endocarditis, wound infections, postoperative bacteremia, and
meningitis, usually in patients with severe underlying illness.
It is highly pathogenic for premature infants and has been associated with neonatal meningitis -
epidemics may occur in nurseries with a mortality rate as high as 55%
E. meningoseptica has increasingly been reported as a cause of healthcare-associated
infections. A cluster of four patients with ventilator- associated pneumonia was reported in an
intensive care unit traced to contaminated humidifier water
Also seen in patients receiving hemodialysis and healthcare-associated bacteriuria primarily in
elderly patients with diabetes.

29. Elizabethkingia – Indole & Oxidase positive NGBNB*

30. Bordetella
Currently the genus Bordetella comprises eight species, four are motile with peritrichous
flagella (B. avium, B. bronchiseptica, B. hinzii, B. trematum and four are nonmotile (B. holmesii,
B. pertussis, B. parapertussis, B. petri
Phenotypically they behave quite differently, however. B. bronchiseptica is motile by means of
peritrichous flagella and grows readily on ordinary media, whereas B. pertussis and B.
parapertussis are both nonmotile. B. pertussis requires special media for growth, whereas B.
parapertussis will grow on blood, chocolate, and MacConkey agars. B. pertussis and B.
parapertussis are the etiologic agents of whooping cough.
B. bronchiseptica has been reported to cause pneumonia in patients with AIDS, acute
leukemia, CF, thoracic trauma, following bone marrow or heart transplantation.
B. Bronchiseptica has the distinguishing biochemical feature of rapidly converting
Christensen’s urea agar

31. Moraxella
The Moraxella species of medical importance are M. lacunata, M. nonliquefaciens, M. osloensis, M.
atlantae (CDC group M-3), and M. catarrhalis.
Blood agar: pinpoint colonies (usually less than 0.5 mm in diameter), with poor or no growth on
MacConkey agar - tiny, gram-negative diplococci or diplobacilli in Gram-stained preparations with a
tendency to resist decolorization. Both oxidase *(Acinetobacter) and catalase (Kingella*)reactions
are positive
Most Moraxella species are extremely susceptible to low concentrations of penicillin. Examination of Gram-stained smears prepared from the outer zone of inhibition
around the penicillin susceptibility disk can be used to distinguish Neisseria species (which retain their coccal morphology) from Moraxella species (which produce
elongated, pleomorphic forms)

32. Shewanella
Shewanella most of which are associated with aquatic and marine habitats; however, the type
species, S. putrefaciens, and another S. algae has been recovered from human clinical specimens
Although they are infrequent clinical isolates, S. putrefaciens and S.algae have been associated
with skin ulcers, ear infections, eye infections, arthritis and osteomyelitis, bacteremia, infective
endocarditis, and peritonitis in patients undergoing continuous ambulatory peritoneal dialysis.
Easily distinguished because they are the only non-fermenters that produce hydrogen sulfide in
KIA and TSI media.
S. algae requires NaCl for growth, while S. putrefaciens does not need it
Shewanellae are generally susceptible to most antimicrobial agents effective against gram-
negative rods, except penicillin and cephalothin.

33. Shewanella

34. Other pigment producing NFGNB - Alcaligenes
Alcaligens fecalis dark greenish agar at periphery
A.faecalis is the most frequently isolated - produce
strong alkaline reactions in all carbohydrate media
Thin, spreading irregular edge of colony - cause a
greenish discoloration of blood agar medium.
A key biochemical feature of this species is its ability
to reduce nitrite but not nitrate.
Rare cause of acute otitis, urinary tract infection and
bacteremia & often found in mixed cultures,
particularly in samples of diabetic ulcers of the feet -
clinical significance is difficult to determine.

35. Pink-red pigment group
Rhizobium radiobacter *L-oxi Roseomonas pink-red Methylobacter – pink/orange

40. TABLE 7-34 Identification of Pigmented Nonfermenters (Oxidase +, Motility +, Fluorescein −, 10% Lactose −, Acetamide −,
Lysine −)

41. Automated systems
1. API 20E and API 20NE Systems for Identifying Nonfermenters
2. RapID NF Plus System for Identifying Nonfermenters
3. The Vitek 2 System
4. The Microscan Walkaway-96, Walkaway-40, and Autoscan-4 Systems
5. The Sensititre AP80 System
6. The Phoenix System

42. 1. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
2. 16S rRNA Gene Sequencing
Molecular methods

43. CREDITS: All tables adapted from Koneman’s Color Altas & Textbook of Diagnostic Microbiology 8th Edition