Oxidase Positive Non-fermenter GNB

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

3. Initial clues to a NFGNB Lack of evidence for glucose fermentation in TSI/KIA Positive cytochrome oxidase reaction* 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)

4. Key tests for identification Hugh Leifson OF medium Oxidase reaction Motility & flagellar stains Pigment production Polymyxin B Urea hydrolysis Nitrate-Nitrite reduction Indole Decarboxylation of amino acids Esculin hydrolysis

5. 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

6. Pseudomonas

7. Non – fluorescent pigmented Pseudomonas Pseudomonas luteola Pseudomonas oryzihabitans

8. Most common & most important NFO*GNB Pseudomonas

9. Pseudomonas

10. 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.

11. Pseudomonas

12. NFO*GNB other than Pseudomonas - Approach Oxidase Pigment Sensitivity to Colistin, Polymyxin B & Cotrimoxazole Motility Morphology – Bacilli/Coccobacilli Indole* TSI* H 2 S

13. New & increasingly common NFO*GNB Burkholderia

14. 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

15. Burkholderia contaminans

16. Burkholderia

17. 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, o pportunistic 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 *

18. 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

19. 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

20. 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

21. Burkholderia mallei & pseudomallei Burkholderia pseudomallei Burkholderia mallei

22. Burkholderia mallei & pseudomallei - key tests

23. Important Yellow pigmented non-fermenters

24. Yellow pigment Myroides odoratus

25. 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.

26. 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.

27. Elizabethkingia – Indole & Oxidase positive NGBNB*

28. 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

29. 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)

30. 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.

31. Shewanella

32. 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.

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

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

35. 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

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

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