Version: 1 / Document code: 002
Section: Microbiology / Title: Identification of NFGNB
Written by: Joanne Letchford / Date: January2012
Updated: 27.01.2013 / Page 1 of 4

Title: Identification of Non Fermenting Gram Negative Bacilli (NFGNB)

Test Summary:

This document outlines techniques used to help identify NFGNB

Principle

Non Fermenting Gram Negative Bacilli (NFGNB) are environmental organisms found in water, soil or on plants.

Because of their ability to survive in aquatic environments these organisms have become a problem in the hospital environment.

NFGNB are associated with a wide range of infections , predominanatly hospital acquired (nosocomial) infections. Such infections usually occur in patients with immunosuppresion.

In clinical specimens the most common NFGNB is Pseudomonas sp, followed by Acinetobacter sp and Stenotrophamonas maltophilia.

Specimen Handling and Preparation

Always ensure you have a pure, fresh culture on sheep blood agar to use for identification testing.

Quality Control

Ensure biochemical testing and media quality control is performed with ATCC organisms.

Reagents, Materials and Equipment

Sheep Blood Agar (SBA)

MacConkey Agar (MAC)

Mueller Hinton Agar (MH)

Kliger Iron Agar

Lysine Iron Agar

Gram stain reagents

Biochemical test reagents

Version:1 / Document code: 002
Section: Microbiology / Title: Identification of NFGNB
Written by: Joanne Letchford / Date: January2012
Updated: 27.01.2013 / Page 2 of 4

Polymixin B discs

Imipenem and Trim/Sulfa discs

Procedure

After performing the usual Gram negative Bacilli identification tests (Indole, Oxidase, KIA, LIA, SIM) consider the following:

Look carefully at colony morphology and colour on Sheep blood agar and MacConkey agar.

Set up Polymixin B and Imipenem and Trim/Sulfa disc diffusion tests.

Always identify to exclude Burkholderia pseudomallei, this is an important pathogen.

Use Standard Operating Procedures to perform the identification tests in the table below.

Use the table below to determine the most likely identification.

Identification TEST / B.cepacia / B.pseudomallei / Acinetobacter / Stenotrophamonas maltophilia / Shewanella putrefaciens
Gram stain / Small GNB / GNB
bipolar / GNCB / Small GNB / GNB
Pigment / yellow/green
(metallic) / Brown/tan
Oxidase / +(weak/slow)/- / + / - / - / +
Mac / LF with prolonged incubation / LF with prolonged incubation / NLF
MAC-Pale pink/purple / NLF / NLF
indole / - / - / - / - / -
motility / + / + / - / + / +
KIA / K/K / K/K / K/K / K/K / K/K
Gas / + / + / + / +
H2S / - / - / - / - / +
LIA / K/K / K/K / K/K / K/K
Catalase / + / + / + / + / +
Polymixin B / R / R / S / S
SXT / S / S (disc diffusion not reliable) / S (drug of choice)
Augmentin / R / S
Imipenem / V / S / S / R
Version: 1 / Document code: 002
Section: Microbiology / Title: Identification of NFGNB
Written by: Joanne Letchford / Date: January 2012
Updated: 27.01.2013 / Page 3 of 4

Reporting Results

Report the organism identified with appropriate antibiotic sensitivity results.

Always include in the report the Gram stain result including the number of polymorphs present.

If it is unclear if the organism is a pathogen, add the comment:

Comment: Significance needs to be assessed clinically

If the organism is difficult to identify:

Report as Non Fermenting Gram Negative Bacilli (based on KIA : K/K), provide an AST and

Comment: Significance needs to be assessed clinically

Or

If the isolate is from a sterile site (eg blood culture) and the clinician considers this a pathogen

Report as above and send the isolate to NAMRU2 for identification.

Expected Values

The table is based on most likely results.

Refer to The Manual of Clinical Microbiology for individual test result likelyhood and further identification testing.

There are many NFGNB and full identification may not be possible, in this case report the organism as: Non Fermenting Gram Negative Bacilli

Limitations

Confirm SIM motility negative with a wet mount. (NFGNB do not grow well in SIM agar)

Check colony morphology and colour changes over 3 days incubation. B.pseudomallei can become more wrinkled and show lactose fermentation on MacConkey agar over time.

Other organisms are resistant to Polymixin B: Achromobacter, Ralstonia

Version: 1 / Document code: 002
Section: Microbiology / Title: Identification of NFGNB
Written by: Joanne Letchford / Date: January 2012
Updated: 27.01.2013 / Page 4 of 4

Reference

Dr Ellen Jo Baron Flowcharts

Manual of Clinical Microbiology 9th Edition P.R.Murray, E.J.Baron, J.H.Jorgensen, M.L.Landry, M.A.Pfaller