Rapid Testing for Gram negative Bacteria- the BioLumix Approach

Gram Negative Bacteria- Cell Wall

Gram negative cell walls are more complex than Gram positive cell walls, both structurally and chemically. Structurally, a Gram negative cell wall contains two layers external to the cytoplasmic membrane. Immediately external to the cytoplasmic membrane is a thin peptidoglycan layer, which accounts for only 5% to 10% of the Gram negative cell wall by weight. External to the peptidoglycan layer is the outer membrane, which is unique to Gram negative bacteria. For Gram negative species, many of the lytic virulence factors such as collagenases, hyaluronidases, proteases, and beta-lactamase are located within the periplasmic space.

The outer layer of the Gram negative bacterial cell wall is made up of lipopolysaccharide and proteins. This outer layer protects a thin layer of peptidoglycan. For the Gram positive bacteria, the outer layer of the cell wall is the peptidoglycan layer and does not contain lipoproteins. Below the outer layer of lipopolysaccharide, there exist layers of periplasmic space and the plasma membrane.

The pathogenic capability of Gram-negative bacteria is often associated with their cell walls, especially, the lipopolysaccharide layer (also known as LPS or endotoxin layer). In humans, LPS can triggers an immune response characterized by cytokine production and immune system activation.

Gram Negative Bacteria- Who are they?

Important gram negative bacteria include: the Enterobacteriaceae family, Pseudomonads, Aeromonas, Plesiomonas, Xahothomonas, Burkholderia, Haemophilus.

The Enterobacteriaceae family contains a number of pathogens including E. coli, Salmonella, Shigella, Klebsiella pneumoniae, Pasteurella, and Yersinia. Pseudomonas aeruginosa is an opportunistic pathogen and can cause urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, etc. Pseudomonas aeruginosa is found in many natural and domestic environments including plants, soils and surface water, especially warm moist environments containing organic material or contaminated by human or animal waste.

Members of the genus Aeromonas and Plesiomonas are involved in human intestinal disease. The species Hemophilus influenzae is a cause of meningitis in children, while Pasteurella. multocida causes cholera in fowl. Burkholderia cepacia, an important pathogen of pulmonary infections in people with cystic fibrosis.

Current methodology

In USP <61-62> the test for Bile-tolerant Gram-negative Bacteria (BTGNB) replaces the “Test for Enterobacteriaceae and Certain Other Gram-Negative Bacteria”. In older USP protocols. USP <62> does require testing for absence of P. aeruginosa and Burkholderia cepacia.

The method usually involves a 1:10 dilution using Soybean–Casein Digest Broth (TSB) as the diluent, and incubation at 20 to 25 C for a time sufficient to resuscitate the bacteria but not sufficient to encourage multiplication of the organisms (usually 2 hours but not more than 5 hours). Thereafter the appropriate amount is sub-cultured on Violet Red Bile Glucose Agar (VRBGA) and incubates at 30 to 35 C for 18 to 24 hours. For the testing of lower numbers of BTGNB, the MPN method can be used. For P. aeruginosa the TSB is pre-incubated for at 30 to 35 C for 18 to 24 hours. Growth is sub-cultured onto a plate of Cetrimide Agar and incubate at 30 to 35 C for 18 to 72 hours

The BioLumix approach

The BioLumix system offers two approaches for the detection of gram negative bacteria:

  1. The Two Vial Approach: An approach similar to the USP procedure where two separate vials are used. One vial detects Bile-tolerant Gram-negative Bacteria and another vial detects P. aeruginosa.
  2. The Single vial approach: An approach where a single vial is used to detect all gram negative bacteria.

A 1:10 dilution of the product is made in TSB. For specified levels of >10 cfu/g, a 1.0 ml sample of this dilution is added to the ENT (Enterobacteriaceae) vial and to a PSE (Pseudomonas) vial. The system automatically monitors the vials and will determine in if the samples are clean or contaminated. When the specified level of absent in 1.0 gram or 10 grams is required, the TSB is pre-incubated overnight as an enrichment step, and then 0.1 ml of the sample is added to the vial.

Single Vial Approach

The GN (Gram Negative) vial has excellent inclusivity for gram negative organisms and does not detect most of the gram positive bacteria. After the first dilution in TSB, with or without pre-incubation depending on the specified level, 0.1 ml of the TSB is added to a single GN vial. This is a very simple and fast procedure to access the absence or presence of gram negative bacteria. This single vial can replace the two vials listed above with better coverage of gram negative bacteria.

ENT Vial: The Enterobacteriaceae vial monitors a change in color due to a pH shift as Enterobacteriaceae organisms ferment glucose in the presence of selective media. The vial medium selectivity is similar to VRBGA.

PSE Vials: The Pseudomonas vial uses the same CO2 sensor as the BioLumnix Total Aerobic Count vial, but with a selective medium. The vial medium selectivity is similar to Centrimide agar.

GN Vial: The hydrolysis of fluorogenic synthetic substrates by bacterial enzymes causes an increase in fluorescence. A fluorogenic synthetic enzyme substrate containing 4-methylumbelliferon, common to all gram negatives, is used in the presence of selective media that inhibits the growth of gram-positive bacteria. The GN vial detects the presence of all Enterobacteriaceae, Pseudomonas, Burkholderia and many other gram negative bacteria. The curves below show the growth of quadruplicate samples for P. aeruginosa (figure on the right: green, lt. blue, red, and purple) and for E. coli (figure on the left: green, lt. blue, red, and purple). A negative control (Bacillus species) is shown in each figure and is represented along the baseline in dark blue.

Advantages: The results of all these assays are available overnight (24 hours), the system is fully automated including achieving of data, data maintenance and report generation, it can be used to create a paperless laboratory. The system is unaffected by product interference, delivering accurate results with faster product release. These assays are simpler to perform than the standard methods saving time, labor, and money.

References

United States Pharmacopeia (2009) Chapter <61> Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests. The National Formulary. Rockville, MD, The United States Pharmaceopeial Convention.

United States Pharmacopeia (2009) Chapter <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms. The National Formulary. Rockville, MD, The United States Pharmaceopeial Convention.

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