Detection of psuedomonads in dairy and water samples using a quantatative one-step testing protocol in just one day

By Roger Brideau*
*Presented in part at the International Association of Food Preservation
‘IAFP’ Conference in Charlotte NC USA

dairy microbiology detection of psuedomonads Introduction- Pseudomonad organisms are a major cause of bacterial spoilage of pasteurized milk and dairy products due to post process contamination.  Early detection of Pseudomonad’s can be a predictor of product shelf-life as they are the predominant psychotropic bacteria present.  BioLumix has developed a rapid method for the detection of Pseudomonad’s in dairy products and the method is also applicable to their detection in process water.

Purpose- To evaluate the ability of the BioLumix system to detect Pseudomonad’s in dairy products, determine the speed to results, sensitivity, selectivity and ability to predict shelf-life.

Methods- the BioLumix system is an optical system that detects growth of Pseudomonad’s using a CO2 sensor in selective growth media.  The BioLumix system was directly compared to the plate count methodology for milk samples stored at refrigerated temperatures and held overnight at room temperatures (enriched).  Testing of water was also accomplished in side by side studies to show the capability of the BioLumix system for quantitation of Pseudomonads.

Results:  Growth of Pseudomonad’s in the BioLumix vial
Table of quantitation of Pseudomonads
A growth comparison was made for detection of each Pseudomonad in the BioLumix system using PSE-B vials and on CFC (Pseudomonas agar) spread plates.  Table 1 summarizes the growth of freshly diluted samples of organisms that were enriched in TSB during the prior 18-24 hrs.  The PSE-B vials are selective, as shown by not allowing growth of unrelated gram positive and gram negative bacteria, yeast or mold.  Four different species of Pseudomonad’s grew in the PSE-B vial and on CFC plates.

Milk Sample Testing
Commercial milk samples were tested upon arrival in the laboratory.  Five of twenty were positive for the presence of Pseudomonad’s using both PSE-B vials and CFC spread agar plates.  After storage for 3-7 days, twelve of twenty samples were positive for Pseudomonad’s including after enrichment at RT for 16-18 hrs.  Thus, refrigerated milk samples have varying incidence of Pseudomonad flora.

Dairy Microbiology Calibration dataMilk Calibration Curve
Organisms from milk samples that grew in PSE-B vials and on CFC plates were used to generate the Calibration Curve shown in Figure 1. These data suggest that low numbers (~10) of Pseudomonad’s should detect within 24 hrs in the PSE-B vial. The Calibration Curve can be embedded into the BioLumix software on the instrument and used to generate a read-out of cfu per gram of milk.  This enables quantitation of the milk sample for the presence of Pseudomonad’s before 24 hr; a distinct advantage over plate methodology taking 48-72 hours.

Dairy Microbiology detection time distributionDistribution of Data
In the dairy settings the goal is to separate a “good” sample that has a potential to maintain quality over a product’s shelf-life from a “bad: sample that will have a shorter shelf life. Criteria for separation between a “good” and “bad” product based upon the Pseudomonad’s numbers can be established. If one selects a count of 1,000 cfu/ml as the separation point: the Histogram shown in Figure 4 indicated at 12.5 hrs all samples with higher counts (in red) detected, while all the samples below 1,000 cfu/ml did not.

Results: Testing of Process Water for the presence of Pseudomonads
Eight different types of process water samples were found to be free of Pseudomonad’s after testing using PSE-B vials and CFC spread plates (data not shown).  Clean process water samples were then inoculated with individual isolates of Pseudomonad’s were used to generate a calibration curves for water, similar to the milk calibration curve. Pseudomonad growth in inoculated process water was measured using PSE-B vials and PA spread plates and was used to generate the Calibration Curve. Detecting vials were confirmed to contain Pseudomonas by the Oxydase test.

Summary
The data presented show equivalency between the BioLumix PSE-B vial and CFC (Pseudomonas agar) plates for the detection of Pseudomonad’s found in commercial milk samples and in inoculated process water samples.  PSE-B vials detected as little as 1-3 organisms (data not shown).
The number of organisms in commercial milk was found to increase over time at refrigerated temperatures and this agreed with a previously published report (Burdova et al 2002) showing the affect of storage temperature on milk shelf-life.
The BioLumix assay is completed in 18 hours and offers an advantage over spread plate methods for time to results and ease of calculation of cfu per gram of milk or water.  A single vial is all that is needed and thus both time and material costs are reduced.  Calibration Curves were easily generated for both milk and water sampling and can be used to generate a cfu/ml of sample in less than 1 day to yield an estimate of cfu/gram.

REFERENCE:  Burdova, O. et al (2002).  Bulletin Vet Med. Poland. 46:325-329. Hygiene of Pasteurized Milk Depending on Psychrotrophic Microorganisms.

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.