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.

Pseudomonads and Their Rapid Detection

Description of Pseudomonad Organisms

Pseudomonas bacteria (Pseudomonads) encompass gram negative, motile, non-fermenting rods. This genus is ubiquitous in nature and these organisms can impact a number of environments and patient populations. The Pseudomonads may be found in soil, on plant material, in water, and can be isolated from various tissues and body fluids from mammals. In human health, some of these organisms, primarily Pseudomonas aeruginosa, can be an opportunist pathogen and cause serious health problems. If allowed to reach unsafe levels, this organism may cause several health problems including skin rash and other skin infections, ear infection, urinary tract infection, and in rare instances, pneumonia. Other Pseudomonads, for example, P. stutzeri can be isolated from wounds but are generally not associated with human disease. Many Pseudomonads found in the soil can damage plant materials by causing spoilage.

Who tests for Pseudomonas and why?

Water Testing: Pseudomonas aeruginosa is a bacterium commonly found in purified water systems. Pseudomonas grows in water. It thrives at warm temperatures, which is why it is so often associated with spas. It can also grow in purified water systems.

Pharmaceutical and Cosmetic Products: Analysis of FDA product recall data for 134 non-sterile pharmaceutical products from 1998 to September 2006 demonstrated that 48% of recalls were due to contamination by either Burkholderia cepacia, or Pseudomonas spp (Jimenez L. 2007). In cosmetic products, P. aeruginosa was recovered from contaminated mascara material and was identified as the agent responsible for corneal ulcers in the 1970s (Ortho 2009). Pseudomonads can survive and grow in DI water—Contaminated DI water may be the source of microbial contamination if it is used for the final rinse of equipment that has been cleaned and sanitized, and it may be the source of contamination for finished products in these industries.

Dairy and Food: The predominant microorganisms limiting the shelf life of processed fluid milk at 4°C are Pseudomonas spp. these species are able to grow to high numbers during refrigerated storage. Pseudomonas species accounted for79% of the psychotropic isolates that spoiled pasteurized milk (Dogan and Boor 2003). Important characteristics of Pseudomonads include their abilities to grow at low temperatures (3–7?C) and to hydrolyze and use large molecules of proteins and lipids for growth.

Biolumix Offers Two Options for Detecting Pseudomonads

For certain industries it is important to detect Pseudomonas aeruginosa, while for others it is important to detect all Pseudomonas spp, including the closely related Burkholderia cepacia. As a result BioLumix offers two different types of vials: the PSE vial for the detection of P. aeruginosa; and the PSB vial for the detection of all strains of Pseudomonas and for B. cepacia.

Detection of Pseudomonas aeruginosa (PSE Vial)

For the Pharma (OTC), Cosmetic and Nutraceutical Industries the primary cause for concern is the absence or presence of Pseudomonas aeruginosa. P. aeruginosa is common and is able to become an opportunistic pathogen in people and may cause severe disease (Hugh and Gilardi 1974). The ability to detect P. aeruginosa is critical in the non sterile Pharmaceutical products, Cosmetic and Nutraceutical Industries to ensure the product material is safe. BioLumix offers a highly selective media in the form of a test vial (PSE) that primarily only allows for the growth of Pseudomonas aeruginosa organisms. Confirmation of the presence of this organism is accomplished using the simple Oxidase reaction on vial contents. The test sample is merely enriched in TSB (Tryptic Soy Broth) per USP instructions and then tested directly in the BioLumix PSE vial. Other common Pseudomonads and closely related organisms, including B. cepacia and P putida, as examples, are excluded from growth due to the use of antibiotic supplements in the BioLumix PSE vial. P. aeruginosa is typically more antibiotic resistant than other Pseudomonas organisms (Blazevic, DL et al 1973). Figure 1 illustrates the growth curve of Pseudomonas aeruginosa ATCC 9027 in the BioLumix PSE vial.

KEY:Dark Blue Curve- P. aeruginosa Green Curve- Negative Control

Detection of other Pseudomonads (PSB Vial)

For many industries including the dairy industry and manufacturers using water, there is a need to test for all Pseudomonads as they impact these industries economically. Other Pseudomonads may include P. fluorescens, P. putida, and P. stutzeri. Burkholderia cepacia, can also be detected using the BioLumix PSE-B vial. Specific to the use of water in manufacturing: Pseudomonas bacteria can be found naturally in the ground and within drinking water sources such as aquifers. Contamination of either dairy products or water systems by Pseudomonads is something to avoid and early detection of goods using a rapid microbiological detection system such as the BioLumix Instrument System, would offer an advantage to the manufacturer. Figure 2 illustrates the growth of many types of Pseudomonads and Burkholderia cepacia in the BioLumix PSE-B vial.

KEY: Dark Blue Curve- B. cepacia; Green Curve -P aeruginosa; Light Blue Curve – P. putida; and Red Curve– P. fluorescens growth

REFERENCES

Blazevic, DJ, Koecke, M.H., and Mastsen J.M. (1973). Incidence and identification of Pseudomonas fluorescens and Pseudomonas putida in the clinical laboratory. Applied Microbiology 25: (1)

Dogan, B. and Boor, K J. (2003). Genetic diversity and spoilage potentials among Pseudomonas ssp. isolated from fluid milk products and dairy processing plants. Appl. Microbiol.,69: 130-138.

Hugh, R. and Gilardi, G. (1974) In “Manual of Clinical Microbiology” Edited by Spaulding, Lennette, Spaulding and Truant. Chapter 23 Pseudomonas.

Jimenez L.(2007). Microbial diversity in pharmaceutical product recalls and environments. Review. PDA J Pharm Sci Technol. 2007 Sep-Oct;61(5):383-99.

Ortho D. (2009). Insight into Cosmetic Microbiology, Chapter 8 263-267

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.

Confirmation Testing of Presumptive Positive Assays Using the BioLumix System

In Microbiology, the initial test result using selective or differential media is called Presumptive Test. Most presumptive tests require confirmation. Confirmation can be accomplished using specific reagents and materials. However, due to the critical importance of testing for pathogens and/or objectionable organisms as contaminants; it may be necessary to perform identification of any organisms isolated from samples. Identification measures microorganisms to the species level.

Initial Testing

For testing of any sample for the presence of microorganisms it is critical to perform a measure of total organism counts (viable organisms). BioLumix provides testing for both Total Aerobic Counts (Bacteria) and for Total Yeast and Mold Counts (Fungi). The BioLumix system in this regard mimics testing for both bacteria and fungi using USP or BAM plate methodology. In these initial tests for total counts there isn’t any discrimination of objectionable organisms from common organisms and common flora. Objectionable organisms for Nutraceutical Samples as an example may include E coli, Staphylococcus aureus, Salmonella or Pseudomonas aeruginosa.

Use of Selective Media

For most samples, it will be necessary to test for at least some objectionable organisms. In order to perform tests for each specific objectionable organism it is necessary to use selective media specifically designed to select for the target organisms. For example, for E coli testing, it is necessary to use selective media that contains both inhibitors that prevent the growth of non-E coli organisms and substrates that can utilized by E coli and not by most other microorganisms. BioLumix make use of such a media, referred to as the EC vial.

Confirmation Test

Unique confirmation tests that can be performed directly from the vials are described for the various objectionable organisms.

E. Coli- Indole Test

For samples that grow and detect in the BioLumix system, a series of Confirmation Tests can be utilized to begin the process of understanding whether the organism(s) are genuine E coli or not. For E coli testing a common initial confirmation test is the Indole Test using the Kovacs reagent. The Indole Test measures the presence of any indole in the growth media as a by-product of tryptophan metabolism by E. coli. Figure 1 depicts a negative (yellow ring) and positive Kovacs Reaction (Red ring) at the top of the media in the test vial.

Staphylococcus aureus – Coagulase Test

The BioLumix vial for testing for the presence of Staphylococcus aureus contains inhibitors of non-Staphylococcus organisms and substrates, such as mannitol as the sole carbon source used by S aureus. If growth is found in the BioLumix STA vial, the analyst can begin to confirm the presence or absence of S. aureus, directly from the vial, using the classic coagulase tests. The coagulase test that has been used for decades uses a known antisera specific for S. aureus epitopes. When S aureus is present, the antiserum reacts with the specific epitopes and forms a lattice of antibody-antigen, and the material coagulates within hours. Figure 2 illustrates the coagulase positive (upper tube) and negative (lower tube) reaction.

Pseudomonas aeruginosa- Oxidase Test

The BioLumix vial for testing for the presence or absence of Pseudomonas aeruginosa contains inhibitors such as Centrimide to prevent the growth of non-pseudomonads and substrates for use by P. aeruginosa. If growth is found in the BioLumix PSE vial, the analyst can begin to confirm the presence or absence of P. aeruginosa using the classic oxidase test. When P. aeruginosa is present, the oxidase test strip reacts with the centrifuged precipitate material (bacterium) and yields a rapid dark blue reaction. This reaction is based on the presence of certain cytochrome oxidase that are found intracellularly in the P. aeruginosa.Figure 3 illustrates the positive oxidase color test ( + ) from the negative reaction ( – ).

Salmonella- Immunoassay Strip

The BioLumix vial for testing for the presence of Salmonella contains inhibitors of non-salmonella bacteria and substrates utilized by Salmonella. If growth is found in the BioLumix SAL vial; the analyst can begin to confirm the presence of absence of Salmonella using commercially available test kits that typically make use of Immunological reactive endpoints. One such kit is shown in the cartoon (Figure 4) and depicts immuno-reactive bands on a test strip.

Identification

Any negative confirmation assay indicates that the target organism is absent and the result is negative. However, in the rare occasions that the vial shows growth and the confirmation assay is positive, it does not necessarily mean that an objectionable organism is present. In these situations further identification of the growing organism might be required. The growing organism could be isolated on selective or non-selective medium and identified by any appropriate identification system.