BioLumix Staphylococcus aureus Vial Assay

Organism of Interest

Staphylococcus aureus is a major pathogen of concern in infectious disease. This organism group may include drug resistant S. aureus, often defined as methicillin resistant Staphylococcus aureus (MRSA). S. aureus is also an objectionable organism for the dietary supplement and nutraceutical industries.

Backgroud

Wikipedia’s description is as follows: S. aureus can cause a range of illnesses fromminor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis,carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases suchas pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS),bacteremia, and sepsis. It is implicated in skin, soft tissue, respiratory, bone, joint,endovascular and wound infections. It is still one of the five most common causes ofnosocomial infections, often causing postsurgical wound infections. Each year, some 500,000 patients in American hospitals contract a staphylococcal infection.

Methicillin-resistant S. aureus, abbreviated MRSA and often pronounced “mer-sa” (in North America), is one of a number of greatly-feared strainsof S. aureus which have become resistant to most antibiotics. MRSA strains are most often found associated with institutions such as hospitals, but are becoming increasingly prevalent in community-acquired infections. Arecent study by the Translational Genomics Research Institute showed that nearly half (47%) of the meat and poultry in U.S. grocery stores were contaminated with S. aureus, withmore than half (52%) of those bacteria resistant to antibiotics.

Current Methodology for S. aureus

Nutraceutical and Dietary Supplement Products: USP <1222> describes the methodrequired to test for the absence of S. aureus (typically in 10 grams) in nutraceutical and dietary supplement products. The method involves mixing of the sample in TSB and pre-incubating the TSB containing product at 30 to 35 degrees for 18 to 24 hours. Followed by streaking a loopful from TSB onto the surface of one or more of the following media:Vogel–Johnson Agar Medium (VJ Agar), Mannitol–Salt–Agar Medium (MS-Agar),and Baird-Parker Agar Medium (BP Agar). If no plates contain colonies having the characteristics described, the test specimen meets the requirement for the absence of Staphylococcus aureus. If characteristic colonies are present, a coagulase test is performed.

Pharmaceutical Products: USP <62> describes a similar method to test for the absence of S. aureus. After the pre-incubation in TSB, Mannitol–Salt–Agar Medium isused for plating.

Food Testing: FDA Bacteriological Analytical Manual (BAM) describes several methods for testing of S. aureus in foods. Methods used to detect and enumerate S. aureus maybe dependent for testing of foods and on the past history of the test material. Processed foods may contain relatively small numbers of debilitated viable cells, whose presence must be demonstrated by appropriate means. Finding food contaminated with S. aureusmay lead to legal action against the party or parties responsible for a contaminated food product. For S. aureus specified levels > 100 cfu/g of S. aureus plating on Baird-Parker agar is recommended. The method involves spreading 1.0 ml on 3 plates and looking for typical colonies. At least 20 colonies must be present on the lowest dilution for reliable results. Typical colonies need to be tested for coagulase. The most probable number (MPN) method is recommended for products in which small numbers of S.aureus are expected to be low and in foods expected to contain a large population of competing species. MPN can be performed in TSB containing 10% NaCl and 1% sodium pyruvate. From each tube showing growth (turbidity) a loopful is transferred to a plate of Baird-Parker medium.

The BioLumix Methodology

Pre-Incubation step Objectionable organisms including S. aureus may be present in very low numbers andmay also be “damaged”. Recovery of these organisms may require growth enrichmentin simple broth media prior to testing in selective media. Enrichment in a media suchas Trypticase soy broth (TSB) is often used as the first step in testing for the presenceor absence of S aureus. In testing of dietary supplement and pharmaceutical samples,enrichment occurs for approximately 18-20 hours. This step is similar to the procedure recommended by USP <1222> and USP <62>. After the pre-incubation in TSB a small amount (0.1 ml, typically) is transferred into the selective media BioLumix STA Vial and inserted into the instrument for 22 hours.

BioLumix Staph Vial Selectivity
The BioLumix STA vial utilizes a combination of inhibitors. These inhibitors target both unrelated organisms such as gram negatives and unrelated gram positives. Theuse of specific carbon sources (Mannitol) to selectively permit growth of primarily only staphylococcal species is also used. In addition, high salt concentrations also inhibitnon-S. aureus organisms including gram negatives and other gram positives. The goal is to slow or inhibit growth of these unrelated organisms including the inhibition of aclosely related organism Staphylococcus epidermidis in this BioLumix media vial.
Growth in the BioLumix STA Vial
A representative BioLumix STA Vial showing growth of S. aureus is shown in the accompanying Figure. The presence of S. aureus causes a detection time (DT) in thecurve (shown as a blue triangle). The figure also includes an un-related organism (Ecoli) tested under the same growth conditions in the BioLumix STA Vial.
Assay Endpoint
If there is no growth and no DT the sample is negative and does notcontain S. aureus. Growth in the
BioLumix STA Vial presumes the presence of S aureus. Confirmationusing a secondary test such ascoagulase is required to verify the presence of S aureus. The coagulase test can be performed directly from the vial.

Organism of Interest

Staphylococcus aureus is a major pathogen of concern in infectious disease. This organism group may include drug resistant S. aureus, often defined as methicillin resistant Staphylococcus aureus (MRSA). S. aureus is also an objectionable organism for the dietary supplement and nutraceutical industries.

Backgroud

Wikipedia’s description is as follows: S. aureus can cause a range of illnesses fromminor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis folliculitis,carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases suchas pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS),bacteremia, and sepsis. It is implicated in skin, soft tissue, respiratory, bone, joint,endovascular and wound infections. It is still one of the five most common causes ofnosocomial infections, often causing postsurgical wound infections. Each year, some 500,000 patients in American hospitals contract a staphylococcal infection.

Methicillin-resistant S. aureus, abbreviated MRSA and often pronounced “mer-sa” (in North America), is one of a number of greatly-feared strainsof S. aureus which have become resistant to most antibiotics. MRSA strains are most often found associated with institutions such as hospitals, but are becoming increasingly prevalent in community-acquired infections. Arecent study by the Translational Genomics Research Institute showed that nearly half (47%) of the meat and poultry in U.S. grocery stores were contaminated with S. aureus, withmore than half (52%) of those bacteria resistant to antibiotics.

Current Methodology for S. aureus

Nutraceutical and Dietary Supplement Products: USP <1222> describes the methodrequired to test for the absence of S. aureus (typically in 10 grams) in nutraceutical and dietary supplement products. The method involves mixing of the sample in TSB and pre-incubating the TSB containing product at 30 to 35 degrees for 18 to 24 hours. Followed by streaking a loopful from TSB onto the surface of one or more of the following media:Vogel–Johnson Agar Medium (VJ Agar), Mannitol–Salt–Agar Medium (MS-Agar),and Baird-Parker Agar Medium (BP Agar). If no plates contain colonies having the characteristics described, the test specimen meets the requirement for the absence of Staphylococcus aureus. If characteristic colonies are present, a coagulase test is performed.

Pharmaceutical Products: USP <62> describes a similar method to test for the absence of S. aureus. After the pre-incubation in TSB, Mannitol–Salt–Agar Medium isused for plating.

Food Testing: FDA Bacteriological Analytical Manual (BAM) describes several methods for testing of S. aureus in foods. Methods used to detect and enumerate S. aureus maybe dependent for testing of foods and on the past history of the test material. Processed foods may contain relatively small numbers of debilitated viable cells, whose presence must be demonstrated by appropriate means. Finding food contaminated with S. aureusmay lead to legal action against the party or parties responsible for a contaminated food product. For S. aureus specified levels > 100 cfu/g of S. aureus plating on Baird-Parker agar is recommended. The method involves spreading 1.0 ml on 3 plates and looking for typical colonies. At least 20 colonies must be present on the lowest dilution for reliable results. Typical colonies need to be tested for coagulase. The most probable number (MPN) method is recommended for products in which small numbers of S.aureus are expected to be low and in foods expected to contain a large population of competing species. MPN can be performed in TSB containing 10% NaCl and 1% sodium pyruvate. From each tube showing growth (turbidity) a loopful is transferred to a plate of Baird-Parker medium.

The BioLumix Methodology

Pre-Incubation step Objectionable organisms including S. aureus may be present in very low numbers andmay also be “damaged”. Recovery of these organisms may require growth enrichmentin simple broth media prior to testing in selective media. Enrichment in a media suchas Trypticase soy broth (TSB) is often used as the first step in testing for the presenceor absence of S aureus. In testing of dietary supplement and pharmaceutical samples,enrichment occurs for approximately 18-20 hours. This step is similar to the procedure recommended by USP <1222> and USP <62>. After the pre-incubation in TSB a small amount (0.1 ml, typically) is transferred into the selective media BioLumix STA Vial and inserted into the instrument for 22 hours.

BioLumix Staph Vial SelectivityThe BioLumix STA vial utilizes a combination of inhibitors. These inhibitors target both unrelated organisms such as gram negatives and unrelated gram positives. Theuse of specific carbon sources (Mannitol) to selectively permit growth of primarily only staphylococcal species is also used. In addition, high salt concentrations also inhibitnon-S. aureus organisms including gram negatives and other gram positives. The goal is to slow or inhibit growth of these unrelated organisms including the inhibition of aclosely related organism Staphylococcus epidermidis in this BioLumix media vial.Growth in the BioLumix STA VialA representative BioLumix STA Vial showing growth of S. aureus is shown in the accompanying Figure. The presence of S. aureus causes a detection time (DT) in thecurve (shown as a blue triangle). The figure also includes an un-related organism (Ecoli) tested under the same growth conditions in the BioLumix STA Vial.Assay EndpointIf there is no growth and no DT the sample is negative and does notcontain S. aureus. Growth in theBioLumix STA Vial presumes the presence of S aureus. Confirmationusing a secondary test such ascoagulase is required to verify the presence of S aureus. The coagulase test can be performed directly from the vial.

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.

Microbiological Testing of Gelatin Capsules

Introduction

Two-piece capsules have been used for almost a century in the pharmaceutical field, and gelatin has been adopted as the main material of these capsules due to its excellent characteristic as a gelling agent. The gelatin dissolves under high concentration into water of a high temperature and quickly gels in room temperature. The thickness of the film made by the gelatin becomes uniform.

The gelatin capsules consist of gelatin, plasticizers and water. Modern day shells may, in addition, consist of preservatives, colors, opacifying agents, flavors, sugars, acids, enteric materials etc. A mixture derived from pork skin and bones is used in capsules.  Pork skin gelatin contributes plasticity while bone gelatin gives firmness.

One important reason for the exclusive use of gelatin for making hard and soft capsules is its solubility characteristics in stomach fluids. It absorbs cold water readily, though the rate of absorption depends upon moisture content of gelatin. Bloom Strength is an empirical gel strength measure which gives an indication of the firmness of gel. The plasticizers used are glycerin, sorbitol etc.  Preservatives, if included, are generally a mixture of methylparaben (4 parts) and propylparaben (1 part) to the extent of 0.2%. Flavors, if added, should not exceed 2% and are generally ethyl vanillin or essential oils. Sugar, if included, may be up to 5% to give the gelatin shell desirable chewable characteristics.

Microbiology Testing

Each incoming lot of capsules needs to be tested using USP <61> and USP<62>. The total aerobic bacterial count should not exceed 3,000 cfu per gram, the combined yeast and mold counts along with Bile-Tolerant Gram-Negative Bacteria should not exceed 300 cfu per gram.  Material must also meet the requirements of the tests for absence in 10 grams of Salmonella species, Escherichia coli, and Staphylococcus aureus.

Challenges of Current Methodology

Testing gelatin capsules for microbiology might result in several challenges.  The 1:10 dilution of the product has high viscosity and is sometimes difficult to pipette. Many capsules have vivid colors that interfere the reading of the plates. 

The current methods used in microbiology originated over 100 years ago.  There have been limited improvements in methods used for microbiological testing in the last decade. The current USP methodology is slow, requiring up to 5-7 days for product release, is manual, and in many cases is inaccurate.  Paper-based QC laboratory processes can be expensive, error-prone, time and labor-intensive.  

Rapid microbiological methods (RMM) offer a cost effective alternative to USP methodology.  With an RMM’s high degree of automation, significant reduction in time to results, faster product release, and improved process control, while providing enhanced accuracy, better repeatability, and total automation

BioLumix Alternative

The BioLumix System simplifies testing, expedites time to results, reduces the testing cost and accelerates product release while providing better control of microbial contamination.  The system can be used to automate microbial testing with a more cost effective and streamline manner. The system reduces the error rates produced by paper-based activity recording and batch data entry.  The BioLumix system also helps automate microbiological quality control processes.

The BioLumix technology is based on continuous monitoring of changes in color or fluorescence as a result of microbial metabolism in ready to use test vials. The results are presented as soon as detection occurs without any involvement of the operator. The fully automated system offers a paperless operation with increase efficiencies.

The key to the technology is the two-zone ready to use vials which eliminates any product interference.  Many types of gelatin capsules were tested in the BioLumix system for total aerobic bacterial count, combined yeast and mold counts, Bile-Tolerant Gram-Negative Bacteria, and for absence in 10 grams of Salmonella species, Escherichia coli, and Staphylococcus aureus.  As can be seen in the figure, there is no product interference even with capsules containing the most vibrant colors.  All assays yielded clear results that correlated well with the USP methodology.

Key: Dark Blue-clear capsule;Green- Brilliant Red Capsule; Light Blue-Brown capsule;Red- Teal capsule; Purple-Inoculated Brilliant Red Capsule.

The BioLumix system is validated against the USP methodology.  All assay results are complete in 48 hours with an automated Certificate of Analysis; resulting in faster product release.