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.

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