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Add 10g of sample to 90mL TSBLT (1:10 dilution) and homogenize as appropriate Incubate at 35°C for 24 ± 2h Transfer 10 mL of enrichment to 90 mL fresh pre-warmed TSBLT Perform 3M ™ Tecra ™ VIA assay following manufacture’s instructions Incubate at 35°C for 24 ± 2h Streak enrichments to MSA* and incubate 35-37°C for 48h Identify and confirm suspect colonies *Under normal use, only presumptive positive assay results are streaked to agar for confirmation Add 10g of sample to 90mL TSBLT (1:10 dilution) and homogenize as appropriate Figure 2. USP <2021/2022> Reference Method for Staphylococcus aureus Testing Incubate at 30-35°C for 18-24h Streak enrichment onto one or more of the following media: Vogel-Johnson Agar Mannitol-Salt Agar Baird-Parker Agar Identify and confirm suspect colonies Incubate at 30-35°C for 24-48h Test Organisms Bacteria for the study were derived from lyophilized preparations from the Quanti- Cult Plus system, ThermoFisher Scientific (Lenexa, KS) – S. aureus isolate ATCC 6538. Following the directions of the Quanti-Cult Plus system, the isolate was hydrated. Preparation of Artificially Inoculated Samples Samples were prepared according to USP 2021. Briefly, a 10 g portion of product was added to a 90 mL container of Fluid Casein Digest-Soy Lecithin-Polysorbate 20 Medium (TSBLT). Products in tablet form were first added to a Whirl-Pak® bag and crushed using a mortar and pestle and then added to the medium. The samples were warmed to 35 ° C for 1 hour and mixed periodically by swirling the container, allowing capsules to dissolve and powders to hydrate. Samples were then adjusted to pH 7.3 ± 0.2 with 1 N NaOH or 1N HCl before inoculating at low levels (<10 cfu/10 grams). Non-inoculated controls were also prepared of each sample tested. Inoculated portions were mixed thoroughly and incubated at 24 ± 2 hours at 35 o C. Tests Mannitol-Salt (MS) agar reference method (Figure 2)—One MS agar plate was inoculated with 0.1 mL of the 24 ± 2 hours-enriched samples. Inocula were spread over the surface of each plate with an inoculating loop, and results – growth (typical S. aureus) or no growth of S. aureus – was recorded after a total of 48 hours. From the enriched samples, several dilutions were made into Butterfields Buffer, Edge Biologicals Inc. (Memphis, TN), and 0.1 mL of each dilution was plated onto one MS agar plate per dilution to determine cfu/mL. Counts of yellow colonies with yellow zones, as based on the morphological characteristics of MS agar, were recorded again after a total of 48 hours. 3M Tecra Staph aureus VIA kit method (Figure 3)—Each sample was tested using the 3M Tecra Staph aureus VIA kit. Results were recorded after both 24 and 48 hours of sample incubation. Each sample was further diluted by taking 10 mL of the enriched sample and diluting into 90 mL fresh TSBLT and incubated for an additional 24 ± 2 hours at 35 o C. Samples were then tested by the 3M Tecra Staph aureus VIA kit, and the results were recorded. 3 ABSTRACT The purpose of this study is to show that the 3M™ Tecra™ Staph aureus Visual Immunoassay (VIA) test kit can be used to obtain a similar result to the United States Pharmacopeia (USP) guideline for the detection of Staphylococcus aureus in dietary supplements. Results from the 3M Tecra Staph aureus VIA kit were recorded at both 24 and 48 hours. In addition, results, from samples that were initially read at 24 hours and further diluted into Fluid Casein Digest-Soy Lecithin-Polysorbate 20 Medium (TSBLT) and then incubated for another 24 hours, were recorded using the 3M Tecra Staph aureus VIA kit. Moreover, results from the USP method were captured at the presumptive stage. At the 24-hour result from the 3M Tecra Staph aureus VIA kit, seven of the twelve samples showed the same results as the USP reference method. Similar results occurred when the 48-hour 3M Tecra Staph aureus VIA kit results were compared to the reference method. When the samples were first read at 24 hours using the 3M Tecra Staph aureus VIA kit and then further diluted into TSBLT, re-incubated for 24 hours, and then read again using the 3M Tecra Staph aureus VIA kit, ten of the twelve samples showed the same results. Because of the results, the 3M Tecra Staph aureus VIA kit can be used to detect S. aureus in dietary supplements. INTRODUCTION Staphylococcus aureus has been reported to be a microbial contaminant in many foods, cosmetic/pharmaceutical products, and dietary supplements. Because of the pathogenicity of S. aureus, the United States Pharmacopeia (USP) requires its absence in certain non-sterile pharmaceutical raw materials and finished products. There is a need to apply new technologies to conduct the microbiological monitoring of raw materials and finished products. These technologies need to be rapid, sensitive, accurate, cost effective, and easy to use. The standard microbiological methods require 4-5 days for the complete isolation and identification of S. aureus. This study compares the 3M™ Tecra™ Staph aureus Visual Immunoassay (VIA) kit (Figure 1) method with the USP method for the detection of S. aureus in dietary supplements. MATERIALS AND METHODS Results with the 3M Tecra Staph aureus VIA kit were similar to those of the USP method in artificially inoculated dietary supplements from one manufacturer in this study. Yet, it is the customer’s responsibility to validate any new method. The 3M Tecra Staph aureus VIA kit gave presumptive results 24 hours earlier than the reference method. Because of the favorable results in this study and the results from another study (D. Hughes et al. 1999), the 3M Tecra Staph aureus VIA kit method can be an easy-to-use alternative method to detect S. aureus in dietary supplements. Figure 1. 3M™ Tecra™ Staph aureus Visual Immunoassay (VIA) Kit RESULTS No growth of S. aureus was observed using both the 3M Tecra Staph aureus VIA kit method and the USP method on the non-inoculated samples. Table 1 lists the results from the 3M Tecra Staph aureus VIA kit method and the USP method for specific dietary supplements. The table lists the supplement, the presumptive result from the 3M Tecra Staph aureus VIA kit after 24 hours of incubation, the presumptive result from the 3M Tecra Staph aureus VIA kit after 48 hours of incubation, the presumptive result from the MS agar method, and the count from the MS agar method. Seven of the twelve results from the 3M Tecra Staph aureus VIA kit recorded after both 24 and 48 hours of incubation were similar. Three of these seven samples showed growth of S. aureus using both methods. Because the actual S. aureus counts were also recorded from the MS agar plate, it was observed that the counts were less than the limit of detection of the 3M Tecra Staph aureus VIA kit (approximately10 6 cfu/mL). Table 2 lists the results from the 3M Tecra Staph aureus VIA kit method and the USP method after the additional dilution in fresh TSBLT. Ten of the samples tested showed exact agreement between the two methods for the detection of S. aureus. Two of the products showed no recovery of the inoculum by the 3M Tecra Staph aureus VIA kit method. Green tea (K. Abascal et al. 2004. “Herbs in Combating Antimicrobial Resistance”) and mustard seeds (Dr. L. Zaika. 1986) exhibit antimicrobial properties that may have affected the results. Further dilution of these types of samples may be required. Comparison of the 3M™ Tecra™ Staph aureus Visual Immunoassay Kit Method to the United States Pharmacopeia Standard Method for the Detection of Staphylococcus aureus in Raw and Finished Dietary Supplements Christine Binsfeld and Enrique Morales, 3M, St. Paul, MN, USA © 3M 2009. All rights reserved. 3M, and Tecra are registered trademarks of 3M. Whirl- Pak is a registered trademark of Nasco. CONCLUSION ACKNOWLEDGEMENTS Marta Henrickson, Brianna Larson, and Kathryn Lindberg are thanked for their technical assistance. REFERENCES USP<2021> Microbial Enumeration Tests – Nutritional and Dietary Supplements USP<2022> Microbiological Procedures for Absence of specified Microorganisms – Nutritional and Dietary Supplements 3M™ Tecra™ Staph aureus Visual Immunoassay Package Insert Figure 3. 3M™ Tecra ™ Staph aureus VIA Kit Method
