What's new with the PathoSEEK® Total Coliform and Entero Bacteria Detection Assay?

In August of 2022 MGC launched the PathoSEEK® Coliform and Entero Detection Assay and published an updated User Guide and Validation document. The new product will replace the Total Coliform and Total Entero Detection Assays.

Here is a summary of the changes to note with this new product release:

  • Affects both flower samples and marijuana infused products (MIPs) 
    • Note: MGC will no longer provide a Cq to CFU conversion recommendation for MIP samples. Due to the low action limit of 100 CFU/g in most jurisdictions, we recommend enriching non flower  samples in TSB and processing as pass/fail as described in the updated Coliform/Entero  v2 user guide to ensure viable Coliform/Entero bacteria is detected if present.  If Coliform/Entero bacteria is detected post enrichment, plating should be performed  to achieve a total viable count for reporting of CFUs.  If no Coliform/Entero bacteria is detected in a sample extracted after enrichment this sample can be reported as non - detect.
  • Requires PathoSEEK Master Kit v3 (SKU 420201) for both flower samples and marijuana infused products (MIPs) 
  • Changes the initial, upfront dilution to 19 mL of Tryptic Soy Broth per 1g of sample for flower samples
  • Adds a centrifugation step to pellet cells prior to lysis (flower only)
  • Adds a back-calculation to the recommended Cq to CFU/g conversion equation to account for the revised upfront dilution (only applies to flower matrix)
  • Requires new reagent SKUs: 
    • PathoSEEK® Total Coliform & Entero Detection Assay (SKU 420153)
      • Reduced concentrations of SCCG internal control and Pseudomonas primers are only differences with v2
    • PathoSEEK BTGN / Coliform / Entero Multiplex Positive Control (SKU 420353)
      • This new positive control can be used with both our Coliform & Entero assay and our BTGN v2 assay
  • The new assay (Part Number 420153) will replace the Total Coliform (420107) and Total Entero (420108) Detection Assays. 
  • The new control (Part Number 420353) will replace the Entero/Coliform Multiplex Control (420314) and the Total Entero Positive Control (420308).

What about the old Cq to CFU conversion equation for MIPs?

When we performed validation studies years ago on our original Total Coliform and Total Entero assays, there were no certified reference materials (CRMs) available for cannabis samples. These validation studies relied instead on bacterial DNA spiked onto sample. In the last couple of years that has changed, and companies like NSI have begun producing cannabis specific products. This has allowed us to redesign to an industry standard, albeit an imperfect one. 

During our very thorough review and validation of this product update we found that continuing to provide Cq/CFU conversions for MIPs was problematic. Action limits set for MIPs in most jurisdictions are so low (100 CFU) that they are essentially pass/fail. 

We recommend enriching non-flower samples in TSB and processing as pass/fail as described above and in the new Coliform/Entero user guide. 

All Coliform bacteria are Enterobacteriaceae bacteria, but not all Enterobacteriaceae are Coliforms. 

So, why does the new PathoSEEK® Coliform assay include Enterobacteriaceae?

Both Coliforms and, more broadly, Enteros are used as indicators of sanitary quality of food and water. They use only slightly different growth conditions and therefore many Coliform specific methods will detect Enteros.

It is important to note that this update includes a change from our previous Coliform assay. In order to achieve better concordance with cannabis CRMs that were not available to the industry at the time of our original validations, we elected to increase assay sensitivity and coverage of the assay design to include all Enterobacteriaceae. Therefore, the Coliform assay will also detect Enteros. However, many Coliform specific plating methods will often detect Enteros as well. We have observed this during our validation studies, reviewed peer reviewed literature that confirms it, and have designed this update accordingly. Coliforms are lactose fermenting while Enteros are mostly glucose fermenting. 3M's own validation studies for Petrifilms show that Enteros are detected on their Coliform specific plates. The study¹ referenced below summarizes the issue best of all.

References:

  1. CJ HervertA,S.,AllesN.H.,MartinK.J., BoorM.Wiedmann, (2016), Evaluation of different methods to detect microbial hygiene indicators relevant to the dairy industry.  Journal of Dairy Science.  Volume 9.9, Issue 9, Pages 7033-7042

https://www.sciencedirect.com/science/article/pii/S0022030216304118