Resolution of the Microbial Identification-system should be considered when identifying QC strains. MicroPhyles Case No. 5

Someone once said, “If you don’t have time to do it right you must have time to do it over.” The ubiquitousness of Quality Control (QC) mechanisms in manufacturing processes and testing laboratories are a testament to this adage. Within the realm of Environmental Monitoring or Clinical or Industrial Microbiology, QC strains (also called reference organisms) are a vital part of a company’s compliance umbrella and their accreditation process.

QC analyses are required for consistent and reliable results. In manufacturing facilities of drug, medical device, food and cosmetic industries, as well as clinical diagnostic laboratories, regular QC analysis ensures the released product is reliable for consumers and test results are accurate for patients. QC strains are also used during process validations, proficiency testing, and instrument qualifications to evaluate performance so that the desired analytical and diagnostic standards can be achieved.

Finding the right species resolution

Cultures of QC strains are available with commercial sources such as ATCC, DSMZ, Microbiologics and ThermoFisher Scientific. Often QC strains are sold as instrument or assay-specific controls. For example, Charles River Laboratories’ Matrix-assisted laser desorption/ionization–time of flight mass spectrometry (otherwise known as MALDI-TOF MS or just MALDI) based Axcess® system for microbial identification (ID) uses a panel of QC organisms available with Microbiologics for system qualification. A key point about using a QC strain for a process that involves confirmation of ID is to ensure the ID system has the correct species level resolution. We do this because species resolution capabilities of ID technologies may be different for closely related species. It is critical, therefore, to understand this before choosing an ID method so that deviations and investigations can be avoided.

At Accugenix, we occasionally receive queries from customers, such as the one mentioned below, who question the MALDI based ID of a known QC strain. Let’s consider the example of Enterobacter hormaechei O’Hara et al. (ATCC 700323), which is a Quality Control strain for Vitek 2 ID-GNB card (bioMerieux). This organism could also make its way into Proficiency Testing or Process Validation cohorts. So when a customer submits an E. hormaechei (ATCC #700323) strain for MALDI-based ID, the result is likely to be correct only to the Species–complex (a group of very closely related species) level due to lack of species level resolution for these organisms.

The Enterobacter cloacae complex

First some background, Enterobacter hormaechei was named after Estenio Hormaeche (a Uruguayan microbiologist who along with P.R.Edwards proposed the genus Enterobacter). E. hormaechei strains have been isolated from human sources such as blood, respiratory tract, and wound.1,2 It belongs to the Enterobacter cloacae complex (ECC) which consists of the additional species E. asburiae, E. cloacae, E. kobei, E. ludwigii and Lelliottia nimipressuralis (Enterobacter nimipressuralis). In general, species within the ECC are well recognized nosocomial pathogens.1,2 Based on our experience with repeatedly testing E. hormaechei (ATCC #700323) by MALDI, it incorrectly identifies as E. cloacae. On the other hand, E. hormaechei (ATCC #700323) correctly identifies as E. hormaechei by our DNA sequencing assay based on the first 500bp of the bacterial ribosomal gene.

But back to that customer query; we reassessed the accuracy of MALDI library entries belonging to ECC (a total of 23 entries representing 6 species). Our review of taxonomic changes of these strains and quality of the spectra used for library entry generation did not point to any inconsistencies, so we knew that erroneous MALDI library entries were not a factor. Therefore, we surmised that MALDI cannot reliably discriminate between species of the ECC most likely due to the lack of resolving power in their abundant protein profiles. A recent study3 that used several culture collection strains of species of ECC observed a similar pattern with MALDI.

An incorrect ID of a QC isolate can cause run failure leading to delayed release of product or test results. However, if the species resolution of the ID system is known in advance, then the appropriate choice of an ID system can be made so that the expected ID matches the system output. In this case, it would be 16S rDNA sequencing.

References:

  1. http://www.futuremedicine.com/doi/full/10.2217/fmb.12.61 [Mezzatesta et al., Future Microbiology, July 2012, Vol. 7, No. 7, Pages 887-902].
  2. http://jcm.asm.org/content/27/9/2046.long [O’Hara et al., J. Clin. Microbiol. 1989 Sep; 27(9):2046-9.]
  3. http://onlinelibrary.wiley.com/doi/10.1111/j.1574-6968.2011.02479.x/full [Pavlovic et al., FEMS Microbiol. Lett. 328 (2012) 46–5].