Selecting and troubleshooting microbial identification methods

BY DR TIM SANDLE | PHARMACEUTICAL MICROBIOLOGY AND CONTAMINATION CONTROL EXPERT

8th July 2024

 

Identifying unknown microbes is complicated. It can be difficult to know where to start, with the variety of different microbial identification systems just the first of many challenges. 

 

Below, Dr Tim Sandle explores the importance of getting your microbial identification right and provides a great grounding on how to make sure you get the results you need.
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What is microbial identification?

 

Microbial identification is concerned with the classification of microorganisms into groups of similar organisms and giving it a name. The outcome of an identification can be either:

 

  • Presumptive – gives an indication of the likely genus
  • Confirmed – designates species level

 

This may be driven by the capabilities of the method used, or by the level of identification required by the microbiologist. A microbiologist may wish to know what an organism is in order to1:

 

  • Gather information that could lead to determining the source of a contamination and take appropriate preventative action to avoid / minimise future recurrence.
  • Understand the normal microbial flora that is inherent to the raw materials used to manufacture products, or those that are present in the manufacturing environment
  • Understand the trends of microbial contamination in the materials used during manufacture
  • Determine the microbiological risk to the product and/or the patient or consumer
  • Build up a library of organisms for culture media testing and method validation

Staphylococcus

 

 

 

 

Objective of identification

 

The objective of microbial identification is to place an unknown organism into a family, genus or species.

 

Let’s use staphylococcus aureus as an example. We can break down this common bacterium into the below classification stages in the adjacent table

 

Value of identification

 

Having an idea of the microbial species helps with our understanding of the origins of the organism. Understanding the origins of the microorganism can aid root cause investigations in response to out-of-limits microbial results. In very general terms:

  • Gram positive rods: typically, bacillus species - environmental origin
  • Gram positive cocci: typically, micrococci and staphylococci – people and skin origin
  • Gram negative cocci: typically, these are not found in product or from the environment. Instead, they are usually from clinical specimens
  • Gram negative rods – could be water in origin, however this is the group that has many examples of ‘objectionable organisms’  

 

 

 

Selecting between microbial identification systems

 

There is a range of different microbial identification systems available. When selecting which technology to use, the applicability to the requirements of the microbiology laboratory and the manufacturing facility, as well as the strengths and weaknesses of the method must be considered.  

 

For most laboratories, basic biochemical or proteomic characterisation methods kits are appropriate 2. As the throughput of samples for confirmed identification increases and/or the need for more accurate identification increases (for example, sites manufacturing sterile products), the need for more advanced methods (such as automated biochemical methods and genotypic methods) may also increase.

 

 

 

Developing a User Requirements Specification (URS)

 

When deciding on the appropriate method to employ, a URS and a business case should be built. The types of things to include in a URS are:

 

When deciding on the appropriate method to employ, a URS and a business case should be built. The types of things to include in a URS are:

 

Purpose

For example, will the technology be used for routine or investigational use?

 

Throughput

Is the technology capable of handling the required sample throughput? Is the workflow required suitable for the laboratory?

 

Time to result

How quickly does the laboratory need the result?

 

Cost per test (including consumable costs, maintenance and calibration costs)

Occasionally reagent costs can outweigh the capital costs. The laboratory needs to be sure the cost per test is calculated according to the throughput.

 

Complexity of the test

The skill level expected of the technicians who will carry out the testing.

 

Capital cost

The initial cost of the equipment required to perform the test.

 

Validation

Consideration of the level of validation required, including computer systems validation and data integrity concerns.

 

Database size and composition

Databases should have adequate coverage in relation to the microorganisms of interest to the laboratory. It is important to review the proportion of ‘industrial isolates’ since many systems were developed for clinical microbiology and have a bias towards clinical isolates.

 

Facility requirements

Consideration should be given to the technology requirements for plumbing, electrical requirements, RNA/DNA containment facilities etc., as well as the size and weight of the system. 

 

Compatibility with existing systems

For example, whether the system is compatible and can interface with existing laboratory information management systems.  Does it need to be compatible with other site systems?

 

Training/Labour requirements

Does the technology require skilled technicians? Are new skills required? What training is required?

 

Preventative maintenance and calibration requirements

 

Data integrity considerations

 

Safety requirements

If there is a need to confirm the similarity of isolates (for example to investigate the similarity of isolates from a sterility test failure and a likely source), then characterisation or strain typing techniques such as RiboPrinting or Rep-PCR should be used. As such situations are uncommon, characterisation identifications will typically be performed by an approved external laboratory.

 

It is good practice to document the appropriateness of the systems used in the laboratory for the types of identifications performed. For example, not all systems work well with anaerobes or Gram negatives, therefore it should be clearly defined in procedures what system should be used for such organisms.

 

This may be in the form of a decision tree that indicates the steps to take, and in what order, to determine which identification system should be selected.

 

When identifications go wrong

 

Despite instrument qualification and the use of defined procedures and trained technicians, identifications do not always produce acceptable results3.

 

Some common issues that need to be considered when choosing an appropriate system and interpreting results include:

 

 

No single identification system is 100% accurate all of the time

As a microbiologist, you need to understand the limitations of the system and the appropriateness of its use, and you should be able to defend the type of system you use at your site. If you have more than one system available, you need to be able to clearly demonstrate which system is used when and why. Choosing the appropriate identification system should be based on the criticality of the identification and the importance of getting the identification right versus the throughput or number of identifications.

 

Over reliance on the method 

It is important that you review all the data available to you for the isolate you are identifying.  This should include where the organism was isolated from, the Gram stain, colony and cell morphology at a minimum. The data should tie together and make sense. If the organism name provided by the identification system does not tie up with the other data you have, review whether the appropriate system was used.

 

Over identification
Do not identify everything. You need to have a clear understanding of what you are going to do with the data and what it actually means. This should be documented so that the microbiologist carrying out and interpreting the identification tests are not tempted to generate data that is inappropriate. For example, if Gram-positive cocci is isolated from a purified water sample, it is likely due to a sampling error rather than a water system failure. Therefore, further identification may not be required.  

 

Not doing the basics

Basic microscopic and macroscopic data are key to determining the appropriate next steps for identification and confirming the result provided by more complex systems. Making sure the Gram stain is correctly executed is critical.

 

Inadequate databases

Some systems are not capable of identifying particular organisms, so it is important that you (as a microbiologist) ensure that the identification system has an adequate database for the types of organisms you are recovering. Many systems are designed for clinical environments, so bear in mind that these may not contain the typical industrial isolates encountered in the pharmaceutical, healthcare and cosmetic manufacturing environments. 


Databases for commercial test systems are maintained by the manufacturer and based on the continuously updated taxonomic status of clinically and industry relevant bacteria 4.  

 

To develop a database, manufacturers need to gather many strains of the same bacterial species and build a characteristic profile of that particular species using their commercial system.  Building the database on a number of different strains of the same species will ensure any interspecies variability that may be present is accounted for in the database development. This is usually built on greater than ten strains per species entry. Because manufacturers build their databases based on the reactions within their system, identification profiles generated by one system should not be used in another system for identification purposes, nor should they be compared.  

 

Taxonomic changes

All new names or reclassified organisms are published in the International Journal of Systematic and Evolutionary Microbiology (IJSEM), which is available online. Manufactures should keep abreast of these changes, however implementation into a commercially available system may be slow. This point is useful to raise in audits of contract test laboratories which undertake identifications. 

 

Closely related species

Different species within the same genera can be very closely related (e.g. bacillus cereus and bacillus anthracis) and difficult to differentiate using a single identification system. Polyphasic approaches to identification, taking multiple data points into consideration, will help 5.

 

Difficult to identify organisms
Some groups are difficult to identify for various reasons. Examples include corynebacteria, non-fermenters, coagulase-negative staphylococci and weak reactors.

 

Data integrity

As with other aspects of microbiology, bacterial identification methods and processes should be subject to a data integrity review. Data integrity controls should be implemented where risks are identified.

 

Summary

 

This article has focused on two key topics relating to microbial identification: the selection of an appropriate microbial identification method and some considerations for troubleshooting methods when spurious results (or no result at all) are obtained.

References

 

1.    Sandle, T. (2013). Automated Microbial Identifications: A comparison of USP and EP approaches, American Pharmaceutical Review, 16 (4): 56-61


2.    Bochner B.R. Global phenotypic characterization of bacteria. FEMS Microbiol. Rev. 2009;33:191–205


3.    Sandle, T., Skinner, K., Sandle, J., Gebala, B., and Kothandaraman, P.  (2013) Evaluation of the GEN III OmniLog® ID System microbial identification system for the profiling of cleanroom bacteria. European Journal of Parenteral & Pharmaceutical Sciences, 18(2):44-50


4.    Donelli G., Vuotto C., Mastromarino P. Phenotyping and genotyping are both essential to identify and classify a probiotic microorganism. Microb. Ecol. Health Dis. 2013;24:1–8. doi: 10.3402/mehd.v24i0.20105


5.    Franco-Duarte R, Černáková L, Kadam S, Kaushik KS, Salehi B, Bevilacqua A, Corbo MR, Antolak H, Dybka-Stępień K, Leszczewicz M, Relison Tintino S, Alexandrino de Souza VC, Sharifi-Rad J, Coutinho HDM, Martins N, Rodrigues CF. Advances in Chemical and Biological Methods to Identify Microorganisms-From Past to Present. Microorganisms. 2019 May 13;7(5):130

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