Traditional cultural techniques versus non targeted molecular detection methods.

Andy Muirhead, ALS company microbiologist, considers established versus more recent and developing testing and detection techniques.

WHAT’S CHANGED?

This year marks the bicentenary of the birth of Louis Pasteur (1822-1895), and if the famous microbiologist would have had access to a time machine and managed to travel forward in time by around 170 years, he could walk into one of our laboratories and still recognise many of the techniques which were performed in his laboratory in the middle of the 19th century.

We still detect and enumerate bacteria by putting them in broths and agars, we place them in optimal growth conditions and demonstrate their presence by allowing the individual cells to multiply to such an extent that we can observe visible colonies on selective or non-selective agars.

Although there has been considerable advances in the agars and cultural techniques which we have at our disposal, it does seem inconceivable that routine diagnostic microbiology (both clinical and food) hasn’t advanced more rapidly than it actually has, and that faster and more sensitive molecular techniques haven’t replaced the more conventional methodologies.

Whilst molecular methods have become the mainstream in virology (we are all familiar with the Polymerase Chain Reaction, or PCR test used for Covid), in bacteriology they are still used very rarely in bacteriology. A possible reason is that traditional cultural virology methods using cell culture lines were difficult to perform. They required a considerable amount of expertise, both in the growth and maintenance of the tissue culture lines and in the inoculation, incubation and the microscopic interpretation of the changes to the tissue cell cultures. It could often take several weeks to get a result, so is no surprise therefore that alternative methods such as ELISA and PCR have gained widespread acceptance in virology, but what about bacteriology?

TRADITIONAL METHODS AUGMENTED

The main reason why bacteriology has fallen behind in terms of embracing new molecular methodologies is that by and large, the traditional methods work, and perhaps more importantly can be delivered at a cost which is acceptable to our clients.

However, we aren’t completely working in the dark ages as over the last 20 years, traditional methods in food microbiology have been augmented by more rapid methods of isolation and identification such as ELISA and MALDI-TOF MS. Performing our Salmonella and Listeria screening tests by ELISA saves an incredible amount of time and money compared to the traditional cultural ISO methodologies.

When we get a presumptive result by ELISA, we then go back to our broth culture and plate it out and look for typical colonial morphology on the selective agar plates. If the colonies look typical for the target organism we obtain a confirmed result by performing conventional biochemical and serological techniques. This however can take another two to three days, which is why we can make use of a mass spectrometry technique known as MALDITOF MS, which enables us to confirm the presumptive isolate much faster.

If MALDI identification leads to a confirmed Salmonella, then we are able to serotype the Salmonella isolates by PCR analysis of the genetic material of the bacteria rather than the antigenic structure of the organism as is used in traditional serological techniques. This gives an accurate and rapid identification of the particular type of Salmonella which can be incredibly useful when attempting to identify a potential cause of contamination of a product. It gives the same result as traditional serological testing, but much quicker and without the requirement to hold expensive banks of antisera.

So, despite my assertion that Louis Pasteur would recognise the basic principles of the traditional cultural techniques which are still in common use in our lab, we have actually embraced many newer methodologies such as ELISA, MALDI-TOF MS and in the serotyping of Salmonella by PCR, which enables us to process large volumes of samples rapidly and cost effectively.

TIME-SAVING

However, even with these techniques we are still relying on initially growing the organisms to levels where they can become detectable, so is it possible to get rid of the requirement to grow the organisms first, as after all that is the time-consuming stage, and instead demonstrate the presence of organisms

ALS Laboratories (UK) Ltd (www.als-testing.co.uk) is one of the UK’s leading providers of food and drink testing services. With six accredited laboratories located across the country, they offer a comprehensive range of high quality, analytical testing services, including microbiological, nutritional, vitamins and minerals, pesticides and contaminants, allergens and speciation. They also provide clients with a wide range of consultancy services and technical support on food safety, labelling requirements, allergens management and sensory testing.

simply by detection of their unique genetic material instead?

The two most likely ways that this “culture free detection” will happen in the future is through PCR and WGS (Whole Gene Sequencing). In food analysis, PCR tests are used in many applications. As well as the detection of pathogenic microorganisms, it can be used in allergen identification, for the detection of genetically modified organisms or for the identification of animal species. PCR, is one of the most well-known techniques in molecular biology, involving the replication of single-stranded DNA from a template using synthetic primers and a DNA polymerase. It still requires an initial “amplification” stage, but it is much shorter than the time required for traditional methods or even ELISA techniques.

Since its inception by Kary Mullis in 1983, the fundamental principles of PCR have remained the same, but methods have evolved with vast performance improvements to DNA polymerases and reagents, as well as innovations in instrumentation. Prior to the introduction of thermal cyclers, PCR was a laborious process involving the transfer of samples between water baths of different temperatures, requiring precise timings of each step. The thermal cycler, together with the discovery of better polymerase enzymes, has made automation of PCR a reality. So, with its increased automation and commercial affordability, will we soon see methods like PCR overtake traditional cultural methods in the microbiology lab, or will we turn to other molecular methods such as Whole Gene Sequencing (WGS) and Next Generational Sequencing (NGS)?

DEVELOPMENTS

Unlike PCR, where a relatively small section of the DNA strand is copied, multiplied and analysed, WGS and NGS does as the name suggests, and sequences the entire microbial genome. Determination of the whole genome sequence of a single cultured isolate offers a higher level of discrimination compared with traditional molecular typing tools such as Multi Locus Sequence Typing (MLST), and means that WGS is now widely used as the preferred surveillance tool for foodborne illness.

Another application of this area of research is “Metagenomics”, where NGS is applied to a food sample generating sequences of all of the microorganisms in that sample. The application of metagenomics for food safety and quality is still in its infancy but it offers exciting opportunities to characterise previously unculturable and therefore unknown microorganisms.

So, are we going to see a radical change in the methods used in contract food microbiology labs anytime soon? Traditional “targeted” methods where we look for specific organisms or groups of organisms have been compared to the school register. The teacher who doesn’t look up from the attendance register is only aware of the pupils who confirm their presence when she shouts out their name and will not be aware of anyone else who may be at the back of the room, but isn’t in the register. We may test a sample for Salmonella, Listeria and STEC and have Not Detected results for all three, but does that guarantee the food safety of the sample? What about all of the organisms we haven’t looked for and what about the organisms for which we have no methods available and are difficult to culture?

WGS/NGS/Metagenomics have enabled many new organisms which live inside our intestines to be discovered and identified.

We also have to remember that in food microbiology many of the organisms we are attempting to isolate have been subjected to the rigors of food processing and may have been heated, chilled, frozen, dried, subjected to acidic conditions, the actions of preservatives and may have had to compete with fermenting bacteria. All of which makes the organisms stressed and makes their detection by conventional means harder. Organisms which are too stressed to grow, or who have complex and fastidious growth requirements are known as viable but non-culturable (VBNC) organisms.

We know that the molecular methods are becoming more affordable and that they are very specific. They also offer much higher discrimination between different strains of the same organism so that they can be a useful diagnostic tool in determining outbreak clusters and identifying the potential source of a contamination. Although they still require an initial “amplification step”, this is much shorter than the resuscitation and enrichment steps in existing methods so they can offer results much faster than traditional cultural methods. They are able to detect organisms which may not be culturable (VBNC) by traditional cultural techniques. Because many of these molecular methods lend themselves to automation, they also require less staffing levels than traditional cultural methodologies.

ADVANTAGES AND DISADVANTAGES

However, the traditional methods still have a lot to be said in their favour. Compared to the molecular methods, the initial investment is low and the return on the investment is relatively short compared to the high initial outlay for the equipment required for PCR/WGS equipment. In the food industry the cost of the test is paramount, and the fact remains that traditional microbiological analysis is still significantly cheaper than most molecular methods. When we grow a target organism on a selective agar plate, we have demonstrated both the presence, but more importantly the viability of the organism. Molecular methods may be able to accurately identify the genetic material of an organism, but does that always the same as the detection of a viable cell which is capable of causing either food spoilage or illness?

There are advantages and disadvantages in both the traditional cultural techniques and the newer molecular methodologies, so it is impossible to say which method is best. Both have their uses and used in conjunction they possibly offer the best solution to food testing labs who need to offer reliable results with a fast turnaround time but at an affordable cost to their clients.

There is no doubt that new technologies will continue to emerge, and that advances in transcriptase enzymes and further improvements in metagenomics will enable make molecular methods such as PCR and WGS even more specific, and as their use increases the costs will hopefully come down and they will become mainstream in many laboratories.

I also have no doubt however that if Louis Pasteur jumped back into his time machine and travelled forward in time for another 100 years he will still see petri dishes, agar plates, autoclaves and incubators in the food testing laboratory of the future.