Identification methods for bacterial isolates

Identification of bacterial isolates provides vital information for companies undertaking environmental monitoring programmes, but methods vary greatly. Vikki Warren from NCIMB discusses

There are a variety of very different approaches to identify bacteria- all of them claim to be rapid and reliable, and they can vary greatly in cost, so which is the best option?

There a number of different factors to be considered, starting with the differences between how the different methods available for identification of bacteria work. The methods can be broadly categorised as genotypic, proteolytic and phenotypic. At one time, the phenotypic approach to identification was the only one available and so phenotypic methods are often viewed as "traditional" microbiology.

Phenotypic methods use the metabolic differences between different species to identify microorganisms. Generally, they involve a combination of Gram staining, culture, and biochemical tests. As the different tests are carried out, the results obtained narrow the possible options until an identification is obtained. Well known examples of phenotypic tests include API strips or VITEK.

The genotypic approach on the other hand identifies microbes on the basis of DNA sequencing, and is sometimes referred to as the "gold standard" for microbial identification, because a DNA sequence has the potential to offer an unambiguous result. Databases of genomes and gene fragments have been created, and this has transformed our ability to understand the relationships between different species, in some cases resulting in their reclassification.

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Proteotypic methods can be seen as sitting between genotypic and phenotypic methods since the genomic information is translated into proteins, and these proteins also reflect the cells' metabolic functions. The MALDI-TOF system is a very commonly used proteotypic method.

NCIMB identifies thousands of environmental isolates every year, and our ID department uses a genotypic approach based on sequencing sections of ribosomal DNA. Of course, all of the identification methods available have their advantages and disadvantages, but we use this method because we believe it is the most accurate and reliable approach - ultimately it is the DNA that defines the species, and this is also the method that we use to confirm the identity of reference strains added to our culture collection - the National Collection of Industrial, Food and Marine Bacteria.

In comparison to phenotypic methods, the genotypic approach has no requirement for growth, so it can easily be used to identify fastidious and uncultivable microorganisms. I also think it is the most flexible in terms of obtaining the best identification possible.

For many of the bacterial identifications that we perform, the customer sends a pure culture, we extract and amplify the DNA then use the Microseq database to give a species or sometimes subspecies level identification. But of course, not every environmental isolate that we receive is included in this database.

By their nature, environmental isolates can be very diverse, and while the Microseq database is extensive, and includes many of the most common environmental isolates, it is not exhaustive in its coverage - no commercial system available for microbial identification is.

When a species-level match cannot be obtained using Microseq, we can usually identify the genus and then search for a species-level match using the much more comprehensive, though unvalidated EMBL-EBI (the European Bioinformatics Institute - part of the European Molecular Biology Laboratory) nucleotide database.

This database is continuously expanding as it allows researchers to share sequence data with the wider scientific community, and usually delivers a species-level match. As this is not a validated database, not all of our customers request this option, but many find the extra information useful. When using public databases to identify unknown isolates, I would always recommend referring to any relevant published papers for supporting information, in order to ensure that the most accurate and reliable identification is obtained.

No commercial system is exhaustive in its list for microbial identification

In any case, the sequence data can be made available for comparison to future isolates, and can help to determine whether successive isolates are the same species. Occasionally, despite an additional search, the genus level Microseq match remains the closest obtained, and this may be an indication that the isolate is a new species that has not yet been sequenced.

Another feature of a genotypic approach is that there is the option of varying the section of DNA that is sequenced to obtain the best identification. Bacterial ribosomes contain a large and a small subunit and 16S rRNA is a structural component of the small subunit. The term 16S rDNA refers to the genes that encode it, and in practice, when people talk about 16S sequencing, they are usually referring to the sequencing of the initial 500 base pairs - approximately a third of the full gene.

This is usually, but not always, enough to identify bacteria at the species level for the purposes of environmental isolates or system contaminants.

NCIMB uses a 16S sequencing to confirm the identity of strains added to their reference collection

Sequencing the full 16S rDNA gene is more commonly used within research projects, or for patenting purposes, but there are some occasions where full 16S sequencing is required for identification of common environmental isolates.

A good illustration of this is with the genus Bacillus. Bacillus is a ubiquitous and diverse genus of bacteria and, an extremely common environmental contaminant. Consequently, it is one of the genera that we are called on to identify most frequently, and 500bp 16S rDNA sequencing is generally our first port of call. While it is a reliable method in the vast majority of cases, we occasionally encounter issues relating to the alignment of the forward and reverse strands of DNA due to the presence of indel mutations.

This creates difficulties for the sequencing software, which will only use sections of the DNA where it can get a good match between the forward and reverse strands. The net result of this is that the system will be trying to identify the bacterial species on the basis of less than 500 base pairs, and this is not a long enough sequence to obtain an accurate identification. However, under these circumstances, we can usually obtain an identification by sequencing the full 16S gene.

When people talk about 16S sequencing, they are usually referring to the initial 500 base pairs

Full 16S sequencing can also be useful for distinguishing between closely related species of bacteria. When undertaking 500 bp sequencing of environmental isolates, we occasionally find that the sequences obtained give a 100% match to more than one species. In this situation, sequencing of the full gene can sometimes give enough additional information to obtain a more precise result.

So if all systems have their advantages and disadvantages - what are the disadvantages of 16S sequencing? Well, it is often viewed as the more expensive option compared to phenotypic tests like API strips or MALDI-TOF. MALDI-TOF is probably the main competing system for environmental isolates and undoubtedly gives fast, low-cost results.

However, the databases are not as comprehensive and the system is more tailored towards clinical isolates, and in my opinion, results are not as reliable.

In conclusion, when choosing between different identification methods, it is important to look not only at price, but at the range of species identified, and the additional information obtained. In practice, pharmaceutical and cleanroom microbiologists may use a combination of approaches, perhaps carrying out phenotypic tests in house before deciding whether another identification method is required.