by Vikki Warren, NCIMB Identification Services Manager
Identification of bacterial isolates provides vital information for pharmaceutical microbiologists undertaking environmental monitoring programmes within GMP production facilities, but some genera and species can be more tricky to identify than others. A commonly encountered example is identification of Bacillus species.
Why 16S sequencing?
At NCIMB we use 16S sequencing and the MicroSeq database to identify environmental isolates for our customers, because it has the potential to offer an unambiguous result – ultimately it is the DNA that defines the species. But what exactly is 16S sequencing?
Prokaryotic ribosomes contain a large (70S) and a small (30S) subunit, and 16S rRNA is a structural component of the 30S small subunit. The term 16S rDNA refers to the genes that encode it, but the method is also sometimes referred to as 16S rRNA sequencing.
The 16S rRNA gene is ideal for identification purposes – it is essential for cell function and consequently, is highly conserved. The variations that exist can be used to determine the relationships between different organisms and build phylogenetic trees.
500bp or full gene?
In practice, when people refer to the 16S ribosomal DNA technique they are usually referring to sequencing of the first 500bp – approximately a third of the full gene. This is usually, though not always, sufficient to identify bacteria at the species level. There are some occasions where full 16S gene sequencing gives the best results. A good illustration of this, from my own experience, is with the genus Bacillus.
Bacillus and indel mutations
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 at NCIMB, and 500bp 16s rDNA sequencing is generally our first port of call. While it is a reliable method in the vast majority of cases, on some occasions, full 16S gene sequencing is required.
Specifically, with Bacillus, issues can arise due to the presence of indel mutations, which create difficulties with respect to the alignment of the forward and reverse strands of DNA. The sequencing software will only use the sections of DNA where it can get a good match between the forward and reverse strands, and so the net result is that the system will be trying to identify the bacterial species on the basis of less than 500 base pairs. This is not a long enough sequence to obtain an accurate bacterial identification.
However, we find that under these circumstances sequencing the full 16s gene provides enough additional data for the MicroSeq system to accurately identify the bacterial species.
The issue of indel mutations in Bacillus species highlights the flexibility of the 16S sequencing approach for obtaining accurate and reliable identifications. Not only are there options for 500bp vs full gene sequencing, but the sequences can be compared with those of previously identified isolates, or searched against other published sources, in addition to the validated MicroSeq data base.
For more information about our full range of ID services is available on our microbial identification pages.
About the author: Vikki Warren joined NCIMB in 2005. She leads a team of scientists responsible for delivering NCIMB’s identification services and sequencing new deposits to the UK’s National Collection of Industrial, Food and Marine Bacteria. Vikki holdas a BSC (hons) degree in Applied Biosciences and Management, and an MSC in Instrumental Analytical Techniques; DNA Analysis, Proteomics and Metabolomics from the Robert Gordon University in Aberdeen
“At NCIMB we use 16S sequencing and the MicroSeq database to identify environmental isolates for our customers, because it has the potential to offer an unambiguous result – ultimately it is the DNA that defines the species.”