Genetic Diversity of the Pneumococcal Capsule: Implications for Molecular-based Serotyping

Mary Catherine McEllistrem

Disclosures

Future Microbiol. 2009;4(7):857-865. 

In This Article

Future Perspective

To circumvent the limitations associated with current PCR- and sequence-based serotyping methods, the use of high-throughput sequencing of the capsular operon will likely be evaluated for pneumococcal serotyping. To date, three vendors provide machines for this service: Roche 454 Life Sciences, Illumina and Applied Biosystems.[51,52] These platforms are also called 'next generation' because they provide exponentially more sequence data at a reasonable cost. For example, the Illumina instrument can produce 1,000,000,000 bp of nucleotide sequence per run, for approximately US$5000.[53] Using high-throughput sequencing, scores of bacteria can now be sequenced in a timely fashion.

The challenge of high-throughput sequencing will be analysis. This technology generates millions of 200–350 bp read lengths per run that must first be assembled into one sequence. Robust analysis programs must then accurately compare the sequences between strains. Finally, the operator must decide which SNPs, deletions or insertions are biologically relevant. For pneumococcus, which has marked interstrain variability, this could become a major roadblock.

Another bacterial typing method that could potentially be exploited for serotyping is matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF MS).[54] This method compares the sizes of small proteins within a bacterial cell to a known dataset for rapid species identification.[55] In addition, MALDI–TOF MS can differentiate subsets of strains within a species. For example, MALDI–TOF MS can differentiate invasive and noninvasive Streptococcus pyogenes isolates that are identical by standard molecular subtyping methods, such as emm typing.[56] Moreover, this tool can quickly ascertain whether a Staphylococcus aureus strain carries the toxin Panton–Valentine leukocidin.[57] Among pneumococci, this technique has been used to generate a library of proteins that are differentially expressed among strains causing conjunctivitis compared with those not associated with this disease.[58] In its current form, MALDI–TOF MS has key limitations that prevent its widespread use. First, the assay may not be reproducible with different MALDI–TOF mass spectrometers. Second, the protein fingerprint could dramatically differ between studies depending on the cultivation conditions. Third, the availability of reference datasets is limited, making strain–strain comparisons untenable. Once these limitations are overcome, MALDI–TOF MS could be used to generate a protein 'fingerprint' for each serotype to replace the quellung reaction.

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