MLST typing: a method in need of standardization
In an effort to better describe the relationships between isolates of scrub typhus, several investigators have accumulated information from multiple loci. However, problems arise because there was no standard concerning the choice of loci. Consequently, several sets of loci have been used, and these sets overlap only slightly. Even when the same loci have been targeted, the choice of PCR primers have resulted in information from different investigators that, again, only partially overlaps between sets of isolates.
When all of the MLST studies are combined, 23 loci have been examined, but only two of these loci are in common between different research groups, and even then, the two loci are in common each between a different pair of research groups. Clearly, the most profitable approach would be to consider a common set of loci that researchers would focus on. The availability of genome sequences make the choice of loci more logical, since loci can be examined to provide a set which includes loci with the greatest degree of phylogenetic information. In the future, it would be hoped that such an approach would be adopted.
In some cases, it might be thought that whole genome sequencing would make the MLST approach obsolete. However, given the very problematic nature of the Orientia genome (with a large number of repeat sequences that make the determination of gene order difficult), an approach that might utilize information from a selected set of loci to constitute an MLST panel may be possible and might even be more cost effective.
The three MLST panels that have been proposed are:
1) A set of seven loci was used by several investigators (Sonthayanon et al. 2010; Phetsouvanh et al. 2015) to compare isolates. (details at Panel 1).
These loci are:
glycerol-3-phosphate dehydrogenase (gpsA)
malate dehydrogenase (Mdh)
ribonucleoside-diphosphate reductase beta subunit (nrdF)
NADH dehydrogenase chain F (nuoF)
pyruvate, phospate dikinase precursor (ppdK)
dihydrolipoamide S-succinyltransferase (sucB)
succinyl-CoA synthase alpha chain (sucD)
Some details about the MLST alleles found in the study of XXX is deposited in the MLSTPub website. The specific subpage home for the sequences from Orientia in MLSTPub is at Orientia tsutsugamushi MLST Databases.
Allele sequences through March 2015 are provided at a subpage, while the MLST allele profiles of 92 MLST types are given at a second subpage.
This set of MLST loci has been examined in 163 isolates (most from Southeast Asia). profiles are given which can be related to the information on the 92 MLST types. It is probable that additional alleles will be uncovered at all of the seven loci in this set, and that consequently, additional MLST types will be defined.
2) A second set of loci that has been studied by Duong et al. 2013a. (Details at Panel 2) The loci in this set include:
Adenosine kinase (adk)
GTP-binding protein (lepB)
lipoyl synthase (lipA)
lipoyltransferase (lipB)
preprotein translocase (secY)
Superoxide dismutase (sodB)
2-oxoglutarate dehydrogenase (sucA) [note: the primer sites provided in the paper seem not to target the sucA gene, but rather target the adjacent sucB gene that is a part of the first MLST set above].
3) Finally, a set of 11 genetic loci were targeted in a report by Nakayama et al. 2010. (Details at Panel 3) These loci included:
ATP synthase beta-chain (atpD)
ATP-dependent Clp protease ATPbinding subunit (clpX)
DnaJ protein (dnaJ)
DnaK protein (dnaK)
malonyl CoA-acyl carrier protein transacylase (fabD)
DNA gyrase subunit B (gyrB)
isocitrate dehydrogenase (icd)
malate dehydrogenase (mdh)
ribonucleoside-diphosphate reductase alphachain (nrdA)
succinyl-CoA synthetase alpha-chain (sucD)
3-octaprenyl-4-hydroxybenzoate carboxylyase (ubiD)
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