Multilocus Sequence Typing Scheme for Staphylococcus aureus: Revision of the gmk Locus

LETTER

Multilocus sequence typing (MLST) is a sequence-based genotyping method based on polymorphisms (each variant is termed an allele) in seven housekeeping genes (loci) (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) in Staphylococcus aureus providing unique allelic profiles known as sequence types (STs) (1). The data obtained by MLST are highly reproducible and are available in the S. aureus MLST database (http://saureus.mlst.net/). Since the publication of Enright and coworkers' article (1), MLST has been extensively used to study evolution and population dynamics among S. aureus lineages (our search identified >1,000 citations of this paper indexed at ISI Web of Knowledge as of 16 April 2012).

We occasionally have had difficulties assigning allele numbers for the gmk locus due to suboptimal DNA sequence quality in the 3′ end of the gmk amplicons, which prompted us to investigate the problem. DNA sequence analysis revealed a 12-bp overlap between the 3′ end of the 429-bp region that is used to define the gmk alleles and the 3′ end of the primer gmk-Dn (5′-TCATTAACTACAACGTAATCGTA-3′ [the overlapping 3′ end is underlined]). This means that the 12 bp (TACGATTACGTT) in the 3′ end of the 429-bp gmk region should always be conserved given that the gmk-Dn primer is integrated into the amplicons.

Nevertheless, among the 183 gmk alleles available in the S. aureus MLST database as of 16 April 2012, we identified 12 gmk alleles with a polymorphism(s) in the 12-bp region corresponding to 48 STs (Table 1). The most common polymorphism (8/12) was a G instead of a T at position 429 (Table 1).

Table 1.

Changes in the S. aureus MLST database due to revision of the gmk locus

Original gmk allele	Polymorphism(s)	Position(s)	New gmk allele	Original STa	New ST	CC change
50	G	429	44	377	152
				1471	1471a
64	G	429	107	1066	1277
				1067	1067a
68	T	426	2	686	489
				1697	121
81	G	429	129	856	1643
88	G	429	1	931	8
				1283	239
				1441	15
				1476	97
				1494	1465
				1595	25
				1738	1
				2129	2129a
				2136	9
				2156	199
				2157	2157a
				2163	2163a
				2278	2278a
121	T	420	2	1452	133
136	G	429	136a	1727	1727a
137	G	429	8	1765	72
				2094	188
149	T, A	420, 429	101	1823	1223
150	T, A	420, 429	34	1824	1824a
				1848	1848a
				1849	2198
				1850	1850a	Singleton to CC75
				2043	2043a
				2044	2044a
154	G	429	18	1868	89
155	G	429	2	1869	30
				1952	59
				2012	2012a	Singleton to CC522
				2073	121
				2076	2057
				2077	398
				2080	433
				2095	1768
				2097	2097a	Singleton to CC398
				2098	2098a
				2099	2099a
				2100	2100a
				2161	2155
				2307	2307a
				2308	2308a
				2348	2348a

^aNew gmk alleles or STs that were unique in the database retained their original allele or ST numbers.

Based on our experience, many of the polymorphisms in the 12-bp region are likely to be “false” due to suboptimal DNA sequence quality toward the end of the gmk amplicons. On the other hand, we acknowledge that some of the polymorphisms may be “true” if they have been identified by PCR amplification using alternative downstream primers or by analysis of whole-genome sequence data.

We argue that exclusion of the 12 bp from the 429-bp gmk region is the most straightforward procedure to ensure the robustness of S. aureus MLST in the future. The required changes in the S. aureus MLST database and the software used to assign gmk alleles based on the new 417-bp region have recently been made, and the issue identified here has been highlighted on the homepage (http://saureus.mlst.net/). Where changes of the ST have occurred, these are noted in the “comments” field of the isolate entry in the database. Those whose isolates were affected by these changes have been notified.

The consequences of these changes are described in Table 1. Where the revision of the gmk locus resulted in a new allele or ST that was unique in the database (1 of 12 affected alleles and 20 of 48 affected STs), the original allele or ST number was maintained. Accordingly, although 12 alleles and 48 STs were affected, only 11 allele numbers and 28 ST numbers had to be changed. In total, 68 of the 4,526 isolates present in the S. aureus MLST database on 16 April 2012 (or 1.5% of all isolates deposited) were affected by these changes. Since the new STs are, by definition, single-locus variants of the 28 original STs, we would expect only slight changes in the population structure. Using the stringent group definition (6/7 shared alleles), eBURST (version 3; http://eburst.mlst.net) analyses of the entire S. aureus MLST database before and after the revision of the gmk locus (conducted on 16 April 2012) showed that the new STs remained within the same clonal complex (CC) as the original STs and that three singletons (ST1850, ST2012, and ST2097) can now be assigned to existing CCs (Table 1).