Short tandem repeat sequences in the Mycoplasma genitalium genome and their use in a multilocus genotyping system

Abstract

BackgroundSeveral methods have been reported for strain typing of Mycoplasma genitalium. The value of these methods has never been comparatively assessed. The aims of this study were: 1) to identify new potential genetic markers based on an analysis of short tandem repeat (STR) sequences in the published M. genitalium genome sequence; 2) to apply previously and newly identified markers to a panel of clinical strains in order to determine the optimal combination for an efficient multi-locus genotyping system; 3) to further confirm sexual transmission of M. genitalium using the newly developed system.ResultsWe performed a comprehensive analysis of STRs in the genome of the M. genitalium type strain G37 and identified 18 loci containing STRs. In addition to one previously studied locus, MG309, we chose two others, MG307 and MG338, for further study. Based on an analysis of 74 unrelated patient specimens from New Orleans and Scandinavia, the discriminatory indices (DIs) for these three markers were 0.9153, 0.7381 and 0.8730, respectively. Two other previously described markers, including single nucleotide polymorphisms (SNPs) in the rRNA genes (rRNA-SNPs) and SNPs in the MG191 gene (MG191-SNPs) were found to have DIs of 0.5820 and 0.9392, respectively. A combination of MG309-STRs and MG191-SNPs yielded almost perfect discrimination (DI = 0.9894). An additional finding was that the rRNA-SNPs distribution pattern differed significantly between Scandinavia and New Orleans. Finally we applied multi-locus typing to further confirm sexual transmission using specimens from 74 unrelated patients and 31 concurrently infected couples. Analysis of multi-locus genotype profiles using the five variable loci described above revealed 27 of the couples had concordant genotype profiles compared to only four examples of concordance among the 74 unrelated randomly selected patients.ConclusionWe propose that a combination of the MG309-STRs and MG191-SNPs is efficient for general epidemiological studies and addition of MG307-STRs and MG338-STRs is potentially useful for sexual network studies of M. genitalium infection. The multi-locus typing analysis of 74 unrelated M. genitalium-infected individuals and 31 infected couples adds to the evidence that M. genitalium is sexually transmitted.

Method comparison for molecular typing of French and Tunisian Mycoplasma genitalium-positive specimens

“…PCR amplification and sequencing of 281 base pairs (bp) of the MG191 semi-conserved region were performed as previously described using the MgPa-1/MgPa-3 primer set (Jensen et al, 1991). A sequence type (ST) type was assigned to each specimen according to previously described types (Hjorth et al, 2006;Ma et al, 2008;Musatovova & Baseman, 2009). We used the numbering system developed originally in 2006 by Hjorth et al (2006) with the description of 56 STs, numbered 1-56.…”

“…We used the numbering system developed originally in 2006 by Hjorth et al (2006) with the description of 56 STs, numbered 1-56. In addition to the 56 STs, we successively included the seven new STs described by Ma et al (2008), numbered 57-63, and the 17 new STs described by Musatovova & Baseman (2009), numbered 64-80. In our study, we then described eight new STs of the mgpB gene, numbered 81-88, and deposited them in GenBank under the accession numbers HM565865-HM565872, respectively.…”

“…After evaluation of the specimens, two of these six VNTR loci were rejected due to lack of adequate discrimination and four VNTR loci were finally chosen for further assessment. Three TR loci, MG307-STR, MG309-STR, and MG338-STR, were named according to a previous study (Ma et al, 2008), and the fourth TR locus, previously identified (Iverson-Cabral et al, 2007) but never used in a typing scheme, was named Mge1.…”

“…Several methods have been developed for the molecular typing of M. genitalium, most of them are based on a specific genomic locus analysis, such as single nucleotide polymorphisms (SNPs) in the rRNA operon (Ma & Martin, 2004) or the semi-conserved region of the MG191 (mgpB) gene (Hjorth et al, 2006;Jensen et al, 2004 genotyping method was proposed in combination with a short tandem repeat (STR) analysis of lipoprotein (LP) genes and analysis of SNPs in rRNA or mgpB genes (Ma et al, 2008). The majority of molecular typing data for M. genitalium clinical specimens is based on the mgpB gene, which encodes the adhesin MgPa (Hjorth et al, 2006;Iverson-Cabral et al, 2006;Musatovova & Baseman, 2009).…”

“…In the present study, we evaluated the two main molecular typing methods, the mgpB SNP typing method (Hjorth et al, 2006) and the combination analysis of a VNTR marker in MG309 and mgpB SNPs (Ma et al, 2008), for the analysis of a large collection of French and Tunisian M. genitalium clinical specimens. We also tried to develop an MLVA scheme, which was compared to the two other methods.…”