BACKGROUND
m. genitalium has been recognized as an important cause of nongonococcal urethritis  in men and is likely to be associated with genital tract inflammatory diseases in women, such as cervicitis  <cit> , endometritis  <cit> , pelvic inflammatory disease  <cit> , and tubal factor infertility  <cit> .
the epidemiologic data suggests that m. genitalium is a sexually transmitted pathogen  <cit> .
a recent molecular study supports these findings  <cit> .
study of m. genitalium presents several unique challenges.
it remains extremely difficult to isolate the organism from clinical specimens, and thus, identification of infected individuals is dependent on the use of polymerase chain reaction  tests.
m. genitalium samples derived directly from patients are usually contaminated with human cells and other microbes, and may contain pcr inhibitors  <cit> .
these difficulties have impeded progress in understanding the epidemiology and pathogenic role of m. genitalium.
during the last few years, several molecular methods have been reported to be useful for strain typing, including: short tandem repeat  analysis of putative lipoprotein  genes  <cit> , single nucleotide polymorphisms  in the rrna genes  <cit> , restriction fragment length polymorphisms  of the mg <dig>  gene  <cit> , and snps in the mg <dig>  conserved gene  <cit> .
the value of these typing techniques has never been comparatively assessed.
the aims of this study were: 1) to identify new potential genetic markers based on an analysis of strs in the published m. genitalium genome sequence; 2) to compare these newly identified markers and those previously described for their utility as components of an efficient multi-locus genotyping system; 3) to provide further evidence for sexual transmission of m. genitalium.
RESULTS
bioinformatics analysis of tandem repeats in the m. genitalium g <dig> genome
although the presence of tandem repeats in the m. genitalium genome has been noted in previous studies  <cit> , a comprehensive list of tandem repeats with accurate information on the location and repeat unit sequence is not available.
we performed a computerized search of the m. genitalium g <dig> genome  <cit>  and identified  <dig> loci containing tandem repeat units ranging from  <dig> to  <dig> bases in length .
none of these loci had repeat unit lengths longer than  <dig> bases .
the repeat copy number for the  <dig> str loci varied from  <dig> to  <dig>  the majority of these strs  consisted of a trinucleotide repeat unit.
for the remaining three loci repeat units consisted of a mononucleotide, tetranucleotide or pentanucleotide in one each.
nine strs were located in coding regions while all others fell in non-coding regions.
six of the non-coding region strs were located in mgpa-related repetitive elements   <cit> , including mgpar  <dig>  mgpar  <dig>  mgpar  <dig> and mgpar  <dig>  <cit> .
a trinucleotide agt repeat motif was present in  <dig> loci, with one in the coding region of mg <dig>  two in the coding region of the adhesin mgpa operon , and three in mgpar regions .
while the agt repeat unit has been previously annotated as tag repeat by other investigators based on analysis of the g <dig> genome sequence only  <cit> , our analyses of multiple m. genitalium strains in the present study and in an earlier study  <cit>  have demonstrated that the correct annotation is agt.
the repeat unit tct identified in mgpars  <dig>   <dig> and  <dig> in this study has been annotated differently in previous reports  <cit> , either as ctt or aag .
we have found evidence that the tct repeat region in these three mgpars can recombine with the mg <dig> gene in which they encode a stretch of serines  <cit> .
thus we believe that the correct repeat unit sequence should be tct.
the str sequence in mg <dig> was shown to be a mixture of agt and aat trinucleotides in our previous study  <cit>  rather than a hexanucleotide repeat  as previously reported  <cit> .
all str regions except for those in mg <dig> and mgpars were present in a single copy in the genome.
agiven as the locus tag annotated in genbank or in reference  <cit> .
use of short tandem repeats in a multilocus genotyping system
previous studies have found that the strs in mg <dig>  mg <dig> and mgpars undergo extensive intrastrain variation due to dna recombination and/or other mechanisms , indicating that these strs are unsuitable for strain typing.
because our earlier study showed that the str in the mg <dig> plp gene was highly variable among clinical strains  <cit> , we targeted the other three plp loci  for further investigation.
the remaining  <dig> str loci were not evaluated due to limited volume of the dna extract available from the specimens.
thirty-one unrelated new orleans specimens were studied initially which revealed significant variation in mg <dig> and mg <dig> but none in mg <dig>  therefore the latter locus was excluded from further study.
the variability of mg <dig>  mg <dig>  and mg <dig> strs was determined in a total of  <dig> unrelated new orleans patients and  <dig> unrelated scandinavian patients .
among the  <dig> patient samples studied, the number of repeats varied from  <dig> to  <dig> for the mg307-strs,  <dig> to  <dig> for the mg309-strs, and  <dig> to  <dig> for the mg338-strs.
twenty-eight  patients contained a single allele at all three str loci.
sequence analysis revealed a mixture of two or more str alleles in the three plp loci as follows: mg <dig> – 30%, mg <dig> – 23%, and mg <dig> – 32%.
a total of  <dig>  of the specimens had mixed str sequences in at least one of the str-containing loci.
in all mixed sequences, the number of repeats varied by only one or two repeat units.
the prevalence of mixed sequences in both mg307-strs and mg338-strs significantly increased as the repeat copy number became larger  while this correlation was not significant for the mg309-strs .
the str numbers in specimens with mixtures of sequences were determined readily by direct sequencing of the pcr products in the forward and reverse directions, in which individual sequences were read by direct observation of the sequence chromatograms .
to verify the presence of mixed sequences as determined by direct sequencing, we performed subcloning of pcr products from  <dig> selected specimens containing mixed sequences, followed by sequencing of 4– <dig> individual plasmid clones per specimen.
the presence of a mixture of  <dig> or more sequences as determined by direct sequencing was verified in all  <dig> specimens .
plasmid cloning revealed the presence of a third and sometimes a fourth sequence in addition to those identified by direct sequencing in a few cases.
in one sample that showed no evidence of a mixed str population by direct sequencing, the  <dig> plasmid clones that were sequenced also showed no evidence of sequence mixtures .
in addition, for  <dig> selected specimens showing mixed sequences we repeated pcr using dna polymerases with proof-reading activity and obtained sequences by direct sequencing that were the same as those provided by taq dna polymerase.
this provided evidence that mixed plp str sequences cannot be attributed to pcr artefacts.
because mg309-strs contain two different repeat units, agt and aat, we examined the distribution patterns of these units among different specimens.
among  <dig> unrelated patient specimens , which showed no evidence of sequence mixtures, the number of mg <dig> repeats varied from  <dig> to  <dig>  two to four different agt/aat distribution patterns were observed in specimens having the same number of strs with the exception of the five specimens with  <dig> repeats, which were all identical.
when both the repeat number and distribution pattern variations were taken into account, there were  <dig> unique mg <dig> sequence types among these  <dig> patients.
figure 3a shows an example of the variation in the distribution of the agt and aat repeat units among patient specimens containing  <dig> and  <dig> repeats.
the distribution patterns of these repeat units were stable in sequential specimens from the same patients  and in plasmid clones that had an identical number of repeats and were obtained from the same patients .
rrna single nucleotide polymorphism  typing
overall rrna-snps types  <dig> and  <dig> are most common .
types  <dig>  and  <dig>  are relatively uncommon.
as in our previous study, type  <dig> is not present.
this type is uniformly found in the currently available atcc strains but apparently nowhere else.
stratifying the data geographically revealed a significant difference between rrna-snp types in new orleans and scandinavia .
in new orleans type  <dig> is most common  while in scandinavia  <dig> is the predominant type  .
thus far types  <dig> and  <dig> are seen only in new orleans.
of interest is that none of the other typing loci studied here differ in prevalence between the two locations.
to increase efficiency of rrna-snp locus typing we developed a simple and rapid method based on single-strand conformational polymorphism  analysis to detect the rrna-snps.
as shown in figure 4b, types  <dig>   <dig> and  <dig> exhibited different sscp patterns.
type  <dig> showed three bands which were well separated.
in contrast, types  <dig> and  <dig> showed two bands with different mobility.
subsequently, we applied the sscp assay blindly to all additional specimens used in this study.
the typing results using the sscp assay were completely consistent with the sequencing data for these three genotypes.
mg191-single nucleotide polymorphism typing
this typing method is based on sequence analysis of the polymorphic sites in an approximately 280-bp fragment of the mg <dig> conserved ab region  <cit> .
previous study of  <dig> specimens by this method has identified  <dig> unique genotypes, designated type  <dig> to  <dig>  <cit> .
in this study, we observed  <dig> and  <dig> different sequence types in  <dig> unrelated new orleans patients and  <dig> unrelated scandinavian patients, respectively .
when the results for both patient populations were combined, there were a total of  <dig> unique sequence types.
twenty-three of these were identical to those reported previously by jensen et al.
<cit> .
among  <dig> previously unstudied specimens from new orleans patient four new types  were identified, which have been deposited into genbank with accession numbers eu <dig> to eu <dig>
stability of various genetic markers
two sequential specimens were obtained from each of  <dig> new orleans patients at an interval of  <dig> to  <dig> days .
none of the  <dig> patients showed a change in mg309-strs, rrna-snps or mg191-snps.
two patients had minor changes in the str number at mg <dig> and/or mg <dig>  these changes involved either contraction or expansion of only one repeat unit.
a underlined numbers indicate repeats confirmed by sequencing of cloned pcr products .
for specimens containing mixed sequences, the genotypes are listed in the order from predominant to minor type.
discriminatory power of various genetic markers
the discriminatory index  for each marker was calculated using the  <dig> specimens having a single allele at all five loci studied .
of the five markers evaluated, the mg191-snps and the mg309-strs loci had the highest dis , followed by the mg338-strs and mg307-strs .
the rrna-snps had the lowest di .
when the variation in both the repeat number and the distribution of the agt and aat repeat units at the mg <dig> locus was considered, the di increased to  <dig> .
we analyzed the di for various combinations of two of the  <dig> markers and found that the combination of mg309-strs and mg191-snps gave the highest .
however, some specimens containing identical mg309-str and mg191-snp genotypes , still could be distinguished by mg307-strs, mg338-strs or rrna-snps .
analysis of sexual transmission by multi-locus typing
to study the sexual transmission of m. genitalium infection, we analyzed the genotype profiles at the five variable loci described above in specimens obtained from  <dig> concurrently infected couples  in comparison with the  <dig> unrelated patients.
figure  <dig> shows the genotype profile at  <dig> loci for  <dig> unrelated patients.
for each patient, the genotypes at all  <dig> loci were used to define the multi-locus genotype profile.
patients were classified as having matching profiles if they had identical alleles at all loci or if they had identical alleles at rrna-snp and mg191-snp loci and shared at least one common allele at each of the three variable str loci when mixed str alleles were present.
among these  <dig> patients, only four sets of two patients had matching profiles and all the remaining patients had mismatching profiles.
among the  <dig> couples studied , matching genotype profiles were identified within  <dig> couples.
among the four couples without complete matches, one  had a mismatch of one repeat unit at a single plp locus  while three  had mismatches at two or three loci.
for all couples showing matching genotypes at mg <dig> strs, the distribution patterns of the agt/aat units were also identical.
discussion
in this study, we carried out a comprehensive analysis of the strs in the genome of the m. genitalium type strain g <dig> and found two new genetic markers that are potentially useful for molecular typing of this organism.
we evaluated the discriminatory power of the new str markers  and three previously described markers, including the mg309-strs  <cit> , rrna-snps  <cit>  and mg191-snps  <cit> , using a collection of  <dig> unrelated patient specimens from both scandinavia and new orleans.
we found that the mg191-snps and the mg309-strs had high discriminatory power  while mg338-strs, mg307-strs and rrna-snps were less so though still useful.
the discriminatory power of mg309-strs was further improved by the variation in the distribution patterns of the two types of repeat units.
a combination of mg309-strs and mg191-snps yielded almost perfect discrimination .
in addition, we found that though the rrna-snps marker had a very low di, its geographical distribution differed strongly between scandinavia and new orleans.
finally, we applied multi-locus typing to study the sexual transmission in concurrently-infected couples from scandinavia and new orleans and further confirmed that m. genitalium is sexually transmitted  <cit> .
molecular typing has proven to be an important tool for understanding the epidemiology and pathogenesis of bacterial infections.
during the past few years, multiple attempts have been made to develop strain typing methods for m. genitalium.
kokotovic et al., were the first to describe a typing method for m. genitalium, which is based on whole-genome fingerprinting involving selective amplification of restriction fragments  <cit> .
this method requires purified dna from cultured strains and is, therefore, not applicable to typing directly from clinical specimens.
to overcome this limitation, several new methods have been reported, all of which rely on pcr amplification of a specific genomic locus followed by dna sequencing or rflp analysis.
they include str analysis of the plp gene mg <dig>  <cit> , snps of the rrna operon  <cit>  and the mg <dig>  gene  <cit> , and rflp of the mg <dig>  gene  <cit> .
the target for the mg192-rflp system developed by musatovova et al.
<cit>  involves a region that has multiple copies  dispersed throughout the m. genitalium genome.
we and others have found that this region is undergoing rapid sequence shifts over time in vitro and in vivo due to homologous recombination with mgpar repeats  <cit> , suggesting that the mg192-rflp system  <cit>  is not suitable for m. genitalium genotyping.
the other three typing methods, including mg309-strs, rrna-snps  <cit>  and mg191-snps  <cit> , appear to have high stability and excellent discriminatory power.
however, they have never been directly compared with respect to discriminatory power and utility in a multilocus genotyping system.
the present study is the first to compare the discriminatory power of different genotyping markers for m. genitalium based on data derived from the same collection of specimens.
we found that mg309-strs and mg191-snps had greater discriminatory power than other markers.
the major advantage of str typing is that it gives the results as numbers allowing for easy interpretation and exchange of data between different laboratories.
a disadvantage of strs typing is the presence of mixed repeat patterns in approximately 20–34% of clinical strains, which may complicate the interpretation of the results for laboratory personnel inexperienced in reading sequence chromatograms.
investigation of the possibility of separating the mixed sequences and quantifying the copy number by other approaches such as denaturing high-resolution gel electrophoresis  <cit>  or fluorescent fragment sizing  <cit>  would be worthwhile.
because snps and strs differ in molecular mechanisms and rates of mutation, combined use of snps and strs can complement each other, provide higher typing efficiency and may define more accurate genetic relationships  <cit> .
of the five markers evaluated in this study, mg191-snps showed the highest di among snp loci while mg <dig> strs showed the highest di among str loci .
the discriminatory power of the mg <dig> strs locus is further improved by the variation in the distribution patterns of the two types of repeat units .
a combination of mg309-strs and mg191-snps achieved almost perfect discrimination .
we propose that a combination of mg <dig> strs and mg191-snps would work efficiently for general epidemiological studies of m. genitalium infection.
the addition of the other three loci results in a small increase in discriminatory power but requires increased effort and expense.
it is of interest that the distribution of the rrna-snps differs strongly between scandinavia and new orleans  whereas mg191-snps and plp gene strs did not .
even with the limited number of specimens studied thus far,  <dig> genotypes of the mg191-snps have been described thus far  <cit> .
in contrast, only  <dig> types of rrna-snps have been found  <cit> .
this is consistent with the known slow rate of rrna mutation making this gene less useful in dynamic sexual network studies but more useful for tracking geographic distribution of m. genitalium.
it would be of considerable interest to determine the rrna-snps distribution in diverse populations from around the world.
moreover, it would be worth determining whether or not rrna-snps associations correlate with environmental factors and/or bacterial virulence.
while the plp strs are stable over a period of up to  <dig> weeks , high frequency of single repeat unit variants resulting in the appearance or disappearance of mixed str alleles suggests the existence of clonal variants resulting from high mutation rates in these str loci.
these are generally believed to be caused by slipped strand mispairing during dna replication  <cit> .
the finding that mixed str alleles are strongly associated with larger repeat copy numbers in mg307-strs and mg338-strs suggests that larger repeat copy numbers have higher mutation rates, consistent with studies of str mutations in other bacteria  <cit> .
in each of those specimens containing mixed str alleles, the individual alleles differed by only one or two repeat units and there was no evidence of mixed alleles in mg191-snps or rrna-snps, further supporting the contention that the presence of mixed str alleles are not due to coinfection with two or more different strains.
therefore, when using the str containing loci for epidemiological purposes, if any two samples are found to share one or more common str alleles they should be considered to have the same genotype at that locus.
sexual transmission of m. genitalium has been suggested by a number of epidemiological studies which have found either high infection concordance rates among sexual partnerships  <cit>  or association with high risk sexual behaviour  <cit> .
however, such epidemiological studies suggest but do not prove sexual transmission of the organism.
other methods are needed to unequivocally establish the sexual transmission of m. genitalium infection.
we have previously addressed this issue by mg191-snp analysis of  <dig> concurrently-infected couples  <cit> .
for all  <dig> couples the sequence type found in specimens from the male partner was identical to that in the female partner.
in one of these couples a new sequence type was documented to have been introduced by a female to her male partner.
in the present study we investigated the genotype profiles at five genomic loci in specimens from  <dig> unrelated patients and  <dig> concurrently infected couples.
among the  <dig> unrelated patients, only four sets of two patients had matching genotype profiles.
in contrast, among the  <dig> couples, matching genotype profiles were identified within  <dig> couples and only  <dig> couples showed mismatching profiles.
furthermore, each of the  <dig> matched couples shared a unique genotype profile.
the degree of concordance  in this study is similar to that  reported for neisseria gonorrhoeae infection from sexual contacts, which was determined by multi-antigen sequence typing  <cit> .
these findings clearly demonstrate that unrelated patients are infected by different strains and that the concurrently infected couples are infected by the same strains thus proving sexual transmission of m. genitalium.
it is noteworthy that, despite the presence of mixed alleles in at least one of the three variable str loci in  <dig> couples, mismatching mg <dig> and mg <dig> str copy numbers were observed between the members of only two couples , while all the remaining couples showed matched repeat copy numbers .
this suggests that the clonal variant populations are continuously transmitted between regular sexual partners through repeated exposure.
the stability of these highly discriminatory genotypes suggests that str analysis might be useful for sexual network studies of m. genitalium infection.
CONCLUSIONS
we evaluated the performance of several genetic markers for typing of m. genitalium and found that the mg191-snps and the mg309-strs had comparable high degrees of discriminatory power.
a combination of mg309-strs and mg191-snps achieved almost perfect discrimination.
the ability to find concordant genotypes within concurrently infected couples and different genotypes among unrelated patients suggests that these typing methods have excellent epidemiological concordance.
we 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.

 m. genitalium has been recognized as an important cause of nongonococcal urethritis in men and is likely to be associated with genital tract inflammatory diseases in women, such as cervicitis, endometritis, pelvic inflammatory disease, and tubal factor infertility. the epidemiologic data suggests that m. genitalium is a sexually transmitted pathogen. a recent molecular study supports these findings. study of m. genitalium presents several unique challenges. it remains extremely difficult to isolate the organism from clinical specimens, and thus, identification of infected individuals is dependent on the use of polymerase chain reaction tests. m. genitalium samples derived directly from patients are usually contaminated with human cells and other microbes, and may contain pcr inhibitors. these difficulties have impeded progress in understanding the epidemiology and pathogenic role of m. genitalium. during the last few years, several molecular methods have been reported to be useful for strain typing, including: short tandem repeat analysis of putative lipoprotein genes, single nucleotide polymorphisms in the rrna genes, restriction fragment length polymorphisms of the mg gene, and snps in the mg conserved gene. the value of these typing techniques has never been comparatively assessed. the aims of this study were: 1) to identify new potential genetic markers based on an analysis of strs in the published m. genitalium genome sequence; 2) to compare these newly identified markers and those previously described for their utility as components of an efficient multi-locus genotyping system; 3) to provide further evidence for sexual transmission of m. genitalium.  bioinformatics analysis of tandem repeats in the m. genitalium g genome although the presence of tandem repeats in the m. genitalium genome has been noted in previous studies, a comprehensive list of tandem repeats with accurate information on the location and repeat unit sequence is not available. we performed a computerized search of the m. genitalium g genome and identified loci containing tandem repeat units ranging from to bases in length. none of these loci had repeat unit lengths longer than bases. the repeat copy number for the str loci varied from to the majority of these strs consisted of a trinucleotide repeat unit. for the remaining three loci repeat units consisted of a mononucleotide, tetranucleotide or pentanucleotide in one each. nine strs were located in coding regions while all others fell in non-coding regions. six of the non-coding region strs were located in mgpa-related repetitive elements, including mgpar mgpar mgpar and mgpar. a trinucleotide agt repeat motif was present in loci, with one in the coding region of mg two in the coding region of the adhesin mgpa operon, and three in mgpar regions. while the agt repeat unit has been previously annotated as tag repeat by other investigators based on analysis of the g genome sequence only, our analyses of multiple m. genitalium strains in the present study and in an earlier study have demonstrated that the correct annotation is agt. the repeat unit tct identified in mgpars and in this study has been annotated differently in previous reports, either as ctt or aag. we have found evidence that the tct repeat region in these three mgpars can recombine with the mg gene in which they encode a stretch of serines. thus we believe that the correct repeat unit sequence should be tct. the str sequence in mg was shown to be a mixture of agt and aat trinucleotides in our previous study rather than a hexanucleotide repeat as previously reported. all str regions except for those in mg and mgpars were present in a single copy in the genome. agiven as the locus tag annotated in genbank or in reference. use of short tandem repeats in a multilocus genotyping system previous studies have found that the strs in mg mg and mgpars undergo extensive intrastrain variation due to dna recombination and/or other mechanisms, indicating that these strs are unsuitable for strain typing. because our earlier study showed that the str in the mg plp gene was highly variable among clinical strains, we targeted the other three plp loci for further investigation. the remaining str loci were not evaluated due to limited volume of the dna extract available from the specimens. thirty-one unrelated new orleans specimens were studied initially which revealed significant variation in mg and mg but none in mg therefore the latter locus was excluded from further study. the variability of mg mg and mg strs was determined in a total of unrelated new orleans patients and unrelated scandinavian patients. among the patient samples studied, the number of repeats varied from to for the mg307-strs, to for the mg309-strs, and to for the mg338-strs. twenty-eight patients contained a single allele at all three str loci. sequence analysis revealed a mixture of two or more str alleles in the three plp loci as follows: mg – 30%, mg – 23%, and mg – 32%. a total of of the specimens had mixed str sequences in at least one of the str-containing loci. in all mixed sequences, the number of repeats varied by only one or two repeat units. the prevalence of mixed sequences in both mg307-strs and mg338-strs significantly increased as the repeat copy number became larger while this correlation was not significant for the mg309-strs. the str numbers in specimens with mixtures of sequences were determined readily by direct sequencing of the pcr products in the forward and reverse directions, in which individual sequences were read by direct observation of the sequence chromatograms. to verify the presence of mixed sequences as determined by direct sequencing, we performed subcloning of pcr products from selected specimens containing mixed sequences, followed by sequencing of 4– individual plasmid clones per specimen. the presence of a mixture of or more sequences as determined by direct sequencing was verified in all specimens. plasmid cloning revealed the presence of a third and sometimes a fourth sequence in addition to those identified by direct sequencing in a few cases. in one sample that showed no evidence of a mixed str population by direct sequencing, the plasmid clones that were sequenced also showed no evidence of sequence mixtures. in addition, for selected specimens showing mixed sequences we repeated pcr using dna polymerases with proof-reading activity and obtained sequences by direct sequencing that were the same as those provided by taq dna polymerase. this provided evidence that mixed plp str sequences cannot be attributed to pcr artefacts. because mg309-strs contain two different repeat units, agt and aat, we examined the distribution patterns of these units among different specimens. among unrelated patient specimens, which showed no evidence of sequence mixtures, the number of mg repeats varied from to two to four different agt/aat distribution patterns were observed in specimens having the same number of strs with the exception of the five specimens with repeats, which were all identical. when both the repeat number and distribution pattern variations were taken into account, there were unique mg sequence types among these patients. figure 3a shows an example of the variation in the distribution of the agt and aat repeat units among patient specimens containing and repeats. the distribution patterns of these repeat units were stable in sequential specimens from the same patients and in plasmid clones that had an identical number of repeats and were obtained from the same patients. rrna single nucleotide polymorphism typing overall rrna-snps types and are most common. types and are relatively uncommon. as in our previous study, type is not present. this type is uniformly found in the currently available atcc strains but apparently nowhere else. stratifying the data geographically revealed a significant difference between rrna-snp types in new orleans and scandinavia. in new orleans type is most common while in scandinavia is the predominant type. thus far types and are seen only in new orleans. of interest is that none of the other typing loci studied here differ in prevalence between the two locations. to increase efficiency of rrna-snp locus typing we developed a simple and rapid method based on single-strand conformational polymorphism analysis to detect the rrna-snps. as shown in figure 4b, types and exhibited different sscp patterns. type showed three bands which were well separated. in contrast, types and showed two bands with different mobility. subsequently, we applied the sscp assay blindly to all additional specimens used in this study. the typing results using the sscp assay were completely consistent with the sequencing data for these three genotypes. mg191-single nucleotide polymorphism typing this typing method is based on sequence analysis of the polymorphic sites in an approximately 280-bp fragment of the mg conserved ab region. previous study of specimens by this method has identified unique genotypes, designated type to. in this study, we observed and different sequence types in unrelated new orleans patients and unrelated scandinavian patients, respectively. when the results for both patient populations were combined, there were a total of unique sequence types. twenty-three of these were identical to those reported previously by jensen et al.. among previously unstudied specimens from new orleans patient four new types were identified, which have been deposited into genbank with accession numbers eu to eu stability of various genetic markers two sequential specimens were obtained from each of new orleans patients at an interval of to days. none of the patients showed a change in mg309-strs, rrna-snps or mg191-snps. two patients had minor changes in the str number at mg and/or mg these changes involved either contraction or expansion of only one repeat unit. a underlined numbers indicate repeats confirmed by sequencing of cloned pcr products. for specimens containing mixed sequences, the genotypes are listed in the order from predominant to minor type. discriminatory power of various genetic markers the discriminatory index for each marker was calculated using the specimens having a single allele at all five loci studied. of the five markers evaluated, the mg191-snps and the mg309-strs loci had the highest dis, followed by the mg338-strs and mg307-strs. the rrna-snps had the lowest di. when the variation in both the repeat number and the distribution of the agt and aat repeat units at the mg locus was considered, the di increased to. we analyzed the di for various combinations of two of the markers and found that the combination of mg309-strs and mg191-snps gave the highest. however, some specimens containing identical mg309-str and mg191-snp genotypes, still could be distinguished by mg307-strs, mg338-strs or rrna-snps. analysis of sexual transmission by multi-locus typing to study the sexual transmission of m. genitalium infection, we analyzed the genotype profiles at the five variable loci described above in specimens obtained from concurrently infected couples in comparison with the unrelated patients. figure shows the genotype profile at loci for unrelated patients. for each patient, the genotypes at all loci were used to define the multi-locus genotype profile. patients were classified as having matching profiles if they had identical alleles at all loci or if they had identical alleles at rrna-snp and mg191-snp loci and shared at least one common allele at each of the three variable str loci when mixed str alleles were present. among these patients, only four sets of two patients had matching profiles and all the remaining patients had mismatching profiles. among the couples studied, matching genotype profiles were identified within couples. among the four couples without complete matches, one had a mismatch of one repeat unit at a single plp locus while three had mismatches at two or three loci. for all couples showing matching genotypes at mg strs, the distribution patterns of the agt/aat units were also identical. discussion in this study, we carried out a comprehensive analysis of the strs in the genome of the m. genitalium type strain g and found two new genetic markers that are potentially useful for molecular typing of this organism. we evaluated the discriminatory power of the new str markers and three previously described markers, including the mg309-strs, rrna-snps and mg191-snps, using a collection of unrelated patient specimens from both scandinavia and new orleans. we found that the mg191-snps and the mg309-strs had high discriminatory power while mg338-strs, mg307-strs and rrna-snps were less so though still useful. the discriminatory power of mg309-strs was further improved by the variation in the distribution patterns of the two types of repeat units. a combination of mg309-strs and mg191-snps yielded almost perfect discrimination. in addition, we found that though the rrna-snps marker had a very low di, its geographical distribution differed strongly between scandinavia and new orleans. finally, we applied multi-locus typing to study the sexual transmission in concurrently-infected couples from scandinavia and new orleans and further confirmed that m. genitalium is sexually transmitted. molecular typing has proven to be an important tool for understanding the epidemiology and pathogenesis of bacterial infections. during the past few years, multiple attempts have been made to develop strain typing methods for m. genitalium. kokotovic et al., were the first to describe a typing method for m. genitalium, which is based on whole-genome fingerprinting involving selective amplification of restriction fragments. this method requires purified dna from cultured strains and is, therefore, not applicable to typing directly from clinical specimens. to overcome this limitation, several new methods have been reported, all of which rely on pcr amplification of a specific genomic locus followed by dna sequencing or rflp analysis. they include str analysis of the plp gene mg, snps of the rrna operon and the mg gene, and rflp of the mg gene. the target for the mg192-rflp system developed by musatovova et al. involves a region that has multiple copies dispersed throughout the m. genitalium genome. we and others have found that this region is undergoing rapid sequence shifts over time in vitro and in vivo due to homologous recombination with mgpar repeats, suggesting that the mg192-rflp system is not suitable for m. genitalium genotyping. the other three typing methods, including mg309-strs, rrna-snps and mg191-snps, appear to have high stability and excellent discriminatory power. however, they have never been directly compared with respect to discriminatory power and utility in a multilocus genotyping system. the present study is the first to compare the discriminatory power of different genotyping markers for m. genitalium based on data derived from the same collection of specimens. we found that mg309-strs and mg191-snps had greater discriminatory power than other markers. the major advantage of str typing is that it gives the results as numbers allowing for easy interpretation and exchange of data between different laboratories. a disadvantage of strs typing is the presence of mixed repeat patterns in approximately 20–34% of clinical strains, which may complicate the interpretation of the results for laboratory personnel inexperienced in reading sequence chromatograms. investigation of the possibility of separating the mixed sequences and quantifying the copy number by other approaches such as denaturing high-resolution gel electrophoresis or fluorescent fragment sizing would be worthwhile. because snps and strs differ in molecular mechanisms and rates of mutation, combined use of snps and strs can complement each other, provide higher typing efficiency and may define more accurate genetic relationships. of the five markers evaluated in this study, mg191-snps showed the highest di among snp loci while mg strs showed the highest di among str loci. the discriminatory power of the mg strs locus is further improved by the variation in the distribution patterns of the two types of repeat units. a combination of mg309-strs and mg191-snps achieved almost perfect discrimination. we propose that a combination of mg strs and mg191-snps would work efficiently for general epidemiological studies of m. genitalium infection. the addition of the other three loci results in a small increase in discriminatory power but requires increased effort and expense. it is of interest that the distribution of the rrna-snps differs strongly between scandinavia and new orleans whereas mg191-snps and plp gene strs did not. even with the limited number of specimens studied thus far, genotypes of the mg191-snps have been described thus far. in contrast, only types of rrna-snps have been found. this is consistent with the known slow rate of rrna mutation making this gene less useful in dynamic sexual network studies but more useful for tracking geographic distribution of m. genitalium. it would be of considerable interest to determine the rrna-snps distribution in diverse populations from around the world. moreover, it would be worth determining whether or not rrna-snps associations correlate with environmental factors and/or bacterial virulence. while the plp strs are stable over a period of up to weeks, high frequency of single repeat unit variants resulting in the appearance or disappearance of mixed str alleles suggests the existence of clonal variants resulting from high mutation rates in these str loci. these are generally believed to be caused by slipped strand mispairing during dna replication. the finding that mixed str alleles are strongly associated with larger repeat copy numbers in mg307-strs and mg338-strs suggests that larger repeat copy numbers have higher mutation rates, consistent with studies of str mutations in other bacteria. in each of those specimens containing mixed str alleles, the individual alleles differed by only one or two repeat units and there was no evidence of mixed alleles in mg191-snps or rrna-snps, further supporting the contention that the presence of mixed str alleles are not due to coinfection with two or more different strains. therefore, when using the str containing loci for epidemiological purposes, if any two samples are found to share one or more common str alleles they should be considered to have the same genotype at that locus. sexual transmission of m. genitalium has been suggested by a number of epidemiological studies which have found either high infection concordance rates among sexual partnerships or association with high risk sexual behaviour. however, such epidemiological studies suggest but do not prove sexual transmission of the organism. other methods are needed to unequivocally establish the sexual transmission of m. genitalium infection. we have previously addressed this issue by mg191-snp analysis of concurrently-infected couples. for all couples the sequence type found in specimens from the male partner was identical to that in the female partner. in one of these couples a new sequence type was documented to have been introduced by a female to her male partner. in the present study we investigated the genotype profiles at five genomic loci in specimens from unrelated patients and concurrently infected couples. among the unrelated patients, only four sets of two patients had matching genotype profiles. in contrast, among the couples, matching genotype profiles were identified within couples and only couples showed mismatching profiles. furthermore, each of the matched couples shared a unique genotype profile. the degree of concordance in this study is similar to that reported for neisseria gonorrhoeae infection from sexual contacts, which was determined by multi-antigen sequence typing. these findings clearly demonstrate that unrelated patients are infected by different strains and that the concurrently infected couples are infected by the same strains thus proving sexual transmission of m. genitalium. it is noteworthy that, despite the presence of mixed alleles in at least one of the three variable str loci in couples, mismatching mg and mg str copy numbers were observed between the members of only two couples, while all the remaining couples showed matched repeat copy numbers. this suggests that the clonal variant populations are continuously transmitted between regular sexual partners through repeated exposure. the stability of these highly discriminatory genotypes suggests that str analysis might be useful for sexual network studies of m. genitalium infection.  we evaluated the performance of several genetic markers for typing of m. genitalium and found that the mg191-snps and the mg309-strs had comparable high degrees of discriminatory power. a combination of mg309-strs and mg191-snps achieved almost perfect discrimination. the ability to find concordant genotypes within concurrently infected couples and different genotypes among unrelated patients suggests that these typing methods have excellent epidemiological concordance. we 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. 
we performed a comprehensive analysis of strs in the genome of the m. genitalium type strain g and identified loci containing strs. we 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. a combination of mg309-strs and mg191-snps yielded almost perfect discrimination. the aims of this study were: 1) to identify new potential genetic markers based on an analysis of short tandem repeat 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. finally we applied multi-locus typing to further confirm sexual transmission using specimens from unrelated patients and concurrently infected couples. the multi-locus typing analysis of unrelated m. genitalium-infected individuals and infected couples adds to the evidence that m. genitalium is sexually transmitted. based on an analysis of unrelated patient specimens from new orleans and scandinavia, the discriminatory indices for these three markers were, and, respectively. an additional finding was that the rrna-snps distribution pattern differed significantly between scandinavia and new orleans. analysis of multi-locus genotype profiles using the five variable loci described above revealed of the couples had concordant genotype profiles compared to only four examples of concordance among the unrelated randomly selected patients. two other previously described markers, including single nucleotide polymorphisms in the rrna genes and snps in the mg gene were found to have dis of and, respectively.
 several 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 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.  we performed a comprehensive analysis of strs in the genome of the m. genitalium type strain g and identified loci containing strs. in addition to one previously studied locus, mg we chose two others, mg and mg for further study. based on an analysis of unrelated patient specimens from new orleans and scandinavia, the discriminatory indices for these three markers were, and, respectively. two other previously described markers, including single nucleotide polymorphisms in the rrna genes and snps in the mg gene were found to have dis of and, respectively. a combination of mg309-strs and mg191-snps yielded almost perfect discrimination. 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 unrelated patients and concurrently infected couples. analysis of multi-locus genotype profiles using the five variable loci described above revealed of the couples had concordant genotype profiles compared to only four examples of concordance among the unrelated randomly selected patients.  we 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 unrelated m. genitalium-infected individuals and infected couples adds to the evidence that m. genitalium is sexually transmitted. 