BACKGROUND
the emergence and transmission of drug-resistant m. tuberculosis strains, especially multidrug-resistant  and extensively drug-resistant  strains pose significant clinical, economic, as well as societal challenges.
according to who report, there were an estimated  <dig>  million incident cases of tb worldwide in  <dig>  most of the estimated number of cases in  <dig> occurred in asia  and the african region .
the five countries with the largest number of incident cases include: india , china , south africa , indonesia  and pakistan .
india and china alone accounted for 26% and 12% of global cases, respectively.
in addition, the global estimate of the burden of mdr-tb was  <dig>   <dig> cases among notified tb patients in  <dig>  india and china were the two countries estimated to have the largest numbers of mdr-tb patients   <cit> .
the latest nationwide baseline survey for tb drug resistance carried out in china for the  <dig> and  <dig> reported that  <dig> % of pulmonary tb patients in china suffered from mdr-tb and  <dig> % from xdr-tb.
in  <dig>  there were an estimated  <dig>  incident cases of mdr-tb and  <dig>  incident cases of xdr-tb  <cit> .
furthermore, most cases of mdr- and xdr-tb were shown to be the result of primary transmission, suggesting that many of the new tb cases suffer from the most intractable types of highly drug-resistant m. tuberculosis strains  <cit> .
antibiotic susceptibility profiles and the corresponding resistance determinants of m. tuberculosis have been extensively reported.
however, the genome variations and evolution of drug resistance in m. tuberculosis are still not well explained.
determining the genome components and variations within natural populations of m. tuberculosis isolates with different antibiotic susceptibility profiles may provide a novel perspective on the evolution of drug resistance in m. tuberculosis and enable us to better understand and control drug-resistant tb.
the mycobacterium tuberculosis complex  lineages were considered to be monomorphic, but more and more studies have confirmed the extensive genetic diversity and genome plasticity of the mycobacterial genome through molecular typing techniques such as is6110-rflp, spoligotyping, and miru-vntr .
with the advent of high throughput next generation sequencing technologies , multiple genome sequences from different strains of a single species can provide comprehensive information for exploring the relationship between genotypes and phenotypes with unprecedented resolution.
in this study, we used the illumina gaiix sequencing platform to generate a high-quality and annotated draft genome for 7 m. tuberculosis clinical isolates with different antibiotic resistance phenotypes in order to better understand the evolution of drug resistance in m. tuberculosis isolates in a clinical context.
comparative genomic analyses of these  <dig> strains as well as  <dig> other previously published m. tuberculosis genomes have revealed some genomic variations which might underlie diverse phenotypes among those strains, but no generalizable differences were identified between drug-susceptible and drug-resistant isolates on the genomic scale.
our study adds some new knowledge on genomic variability and evolution of drug-resistant m. tuberculosis.
RESULTS
whole genome sequencing statistics
the detailed epidemiologic and clinical data of the selected m. tuberculosis isolates were summarized in table  <dig>  the basic whole genome sequencing statistics are shown in additional file 1: table s <dig>  the coverage ranged between 200× and 560×, and the completion was  <dig> - <dig> %.
by comparing the sequenced m. tuberculosis clinical isolates to h37rv, we observed that all  <dig> isolates with different levels and profiles of drug resistance harbored similar numbers of snps, ranging from 1409– <dig>  the numbers of insertion/deletions  identified in the  <dig> isolates were also similar, ranging from  <dig> to  <dig> table  <dig>
epidemiologic and clinical data of clinical
m. tuberculosis
isolates
n.a.
= not available.
ainh, isoniazid; rmp, rifampicin; str, streptomycin; emb, ethambutol; pza, pyrazinamide; ofx, ofloxacin; lvx, levofloxacin; kan, kanamycin; cap, capreomycin; amk, amikacin; pas, para-amino salicylic acid; eth, ethionamide.
snp clustering and distribution in the m. tuberculosis genomes
further comparative genomic analysis identified a total of  <dig> non repetitive snps, among which a common pool of  <dig> snps were shared by the  <dig> isolates.
more detailed information on total snps as well as snps in each isolate relative to h37rv are summarized in additional file 2: table s <dig>  to identify regions of snp clustering, snp density was estimated throughout the genomes using a sliding window of 5 kb.
the resulting snp density map shows a non-random distribution of snps, with  <dig> regions having statistically significant clusters .
the detailed information on the  <dig> regions with significantly high snp density is shown in additional file 2: table s <dig>  we further analyzed the distribution of snps according to the different classes of the clusters of orthologous groups  .
we found that snps were significantly under-represented in genes belonging to secondary metabolites biosynthesis, transport, and catabolism , while genes whose functions were unknown  were significantly enriched in snps  .
snps were also slightly over-represented in genes belonging to several other classes such as class m , class r , class v , class j , class k , class t , and class n .figure  <dig>
snp density map constructed using circos.
note: green bars means the density of snps in non-overlapping 5-kb regions; red bars means the regions with significantly high snp density.
genomic insertions and deletions
we further analyzed large insertions and deletions  in clinical m. tuberculosis relative to m. tuberculosis h37rv.
in total,  <dig> non strain-specific and  <dig> strain-specific large insertions as well as  <dig> non strain-specific and  <dig> strain-specific large deletions were identified.
sixteen of those indels spanned pe-ppe-pgrs genes, which have been considered a major source of antigenic variability  <cit> .
many indels were identified both in drug-susceptible and drug-resistant strains.
crispr distribution in the m. tuberculosis genomes
crisprfinder was used to identify putative crispr loci in the genomes of the 7 m. tuberculosis isolates.
in contrast to the m. tuberculosis lab strain h37rv, which was predicted to have two crispr loci, all the  <dig> clinical m. tuberculosis isolates sequenced in this study as well as two other previously sequenced clinical m. tuberculosis isolates  were predicted to have only one of the two crispr loci.
while the spacers in the crispr were identical among  <dig> clinical isolates including ccdc <dig>  ccdc <dig> and three of our clinical isolates , other isolates had high variability in the spacers .
no correlation between antibiotic resistance and the presence of crispr-cas locus was observed.
gene mutations associated with drug resistance in m. tuberculosis
the detailed information on mutations identified in drug resistance-associated loci of the  <dig> chinese clinical isolates is summarized in table  <dig> and  <dig>  a total of  <dig> types of mutations were identified in drug resistance-associated loci, including  <dig> previously reported ones and  <dig> newly identified ones.
the levels of correlation between phenotypic drug resistance and drug resistance-associated mutations varied greatly for different drugs, ranging from 0%  to 100% .
we also identified  <dig> known or putative drug efflux pumps with non-synonymous snps in mdr, pre-xdr and xdr m. tuberculosis isolates but not in h37rv strain .
we further over expressed the mutated drug efflux pump genes in the drug-susceptible reference h37rv strain and determined mics of those recombinant strains.
no increased drug resistance was observed for all examined strains over expressing mutated drug efflux pump genes.
we also performed genetic studies by creating point mutations in the susceptible reference strain h37rv using the pjv53k system for some other potential drug resistance-associated mutations identified in this study  <cit> , but also could not confirm their function in causing drug resistance .table  <dig>
snps and indels identified in antibiotic resistance-associated regions in
m. tuberculosis
isolates
a“r”, resistance of isolates to the corresponding anti-tb drug; “s”, sensitivity of isolates to the corresponding anti-tb drug; “del”, deletion; inh, isoniazid; rmp, rifampicin; str, streptomycin; emb, ethambutol; pza, pyrazinamide; ofx, ofloxacin; lvx, levofloxacin; kan, kanamycin; cap, capreomycin; amk, amikacin; eth, ethionamide.
bintergenic regions.
cnucleotide mutational position is relative to mycobacterium tuberculosis h37rv rpob, and amino acid position is relative to escherichia coli numbering.
ddrug resistance-asssociated mutations with high confidence.
esynonymous.
fnewly identified mutations.
gnon-synonymous.
snps and identified in antibiotic resistance-associated regions in
m. tuberculosis
isolate
s
a“r”, resistance of isolates to the corresponding anti-tb drug; “s”, sensitivity of isolates to the corresponding anti-tb drug; “del”, deletion; inh, isoniazid; rmp, rifampicin; str, streptomycin; emb, ethambutol; pza, pyrazinamide; ofx, ofloxacin; lvx, levofloxacin; kan, kanamycin; cap, capreomycin; amk, amikacin; eth, ethionamide.
bintergenic regions.
cnucleotide mutational position is relative to mycobacterium tuberculosis h37rv rpob, and amino acid position is relative to escherichia coli numbering.
ddrug resistance-asssociated mutations with high confidence.
esynonymous.
fnewly identified mutations.
gnon-synonymous.
genetic diversity and selection intensity in the m. tuberculosis genomes
we used the whole genome sequences of the m. tuberculosis isolates for genetic diversity and selection intensity analysis and the data were shown in additional file 6: table s <dig>  the nucleotide diversity  for the whole genome sequences of the  <dig> newly sequenced clinical isolates were similar, ranging from  <dig>  to  <dig> .
there was no significant differences in Л values between drug-susceptible isolates and drug-resistant isolates , while the Л value was significantly higher among clinical isolates  as compared with lab strains .
the dn/ds values for the whole genome sequences were similar among isolates with different drug resistance profiles, ranging among  <dig>  to  <dig> .
there was no significant differences in dn/ds values between drug-susceptible isolates and drug-resistant isolates , while the dn/ds value was significantly lower among clinical isolates  as compared with lab strains .
we observed significant differences in Л values between our  <dig> clinical isolates and  <dig> previously described clinical isolates .
but when we analyzed our  <dig> isolates together with the two beijing lineage strains  from the  <dig> previously described clinical isolates, the Л value increased from  <dig>  to  <dig> .
phylogenetic analysis of m. tuberculosis isolates
two phylogenetic trees including a neighbor-joining  tree and a maximum-likelihood  tree were created based on snps from whole genome sequences of the  <dig> clinical m. tuberculosis isolates and other  <dig> completely sequenced m. tuberculosis strains.
the phylogenetic relationships among different clinical isolates were similar in two phylogenetic trees .
the  <dig> newly sequenced chinese clinical isolates as well as the two previously sequenced beijing lineage strains ccdc <dig> and ccdc <dig> formed a single clade.figure  <dig>
phylogenetic relationships of
m. tuberculosis
isolates based on snps from whole genome sequences.
the trees were constructed by the neighbor-joining method  or maximum-likelihood method .
only bootstrap values >50% are shown .
discussion
to determine the genome components and variations within natural populations of m. tuberculosis isolates and to better understand the evolution of drug resistance among those isolates, we explored the feasibility of using deep genome sequencing to characterize variations in clinical m. tuberculosis isolates with different drug susceptibility profiles.
our results suggest that the level of genetic diversity is independent of the drug resistance phenotype, since the isolates with different drug resistance profiles harbored similar numbers of snps, nucleotide diversity , and selection intensity .
the relatively high number of snps we identified in all isolates could be partially caused by natural variation, as we included all genes from  <dig> strains isolated from patients diversely located.
selective diversification of m. tuberculosis isolates might also explain an association between host response and strain genetic background as previously reported  <cit> .
several lines of evidence in this study support a significant role of natural selection in shaping m. tuberculosis genomes.
first, the snp distribution in genomes is not random, suggesting that diversifying selection is at work notably in certain genes such as those that play a role in cell wall/membrane/envelope biogenesis  and in general function , which tend to accumulate an excess of snps  <cit> .
second, in the snp density map, many genes located in the regions with significantly high snp density are involved in host–pathogen interactions and may contribute to strain-specific virulence attributes.
for example, one region corresponds to a previously reported virulence operon including the genes rv0986–rv <dig> that are present in one of horizontal genetic transfer  regions  <cit> .
another region with high density of snps was found in the esx- <dig> locus , which includes a type vii secretion system  <cit> .
but in the absence of the information about strain-specific differences in virulence, the high number of snps could also be the result of lateral gene transfer.
third, the dn/ds values were less than  <dig> for the genomes of all  <dig> isolates analyzed, consistent with genome-wide purifying selection.
we have previously shown that the dn/ds values for coding regions of drug resistance-associated genes in mdr and xdr isolates were higher than  <dig>  suggesting that exposure to drugs is among the major forces driving the high dn/ds ratios in those drug resistance-associated genes  <cit> .
but as suggested in this study, on the genome-wide scale, the clinical m. tuberculosis strains with different drug resistance profiles undergo similar levels of purifying selection.
consistently, results from a recent study suggest that the dominant effect of selection on natural m. tuberculosis population is removal of novel variants, with exceptions in certain group of genes such as those involved in defense  <cit> .
indels have a wide range of effects as a very important cause of phenotypic variability.
the acquisition and loss of certain genes could provide pathogens with some advantages during infection and transmission.
thus, the indel loci identified in this study are candidates for drug resistance or virulence-associated factors that may represent evolutionary signatures during the co-evolution of humans and pathogens.
for example, the deletion of a polyketide synthase gene  with high homology to mycocerosic acid synthase is particularly intriguing because the product of this gene may be involved in the production of multimethylated branched lipids  <cit> .
in addition, the pks <dig> mutant strain of m. tuberculosis h37rv was shown to display severe growth defects in mice  <cit> .
it is also worth noting that sixteen of those indels spanned pe-ppe-pgrs genes, which have been considered a major source of antigenic variability  <cit> .
in addition, two of those unique proteins code for putative membrane proteins  and may directly alter the interactions between pathogens and their hosts  <cit> .
since we identified many indels including some of those above-mentioned virulence-associated genes within both drug-susceptible and drug-resistant strains, our results suggest that drug resistance in m. tuberculosis is not necessarily an indication of increased virulence.
our findings are consistent with the notion that the virulence of individual clinical m. tuberculosis isolate is dependent on multiple factors including strain genetic background and host immune responses  <cit> .
a highly significant inverse correlation between the presence of crispr-cas locus and acquired antibiotic resistance was observed in e. faecalis, suggesting that antibiotic use inadvertently selects for enterococcal strains with compromised genome defense  <cit> .
but in this study, no functional genes were identified in crispr locus and no correlation between antibiotic resistance and the presence of crispr-cas locus was observed in clinical m. tuberculosis isolates.
using our previously established method of automatic tbdreamdb-coupled analysis for drug resistance-associated mutations in m. tuberculosis isolates  <cit> , we detected  <dig> types of unreported mutations, as well as  <dig> known or putative drug efflux pumps with non-sense snps in mdr, pre-xdr and xdr m. tuberculosis isolates, but we could not establish the association between over expression of those mutated drug efflux pumps with increased drug resistance in m. tuberculosis.
it was reported previously that mutations or overexpression of rv <dig> and rv2686c are associated with increased resistance to multiple drugs in m. tuberculosis <cit> .
but according to another recent study which aimed to compare the differences of the expression of  <dig> putative multidrug efflux pump genes in clinically isolated drug sensitive and mdr m. tuberculosis isolates, all the tested putative multidrug efflux pump genes in the drug-sensitive and mdr m. tuberculosis isolates have similar rates of expression  <cit> .
thus, the existence of mutations and over expression of the efflux pump genes might not be necessarily associated with increased drug resistance.
by closely examining the correlation of the phenotypic drug susceptibility profiles of the strains with mutations identified in their drug resistance-associated genes, we identified a few potential new genetic determinants of drug resistance.
for example, while  <dig>  of the  <dig> strains exhibited phenotypic resistance to ofloxacin and levofloxacin, only  <dig> of them  had gyra d94g mutation known to confer resistance to fluoroquinolones.
the other  <dig> had the same gyra e21q, g668d, and s95t mutations seen in fluoroquinolone susceptible strain mtb <dig>  indicating that these mutations are not the source of fluoroquinolone resistance.
similarly, among the  <dig> strains showing phenotypic resistance to rifampicin,  <dig>  only had the rpob a <dig> mutation, which was also present in the susceptible strain, suggesting the presence of other unknown mechanisms for rifampicin resistance in them.
since we identified no mutations by further examining other drug resistance-associated genes such as gyrb, gidb and eis in those strains  <cit> , we then performed genetic studies for those newly identified potential drug resistance-associated mutations, but failed to confirm their function in causing drug resistance either .
thus, our observations demonstrated that though certain drug resistance-associated mutations such as rpob s531l, katg s315t, gyra d94g, embb m306v, rpsl k43r, and rrs a1401g could serve as useful markers for rapid detection of resistance in the clinical m. tuberculosis isolates, the accuracy and sensitivity of genetic-based drug resistance assays still need to be increased by further elucidation of unknown mechanisms of drug resistance, especially for second-line drugs .
it should also be pointed out that confirming drug resistance-associated mutations by genetic study could only examine the function of individual gene mutation without taking into consideration the whole genetic background of the strain, while based on the whole genome sequencing studies by us and a few others, there might be no common causes of drug resistance to multiple drugs.
rather, the mdr and xdr phenotypes could result from a combination of mutations in the genomes  <cit> .
the phylogenetic relationships among different clinical isolates were similar in two phylogenetic trees based on whole genome snps.
the whole genome sequencing has been proposed as a sort of “gold standard” for strain typing in m. tuberculosis since it clarifies previous strain typing approaches used for phylogenetic and epidemiologic studies and provides more detailed genomic variation information.
the observation that mdr, pre-xdr, and xdr isolates were located sporadically on different branches in phylogenetic trees based on snps from whole genome sequences of the 14 m. tuberculosis isolates further confirms our previous observation that they have evolved and acquired mutations independently on multiple occasions.
the observation that isolates from china were phylogenetically distant from the isolates from other regions such as the kzn strain from south africa in the phylogenetic trees also confirmed our previous observation that drug-resistant m. tuberculosis strains from different geographic regions have distinct evolutionary pathways  <cit> .
the close phylogenetic relatedness among the  <dig> clinical m. tuberculosis isolates could also be best supported by the analysis of specific snps in drug resistance-associated genes.
the presence of identical uncommon mutations in many of those genes among the  <dig> strains  is indicative of a single cluster of strains circulating in the population.
the finding of high levels of clustering and minimal strain diversity among mdr/xdr m. tuberculosis strains within a population has been described previously  <cit> .
this study has several limitations.
firstly, since the  <dig> clinical m. tuberculosis isolates included in the analysis all belonged to the beijing lineage, thus it is possible that similarities and differences between different strain groups may be explained by phylogenetic lineages, rather than phenotypic differences.
by comparing our  <dig> clinical isolates with  <dig> previously described clinical isolates from diverse lineages and countries of origin, we did observe significantly higher Л value for those  <dig> previously described clinical isolates.
however, when we analyzed our  <dig> beijing lineage isolates together with two previously described beijing lineage isolates , the Л value increased significantly, we thus suggest that genomic variations we observed among different groups of isolates are unlikely caused completely by phylogenetic lineages, but rather associated with diverse phenotypes of the isolates.
secondly, this study was limited by the relatively small number of isolates included in the analysis.
it is likely that a larger sample with diverse lineages and countries of origin would probably reveal more information on genomic variations and evolution of drug-resistant m. tuberculosis strains.
CONCLUSIONS
in this study, by performing whole genome sequencing study, we show that though clinical m. tuberculosis isolates have a certain degree of similarity in their genetic make-up, they exhibit distinct variations in terms of the distribution of snp, indels, crispr-cas locus, as well as the nucleotide diversity and selection intensity.
no generalizable differences were identified between drug-susceptible and drug-resistant isolates on the genomic scale.
our study provides evidence strengthening the notion that the evolution of drug resistance among clinical m. tuberculosis isolates is clearly a complex and diversified process.
several questions remain further in-depth investigations, such as whether drug susceptibility is affected by the deletion of specific genes and disabling of specific metabolic pathways.
in addition, further studies using a larger sampling of m. tuberculosis isolates from diverse lineages are warranted to better understand the evolution of drug-resistant m. tuberculosis strains.

 the emergence and transmission of drug-resistant m. tuberculosis strains, especially multidrug-resistant and extensively drug-resistant strains pose significant clinical, economic, as well as societal challenges. according to who report, there were an estimated million incident cases of tb worldwide in most of the estimated number of cases in occurred in asia and the african region. the five countries with the largest number of incident cases include: india, china, south africa, indonesia and pakistan. india and china alone accounted for 26% and 12% of global cases, respectively. in addition, the global estimate of the burden of mdr-tb was cases among notified tb patients in india and china were the two countries estimated to have the largest numbers of mdr-tb patients. the latest nationwide baseline survey for tb drug resistance carried out in china for the and reported that % of pulmonary tb patients in china suffered from mdr-tb and % from xdr-tb. in there were an estimated incident cases of mdr-tb and incident cases of xdr-tb. furthermore, most cases of mdr- and xdr-tb were shown to be the result of primary transmission, suggesting that many of the new tb cases suffer from the most intractable types of highly drug-resistant m. tuberculosis strains. antibiotic susceptibility profiles and the corresponding resistance determinants of m. tuberculosis have been extensively reported. however, the genome variations and evolution of drug resistance in m. tuberculosis are still not well explained. determining the genome components and variations within natural populations of m. tuberculosis isolates with different antibiotic susceptibility profiles may provide a novel perspective on the evolution of drug resistance in m. tuberculosis and enable us to better understand and control drug-resistant tb. the mycobacterium tuberculosis complex lineages were considered to be monomorphic, but more and more studies have confirmed the extensive genetic diversity and genome plasticity of the mycobacterial genome through molecular typing techniques such as is6110-rflp, spoligotyping, and miru-vntr. with the advent of high throughput next generation sequencing technologies, multiple genome sequences from different strains of a single species can provide comprehensive information for exploring the relationship between genotypes and phenotypes with unprecedented resolution. in this study, we used the illumina gaiix sequencing platform to generate a high-quality and annotated draft genome for 7 m. tuberculosis clinical isolates with different antibiotic resistance phenotypes in order to better understand the evolution of drug resistance in m. tuberculosis isolates in a clinical context. comparative genomic analyses of these strains as well as other previously published m. tuberculosis genomes have revealed some genomic variations which might underlie diverse phenotypes among those strains, but no generalizable differences were identified between drug-susceptible and drug-resistant isolates on the genomic scale. our study adds some new knowledge on genomic variability and evolution of drug-resistant m. tuberculosis.  whole genome sequencing statistics the detailed epidemiologic and clinical data of the selected m. tuberculosis isolates were summarized in table  the basic whole genome sequencing statistics are shown in additional file 1: table s the coverage ranged between 200× and 560×, and the completion was - %. by comparing the sequenced m. tuberculosis clinical isolates to h37rv, we observed that all isolates with different levels and profiles of drug resistance harbored similar numbers of snps, ranging from 1409– the numbers of insertion/deletions identified in the isolates were also similar, ranging from to table epidemiologic and clinical data of clinical m. tuberculosis isolates n.a. = not available. ainh, isoniazid; rmp, rifampicin; str, streptomycin; emb, ethambutol; pza, pyrazinamide; ofx, ofloxacin; lvx, levofloxacin; kan, kanamycin; cap, capreomycin; amk, amikacin; pas, para-amino salicylic acid; eth, ethionamide. snp clustering and distribution in the m. tuberculosis genomes further comparative genomic analysis identified a total of non repetitive snps, among which a common pool of snps were shared by the isolates. more detailed information on total snps as well as snps in each isolate relative to h37rv are summarized in additional file 2: table s to identify regions of snp clustering, snp density was estimated throughout the genomes using a sliding window of 5 kb. the resulting snp density map shows a non-random distribution of snps, with regions having statistically significant clusters. the detailed information on the regions with significantly high snp density is shown in additional file 2: table s we further analyzed the distribution of snps according to the different classes of the clusters of orthologous groups. we found that snps were significantly under-represented in genes belonging to secondary metabolites biosynthesis, transport, and catabolism, while genes whose functions were unknown were significantly enriched in snps. snps were also slightly over-represented in genes belonging to several other classes such as class m, class r, class v, class j, class k, class t, and class n.figure snp density map constructed using circos. note: green bars means the density of snps in non-overlapping 5-kb regions; red bars means the regions with significantly high snp density. genomic insertions and deletions we further analyzed large insertions and deletions in clinical m. tuberculosis relative to m. tuberculosis h37rv. in total, non strain-specific and strain-specific large insertions as well as non strain-specific and strain-specific large deletions were identified. sixteen of those indels spanned pe-ppe-pgrs genes, which have been considered a major source of antigenic variability. many indels were identified both in drug-susceptible and drug-resistant strains. crispr distribution in the m. tuberculosis genomes crisprfinder was used to identify putative crispr loci in the genomes of the 7 m. tuberculosis isolates. in contrast to the m. tuberculosis lab strain h37rv, which was predicted to have two crispr loci, all the clinical m. tuberculosis isolates sequenced in this study as well as two other previously sequenced clinical m. tuberculosis isolates were predicted to have only one of the two crispr loci. while the spacers in the crispr were identical among clinical isolates including ccdc ccdc and three of our clinical isolates, other isolates had high variability in the spacers. no correlation between antibiotic resistance and the presence of crispr-cas locus was observed. gene mutations associated with drug resistance in m. tuberculosis the detailed information on mutations identified in drug resistance-associated loci of the chinese clinical isolates is summarized in table  and a total of types of mutations were identified in drug resistance-associated loci, including previously reported ones and newly identified ones. the levels of correlation between phenotypic drug resistance and drug resistance-associated mutations varied greatly for different drugs, ranging from 0% to 100%. we also identified known or putative drug efflux pumps with non-synonymous snps in mdr, pre-xdr and xdr m. tuberculosis isolates but not in h37rv strain. we further over expressed the mutated drug efflux pump genes in the drug-susceptible reference h37rv strain and determined mics of those recombinant strains. no increased drug resistance was observed for all examined strains over expressing mutated drug efflux pump genes. we also performed genetic studies by creating point mutations in the susceptible reference strain h37rv using the pjv53k system for some other potential drug resistance-associated mutations identified in this study, but also could not confirm their function in causing drug resistance.table snps and indels identified in antibiotic resistance-associated regions in m. tuberculosis isolates a“r”, resistance of isolates to the corresponding anti-tb drug; “s”, sensitivity of isolates to the corresponding anti-tb drug; “del”, deletion; inh, isoniazid; rmp, rifampicin; str, streptomycin; emb, ethambutol; pza, pyrazinamide; ofx, ofloxacin; lvx, levofloxacin; kan, kanamycin; cap, capreomycin; amk, amikacin; eth, ethionamide. bintergenic regions. cnucleotide mutational position is relative to mycobacterium tuberculosis h37rv rpob, and amino acid position is relative to escherichia coli numbering. ddrug resistance-asssociated mutations with high confidence. esynonymous. fnewly identified mutations. gnon-synonymous. snps and identified in antibiotic resistance-associated regions in m. tuberculosis isolate s a“r”, resistance of isolates to the corresponding anti-tb drug; “s”, sensitivity of isolates to the corresponding anti-tb drug; “del”, deletion; inh, isoniazid; rmp, rifampicin; str, streptomycin; emb, ethambutol; pza, pyrazinamide; ofx, ofloxacin; lvx, levofloxacin; kan, kanamycin; cap, capreomycin; amk, amikacin; eth, ethionamide. bintergenic regions. cnucleotide mutational position is relative to mycobacterium tuberculosis h37rv rpob, and amino acid position is relative to escherichia coli numbering. ddrug resistance-asssociated mutations with high confidence. esynonymous. fnewly identified mutations. gnon-synonymous. genetic diversity and selection intensity in the m. tuberculosis genomes we used the whole genome sequences of the m. tuberculosis isolates for genetic diversity and selection intensity analysis and the data were shown in additional file 6: table s the nucleotide diversity for the whole genome sequences of the newly sequenced clinical isolates were similar, ranging from to. there was no significant differences in Л values between drug-susceptible isolates and drug-resistant isolates, while the Л value was significantly higher among clinical isolates as compared with lab strains. the dn/ds values for the whole genome sequences were similar among isolates with different drug resistance profiles, ranging among to. there was no significant differences in dn/ds values between drug-susceptible isolates and drug-resistant isolates, while the dn/ds value was significantly lower among clinical isolates as compared with lab strains. we observed significant differences in Л values between our clinical isolates and previously described clinical isolates. but when we analyzed our isolates together with the two beijing lineage strains from the previously described clinical isolates, the Л value increased from to. phylogenetic analysis of m. tuberculosis isolates two phylogenetic trees including a neighbor-joining tree and a maximum-likelihood tree were created based on snps from whole genome sequences of the clinical m. tuberculosis isolates and other completely sequenced m. tuberculosis strains. the phylogenetic relationships among different clinical isolates were similar in two phylogenetic trees. the newly sequenced chinese clinical isolates as well as the two previously sequenced beijing lineage strains ccdc and ccdc formed a single clade.figure phylogenetic relationships of m. tuberculosis isolates based on snps from whole genome sequences. the trees were constructed by the neighbor-joining method or maximum-likelihood method. only bootstrap values >50% are shown. discussion to determine the genome components and variations within natural populations of m. tuberculosis isolates and to better understand the evolution of drug resistance among those isolates, we explored the feasibility of using deep genome sequencing to characterize variations in clinical m. tuberculosis isolates with different drug susceptibility profiles. our results suggest that the level of genetic diversity is independent of the drug resistance phenotype, since the isolates with different drug resistance profiles harbored similar numbers of snps, nucleotide diversity, and selection intensity. the relatively high number of snps we identified in all isolates could be partially caused by natural variation, as we included all genes from strains isolated from patients diversely located. selective diversification of m. tuberculosis isolates might also explain an association between host response and strain genetic background as previously reported. several lines of evidence in this study support a significant role of natural selection in shaping m. tuberculosis genomes. first, the snp distribution in genomes is not random, suggesting that diversifying selection is at work notably in certain genes such as those that play a role in cell wall/membrane/envelope biogenesis and in general function, which tend to accumulate an excess of snps. second, in the snp density map, many genes located in the regions with significantly high snp density are involved in host–pathogen interactions and may contribute to strain-specific virulence attributes. for example, one region corresponds to a previously reported virulence operon including the genes rv0986–rv that are present in one of horizontal genetic transfer regions. another region with high density of snps was found in the esx- locus, which includes a type vii secretion system. but in the absence of the information about strain-specific differences in virulence, the high number of snps could also be the result of lateral gene transfer. third, the dn/ds values were less than for the genomes of all isolates analyzed, consistent with genome-wide purifying selection. we have previously shown that the dn/ds values for coding regions of drug resistance-associated genes in mdr and xdr isolates were higher than suggesting that exposure to drugs is among the major forces driving the high dn/ds ratios in those drug resistance-associated genes. but as suggested in this study, on the genome-wide scale, the clinical m. tuberculosis strains with different drug resistance profiles undergo similar levels of purifying selection. consistently, results from a recent study suggest that the dominant effect of selection on natural m. tuberculosis population is removal of novel variants, with exceptions in certain group of genes such as those involved in defense. indels have a wide range of effects as a very important cause of phenotypic variability. the acquisition and loss of certain genes could provide pathogens with some advantages during infection and transmission. thus, the indel loci identified in this study are candidates for drug resistance or virulence-associated factors that may represent evolutionary signatures during the co-evolution of humans and pathogens. for example, the deletion of a polyketide synthase gene with high homology to mycocerosic acid synthase is particularly intriguing because the product of this gene may be involved in the production of multimethylated branched lipids. in addition, the pks mutant strain of m. tuberculosis h37rv was shown to display severe growth defects in mice. it is also worth noting that sixteen of those indels spanned pe-ppe-pgrs genes, which have been considered a major source of antigenic variability. in addition, two of those unique proteins code for putative membrane proteins and may directly alter the interactions between pathogens and their hosts. since we identified many indels including some of those above-mentioned virulence-associated genes within both drug-susceptible and drug-resistant strains, our results suggest that drug resistance in m. tuberculosis is not necessarily an indication of increased virulence. our findings are consistent with the notion that the virulence of individual clinical m. tuberculosis isolate is dependent on multiple factors including strain genetic background and host immune responses. a highly significant inverse correlation between the presence of crispr-cas locus and acquired antibiotic resistance was observed in e. faecalis, suggesting that antibiotic use inadvertently selects for enterococcal strains with compromised genome defense. but in this study, no functional genes were identified in crispr locus and no correlation between antibiotic resistance and the presence of crispr-cas locus was observed in clinical m. tuberculosis isolates. using our previously established method of automatic tbdreamdb-coupled analysis for drug resistance-associated mutations in m. tuberculosis isolates, we detected types of unreported mutations, as well as known or putative drug efflux pumps with non-sense snps in mdr, pre-xdr and xdr m. tuberculosis isolates, but we could not establish the association between over expression of those mutated drug efflux pumps with increased drug resistance in m. tuberculosis. it was reported previously that mutations or overexpression of rv and rv2686c are associated with increased resistance to multiple drugs in m. tuberculosis. but according to another recent study which aimed to compare the differences of the expression of putative multidrug efflux pump genes in clinically isolated drug sensitive and mdr m. tuberculosis isolates, all the tested putative multidrug efflux pump genes in the drug-sensitive and mdr m. tuberculosis isolates have similar rates of expression. thus, the existence of mutations and over expression of the efflux pump genes might not be necessarily associated with increased drug resistance. by closely examining the correlation of the phenotypic drug susceptibility profiles of the strains with mutations identified in their drug resistance-associated genes, we identified a few potential new genetic determinants of drug resistance. for example, while of the strains exhibited phenotypic resistance to ofloxacin and levofloxacin, only of them had gyra d94g mutation known to confer resistance to fluoroquinolones. the other had the same gyra e21q, g668d, and s95t mutations seen in fluoroquinolone susceptible strain mtb indicating that these mutations are not the source of fluoroquinolone resistance. similarly, among the strains showing phenotypic resistance to rifampicin, only had the rpob a mutation, which was also present in the susceptible strain, suggesting the presence of other unknown mechanisms for rifampicin resistance in them. since we identified no mutations by further examining other drug resistance-associated genes such as gyrb, gidb and eis in those strains, we then performed genetic studies for those newly identified potential drug resistance-associated mutations, but failed to confirm their function in causing drug resistance either. thus, our observations demonstrated that though certain drug resistance-associated mutations such as rpob s531l, katg s315t, gyra d94g, embb m306v, rpsl k43r, and rrs a1401g could serve as useful markers for rapid detection of resistance in the clinical m. tuberculosis isolates, the accuracy and sensitivity of genetic-based drug resistance assays still need to be increased by further elucidation of unknown mechanisms of drug resistance, especially for second-line drugs. it should also be pointed out that confirming drug resistance-associated mutations by genetic study could only examine the function of individual gene mutation without taking into consideration the whole genetic background of the strain, while based on the whole genome sequencing studies by us and a few others, there might be no common causes of drug resistance to multiple drugs. rather, the mdr and xdr phenotypes could result from a combination of mutations in the genomes. the phylogenetic relationships among different clinical isolates were similar in two phylogenetic trees based on whole genome snps. the whole genome sequencing has been proposed as a sort of “gold standard” for strain typing in m. tuberculosis since it clarifies previous strain typing approaches used for phylogenetic and epidemiologic studies and provides more detailed genomic variation information. the observation that mdr, pre-xdr, and xdr isolates were located sporadically on different branches in phylogenetic trees based on snps from whole genome sequences of the 14 m. tuberculosis isolates further confirms our previous observation that they have evolved and acquired mutations independently on multiple occasions. the observation that isolates from china were phylogenetically distant from the isolates from other regions such as the kzn strain from south africa in the phylogenetic trees also confirmed our previous observation that drug-resistant m. tuberculosis strains from different geographic regions have distinct evolutionary pathways. the close phylogenetic relatedness among the clinical m. tuberculosis isolates could also be best supported by the analysis of specific snps in drug resistance-associated genes. the presence of identical uncommon mutations in many of those genes among the strains is indicative of a single cluster of strains circulating in the population. the finding of high levels of clustering and minimal strain diversity among mdr/xdr m. tuberculosis strains within a population has been described previously. this study has several limitations. firstly, since the clinical m. tuberculosis isolates included in the analysis all belonged to the beijing lineage, thus it is possible that similarities and differences between different strain groups may be explained by phylogenetic lineages, rather than phenotypic differences. by comparing our clinical isolates with previously described clinical isolates from diverse lineages and countries of origin, we did observe significantly higher Л value for those previously described clinical isolates. however, when we analyzed our beijing lineage isolates together with two previously described beijing lineage isolates, the Л value increased significantly, we thus suggest that genomic variations we observed among different groups of isolates are unlikely caused completely by phylogenetic lineages, but rather associated with diverse phenotypes of the isolates. secondly, this study was limited by the relatively small number of isolates included in the analysis. it is likely that a larger sample with diverse lineages and countries of origin would probably reveal more information on genomic variations and evolution of drug-resistant m. tuberculosis strains.  in this study, by performing whole genome sequencing study, we show that though clinical m. tuberculosis isolates have a certain degree of similarity in their genetic make-up, they exhibit distinct variations in terms of the distribution of snp, indels, crispr-cas locus, as well as the nucleotide diversity and selection intensity. no generalizable differences were identified between drug-susceptible and drug-resistant isolates on the genomic scale. our study provides evidence strengthening the notion that the evolution of drug resistance among clinical m. tuberculosis isolates is clearly a complex and diversified process. several questions remain further in-depth investigations, such as whether drug susceptibility is affected by the deletion of specific genes and disabling of specific metabolic pathways. in addition, further studies using a larger sampling of m. tuberculosis isolates from diverse lineages are warranted to better understand the evolution of drug-resistant m. tuberculosis strains. 
to better understand the evolution of drug-resistant m. tuberculosis strains, we performed whole genome sequencing for 7 m. tuberculosis clinical isolates with different antibiotic resistance profiles and conducted comparative genomic analysis of gene variations among them. in this study, we show that clinical m. tuberculosis isolates exhibit distinct variations in terms of the distribution of snp, indels, crispr-cas locus, as well as the nucleotide diversity and selection intensity, but there are no generalizable differences between drug-susceptible and drug-resistant isolates on the genomic scale. aside from snps and indels, a crispr locus with varied spacers was observed in all clinical isolates, suggesting that they might play an important role in plasticity of the m. tuberculosis genome. the dn/ds values were less than for all isolates, supporting the notion that m. tuberculosis genomes undergo purifying selection. the Л values and dn/ds values were comparable between drug-susceptible and drug-resistant strains. our study provides evidence strengthening the notion that the evolution of drug resistance among clinical m. tuberculosis isolates is clearly a complex and diversified process. we observed that all 7 m. tuberculosis clinical isolates with different levels of drug resistance harbored similar numbers of snps, ranging from 1409– the numbers of insertion/deletions identified in the isolates were also similar, ranging from to a total of types of mutations were identified in drug resistance-associated loci, including previously reported ones and newly identified ones. sixteen of the identified large indels spanned pe-ppe-pgrs genes, which represents a major source of antigenic variability. the nucleotide diversity and selection intensity of the whole genome sequences of the isolates were similar. mycobacterium tuberculosisdrug resistancesingle nucleotide polymorphismswhole genome sequencingevolutionissue-copyright-statement© the author 2014
 due to excessive antibiotic use, drug-resistant mycobacterium tuberculosis has become a serious public health threat and a major obstacle to disease control in many countries. to better understand the evolution of drug-resistant m. tuberculosis strains, we performed whole genome sequencing for 7 m. tuberculosis clinical isolates with different antibiotic resistance profiles and conducted comparative genomic analysis of gene variations among them.  we observed that all 7 m. tuberculosis clinical isolates with different levels of drug resistance harbored similar numbers of snps, ranging from 1409– the numbers of insertion/deletions identified in the isolates were also similar, ranging from to a total of types of mutations were identified in drug resistance-associated loci, including previously reported ones and newly identified ones. sixteen of the identified large indels spanned pe-ppe-pgrs genes, which represents a major source of antigenic variability. aside from snps and indels, a crispr locus with varied spacers was observed in all clinical isolates, suggesting that they might play an important role in plasticity of the m. tuberculosis genome. the nucleotide diversity and selection intensity of the whole genome sequences of the isolates were similar. the dn/ds values were less than for all isolates, supporting the notion that m. tuberculosis genomes undergo purifying selection. the Л values and dn/ds values were comparable between drug-susceptible and drug-resistant strains.  in this study, we show that clinical m. tuberculosis isolates exhibit distinct variations in terms of the distribution of snp, indels, crispr-cas locus, as well as the nucleotide diversity and selection intensity, but there are no generalizable differences between drug-susceptible and drug-resistant isolates on the genomic scale. our study provides evidence strengthening the notion that the evolution of drug resistance among clinical m. tuberculosis isolates is clearly a complex and diversified process. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mycobacterium tuberculosisdrug resistancesingle nucleotide polymorphismswhole genome sequencingevolutionissue-copyright-statement© the author 2014 