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Recently, World Health Organization (WHO) has listed streptomycin (STR) in the list of secondline injectable drugs in the multidrug-resistant tuberculosis (MDR-TB) regimens and may replace amikacin under the same conditions. Nevertheless, molecular characterizations associated with STR resistance in the Mycobacterium tuberculosis population have not been fully investigated in Vietnam. The present study aimed to explore the variation and frequency of mutations in rpsL and rrs genes and their relationship with drug-resistant patterns and M. tuberculosis genotypes in 163 STR-resistant strains from Vietnam. The mutation frequency of the rpsL and rrs genes were 62% and 20.9%, respectively, and the mutation combination in both genes covered 81% of STRresistant strains. The most prevalent mutations included rpsL Lys43Arg (38.7%), Lys88Arg (19.6%), rrs A514C (10.4%) and A517C (5.5%). Thus, sequence analysis of rpsL and rrs exhibited a sensitivity of 81% and specificity of 100% for the prediction of STR resistance in Vietnamese M. tuberculosis strains. The prevalence of STR-resistant mutations in double, triple and quadruple resistance strains was significantly different, compared with mono STR-resistant ones. Similarly, mutation frequency associated with STR resistance in MDR strains was significantly higher than that in non-MDR strains. In addition, the lineage 2 genotype was significantly correlated with a high rate of STR resistance-conferring mutation, as well as the mutation rpsL Lys43Arg (P < 0.01), while the lineage 1 genotype was associated with a low rate of STR resistance-conferring mutation and rrs mutations (P<0.05). In conclusion, sequence analysis may be useful for the rapid detection of STR resistance in MDR M. tuberculosis strains, which in turn could contribute to better control strategies of TB in Vietnam. Other molecular mechanisms associated with STR resistance in STR-resistant strains without mutations in the rpsL and rrs genes need to be further investigated. [ABSTRACT FROM AUTHOR]

ABSTRACT Strains of the Mycobacterium tuberculosis complex (MTBC) Beijing family aroused concern because they were often found in clusters and appeared to be exceptionally transmissible. However, it was later found that strains of the Beijing family were heterogeneous, and the transmission advantage was restricted to sublineage L2.3 or modern Beijing. In this study, we analyzed the previously published genome sequences of 7,896 L2.3 strains from 51 different countries. Using BEAST software to approximate the temporal emergence of L2.3, our calculations suggest that L2.3 initially emerged in northern East Asia during the early 15th century and subsequently diverged into six phylogenetic clades, identified as L2.3.1 through L2.3.6. Using terminal branch length and genomic clustering as proxies for transmissibility, we found that the six clades displayed distinct population dynamics, with the three recently emerged clades (L2.3.4 to L2.3.6) exhibiting significantly higher transmissibility than the older three clades (L2.3.1 to L2.3.3). Of the Beijing family strains isolated outside East Asia, 83.1% belonged to the clades L2.3.4 to L2.3.6, which were also associated with more cross-border transmission. This work reveals the heterogeneity in sublineage L2.3 and demonstrates that the global success of Beijing family strains is driven by the three recently emerged L2.3 clades. IMPORTANCE The recent population dynamics of the global tuberculosis epidemic are heavily shaped by Mycobacterium tuberculosis complex (MTBC) strains with enhanced transmissibility. The infamous Beijing family strain stands out because it has rapidly spread throughout the world. Identifying the strains responsible for the global expansion and tracing their evolution should help to understand the nature of high transmissibility and develop effective strategies to control transmission. In this study, we found that the L2.3 sublineage diversified into six phylogenetic clades (L2.3.1 to L2.3.6) with various transmission characteristics. Clades L2.3.4 to L2.3.6 exhibited significantly higher transmissibility than clades L2.3.1 to L2.3.3, which helps explain why more than 80% of Beijing family strains collected outside East Asia belong to these three clades. We conclude that the global success of L2.3 was not caused by the entire L2.3 sublineage but rather was due to the rapid expansion of L2.3.4 to L2.3.6. Tracking the transmission of L2.3.4 to L2.3.6 strains can help to formulate targeted TB prevention and control.

Background and Objectives: The Beijing family of Mycobacterium tuberculosis has been identified as a severe pathogen among this species and found in many clinical isolates during the last decade. Early identification of such genotype is important for better prevention and treatment of tuberculosis. The present study performed to compare the efficiency of Real-Time PCR and IS6110-Based Inverse PCR methods to identify the Beijing family. Materials and Methods: This study was carried out on 173 clinical isolates of Mycobacterium tuberculosis complex in Golestan Province, northern Iran. DNA extraction performed by boiling and determining the Beijing and non-Beijing strains carried out using Real-Time PCR and IS6110-Based Inverse PCR. Results: In both Real-Time PCR and IS6110-Based Inverse PCR method, 24 specimens (13.9%) of the Beijing family were identified and the result of the IS6110-Based Inverse PCR method showed that all the Beijing strains in this region belonged to the Ancient Beijing sub-lineage. Conclusion: Although the efficacy of the two methods in the diagnosis of the Beijing family is similar, the IS6110-Based Inverse PCR is more applicable to the ability to detect new and old Beijing family.

Background and Objectives: The Beijing family of Mycobacterium tuberculosis has been identified as a severe pathogen among this species and found in many clinical isolates during the last decade. Early identification of such genotype is important for better prevention and treatment of tuberculosis. The present study performed to compare the efficiency of Real-Time PCR and IS6110-Based Inverse PCR methods to identify the Beijing family. Materials and Methods: This study was carried out on 173 clinical isolates of Mycobacterium tuberculosis complex in Golestan Province, northern Iran. DNA extraction performed by boiling and determining the Beijing and non-Beijing strains carried out using Real-Time PCR and IS6110-Based Inverse PCR. Results: In both Real-Time PCR and IS6110-Based Inverse PCR method, 24 specimens (13.9%) of the Beijing family were identified and the result of the IS6110-Based Inverse PCR method showed that all the Beijing strains in this region belonged to the Ancient Beijing sub-lineage. Conclusion: Although the efficacy of the two methods in the diagnosis of the Beijing family is similar, the IS6110-Based Inverse PCR is more applicable to the ability to detect new and old Beijing family. [ABSTRACT FROM AUTHOR]

The American state of Hawaii presents a tuberculosis (TB) burden more consistent with that of the Philippines and the Pacific Islands than that with the United States (US) or Europe. This study seeks to determine if the genetic families of Mycobacterium tuberculosis (Mtb) that are prevalent in Hawaii display differences in host demographics that may be of use for TB control in Hawaii and the Pacific. This retrospective study was conducted by analyzing data from the Hawaii State Department of Health to investigate the demographics associated with the Beijing (global lineage 2) and Manila (lineage 1) families of Mtb in Hawaii. Deidentified records of all culture-positive TB cases reported by the Hawaii State Department of Health Tuberculosis Control Program from 2004 to 2016 were analyzed to identify lineage-specific demographic differences and trends. Patients' countries of origin, age, sex, and time in the US before TB diagnosis were included in this analysis. Manila family isolates were found to predominantly enter Hawaii through Filipino immigrants, whereas Beijing family isolates originated from a diverse set of countries. Both families exhibited significant differences in age and sex demographics. In addition, Manila family cases presented from patients with significantly longer average time of residence in the US than non-Manila cases, whereas Beijing family cases presented from patients with significantly shorter time of residence in the US than non-Beijing cases. Both the Beijing and Manila families of Mtb demonstrated demographic differences in Hawaii that may prove important for improving TB control and surveillance policy in Hawaii and throughout the Pacific. Areas with heavy Filipino immigration may benefit from directing more resources toward screening and education efforts for middle-aged men and those who have resided in the country longer, whereas other areas of the Pacific should consider a younger and more sex-balanced allocation. Specific to the US and Hawaii, effective screening of youths emigrating from the Compact of Free Association states remains vital. • Hawaii's genetic lineages of Mycobacterium tuberculosis show distinct epidemiology. • Beijing family cases in the Pacific originate from a diverse set of countries. • Manila family TB is most common in older persons who have been in the USA longer. • Beijing family TB has younger, diverse demographic and rapid disease presentation. [ABSTRACT FROM AUTHOR]

Background: Strains of the Beijing sublineage of Mycobacterium tuberculosis have caused large outbreaks of tuberculosis, often involving multidrug resistance strains and this genetically highly conserved family of strains predominates in some geographic areas. For most of the countries of Latin America, no country-wide studies about the prevalence of the Beijing lineage are available. Methods: In this study, we determine the prevalence of the Beijing sublineage in Ecuador, using a large nation-wide sample of 991 isolates from the years 2014–2016 and with the strains, in case-related-proportional representation, emerging from most of the provinces of the country. The isolates were genotyped with a singlenucleotidespecific polymorphism (SNP) polymerase chain reaction for the Beijing sublineage. SNPpositive strains were confirmed as belonging to this lineage with 24 mycobacterial interspersed repetitive unitvariable number of tandem repeat and DNA sequencing. Results: We identified only four Beijing isolates in this collection of 991 strains and calculated a prevalence rate of 0.43%. Conclusions: Our study shows a limited dissemination of the Beijing strains in the Ecuadorian population. This in contrast  with the neighbor countries of Peru and Colombia were locally a prevalence of up to 16% has been reported. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a severe and wide-spread infectious disease worldwide. The modern Beijing subfamily, one lineage of M. tuberculosis , reportedly has high pathogenicity and transmissibility. This study used a molecular epidemiological approach to investigate the transmissibility of the modern Beijing subfamily in the Airin area of Osaka City, Japan. During 2006–2016, we collected 596 M. tuberculosis clinical isolates in the Airin area, Osaka city, Japan. We analyzed the 24-locus variable number of tandem repeats typing optimized for the Beijing family of isolates, M. tuberculosis lineage, and patient epidemiological data. The proportion of the modern Beijing subfamily was significantly higher not only than previously obtained data for the Airin area: it was also higher than the nationwide in Japan. The rate of recent clusters, defined as a variable number of tandem repeats profile identified within two years, of the modern Beijing subfamily was significantly higher than that the rate of recent clusters of the ancient Beijing subfamily. Results suggest that TB control measures formulated with attention to the modern Beijing subfamily might be an important benchmark to understanding recent TB transmission in the area. • Tuberculosis (TB) is one of the top 10 causes of death worldwide. • All countries must use country-specific measures to end TB. • The Airin area of Osaka has the highest TB incidence in Japan. • VNTR typing was used for M. tuberculosis cluster analysis and genetic estimation. • The modern Beijing subfamily showed successful transmission in the Airin area. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (MTB) Beijing strains have caused a great concern because of their rapid emergence and increasing prevalence in worldwide regions. Great efforts have been made to investigate the pathogenic characteristics of Beijing strains such as hypervirulence, drug resistance and favoring transmission. Phylogenetically, MTB Beijing family was divided into modern and ancient sublineages. Modern Beijing strains displayed enhanced virulence and higher prevalence when compared with ancient Beijing strains, but the genetic basis for this difference remains unclear. In this study, by analyzing previously published sequencing data of 1082 MTB Beijing isolates, we determined the genetic changes that were commonly present in modern Beijing strains but absent in ancient Beijing strains. These changes include 44 single-nucleotide polymorphisms (SNPs) and two short genomic deletions. Through bioinformatics analysis, we demonstrated that these genetic changes had high probability of functional effects. For example, 4 genes were frameshifted due to premature stop mutation or genomic deletions, 19 nonsynonymous SNPs located in conservative codons, and there is a significant enrichment in regulatory network for all nonsynonymous mutations. Besides, three SNPs located in promoter regions were verified to alter downstream gene expressions. Our study precisely defined the genetic features of modern Beijing strains and provided interesting clues for future researches to elucidate the mechanisms that underlie this sublineage’s successful expansion. These findings from the analysis of the modern Beijing sublineage could provide us a model to understand the dynamics of pathogenicity of MTB.

Mutations in genes involved in drug metabolism have been well-associated with drug resistance. Sequence analysis of known antimycobacterial drug-resistant genes is often used to predict resistance to antibiotics. However, some polymorphisms in such genes may serve a phylogenetic purpose rather than resistance to drugs. The Beijing family of Mycobacterium tuberculosis (MTB) is prevalent worldwide and has been associated with the emergence of multidrug resistance. Sequence type (ST) 10 of the Beijing family is the most predominant in countries like Peru, Taiwan and Thailand. A sequence analysis was performed of 81 previously reported drug-resistant associated genes in multidrug-resistant and pan-susceptible strains of the Beijing family sequence type 10 of MTB. This analysis revealed 10 synonymous and 12 nonsynonymous single nucleotide polymorphisms (SNPs) that are shared by all strains under study. One frameshift mutation was also observed to be common to all. These data might be useful in excluding some observed SNPs in drug-resistant-associated genes of MTB Beijing ST 10 when performing genotypic drug susceptibility assay.

Strains of the Mycobacterium tuberculosis complex (MTBC) Beijing family aroused concern because they were often found in clusters and appeared to be exceptionally transmissible. However, it was later found that strains of the Beijing family were heterogeneous, and the transmission advantage was restricted to sublineage L2.3 or modern Beijing. In this study, we analyzed the previously published genome sequences of 7,896 L2.3 strains from 51 different countries. Using BEAST software to approximate the temporal emergence of L2.3, our calculations suggest that L2.3 initially emerged in northern East Asia during the early 15th century and subsequently diverged into six phylogenetic clades, identified as L2.3.1 through L2.3.6. Using terminal branch length and genomic clustering as proxies for transmissibility, we found that the six clades displayed distinct population dynamics, with the three recently emerged clades (L2.3.4 to L2.3.6) exhibiting significantly higher transmissibility than the older three clades (L2.3.1 to L2.3.3). Of the Beijing family strains isolated outside East Asia, 83.1% belonged to the clades L2.3.4 to L2.3.6, which were also associated with more cross-border transmission. This work reveals the heterogeneity in sublineage L2.3 and demonstrates that the global success of Beijing family strains is driven by the three recently emerged L2.3 clades. IMPORTANCE The recent population dynamics of the global tuberculosis epidemic are heavily shaped by Mycobacterium tuberculosis complex (MTBC) strains with enhanced transmissibility. The infamous Beijing family strain stands out because it has rapidly spread throughout the world. Identifying the strains responsible for the global expansion and tracing their evolution should help to understand the nature of high transmissibility and develop effective strategies to control transmission. In this study, we found that the L2.3 sublineage diversified into six phylogenetic clades (L2.3.1 to L2.3.6) with various transmission characteristics. Clades L2.3.4 to L2.3.6 exhibited significantly higher transmissibility than clades L2.3.1 to L2.3.3, which helps explain why more than 80% of Beijing family strains collected outside East Asia belong to these three clades. We conclude that the global success of L2.3 was not caused by the entire L2.3 sublineage but rather was due to the rapid expansion of L2.3.4 to L2.3.6. Tracking the transmission of L2.3.4 to L2.3.6 strains can help to formulate targeted TB prevention and control., Competing Interests: The authors declare no conflict of interest.

A high prevalence of tuberculosis (TB) isolates that are genetically homogenous and from the Beijing family has been reported in Russia. To map TB transmission caused by these strains, new genotyping systems are needed. Mycobacterial interspersed repetitive units (MIRUs) offer the possibility of rapid PCR-based typing with comparable discrimination to IS6110 restriction fragment length polymorphism techniques. Spoligotyping and detection of IS6110 insertion in the dnaA-dnaN region were used to identify Beijing strains in 187 Mycobacterium tuberculosis isolates from Samara, Russia. The Beijing isolates were analyzed by using 12-MIRU and 3–exact tandem repeats (ETR) loci and by an expanded set of 10 additional variable number tandem repeats loci. The expanded set of 25 MIRUs provided better discrimination than the original set of 15 (Hunter-Gaston diversity index 0.870 vs 0.625). Loci MIRU 26, 1982, and 3232 were the most polymorphic in Beijing isolates.

Among the most prevalent Mycobacterium tuberculosis (Mtb) strains worldwide is the Beijing genotype, which has caused large outbreaks of tuberculosis (TB). Characteristics facilitating the dissemination of Beijing family strains remain unknown, but they are presumed to have been acquired through evolution of the lineage. To explore the genetic diversity of the Beijing family Mtb and explore the discriminatory ability of mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) loci in several regions of East Asia, a cross-sectional study was conducted with a total of 163 Beijing strains collected from registered TB patients between 1 June 2009 and 31 November 2010 in Funing County, China. The isolated strains were analysed by 15-MIRU-VNTR loci typing and compared with published MIRU-VNTR profiles of Beijing strains. Synonymous single nucleotide polymorphisms at 10 chromosomal positions were also analysed. The combination of SNP and MIRU-VNTR typing may be used to assess Mtb genotypes in areas dominated by Beijing strains. The modern subfamily in Shanghai overlapped with strains from other countries, whereas the ancient subfamily was genetically differentiated across several countries. Modern subfamilies, especially ST10, were prevalent. Qub11b and four other loci (MIRU 26, Mtub21, Qub26, Mtub04) could be used to discriminate Beijing strains. [ABSTRACT FROM AUTHOR]

The majority of tuberculosis (TB) vaccine candidates advanced to clinical trials have been evaluated preclinically using laboratory-adapted strains. However, it has been proposed that challenge with clinical isolates in preclinical vaccine testing could provide further and more practical validation. Here, we tested the ID93/GLA-SE TB vaccine candidate against the clinical Mycobacterium tuberculosis (Mtb) strain K (Mtb K) belonging to the Beijing family, the most prevalent Mtb strain in South Korea. Mice immunized with ID93/GLA-SE exhibited a significant reduction in bacteria and reduced lung inflammation against Mtb K when compared to non-immunized controls. In addition, we analyzed the immune responses in the lungs of ID93/GLA-SE-immunized mice, and showed that ID93/GLA-SE was able to elicit sustained Th1-biased immune responses including antigen-specific multifunctional CD4 + T cell co-producing IFN-γ, TNF-α, and IL-2 as well as a high magnitude of IFN-γ response for up to 10 weeks post-challenge. Notably, further investigation of T cell subsets in the lung following challenge showed remarkable generation of CD8 + central memory T cells by ID93/GLA-SE-immunization. Our findings showed that ID93/GLA-SE vaccine confers a high level of robust protection against the hypervirulent Mtb Beijing infection which was characterized by pulmonary Th1-polarized T-cell immune responses. These findings may also provide relevant information for potential utility of this vaccine candidate in East-Asian countries where the Beijing genotype is highly prevalent. [ABSTRACT FROM AUTHOR]

Background: The ability of Mycobacterium tuberculosis to establish a latent infection (LTBI) in humans confounds the treatment of tuberculosis. Consequently, there is a need to discover new therapeutic agents that can kill M. tuberculosis both during active disease and LTBI. The streptomycin-dependent strain of M. tuberculosis, 18b, provides a useful tool for this purpose since upon removal of streptomycin (STR) it enters a non-replicating state that mimics latency both in vitro and in animal models. Results: The 4.41 Mb genome sequence of M. tuberculosis 18b was determined and this revealed the strain to belong to clade 3 of the ancient ancestral lineage of the Beijing family. STR-dependence was attributable to insertion of a single cytosine in the 530 loop of the 16S rRNA and to a single amino acid insertion in the N-terminal domain of initiation factor 3. RNA-seq was used to understand the genetic programme activated upon STR-withdrawal and hence to gain insight into LTBI. This revealed reconfiguration of gene expression and metabolic pathways showing strong similarities between non-replicating 18b and M. tuberculosis residing within macrophages, and with the core stationary phase and microaerophilic responses. Conclusion: The findings of this investigation confirm the validity of 18b as a model for LTBI, and provide insight into both the evolution of tubercle bacilli and the functioning of the ribosome. [ABSTRACT FROM AUTHOR]

Summary Background Highly lethal outbreaks of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are increasing. Mycobacterium tuberculosis variant Beijing family and its members is regarded as a successful clone of M. tuberculosis that is associated with drug resistance in China. Understanding the genetic characteristics and molecular mechanism of drug resistant tuberculosis within Beijing family may help to clarify its origin and evolutionary history and the driving forces behind its emergence and current dissemination. Methods Totally of 1222 Mycobacterium tuberculosis isolates were recovered from patients in six counties of two provinces in eastern China within 2010/2012. Strain lineage and its major subgroups were studied respectively by using Spoligotyping and MIRU-VNTR. The 1 st -line drug susceptibility was analyzed by proportional method and 2 nd -line drug susceptibility was determined by the HAINs MTBDRsl test. The genetic characterization of drug resistance was analyzed by sequencing the previously reported genes and loci associated with drug resistance together with the multiple genotyping including MIRU-VNTR, Spoligotyping and LSP genotyping. Results Of the 1222 Mtb isolates, 298 (24.4%) were resistant to 1 st -line drug and 73 (5.9%) were simultaneously resistant to INH and RIF namely MDR-TB. Respectively 23.8% of 1 st -line drug resistant TB and 12.0% of the drug susceptible TB contained the mutation associated with 2 nd -line drugs by HAINs test. The Spoligotyping of 1222 Mtb isolates revealed the 967 (79.1%) of the isolates belonged to the W-Beijing family. Within W-Beijing family, 78.8% MDR-TB were observed in the isolates with simultaneous deletion of RD105 and RD207, with sub-lineage 181 accounting for 75% of MDR-TB. Analysis of 24 MIRU-VNTR loci revealed that 88.2% (15/17) of MDR and extensively drug resistant (XDR) clustered isolates were sub-lineage 181. Conclusions Sublineage 181 might have the capacity to spread throughout the general community in rural China. This is the first report on the extensive association of sub-lineage 181 with MDR TB and possibly pre-XDR TB and XDR TB. It is important to monitor sublineage 181 to verify its heightened transmission and understand its importance in the global MDR–TB and XDR–TB epidemics. [ABSTRACT FROM AUTHOR]

Molecular epidemiologic studies of strains of Mycobacterium tuberculosis are currently conducted worldwide. The genetically distinct Beijing family of strains has been associated with large outbreaks of tuberculosis, increased virulence, and multidrug resistance. However, in this first population-based search for Beijing strains in the Danish DNA fingerprint database, analysis of 97% of all culture-positive tuberculosis patients in 1992 to 2001, showed that 2.5% of 3,844 patients, 1.0% of Danish-born patients and 3.6% of immigrants (from 85 countries) had Beijing strains. No Beijing strains were found among 201 strains from Danish-born patients sampled in the 1960s, and no evidence of an increase in Beijing strains was found over time. The true prevalence of Beijing strains worldwide is unknown because only a fraction of global strains have been analyzed.

Strains of the Beijing/W genotype family of Mycobacterium tuberculosis have caused large outbreaks of tuberculosis, sometimes involving multidrug resistance. This genetically highly conserved family of M. tuberculosis strains predominates in some geographic areas. We have conducted a systematic review of the published reports on these strains to determine their worldwide distribution, spread, and association with drug resistance. Sixteen studies reported prevalence of Beijing strains defined by spoligotyping; another 10 used other definitions. Beijing strains were most prevalent in Asia but were found worldwide. Associations with drug resistance varied: in New York, Cuba, Estonia, and Vietnam, Beijing strains were strongly associated with drug resistance, but elsewhere the association was weak or absent. Although few reports have measured trends in prevalence, the ubiquity of the Beijing strains and their frequent association with outbreaks and drug resistance underline their importance.

Mutations in genes involved in drug metabolism have been well-associated with drug resistance. Sequence analysis of known antimycobacterial drug-resistant genes is often used to predict resistance to antibiotics. However, some polymorphisms in such genes may serve a phylogenetic purpose rather than resistance to drugs. The Beijing family of Mycobacterium tuberculosis (MTB) is prevalent worldwide and has been associated with the emergence of multidrug resistance. Sequence type (ST) 10 of the Beijing family is the most predominant in countries like Peru, Taiwan and Thailand. A sequence analysis was performed of 81 previously reported drug-resistant associated genes in multidrug-resistant and pan-susceptible strains of the Beijing family sequence type 10 of MTB . This analysis revealed 10 synonymous and 12 nonsynonymous single nucleotide polymorphisms (SNPs) that are shared by all strains under study. One frameshift mutation was also observed to be common to all. These data might be useful in excluding some observed SNPs in drug-resistant-associated genes of MTB Beijing ST 10 when performing genotypic drug susceptibility assay. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis Beijing family includes a variety of sublineages. Knowledge of the distribution of a certain sublineage of the Beijing family may help to understand the mechanisms of its rapid spread and to establish an association between a certain genotype and the disease outcome. We have previously found that M. tuberculosis Beijing family clinical isolates represent approximately 90% of the clinical isolates from Heilongjiang Province, China. To clarify the distribution of M. tuberculosis Beijing family sublineages in Heilongjiang Province, China and to investigate the regularity rule for their evolution, we examined single nucleotide polymorphisms (SNPs) of 250 M. tuberculosis Beijing family clinical isolates using 10 SNP loci that have been identified as appropriate for defining Beijing sublineages. After determining the sequence type (ST) of each isolate, the sublineages of all M. tuberculosis Beijing family isolates were determined, and phylogenetic analysis was performed. We found that 9 out of the 10 SNP loci displayed polymorphisms, but locus 1548149 did not. In total, 92.8% of the isolates in Heilongjiang Province are modern sublineages. ST10 is the most prevalent sublineage (ST10 and ST22 accounted for 63.2% and 23.6% of all the Beijing family isolates, respectively). A new ST, accounting for 4% of the Beijing family isolates in this area, was found for the first time. Each new ST isolate showed a unique VNTR pattern, and none were clustered. The present findings suggest that controlling the spread of these modern sublineages is important in Heilongjiang Province and in China. [ABSTRACT FROM AUTHOR]

Highlights: [•] The Beijing family of M. tuberculosis is an emerging lineage in the area of Milan. [•] Beijing strains affect mainly young foreign-born patients. [•] Beijing strains are associated with MDR, but not with HIV infection. [•] Constant prevalence over time of the founder Beijing sublineages. [Copyright &y& Elsevier]

Background: The genotypes of Mycobacterium tuberculosis (MTB) have been found to be related to the risk of transmission and the development of drug resistance of this pathogen. Thus, exploring the molecular characteristics of MTB is helpful for understanding and controlling the spread of strains in areas with a high incidence of tuberculosis. Methods: We recruited 512 sputum smear-positive tuberculosis patients from 30 counties from 1 April to 30 June 2010; 503 MTB strains were isolated and 497 were successfully genotyped. We genotyped the strains based on a new 15-locus mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) method in combination with spacer-oligonucleotide typing (spoligotyping) technology. Results: Based on spoligotyping, 487 strains displayed known patterns, and 10 were absent from the current global spoligotyping database (SpolDB4). The predominant spoligotypes belonged to the Beijing or Beijing-like family (81.1%). When we used the new 15-locus (MIRU-15) set for the MIRU-VNTR analysis, 388 different patterns were identified, including 46 clusters and 342 unique patterns. The combination of spoligotyping and MIRU-15 demonstrated a high discriminatory power. The proportion of clusters varied significantly between the Beijing and non-Beijing family strains, but no significant association was observed between multidrug resistance and Beijing family strains. Conclusions: The present study demonstrated that the Beijing family strains are the most prevalent in rural China. Spoligotyping in combination with the new MIRU-15 technique is useful for the epidemiological analysis of MTB transmission and could be used as a first-line method for the large-scale genotyping of MTB. [ABSTRACT FROM AUTHOR]

Abstract: Genotypic classification in Mycobacterium tuberculosis has greatly contributed to the comprehension of phylogenetic and population genetic relationships. It is, therefore, necessary to verify the robustness of the genetic markers for phylogenetic classification. In this study, we report some examples of homoplasy for two molecular markers, the IS6110 insertion at the NTF region, and a single nucleotide polymorphism (SNP) at locus 909166, through genotyping of 1054 Beijing family strains. Our data revealed that a small fraction of strains traditionally classified into modern sublineages by IS6110 insertion at NTF actually belong to an ancient sublineage. We also proved that the robustness of branches in the evolutionary tree established using the putative homoplasious SNP 909166 is relatively low. Our findings highlight the importance of validating genetic markers used to establish phylogeny, evolution, and phenotypic characteristics. [Copyright &y& Elsevier]

Abstract: The Beijing family is an endemic lineage of Mycobacterium tuberculosis in eastern Asia. In Japan, five evolutionarily sequential sublineages composing the lineage have predominated. Comparative genomic sequencing based on a microarray technique was conducted for five representative strains of those respective sublineages. Results revealed approximately 200 point mutations specific to each strain. Subsequently, to investigate the genetic diversity of each sublineage, we analysed the phylogenetic divergence of 103 domestic strains belonging to them using genetic markers derived from the mutation information. Results show that the five sublineages have comprised smaller lineages which had diverged at various points. The smaller sub-sublineages have emerged with respective bottlenecks, which are reflected in the excessive monophyletic evolution of the species. Our data provide necessary information to grasp a comprehensive picture of genetic diversity of the lineage constructed in its evolution. [Copyright &y& Elsevier]

Background: Tuberculosis is one of the most infectious diseases in the world. Molecular typing methods such as spoligotyping, and VNTR (variable number tandem repeats), IS6110 in the NTF region and LSP (large sequence polymorphisms) analysis are generally useful tools for the resolution of various issues related to the classical epidemiology of Mycobacterium tuberculosis (M. tuberculosis). Methods: To determine the transmission characteristics of M. tuberculosis strains isolated in Beijing, China, and their genetic relationships, especially those among Beijing family strains, 260 M. tuberculosis strains isolated from patients presenting pulmonary tuberculosis were analyzed by spoligotyping, and by examining 22 VNTR loci and the presence/absence of IS6110 in the NTF region, RD105 and RD181. Results: 81% (211 strains) of the isolates studied were Beijing family strains, 174 (82.5%) of which were identified as modern Beijing strains based on the presence of IS6110 upstream of the NTF region. RD181 was intact in 9 of the other 37 (17.5%) ancestral Beijing strains. The percentage of Beijing family strains in this study was consistent with previous reports. There are many differences, however, in allele diversity among VNTR loci between reports on strains from different areas. Conclusions: The Beijing family is the most prevalent genotype in Beijing city and the predominance of Beijing family strains has not altered in almost twenty years. Differences in the alleles and discrimination ability of VNTR loci between different regions is likely due to population differences in the regions where these M. tuberculosis strains were isolated or to differences in sampling times. [ABSTRACT FROM AUTHOR]

Objective: Tuberculosis remains a severe public health issue, and the Beijing family of mycobacterium tuberculosis (M. tuberculosis) is widespread in East Asia, especially in some areas in China, like Beijing and Tianjin. This study aimed at determining the mutation patterns of drug-resistant Beijing strains of M. tuberculosis isolated from Tianjin, China. Methods: A total of 822 M. tuberculosis isolates were screened for drug resistance by an absolute concentration method and the genotype was identified by PCR. 169 drug-resistant isolates of the Beijing family were analyzed for the potential mutations in the rpoB, katG, inhA promoter region and in rpsL, rrs and embB genes, which are associated with resistance to rifampin (RFP), isoniazid (INH), streptomycin (SM) and ethambutol (EMB) respectively by PCR and DNA sequencing. Results: Fifty-eight out of 63 RFP-resistant isolates were found to carry the mutations within the 81-bp RFP resistance determining region (RRDR) of the rpoB gene and the most frequent mutations occurred at codon 531 (44.4%), 526 (28.6%), and 516 (7.9%) respectively. 16 mutation patterns affecting 12 different codons around the RRDR of rpoB were found. Of 116 INH-resistant isolates, 56 (48.3%) had the mutation of katG 315 (AGC→ACC) (Ser→Thr), 3 (2.6%) carried S315N (AGC→AAC) and 27 (16.0%) had the mutation of inhA–15A→T. 84 out of 122 SM-resistant isolates (68.9%) displayed mutations at the codons 43 or 88 with AAG→AGG (Lys→Arg) of the rpsL gene and 22 (18.0%) with the mutations at positions 513A→C, 516C→T or 905 A→G in the rrs gene. Of 34 EMB-resistant isolates, 6 had mutation with M306V (ATG→GTG), 3 with M306I (ATG→ATT), 1 with M306I (ATG→ATA), 1 with D328Y (GAT→TAT), 1 with V348L (GTC→CTC), and 1 with G406S (GGC→AGC) in the embB gene. Conclusion: These novel findings extended our understanding of resistance-related mutations in the Beijing strains of M. tuberculosis and may provide a scientific basis for development of new strategies for diagnosis and control of tuberculosis in China and other countries where Beijing strains are prevalent. [Copyright &y& Elsevier]

Abstract: The Beijing family is the putative hypervirulent lineage of Mycobacterium tuberculosis that has been endemic in East Asia and has disseminated worldwide. The genetic population structure of Beijing family strains with regard to Japan is notable in its high diversity and dominance of the ancestral sublineage, in contrast to the modern sublineage found worldwide. Therefore, it is expected to be a suitable population model for investigating the microevolutionary process of the lineage. Variable number of tandem repeats (VNTR) has become a reliable genotyping method for M. tuberculosis, but its dynamics in the phylogenetic process remains unclear. Using 355 clinical Beijing family isolates in Japan, genetic traits, including VNTR, were analyzed and subjected to minimum spanning tree (MST) reconstruction. In the results, the topology of the tree was tightly related to other genotypic characters. We also found that some VNTR alleles were specific to each sublineage and provided clues for reconstructing a valid MST topology for the population. It is suggested that VNTR typing can elucidate genetic markers representing the phylogenetic classification in the lineage. [Copyright &y& Elsevier]

Summary: A population-based study of Mycobacterium tuberculosis isolated from homeless tuberculosis patients was performed during 2002–2004 in Osaka City, Japan. The data show that the ancient Beijing subfamily was predominant, whereas clustered isolates based on refined variable number of tandem repeats genotyping (19 loci) mainly belonged to the modern Beijing subfamily, suggesting its increased transmissibility. [Copyright &y& Elsevier]

The Beijing/W family is the endemic lineage of Mycobacterium tuberculosis in East Asia: it has disseminated worldwide. To elucidate its genetic diversity in Japan, phylogenetic reconstruction was performed using 403 M. tuberculosis Beijing family clinical isolates. Variable number of tandem repeats analysis revealed the strains from Japan to be dispersed mainly among five subgroups in a phylogenetic tree. Interestingly, the genotypes of the strains from China and Mongolia were restricted mainly to a single branch; they exhibited high clonality. IS 6110 insertion in the NTF region was also analyzed. The majority (78.6%) of Japanese isolates belonged to the ancient sublineage. The modern Beijing strains were observed to correspond to the branch containing the foreign strains, although the ancient Beijing strains were dispersed among the tree's other branches. Our results reflect the singular genetic diversity and the epidemiological pattern of Beijing M. tuberculosis in Japan. [ABSTRACT FROM AUTHOR]

Variable number tandem repeat (VNTR) typing of Mycobacterium tuberculosis, as presently used, has often failed to differentiate clonal strains especially in areas where the Beijing family genotype is predominant. We have evaluated mycobacterial interspersed repetitive units (MIRUs), Queen's University of Belfast (QUB) loci and exact tandem repeat (ETR) loci individually for their abilities to differentiate the Beijing and non-Beijing genotype families of M. tuberculosis. The best locus was QUB 3232 followed by QUB 11b. Available MIRU, QUB and ETR loci were then consecutively arranged in declining order of discriminatory power, and combined to form an optimal set of 12-loci VNTR typing (MIRU 10, 26, 39, 40; QUB 11a, 11b, 15, 18, 26, 3232, 3336; ETR A) that possessed comparable discriminatory ability to IS 6110 restriction fragment length polymorphism. This optimized 12-loci VNTR typing set can become an important tool for tracking M. tuberculosis of the Beijing family. [ABSTRACT FROM AUTHOR]

Abstract: The frequency of resistance genotypes among Beijing and non-Beijing strains was compared using a reverse blot hybridization assay to detect mutations within genes associated with rifampicin (rpoB) and isoniazid (katG, inhA, and ahpC) resistance. Of the 743 Mycobacterium tuberculosis isolates, 569 (77%) belonged to Beijing family. The proportion of Beijing strains was significantly higher among MDR-TB isolates than among drug-susceptible strains (82% vs. 72%, p <0.01). Genotype analysis of the rpoB gene revealed significantly lower rates of the Ser531Leu mutation rate among Beijing vs. non-Beijing MDR-TB strains (41% vs. 66%, p <0.005). While the mutation for Ser315Thr in the katG gene was more common among Beijing vs. non-Beijing family strains (65% vs. 50%, p <0.01), the mutation rate of promoter region of the inhA gene was lower among Beijing strains compared with non-Beijing strains (14% vs. 25%, p <0.05). Reverse hybridization successfully detected over 80% of isoniazid-resistant strains and over 92% of rifampicin-resistant strains among Korean isolates. Significant differences in mutation rates in the rpoB, katG, and inhA genes between Beijing strains and non-Beijing strains could explain discrepancies in mutation rates of genotypes in different countries. Reverse hybridization was useful for rapid detection of isoniazid and rifampicin resistant strains. [Copyright &y& Elsevier]

A selection of genetic markers was used to study the evolution of Mycobacterium tuberculosis Beijing family strains in northwestern Russia. A total of 221 of 434 epidemiologically unlinked isolates studied in 1996–2001 belonged to the Beijing family as determined by standard spoligotyping (signals 35–43). Ninety-six percent of these Beijing isolates (“typical”) were closely related in IS6110-RFLP (D > 0.85) while 9 remaining isolates (2 different profiles, “atypical”) were more distant from the rest (D = 0.6–0.7). Further analysis was performed on a selection of 12 typical and both atypical Beijing strains with different IS6110-RFLP profiles (2 isolates each). All 28 Beijing isolates studied had the KatG 463Leu allele, an intact mtp40 fragment of the mpcA gene, and an identical structure of the DR locus (15 DVRs) with an upstream IS6110 copy in opposite orientation. The IS6110-RFLP based neighbor-joining (distance) and quartet-puzzling (maximum-likelihood) trees showed that the branch lengths were considerably longer for atypical Beijing strains. Typical Beijing strains had the 1.02 kb Rv3135 PPE-family gene and two IS1547 copies (iplA and iplB), one of them (iplB) disrupted by IS6110 insertion. Atypical Beijing strains had the 1.97 kb Rv3135 gene and a single intact IS1547/iplA copy. We suggest that the M. tuberculosis Beijing family strains currently circulating in the northwest of Russia are relatively ancient and thus appear to be endemic in this region since evolutionarily distant time. The prevalent typical Beijing strains (96%) are likely to be of monophyletic origin and their ongoing dissemination has started recently: these strains differ in rapidly evolving IS6110-RFLP but have identical structure of other polymorphic genome regions studied. The atypical Beijing strains (4%) are evolutionary older; they probably had a common (unknown) ancestor with typical Beijing strains. [Copyright &y& Elsevier]

Abstract Background Tuberculosis is one of the most infectious diseases in the world. Molecular typing methods such as spoligotyping, and VNTR (variable number tandem repeats), IS6110 in the NTF region and LSP (large sequence polymorphisms) analysis are generally useful tools for the resolution of various issues related to the classical epidemiology of Mycobacterium tuberculosis (M. tuberculosis). Methods To determine the transmission characteristics of M. tuberculosis strains isolated in Beijing, China, and their genetic relationships, especially those among Beijing family strains, 260 M. tuberculosis strains isolated from patients presenting pulmonary tuberculosis were analyzed by spoligotyping, and by examining 22 VNTR loci and the presence/absence of IS6110 in the NTF region, RD105 and RD181. Results 81% (211 strains) of the isolates studied were Beijing family strains, 174 (82.5%) of which were identified as modern Beijing strains based on the presence of IS6110 upstream of the NTF region. RD181 was intact in 9 of the other 37 (17.5%) ancestral Beijing strains. The percentage of Beijing family strains in this study was consistent with previous reports. There are many differences, however, in allele diversity among VNTR loci between reports on strains from different areas. Conclusions The Beijing family is the most prevalent genotype in Beijing city and the predominance of Beijing family strains has not altered in almost twenty years. Differences in the alleles and discrimination ability of VNTR loci between different regions is likely due to population differences in the regions where these M. tuberculosis strains were isolated or to differences in sampling times.