Genomic Analysis of Mycobacterium tuberculosis Isolates and Construction of a Beijing Lineage Reference Genome.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, kills over 1 million people worldwide annually. Development of drug resistance (DR) in the pathogen is a major challenge for TB control. We conducted whole-genome analysis of seven Taiwan M. tuberculosis isolates: One drug susceptible (DS) and five DR Beijing lineage isolates and one DR Euro-American lineage isolate. Developing a new method for DR mutation identification and applying it to the next-generation sequencing (NGS) data from the 6 Beijing lineage isolates, we identified 13 known and 6 candidate DR mutations and provided experimental support for 4 of them. We assembled the genomes of one DS and two DR Beijing lineage isolates and the Euro-American lineage isolate using NGS data. Moreover, using both PacBio and NGS sequencing data, we obtained a high-quality assembly of an extensive DR Beijing lineage isolate. Comparative analysis of these five newly assembled genomes and two published complete genomes revealed a large number of genetic changes, including gene gains and losses, indels and translocations, suggesting rapid evolution of M. tuberculosis. We found the MazEF toxin-antitoxin system in all the seven isolates studied and several interesting mutations in MazEF proteins. Finally, we used the four assembled Beijing lineage genomes to construct a high-quality Beijing lineage reference genome that is DS and contains all the genes in the four genomes. It contains 212 genes not found in the standard reference H37Rv, which is Euro-American. It is therefore a better reference than H37Rv for the Beijing lineage, the predominant lineage in Asia.

Effect of ventilation improvement during a tuberculosis outbreak in underventilated university buildings.

The role of ventilation in preventing tuberculosis (TB) transmission has been widely proposed in infection control guidance. However, conclusive evidence is lacking. Modeling suggested the threshold of ventilation rate to reduce effective reproductive ratio (ratio between new secondary infectious cases and source cases) of TB to below 1 is corresponding to a carbon dioxide (CO2 ) level of 1000 parts per million (ppm). Here, we measured the effect of improving ventilation rate on a TB outbreak involving 27 TB cases and 1665 contacts in underventilated university buildings. Ventilation engineering decreased the maximum CO2 levels from 3204 ± 50 ppm to 591-603 ppm. Thereafter, the secondary attack rate of new contacts in university dropped to zero (mean follow-up duration: 5.9 years). Exposure to source TB cases under CO2 >1000 ppm indoor environment was a significant risk factor for contacts to become new infectious TB cases (P < .001). After adjusting for effects of contact investigation and latent TB infection treatment, improving ventilation rate to levels with CO2 <1000 ppm was independently associated with a 97% decrease (95% CI: 50%-99.9%) in the incidence of TB among contacts. These results show that maintaining adequate indoor ventilation could be a highly effective strategy for controlling TB outbreaks.

Using genotyping to delineate tuberculosis transmission in long-term care facilities: single facility 4-year experience.

BACKGROUND: Residents in long-term care facilities (LTCFs) are vulnerable to tuberculosis (TB) transmission; however, to delineate possible routes of TB transmission in LTCFs is difficult. This study aimed to address the use of regular genotyping surveillance to delineate TB transmission in LTCFs. METHODS: All of Mycobacterium tuberculosis isolates in the reported 620-bed LTCF between July 2011 and August 2015 were genotyped, and we retrospectively compared epidemiological data and genotyping results. RESULTS: A total of 42 subjects were diagnosed with culture-positive pulmonary TB infection during the 4-year period. Their median age was 76.5 years, and 64.3% (27/42) of them were male. Genotyping identified 5 clustered TB infections involving 76.2% (32/42) of all TB subjects. In a multivariate logistic regression model adjusted for age, sex, chronic obstructive pulmonary disease, and body mass index, subjects with clustered TB infection were less likely to be Activities of Daily Living (ADL)-dependence (adjOR 0.073, 95% CI 0.007-0.758) when compared with subjects having individual TB infections. Prolonged surveillance is essential given that the median interval to diagnose secondary subjects was 673 days. Finally, only 63.0% (17/27) of the 27 secondary TB subjects in this study had contact history with index subject in the same ward. CONCLUSIONS: In conclusion, possible routes of TB transmission in a complex TB outbreak at LTCFs might be delineated by routine genotyping surveillance and regular health check-up.

European 1: a globally important clonal complex of Mycobacterium bovis.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.

Characteristics of multidrug-resistant Mycobacterium tuberculosis in Taiwan: a population-based study.

A population-based study was performed to characterize the genotype and drug-resistant patterns of multidrug-resistant tuberculosis (MDR-TB) in Taiwan. From 2007 to 2008, we analyzed 494 MDR Mycobacterium tuberculosis complex isolates using spacer oligonucleotide typing and drug susceptibility testing. The majority of cases occurred in the age groups of 45-54 (24.3%) and ≥65 (23.1%). Of the 494 MDR isolates, 25.1% were resistant to ethambutol, 15.6% were resistant to streptomycin, 27.1% were resistant to all four first-line anti-tuberculosis drugs, 28.9% were resistant to ofloxacin, and 8.7% were extensively drug-resistant (XDR). Compared with the SpolDB4, 86 spoligotypes were identified in 492 isolates. We observed 427 (86.8%) isolates belonging to 49 known spoligotypes and 65 isolates (13.2%) in 37 undesignated spoligotypes. Beijing lineages (50.0%) were the predominant genotype, followed by Haarlem (18.2%) and East-African-Indian (EAI) (5.7%). Geographically, Beijing lineages were predominant in all regions, whereas Haarlem lineages were predominant only in the east (28.1%) and EAI (11.3%) only in the south. Beijing lineages are statistically associated with MDR in younger age groups and eastern Taiwan. Furthermore, we found that Beijing ST1 (46.1%), Haarlem3 ST50 (7.1%) and ST742 (4.7%), and EAI2_MANILA ST19 (3.9%) were the prevalent groups. Thus, continuous surveillance with more thorough genotyping and epidemiological investigation is crucial for the prevention of further dissemination, the determination of the temporal and spatial trends of multi-drug resistance, and the emergence of XDR-TB in Taiwan.

Single-nucleotide polymorphism in the fadD28 gene as a genetic marker for East Asia Lineage Mycobacterium tuberculosis.

Mycobacterium tuberculosis isolates with a region of difference 105 (RD105) deletion, mainly Beijing family spoligotypes, were phylogenetically grouped into the East Asia lineage. We identified a single nucleotide polymorphism in codon 507, ATC to ATT, of the fadD28 gene as a robust marker and developed a rapid assay for East Asia lineage M. tuberculosis.

Single nucleotide polymorphisms in cell wall biosynthesis-associated genes and phylogeny of Mycobacterium tuberculosis lineages.

To investigate specific single nucleotide polymorphisms (SNPs) of different lineages of Mycobacterium tuberculosis, cell wall biosynthesis-associated genes encoding antigen 85 complex (fbpA, fbpB, and fbpC) and mannosyltransferase (pimB) were analyzed. Genetically diversified and predominant M. tuberculosis and Mycobacterium bovis genotypes identified in Taiwan (26 Beijing and 44 non-Beijing) were included in the study. Sequence analyses revealed that nine novel SNPs were found within main lineages of M. tuberculosis complex, including East-African-Indian (EAI), Beijing, Central-Asian (CAS), Bovis, and one lineage containing Latin American and Mediterranean (LAM), Haarlem and T. Specifically, a SNP in pimB codon 270 was identified in EAI, fbpA codon 156 in ancestral Beijing, fbpB codon 238 in modern Beijing, fbpA codon 4 and fbpC codon 158 in CAS, fbpA codon 311 in M. bovis and an additional SNP in fbpB codon 140 in M. bovis-BCG, and pimB codon 107 in other spoligotypes lineages including an additional SNP in fbpC codon 103 in LAM. In addition, we proved that isolates with spoligotype shared type (ST) no. 523 (carrying all 43 spacers), designated as unknown lineage in an international spoligotyping database (SpolDB4), belong to an early ancestral Beijing sublineage. Accordingly, a phylogenetic tree was constructed using those SNPs, and an evolutionary hypothesis for lineages of M. tuberculosis was proposed. These novel lineage-specific SNPs will be informative genetic markers in molecular epidemiological and evolutionary studies of M. tuberculosis.

Characterization of a glycine N-methyltransferase gene knockout mouse model for hepatocellular carcinoma: Implications of the gender disparity in liver cancer susceptibility.

Hepatocellular carcinoma (HCC) is the fifth common cancer in the world and it mainly occurs in men. Glycine N-methyltransferase (GNMT) participates in one-carbon metabolism and affects DNA methylation by regulating the ratio of S-adenosylmethionine to S-adenosylhomocystine. Previously, we described that the expression of GNMT was diminished in human HCC. Here, we showed that 50% (3/6) male and 100% (7/7) female Gnmt-/- mice developed HCC, and their mean ages of HCC development were 17 and 16.5 months, respectively. In addition, 42.9% (3/7) of female Gnmt-/- mice had hemangioma. Wnt reporter assay demonstrated that Gnmt is a negative regulator for canonical Wnt signaling pathway. Beta-catenin, cyclin D1 and c-Myc, genes related to Wnt pathway, were upregulated in the liver tissues from both 11 weeks and HCC stage of Gnmt-/- mice. Furthermore, global DNA hypomethylation and aberrant expression of DNA methyltransferases 1 and 3b were found in the early and late stages of HCC development. Hierarchical cluster analysis of 6,023 transcripts from microarray data found that gene expression patterns of HCC tumors from male and female Gnmt-/- mice were distinctively different. Real-time PCR confirmed that Gadd45a, Pak1, Mapk3 and Dsup3 genes of mitogen-activated protein kinase (MAPK) pathway were activated in Gnmt-/- mice, especially in the female mice. Therefore, GNMT is a tumor suppressor gene for liver cancer, and it is associated with gender disparity in liver cancer susceptibility.

Spoligotypes of Mycobacterium tuberculosis isolates of a high tuberculosis burden aboriginal township in Taiwan.

To understand the genotype of Mycobacterium tuberculosis isolates circulating in the aboriginal Sioulin Township, the highest tuberculosis (TB) endemic area in Taiwan, a total of 138 isolates were collected between January 2003 and December 2004 for genotyping. Genotyping consisted of spacer oligonucleotide typing (spoligotyping), IS6110-restriction fragment length polymorphism (RFLP) and mgtC and ogt single nucleotide polymorphisms (SNPs) characterizations. Spoligotyping data were compared with those from the fourth international spoligotyping database, SpolDB4. Of 27 resolved spoligotypes, 14 spoligotype patterns matched those found in SpolDB4 and 13 (TW1-13) were identified as novel. The most common among the 14 defined spoligotypes was Beijing ST1 (35.5%, 49/138), followed by Haarlem ST742 (10.9%, 15/138), Latin-American-Mediterranean (LAM) ST33 (5.8%, 8/138) and Haarlem ST50 (3.6%, 5/138). Of the 13 novel spoligotypes, 5 (TW 6-8, 12 and 13) were identified as "Haarlem-like" lineages according to clade analyses of spoligotyping and RFLP dendrograms. Overall, major spoligotypes found in Sioulin Township were Haarlem and Haarlem-like (39.1%), Beijing (38.4%), and LAM (5.8%) lineages. Interestingly, the results did not indicate any East-African-Indian lineages, which are highly prevalent in Far-East Asia. Our data also contained the first evidence of ST33 (LAM3 lineage) in Asia. This study provides first depiction of molecular epidemiology of M. tuberculosis in this isolated aboriginal population and further elucidation of the global historical expansion of the isolates.

Transmission of Mycobacterium tuberculosis in a family proved by genotyping.

BACKGROUND/PURPOSE: Molecular genetic methods have been applied in various epidemiologic studies including investigations of disease acquisition by contact. This report describes the use of various molecular genetic methods in tracing possible household transmission of tuberculosis by contact. METHODS: Four Mycobacterium tuberculosis strains, each from four members of a family, were first isolated and identified in the clinical laboratory of the Chest Hospital and were submitted to the National Reference Laboratory of Mycobacteriology for further confirmation and genotyping. In this study, IS6110 restriction fragment length polymorphism (RFLP), spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR), and rpoB gene sequencing were used for genotyping. RESULTS: All four strains were found to have identical spoligotypes, MIRU-VNTR patterns, and similar IS6110 RFLP profiles. The results of the drug susceptibility test and of rpoB sequencing showed that all four strains were rifampicin resistant. CONCLUSION: Household transmission through close contact was thus proved by genotyping. We conclude that all four family members were infected with the same lineage of M. tuberculosis.