Beijing Genotype Research Articles

Screening of compounds containing aminobutanol and camphane moieties against mycobacterium tuberculosis clinical isolates of different genotypes

Aim and objectives: Despite the availability of sufficiently effective treatment regimens, prevention and control, the widespread transmission and prevalence of resistant variants of Mycobacterium tuberculosis, which do not respond to any of the commercial drugs, remains a serious health problem for both developed and developing countries. Multidrug-resistant (MDR) strains present new threat to health security. This situation is of global concern due to increased human mobility, partly illegal and spontaneous. Patients infected with drug resistant strains are hard or impossible to be cured, and their treatment is more toxic and expensive. The urgent need of new antimycobacterial agents and development pathways is becoming more and more apparent. Methods: In this study, we synthesized new compounds containing 2-aminobutanol and camphane moieties and evaluated them with the reference strain and clinical M. tuberculosis isolates from a high-burden country, Russian Federation. Results: The isolates represented different clusters within clinically/epidemiologically important Beijing genotype (B0/W148, 94-32, Central Asian Outbreak, ancestral sublineage) and differed in phenotypic drug resistance (one extensively drug resistant, six multidrug resistant, two polyresistant, and two susceptible to all tested drugs). The most efficient compound showed a wide range of MIC that varied from 3.1 μg/ml (H37Rv, ancestral Beijing, most of B0/W148 isolates) to 100 μg/ml (other isolates). Conclusions: To conclude, this study highlighted a key importance of testing new anti-TB compounds not only with reference laboratory strain H37Rv that belongs to the phylogenetically marginal branch of M. tuberculosis, but on the clinical drug-resistant isolates circulating in the countries with high burden of MDR-TB. Acknowledgements. This study was supported by the Russian Science Foundation (grant 19-15-00028). Partial support was received from the Bulgarian National Science Fund (grant KP-06-H39-7).

Genotyping and Molecular Characterization of Fluoroquinolone's Resistance Among Multidrug-Resistant Mycobacterium tuberculosis in Southwest of China.

Although fluoroquinolones (FQs) are the backbone drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB), the knowledge about the resistance pattern and molecular characterization of new-generation FQs in Chongqing is limited. This study aimed to investigate the resistance rate and mutation types of later-generation FQs against MDR-TB in Chongqing, and further to explore the relationship between different genotypes and phenotypes. A total of 967 clinical strains were characterized using multilocus sequence typing and drug susceptibility testing, followed by analysis of genotype/phenotype association. The 229 (23.7%, 229/967) isolates were identified as MDR-TB. The most effective agent against MDR-TB was gatifloxacin (GFX) (20.1%, 46/229), and the highest resistant rate was observed in ofloxacin (OFX) (41.0%, 94/229). Of the 190 strains (83.0%) identified as Beijing genotype, 111 isolates were modern Beijing genotype (58.4%) and 79 isolates were ancient Beijing genotype (41.6%). By analyzing 94 OFX-resistant isolates, 13 isolates were clustered with the cumulative clustering rate of 13.8% (13/94). Of the 91 isolates (39.7%, 91/229) with a mutation in gyrA gene, mutation in codon 94 was the most prevalent. Only 15 isolates (6.6%, 15/229) harbored a mutation in gyrB gene. There was no significant difference in the mutation rate of gyrA gene between Beijing and non-Beijing genotype, clustered isolates, and nonclustered isolates (p > 0.05).

Socio-demographic associations of Mycobacterium tuberculosis molecular genotypes in Hong Kong

Background: Different Mycobacterium tuberculosis (MTB) genotypes are circulating in the population, the transmission patterns of which may vary. We studied the association between MTB molecular genotypes and socio-demographics in Hong Kong, a city with intermediate TB burden (60.5/100,000 persons). Methods and materials: MTB clinical isolates and hospital data of patients with positive sputum culture were collected from one major hospital cluster region in Hong Kong between 2015–2016. Residential addresses of patients were geocoded and aggregated in administrative units for mapping. Polymerase chain reaction test was performed for genotype classification. In logistic regression models, sociodemographic characteristics were compared between Beijing and non-Beijing genotype, and the latter's association with drug resistance were examined. Results: Out of 383 unique isolates collected, 263(69%) were from male, with 192 (50%) aged ≥60 years, 42 (11%) born locally, 53 (14%) permanent residents, 24 (6%) resided in elderly homes, and 319 (83%) living in 67 (89%) administrative units within the hospital cluster boundary. Twenty-nine strains (8%) were resistant to one of the four first line anti-TB drugs, while 14 (4%) were resistant to more than one. Overall, 286 (75%) belonged to Beijing genotype family, 52 (14%) to non-Beijing genotype, and 44 (11%) undetermined. Isolates of male patients (OR = 2.25), born in Hong Kong (OR = 6.4, born outside Hong Kong as reference), and permanent residents (OR = 4.18) were more likely to belong to Beijing genotype. However, smoking, alcoholic, drug abuse, co-morbidities and drug resistance were not significantly different between Beijing and non-Beijing genotypes. Correlation between the proportion of Beijing genotype isolates and proportion of male general population in corresponding administrative units was marginally significant (correlation coefficient = 0.23, p = 0.058). In sensitivity analysis combining undetermined with non-Beijing genotype, male gender and local residency were more likely to be associated with Beijing genotype. Single drug resistance (OR = 4.42, no resistance as reference) was more likely while multidrug (OR = 0.34) and isoniazid resistance (OR = 0.35) were less likely with Beijing genotype. Conclusion: In Hong Kong, Beijing genotype was the predominant lineage, which was characterised by higher proportion of male gender and local residents. Consistent with previous studies in China, Beijing genotype was not significantly associated with drug resistance.

Genotyping of Mycobacterium tuberculosis in Liaoning with variable number of tandem repeats method

Objective To explore the genotype distribution and transmission patterns of 81 Mycobacterium tuberculosis (MTB) clinical isolates from Liaoning province with the variable number tandem repeats (VNTR). Methods The 81 strains were isolated from each city of the province in 2018, the p-nitrobenzoic acid (PNB) was used to identify the flora, so as to distinguish Mycobacterium tuberculosis complex from non-tuberculous mycobacteria. The solid ratio method was used to conduct drug susceptibility tests on four first-line anti-tuberculosis drugs using Roche's medium. Based on PCR, the standard 24 sites were detected and analyzed, and the Beijing genotype was identified by the RD105 gene deletion method. Microsoft Excel software was used for polymorphism analysis, and website ( https://www.miru-vntrplus.org/MIRU/index ) was used to get the VNTR genotyping results, the locus polymorphism and clustering rate at the same time was calculated. SPSS 23.0 software was used for statistical description of data, and χ 2 test carried out on count data or drug resistance in each year; when the theoretical frequency is less than 5, the Fisher exact probability method is used to calculate the two-sided P value, and the test level is α=0.05. Results The clustering analysis of genotype showed that 81 strains were categorized into 3 gene clusters (genogroup I, II and III), in which 1.23% (1/81) was belong to genogroup I, 4.94% (4/81) was genogroup II, and 93.83% (76/81), one of the largest group of genes for III group. The clustering rate was 4.94% (4/81) and the least estimated proportion of the recent infection rate was 2.47% (2/81). The polymorphism of 5 in 24 VNTR loci was better, 3 loci was acceptable, the remaining 16 loci was less polymorphic. Conclusion The drug resistant Mycobacterium tuberculosis shows obvious gene polymorphisms in Liaoning province and Beijing genotype is the predominant prevalent strain (III group). 摘要 ：目的了解辽宁省81例结核分枝杆菌多位点数目可变串联重复序列分析(variable number of tandem repeats. VNTR)的基因多态性及近期传播情况。 方法 对2018年辽宁省内送至辽宁省疾病预防控制中心实验室的菌株进行传 代后米用对硝基苯甲酸(p-nitrobenzok: acid, PNB)培养基进行菌群鉴定, 以区分结核分枝杆菌复合体和非结核分枝杆 菌。采用固体比例法利用罗氏培养基对4种一线抗结核药物进行药敏实验。以PCR为基础对标准24位点进行检测分 析, 利用RD105基因缺失法鉴定北京基因型。利用Microsoft Excel软件进行多态性分析, 利用 https://www.miru-vntrplus.org/MIRU/index 网站得到VNT R基因分型结果, 同时进行位点多态性和成簇率的计算。采用SPSS 23.0软件进行数据的 统计描述 , 对计数资料或各年度耐药情况进行 χ 2 检验；当理论频数<5时 , 采用Fisher确切概率法计算双侧 P 值, 检验水 准α=0.05。 结果 经分析81株菌株可分为3个基因群（I、Ⅱ、Ⅲ), 菌株数分别为1(1.23%)、4(4.94%)和76(93.83%), 其中最大的基因群为Ⅲ群。成簇率为4.94%(4/81)。近期感染率最小估计值为2.47%(2/81)。24个VNTR位点中有5 个位点的多态性较好, 3个位点的多态性尚可, 16个位点的多态性较差。 结论 初步证实辽宁省耐药结核分枝杆菌菌 株存在明显的基因多态性, 其主要流行型为北京基因型（Ⅲ群）。

Probable long-term prevalence for a predominant Mycobacterium tuberculosis clone of a Beijing genotype in Colon, Panama.

Beijing genotype Mycobacterium tuberculosis strains associate with increased virulence, resistance and/or higher transmission rates. This study describes a specific Beijing strain predominantly identified in the Panamanian province of Colon with one of the highest incidences of tuberculosis in the country. Retrospective mycobacterial interspersed repetitive unit/variable number of tandem repeats analysis of 42 isolates collected between January and August 2018 allowed to identify a cluster (Beijing A) with 17 (40.5%) Beijing isolates. Subsequent prospective strain-specific PCR-based surveillance from September 2019 to March 2020 confirmed the predominance of the Beijing A strain (44.1%) in this province. Whole-genome sequencing revealed higher-than-expected diversity within the cluster, suggesting long-term prevalence of this strain and low number of cases caused by recent transmission. The Beijing A strain belongs to the Asian African 3 (Bmyc13, L2.2.5) branch of the modern Beijing sublineage, with their closest isolates corresponding to cases from Vietnam, probably introduced in Panama between 2000 and 2012.

Detection of Beijing strains of MDR M. tuberculosis and their association with drug resistance mutations in katG, rpoB, and embB genes

BackgroundMolecular epidemiological studies of Mycobacterium tuberculosis (MTB) are the core of current research to find out the association of the M. tuberculosis genotypes with its outbreak and transmission. The high prevalence of the Beijing genotype strain among multidrug resistance (MDR) TB has already been reported in various studies around India. The overall objective of this study was to detect the prevalence of Beijing genotype strains of MDR M. tuberculosis and their association with the clinical characteristics of TB patients.MethodsIn this study 381 M. tuberculosis clinical isolates were obtained from sputum samples from 2008 to 2014. The multiplex-PCR and Spoligotyping (n = 131) methods were used to investigate the prevalence of the Beijing genotype strain by targeting the Rv2820 gene and their association with drug resistance and clinical characteristics of TB patients. The drug susceptibility testing of first-line anti-TB drugs was performed by using the proportion method and MGIT960. A collection of isolates having Beijing and non-Beijing strains were also characterized to see if Beijing genotype strains had a higher rate of mutations at codons 516, 526 and 531 of the 81-bp region of the rpoB gene, codon 315 of the katG gene, and codon 306 of the embB gene.ResultsThe sensitivities and specificities of multiplex-PCR assay compared to that of standard Spoligotyping was detected to be 100%. Further, we observe that the multi drug-resistance was significantly associated with Beijing genotype strains (p = 0.03) and a strong correlation between Beijing genotype strains and specific resistance mutations at the katG315, rpoB531, and embB306 codons (p = < 0.0001, < 0.0001 & 0.0014 respectively) was also found.ConclusionsThis rapid, simple, and cost-effective multiplex PCR assay can effectively be used for monitoring the prevalence of Beijing genotype strains in low resource settings. Findings of this study may provide a scientific basis for the development of new diagnostic tools for detection and effective management of DR-TB in countries with a higher incidence rate of Beijing genotype strains.

Assessment of the Prevalence and Epidemic Spread of Strains of Ancient, and Modern Sublineages of the Mycobacterium tuberculosis Beijing Genotype in Omsk Region

Relevance. The spread of the epidemic and resistant Mycobacterium tuberculosis genotypes presents a serious threat to the implementation of the national tuberculosis control program in Russia. The study aimed to assess the trends in the development of the epidemic process of tuberculosis and the prevalence of strains of different sublineages and clusters of the M. tuberculosis Beijing genotype in the Omsk region. Materials & Methods. Following the generally accepted algorithm of descriptive and evaluation epidemiological research, the incidence, prevalence, and mortality from tuberculosis in the Omsk region for the period 2009-2019 were studied. The structure of the M. tuberculosis population in the Omsk region was studied (n = 483). M. tuberculosis culture, drug susceptibility testing, DNA extraction were carried out by standard methods. The M. tuberculosis Beijing genotype and its sublineages and clusters B0/W148 and 94-32 were detected based on the PCR analysis of specific markers. Results. The incidence of tuberculosis decreased by 1.9 times and was 67.3 per100 ths in 2019. The dynamics of tuberculosis prevalence and mortality were also characterized by a pronounced decreasing trend. 65.6% (n = 317) of the isolates belonged to the Beijing genotype, which was represented by strains of ancient (n = 44, 13.9%) and modern (n = 273, 86.1%) sublineages. The latter one included two main clusters: B0/W148 (n = 94, 29.6%) and 94-32 (n = 178, 56.2%). Almost all ancient Beijing strains were MDR (97.7%), and were characterized by extensive drug resistance (XDR) and pre-XDR (52.2% versus 19.4% in modern sublineage (PR = 0.59; [95% CI 0.35÷0, 99]). The high level of clustering of strains of ancient sublineage (CR = 0.84) allows us to consider them as potential epidemics for the Omsk region. Conclusions. Despite the general improvement of the epidemiological situation with tuberculosis in the Omsk region, the circulation of MDR strains of the Beijing B0/W148 cluster and pre-XDR/XDR strains of an ancient sublineage of the Beijing genotype points to the epidemic potential of these M. tuberculosis genovariants and emphasizes the need for their careful monitoring in the epidemiological surveillance system.

Spatiotemporal dynamics of drug-resistant Mycobacterium tuberculosis: Contrasting trends and implications for tuberculosis control in EU high-priority country.

Different and contrasting trends related to human migration and the implementation of health control programmes influence the spread of drug-resistant tuberculosis (TB). We analysed the Mycobacterium tuberculosis population structure in Estonia, a high-priority EU country for TB control, to detect the dynamic changes and underlying factors. The study collection included 278 M.tuberculosis isolates recovered in 1999 and 2014-2015. The isolates were subjected to drug susceptibility testing, genotyping and analysis of sublineage/cluster-specific markers and drug resistance mutations. The Beijing genotype was the most prevalent, and its rate increased from 28.6% in 1999 to 38.5% in 2015 (p=.09). The non-Beijing strains represented Euro-American lineage (Latin American Mediterranean [LAM], Ural, Haarlem, T, X genotypes) and Indo-Oceanic lineage (one EAI-IND isolate). The proportion of isolates resistant to two or more drugs increased from 22.4% to 29.1% (p=.1). The pre-XDR/XDR isolates were identified only within the Beijing genotype. In contrast, the drug resistance rate decreased in the LAM genotype from 42.1% to 11.8% (p=.05). The Beijing B0/W148-cluster ('successful Russian strain') included only MDR, pre-XDR or XDR isolates. All B0/W148-cluster isolates were resistant to two or more drugs compared to 28% of the Beijing 94-32-cluster (p=.0002). The Beijing genotype was not identified in the isolates from patients born in Estonia before 1940 compared to its 35.2% rate among other patients. In summary, the circulation of the highly drug-resistant isolates of the Beijing B0/W148 subtype, the increased prevalence of the Beijing genotype among HIV-coinfected patients and the increased number of patients with alcohol abuse (47.5%) present major challenges of the current TB control in Estonia. The Beijing genotype was likely brought to Estonia after 1945 due to the massive human influx from the Soviet Union. In contrast, the main genotypes of the Euro-American lineage were likely endemic in Estonia during all 20th century.

Molecular analysis of streptomycin-resistance associating genes in Mycobacterium tuberculosis isolates from Nepal

Mutation in rpsL (encoding ribosomal protein S12), rrs (encoding 16S ribosomal RNA) and gidB (encoding 7-methylguanosine methyltransferase) are associated with resistance to streptomycin (STR), which is used for the treatment of multi-drug resistant tuberculosis (MDR-TB) in Nepal. The aim of our study is to analyze the correlation between mutations in the target genes and STR-resistance in 197 Mycobacterium tuberculosis (MTB) isolates from Nepal. Mutations in rpsL was harbored by 65.9% of isolates, in which the most common mutation in rpsL is caused by K43R (58.8%) and were significantly associated with Beijing genotype (P < 0.001). About 13.2% of isolates harbored mutations in two highly mutable regions of rrs, the 530 loop and the 912 region. About 13.2% of gidB mutants do not show any mutation in rpsL and rrs, which might suggest the role of gidB mutations in STR-resistance in MTB. In addition, 5.6% of isolates do not show any mutations in three genes examined, suggesting the involvement of other mechanism in STR-resistance in MTB. Our findings can be implemented for the establishment of molecular STR-susceptibility testing, in which tuberculosis can be treated with appropriate drugs and can improve control strategies for DR-TB.

Genomic Analysis Identifies Mutations Concerning Drug-Resistance and Beijing Genotype in Multidrug-Resistant Mycobacterium tuberculosis Isolated From China

Development of modern genomics provides us an effective method to understand the molecular mechanism of drug resistance and diagnose drug-resistant Mycobacterium tuberculosis. In this study, mutations in 18 genes or intergenic regions acquired by whole-genome sequencing (WGS) of 183 clinical M. tuberculosis strains, including 137 multidrug-resistant and 46 pan-susceptible isolates from China, were identified and used to analyze their associations with resistance of isoniazid, rifampin, ethambutol, and streptomycin. Using the proportional method as the gold standard method, the accuracy values of WGS to predict resistance were calculated. The association between synonymous or lineage definition mutations with different genotypes were also analyzed. The results show that, compared to the phenotypic proportional method, the sensitivity and specificity of WGS for resistance detection were 94.2 and 100.0% for rifampicin (based on mutations in rpoB), 90.5 and 97.8% for isoniazid (katG), 83.0 and 97.8% for streptomycin (rpsL combined with rrs 530 loop and 912 loop), and 90.9 and 65.1% for ethambutol (embB), respectively. WGS data also showed that mutations in the inhA promoter increased only 2.2% sensitivity for INH based on mutations in katG. Synonymous mutation rpoB A1075A was confirmed to be associated with the Beijing genotype. This study confirmed that mutations in rpoB, katG, rrs 530 loop and 912 loop, and rpsL were excellent biomarkers for predicting rifampicin, isoniazid, and streptomycin resistance, respectively, and provided clues in clarifying the drug-resistance mechanism of M. tuberculosis isolates from China.

Molecular epidemiology of multidrug resistant tuberculosis in Mongolia and Eastern Siberia: two independent dissemination processes for dominant strains

Mongolia and Russia are among the countries with the high tuberculosis (TB) burden. The prevalence of tuberculosis, including multidrug-resistant tuberculosis (MDR), in Eastern Siberia bordering Mongolia is significantly higher than in the European part of Russia. In addition, unlike Mongolia, Eastern Siberia is characterized by a high prevalence of HIV infection. The cross-border spread of socially significant infections in these countries seems to occur due to their wide-range cooperation and cultural exchange. Whereas the HIV infection has no epidemiological significance for Mongolia at the moment, tuberculosis, however, has a similar prevalence on both sides of the border. The aim was to evaluate the cross-border MDR M. tuberculosis distribution in Mongolia and Eastern Siberia by using molecular genetic data. Materials and methods. A total of 1045 M. tuberculosis strains isolated in Mongolia (291) and the three regions of Eastern Siberia (754) were studied by using the MIRU-VNTR-24 loci genotyping. The CC2/W148 and CC1 subtypes were identified by the specific deletion in the kdpD gene and SNP in the pks17 gene at position 1887060, respectively. Phylogenetic analysis of MIRU-VNTR patterns was carried out by generating UPGMA tree and maximum likelihood tree. Results. The Beijing genotype was found in 75.3% (219/291) and 69.0% (520/754) from Mongolian and East Siberian collection, respectively. Common minor genotypes were LAM (11.0% and 15.1%), T (10.3% and 4.5%), and Haarlem (1.4% and 2.4%) found in Mongolia and Eastern Siberia, respectively. The genotypes S (1.3%) and Ural (5.0%) were found solely in the Russia-derived samples. The main epidemic Beijing subtypes in each country belonged to different clonal complexes (CC): the majority of Mongolian Beijing strains displayed profiles 342-32, 3819-32, 1773-32 MLVA types and belonged to the CC4 subtype; Russian Beijing strains mainly belonged to the CC1 (43.7% — 227/520) and CC2/W148 (34.8% — 181/520) subtypes. The MDR level and distribution patterns differed significantly between Mongolia and Eastern Siberia. Modeling of Beijing strain expansion evidences about extremely subtle contribution of the M. tuberculosis cross-border transmission between Mongolia and Russia. The phylogenetic reconstruction of Beijing CC4 subtype evolution in Mongolia suggests that its distribution is primarily associated with China and other countries of the Western Pacific Region. Three main phylogenetic branches of CC4 subtype were traced, which probably spread throughout Mongolia in the 11—12th centuries. It may be assumed that spreading of the epidemic Beijing CC4 subtype might occur in two stages: early period — emergence of ancestral CC4 variants in Mongolia or their introduction from China (they are homologous to the strains preserved in the Chinese population), later period — dissemination due to the active exchange of M. tuberculosis with countries of Southeast Asia, but not Russia. Conclusion. Using MIRU-VNTR-24 genotyping as well as classification according to specific single nucleotide polymorphisms specific to certain Beijing subtypes, it allowed to describe separate patterns of the epidemic variants spread in Mongolia and Russia. It has been demonstrated that emergence and spread of MDR-TB in Mongolia are entirely iatrogenic in nature, while the epidemic subtypes of the Beijing genotype (subtypes CC1 and CC2/W148) contribute markedly into the MDR-TB spreading in Eastern Siberia.

Comparison of two molecular diagnostic methods for identifying Beijing genotype of Mycobacterium tuberculosis

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. 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. 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. 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.

Genomic signatures of drug resistance in highly resistant Mycobacterium tuberculosis strains of the early ancient sublineage of Beijing genotype in Russia

The Mycobacterium tuberculosis Beijing genotype is a clinically and epidemiologically important lineage that is subdivided into ancient/ancestral and modern strains. In our previous study in western Siberia, we identified variable number of tandem repeats (VNTR)-based clusters within the early ancient sublineage of the Beijing genotype characterized by an unexpectedly high rate of extensive drug resistance (XDR). In the current study, next generation sequencing data were analysed to gain insight into genomic signatures underlying drug resistance of these strains. A total of 184 genomes of the Beijing early ancient sublineage from Russia (16), China (15), Japan (36), Korea (25), Vietnam (18), Thailand (73), and the USA (1) were used for phylogenetic analysis. The drug-resistant profile was deduced genotypically. The Russian isolates were distributed into two clusters and were all drug resistant, mainly pre-XDR and XDR. The largest of these clusters included only Russian isolates from remote locations in both Asian and European parts of the country. All its isolates had a quadruple drug resistance (to isoniazid, rifampin, ethambutol and streptomycin) due to the 6-mutation signature (KatG Ser315Thr, KatG Ile335Val, RpoB Ser450Leu, RpoC Asp485Asn, EmbB Gln497Arg, and RpsL Lys43Arg). In most samples, it was complemented with additional and different pncA, gyrA and rrs mutations leading to the pre-XDR/XDR genotype. Phylogenomic analysis indicates a distant origin of this Russian resistant cluster in the early 1970s but location and circumstances are yet to be clarified.

Evidence for the Effect of Vaccination on Host-Pathogen Interactions in a Murine Model of Pulmonary Tuberculosis by Mycobacterium tuberculosis.

The global control of Tuberculosis remains elusive, and Bacillus Calmette-Guérin (BCG) -the most widely used vaccine in history—has proven insufficient for reversing this epidemic. Several authors have suggested that the mass presence of vaccinated hosts might have affected the Mycobacterium tuberculosis (MTB) population structure, and this could in turn be reflected in a prevalence of strains with higher ability to circumvent BCG-induced immunity, such as the recent Beijing genotype. The effect of vaccination on vaccine-escape variants has been well-documented in several bacterial pathogens; however the effect of the interaction between MTB strains and vaccinated hosts has never been previously described. In this study we show for the first time the interaction between MTB Beijing-genotype strains and BCG-vaccinated hosts. Using a well-controlled murine model of progressive pulmonary tuberculosis, we vaccinated BALB/c mice with two different sub-strains of BCG (BCG-Phipps and BCG-Vietnam). Following vaccination, the mice were infected with either one of three selected MTB strains. Strains were selected based on lineage, and included two Beijing-family clinical isolates (strains 46 and 48) and a well-characterized laboratory strain (H37Rv). Two months after infection, mice were euthanized and the bacteria extracted from their lungs. We characterized the genomic composite of the bacteria before and after exposure to vaccinated hosts, and also characterized the local response to the bacteria by sequencing the lung transcriptome in animals during the infection. Results from this study show that the interaction within the lungs of the vaccinated hosts results in the selection of higher-virulence bacteria, specifically for the Beijing genotype strains 46 and 48. After exposure to the BCG-induced immune response, strains 46 and 48 acquire genomic mutations associated with several virulence factors. As a result, the bacteria collected from these vaccinated hosts have an increased ability for immune evasion, as shown in both the host transcriptome and the histopathology studies, and replicates far more efficiently compared to bacteria collected from unvaccinated hosts or to the original-stock strain. Further research is warranted to ascertain the pathways associated with the genomic alterations. However, our results highlight novel host-pathogen interactions induced by exposure of MTB to BCG vaccinated hosts.

Analysis on drug sensitivity spectrum of 167 multidrug-resistant Mycobacterium tuberculosis in China

Objective: To investigate the drugs-sensitivity spectrum of multidrug-resistant tuberculosis (MDR-TB) in China and provide a scientific evidence for the drug selection in clinical therapy and the control of MDR-TB. Methods: A total of 167 strains of MDR-TB were included in this study. Every strain was genotyped by lysX gene sequencing and their sensitivity to 13 different anti-TB drugs was tested by using MicroDST(TM) and BACTEC(TM) MGIT 960(TM) liquid-culturing method. The association between drug resistance and genotypes as well as cross drug resistance was also analyzed. The results were analyzed by means of the comparison of enumeration data between two groups with χ(2) test. Results: The overall resistance rate of 167 MDR-TB strains to 11 anti-TB drugs, except isoniazide and rifampicin, was 95.81%, the rates of pre-extensive drug-resistance (pre-XDR) and extensive drug-resistance were 31.14%(52/167) and 6.59% (11/167), respectively. The streptomycin resistance rate of Beijing genotypes was significantly higher than that of the non-Beijing genotypes ( χ(2)=30.682, P<0.05), while the pre-XDR proportion in Beijing genotypes was lower than that in non-Beijing genotypes (χ(2)=5.332, P<0.05). The resistance rates of Ofloxacin and Pyrazinamide in the modern Beijing genotype were significantly higher than those in classical ones (χ(2)=4.105 and χ(2)=3.912, P<0.05). In addition, the cross-resistance rate to rifampicin and rifabutin was 86.23%. A significant difference in drug-resistance rate to rifabutin was seen among groups with different levels of rifampicin resistance (χ(2)=45.912, P<0.05). There was positive correlation not only between ofloxac resistance and moxifloxac resistance, but also between amikacin resistance and kanamycin resistance, with the coefficient of 0.87 and 0.91, respectively. Conclusions: In this study, we observed that there were high incidences of the resistance to 11 anti-TB drugs in 167 clinical MDR-TB strains and the cross resistance phenomena between drugs of the same type were quite serious. The majority of MDR-TB strains belonged to Beijing genotype, which was highly associated with streptomycin resistance.

Analysis on drug resistance-associated mutations of multi-drug resistant Mycobacterium tuberculosis based on whole-genome sequencing in China

Objective: To analyze the resistance mutational profiles of multi-drug resistant Mycobacterium tuberculosis in China and the correlation between major mutation types and genotypes based on the whole-genome sequencing data. Methods: Search and download of the genome-wide sequencing data of M. tuberculosis published in China by August 2019 on NCBI database were conducted. Mutation frequency of drug resistance-related gene loci based on whole-genome sequencing was used to predict the molecular susceptibility of strains, and the correlation between mutation types and genotypes was analyzed. Results: According to the results of molecular resistance and susceptibility profiles, 1 024 MDR strains were identified from 2 019 M. tuberculosis strains. The major mutation types of resistance-related genes to common drugs were katG S315T (73.2%, isoniazid), rpoB S450L (63.1%, rifampicin), rpsL K43R (70.0%, streptomycin), embB M306V (37.4%, ethambutol), pncA_promoter T (-11)C (7.9%, pyrazinamide), gyrA A90V (32.3%, fluoroquinolones), rrs A1401G (67.7%, second-line injection drugs), fabG1_promoter C (-15) T (87.0%, Ethionamide), folC I43T (30.4%, P-aminosalicylic acid). Among them, the frequencies of katG S315T, embB M306V, rpsL K43R, gyrA A90V in lineage 2 were significantly higher than those in lineage 4, and folC I43T was only found in lineage 2. The proportion of katG S315T was significantly higher in the ancient Beijing genotype compared to the modern genotype, in contrast, the proportion of rpsL K43R was significantly higher in modern Beijing genotype, the differences were significant (all P<0.05). Conclusions: The results showed the main mutation types of resistance-related genes of MDR strains to many commonly used anti-tuberculosis drugs in China based on whole-genome sequencing, providing a basis for the development of sensitive and specific rapid molecular detection methods. At the same time, it was also found that the major mutation types of MDR-related genes were related to the genotype of the strains.

Genotyping, Assessment of Virulence and Antibacterial Resistance of the Rostov Strain of Mycobacterium tuberculosis Attributed to the Central Asia Outbreak Clade.

The Central Asia Outbreak (CAO) clade is a growing public health problem for Central Asian countries. Members of the clade belong to the narrow branch of the Mycobacterium tuberculosis Beijing genotype and are characterized by multidrug resistance and increased transmissibility. The Rostov strain of M. tuberculosis isolated in Russia and attributed to the CAO clade based on PCR-assay and whole genome sequencing and the laboratory strain H37Rv were selected to evaluate the virulence on C57Bl/6 mice models by intravenous injection. All mice infected with the Rostov strain succumbed to death within a 48-day period, while more than half of the mice infected by the H37Rv strain survived within a 90-day period. Mice weight analysis revealed irreversible and severe depletion of animals infected with the Rostov strain compared to H37Rv. The histological investigation of lung and liver tissues of mice on the 30th day after injection of mycobacterial bacilli showed that the pattern of pathological changes generated by two strains were different. Moreover, bacterial load in the liver and lungs was higher for the Rostov strain infection. In conclusion, our data demonstrate that the drug-resistant Rostov strain exhibits a highly virulent phenotype which can be partly explained by the CAO-specific mutations.

Study on the prevalence of Beijing genotype Mycobacterium tuberculosis and its relationship with second-line anti-tuberculosis drug resistance

Objective: To explore the prevalence risk factors of Beijing genotype Mycobacterium tuberculosis (MTB) in Beijing and its correlation with second-line anti tuberculosis drug resistance. Methods: A total of 1 140 clinical MTB positive strains were collected from various districts in Beijing, and the drug sensitivity was detected by proportion method. Beijing genotype and non Beijing genotype MTB were identified by the method of Spoligotyping. Using SPSS 22.0 statistical software, chi square test or Fisher exact probability test was used to analyze the experimental data. Results: Among 1 140 MTB clinical isolates, 941 (82.5%) were Beijing genotype MTB, 199 were non Beijing genotype MTB. There were 663 males (70.5%) in Beijing genotype and 124 males (62.3%) in non Beijing genotype strains. There were significant differences in the proportion of males between the two genotypes [P=0.021, OR (95% CI):1.442 (1.048-1.985)]. There were 441 floating population (46.9%) in Beijing genotype MTB and 78 floating population (39.2%) in non Beijing genotype MTB. There was a significant difference in the proportion of floating population between the two genotypes [P=0.048,OR (95%CI):1.368(1.001-1.869)]. There were 129 patients (13.7%) aged 65 or older in Beijing genotype MTB, 40 patients (20.1%) aged 65 or older in non Beijing genotype MTB. The difference was statistically significant [P=0.021, or (95% CI): 0.631 (0.426-0.936)]. The resistance drug rates of Levofloxacin (Lfx), Amikacin (Am), Capreomycin (Cm), Para-aminosalicylic (PAS) in Beijing genotypes were 5.5% (52/941), 1.3% (12/941), 3.2% (30/941) and 3.0% (28/941), respectively, and those of non Beijing genotypes were 10.6% (21/199), 8.5% (17/199, 12.6% (25/199) and 11.6% (23/199), the difference was statistically significant (all P<0.05). There were 58 (6.2%) multidrug-resistant (MDR) strains in Beijing genotype MTB and 19 (9.5%) multidrug-resistant strains in non Beijing genotype. There was no significant difference in the proportion of MDR strains between Beijing genotype and non Beijing genotype (P>0.05). Conclusions: Beijing genotype MTB is widespread in Beijing and has a higher proportion in male population and floating population. Compared with non Beijing genotype, Beijing genotype MTB has a lower resistance rate to Lfx, Am, Cm and PAS, and there is no significant difference in the proportion of MDR-TB patients between the two genotypes.

INFLUENCE OF GENOTYPIC VARIABILITY OF M. TUBERCULOSIS ON THE COURSE OF TUBERCULOSIS WITH MULTIPLE DRUG RESISTANCE

Tuberculosis is infectious and socially dependent disease, being now one of the most pressing issues in practical health care. As well the usual types of tuberculosis infection, chemoresistant tuberculosis is spreading rapidly in the world. The WHO estimates that about 500,000 people on the planet are infected with M. tuberculosis, which is resistant to standard anti−tuberculosis drugs. The probability of successful treatment decreases with emergence of new genotypes of M. tuberculosis with total resistance. In the modern epidemiology of tuberculosis, it is important to identify genotypes on certain signs, allowing to address issues such as their origin, identification of the infection source, possible routes and factors of transmission, as well as to reveal cases and spread of resistance to anti−tuberculosis drugs. To evaluate the therapy efficiency of multidrug−resistant tuberculosis patients with revealed genotypic variability during treatment, 10 patients with chemoresistant pulmonary tuberculosis having M. tuberculosis genotypic variability were treated. In these patients, the clinical, laboratory and radiological dynamics of disease in intensive phase of treatment were studied. Analysis of treatment results for patients with chemoresistant tuberculosis with genotypic variability of M. tuberculosis was evaluated by the intoxication syndrome dynamics of, the timing of closure of the decay cavities and cessation of bacterial excretion. The study found that the genotypic variability of M. tuberculosis is characterized by the change of less virulent genotypes of M. tuberculosis to more virulent. Signs of intoxication have been shown to change from less virulent M. tuberculosis genotypes to M. tuberculosis Beijing genotypes. Genotypic variability of mycobacteria in hospital suggests that hospitalization in tuberculosis facilities is a risk of exogenous tuberculosis superinfection. Studying the influence of genotypic variability of M. tuberculosis on the course of multidrug−resistant tuberculosis requires more extensive research, being a very relevant and promising area in phthisiology. Key words: Mycobacterium tuberculosis, genotypic variability, VNTR−genotyping, treatment.

A landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic

BackgroundAtypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations.MethodsWe conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants.ResultsPhylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role.ConclusionThe presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.