Tuberculous Spondylitis in Russia and Prominent Role of Multidrug-Resistant Clone Mycobacterium tuberculosis Beijing B0/W148

ABSTRACTExtrapulmonary and, in particular, spinal tuberculosis (TB) constitutes a minor but significant part of the total TB incidence. In spite of this, almost no studies on the genetic diversity and drug resistance ofMycobacterium tuberculosisisolates from spinal TB patients have been published to date. Here, we report results of the first Russian and globally largest molecular study ofM. tuberculosisisolates recovered from patients with tuberculous spondylitis (TBS). The majority of 107 isolates were assigned to the Beijing genotype (n= 80); the other main families were T (n= 11), Ural (n= 7), and LAM (n= 4). Multidrug resistance (MDR) was more frequently found among Beijing (90.5%) and, intriguingly, Ural (71.4%) isolates than other genotypes (5%;P< 0.001). The extremely drug-resistant (XDR) phenotype was exclusively found in the Beijing isolates (n= 7). A notable prevalence of therpoB531andkatG315mutations in Beijing strains that were similarly high in both TBS (this study) and published pulmonary TB (PTB) samples from Russia shows that TBS and PTB Beijing strains follow the same paradigm of acquisition of rifampin (RIF) and isoniazid (INH) resistance. The 24-locus mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) subtyping of 80 Beijing isolates further discriminated them into 24 types (Hunter Gaston index [HGI] = 0.83); types 100-32 and 94-32 represented the largest groups. A genotype of Russian successful clone B0/W148 was identified in 30 of 80 Beijing isolates. In conclusion, this study highlighted a crucial impact of the Beijing genotype and the especially prominent role of its MDR-associated successful clone B0/W148 cluster in the development of spinal MDR-TB in Russian patients.

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Compensatory Mutations of Rifampin Resistance Are Associated with Transmission of Multidrug-Resistant Mycobacterium tuberculosis Beijing Genotype Strains in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02358-15 ◽

2016 ◽

Vol 60 (5) ◽

pp. 2807-2812 ◽

Cited By ~ 23

Author(s):

Qin-jing Li ◽

Wei-wei Jiao ◽

Qing-qin Yin ◽

Fang Xu ◽

Jie-qiong Li ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Variable Number Tandem Repeat ◽

Northern China ◽

Multidrug Resistant ◽

Variable Number ◽

Beijing Genotype ◽

Content Type ◽

Resistant Tuberculosis ◽

Rifampin Resistance

ABSTRACTMycobacterium tuberculosiscan acquire resistance to rifampin (RIF) through mutations in therpoBgene. This is usually accompanied by a fitness cost, which, however, can be mitigated by secondary mutations in therpoAorrpoCgene. This study aimed to identifyrpoAandrpoCmutations in clinicalM. tuberculosisisolates in northern China in order to clarify their role in the transmission of drug-resistant tuberculosis (TB). The study collection included 332 RIF-resistant and 178 RIF-susceptible isolates. The majority of isolates belonged to the Beijing genotype (95.3%, 486/510 isolates), and no mutation was found inrpoAorrpoCof the non-Beijing genotype strains. Among the Beijing genotype strains, 27.8% (89/320) of RIF-resistant isolates harbored nonsynonymous mutations in therpoA(n= 6) orrpoC(n= 83) gene. The proportion ofrpoCmutations was significantly higher in new cases (P= 0.023) and in strains with therpoBS531L mutation (P< 0.001). In addition, multidrug-resistant (MDR) strains withrpoCmutations were significantly associated with 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat clustering (P= 0.016). In summary, we believe that these findings indirectly suggest an epistatic interaction of particular mutations related to RIF resistance and strain fitness and, consequently, the role of such mutations in the spread of MDRM. tuberculosisstrains.

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Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage, Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil

Journal of Clinical Microbiology ◽

10.1128/jcm.02012-15 ◽

2015 ◽

Vol 53 (12) ◽

pp. 3805-3811 ◽

Cited By ~ 10

Author(s):

Elis R. Dalla Costa ◽

Sidra E. G. Vasconcelos ◽

Leonardo S. Esteves ◽

Harrison M. Gomes ◽

Lia L. Gomes ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Latin American ◽

Variable Number Tandem Repeat ◽

Multidrug Resistant ◽

Variable Number ◽

Compensatory Mutation ◽

Single Nucleotide ◽

Content Type ◽

A Site ◽

South Brazil

We recently detected the spoligotype patterns of strains ofMycobacterium pinnipedii, a species of theMycobacterium tuberculosiscomplex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9,pks15/1, RDRio, andfbpC, the insertion of IS6110at a site specific to theM. tuberculosisLatin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in factM. tuberculosisand more specifically belong to the LAM genotype. Most of these isolates (n= 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of therpoA,rpoB,rpoC,katG, andinhAgenes. Seven isolates (77.8%) carried the S315T mutation inkatG, and one of these (11%) also presented the C(−17)T single-nucleotide polymorphism (SNP) ininhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 ofrpoB. No putative compensatory mutation was found in eitherrpoAorrpoC. This is the first report of anM. tuberculosisLAM family strain with a convergentM. pinnipediispoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in theM. tuberculosiscomplex on the basis of single genetic markers.

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Prediction of Local Transmission of Mycobacterium tuberculosis Isolates of a Predominantly Beijing Lineage by Use of a Variable-Number Tandem-Repeat Typing Method Incorporating a Consensus Set of Hypervariable Loci

Journal of Clinical Microbiology ◽

10.1128/jcm.01016-17 ◽

2017 ◽

Vol 56 (1) ◽

Author(s):

Yoshiro Murase ◽

Kiyohiko Izumi ◽

Akihiro Ohkado ◽

Akio Aono ◽

Kinuyo Chikamatsu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Tandem Repeat ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Trend Test ◽

Typing Method ◽

Content Type ◽

Tuberculosis Transmission ◽

High Prevalence ◽

Hypervariable Loci

ABSTRACT Strain genotyping based on the variable-number tandem repeat (VNTR) is widely applied for identifying the transmission of Mycobacterium tuberculosis. A consensus set of four hypervariable loci (1982, 3232, 3820, and 4120) has been proposed to improve the discrimination of Beijing lineage strains. Herein, we evaluated the utility of these four hypervariable loci for tracing local tuberculosis transmission in 981 cases over a 14-month period in Japan (2010 to 2011). We used six different VNTR systems, with or without the four hypervariable loci. Patient ages and weighted standard distances (a measure of the dispersion of genotype-clustered cases) were used as proxies for estimating local tuberculosis transmission. The highest levels of isolate discrimination were achieved with VNTR systems that incorporated the four hypervariable loci (i.e., the Japan Anti-Tuberculosis Association [JATA]18-VNTR, mycobacterial interspersed repetitive unit [MIRU]28-VNTR, and 24Beijing-VNTR). The clustering rates by JATA12-VNTR, MIRU15-VNTR, JATA15-VNTR, JATA18-VNTR, MIRU28-VNTR, and 24Beijing-VNTR systems were 52.2%, 51.0%, 39.0%, 24.1%, 23.1%, and 22.0%, respectively. As the discriminative power increased, the median weighted standard distances of the clusters tended to decrease (from 311 to 80 km, P < 0.001, Jonckheere-Terpstra trend test). Concurrently, the median ages of patients in the clusters tended to decrease (from 68 to 60 years, P < 0.001, Jonckheere-Terpstra trend test). These findings suggest that strain typing using the four hypervariable loci improves the prediction of active local tuberculosis transmission. The four-locus set can therefore contribute to the targeted control of tuberculosis in settings with high prevalence of Beijing lineage strains.

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Severe Clinical Outcomes of Tuberculosis in Kharkiv Region, Ukraine, Are Associated with Beijing Strains of Mycobacterium tuberculosis

Pathogens ◽

10.3390/pathogens8020075 ◽

2019 ◽

Vol 8 (2) ◽

pp. 75 ◽

Cited By ~ 1

Author(s):

Olha Konstantynovska ◽

Mariia Rekrotchuk ◽

Ivan Hrek ◽

Anton Rohozhyn ◽

Nataliia Rudova ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Clinical Outcomes ◽

Tandem Repeats ◽

Genotypic Variation ◽

Variable Number Tandem Repeat ◽

Severe Infection ◽

Variable Number ◽

Beijing Genotype ◽

Multiple Drug ◽

Public Health Response

Genotypic variation in Beijing lineages of Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), has been associated with hyper virulence and the spread of extensively and multiple drug (X/MDR) resistant MTB strains in Eastern Europe, Central Asia, and East Asia. The clinical outcomes of 215 new cases of TB among the population of the Kharkiv region of Eastern Ukraine were analyzed to uncover factors associated with severe infection. Infecting MTB strains were profiled by 5 locus exact tandem repeats (ETRs) and 15 locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) genotyping. Among diverse MTB genotypes discovered in Ukraine, the Beijing genotype (MIRU-VNTR 42425) was significantly associated with risk factors for severe outcomes of disease in the study population, including TB/HIV co-infection and treatment failure. Strain replacement (superinfection) was observed in 10 patients, suggesting repeated exposure to novel MTB strains in hospital or community settings. Inclusion of MTB genotyping data may identify at-risk patients and improve treatment adherence to prevent X/MDR development for effective public health response against tuberculosis in Ukraine.

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Mycobacterium tuberculosis Beijing Genotype in Russia: in Search of Informative Variable-Number Tandem-Repeat Loci

Journal of Clinical Microbiology ◽

10.1128/jcm.00414-08 ◽

2008 ◽

Vol 46 (11) ◽

pp. 3576-3584 ◽

Cited By ~ 54

Author(s):

I. Mokrousov ◽

O. Narvskaya ◽

A. Vyazovaya ◽

J. Millet ◽

T. Otten ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Tandem Repeat ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Beijing Genotype

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Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates

Journal of Clinical Microbiology ◽

10.1128/jcm.03507-14 ◽

2015 ◽

Vol 53 (7) ◽

pp. 2138-2147 ◽

Cited By ~ 20

Author(s):

Chao Zheng ◽

Song Li ◽

Zhongyue Luo ◽

Rui Pi ◽

Honghu Sun ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Odds Ratio ◽

Variable Number Tandem Repeat ◽

Risk Groups ◽

Variable Number ◽

Mixed Infections ◽

Proper Solution ◽

Content Type ◽

And Control ◽

First Time

Mixed infections and heteroresistance ofMycobacterium tuberculosiscontribute to the difficulty of diagnosis, treatment, and control of tuberculosis. However, there is still no proper solution for these issues. This study aimed to investigate the potential relationship between mixed infections and heteroresistance and to determine the high-risk groups related to these factors. A total of 499 resistant and susceptible isolates were subjected to spoligotyping and 24-locus variable-number tandem repeat methods to analyze their genotypic lineages and the occurrence of mixed infections. Two hundred ninety-two randomly selected isolates were sequenced on theirrpoBgene to examine mutations and heteroresistance. The results showed that 12 patients had mixed infections, and the corresponding isolates belonged to Manu2 (n= 8), Beijing (n= 2), T (n= 1), and unknown (n= 1) lineages. Manu2 was found to be significantly associated with mixed infections (odds ratio, 47.72; confidence interval, 9.68 to 235.23;P< 0.01). Four isolates (1.37%) were confirmed to be heteroresistant, which was caused by mixed infections in three (75%) isolates; these belonged to Manu2. Additionally, 3.8% of the rifampin-resistant isolates showing no mutation in therpoBgene were significantly associated with mixed infections (χ2, 56.78;P< 0.01). This study revealed for the first time that Manu2 was the predominant group in the cases of mixed infections, and this might be the main reason for heteroresistance and a possible mechanism for isolates without any mutation in therpoBgene to become rifampin resistant. Further studies should focus on this lineage to clarify its relevance to mixed infections.

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Effect of Mutation and Genetic Background on Drug Resistance in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.06460-11 ◽

2012 ◽

Vol 56 (6) ◽

pp. 3047-3053 ◽

Cited By ~ 63

Author(s):

Lukas Fenner ◽

Matthias Egger ◽

Thomas Bodmer ◽

Ekkehardt Altpeter ◽

Marcel Zwahlen ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

Hot Spot ◽

Multidrug Resistant ◽

Beijing Genotype ◽

Drug Resistant ◽

Resistance Mutations ◽

Content Type ◽

Drug Resistance Mutations ◽

Resistant Strains

ABSTRACTBacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistantMycobacterium tuberculosisisolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains,katGmutations were associated with high-level andinhApromoter mutations with low-level drug resistance. OnlykatG(S315T) (65.6% of all isoniazid-resistant strains) andinhApromoter −15C/T (22.7%) were found in molecular clusters.M. tuberculosislineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6;P< 0.0001). Lineage 1 was associated withinhApromoter −15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7;P= 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages;P< 0.0001). In conclusion,M. tuberculosisdrug resistance mutations were associated with various levels of drug resistance and transmission, andM. tuberculosislineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds inM. tuberculosisdrug resistance.

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A Predominant Variable-Number Tandem-Repeat Cluster of Mycobacterium tuberculosis Isolates among Asylum Seekers in the Netherlands and Denmark, Deciphered by Whole-Genome Sequencing

Journal of Clinical Microbiology ◽

10.1128/jcm.01100-17 ◽

2017 ◽

Vol 56 (2) ◽

Cited By ~ 5

Author(s):

Rana Jajou ◽

Albert de Neeling ◽

Erik Michael Rasmussen ◽

Anders Norman ◽

Arnout Mulder ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

The Netherlands ◽

Genetic Distance ◽

Genome Sequencing ◽

Tandem Repeat ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Whole Genome ◽

Average Genetic Distance ◽

Content Type

ABSTRACT In many countries, Mycobacterium tuberculosis isolates are routinely subjected to variable-number tandem-repeat (VNTR) typing to investigate M. tuberculosis transmission. Unexpectedly, cross-border clusters were identified among African refugees in the Netherlands and Denmark, although transmission in those countries was unlikely. Whole-genome sequencing (WGS) was applied to analyze transmission in depth and to assess the precision of VNTR typing. WGS was applied to 40 M. tuberculosis isolates from refugees in the Netherlands and Denmark (most of whom were from the Horn of Africa) that shared the exact same VNTR profile. Cluster investigations were undertaken to identify in-country epidemiological links. Combining WGS results for the isolates (all members of the central Asian strain [CAS]/Delhi genotype), from both European countries, an average genetic distance of 80 single-nucleotide polymorphisms (SNPs) (maximum, 153 SNPs) was observed. The few pairs of isolates with confirmed epidemiological links, except for one pair, had a maximum distance of 12 SNPs. WGS divided this refugee cluster into several subclusters of patients from the same country of origin. Although the M. tuberculosis cases, mainly originating from African countries, shared the exact same VNTR profile, most were clearly distinguished by WGS. The average genetic distance in this specific VNTR cluster was 2 times greater than that in other VNTR clusters. Thus, identical VNTR profiles did not represent recent direct M. tuberculosis transmission for this group of patients. It appears that either these strains from Africa are extremely conserved genetically or there is ongoing transmission of this genotype among refugees on their long migration routes from Africa to Europe.

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Standard Genotyping Overestimates Transmission of Mycobacterium tuberculosis among Immigrants in a Low-Incidence Country

Journal of Clinical Microbiology ◽

10.1128/jcm.00126-16 ◽

2016 ◽

Vol 54 (7) ◽

pp. 1862-1870 ◽

Cited By ~ 58

Author(s):

David Stucki ◽

Marie Ballif ◽

Matthias Egger ◽

Hansjakob Furrer ◽

Ekkehardt Altpeter ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Nucleotide Polymorphisms ◽

Foreign Born ◽

Single Nucleotide ◽

Content Type ◽

Weak Evidence ◽

High Incidence ◽

Low Incidence

Immigrants from regions with a high incidence of tuberculosis (TB) are a risk group for TB in low-incidence countries such as Switzerland. In a previous analysis of a nationwide collection of 520Mycobacterium tuberculosisisolates from 2000 to 2008, we identified 35 clusters comprising 90 patients based on standard genotyping (24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat [MIRU-VNTR] typing and spoligotyping). Here, we used whole-genome sequencing (WGS) to revisit these transmission clusters. Genome-based transmission clusters were defined as isolate pairs separated by ≤12 single nucleotide polymorphisms (SNPs). WGS confirmed 17/35 (49%) MIRU-VNTR typing clusters; the other 18 clusters contained pairs separated by >12 SNPs. Most transmission clusters (3/4) of Swiss-born patients were confirmed by WGS, as opposed to 25% (4/16) of the clusters involving only foreign-born patients. The overall clustering proportion was 17% (90 patients; 95% confidence interval [CI], 14 to 21%) by standard genotyping but only 8% (43 patients; 95% CI, 6 to 11%) by WGS. The clustering proportion was 17% (67/401; 95% CI, 13 to 21%) by standard genotyping and 7% (26/401; 95% CI, 4 to 9%) by WGS among foreign-born patients and 19% (23/119; 95% CI, 13 to 28%) and 14% (17/119; 95% CI, 9 to 22%), respectively, among Swiss-born patients. Using weighted logistic regression, we found weak evidence of an association between birth origin and transmission (adjusted odds ratio of 2.2 and 95% CI of 0.9 to 5.5 comparing Swiss-born patients to others). In conclusion, standard genotyping overestimated recent TB transmission in Switzerland compared to WGS, particularly among immigrants from regions with a high TB incidence, where genetically closely related strains often predominate. We recommend the use of WGS to identify transmission clusters in settings with a low incidence of TB.

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First insight into the genotype and molecular epidemiology of MDR-TB isolates from Chongqing Municipality of China using 24-loci MIRU-VNTR technique

10.21203/rs.2.10166/v1 ◽

2019 ◽

Author(s):

Lei Xu ◽

Yan Hu ◽

Tongxin Li ◽

Chun Yang ◽

Yonglin He ◽

...

Keyword(s):

Variable Number Tandem Repeat ◽

Melting Curve ◽

Multidrug Resistant ◽

Variable Number ◽

Beijing Genotype ◽

Molecular Characteristics ◽

Chongqing Municipality ◽

Resistant Tuberculosis ◽

Mdr Tb ◽

High Discriminatory Power

Abstract Background: Multidrug-resistant tuberculosis (MDR-TB) has become a great threat to TB control around the world. In 2017, there were 889,000 new tuberculosis cases in China, and 31.1% were MDR/RR-TB (TB resistant to rifampicin). This study aims to explore the the molecular characteristics and factors associated with infection among different genotype MDR strains in Chongqing, China. Methods: All the 230 MDR-TB isolates were genotyped using 24-loci mycobacterial interspersed repetitive unite variable number tandem repeat (MIRU-VNTR) method and multiplex real-time PCR melting curve assay of Rv2952 gene and mutT2 gene. Polymorphism and clustering analysis of each locus was carried out by BioNumerics Version 5.0. Results: By genotyping, 83.0% (191/230) of the MDR-TB isolates were Beijing strains, among which 41.9% from the ancient Beijing genotype and 58.1% from the modern Beijing genotype. Based on 24-loci, the 230 MDR isolates were classified into 208 genotypes, among which 38 isolates belonged to 16 clusters. The clustering rate was 16.5%. The percentages of SM-resistant and EMB-resistant in Beijing genotype were significantly higher than those in non-Beijing genotype (P < 0.01). The Beijing genotype had a significantly high risk to be clustered than non-Beijing genotype (p < 0.01). The percentages of SM-resistant isolates in clustered group were significantly higher than non-clustered group (P < 0.01). According to 24-loci, the HGDI was 0.9988, and five loci (Qub11b, Qub26, Mtub21, MIRU26, Mtub04) have high discriminatory power (HGDI > 0.6), while 7 loci (MIRU4, MIRU23, Mtub34, MIRU20, Mtub29, MIRU2, MIRU24) showed negligible diversity (HGDI < 0.1). The percentage of clustered rate showed no difference between 24-loci and 15-loci (p = 0.19). Conclusion: Among MDR isolates in Chongqing China, Beijing genotype is more likely to be drug resistant and clustered, and SM-resistant isolates are more likely to be clustered that may related to the MDR epidemic. Although 24-loci had a high resolution of genotype, some new loci should be found to replace the poor diversity loci in 24-loci and additional analysis of specific sublineage of Beijing genotype is needed in order to better understand the relations between the molecular characteristics of strains and MDR-TB epidemic.

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